Purinergic and Immune Signaling

#### **Chapter 1**

## Purinergic System in Immune Response

*Yerly Magnolia Useche Salvador*

#### **Abstract**

In mammalian cells, the purinergic signaling and inflammatory mediators regulate each other. During microbial infection, nucleotides and nucleosides from both dying host cells and pathogens may be recognized by the host receptors. These receptors include purinergic receptors such P2X, P2Y, and A2A, as well Toll-like receptors, and NOD-like receptors. The interaction with most of these receptors activates immune responses, including inflammasome activation, releasing of pro-inflammatory cytokines, reactive nitrogen and oxygen species production, apoptosis induction, and regulation of T cell responses. Conversely, activation of adenosine receptors is associated with anti-inflammatory responses. The magnitude of resultant responses may contribute not only to the host defense but also to the homeostatic clearance of pathogens, or even to the severe progression of infectious diseases. In this chapter, we discuss how the purinergic signaling activation upregulates or downregulates mechanisms in infectious diseases caused by the bacterial, parasite, and viral pathogens, including SARS-CoV-2. As a concluding remark, purinergic signaling can modulate not only infectious diseases but also cancer, metabolic, and cardiovascular diseases, constituting a strategy for the development of treatments.

**Keywords:** purinergic receptors, immune responses, inflammation, infectious disease

#### **1. Introduction**

The purinergic signaling modulates pathways of both neural and non-neural physiological processes, including immune responses, inflammation, pain, platelet aggregation, endothelium-mediated vasodilation, proliferation, and cell death [1]. Three main components are part of the system. Purinergic: extracellular nucleotides and nucleosides, their receptors, and the ectoenzymes responsible for regulating the levels of these molecules [2]. In addition, nucleotides, nucleosides, and uric acid resulting from the death of infected or injured cells are also recognized by other receptors better known for their role in pathogen recognition as Toll-like receptors (TLRs), and NOD-like receptors (NLRs) [3]. Other immune innate receptors are able to detect nucleic acids (RNA or DNA) from either phagocyted or circulating microbes, including retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), the C-type lectin receptors (CLRs), and cytosolic sensors [4,5]. All immune cells are able to recognize nucleotides as a danger signal throughout either purinergic or no purinergic receptors. Immediately various immune responses can be activated,

such as pro-inflammatory cytokines secretion by macrophages, quimocine production by eosinophils, maturation of dendritic cells (DCs), as well as T and B cells costimulation [6].

Extracellular nucleotides and nucleosides are released, along with many other molecules, from dead cells. Apoptosis and necrosis are the cell death mechanisms that

#### **Figure 1.**

*Infected cells release nucleotides and nucleosides. High ATP concentration can induce both dead cell (dashed line, in dendritic cell) and several functions over immune cells as maturation, and quimiotaxis. Low ATP concentration ([ATP]) can induce (arrow) or inhibit (bar-headed line) responses depending on the immune cell. Low adenosine concentration ([ADO]) can induce ROS and NO. High adenosine concentration ([ADO]) inhibit pro-inflammatory cytokine (\*Cytokines) expression. Reddish and bluish background means pro-inflammatory and anti-inflammatory context, respectively. Source: The figure 1 design is original and cell vectors were modified from freepik's: https://www.freepik.es/vectores/personas, People Vector created by brgfx - http://www.freepik.es.*

#### *Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

can operate in physiological conditions. Apoptosis is activated by genetically controlled cell signals to modulate cell growth and development, as it is a programmed event. It is an ordered process that does not trigger inflammation. Conversely, necrosis is a not regulated cell death, characterized by the cell content release as a consequence of the effect of diverse environmental factors, leading to higher inflammation around [7]. Furthermore, during infections, some intracellular pathogens require cell lysis, while others have developed mechanisms to prevent cell death during their replication and dissemination outside the infected cell.

ATP is known as a damage signal, released or leaked by injured cells, or a molecular pattern associated with damage [8]. Necrotic cells may use either pannexin channels or connexin hemichannels to release intracellular ATP, and the P2X7R may be involved in this process [6]. Adenosine is a nucleoside that mediates antiinflammatory and immunosuppressive actions, such as inhibiting the production of pro-inflammatory cytokines and lymphocyte proliferation [9]. In pathological conditions, adenosine plays a protective role acting as an endogenous regulator of innate immunity and in host defense against excessive tissue damage associated with inflammation [10]. The A1 and A2A receptors (A1R and A2AR) are activated by adenosine concentrations in the nanomolar range, while the A2B and A3 receptors (A2BR and A3R) become active only when the extracellular levels of adenosine rise in the micromolar range during periods of inflammation, hypoxia or ischemia [11] (**Figure 1**). Other nucleosides are recognized by several P2 receptors, which are going to explain later.

#### **2. Regulation of extracellular nucleotides and nucleosides**

In the context of the immune response, as mentioned, while the extracellular ATP (eATP) exhibits pro-inflammatory and stimulatory effects in the immune system, either appropriate or exacerbated responses [6], adenosine has primarily antiinflammatory and inhibitory effects [9]. Therefore, the balance between ATP versus adenosine levels is important in modulating cellular immune responses and pathogen survival [12]. The concentrations of extracellular nucleotides and nucleosides are regulated by immune and non-immune cells through the action of enzymes anchored to the cell membrane, with their catalytic site facing the extracellular environment [13]. These enzymes, called ectonucleotidases, hydrolyze extracellular nucleotides into their respective nucleosides to control exacerbated levels of nucleotides and maintain steady-state conditions [12]. Among them, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, CD39, or apyrases), hydrolyzes both ATP and ADP to AMP, in the presence of divalent cations such as calcium and magnesium. Sequentially, the E-5′-nucleotidase (CD73) terminates the ectonucleotidase cascade with the hydrolysis of monophosphate nucleotides, resulting in adenosine. This in turn is hydrolyzed by the enzyme adenosine deaminase (E-ADA), transforming adenosine into inosine, its inactive metabolite [13]. In addition, the ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) yield free nucleosides. The nitrogenous bases are hydrolyzed from nucleosides by the action of phosphorylases that yield ribose-1-P and free bases. If the nucleosides and/or bases are not re-utilized, the purine bases are further degraded to uric acid [3].

Two important environments for the highly reported purinergic signaling activation are the central nervous system (CNS) and the blood. The principal cell type in the brain involved in ATP degradation is the microglia through the expression of

CD39 [14]. The ATP-regulation in the blood is mediated by the red blood cells, which may regulate tissue circulation and O2 delivery by releasing the vasodilator ATP in response to hypoxia. When released extracellularly, ATP is rapidly degraded to ADP in the circulation by ectonucleotidases. Moreover, ADP acting on P2Y13 receptors on red blood cells serves as a negative feedback pathway for the inhibition of ATP release [15].

Nucleosides and nucleotides are recognized by purinergic receptors P1, P2, TLRs, and NLRs. The recognition implies that nucleosides and nucleotides are temporarily held at different concentrations to activate their respective receptors. Purinergic receptors are divided into two families: P1 and P2 receptors [1]. The G-protein coupled metabotropic P1 receptors recognize exclusively extracellular adenosine and can be subdivided into A1R, A2AR, A2BR, and A3R. The P2 receptors can be subdivided into two subtypes: non-selective ion-gated channel P2X receptors (that recognize ATP) and G-coupled P2Y receptors (that recognize ATP, ADP, UTP, UDP, and UDP-glucose) [6]. Actually, it has been described seven P2X receptors (from P2X1 to P2X7) with different affinities for ATP. The P2X7 receptor has a low affinity for ATP (requiring ≥100 mM to be activated; while others can be activated at lower concentrations) [12].

The consensus about the relationship between purinergic signaling and the immune system can be summed up by the opposing effects of ATP and adenosine. The ATP contributes to triggering the inflammatory response along with molecular patterns associated with pathogens [5]. Conversely, the adenosine nucleoside mediates anti-inflammatory and immunosuppressive actions, such as inhibiting the production of pro-inflammatory cytokines and lymphocyte proliferation [9]. However, there are other nucleotides and nucleosides modulating the immune system.

#### **3. Effects of purinergic signaling on the innate and adaptive immune system**

The eATP released from stressed, dying or infected cells bind to P2 receptors (as P2X7R) and may lead to pathogen elimination through several mechanisms: (1) host cell death; (2) inflammasome activation and IL-1β secretion; and (3) production of reactive oxygen species (ROS) and nitric oxide (NO); promoting lysosome and phagosome fusion [16].

P2X7R activation is associated with pore formation, which depends on the concentration and duration of ATP treatment [17], as well as leads to the opening of pores that allow the passage of small molecules (< 900 Da) [18] as dinucleotides or nucleosides, increasing the extracellular concentration of these purinergic ligands. Inflammasomes are multi-protein complexes assembled in the host cell in response to infection or cellular stress, leading to non-homeostatic and lytic cell death, called pyroptosis. P2X7R was shown to activate NLRP3 inflammasomes [19]; and recently, caspase-11-induced pyroptosis was shown to require pannexin-1 channels and the P2X7R activation [20]. Pyroptosis is important because of the cytokines, chemokines, and damage-associated molecular patterns (DAMPs) which are released to the extracellular compartment, and also because intracellular pathogens are exposed to extracellular immune response, thus allowing their destruction [21]. At the same time, the inflammasomes lead to the maturation and secretion of pro-inflammatory cytokines, such as IL-1b and IL-18 [21]. IL-1β affects virtually all cells and organs of the body and is one of the most important cytokines that mediate autoimmunity,

#### *Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

infections, and degenerative diseases [22]. This cytokine has a role in the CNS as an endogenous pyrogenic agent, and it can also induce inflammation, leukocyte recruitment, and Th17 profile immune responses [23]. In addition to eATP, uridin diphosphate (UDP) is released by the cleavage of pannexin-1 channels via caspase in apoptotic cells resulting from the vesicular stomatitis virus infection. Then, the UDP-P2Y6R signaling is able to protect both cells and mice from infection through an increase in IFN-β production, in acute neurotropic infection [24].

Purinergic receptors P1 (P1R) and P2 (P2R) can be expressed simultaneously in almost all immune cells, apparently depending on their ligand concentrations in the extracellular space [25]. Therefore, eATP-P2R interactions also activate proinflammatory responses in immune cells [26], as we have seen in infected or dying non-immune cells.

Neutrophils, granulocytes participating in both immune systems innate and adaptative, are the first immune cells in arrived at the inflammation site, constituting the main acute inflammatory response against pathogens by both phagocytosis and the oxidative microbicidal molecules production [25]. Neutrophils are the more affected cells by the purinergic signaling, probably because they express several purinergic receptors [26]. For instance, P2Y11 receptor (P2Y11R) is responsible for the ATP-mediated differentiation and maturation of granulocytic progenitors in the bone marrow [27]. Also, the interaction between eATP-P2Y11R mediates the inhibition of neutrophil apoptosis [28] and increases the chemotactic response of neutrophils [29]. In addition to eATP, other nucleotide released by damaged cells is the uridine 5′-diphosphoglucose (UDP-glucose), which activates the P2Y14R signaling. UDP-glucose promotes chemotaxis of freshly isolated human neutrophils through P2Y14R activation [30]. Moreover, some inflammatory diseases have been related to P2Y14R activation. During pelvic inflammatory disease, in the endometria in both women and female mice, the P2Y14R and pro-inflammatory cytokines as IL-8 are upregulated in the epithelium [31]. Then, the design of therapies to modulate mucosal immunity may be done by targeting P2Y14R [30].

Occasionally, some nucleosides are more concentrated than the ATP in the extracellular matrix. When uridine triphosphate (UTP) is more available than ATP, P2Y2 receptors (P2Y2R) may be activated by either ATP or UTP mediating several activities in the P2Y2R expressing cells. For instance, fibrotic lung disease is related to some activities mediated by P2Y2R such as the lung fibroblast's proliferation and migration, the recruitment of neutrophils, and IL-6 secretion in the lungs [32]. By the other way, when the eATP-P2X1R signaling is activated in neutrophils and platelets, activated neutrophils are recruited to the injury site and their adherence to vessel walls together with the platelets occurred, promoting both thrombosis and fibrinogenesis [33].

In addition to neutrophils, the eosinophils, and basophils, other granulocyte cells, which are activated during parasitic infections and allergies, are also regulated by the purinergic system. The accumulation of eosinophils during lung inflammation is triggered by UTP-P2Y2R interaction that induces the expression of VCAM-1, an adhesion molecule, which in turn, induces changes in endothelial cell shape for the opening of passageways through which eosinophils migrate [34]. Moreover, P2Y2R activation by ATP in eosinophils has been reported to induce chemotaxis in allergic lung inflammation [35]. In other circumstances, when UDP have more concentrated than UTP or ATP, the UDP-P2Y6R signaling induces IgE-dependent degranulation in human basophils [36].

During infections, dendritic cells (DCs) are responsible for presenting antigens to naive T cells and activating them, making a link between the innate and adaptive immune response [37]. The ATP-P2R interaction is involved in the migration and differentiation of DCs [38]. Specifically, the eATP-P2Y11R interaction modulates the maturation of human monocyte-derived dendritic cells (MoDCs) [39]. Moreover, P2Y2R activation by ATP promotes chemotaxis of MoDCs [35]. In fact, the eATP-P2Y11R interaction mediates the migration of DCs accordingly with the DC type, although all DC populations express P2Y11R. MoDCs down-regulate the P2Y11R expression, decreasing the inhibition of migration triggered by ATP. While either interleukin-3 receptor-positive plasmacytoid DCs or CD1c + peripheral blood DCs do not inhibit their migration by ATP. Then, the possibility of a meeting between DCs and antigens may be mediated by gradients of ATP formed in and around inflamed areas. Therefore, after vaccination, the migration of DCs charged with antigens to near lymph nodes may be increased with the inhibition of P2Y11R expression. This strategy could improve the time of response after vaccination [38]. In addition, P2Y12 receptor (P2Y12R) modulates murine DCs function by ADP, including induction of intracellular Ca2+ transportation, macropinocytosis, and T-cell stimulation [40]. However, the stimulation of the P2X7R with ATP can induce cell death; such as in murine spleen-derived DCs, which increase the permeabilization and the intracellular calcium, resulting in apoptosis [41].

As reviewed, the T-cell activation by DCs can be modulated by the purinergic system. Additionally, lymphocytes (mainly T and B cells) that are characterized by expressing antigen receptors, allowing the activation of anti-microbial responses [25], also express purinergic receptors which modulates lymphocyte proliferation, differentiation, and functioning. Immature T cells pass through the thymus for differentiation, where stromal epithelial cells are in charge of both the positive and negative selection processes, which in turn defines the T cell functional profile between CD8+ or CD4+ cells [42]. In the thymus, P2X7R and P2Y2R are expressed in several cells as murine thymic epithelial cells (TECs), leading to the release of calcium from intracellular stores and increasing the permeabilization membrane [43]; possibly leading to TECs apoptosis as well as reported in DCs [41], and ending in the alteration of both T-cell differentiation and their peripheral functioning. Similarly, the eATP-P2X7R signaling leads to the opening of a transmembrane cationic channel that allows K+ efflux and Na + and Ca2+ influx and promotes cytoplasmic membrane depolarization in the phagocytic cell of the thymic reticulum [18], leading to increase permeabilization and apoptosis, and impairing of T-cell precursors proliferation.

During immune synapse, naïve T-cells release ATP throughout pannexin-1 channels, then the eATP interaction with P2X1R and P2X4R receptors regulates T-cell activation, calcium entry, and IL-2 release [44]. Also, γδ T-cells, abundant at barrier sites such as the skin, gut, lung, and reproductive tract, are activated and upregulated tumor necrosis factor-alpha (TNF-α), and interferon-γ (IFN-γ) release through the eATP-P2X4R interaction [45]. Moreover, P2X4Rs participate in the migration process of CD4+ T-cells. This migration is mediated by the chemokine stromal-derived factor-1α (SDF-1α), which triggers the T-cell polarization by the accumulation of ATP producing mitochondria near ATP-releasing pannexin-1 channels and newly expressed P2X4Rs. This set of molecules promotes both the Ca2+ influx and sustained mitochondrial ATP production required for the pseudopod protrusion and T-cell migration [46]. Conversely, exogenous activation of P2Y11R with eATP blocks T-cell trafficking [47].

Specifically, the purinergic system may modulate inflammatory severe clinical conditions like sepsis. This condition has two phases, first is the hyperinflammatory

#### *Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

phase, which may be later restricted by the immunosuppressive phase. However, this last phase characterized by high blood levels of regulatory T cells (Tregs) is strongly associated with mortality. Tregs proliferation is controlled by P2Y12R activation in both Tregs and platelets, and P2Y12R blockade restores the immunological homeostasis. Therefore, this strategy may guide pharmacological treatment for sepsis and increase patient survival [48].

Among innate immune cells acting mainly in chronic inflammatory responses are the mononuclear phagocytes, which are in circulation as monocytes or in several tissues as macrophages, or in specific tissues as microglial cells in the brain. These phagocytes may have either a pro-inflammatory or anti-inflammatory role depending on the type of cytokines around them and express several P2 receptors [25]. For instance, the major pathway of macrophage activation is the eATP-P2Y11R signaling, which leads to cytokine release [49]. Moreover, macrophages exposed to LPS, increase the P2YR and P2X7R activation mediated by eATP, modulating the IL-1β, TNF-α, and NO production [50]. Monocyte adhesion process is also regulated by the ATP-P2R interaction [51]. For example, activated P2Y12R induces both vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte adhesion into the vascular wall, promoting atherosclerotic lesions [52].

Microglia is the cell responsible for the immune function of the nervous system in both physiological and pathological conditions [53]. Increased P2X7R expression and its ATP-mediated activation in microglia are observed after the LPS brain challenge, leading to increased immune response associated with NO and ROS, along with reduced neuronal viability. Inhibition of this purinergic response may be a neuroprotective strategy in brain inflammatory diseases [54]. In addition, the upregulation of P2Y6, P2Y12, P2Y13, and P2Y14 receptors in spinal microglia is associated with the development of neuropathic pain [55]. In an inflammatory context, ADP acting on P2Y12R induces extension of microglia processes thereby attracting this cell to the site of ATP/ADP leaking or release. Moreover, the ADP-P2Y12R activation in microglia induces intracellular calcium accumulation, which in turn causes the increase of CC chemokine ligand 3 (CCL3) expression in the peripheral injured site and also in the spinal cord, inducing neuropathic pain. Since the inhibition of CCL3-CCR5 signaling suppresses the development of neuropathic pain, treatments based on inhibition of CCL3 expression can be promising to control this kind of inflammatory disorder [56].

The P2Y6R is also upregulated in microglia when neurons are damaged, then the UDP-P2Y6R signaling facilitates microglial phagocytosis [57]. Consistently, the brain injury caused by ischemic accidents is increased by the inhibition of both P2Y6R expression and the microglia-phagocytic activity [58]. The UDP-P2Y6R signaling is also associated with neuropathic pain and is partially explained by the induction of CCL2 production through the MAP kinases-NF-kappaB pathway in microglia [59]. CCL2 is a recruitment factor of myeloid cells to the regions with injured neurons. In the spinal cord, CCL2 released from primary afferent neurons and reactive astrocytes could contribute to either the induction or maintenance of chronic pain [60], neurodegenerative and neuroinflammatory diseases [61].

As mentioned above, in addition to purinergic receptors, other receptors are able to recognize eATP such as the NLRs and TLRs. These receptors recognize both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (including eATP and uric acid). The relationship between these receptors and nucleotides or nucleosides, and the structure and functions of NLRs will be addressed immediately. TLRs will be explained through the case of gout disease.

The NLRs are cytoplasmic receptors and the structure has three domains: a common domain organization with a central conserved domain NOD (NACHT: NAIP, CIITA, HET-E, and TP-2), N-terminal effector domain, and C-terminal leucine-rich repeats (LRRs) [62]. The NAIP motif (neuronal apoptosis inhibitor protein) and the CIITA motif (MHC class II transcription activator) contain a distinct predicted nucleoside triphosphatase (NTPase) domain. In addition, NTPase domain in CIITA shows highly significant sequence similarity to CARD4 (pro-apoptotic protein). Therefore, the NACHT family includes both pro-apoptotic (e.g. CARD4) and anti-apoptotic (e.g. NAIP) predicted NTPases [63]. In consequence, all NLRs could modulate apoptotic process. However, either the possible apoptotic effect or the ATPase activity of the NATCH domain and the consequence on the concentration of nucleotides and derivatives in the cytoplasm must be the subject of study.

Most NLRs recognize various ligands activating inflammatory responses. These ligands come from different sources, including microbial pathogens (peptidoglycan, flagellin, viral RNA, fungal hyphae, etc.), host cells (ATPs, uric acid, etc.), and esteril activators (alum, silica, UV radiation, skin irritants, etc.). In addition, some NLRs respond to cytokines such as interferons. The activated NLRs show various functions that can be divided into four broad categories: inflammasome formation, signaling transduction, transcription activation, and autophagy [64]. Several NLRs have been used to identify inflammasomes, depending on the receptor that recognizes the PAMPs (for example, NLRP1, NLRP3, AIM2, NLRC4), while the other group of inflammasomes can be activated by cytosolic lipopolysaccharides (LPS) derived from gram-negative bacteria. The NLRP3 inflammasome can be activated by different stimuli, such as bacterial, viral, and fungal pathogens, pore-forming toxins, crystals, silica, and DAMPs (for example, eATP) [65]. The activation of the NLRP3 inflammasome requires two signals: (1) a PAMP, such as LPS, leading to transcription of NF-kB, upregulating genes encoding proinflammatory cytokines, chemokines, and proteins involved in the inflammasome platform; and (2) a DAMP, such as eATP, which induces inflammasome activation after ligation to the P2X7R. Once activated, these complexes promote activation of the protease caspase-1, which cleaves pro-IL-1β and pro-IL-18 into their active forms: IL-1β and IL-18 [12].

In addition to the inflammasome activation, eATP induces ROS production. ROS are highly reactive chemicals formed from O2 (such as peroxides, superoxides, and hydroxyl radicals) [66]. For instance, respiratory epithelial cells induce mitochondrial ROS in response to influenza infection. ROS induces the expression of type III interferon, a response associated with viral infection control [67]. Moreover, *Porphyromonas gingivalis (P. gingivalis)* infection of gingival epithelial cells induces assembly of the P2X4R and P2X7R to form a pore, pannexin-1, promoting ROS production triggered by ATP-P2X7R activation. Later the ROS can activate the NLRP3 inflammasome and caspase-1, resulting in bacteria death [68].

Some TLRs recognize in addition to PAMPs from intracellular or extracellular pathogens, others molecules like ATP and uric acid. TLRs recognizing uric acid are involved in several metabolic and cardiovascular diseases, including gout, chronic renal tubular damage, autosomal dominant polycystic kidney disease, and cartilage degeneration. Among them, the accumulation of uric acid crystals (monosodium urate - MSU -) in the joints causes arthritis which is the base of the metabolic disease called Gout [64]. The uric acid also could cause inflammatory events by the activation of the NLRP3 inflammasome and its activation induces the IL-1β formation, which leads to the development of gouty arthropathy [69]. Moreover, when the level of uric

#### *Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

acid is higher than 6.8 mg/dL, MSU crystals are formed and are recognized by TLRs. These TLRs then activate the NALP3 inflammasome. MSU also triggers neutrophil activation and further produces immune mediators, which lead to a pro-inflammatory response [70]. In the mice model of Gout, it was found that MSU crystals are recognized by TLR2 and TLR4 with the participation of the TLR adapter protein myeloid differentiation factor 88 (MyD88) in bone marrow-derived macrophages. After recognition by these TLRs, MSU induced the production of pro-inflammatory cytokines as IL-1β, TNF-α, keratinocyte-derived cytokine/growth-related oncogene alpha (KDC/GROα), and transforming growth factor beta1(TGF-β1). Moreover, neutrophil influx, local induction of IL-1β, and more pro-inflammatory reaction were promoted [71].

In addition, patients with hyperuricemia (high levels of uric acid in the blood) develop vascular diseases associated with the formation of aminocarbonyl radicals from excess uric acid with the concomitant oxidative effect [72]. Additionaly, metabolic and cardiovascular diseases are associated with the activation of the reninangiotensin system (RAS) mediated by uric acid, in adipose tissue. For instance, high blood pressure and increased expression of both TLR2/4, pro-inflammatory cytokines (TNF-α and IL-6), and RAS activation in adipocytes were found in hyperuricemic rats. These high levels of cytokines and RAS components were reverted by TLR2/4 RNA silencing [73]. Proinflammatory pathways are induced by uric acid and angiotensin II-mediated by TLR4 in renal proximal tubular cells developing chronic tubular damage [74]. Moreover, TLR-2 and TLR-4 gene expressions are associated with rapid progression in autosomal dominant polycystic kidney disease (ADPKD) patients [75]. Furthermore, in human chondrocytes, the accumulation of both calcium pyrophosphate dihydrate (CPPD) and MSU crystals was associated with increased expression of TLR2 and the NO generation triggered by TLR2 signaling, inducing inflammation and cartilage deterioration. Other TLR signaling pathways producing NO release are induced by both MSU and CPPD crystals, including the Pl3K/Akt/NF-κB signaling pathway and other mediators as MyD88, IRAK1, and TRAF6 [76].

#### **4. Downregulation of immune responses by purinergic signaling**

In the lymph nodes and spleen, lymphocytes are stimulated through eATP-P2X7R interaction to promote the Th1 pro-inflammatory response [77]. However, the eATP may also play an immunosuppressive role. This mechanism occurs mainly when eATP is in low (micromolar) concentrations, increasing its affinity for P2YR, located on the surface of lymphocytes. When stimulated, P2YRs promote the downregulation in the expression and release of pro-inflammatory cytokines, promoting a protective effect against excessive tissue damage [25]. Moreover, chronic exposure of DCs to low ATP doses reduces the capacity to stimulate the Th1 response, while Th2 response is favored [78], which induces the activation of T-cells with an anti-inflammatory profile. However, micromolar levels of eATP through P2Y2R induce the mechanisms of phagocytosis and increase ROS and NO production by macrophages and neutrophils [25].

The most recognized effector of anti-inflammatory responses of the purinergic system is adenosine. Extracellular adenosine is recognized by P1 receptors (A2AR and A2BR). High concentrations of adenosine activate A2AR, inhibiting the production of pro-inflammatory cytokines by macrophages [79], and also decreasing the production of ROS and NO by neutrophils, monocytes, and macrophages. However, low concentrations of adenosine (lower than micromoles) increase phagocytosis and ROS production by activation of A1R in neutrophils [25]. Also, adenosine acts on A2AR inhibiting the production of IL-12 and TNF-α in mice liver and preventing the damage by injury [10].

A1AR and A2AR are abundantly expressed at synapses in the CNS, modulating the synaptic efficacy [80]. A1AR and A2AR receptors are also expressed in the microglia and their activation promotes anti-inflammatory and migration activities, respectively [81]. In the presence of mild alterations of CNS high amounts of ATP can be release, then the activation of P2X7R induces both activation and pro-inflammatory response by microglia, leading to surrounding neuronal death [82]. Therefore, the ATP regulation in the CNS is critical; it has been suggested that CD39 expression has an essential role in cell proliferation and growth, inflammatory processes, and triggering cellular responses from ATP-induced contribute to apoptosis and host defense [83]. Moreover, *in vivo* studies on brain trauma and Alzheimer's disease, neuroinflammation has been detected associated with ATP release from microglia, occurring in an uncontrolled way mainly through pannexin/connexin hemichannels [84]. While adenosine binding to A1R or A2AR during brain disorder exerts neuroprotective and immunosuppressive capacities, respectively [85].

#### **5. Pathogens and purinergic signaling mediating immune response**

Some obligated intracellular bacterial pathogens have diverse target organs. For instance, *Mycobacterium tuberculosis* (*M. tuberculosis*) invades lungs, kidney, spinal cord, and brain, while *Chlamydia trachomatis* (*C. trachomatis*) infects genital and ocular tissue. It has been reported that these pathogens may be controlled by eATP treatment. The eATP treatment of macrophages enhances their antimicrobial properties in a P2X7R-dependent manner. For instance, eATP-related killing of *M. tuberculosis* and *C. trachomatis* within human and murine macrophages is associated with mobilization of intracellular Ca+2 and consequently lysosomal fusion and acidification of the containing-pathogen phagosomes [86, 87]. Moreover, adenine nucleotides (AMP and ATP) and adenosine can inhibit *C. trachomatis* growth in epithelial cells; for instance, micromolar eATP concentrations reversibly inhibit chlamydial infection via the P2X4 receptor in epithelial cells [88]. While millimolar eATP concentrations are sufficient to inhibit chlamydial infection via P2X7 receptor in macrophages [89].

Another bacteria controlled by eATP-triggered mechanisms is *P. gingivalis*, an intracellular bacterium that infects gingival epithelial cells (GECs) and the oral mucosa, causing periodontitis [90]. The mechanisms described to control *P. gingivalis* via eATP activation are (1) P2X7R-mediated apoptosis [91]; (2) ROS production via P2X7R-NADPH oxidase signaling [92] inflammasome activation and IL-1β release [93]. Conversely, adenosine-receptors signaling downregulates the immune response. For example, A2AR stimulated by its agonist CGS21680 increases the *P. gingivalis* proliferation in GECs [94]. On the other hand, *P. gingivalis* can inhibit eATP-induced apoptosis in GECs through the secretion of the enzyme nucleoside-diphosphatekinase (NDK), which can hydrolyze eATP [92], inhibiting the three eATP-triggered mechanisms to control the bacteria.

Intracellular protozoan parasites as *Leishmania amazonensis (L. amazonensis), Toxoplasma gondii (T. gondii)*, and *Trypanosoma cruzi (T. cruzi)* may also be controlled by eATP. Murine macrophages infected with *L. amazonensis* and cells from established cutaneous lesions enhanced P2X7R expression and were more responsive to

#### *Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

eATP activation, inhibiting parasite growth [95]. Also, UTP inhibits *L. amazonensis* infection in murine macrophages, probably by P2Y2R or P2Y4R activation, inducing morphological damage inside the parasite promoting apoptosis of macrophages, producing ROS and reactive nitrogen species (RNS), and increasing intracellular Ca+2 concentrations [96]. Nonetheless, several species of *Leishmania* modulate eATP and adenosine levels by directly acting on these molecules or by inducing CD39 and CD73 expression on the infected cells, influencing the immune response and contributing to parasite growth or survival [97]. First, saliva from phlebotomine sand flies, *Leishmania* promastigotes vector, is rich in adenosine and AMP, which levels are mediated by the enzymatic activity of apyrases and 5′-nucleotidases present in saliva [98]. Therefore, low levels of ATP decrease the activation of P2X and P2Y, inhibiting platelet aggregation [99], leading to the free spread of the parasite. Second, parasite-infected cells increase the expression of ectonucleotidases (CD39 and CD73) on their surface and, therefore, also the production of extracellular adenosine. Later, adenosine mediates the activation of A2BR, necessary for the expression of CD40 (DC activation marker). Conversely, the blockade of the A2BR inhibits the DC activation and interferes with T cell proliferation [100]. Third, the A2BR activation inhibits the production of NO and IL-12 by infected macrophages [101], allowing the parasite survive. Moreover, the increased A2BR expression involved IL-10 production by infected cells, in monocytes from patients with visceral leishmaniasis [102], inducing an anti-inflammatory response.

During the acute toxoplasmosis, in the mice brain, occurs an increase in purines (ATP, ADP, AMP, adenosine, xanthine, hypoxanthine, and uric acid), while in chronic toxoplasmosis reduction of the same purines, except the antioxidant, uric acid, occurs [103]. Specifically, the high levels of xanthine and hypoxanthine are associated with the inhibition of the enzyme xanthine oxidase, which catalyzes the production of uric acid, reported in *T. gondii* infected mice [104]. Moreover, in mice with toxoplasmosis, the elevated ATP levels promote increased levels of calcium inside infected cells mediated by P2X receptors, causing damage to the cells and contributing to nervous disorders and behavioral alterations [105]. Besides, *T. gondii is* eliminated by the activation of eATP-P2X7R signaling in infected macrophages mediated by the acidification of the parasitophorous vacuole and ROS production [106]. Additionally, UTP and UDP treatment in murine macrophages infected with *T. gondii* promotes 90% parasite elimination without inducing NO, ROS or apoptosis in the host cell [107]. Interestingly, UTP and UDP induced prematurely parasite egress from the host cell via P2Y2R, P2Y4R, and P2Y6R thus compromising infectivity and replication of the egressed parasites [107].

Interestingly, *T. gondii* does not produce adenosine, then the efficient transformation to the bradyzoite or long-lived cyst stage depends on the extracellular adenosine produced by ectonucleotidases expressed by infected cells [108]. CD73 expression promotes *T. gondii* differentiation and cyst formation by a mechanism dependent on adenosine generation, but independent of adenosine-receptor signaling [108]. In fact, some pathogens stimulate extracellular adenosine generation independently of the host. *Staphylococcus aureus* produces adenosine synthase A (AdsA), a cell wallanchored enzyme which allows the bacteria to escape from clearance by phagocytosis and favoring the formation of organ abscesses. Moreover, bacteria from the gastrointestinal tract as *Enterococcus faecalis* and *Streptococcus mutans* possess homologs of adenosine synthase [109].

During acute *T. cruzi* infection in CNS, the parasite stimulates P2X7R expression in the cerebral cortex, being activated by the available eATP. As a consequence, P2X7R activation induces ATP release, from immune and non-immune cells, chiefly via pannexin hemichannels-boosting inflammation [83]. Besides, the P2X7R activation by the parasite transialidase is involved in the massive loss of immature CD4/CD8 double-positive cells, which determine the prominent thymus atrophy in acute *T. cruzi* infection [110]. In addition, eATP mediates mechanisms to control the parasite as astrocyte proliferation and differentiation, cytokine release, and the ROS and RNS formation. Furthermore, ATP, ADP, and AMP hydrolysis occur in infected animals, related to the enzymatic modulation in the presence of high parasitism [111]. As mentioned, adenosine has a neuroprotective role; however, E-ADA activity is augmented in infected animals, producing iosine which is later used in the purine rescue pathway of *T. cruzi* [83], and in other parasites such as *Trypanosoma evansi* [112] and *Plasmodium falciparum* [113].

Interestingly, some pathogens have evolved extracellular nucleotide-hydrolyzing enzymes that mimic the ectonucleotidases expressed in the host, probably inhibiting the ATP-driven immune response [114]. For instance, the surface of *T. cruzi* expresses an Mg2+-dependent ecto-ATPase enzyme (Mg-eATPase), which have higher activity in trypomastigotes, maybe promoting the host infection [115]. Another parasite with Mg-eATPase is *L. amazonensis*, whose virulent promastigotes are very efficient in hydrolyzing eATP and acquiring adenosine, which is used by the parasite [116].

The participation of the purinergic system in the immune response and pathogenesis as a consequence of SARS-CoV-2 virus infection has also been reported. SARS-CoV-2 induces the IFN response in patients, through MDA5-mediated RNA sensing with the participation of IRF3, IRF5, and NF-κB/p65 pro-inflammatory transcription factors [117]. However, Coronaviruses can evade the MDA5 recognition by forming endoplasmic reticulum-derived membrane vesicles around their RNA [118], delaying the IFN production; and in consequence, allowing higher viral replication. Viral load is highly correlated with the levels of IFNs and TNF-α, suggesting that viral load may drive high cytokine production [119]. Increased levels of TNF-α during inflammation induce ATP release via pannexin-1 channels [120]. ATP exportation out of the cell implies a deficit of intracellular ATP available for the ATP-dependent enzymes in the JAK–STAT pathway induced by IFN-I, limiting the cytokine expression and T helper cell activation [121].

At the same time, a pro-inflammatory immune response is initiated by the increase in the extracellular ATP and ADP levels in the microenvironment of immune cells activating the P2XRs and P2YRs [122]. The eATP-P2X7R signaling activation is a key process in the hyper inflammation resulting from the severe pro-inflammatory immune response against SARS-CoV-2 [123]. High levels of eATP are accompanied by the desensitization of all P1 and P2 purinergic receptors, except P2X7R, inducing more hyper inflammation [124], the worst scenario for a COVID-19 patient.

Shortly after the inflammatory explosion or simultaneously, the eATP concentration could decrease by the CD39-mediated transformation into eADP and eAMP, while adenosine quickly increases by the CD73-mediated eAMP conversion [125]. Then, immunosuppressive responses are activated by the adenosine excess in interaction with their A2AR and A2BR, including inhibition of macrophages and lymphocytes [10].

Moreover, increased eADP levels promote platelet activation and intravascular thrombosis mediated by P2YRs [126], and COVID-19 patients with pneumonia frequently developed microvascular thrombosis in their lungs [127]. In summary, the degree of involvement of purinergic receptors and their ligands in the response to SARS-CoV-2 virus infection may partially explain, the presence of asymptomatic infected people and the variation in the severity among the COVID-19 patients.

#### **6. Perspectives**

During inflammation, macrophages, NK cells, and some lymphocytes activities are impaired by the interaction of their adenosine receptors and the high extracellular levels of adenosine [10]. Therefore, the factors involved in extracellular adenosine production may be used in anti-inflammatory strategies, including the ectonucleotidases CD39 which degrades ATP into AMP, and the ectonucleotidase CD73 which converts AMP into adenosine. Following this rationale, several monoclonal antibodies (mAb) have been developed using CD73, CD39, and A2AR receptors as a target [128]. For instance, a humanized anti-CD39 mAb prevents the ATP-ADP conversion. Moreover, the enhancement of T cells and NK cells function was found, when CD39 was blocked by either antibodies or inhibitors such as POM-1; aside from increased T cell proliferation by the lack of suppression exerted by Treg cells [129].

Moreover, the prevention of AMP to adenosine conversion is also achieved using the mAb anti-CD73 which leads to its internalization [130]. As consequence, the adenosine low levels can not inhibit lymphocytes, therefore CD8 and macrophages activities are enhanced, while both myeloid suppressor cells and Treg lymphocytes are inhibited [128]. Lymphocyte proliferation is also promoted with the administration of an A2AR antagonist in two ways, removing checkpoints on both CD4+ FoxP3+ Tregs and CD8+ effector T cells development, and inhibiting the expression of the programmed death-1 receptor (PD-1) in draining lymph nodes [131]. Some of these drugs have been used as anti-cancer therapies, nevertheless, they have a potential action in many diseases based on immunosuppressive mechanisms.

On the other hand, antagonists of P2X7R as lidocaine can disrupt hyperinflammation, leading to the activation of anti-inflammatory responses. For instance, the clonal expansion of Tregs in lymph nodes is promoted by the P2X7Rs-mediated inhibition of the immune cells in the lymphatic system. Later, the Tregs control the hyperinflammation throughout their anti-inflammatory mechanisms [16]. Also, since eATP-P2Y11R signaling is highly activated in macrophages, P2Y11R antagonists maybe they can be used for the treatment of inflammatory diseases [40]. These strategies may constitute immunotherapy with promising results for inflammatorybased diseases, such as severe forms of various viral or bacterial infections, or even autoimmune diseases.

#### **7. Conclusion**

PX and PY receptors are involved in the inflammasome activation, apoptosis induction, oxidant production and activation of several immune cells, mechanisms that can control the infection of several pathogens. Conversely, adenosine is generally associated with the downregulation of inflammation. However, the effects triggered by eATP and nucleosides and their respective purinergic receptors in infected cells, depend on several aspects. These include first, the ability of the receptor expression by infected cells; second, the mechanisms to maintain the balance of nucleotide and nucleoside concentrations in the extracellular environment; and third, the survival strategies of specific pathogens.

The purinergic signaling can modulate infections by different intracellular pathogens, including viruses, bacteria, and parasites, and mediates inflammatory processes in metabolic, cardiovascular, and cancer diseases. For this reason, this knowledge field represents an important focus for future research regarding the survival and elimination of different pathogens and the maintenance of the homeostasis of the diseases related to hyper-inflammation.

#### **Acknowledgements**

Post-doctoral Fellowship of Research Support Foundation of the State of Rio de Janeiro–FAPERJ—Health Research Networks Program in the State of Rio de Janeiro—2019, Brazil. Processo E-26/ 202.139/2020.

#### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Yerly Magnolia Useche Salvador Laboratory of Thymus Research, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil

\*Address all correspondence to: yerly.useches@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Burnstock G. Purine and pyrimidine receptors. Cellular and Molecular Life Sciences. 2007;**64**:1471-1483

[2] Yegutkin G. Nucleotide and nucleoside converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochimica et Biophysica Acta. 1783;**2008**:673-694

[3] Ishii KJ, Akira S. Potential link between the immune system and metabolism of nucleic acids. Current Opinion in Immunology. 2008;**20**:524- 529. DOI: 10.1016/j.coi.2008.07.002

[4] Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007;**449**:819e26

[5] Ishii KJ, Koyama S, Nakagawa A, Coban C, Akira S. Host innate immune receptors and beyond: Making sense of microbial infections. Cell Host & Microbe. 2008;**3**:352-363

[6] Idzko M, Ferrari D, Eltzschig HK. Nucleotide signalling during inflammation. Nature. 2014;**509**:310e7

[7] Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: Mechanistic description of dead and dying eukaryotic cells. Infection and Immunity. 2005;**73**:1907-1916. DOI: 10.1128/ IAI.73.4.1907-1916.2005

[8] Di Virgilio F. Purinergic mechanism in the immune system: A signal of danger for dendritic cells. Purinergic Signalling. 2005;**1**:205-209

[9] Gessi S, Merighi S, Varani K, Cattabriga E, Benini A, Mirandola P, et al. Adenosine receptors in colon carcinoma

tissues and colon tumoral cell lines: Focus on the a(3) adenosine subtype. Journal of Cellular Physiology. 2007;**211**:826-836. DOI: 10.1002/jcp.20994

[10] Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature. 2001;**414**:916-920. DOI: 10.1038/414916a

[11] Olah ME, Stiles GL. Adenosine receptor subtypes: Characterization and therapeutic regulation. Annual Review of Pharmacology and Toxicology. 1995;**35**:581-606

[12] Coutinho-Silva R, Ojcius DM. Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites. Microbes and Infection. 2012;**14**:1271e7

[13] Zimmermann H. Biochemistry, localization and functional roles of ectonucleotidases in the nervous system. Progress in Neurobiology. 1996;**49**:589-618

[14] Braun N, Sévigny J, Robson SC, Enjyoji K, Guckelberger O, Hammer K, et al. Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. The European Journal of Neuroscience. 2000;**12**:4357-4366

[15] Wang L, Olivecrona G, Gotberg M, Olsson ML, Winzell MS, Erlinge D. ADP acting on P2Y13 receptors is a negative feedback pathway for ATP release from human red blood cells. Circulation Research. 2005;**96**:189-196. DOI: 10.1161/01.res.0000153670.07559.e4

[16] Almeida-da-Silva CLC, Morandini AC, Ulrich H, Ojcius DM, Coutinho-Silva R. Purinergic signaling during Porphyromonas gingivalis infection. Biomedical Journal. 2016;**39**:251-260. DOI: 10.1016/j. bj.2016.08.003

[17] Morandini AC, Savio LE, Coutinho-Silva R. The role of P2X7 receptor in infectious inflammatory diseases and the influence of ectonucleotidases. Biomedical Journal. 2014;**37**:169e77

[18] Coutinho-Silva R, Alves LA, de Carvalho AC, Savino W, Persechini PM. Characterization of P2Z purinergic receptors on phagocytic cells of the thymic reticulum in culture. Biochimica et Biophysica Acta. 1996;**1280**(2):217- 222. DOI: 10.1016/0005-2736(95)00293-6

[19] Pelegrin P. P2X7 receptor and the NLRP3 inflammasome: Partners in crime. Biochemical Pharmacology. 2021;**187**:114385. DOI: 10.1016/j. bcp.2020.114385

[20] Yang D, He Y, Munoz-Planillo R, Liu Q, Nunez G. Caspase-11 requires the Pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock. Immunity. 2015;**43**:923e32

[21] Lamkanfi M, Dixit VM. Modulation of inflammasome pathways by bacterial and viral pathogens. Journal of Immunology. 2011;**187**:597e602

[22] Garlanda C, Dinarello CA, Mantovani A. The interleukin-1 family: Back to the future. Immunity. 2013;**39**:1003e18

[23] Dinarello CA. Anti-inflammatory agents: Present and future. Cell. 2010;**140**:935e50

[24] Li R, Tan B, Yan Y, Ma X, Zhang N, Zhang Z, et al. Extracellular UDP and

P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-beta production. Journal of Immunology. 2014;**193**:4515-4526. DOI: 10.4049/jimmunol.1301930

[25] Bours MJ, Swennen EL, Di Virgilio F, Cronstein BN, Dagnelie PC. Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacology & Therapeutics. 2006;**112**:358-404. DOI: 10.1016/j. pharmthera.2005.04.013

[26] Hasan D, Shono A, van Kalken CK, van der Spek PJ, Krenning EP, Kotani T. A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling. Purinergic Signal. 2021;**10**:1-47. DOI: 10.1007/ s11302-021-09814-6

[27] van der Weyden L, Conigrave AD, Morris MB. Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor. Immunology and Cell Biology. 2000;**78**:369-374. DOI: 10.1046/j.1440-1711.2000.00918.x

[28] Vaughan KR, Stokes L, Prince LR, Marriott HM, Meis S, Kassack MU, et al. Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor. Journal of Immunology. 2007;**179**:8544-8553

[29] Alkayed F, Kashimata M, Koyama N, Hayashi T, Tamura Y, Azuma Y. P2Y11 purinoceptor mediates the ATPenhanced chemotactic response of rat neutrophils. Journal of Pharmacological Sciences. 2012;**120**:288-295

[30] Barrett MO, Sesma JI, Ball CB, Jayasekara PS, Jacobson KA, Lazarowski ER, et al. A selective highaffinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated

*Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

chemotaxis of human neutrophils. Molecular Pharmacology. 2013;**84**:41-49. DOI: 10.1124/mol.113.085654

[31] Arase T, Uchida H, Kajitani T, Ono M, Tamaki K, Oda H, et al. The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8. Journal of Immunology. 2009;**182**:7074- 7084. DOI: 10.4049/jimmunol.0900001

[32] Muller T, Fay S, Vieira RP, Karmouty-Quintana H, Cicko S, Ayata K, et al. The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease. Oncotarget. 2017;**8**:35962-35972. DOI: 10.18632/oncotarget.16414

[33] Oury C, Lecut C, Hego A, Wera O, Delierneux C. Purinergic control of inflammation and thrombosis: Role of P2X1 receptors. Computational and Structural Biotechnology Journal. 2015;**13**:106-110. DOI: 10.1016/j. csbj.2014.11.008

[34] Vanderstocken G, Bondue B, Horckmans M, Di Pietrantonio L, Robaye B, Boeynaems JM, et al. P2Y2 receptor regulates VCAM-1 membrane and soluble forms and eosinophil accumulation during lung inflammation. Journal of Immunology. 2010;**185**:3702- 3707. DOI: 10.4049/jimmunol.0903908

[35] Muller T, Robaye B, Vieira RP, Ferrari D, Grimm M, Jakob T, et al. The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation. Allergy. 2010;**65**:1545- 1553. DOI: 10.1111/j.1398-9995. 2010.02426.x

[36] Nakano M, Ito K, Yuno T, Soma N, Aburakawa S, Kasai K, et al. UDP/P2Y6 receptor signaling regulates IgEdependent degranulation in human

basophils. Allergology International. 2017;**66**:574-580. DOI: 10.1016/j. alit.2017.02.014

[37] Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annual Review of Immunology. 2000;**18**:767-811. DOI: 10.1146/annurev. immunol.18.1.767

[38] Schnurr M, Toy T, Stoitzner P, Cameron P, Shin A, Beecroft T, et al. ATP gradients inhibit the migratory capacity of specific human dendritic cell types: Implications for P2Y11 receptor signaling. Blood. 2003;**102**:613-620. DOI: 10.1182/blood-2002-12-3745

[39] Wilkin F, Duhant X, Bruyns C, Suarez-Huerta N, Boeynaems JM, Robaye B. The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells. Journal of Immunology. 2001;**166**:7172-7177

[40] Ben Addi A, Cammarata D, Conley PB, Boeynaems JM, Robaye B. Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP. Journal of Immunology. 2010;**185**:5900-5906. DOI: 10.4049/jimmunol.0901799

[41] Nihei OK, de Carvalho AC, Savino W, Alves LA. Pharmacologic properties of P(2Z)/P2X(7) receptor characterized in murine dendritic cells: Role on the induction of apoptosis. Blood. 2000;**96**:996-1005

[42] Wang HX, Pan W, Zheng L, et al. Thymic Epithelial Cells Contribute to Thymopoiesis and T Cell Development. Frontiers in Immunology. 2020;**10**:3099. DOI: 10.3389/fimmu.2019.03099

[43] Bisaggio RD, Nihei OK, Persechini PM, Savino W, Alves LA. Characterization of P2 receptors in

thymic epithelial cells. Cellular and Molecular Biology. 2001;**47**:19-31

[44] Woehrle T, Yip L, Elkhal A, Sumi Y, Chen Y, Yao Y, et al. Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse. Blood. 2010;**116**:3475-3484. DOI: 10.1182/blood-2010-04-277707

[45] Manohar M, Hirsh MI, Chen Y, Woehrle T, Karande AA, Junger WG. ATP release and autocrine signaling through P2X4 receptors regulate gammadelta T cell activation. Journal of Leukocyte Biology. 2012;**92**:787-794. DOI: 10.1189/ jlb.0312121

[46] Ledderose C, Liu K, Kondo Y, Slubowski CJ, Dertnig T, Denicoló S, et al. Purinergic P2X4 receptors and mitochondrial ATP production regulate T cell migration. The Journal of Clinical Investigation. 2018;**128**:3583-3594. DOI: 10.1172/jci120972

[47] Ledderose C, Bromberger S, Slubowski CJ, Sueyoshi K, Aytan D, Shen Y, et al. The purinergic receptor P2Y11 choreographs the polarization, mitochondrial metabolism, and migration of T lymphocytes. Science Signaling. 2020;**13**:651. DOI: 10.1126/ scisignal.aba3300

[48] Albayati S, Vemulapalli H, Tsygankov AY, Liverani E. P2Y(12) antagonism results in altered interactions between platelets and regulatory T cells during sepsis. Journal of Leukocyte Biology. 2020;**110**:141-153. DOI: 10.1002/ jlb.3a0220-097r

[49] Sakaki H, Tsukimoto M, Harada H, Moriyama Y, Kojima S. Autocrine regulation of macrophage activation via exocytosis of ATP and activation of P2Y11 receptor. PLoS One. 2013;**8**:e59778. DOI: 10.1371/journal.pone.0059778

[50] Guerra AN, Fisette PL, Pfeiffer ZA, Quinchia-Rios BH, Prabhu U, Aga M, et al. Purinergic receptor regulation of LPS-induced signaling and pathophysiology. Journal of Endotoxin Research. 2003;**9**:256-263. DOI: 10.1179/096805103225001468

[51] Ventura MA, Thomopoulos P. ATP and ADP activate distinct signalling pathways in human promonocyte U-937 cells differentiated with 1,25-dihydroxyvitamin D3. Molecular Pharmacology. 1995;**47**:104-114

[52] Satonaka H, Nagata D, Takahashi M, Kiyosue A, Myojo M, Fujita D, et al. Involvement of P2Y12 receptor in vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte adhesion. American Journal of Physiology. Heart and Circulatory Physiology. 2015;**308**:H853-H861. DOI: 10.1152/ ajpheart.00862.2013

[53] Calovi S, Mut-Arbona P, Sperlágh B. Microglia and the purinergic Signaling system. Neuroscience. 2019;**405**:137-147. DOI: 10.1016/j.neuroscience.2018.12.021

[54] Choi HB, Ryu JK, Kim SU, McLarnon JG. Modulation of the purinergic P2X7 receptor attenuates lipopolysaccharide-mediated microglial activation and neuronal damage in inflamed brain. The Journal of Neuroscience. 2007;**27**:4957-4968

[55] Kobayashi K, Yamanaka H, Yanamoto F, Okubo M, Noguchi K. Multiple P2Y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury. Glia. 2012;**60**:1529e1539

[56] Tozaki-Saitoh H, Miyata H, Yamashita T, Matsushita K, Tsuda M, Inoue K. P2Y12 receptors in primary microglia activate nuclear factor of

*Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

activated T-cell signaling to induce C-C chemokine 3 expression. Journal of Neurochemistry. 2017;**141**:100-110. DOI: 10.1111/jnc.13968

[57] Inoue K. UDP facilitates microglial phagocytosis through P2Y6 receptors. Cell Adhesion & Migration. 2007;**1**:131-132

[58] Wen RX, Shen H, Huang SX, Wang LP, Li ZW, Peng P, et al. P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis. CNS Neuroscience & Therapeutics. 2020;**26**:416-429. DOI: 10.1111/cns.13296

[59] Morioka N, Tokuhara M, Harano S, Nakamura Y, Hisaoka-Nakashima K, Nakata Y. The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2 through the MAP kinases-NF-kappaB pathway. Neuropharmacology. 2013;**75**:116-125. DOI: 10.1016/j.neuropharm.2013.07.017

[60] Van Steenwinckel J, Reaux-Le GA, Pommier B, Mauborgne A, Dansereau MA, Kitabgi P, et al. CCL2 released from neuronal synaptic vesicles in the spinal cord is a major mediator of local inflammation and pain after peripheral nerve injury. The Journal of Neuroscience. 2011;**31**:5865e5875

[61] Rostène W, Dansereau MA, Godefroy D, Van Steenwinckel J, Reaux-Le GA, Mélik-Parsadaniantz S, et al. Neurochemokines: A menage a trois providing new insights on the functions of chemokines in the central nervous system. Journal of Neurochemistry. 2011;**118**:680e694

[62] Ting JP, Lovering RC, Alnemri ES, Bertin J, Boss JM, Davis BK, et al. The NLR gene family: A standard nomenclature. Immunity. 2008;**28**:285-287

[63] Koonin EV, Aravind L. The NACHT family - a new group of predicted NTPases implicated in apoptosis and MHC transcription activation. Trends in Biochemical Sciences. 2000;**25**:223-224

[64] Pillinger MH, Rosenthal P, Abeles AM. Hyperuricemia and gout: New insights into pathogenesis and treatment. Bulletin of the NYU Hospital for Joint Diseases. 2007;**65**:215-221

[65] Lamkanfi M, Dixit VM. Mechanisms and functions of inflammasomes. Cell. 2014;**157**:1013e22

[66] Hayyan M, Hashim MA, AlNashef IM. Superoxide ion: Generation and chemical implications. Chemical Reviews. 2016;**116**:3029-3085. DOI: 10.1021/acs.chemrev.5b00407

[67] Kim HJ, Kim CH, Ryu JH, Kim MJ, Park CY, Lee JM, et al. Reactive oxygen species induce antiviral innate immune response through IFN-λ regulation in human nasal epithelial cells. American Journal of Respiratory Cell and Molecular Biology. 2013;**49**:855-865. DOI: 10.1165/ rcmb.2013-0003OC

[68] Hung SC, Choi CH, Said-Sadier N, Johnson L, Atanasova KR, Sellami H, et al. P2X4 assembles with P2X7 and pannexin-1 in gingival epithelial cells and modulates ATP-induced reactive oxygen species production and inflammasome activation. PLoS One. 2013;**8**:e70210

[69] Martinon F, Pétrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;**440**:237-241

[70] Jin M, Yang F, Yang I, Yin Y, Luo JJ, Wang H, et al. Uric acid, hyperuricemia and vascular diseases. Frontiers in Bioscience. 2012;**1**:656-669. DOI: 10.2741/3950

[71] Liu-Bryan R, Scott P, Sydlaske A, Rose DM, Terkeltaub R. Innate immunity conferred by toll-like receptors 2 and 4 and myeloid differentiation factor 88 expression is pivotal to monosodium urate monohydrate crystalinduced inflammation. Arthritis and Rheumatism. 2005;**52**:2936-2946. DOI: 10.1002/art.21238

[72] Patterson RA, Horsley ET, Leake DS. Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: Important role of uric acid. Journal of Lipid Research. 2003;**44**:512-521

[73] Zhang J, Diao B, Lin X, Xu J, Tang F. TLR2 and TLR4 mediate an activation of adipose tissue renin-angiotensin system induced by uric acid. Biochimie. 2019;**162**:125-133. DOI: 10.1016/j. biochi.2019.04.013

[74] Milanesi S, Verzola D, Cappadona F, Bonino B, Murugavel A, Pontremoli R, et al. Uric acid and angiotensin II additively promote inflammation and oxidative stress in human proximal tubule cells by activation of toll-like receptor 4. Journal of Cellular Physiology. 2019;**234**:10868- 10876. DOI: 10.1002/jcp.27929

[75] Kocyigit I, Sener EF, Taheri S, Eroglu E, Ozturk F, Unal A, et al. Tolllike receptors in the progression of autosomal dominant polycystic kidney disease. Therapeutic Apheresis and Dialysis. 2016;**20**:615-622. DOI: 10.1111/1744-9987.12458

[76] Liu-Bryan R, Pritzker K, Firestein GS, Terkeltaub R. TLR2 signaling in chondrocytes drives calcium pyrophosphate dihydrate and monosodium urate crystal-induced nitric oxide generation. Journal of Immunology. 2005;**174**:5016-5023. DOI: 10.4049/jimmunol.174.8.5016

[77] Grassi F. The P2X7 receptor as regulator of T cell development and function. Frontiers in Immunology. 2020;**11**:1179. DOI: 10.3389/ fimmu.2020.01179

[78] La Sala A, Sebastiani S, Ferrari D, Di Virgilio F, Idzko M, Norgauer J, et al. Dendritic cells exposed to extracellular adenosine triphosphate acquire the migratory properties of mature cells and show a reduced capacity to attract type 1 T lymphocytes. Blood. 2002;**99**:1715-1722

[79] Kreckler LM, Gizewski E, Wan TC, Auchampach JA. Adenosine suppresses lipopolysaccharide-induced tumor necrosis factor-alpha production by murine macrophages through a protein kinase A- and exchange protein activated by cAMP-independent signaling pathway. The Journal of Pharmacology and Experimental Therapeutics. 2009;**331**:1051-1061. DOI: 10.1124/ jpet.109.157651

[80] Cunha RA. How does adenosine control neuronal dysfunction and neurodegeneration? Journal of Neurochemistry. 2016;**139**:1019-1055

[81] Luongo L, Guida F, Imperatore R, Napolitano F, Gatta L, Cristino L, et al. The A1 adenosine receptor as a new player in microglia physiology. Glia. 2014;**62**:122-132. DOI: 10.1002/glia.22592

[82] Savio LEB, de Andrade MP, da Silva CG, Coutinho-Silva R. The P2X7 receptor in inflammatory diseases: Angel or demon? Frontiers in Pharmacology. 2018;**9**:52. DOI: 10.3389/ fphar.2018.00052

[83] Fracasso M, Reichert K, Bottari NB, da Silva AD, Schetinger MRC, Monteiro SG, et al. Involvement of ectonucleotidases and purinergic receptor expression during acute Chagas disease in the cortex of mice treated with resveratrol and benznidazole. Purinergic Signal.

*Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

2021;**17**:493-502. DOI: 10.1007/s11302- 021-09803-9

[84] Yi C, Ezan P, Fernández P, Schmitt J, Sáez JC, Giaume C, et al. Inhibition of glial hemichannels by boldine treatment reduces neuronal suffering in a murine model of Alzheimer's disease. Glia. 2017;**65**:1607-1625

[85] Stone TW, Ceruti S, Abbracchio MP. Adenosine receptors and neurological disease: Neuroprotection and neurodegeneration. Handbook of Experimental Pharmacology. 2009;**193**:535-587. DOI: 10.1007/ 978-3-540-89615-9\_17

[86] Kusner DJ, Adams J. ATP-induced killing of virulent mycobacterium tuberculosis within human macrophages requires phospholipase D. Journal of Immunology. 2000;**164**:379e88

[87] Kusner DJ, Barton JA. ATP stimulates human macrophages to kill intracellular virulent mycobacterium tuberculosis via calcium-dependent phagosomelysosome fusion. Journal of Immunology. 2001;**167**:3308e15

[88] Pettengill MA, Marques-da-Silva C, Avila ML, d'Arc dos Santos OS, Lam VW, Ollawa I, et al. Reversible inhibition of chlamydia trachomatis infection in epithelial cells due to stimulation of P2X(4) receptors. Infection and Immunity. 2012;**80**:4232e8

[89] Coutinho-Silva R, Stahl L, Raymond MN, Jungas T, Verbeke P, Burnstock G, et al. Inhibition of chlamydial infectious activity due to P2X7R-dependent phospholipase D activation. Immunity. 2003;**19**:403e22

[90] Almeida-da-Silva CLC, Morandini AC, Ulrich H, Ojcius DM, Coutinho-Silva R. Purinergic signaling during Porphyromonas gingivalis

infection. Biomedical Journal. 2016;**39**:251-260. DOI: 10.1016/j. bj.2016.08.003

[91] Yilmaz O, Yao L, Maeda K, Rose TM, Lewis EL, Duman M, et al. ATP scavenging by the intracellular pathogen Porphyromonas gingivalis inhibits P2X7 mediated host-cell apoptosis. Cellular Microbiology. 2008;**10**:863e75

[92] Choi CH, Spooner R, DeGuzman J, Koutouzis T, Ojcius DM, Yilmaz O. Porphyromonas gingivalis-nucleosidediphosphate-kinase inhibits ATP-induced reactive-oxygen-species via P2X7 receptor/NADPH-oxidase signalling and contributes to persistence. Cellular Microbiology. 2013;**15**:961e76

[93] Johnson L, Atanasova KR, Bui PQ, Lee J, Hung SC, Yilmaz O, et al. Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase. Microbes and Infection. 2015;**17**:369e77

[94] Spooner R, De Guzman J, Lee KL, Yilmaz O. Danger signal adenosine via adenosine 2a receptor stimulates growth of Porphyromonas gingivalis in primary gingival epithelial cells. Molecular Oral Microbiology. 2014;**29**:67e78

[95] Chaves SP, Torres-Santos EC, Marques C, Figliuolo VR, Persechini PM, Coutinho-Silva R, et al. Modulation of P2X(7) purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination. Microbes and Infection. 2009;**11**:842e9

[96] Marques-da-Silva C,

Chaves MM, Chaves SP, Figliuolo VR, Meyer-Fernandes JR, Corte-Real S, et al. Infection with Leishmania amazonensis upregulates purinergic receptor expression and induces host-cell susceptibility to UTP-mediated

apoptosis. Cellular Microbiology. 2011;**13**:1410e28

[97] Figueiredo AB, Souza-Testasicca MC, Afonso LCC. Purinergic signaling and infection by Leishmania: A new approach to evasion of the immune response. Biomedical Journal. 2016;**39**:244-250. DOI: 10.1016/j.bj.2016.08.004

[98] Katz O, Waitumbi JN, Zer R, Warburg A. Adenosine, AMP, and protein phosphatase activity in sandfly saliva. The American Journal of Tropical Medicine and Hygiene. 2000;**62**:145e50

[99] Vial C, Rolf MG, Mahaut-Smith MP, Evans RJ. A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors. British Journal of Pharmacology. 2002;**135**:363e72

[100] Figueiredo AB, Serafim TD, Marquesda-Silva EA, Meyer-Fernandes JR, Afonso LC. Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation. European Journal of Immunology. 2012;**42**:1203e15

[101] Gomes RS, de Carvalho LC, de Souza VR, Fietto JL, Afonso LC. E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) of Leishmania amazonensis inhibits macrophage activation. Microbes and Infection. 2015;**17**:295e303

[102] Amit A, Kumar S, Dikhit MR, Jha PK, Singh AK, et al. Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani. Cytokine. 2016;**79**:38e44

[103] Tonin AA, Da Silva AS, Casali EA, Silveira SS, Moritz CE, Camillo G, et al. Influence of infection by toxoplasma gondii on purine levels and E-ADA activity in the brain of mice experimentally infected mice. Experimental Parasitology. 2014;**142**:51- 58. DOI: 10.1016/j.exppara.2014. 04.008

[104] Gherardi A, Sarciron ME, Francoise A, Peyron F. Purine pathway enzymes in a cyst forming strain of toxoplasma gondii. Life sciences. 1999;**65**:1733-1738

[105] Edwards FA, Gibb AJ, Colquhoun D. ATP receptor-mediated synaptic currents in the central nervous system. Nature. 1992;**359**:144-147

[106] Correa G, da SC M, de Abreu Moreira-Souza AC, Vommaro RC, Coutinho-Silva R. Activation of the P2X(7) receptor triggers the elimination of toxoplasma gondii tachyzoites from infected macrophages. Microbes and Infection. 2010;**12**:497e504

[107] Moreira-Souza AC, Marinho Y, Correa G, Santoro GF, Coutinho CM, Vommaro RC, et al. Pyrimidinergic receptor activation controls toxoplasma gondii infection in macrophages. PLoS One. 2015;**10**:e0133502

[108] Mahamed DA, Mills JH, Egan EE, Denkers EY, Bynoe MS. CD73-generated adenosine facilitates toxoplasma gondii differentiation to long-lived tissue cysts in the central nervous system. Proceedings. National Academy of Sciences. United States of America. 2012;**109**:16312-16317

[109] Thammavongsa V, Kern JW, Missiakas DM, Schneewind O. Staphylococcus aureus synthesizes adenosine to escape host immune responses. The Journal of Experimental Medicine. 2009;**206**:2417e27

[110] Henriques-Pons A, DeMeis J, Cotta-De-Almeida V, Savino W,

*Purinergic System in Immune Response DOI: http://dx.doi.org/10.5772/intechopen.104485*

Araújo-Jorge TC. Fas and perforin are not required for thymus atrophy induced by Trypanosoma cruzi infection. Experimental Parasitology. 2004;**107**:1-4. DOI: 10.1016/j.exppara.2004.04.010

[111] Santos EC, Novaes RD, Cardoso SA. Oliveira LL implication of purinergic signalling pathways in clinical management of Chagas disease. OA Biotechnology. 2013;**2**:27

[112] Da Silva AS, Bellé LP, Bitencourt PE, Perez HA, Thomé GR, Costa MM, et al. Trypanosoma evansi: Adenosine deaminase activity in the brain of infected rats. Experimental Parasitology. 2011;**127**:173-177. DOI: 10.1016/j. exppara.2010.07.010

[113] Ivanov A, Matsumura I. The adenosine deaminases of plasmodium vivax and plasmodium falciparum exhibit surprising differences in ligand specificity. Journal of Molecular Graphics & Modelling. 2012;**35**:43-48. DOI: 10.1016/j.jmgm.2012.2.004

[114] Almeida-da-Silva CLC, Morandini AC, Ulrich H, Ojcius DM, Coutinho-Silva R. Purinergic signaling during Porphyromonas gingivalis infection. Biomedical Journal. 2016;**39**:251-260. DOI: 10.1016/j. bj.2016.08.003

[115] Bisaggio DF, Peres-Sampaio CE, Meyer-Fernandes JR, Souto-Padron T. Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite-host cell interaction. Parasitology Research. 2003;**91**:273e82

[116] Berredo-Pinho M, Peres-Sampaio CE, Chrispim PP, Belmont-Firpo R, Lemos AP, Martiny A, et al. A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Archives of Biochemistry and Biophysics. 2001;**391**:16e24

[117] Yin X, Riva L, Pu Y, Martin-Sancho L, Kanamune J, Yamamoto Y, et al. MDA5 governs the innate immune response to SARS-CoV-2 in lung epithelial cells. Cell Reports. 2021;**34**:108628. DOI: 10.1016/j. celrep.2020.108628

[118] Kindler E, Thiel V. To sense or not to sense viral RNA–essentials of coronavirus innate immune evasion. Current Opinion in Microbiology. 2014;**20**:69-75. DOI: 10.1016/j. mib.2014.05.005

[119] Lucas C, Wong P, Klein J, Castro TBR, Silva J, Sundaram M, et al. Longitudinal analyses reveal immunological misfiring in severe COVID-19. Nature. 2020;**584**:463-469

[120] Lohman AW, Leskov IL, Butcher JT, Johnstone SR, Stokes TA, Begandt D, et al. Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation. Nature Communications. 2015;**6**:7965. DOI: 10.1038/ncomms8965

[121] Seif F, Khoshmirsafa M, Aazami H, Mohsenzadegan M, Sedighi G, Bahar M. The role of JAK-STAT signaling pathway and its regulators in the fate of T helper cells. Cell Communication and Signaling: CCS. 2017;**15**:23. DOI: 10.1186/ s12964-017-0177-y

[122] Eltzschig HK, Sitkovsky MV, Robson SC. Purinergic signaling during inflammation. The New England Journal of Medicine. 2012;**367**:2322-2333

[123] Di Virgilio F, Tang Y, Sarti AC, Rossato M. A rationale for targeting the P2X7 receptor in coronavirus disease 19 (Covid-19). British Journal of Pharmacology. 2020;**177**:4990-4994. DOI: 10.1111/bph.15138

[124] Klaasse EC, Ijzerman AP, de Grip WJ, Beukers MW. Internalization and desensitization of adenosine receptors. Purinergic Signal. 2008;**4**: 21-37. DOI: 10.1007/s11302-007-9086-7

[125] Abdel-Magid AF. Inhibitors of CD73 may provide a treatment for Cancer and autoimmune diseases. ACS Medicinal Chemistry Letters. 2017;**8**:781-782. DOI: 10.1021/ acsmedchemlett.7b00255

[126] Nylander S, Mattsson C, Ramstrom S, Lindahl TL. Synergistic action between inhibition of P2Y12/ P2Y1 and P2Y12/thrombin in ADPand thrombin-induced human platelet activation. British Journal of Pharmacology. 2004;**142**:1325-1331. DOI: 10.1038/sj.bjp.0705885

[127] McGonagle D,

O'Donnell JS, Sharif K, Emery P, Bridgewood C. Immune mechanisms of pulmonary intravascular coagulopathy in COVID-19 pneumonia. The Lancet Rheumatology. 2020;**2**:e437-e445. DOI: 10.1016/s2665-9913(20)30121-1

[128] Abouelkhair MA. Targeting adenosinergic pathway and adenosine A2A receptor signaling for the treatment of COVID-19: A hypothesis. Medical Hypotheses. 2020;**144**:110012. DOI: 10.1016/j.mehy.2020.110012

[129] Vigano S, Alatzoglou D, Irving M, Ménétrier-Caux C, Caux C, Romero P. Targeting adenosine in cancer immunotherapy to enhance T-cell function. Frontiers in Immunology. 2019;**10**:925

[130] Terp MG, Olesen KA, Arnspang EC, Lund RR, Lagerholm BC, Ditzel HJ. Anti-human CD73 monoclonal antibody inhibits metastasis formation in human breast cancer by inducing clustering and internalization of CD73 expressed on the surface of cancer cells. Journal of Immunology. 2013;**191**:4165-4173

[131] Leone RD, Lo Y-C, Powell JD. A2aR antagonists: Next generation checkpoint blockade for cancer immunotherapy. Computational and Structural Biotechnology Journal. 2015;**13**:265-272

#### **Chapter 2**

## Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic Diseases

*Richa Rai*

#### **Abstract**

Purine derivatives like adenosine 5′-triphosphate (ATP) is the powerhouse of the cell and is essential to maintain the cellular homeostasis and activity. Besides this they also act as a chemical messenger when released into the extracellular milieu because of stress and cellular insult. The extracellular ATP (eATP) as well as its metabolite adenosine triggers purinergic signaling affecting various cellular processes such as cytokine and chemokine production, immune cell function, differentiation, and maturation, and mediates inflammatory activity. Aberrant purinergic signaling had been implicated in several diseased conditions. This chapter will focus on the dynamics of purinergic signaling and immune signaling in driving under various diseased conditions like autoimmunity and infectious disease.

**Keywords:** ATP, adenosine, ectonucleotidases, CD39, CD73, purinergic signaling, systemic lupus erythematosus, rheumatoid arthritis, infectious disease, SARS-CoV2

#### **1. Introduction**

Adenosine 5′-triphosphate (ATP) is abundantly generated in the cytosol through respiration and glycolysis. Primarily, these are the "energy currency" of the cell as ATP hydrolysis release energy and is essential to maintain the cellular homeostasis and activity [1]. Extracellular activity of ATP was first described by Drury and Szent-Györgyi in 1929 [2]. Later, in 1970s, ATP was shown to be involved in non-adrenergic, non-cholinergic nerve-mediated responses, and further its function as a neurotransmitter was established that led to introduction of the term "purinergic signaling" [3]. Burnstock has described about the purinergic signaling and purinergic systems in very detail, which consists of (a) purine or pyrimidine derivatives that serve as an "extracellular messenger," (b) "membrane transporter" that are responsible for the extracellular release of these nucleotides or nucleosides, (c) "metabolizing enzymes" present on the cell surface that hydrolyze the ATP to adenosine diphosphate (ADP) then to adenosine monophosphate (AMP) and adenosine and, (d) "purinergic receptors" that sense the extracellular purine or pyrimidine derivatives [3–7].

In the beginning, purinergic signaling was determined to have a role in neuronal signaling but now, their role in immune responses, inflammation, pain, exocrine and endocrine secretion, platelet aggregation, and endothelial-mediated vasodilatation had been explored and established [3, 4, 6, 8]. Additionally, cross talk of purinergic signaling with other signaling network also associates with the impact on cell proliferation, differentiation, and death that occur during the development and regeneration processes. Under normal condition, purinergic signaling operate in a very wellregulated manner to maintain the physiological function of different organ systems. Dysregulation in any component of the purinergic signaling network depending on the expression or activation of purinergic receptors, ectonucleotidases or release of agonist from damaged cell resulting from stress, inflammation serves as a potent modulator of inflammation and key promoters of host defenses, immune cells activation, pathogen clearance, and tissue repair that contributes to the disease pathogenesis [9]. Thus, their knowledge is of great importance for a full understanding of the pathophysiology of acute and chronic inflammatory diseases and will give an insight on novel therapeutic approaches to overcome inflammation. This chapter describes the component of purinergic system, its cross talk with immune signaling. Major focus of this chapter is to present the dynamics of purinergic signaling under normal physiological condition and its role in modulating the immune and inflammatory response under various diseased conditions like autoimmunity, and microbial infection.

#### **2. Purinergic system and its component on immune cells: an immunomodulator**

#### **2.1 Mechanism of release of nucleotides**

Extracellular ATP (eATP) has been well established as a ligand for autocrine and paracrine signaling that has a pathophysiological role. In addition, to eATP other nucleotides and nucleosides such as the adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), uridine diphosphate (UDP), uridine triphosphate (UTP), and nicotinamide adenine dinucleotide (NAD+) also serve as a potent purinergic signaling modulator. The release of nucleotides into the extracellular space occurs via regulated and unregulated mechanisms. Regulated mode of release of nucleotide is mediated through classical exocytosis [10] or conductive ATP release through ATP-permeable channels [11]. Currently, five groups of ATP-release channels are known such as: connexin hemichannels, Pannexin (PANX), calcium homeostasis modulator 1 (CALHM1), volume-regulated anion channels (VRACs), and maxi-anion channels (MACs) [12].

The ATP release by exocytosis is an active release mechanism that involves vesicular nucleotide transporter (VNUT). It is responsible for the accumulation and exocytosis of ATP from exocytotic vesicles that occurs in a proton-dependent electrochemical gradient manner generated by a vacuolar-ATPase (v-ATPase). Further, intracellular Ca+2 level and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) drives the fusion of the exocytotic vesicles with the plasma membrane ultimately resulting in the release of nucleotides into the extracellular space [13, 14]. Hemichannels are the ATP permeable channels that support the release of ATP under specific pathological condition. Primarily, these channels contribute to various cellular and physiological functions by forming gap junctions or hemichannels, to allow intercellular communication. They are categorized into two based on

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

their functions, [1] connexins that has both gap junction and form hemichannel function whereas [2] PANX only form hemichannel [15]. These channels are in closed state under normal condition to avoid the loss of vital ionic, energetic, and metabolic gradients. However, chemical and biochemical stimuli resulting from pathological conditions trigger their opening and lead to release of ATP. Till date, 21 isoforms of connexins are reported in human of which connexin-43, -37, -26, and -36 have been shown to support ATP release [16]. Connexin-43 is widely expressed and very well studied. It is activated by increase in the intracellular Ca+2 concentration, plasma membrane depolarization, reactive oxygen species (ROS) or nitric oxide (NO) [17, 18]. The PANX (PANX) family is comprised of three members, PANX-1, -2, and -3 of which PANX-1 and -3 are widely expressed in different tissues, while PANX-2 is exclusively found in the brain [19]. In resting state, PANX channels are closed, mainly due to the blockage of the pore by C-terminal tail from the intracellular side [18]. However, in response to apoptosis or pyroptosis, C-terminal tail gets cleaved by caspase-3, -7, or -11 leading to opening of PANX-1 and allows nucleotides to cross the plasma membrane [20, 21]. Additionally, other stimuli such as intracellular calcium increase, redox potential changes, mechanical stress, and activation of the P2X7R can trigger PANX-1 channel opening [22].

Another mechanism involves the disruption of the cell membrane by apoptosis, necrosis, pyroptosis, or netosis, which leads to the unregulated leakage of ATP as well as other large cytosolic molecules including enzymes [11, 23, 24].

#### **2.2 Metabolism of extracellular nucleotides and nucleosides**

The life span of eATP is controlled by purinergic ectoenzymes that coordinate a sequential two-step process of hydrolyzing ATP into AMP and then into the potent anti-inflammatory adenosine. This make ectonucleotidases enzymes a crucial component of the purinergic system, which balance the level of eATP as well as other nucleotide derivatives UTP, NAD+, and their metabolites, thereby controlling the activation of purinergic receptors and biochemical composition of the inflammatory microenvironment. These enzymes are classified into four major families: (a) *ectonucleoside triphosphate phosphohydrolases (NTPDases)*: This group of enzymes are further classified into 8 subfamilies—NTPDase 1 (CD39), 2, 3, and 8, which are expressed on the cell surface, whereas NTPDases 4–7 are present in the intracellular organelles. Of these 8 subfamilies, NTPDase1 (CD39) is the best characterized that hydrolyses ATP to ADP and further to AMP. CD39 is expressed on wide variety of immune cell, e.g., monocytes, dendritic cells (DCs), T regulatory (Treg) cells, and natural killer (NK) cells. (b) *nicotinamide adenine dinucleotide glycohydrolase (NAD glycohydrolase/CD38)*: CD38 is a cell surface glycoprotein highly expressed in hematopoietic tissues such as the bone barrow and lymph nodes. Among immune cells, CD38 is highly expressed on monocytes, macrophages, DCs, neutrophils, innate lymphoid cells (ILC), NK cells, T and B cells. It hydrolyses NAD+ to cyclic-ADP ribose (cADPR) and then to AMP. (c) *ecto-5*′*-nucleotidase (NT5E/CD73)*: CD73 degrades AMP generated by CD39 or CD38 to adenosine. It is expressed on stromal cells, follicular DCs, endothelial cells, neutrophils, macrophages, and subpopulations of T cells. and (d) *ectonucleotide pyrophosphatase/phosphodiesterase (NPPs)*: NPPs include 7 members NPP 1–7. NPP1–3 degrade nucleoside triphosphates and diphosphates, NAD+, UDP-sugars, and dinucleoside polyphosphates. NPP2 also known as autotaxin (ATX) has unique property of hydrolyzing nucleotide as well as phospholipids but acts more efficiently on later to generate the bioactive phospholipid mediator's lysophosphatidic acid (LPA)

and sphingosine-1-phosphate (S1P). NPP6 and 7 hydrolyzes phospholipids only, whereas catalytic properties of NPP4 and 5 are not known. Some NPPs are expressed on liver and intestinal epithelia, neuronal cells; NPP1 is also expressed on B and T cells [25–28]. The ectonucleotidases are present on almost all types of immune cells, but their expression pattern changes in a function dependent manner and controls the pro-inflammatory and anti-inflammatory condition to avoid any pathological conditions like autoimmunity, cancer, and infectious disease.

Briefly, CD39 has an anti-inflammatory property that controls the extracellular level of ATP by converting it into adenosine in conjunction with CD73. CD39 and CD73 exhibit an immunosuppressive activity as shown by its expression on Tregs cells [29–31]; CD8 T cells [32] and B cells [33] and inhibits the pathogenic T cells. Breakdown of eATP by CD39 prevents the activation of P2X7R and attenuates the secretion of IL-1β and IL-18 [34]. The expression pattern of CD38 varies during the differentiation and maturation of B and T cells [35, 36]. The enzymatic activity of CD38 generates cADPR/ADPR and triggers Ca+2 release from intracellular stores and Ca+2 influx from the extracellular space that have role in transmigration and chemotaxis of neutrophils, monocytes and DCs, and cytokine release [37]. Elevated level of cADPR/ADPR and intracellular Ca+2 regulates cellular chemotaxis [38], phagocytosis [39], and antigen presentation [40] in a CD38 dependent manner. Thus, dysregulation of CD38 has been implicated in several inflammatory pathologies such as autoimmunity and cancer [37, 41]. It is important to note that cADPR is generated by hydrolysis of NAD+, disruption in the metabolism of NAD+ has been associated with multiple pathological conditions [42]. Different types of NPPs have been implicated in a various of pathologic conditions such as tumor invasion and metastasis, inflammation, and angiogenesis (NPP2), tissue calcification and bone development (NPP1), and hemostasis and platelet aggregation (NPP4) [43]. However, NPP2 (ATX) is widely studied, ATX-LPA signaling axis induces inflammatory mediators such as IL-8, IL-6, TNF-α, and growth factors such as the vascular endothelial growth factor (VEGF) and the granulocyte colony-stimulating factor (G-CSF) thereby augmenting the cytokine production and lymphocyte infiltration that ultimately aggravates the inflammation in conditions such as asthma, pulmonary fibrosis, and rheumatoid arthritis [44, 45].

#### **2.3 Purinergic receptors**

Purinergic receptors are divided into two subtypes based on their binding tendency to different purine derivatives—P1 receptor (P1R) has affinity to bind adenosine only, whereas P2 can bind ATP, ADP, UDP-glucose, UDP and UTP [46]. Adenosine receptors (AR) belong to rhodopsin-like family of G protein receptors and consist of four subtypes such as A1, A2A, A2B, and A3. Adenosine generated by the hydrolysis of extracellular ATP, ADP, or AMP are either metabolized by adenosine deaminase (ADA) or shuttled back to the cells via two types of transporters, the equilibrate nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs) to stimulate various intracellular pathways like AMP-activated protein kinase, adenosine kinase and S-adenosyl homocysteine hydrolase [47]. Although it may depend on the concentration of adenosine and the given P1 receptor subtype engaged, but adenosine primarily, have anti-inflammatory and immune suppressive functions. The immunosuppressant activity of adenosine relies on the inhibition of virtually all immune cell populations such as T and B lymphocytes, NK cells, DCs, granulocytes, monocytes, and macrophages.

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

P2 receptors further categorized into two families based on molecular structure and second messenger systems, namely P2X ionotropic ligand-gated ion channel receptors that only binds to ATP and P2Y metabotropic G protein-coupled receptors (GPCR) can bind to ADP, UDP- glucose, UDP, and UTP [46]. The family of P2X receptors comprises seven members (P2X1–7), which perform tissue-specific functions by forming homo- or hetero-trimeric complexes. At least three P2X subunits assemble to form hetero- (e.g., P2X2/3 and P2X1/5) or homo-trimeric (P2X7) channels. This kind of assembly confers to P2X receptors a large repertoire of physiological functions in different tissues. Among P2XRs, the P2X7R has a special place in inflammation since its stimulation promotes NLRP3 inflammasome assembly and the associated IL-1β secretion. There are eight subtypes of P2Y receptors, which is further characterized into two subfamilies P2Y1 and P2Y12 based on their coupling to Gq and Gi, respectively. P2Y1 subfamily includes P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors. The second subfamily is P2Y12, which contains P2Y12, P2Y13, and P2Y14 receptors. Each P2Y receptors has different affinity towards different nucleotides and has a tissue-specific function. For instance, P2YR11 has affinity for ATP; P2YR1, P2YR12, and P2YR13 for ADP; P2YR2 and P2YR4 for UTP; P2YR6 for UDP; and P2YR14 for UDP-glucose and UDP-galactose [5].

The purinoceptors are expressed on almost all kinds of peripheral tissues and are involved in short-term as well as long-term regulation of variety of functions, ranging from neuromuscular and synaptic transmission to secretion in gut, kidney, liver, and reproductive systems. Their contribution in immune signaling is enormous, as these receptors are expressed on almost all types of immune cells. The purine nucleotides orchestrate the onset, magnitude duration, and resolution of the inflammatory response through the activation of purinergic receptors, which is also governed by the activity of ectonucleotidases (**Figures 1** and **2**). Any alterations in the purinergic machinery could contribute to the pathophysiological processes underlying the onset

#### **Figure 1.**

*Cartoon depicting the components of purinergic system and their functions. Mechanism of nucleotide release from the intact cells via exocytosis or transport channels as well as leakage of ATP from apoptotic and netosis (bottom of the image). The nucleotide triggers the activation of immune cells via specific purinergic receptor (top of the image). Activation of purinergic receptors ATP or their hydrolyzed metabolites ADP/AMP and adenosine by ectonucleotidases (middle of the image).*

#### **Figure 2.**

*Pictorial representation of cross talk of purinergic and immune signaling during normal physiological condition and inflammatory condition.*

and development of immunological diseases, neurodegeneration, cancer, diabetes, and hypertension [7, 46, 48].

#### **3. Interplay of purinergic signaling and immune signaling on inflammatory response**

Beyond the physical and chemical barrier of skin and mucous lining, our body is guarded from the pathogens as well as self-attacking/cancerous cells by two different kinds of immune responses that acts in a coordinated manner. This includes (a) innate immune response comprising myeloid lineage derived cells (monocytes, macrophages, neutrophils, and DCs) and NK cells derived from lymphoid progenitors, and (b) adaptive immune response consists of B and T cells. Innate immune response provides the first line of defense against pathogens. It is an antigen-independent defense mechanism that is elicited when immune cells encounter pathogens. This response has no memory and remains similar during the lifetime. On the other hand, adaptive immune response is an antigen dependent, antigen specific, and has the tendency to form memory cells to elicit rapid response based on the previous encounter with the similar kind of antigen or pathogenic exposure. Innate and adaptive immune responses are not mutually exclusive defense mechanisms. They work in a very organized fashion and complement the functions, as activation of T cells requires antigen presentation by professional antigen presenting cells (dendritic cells, B-cells, or macrophages), together with the major histocompatibility complex (MHC) type I or II [49]. Defects in any of the component increases the vulnerability towards infection and disease.

ATP and adenosine are the key modulators of the immune response, ATP being an immunostimulant, whereas adenosine has an immunosuppressive effect thus balance between the two is crucial for the proper functioning of immune system. Extracellular signals by ATP and adenosine are detected and transduced by P2 and P1 receptors (**Figure 2**), respectively which is present on all kinds of immune cells, thus

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

purinergic signaling affects all aspects of immunity and inflammation [50], which is described in detail in further section.

#### **3.1 Effect on innate immune signaling**

#### *3.1.1 Monocytes/macrophages*

Macrophages are the subset of myeloid cells that have immune surveillance function and sense even a minute changes in the tissue microenvironment. They express a variety of pattern recognition receptors (PRRs) that are present either on the surface, cytosol or in the endosome such as toll like receptors (TLRs), NOD like receptor (NLRs), retinoic acid inducible gene I like receptors (RLR), transmembrane C-type lectin receptors, and absent in melanoma (AIM)2-like receptors (ALRs) that recognize either pathogen associated or damage associated molecular patterns (PAMP and DAMP, respectively). These cells are highly plastic that could undergo profound metabolic modifications after sensing the pathogens or damage signal via PRRs to elicit the immune response. In addition, macrophages are endowed with purinergic P1, P2X, and P2Y receptors that also respond to damage associated molecules, extracellular nucleotides, and their derivatives, and undergo reprogramming from pro-inflammatory profile M1-like phenotype to an anti-inflammatory M2-like phenotype. As indicated by Elliott et al., that monocytes or macrophages sense extracellular nucleotide as a danger signal for "find me" or "eat me" to engulf and phagocytose the dying cells [24]. These cells not only sense the distant signal but also amplify the signaling for chemotaxis by releasing ATP by "autocrine purinergic loop" via P2Y2 and A3 receptors [51, 52]. Macrophages control their activation state in an autoregulatory mechanism by inducing the production of ATP and extracellular degradation to adenosine. Deficiency of CD39 promotes a sustained inflammatory activation state and inhibits the switch to an immunosuppressive phenotype [53]. Presence of extracellular adenosine stimuli in macrophages drives the polarization towards M2 phenotype with diminished expression of inflammatory genes TNF-α and IL-6 and increased expression of anti-inflammatory cytokines such as IL-10 and VEGF via A2A and A2B receptors [54]. Furthermore, macrophages exhibit a unique repertoire of P2X receptors such as expression of P2X1, P2X4, as well as P2X7 [55]. Among P2Y receptors, P2Y1 and P2Y4 receptors play minor roles, whereas the functions of P2Y2, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14 are more established in the macrophage biology as described elsewhere [56]. A recent study demonstrated that bone marrow derived macrophages display unique expression pattern of purinergic receptors that correlates with a M1or M2 inflammatory phenotype. M1 phenotype exhibit a unique and more pronounced P2X7 negative macrophage population, which associates with decreased inflammasome formation. P1 receptors A2A and A2B are upregulated in M1 and M2. P2Y1 and P2Y6 exclusively upregulated in M2, whereas P2Y13 and P2Y14 are overexpressed in M1 [57]. This unique feature demonstrates capability of purinergic receptors on macrophages to adapt to pro- and anti-inflammatory macrophage differentiation with functional consequences to nucleotide stimulation.

#### *3.1.2 Dendritic cells*

DCs are professional antigen-presenting cells (APCs), which has a crucial role in initiating and regulating the adaptive immune response by directing the activation and differentiation of naive T cells. Immature DCs (iDCs) sense the danger signals in the similar fashion as monocytes and macrophages do, however upon exposure, DCs lose their phagocytotic capacity, migrate to secondary lymphoid organs and transition to a mature DC (mDC) by acquiring MHC and costimulatory molecules, such as CD54, CD80, CD83, and CD86. Migration of DCs to the inflamed tissue is mediated by A1 and A3 ARs [58]. Adenosine upregulates the expression of co-stimulatory molecules on mDCs [59]. Both, A2A and A2B ARs suppress maturation of DCs as well as their capacity to initiate Th1 response, however, it increases pro-angiogenic VEGF, IL-10 and cytokines that contribute to Th17 cell polarization [59, 60]. Adenosine also mediates the attraction of DC and Treg cells, which is crucial for the immunosuppressive activity of Treg cells [61]. Similarly, ATP also acts as a chemoattractant for iDCs, and enhance the migration by autocrine signaling loop mechanism via P2X7. This signaling is further amplified by the release of ATP by PANX-1 channels [62]. Furthermore, eATP had been shown to activate P2X7R to promote the maturation of dendritic cells via NF-κB (p65) pathway [63]. On the other hand, P2Y6 has inhibitory role in the maturation and activation of DCs via NF-κB by inhibiting the production of IL-12 and IL-23 and the polarization of Th1 and Th17. Loss of P2Y6 enhances the DC mediates differentiation of Th1 and Th17 subsets [64]. The ATP-P2X7 signaling axis of DCs also promotes interleukin (IL)-1β and IL-18 secretion by activating NLRP3 inflammasome and induces Th2/Th17 differentiation [65]. P2X4 acts in conjunction with P2X7 to regulate IL-1β production by DCs [66]. As described previously, the balance of proinflammatory-ATP and anti-inflammatory adenosine is regulated by CD39 and CD73 present on the immune cells. In context of DCs, their expression fine tunes the DCs function either as tolerance (higher expression) or as immunity (lower expression) ensues [67, 68].

#### *3.1.3 Neutrophils*

Neutrophils belongs to the granulocyte family, which has a major role during the early stages of the inflammatory response. They are the first cell to arrive at the inflammation site, which employ an extracellular ATP-dependent mechanism to generate a chemotactic gradient and orientate its migration. Remarkably, the purinergic system regulates many effector functions of neutrophils such as phagocytosis, oxidative burst, degranulation, and neutrophil extracellular traps (NETs) formation via Netosis [69, 70]. Apoptotic neutrophils release ATP to stimulate mononuclear phagocytic cell influx and promote engulfment and clearance functions. Nucleotides released as a result of the apoptosis and netosis serve as danger or find me signal to initiate immune cell chemotaxis via P2Y2 receptor towards inflamed tissue and finetuned control local inflammation and promote phagocytosis and clearance [24, 71]. Similar to other phagocytic cells such as monocytic and dendritic cells, neutrophils in the immune microenvironment also release ATP via PANX-1 to induce chemotaxis by autocrine stimulation of P2Y2 [51, 52, 62, 72]. On the other hand, P1 receptor, A2A (activated by adenosine) blocks the chemoattractant signaling, whereas alternative binding of adenosine to A3 receptors, stimulate immune migration. Thus, P2Y2 and A3 receptors are responsible for the amplification of the chemotaxis signal via feedback loop mechanism [52]. P2Y2 receptors play crucial role in neutrophil activation by regulating the release of IL-8, a major chemokine for neutrophils chemotaxis [73]. IL-8 secretion is in turn controlled by CD39 [74]. Thus, the local microenvironment composed by ATP and the consequent degradation to adenosine by CD39 and CD73 ectoenzymes influence reprogramming of the innate immune cells and their response towards pathogens and other diseased condition.

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

#### *3.1.4 Natural killer cells*

NK cells are considered as a component of innate immune system due to the lack antigen-specific cell surface receptors but morphologically they resemble lymphocytes as they originate from the common lymphoid progenitor cell in the bone marrow. NK cells exert sophisticated biological functions that attribute to both innate and adaptive immunity, thus the functional boundary between these two arms of the immune response is obscure [75]. These cells express a repertoire of activating (NKG2 C-H) and inhibitory receptors (NKG2 A and B) through which it interacts with pathogens by recognizing MHC-I molecule [76]. Activation of the NK cell leads to cytolytic killing of infected cells. Adenosine receptors A1, A3 and A2A, A2B have an antagonistic effect in controlling the intracellular cAMP levels. A1, A3 inhibits the adenylyl cyclase and decreases the intracellular cAMP level, which has a stimulatory effect on NK cell and promote the cytotoxic activity whereas, A2A and A2B has the immunosuppressive effects on NK cells [76, 77]. NAD+ and ADP-ribose inhibited human NK proliferation [78]. Nucleotide triphosphates (ATP, GTP) have high potency in inhibiting NK cell-mediated cytotoxicity, this tendency however decreases with reduced negative charge due to less phosphate group [(ADP, GDP) > (AMP, GMP)]. Ectonucleotidases do not have any significant role in modulating the cytolytic effect of NK cells by extracellular ATP/ADP/AMP [79]. NK cells express lower level of CD73 even with IL-15 and IL-12 priming [74]. Decreased expression of P2Y6 promotes the development of the NK precursor cells into immature NK and mature NK cells suggesting P2Y6 as a negative regulator of NK cell maturation and function [80]. Among other extracellular purine derivatives, NKT cells display higher sensitivity to NAD+ and induce cell death via P2X7 pathway [81, 82]. Furthermore, another phenotypically heterogeneous NKT cells subset includes invariant natural killer T (iNKT), which are CD4 and CD8 negative but express NK cell marker and produce IL-4 and IFNγ. iNKT recognizes lipid antigens combined with CD1d on the surface [83]. Activation of iNKTs *in vitro* induces the expression of purinergic signaling genes A2A, P2X7R, CD38, CD39, NPP1, CD73, PANX-1, and ENT1, which has an anti-inflammatory role [84]. iNKT cells interact with DCs and monocytes via P2X7 dependent and an independent manner, respectively [85, 86]. Overall, NK cells alter their functional responses to adenosine signaling via mechanisms that are sensitive to specific cytokine activation programs.

#### **3.2 Effect on adaptive immune signaling**

#### *3.2.1 T cells*

Activation of T cell immune response is the key in adaptive immune system functions, which elicits both cellular and humoral immunity. Naïve T cells are activated by APCs, but they require two subsequent signals, first one is the binding of TCR to peptide–MHC complex and the second one is the co-stimulatory interaction at the interface between APCs and T cells via B7/CD28, LFA-1/ICAM-1 and ICAM2, and CD2/LFA-3 ligand and receptor complex [87]. Di Virgilio et al. was the first to show T cell responsiveness to extracellular ATP (eATP), back to 1989 [88]. Once T cells are activated, they release ATP via PANX-1 channels, resulting in the activation of P2X1, P2X4, and P2X7 receptors that promotes downstream signal transduction pathways leading to IL-2 expression and T cell proliferation via Ca+2 influx [89]. P2X7 receptor stands out among P2X family members as the most important regulator

of T cell function [90]. The released ATP stimulates purinergic receptors that also contributes to the amplification of co-stimulatory TCR/CD28 signal at the immune synapse by autocrine stimulation of P2X7 [91] and P2Y1 receptors [92]. In addition, the T cell activation via P2X7R inhibits the immunosuppressive Tregs cells [93]. P2X7R is also crucial for the activation of CD8 T cells, and its expression increases as they differentiate to TCM (central memory) and TRM (tissue-resident memory) suggesting its key role in generating long-lived memory CD8T cells [94]. However, another study demonstrated that eATP treatment can trigger cell death in the naive CD8 (CD44loCD45RBhi) subset, but it is unable to induce these cellular activities in the effector/memory CD8 (CD44hiCD45RBhi) subset. Even though both subsets express similarly low levels of P2X7R, but they demonstrate different sensitivity to ATP depending on the stage of differentiation instead of P2X7R expression levels [95]. Importantly, expression of CD39 and CD73, the ecto-5′-nucleotidase that degrades extracellular AMP into adenosine, by other immune and tissue-resident cells can dramatically condition the outcome of T cell responses [96]. On the other hand, A2A receptor signal inhibits Th1 cell generation and IFN-γ production, triggering the induction of FoxP3 + Treg cell subset and the production of TGF-β. ATP catabolism and generation of retaliatory metabolite adenosine is a typical suppression mechanism of regulatory cells involving Treg, type-1 regulatory (Tr1) T cells, and myeloid-derived suppressor cells (MDSCs) [97]. These regulatory cells express CD39 and CD73 to abrogate ATP-related effects and enable the inhibitory properties. P2X7R can imprint distinct outcomes to the T cell depending on the metabolic fitness and/ or developmental stage via autocrine signaling or microenvironment's clues. The peculiarity of P2X7R function as cationic channel and cytolytic pore could be responsible for some apparently contradictory findings on P2X7R dependent responses in particular T cell subsets in different experimental settings [94–96].

#### *3.2.2 B cells*

Another important arm of adaptive immune response is the humoral immunity, which is mediated by B cells. These cells are also necessary for the development of T-cell immunity because they serve as an APC, providing costimulatory signals and producing cytokines necessary for effector functions of T cells. B cells exhibit expression of the membrane B cell receptor (BCR), which can recognize antigens in their native forms, thus B cells do not need antigen presentation for activation. Antigen recognition, together with signals from activated Th2 cells, induces B cells to proliferate and generate effector plasma cells and memory B cells. B cells expresses ectonucleotidases—CD39 and CD73, P1 receptors—A1, A2A, and A3, and P2 receptors—P2X1, P2X2, P2X4, and P2X7 [33, 98, 99]. The function and activity of B cells are largely governed by the concentration of adenosine and ATP in the microenvironment. Adenosine imposes suppressive effect on B cells, whereas increased ATP release and production are associated with activated B cells thereby exerting pro-inflammatory effect on the target tissue and IgM release [100]. *In vitro* activated B cells exhibit downregulation of CD73, which mainly produces AMP, and inhibits T-cell proliferation and cytokine production, whereas overexpress A3 receptor in activated state [98]. Accumulation of pericellular ATP occurring in B cells activates the P2X7 receptor, which results in shedding of CD21, CD23, and CD62L from the cell surface [101, 102]. This process is involved in transendothelial migration of B cells. There is also evidence showing that P2X7 is directly involved in the release of IgM from B cells after T cell independent activation [103]. Moreover, CD73 is progressively

#### *Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

upregulated on germinal center (GC) B cells following immunization, and is expressed at even higher levels among T follicular helper cells but is absent among plasma cells and plasmablasts. CD73-dependent adenosine signaling is prominent in the mature GC, maintenance of plasma cell compartment and necessary for immunoglobulin class switching [100, 104]. Thus, any disruption in the balance of ATP signaling that is dominant in activated B cells and adenosine signaling, which seems crucial in achieving immunocompetence by activated cells [100] could lead to severe immunological disorder.

#### **4. Implication of purinergic signaling in various pathological conditions**

A healthy individual has a practically insignificant amount ATP in the extracellular microenvironment (at the nanomolar range), whereas, they have significantly higher concentration of ATP in the intracellular environment (reaching several millimolar), as ATPs are the powerhouse of the cell. Inflammatory stress due to the increased production of proinflammatory mediators associates with release of ATP and other nucleotides into the extracellular space (**Figure 1**). These extracellular nucleotides trigger a stimulation of purinergic receptors, which is a normal physiological phenomenon and beneficial for preventing tissue damage ensuring host survival, it may also be detrimental for clearance of pathogens or dying cells. However, failure in the fine tuning of the immune response alters inflammatory and regulatory microenvironments, leading to unbalanced stimulation and culminates a hyperinflammatory condition generating numerous pathologies such as autoimmunity, chronic infectious diseases, and cancer (**Figure 2**).

#### **4.1 Purinergic signaling in autoimmune disease**

Autoimmune diseases are characterized by diverse clinical manifestations including dysregulated innate and adaptive immune signaling, chronic inflammation, autoreactive immune cells, generation of autoantibodies to self-nuclear and cytoplasmic component. Based on the target organ and tissues, they are represented as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome (SS), systemic sclerosis (SSc), etc. As described previously that some of the purinergic receptors are coupled with inflammasome assembly, pro-inflammatory cascades, secretion of IL-1β, IL-18, and T and B cell activation, and maturation, all these events play a pivotal role in autoimmunity [105].

#### *4.1.1 Systemic lupus erythematosus*

SLE is an inflammatory autoimmune disease that affects many organs, including the skin, joints, the central nervous system, and the kidneys. A frequent and serious manifestation of SLE includes glomerulonephritis (GN), a condition that can cause proteinuria and progresses to kidney failure. These diverse clinical features include hematological and serological abnormalities, such as decreased levels of complement and increased levels of autoantibodies [106, 107]. SLE has multiple etiology like genetic, environmental, and hormonal factor but involvement of dysfunctional innate and the adaptive system is prominent [108]. Purinergic signaling is another key pathway that connects with the inflammatory signaling cascade and contributes to the immunopathogenesis of SLE.

Till date, more than 180 autoantibodies have been documented in SLE patients [107]. Source of the diverse pool of autoantigens are apoptosis [109], netosis [110], and pyroptosis [111]. Simultaneously, SLE patient also exhibit impairment of phagocytotic clearance and NET degradation [112, 113], which together represent a mechanism that trigger to breakdown of the self-tolerance against autoantigens and leading to initiation of SLE. Defects in the purinergic signaling and its role in SLE pathogenesis and disease severity had been described at several instances. Therefore, P2X7R activation by ATP or by extracellular complexes, such as NETs, might have a dual pathogenetic role in promoting inflammation in lupus: on one hand, it directly triggers inflammation by stimulating the NLRP3 inflammasome, and on the other it has an indirect pro-inflammatory effect by inducing pyroptotic cell death [114]. Presence of the NETs in the microenvironment induce NLRP3 inflammasome, in macrophages and results in the amplification of inflammation by releasing of IL-1β and IL-18, which is mediated via P2X7R [115]. Induction of inflammasome and IL-1β and IL-18 release have been shown to contribute to the cardiovascular, skin, and nephritis manifestations [116–118]. Evidence suggests the higher P2X7R in renal tissue of lupus nephritis patients [119]. In that context, a study demonstrated substantial up-regulation of P2X7R, NLRP3, and ASC, in the kidneys of MLR/lpr mice compared to control mice and inhibition of P2X7R ameliorates the disease phenotype mainly diminished both the severity of nephritis and levels of circulating anti-dsDNA antibodies [120, 121]. The presence of single nuclear polymorphism (SNP) 489C>T in P2X7 receptor had been associated with increased inflammasome activation in SLE patients and shows involvement in pericarditis [122, 123]. Th1, Th17, and Regulatory T (Treg) cells in SLE patients display higher expression of P2X7 receptor, which correlates with active SLE disease and increased levels of IFN γ , IL-1 β , IL-6, IL-17A, and IL-23 cytokines [124]. Monocytes and lymphocytes from SLE patients and RA patients show reduced expression of P2X7R gene. They show reduced tendency to induce apoptosis and cytokine release *in vitro* compared to cells from healthy individual [125].

Furthermore, P2X7R has an important function of restricting the expansion of T follicular helper (Tfh) cells by pyroptosis and controls the development of pathogenic ICOS+ IFN-γ–secreting cells and in turn prevents the overproduction of autoantibodies and activation of T cells that ultimately controls the production of autoantibodies conditions [126]. SLE patients exhibit deletion of P2X7R genes that have deleterious effect of autoantibody generation [126]. Another study had reported that deletion of P2X7R could amplify the defect in peripheral T cell homeostasis due to the FAS mutation and thus contribute to the autoimmune pathology [127]. In similar way, another purinergic receptor P2Y8R restricts the proliferation of self-tolerant B cells. Distinct variant of P2Y8R had been shown to be downregulated in SLE patients and these are associated with the loss of function, which leads to increased expansion of self-reactive B cells, resulting in the increased autoantibody production. P2Y8R correlated with lupus nephritis and increased age-associated B cells and plasma cells indicating a role of P2Y8R in immunological tolerance and lupus pathogenesis [128].

The role of CD39 in the maintenance of immune tolerance is associated with its capacity of degrading ATP and consequently inhibiting the production of IL-17, which stimulates B cells to produce autoantibodies. Ectonucleotide provides protection in by converting eATP to adenosine. Deletion of ectonucleotides mainly, CD39 and CD73 lead to higher levels of anti-RNP antibodies in response to pristane, with

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

CD73 deletion in particular promoting expansion of splenic B cell and T cell populations that likely contribute to autoantibody production [129]. B cells show the highest CD73 surface expression among human circulating immune cells. In SLE patients, the activity of CD73 and CD38 was found to be selectively silenced in B cells. Since CD73 is the bottleneck of extracellular nucleotide degradation to anti-inflammatory adenosine, this pathway is likely to be a crucial step in the pathophysiology of SLE involving B cell immune cell interactions [130].

#### *4.1.2 Rheumatoid arthritis*

RA is a chronic inflammatory disease of joints characterized by damage of bone and cartilage, which leads to joint destruction and disability. Primarily, it is driven by proliferation of synovial fibroblasts, inflammatory response of innate and adaptive immune response, differentiation of macrophage into osteoclasts, and impaired differentiation of mesenchymal stem cells into osteoblasts. The incidence is about 5 per 1000 people and can lead to severe joint damage and disability [131]. Studies have shown a critical role for P2 receptors in osteoblastogenesis and mineralization, synoviocytes proliferation, inflammation of immune cells, and differentiation of macrophages into osteoclasts [132]. Specifically, P2X7, P2Y14, P2Y12, P2Y6, P2Y1, P2Y2, and P2X4 receptors are involved in modulating bone and joint biology [133]. Pain is the major symptom of RA, which associates with the involvement of P2X4R had been reported in chronic arthritis [134]. Knockout of this gene in mice model alleviates the pain [135]. P2X4R control the production of Th17 cells, as shown by the inhibition of P2X4 receptor which reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis [136]. Inhibition of P2X4R associated with the attenuation of synovial inflammation and joint destruction as well as decreased the levels of serum IL-1β, TNF-α, IL-6, and IL-17 via NLRP1 [137]. Similar to SLE, SNP in P2X7 is associated with increased inflammatory response and susceptibility to RA [123, 125]. P2X7 receptor-mediates the release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases and is important for osteoclastogenesis [138]. It also regulates the differentiation of Th17 cells and type II collagen-induced arthritis in mice [139]. P2Y receptor also contribute to the development of RA such as P2Y11 receptor induce inflammation in primary fibroblast-like synoviocytes [140], P2Y12 and P2Y14 receptors induce bone lysis by activating osteoclasts [141–143]. RA patients demonstrate differential expression of adenosine receptors on synovium with preferential expression of A3 and its variant. However, in a separate RA cohort treated with methotrexate shows overexpression A2A and A2B indicating the anti-inflammatory property via these adenosine receptors [144]. Under hypoxia condition, bone resorption is increased in RA patients via A2B receptors. Inhibition of A2B receptors potentially prevent the hypoxia-mediated pathological osteolysis in RA [145].

The expression of CD39 in Tregs is limited by single nucleotide polymorphisms (SNP). It has been shown that AA genotype of the rs10748643 SNP, a low-expressing CD39 variant, is involved in the regulation of the immune system in autoimmunity [146]. A reduced response to methotrexate (MTX) in patients with rheumatoid arthritis was also shown to be related to an SNP that decreases the frequencies of CD39-expressing Tregs, the rs7071836 SNP [147]. Lower expression of CD73 in lymphocytes at the sites of inflammation has been associated with disease severity in juvenile idiopathic arthritis [148].

#### *4.1.3 Multiple sclerosis*

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system, characterized by the presence of focal lesions in white and gray matter, which is associated with pathological and progression neurological dysfunction. Presence of peripheral immune cells infiltration is a main diagnostic hallmark of the disease. Purinergic receptors control immune cell function as well as neuronal and oligodendroglia survival, and the activation of astrocytes and microglia, the endogenous brain immune cells. Genetic variation in P2X4 and P2X7 receptors show susceptibility to MS. Functionally, the variants impair the expression of P2X7 on the surface resulting in the inhibition of ATP-induced pore function and phagocytic activity [149]. Cortical microglia from MS patient exhibit loss of P2Y12 receptor, which associates with the pro-inflammatory and neuronal damaging profile in MS [150]. On the other hand, P2Y12 is the markers of platelet and megakaryocyte activation. Its increased expression in MS patients associates with cardiovascular disease [151]. A study in mice model show that the loss of P2Y6 develop more severe experimental autoimmune encephalomyelitis compared with wild-type mice as it has pivotal role in DCs regulation [64]. Lymphocytes from MS patients also exhibit upregulation of A2A receptor, which modulates the release of proinflammatory cytokine TNF-α, IFN-γ, IL-6, IL-1β, IL-17 via NF-κB. A2A receptor upregulation was observed in lymphocytes from MS patients in comparison with healthy subjects. The stimulation of these receptors mediated a significant inhibition of TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and cell proliferation as well as very late antigen (VLA)-4 expression and NF-κB activation [152].

CD39 expressing Treg cells controls the neuroinflammation in MS by suppressing the pathogenic Th17 cells and IL-17 production [31]. Its activity and the frequency were elevated in relapsing MS patients [153]. Furthermore, a study on animal model demonstrated that overexpression of CD39 on reactive microglia/macrophages that associates with either pro-inflammatory (M1-subtype) or neuroprotective (M2-subtype) at different stages of the disease. At the peak of EAE, CD39 immunoreactivity showed much higher co-occurrence with Arg1 immunoreactivity in microglia and macrophages, compared to iNOS, implying its stronger association with M2-like reactive phenotype [154]. Thus, modulation of purinergic signaling using an agonist or antagonist provides a new avenue for treatment of disease [155, 156].

#### **4.2 Purinergic signaling during bacterial and viral infection**

Infectious diseases are caused by the invasion of pathogenic microorganisms. After infection, host immune system elicits the anti-microbial immune response and at the same time microorganisms develop strategies to evade host defense mechanism. This involves generation of a variety of inflammatory and suppressive responses along with regulatory feedback systems to eliminate the pathogens but also to restore the homeostatic condition following infection or injury [157]. The purinergic system has the dual function of regulating the immune response and triggering effector antimicrobial response against bacterial and viral infections. During the infections, the ATP release initiates a cascade that activates purinergic receptors. This receptor activation enhances the secretion of pro-inflammatory cytokines and performs the chemotaxis of macrophages and neutrophils, generating an association between the immune and the purinergic systems. Immunomodulation by purinergic signaling has been widely discussed elsewhere [26, 158]. Some instances of involvement of

#### *Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

purinergic signaling in bacterial infection include, reduced CD73 expression was associated with macrophage phagocytosis and an efficient clearance of *Salmonella* infection [159]. Likewise, depletion of CD39 on CD4, CD8, and Treg cells augments the T cells response to *Listeria* and *Mycobacterium* infections [160, 161]. On the other hand, transgenic mice with overexpression of CD39 in lung epithelia shows increased recruitment of neutrophils and macrophages in lungs upon *Pseudomonas aeruginosa* infection. The CD39 activity associates with efficient clearance of infection [162]. CD39, due to ATP-scavenging property it limits P2X7 receptor mediated pro-inflammatory responses. Thus, deletion of CD39 exacerbates sepsis-induced liver injury [163]. P2X7R signaling has a detrimental role in severe tuberculosis infection. ATP release and activation of P2X7R cause macrophage necrosis resulting in the spread of bacterial particles, leukocyte infiltration, and tissue damage [164]. Deletion of P2X7 receptor or blockage of P2X7R, or scavenging of eATP may attenuated inflammation, largely preventing increased cytokine secretion and tissue damage [163, 165].

Immunomodulation of purinergic signaling had been implicated in wide variety of viral infections such as human immunodeficiency virus (HIV)-1, hepatitis virus, dengue virus, and SARS-CoV2 [166–169]. HIV-1 primarily infects CD4 T cells, but also affects myeloid dendritic cells and monocyte, macrophages populations that express CD4 receptor. Infected patients exhibit decreased CD4 T cell counts and a reversed CD4/CD8 T cells ratio. Adenosine has an immunosuppressive effect, patients with HIV infection show upregulated CD39 on Treg cells which is inversely related with the CD4 T cell count [167, 170]. In contrast to CD39, CD73 expression was diminished on CD4 T cells, which represent a phenotypically and functionally different subpopulation of CD73+ CD4 T cells. This T cell subsets are preferentially reduced in HIV patients, which suggests the effect of an adenosine diminished microenvironment that cannot prevent persistent immune activation. CD73+ CD4+ T cell counts were inversely associated with T cell activation, as well as plasma C reactive protein levels [171]. Besides, CD73 is involved in the expansion of HIV-specific CD8 T cells, whereas CD73 expression is higher in memory CD8+ T cell subset. The frequency of CD73+ CD8+ T cells is inversely associated with cell activation and plasma viral load [172]. PANX-1 hemichannel opening, activation of P2Y2R, P2X1R are involved in the mediating the effective viral entry and replication in CD4 or target cells [173–175]. Blocking the P2X1 and P2X7 receptors inhibits the viral entry and fusion [176]. Similarly, the P2X1R, P2X4R and P2X7R expression increased in during hepatitis C virus infection and Dengue virus infection [168, 177]. Blocking P2X receptor with antagonist improves the anti-viral response and T cell function [168, 178].

Given the pathophysiological role of purinergic signaling in highly prevalent viral infections has developed a potential interest in investigating the effects of purinergic system in severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2). SARS-CoV-2 infection had impacted more than millions of people worldwide since its emergence in December 2019, in Wuhan, China. The clinical manifestations of SARS-CoV-2 include pneumonia, acute respiratory distress syndrome (ARDS), and hyperinflammation. SARS-CoV-2 primarily invade the alveolar epithelia of respiratory tract and lungs where they replicate, triggers the activation of the immune system resulting in the release of cytokines as a defense mechanism, but the response become exaggerated and prompt the so-called "cytokine storm." This is a state of hyperinflammatory response, which develops acute respiratory syndrome (SARS). This is characterized by fever, cough, and difficulty breathing, which can progress to pneumonia, failure of different organs, and death. Patients with SARS-CoV-2 infection exhibit increased purinergic signaling, which has been suggested to have a role in hyperinflammatory

state [179]. The mechanisms have been described in very detail in review articles [169, 180]. The increased inflammations resulting from activated purinergic signaling in SARS-Cov-2 infections are also associated with different pathological conditions such as neuropathy [181], thrombopathy [182, 183]. As observed in other viral infections patients with SARS-CoV-2 shows reduced expression of CD73 on circulating CD8, NK, and NKT cells. However, cells lacking CD73 exhibit increased cytotoxic effector capacity compared to their counterpart CD73+ [184]. P2X7R-NLRP3 signaling axis are the key driver of inflammation in SARS-CoV-2 [185]. Therefore, P2X7R could serve as a potential therapeutic target to control the inflammation [186]. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function [169]. Understanding this biology is very crucial as anti-inflammatory drugs are not effective and sometimes accompanied by serious adverse effects.

### **5. Conclusion**

This chapter has highlighted the importance of purinergic signaling in modulating the immune system in various therapeutic areas. Purinergic system is capable of fine tuning the levels of nucleotides and their derivative in the extracellular space thereby controlling the chemotaxis, proliferation, differentiation of various immune cell presents locally or far from the infectious site. Dysregulation of purinergic signaling because of genetic factor or escape mechanism employed by the microbes or regulatory cell leads to overt inflammation that contributes to the disease. Special attention has been paid to the mechanisms through which alterations in the various compartments of the purinergic system could contribute to the patho-pathophysiology of autoimmune disease and microbial infection. This chapter could help in gaining insight on the possibility of counteracting such dysfunctions by means of pharmacological interventions on purinergic molecular targets.

### **Author details**

Richa Rai Division of Hematology and Oncology, Department of Medicine, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

\*Address all correspondence to: richa.23m@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

#### **References**

[1] Dunn J, Grider MH. Physiology, adenosine triphosphate. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021

[2] Drury AN, Szent-Györgyi A. The physiological activity of adenine compounds with especial reference to their action upon the mammalian heart. The Journal of Physiology. 1929;**68**(3):213-237

[3] Burnstock G. Purinergic nerves. Pharmacological Reviews. 1972;**24**(3):509-581

[4] Burnstock G. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology. 1997;**36**(9):1127-1139

[5] Burnstock G. Purinergic system. In: Offermanns S, Rosenthal W, editors. Encyclopedia of Molecular Pharmacology. Berlin, Heidelberg, Springer; 2008. pp. 1047-1053

[6] Burnstock G. Short- and longterm (trophic) purinergic signalling. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2016;**371**(1700):20150422

[7] Burnstock G. The therapeutic potential of purinergic signalling. Biochemical Pharmacology. 2018;**151**:157-165

[8] Burnstock G, Verkhratsky A. Longterm (trophic) purinergic signalling: Purinoceptors control cell proliferation, differentiation and death. Cell Death & Disease. 2010;1(1):e9-e

[9] Huang Z, Xie N, Illes P, Di Virgilio F, Ulrich H, Semyanov A, et al. From purines to purinergic signalling:

Molecular functions and human diseases. Signal Transduction and Targeted Therapy. 2021;**6**(1):162

[10] Fitz JG. Regulation of cellular ATP release. Transactions of the American Clinical and Climatological Association. 2007;**118**:199-208

[11] Dosch M, Gerber J, Jebbawi F, Beldi G. Mechanisms of ATP release by inflammatory cells. International Journal of Molecular Sciences. 2018;**19**(4):1222

[12] Taruno A. ATP release channels. International Journal of Molecular Sciences. 2018;**19**(3):808

[13] Imura Y, Morizawa Y, Komatsu R, Shibata K, Shinozaki Y, Kasai H, et al. Microglia release ATP by exocytosis. Glia. 2013;**61**(8):1320-1330

[14] Südhof TC, Rothman JE. Membrane fusion: Grappling with SNARE and SM proteins. Science. 2009;**323**(5913):474-477

[15] Wang N, De Bock M, Decrock E, Bol M, Gadicherla A, Vinken M, et al. Paracrine signaling through plasma membrane hemichannels. Biochimica et Biophysica Acta. 2013;**1828**(1):35-50

[16] Kar R, Batra N, Riquelme MA, Jiang JX. Biological role of connexin intercellular channels and hemichannels. Archives of Biochemistry and Biophysics. 2012;**524**(1):2-15

[17] Wang N, De Bock M, Decrock E, Bol M, Gadicherla A, Bultynck G, et al. Connexin targeting peptides as inhibitors of voltage- and intracellular Ca+2 triggered Cx43 hemichannel opening. Neuropharmacology. 2013;**75**:506-516

[18] Dourado M, Wong E, Hackos DH. Pannexin-1 is blocked by its C-terminus through a delocalized non-specific interaction surface. PLoS One. 2014;**9**(6): e99596

[19] Penuela S, Gehi R, Laird DW. The biochemistry and function of pannexin channels. Biochimica et Biophysica Acta. 2013;**1828**(1):15-22

[20] Chekeni FB, Elliott MR, Sandilos JK, Walk SF, Kinchen JM, Lazarowski ER, et al. Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis. Nature. 2010;**467**(7317):863-867

[21] Sandilos JK, Chiu YH, Chekeni FB, Armstrong AJ, Walk SF, Ravichandran KS, et al. Pannexin 1, an ATP release channel, is activated by caspase cleavage of its pore-associated C-terminal autoinhibitory region. The Journal of Biological Chemistry. 2012;**287**(14):11303-11311

[22] Yang D, He Y, Muñoz-Planillo R, Liu Q, Núñez G. Caspase-11 requires the Pannexin-1 channel and the purinergic P2X7 pore to mediate pyroptosis and endotoxic shock. Immunity. 2015;**43**(5):923-932

[23] Iyer SS, Pulskens WP, Sadler JJ, Butter LM, Teske GJ, Ulland TK, et al. Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome. Proceedings of the National Academy of Sciences of the United States of America. 2009;**106**(48):20388-20393

[24] Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature. 2009;**461**(7261):282-286

[25] Stefan C, Jansen S, Bollen M. Modulation of purinergic signaling by NPP-type ectophosphodiesterases. Purinergic Signalling. 2006;**2**(2):361-370

[26] Giuliani AL, Sarti AC, Di Virgilio F. Ectonucleotidases in acute and chronic inflammation. Frontiers in Pharmacology. 2021;**11**:619458

[27] Haas CB, Lovászi M, Pacher P, de Souza PO, Pelletier J, Leite RO, et al. Extracellular ectonucleotidases are differentially regulated in murine tissues and human polymorphonuclear leukocytes during sepsis and inflammation. Purinergic Signalling. 2021;**17**(4):713-724

[28] Haas CB, Lovászi M, Braganhol E, Pacher P, Haskó G. Ectonucleotidases in inflammation, immunity, and cancer. Journal of Immunology. 2021;**206**(9):1983-1990

[29] Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R, et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: Hydrolysis of extracellular ATP and immune suppression. Blood. 2007;**110**(4):1225-1232

[30] Mandapathil M, Hilldorfer B, Szczepanski MJ, Czystowska M, Szajnik M, Ren J, et al. Generation and accumulation of immunosuppressive adenosine by human CD4+ CD25highFOXP3+ regulatory T cells. The Journal of Biological Chemistry. 2010;**285**(10):7176-7186

[31] Fletcher JM, Lonergan R, Costelloe L, Kinsella K, Moran B, O'Farrelly C, et al. CD39+Foxp3+ regulatory T cells suppress pathogenic Th17 cells and are impaired in multiple sclerosis. Journal of Immunology. 2009;**183**(11):7602-7610

[32] Schneider E, Winzer R, Rissiek A, Ricklefs I, Meyer-Schwesinger C, Ricklefs FL, et al. CD73-mediated

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

adenosine production by CD8 T cellderived extracellular vesicles constitutes an intrinsic mechanism of immune suppression. Nature Communications. 2021;**12**(1):5911

[33] Saze Z, Schuler PJ, Hong C-S, Cheng D, Jackson EK, Whiteside TL. Adenosine production by human B cells and B cell-mediated suppression of activated T cells. Blood. 2013;**122**(1):9-18

[34] Ferrari D, Pizzirani C, Adinolfi E, Lemoli RM, Curti A, Idzko M, et al. The P2X7 receptor: A key player in IL-1 processing and release. Journal of Immunology. 2006;**176**(7):3877-3883

[35] Clavarino G, Delouche N, Vettier C, Laurin D, Pernollet M, Raskovalova T, et al. Novel strategy for phenotypic characterization of human B lymphocytes from precursors to effector cells by flow cytometry. PLoS One. 2016;**11**(9):e0162209

[36] Sandoval-Montes C, Santos-Argumedo L. CD38 is expressed selectively during the activation of a subset of mature T cells with reduced proliferation but improved potential to produce cytokines. Journal of Leukocyte Biology. 2005;**77**(4):513-521

[37] Piedra-Quintero ZL, Wilson Z, Nava P, Guerau-de-Arellano M. CD38: An immunomodulatory molecule in inflammation and autoimmunity. Frontiers in Immunology. 2020;**11**: 597959

[38] Partida-Sánchez S, Cockayne DA, Monard S, Jacobson EL, Oppenheimer N, Garvy B, et al. Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo. Nature Medicine. 2001;**7**(11):1209-1216

[39] Kang J, Park KH, Kim JJ, Jo EK, Han MK, Kim UH. The role of CD38 in Fcγ receptor (FcγR)-mediated phagocytosis in murine macrophages. The Journal of Biological Chemistry. 2012;**287**(18):14502-14514

[40] Muñoz P, Mittelbrunn M, de la Fuente H, Pérez-Martínez M, García-Pérez A, Ariza-Veguillas A, et al. Antigen-induced clustering of surface CD38 and recruitment of intracellular CD38 to the immunologic synapse. Blood. 2008;**111**(7):3653-3664

[41] Linden J, Koch-Nolte F, Dahl G. Purine release, metabolism, and signaling in the inflammatory response. Annual Review of Immunology. 2019;**37**:325-347

[42] Zeidler JD, Hogan KA, Agorrody G, Peclat TR, Kashyap S, Kanamori KS, et al. The CD38 glycohydrolase and the NAD sink: Implications for pathological conditions. American Journal of Physiology. Cell Physiology. 2022; **322**(3):C521-C545

[43] Albright RA, Ornstein DL, Cao W, Chang WC, Robert D, Tehan M, et al. Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke. The Journal of Biological Chemistry. 2014;**289**(6):3294-3306

[44] Knowlden S, Georas SN. The autotaxin-LPA axis emerges as a novel regulator of lymphocyte homing and inflammation. Journal of Immunology (Baltimore, MD: 1950). 2014;**192**(3):851-857

[45] Benesch MG, Tang X, Dewald J, Dong WF, Mackey JR, Hemmings DG, et al. Tumor-induced inflammation in mammary adipose tissue stimulates a vicious cycle of autotaxin expression and breast cancer progression. The FASEB Journal. 2015;**29**(9):3990-4000

[46] Burnstock G. Purine and purinergic receptors. Brain and Neuroscience Advances. 2018;**2**:2398212818817494

[47] Pastor-Anglada M, Pérez-Torras S. Who is who in adenosine transport. Frontiers in Pharmacology. 2018;**9**:627

[48] Antonioli L, Colucci R, Pellegrini C, Giustarini G, Tuccori M, Blandizzi C, et al. The role of purinergic pathways in the pathophysiology of gut diseases: Pharmacological modulation and potential therapeutic applications. Pharmacology & Therapeutics. 2013;**139**(2):157-188

[49] Marshall JS, Warrington R, Watson W, Kim HL. An introduction to immunology and immunopathology. Allergy, Asthma & Clinical Immunology. 2018;**14**(2):49

[50] Cekic C, Linden J. Purinergic regulation of the immune system. Nature Reviews Immunology. 2016;**16**(3):177-192

[51] Kronlage M, Song J, Sorokin L, Isfort K, Schwerdtle T, Leipziger J, et al. Autocrine purinergic receptor signaling is essential for macrophage chemotaxis. Science Signaling. 2010;**3**(132):ra55

[52] Chen Y, Corriden R, Inoue Y, Yip L, Hashiguchi N, Zinkernagel A, et al. ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science. 2006;**314**(5806):1792-1795

[53] Cohen HB, Briggs KT, Marino JP, Ravid K, Robson SC, Mosser DM. TLR stimulation initiates a CD39-based autoregulatory mechanism that limits macrophage inflammatory responses. Blood. 2013;**122**(11):1935-1945

[54] Ferrante CJ, Pinhal-Enfield G, Elson G, Cronstein BN, Hasko G, Outram S, et al. The

adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Rα) signaling. Inflammation. 2013;**36**(4):921-931

[55] Wang L, Jacobsen SE, Bengtsson A, Erlinge D. P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells. BMC Immunology. 2004;**5**:16

[56] Klaver D, Thurnher M. Control of macrophage inflammation by P2Y purinergic receptors. Cell. 2021;**10**(5):109

[57] Merz J, Nettesheim A, von Garlen S, Albrecht P, Saller BS, Engelmann J, et al. Pro- and anti-inflammatory macrophages express a sub-type specific purinergic receptor profile. Purinergic Signalling. 2021;**17**(3):481-492

[58] Schnurr M, Toy T, Shin A, Hartmann G, Rothenfusser S, Soellner J, et al. Role of adenosine receptors in regulating chemotaxis and cytokine production of plasmacytoid dendritic cells. Blood. 2004;**103**(4):1391-1397

[59] Panther E, Corinti S, Idzko M, Herouy Y, Napp M, la Sala A, et al. Adenosine affects expression of membrane molecules, cytokine and chemokine release, and the T-cell stimulatory capacity of human dendritic cells. Blood. 2003;**101**(10):3985-3990

[60] Wilson JM, Kurtz CC, Black SG, Ross WG, Alam MS, Linden J, et al. The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6. Journal of Immunology. 2011;**186**(12):6746-6752

[61] Ring S, Pushkarevskaya A, Schild H, Probst HC, Jendrossek V, Wirsdörfer F, et al. Regulatory T cell-derived adenosine induces dendritic cell migration through

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

the Epac-Rap1 pathway. Journal of Immunology. 2015;**194**(8):3735-3744

[62] Sáez PJ, Vargas P, Shoji KF, Harcha PA, Lennon-Duménil AM, Sáez JC. ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X(7) receptors. Science Signaling. 2017;**10**(506):eaah7107

[63] Yu Y, Feng S, Wei S, Zhong Y, Yi G, Chen H, et al. Extracellular ATP activates P2X7R-NF-κB (p65) pathway to promote the maturation of bone marrow-derived dendritic cells of mice. Cytokine. 2019;**119**:175-181

[64] Li Z, He C, Zhang J, Zhang H, Wei H, Wu S, et al. P2Y(6) deficiency enhances dendritic cell-mediated Th1/Th17 differentiation and aggravates experimental autoimmune encephalomyelitis. Journal of Immunology. 2020;**205**(2):387-397

[65] Li R, Wang J, Li R, Zhu F, Xu W, Zha G, et al. ATP/P2X7-NLRP3 axis of dendritic cells participates in the regulation of airway inflammation and hyper-responsiveness in asthma by mediating HMGB1 expression and secretion. Experimental Cell Research. 2018;**366**(1):1-15

[66] Sakaki H, Fujiwaki T, Tsukimoto M, Kawano A, Harada H, Kojima S. P2X4 receptor regulates P2X7 receptordependent IL-1β and IL-18 release in mouse bone marrow-derived dendritic cells. Biochemical and Biophysical Research Communications. 2013;**432**(3):406-411

[67] Silva-Vilches C, Ring S, Mahnke K. ATP and its metabolite adenosine as regulators of dendritic cell activity. Frontiers in Immunology. 2018;**9**:2581

[68] Zhao R, Qiao J, Zhang X, Zhao Y, Meng X, Sun D, et al. Toll-like receptor-mediated activation of CD39 internalization in BMDCs leads to extracellular ATP accumulation and facilitates P2X7 receptor activation. Frontiers in Immunology. 2019;**10**:2524

[69] Mantovani A, Cassatella MA, Costantini C, Jaillon S. Neutrophils in the activation and regulation of innate and adaptive immunity. Nature Reviews. Immunology. 2011;**11**(8):519-531

[70] Amulic B, Cazalet C, Hayes GL, Metzler KD, Zychlinsky A. Neutrophil function: From mechanisms to disease. Annual Review of Immunology. 2012;**30**:459-489

[71] la Sala A, Ferrari D, Di Virgilio F, Idzko M, Norgauer J, Girolomoni G. Alerting and tuning the immune response by extracellular nucleotides. Journal of Leukocyte Biology. 2003;**73**(3):339-343

[72] Chen Y, Yao Y, Sumi Y, Li A, To UK, Elkhal A, et al. Purinergic signaling: A fundamental mechanism in neutrophil activation. Science Signaling. 2010;**3**(125):ra45

[73] Kukulski F, Ben Yebdri F, Lecka J, Kauffenstein G, Lévesque SA, Martín-Satué M, et al. Extracellular ATP and P2 receptors are required for IL-8 to induce neutrophil migration. Cytokine. 2009;**46**(2):166-170

[74] Kukulski F, Bahrami F, Ben Yebdri F, Lecka J, Martín-Satué M, Lévesque SA, et al. NTPDase1 controls IL-8 production by human neutrophils. Journal of Immunology. 2011;**187**(2):644-653

[75] Pierce S, Geanes ES, Bradley T. Targeting natural killer cells for improved immunity and control of the adaptive immune response. Frontiers in cellular and infection. Microbiology. 2020;**10**:231

[76] Chambers AM, Wang J, Lupo KB, Yu H, Atallah Lanman NM, Matosevic S. Adenosinergic Signaling alters natural killer cell functional responses. Frontiers in Immunology. 2018;**9**:2533

[77] Hoskin DW, Mader JS, Furlong SJ, Conrad DM, Blay J. Inhibition of T cell and natural killer cell function by adenosine and its contribution to immune evasion by tumor cells (review). International Journal of Oncology. 2008;**32**(3):527-535

[78] Miller JS, Cervenka T, Lund J, Okazaki IJ, Moss J. Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor. The Journal of Immunology. 1999;**162**(12):7376-7382

[79] Bajpai A, Brahmi Z. Regulation of resting and IL-2-activated human cytotoxic lymphocytes by exogenous nucleotides: Role of IL-2 and ecto-ATPases. Cellular Immunology. 1993;**148**(1):130-143

[80] Li Z, Gao Y, He C, Wei H, Zhang J, Zhang H, et al. Purinergic receptor P2Y(6) is a negative regulator of NK cell maturation and function. Journal of Immunology. 2021;**207**(6):1555-1565

[81] Rissiek B, Danquah W, Haag F, Koch-Nolte F. Technical advance: A new cell preparation strategy that greatly improves the yield of vital and functional Tregs and NKT cells. Journal of Leukocyte Biology. 2014;**95**(3):543-549

[82] Rissiek B, Haag F, Boyer O, Koch-Nolte F, Adriouch S. ADP-ribosylation of P2X7: A matter of life and death for regulatory T cells and natural killer T cells. In: Koch-Nolte F, editor. Endogenous ADP-Ribosylation. Cham: Springer International Publishing; 2015. pp. 107-126

[83] Krovi SH, Gapin L. Invariant natural killer T cell subsets—More than just

developmental intermediates. Frontiers in Immunology. 2018;**9**:1393

[84] Yu JC, Lin G, Field JJ, Linden J. Induction of antiinflammatory purinergic signaling in activated human iNKT cells. JCI Insight. 2018;**3**(17):e91954

[85] Felley LE, Sharma A, Theisen E, Romero-Masters JC, Sauer JD, Gumperz JE. Human invariant NKT cells induce IL-1β secretion by peripheral blood monocytes via a P2X7-independent pathway. Journal of Immunology. 2016;**197**(6):2455-2464

[86] Xu X, Pocock GM, Sharma A, Peery SL, Fites JS, Felley L, et al. Human iNKT cells promote protective inflammation by inducing oscillating purinergic signaling in monocytederived DCs. Cell Reports. 2016;**16**(12): 3273-3285

[87] Tai Y, Wang Q, Korner H, Zhang L, Wei W. Molecular mechanisms of T cells activation by dendritic cells in autoimmune diseases. Frontiers in Pharmacology. 2018;**9**:642

[88] Di Virgilio F, Bronte V, Collavo D, Zanovello P. Responses of mouse lymphocytes to extracellular adenosine 5′-triphosphate (ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effects of ATP. Journal of Immunology. 1989;**143**(6):1955-1960

[89] Woehrle T, Yip L, Elkhal A, Sumi Y, Chen Y, Yao Y, et al. Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse. Blood. 2010;**116**(18): 3475-3484

[90] Grassi F. The P2X7 receptor as regulator of T cell development and function. Frontiers in Immunology. 2020;**11**:1179

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

[91] Yip L, Woehrle T, Corriden R, Hirsh M, Chen Y, Inoue Y, et al. Autocrine regulation of T-cell activation by ATP release and P2X7 receptors. The FASEB Journal. 2009;**23**(6):1685-1693

[92] Woehrle T, Ledderose C, Rink J, Slubowski C, Junger WG. Autocrine stimulation of P2Y1 receptors is part of the purinergic signaling mechanism that regulates T cell activation. Purinergic Signalling. 2019;**15**(2):127-137

[93] Schenk U, Frascoli M, Proietti M, Geffers R, Traggiai E, Buer J, et al. ATP inhibits the generation and function of regulatory T cells through the activation of purinergic P2X receptors. Science Signaling. 2011;**4**(162):ra12

[94] Borges da Silva H, Beura LK, Wang H, Hanse EA, Gore R, Scott MC, et al. The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8(+) T cells. Nature. 2018;**559**(7713):264-268

[95] Mellouk A, Bobé P. CD8(+), but not CD4(+) effector/memory T cells, express the CD44(high)CD45RB(high) phenotype with aging, which displays reduced expression levels of P2X(7) receptor and ATP-induced cellular responses. The FASEB Journal. 2019;**33**(3):3225-3236

[96] Safya H, Mellouk A, Legrand J, Le Gall SM, Benbijja M, Kanellopoulos-Langevin C, et al. Variations in cellular responses of mouse T cells to Adenosine-5′-triphosphate stimulation do not depend on P2X7 receptor expression levels but on their activation and differentiation stage. Frontiers in immunology. 2018;**9**:360

[97] Ohta A, Ohta A, Madasu M, Kini R, Subramanian M, Goel N, et al. A2A adenosine receptor may allow expansion of T cells lacking effector

functions in extracellular adenosinerich microenvironments. Journal of Immunology. 2009;**183**(9):5487-5493

[98] Gessi S, Varani K, Merighi S, Cattabriga E, Avitabile A, Gavioli R, et al. Expression of A3 adenosine receptors in human lymphocytes: Up-regulation in T cell activation. Molecular Pharmacology. 2004;**65**(3):711-719

[99] Sluyter R, Barden JA, Wiley JS. Detection of P2X purinergic receptors on human B lymphocytes. Cell and Tissue Research. 2001;**304**(2):231-236

[100] Przybyła T, Sakowicz-Burkiewicz M, Pawełczyk T. Purinergic signaling in B cells. Acta Biochimica Polonica. 2018;**65**(1):1-7

[101] Sengstake S, Boneberg E-M, Illges H. CD21 and CD62L shedding are both inducible via P2X7Rs. International Immunology. 2006;**18**(7):1171-1178

[102] Pupovac A, Geraghty NJ, Watson D, Sluyter R. Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells. Immunology and Cell Biology. 2015;**93**(1):77-85

[103] Sakowicz-Burkiewicz M, Kocbuch K, Grden M, Maciejewska I, Szutowicz A, Pawelczyk T. High glucose concentration impairs ATP outflow and immunoglobulin production by human peripheral B lymphocytes: Involvement of P2X7 receptor. Immunobiology. 2013;**218**(4):591-601

[104] Conter LJ, Song E, Shlomchik MJ, Tomayko MM. CD73 expression is dynamically regulated in the germinal center and bone marrow plasma cells are diminished in its absence. PLoS One. 2014;**9**(3):e92009

[105] Cao F, Hu L-Q, Yao S-R, Hu Y, Wang D-G, Fan Y-G, et al. P2X7 receptor: A potential therapeutic target for autoimmune diseases. Autoimmunity Reviews. 2019;**18**(8):767-777

[106] Cojocaru M, Cojocaru IM, Silosi I, Vrabie CD. Manifestations of systemic lupus erythematosus. Maedica. 2011;**6**(4):330-336

[107] Yaniv G, Twig G, Shor DB, Furer A, Sherer Y, Mozes O, et al. A volcanic explosion of autoantibodies in systemic lupus erythematosus: A diversity of 180 different antibodies found in SLE patients. Autoimmunity Reviews. 2015;**14**(1):75-79

[108] Rai G, Rai R, Saeidian AH, Rai M. Microarray to deep sequencing: Transcriptome and miRNA profiling to elucidate molecular pathways in systemic lupus erythematosus. Immunologic Research. 2016;**64**(1):14-24

[109] Rai R, Chauhan SK, Singh VV, Rai M, Rai G. Heat shock protein 27 and its regulatory molecules express differentially in SLE patients with distinct autoantibody profiles. Immunology Letters. 2015;**164**(1):25-32

[110] Darrah E, Andrade F. NETs: The missing link between cell death and systemic autoimmune diseases? Frontiers in Immunology. 2012;**3**:428

[111] Magna M, Pisetsky DS. The role of cell death in the pathogenesis of SLE: Is pyroptosis the missing link? Scandinavian Journal of Immunology. 2015;**82**(3):218-224

[112] Chauhan SK, Rai R, Singh VV, Rai M, Rai G. Differential clearance mechanisms, neutrophil extracellular trap degradation and phagocytosis, are operative in systemic lupus erythematosus patients with distinct autoantibody specificities. Immunology Letters. 2015;**168**(2):254-259

[113] Mahajan A, Herrmann M, Muñoz LE. Clearance deficiency and cell death pathways: A model for the pathogenesis of SLE. Frontiers in Immunology. 2016;**7**:35

[114] Di Virgilio F, Giuliani AL. Purinergic signalling in autoimmunity: A role for the P2X7R in systemic lupus erythematosus? Biomedical Journal. 2016;**39**(5):326-338

[115] Kahlenberg JM, Carmona-Rivera C, Smith CK, Kaplan MJ. Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in lupus macrophages. Journal of Immunology. 2013;**190**(3):1217-1226

[116] Wang D, Drenker M, Eiz-Vesper B, Werfel T, Wittmann M. Evidence for a pathogenetic role of interleukin-18 in cutaneous lupus erythematosus. Arthritis and Rheumatism. 2008;**58**(10):3205-3215

[117] Hu D, Liu X, Chen S, Bao C. Expressions of IL-18 and its binding protein in peripheral blood leukocytes and kidney tissues of lupus nephritis patients. Clinical Rheumatology. 2010;**29**(7):717-721

[118] Kahlenberg JM, Thacker SG, Berthier CC, Cohen CD, Kretzler M, Kaplan MJ. Inflammasome activation of IL-18 results in endothelial progenitor cell dysfunction in systemic lupus erythematosus. Journal of Immunology. 2011;**187**(11):6143-6156

[119] Turner CM, Tam FW, Lai PC, Tarzi RM, Burnstock G, Pusey CD, et al. Increased expression of the proapoptotic ATP-sensitive P2X7 receptor in experimental and human glomerulonephritis. Nephrology, Dialysis, Transplantation. 2007;**22**(2):386-395

[120] Zhao J, Wang H, Dai C, Wang H, Zhang H, Huang Y, et al. P2X7 blockade *Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

attenuates murine lupus nephritis by inhibiting activation of the NLRP3/ ASC/caspase 1 pathway. Arthritis and Rheumatism. 2013;**65**(12):3176-3185

[121] Taylor SR, Turner CM, Elliott JI, McDaid J, Hewitt R, Smith J, et al. P2X7 deficiency attenuates renal injury in experimental glomerulonephritis. The Journal of the American Society of Nephrology. 2009;**20**(6):1275-1281

[122] Hu S, Yu F, Ye C, Huang X, Lei X, Dai Y, et al. The presence of P2RX7 single nuclear polymorphism is associated with a gain of function in P2X7 receptor and inflammasome activation in SLE complicated with pericarditis. Clinical and Experimental Rheumatology. 2020;**38**(3):442-449

[123] Portales-Cervantes L, Niño-Moreno P, Salgado-Bustamante M, García-Hernández MH, Baranda-Candido L, Reynaga-Hernández E, et al. The His155Tyr (489C>T) single nucleotide polymorphism of P2RX7 gene confers an enhanced function of P2X7 receptor in immune cells from patients with rheumatoid arthritis. Cellular Immunology. 2012;**276**(1-2):168-175

[124] Li M, Yang C, Wang Y, Song W, Jia L, Peng X, et al. The expression of P2X7 receptor on Th1, Th17, and regulatory T cells in patients with systemic lupus erythematosus or rheumatoid arthritis and its correlations with active disease. Journal of Immunology. 2020;**205**(7):1752-1762

[125] Portales-Cervantes L, Niño-Moreno P, Doníz-Padilla L, Baranda-Candido L, García-Hernández M, Salgado-Bustamante M, et al. Expression and function of the P2X7 purinergic receptor in patients with systemic lupus erythematosus and rheumatoid arthritis. Human Immunology. 2010;**71**(8):818-825

[126] Faliti CE, Gualtierotti R, Rottoli E, Gerosa M, Perruzza L, Romagnani A,

et al. P2X7 receptor restrains pathogenic Tfh cell generation in systemic lupus erythematosus. The Journal of Experimental Medicine. 2019;**216**(2):317- 336

[127] Le Gall SM, Legrand J, Benbijja M, Safya H, Benihoud K, Kanellopoulos JM, et al. Loss of P2X7 receptor plasma membrane expression and function in pathogenic B220+ double-negative T lymphocytes of autoimmune MRL/lpr mice. PLoS One. 2012;**7**(12):e52161

[128] He Y, Gallman AE, Xie C, Shen Q, Ma J, Wolfreys FD, et al. P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance. The Journal of Experimental Medicine. 2022;**219**(1):e20211004

[129] Knight JS, Mazza LF, Yalavarthi S, Sule G, Ali RA, Hodgin JB, et al. Ectonucleotidasemediated suppression of lupus autoimmunity and vascular dysfunction. Frontiers in Immunology. 2018;**9**:1322

[130] Hesse J, Siekierka-Harreis M, Steckel B, Alter C, Schallehn M, Honke N, et al. Profound inhibition of CD73-dependent formation of anti-inflammatory adenosine in B cells of SLE patients. eBioMedicine. 2021;**73**:103616

[131] Aletaha D, Smolen JS. Diagnosis and management of rheumatoid arthritis: A review. Journal of the American Medical Association. 2018;**320**(13):1360-1372

[132] Idzko M, Ferrari D, Eltzschig HK. Nucleotide signalling during inflammation. Nature. 2014;**509**(7500):310-317

[133] Bhagavatham SKS, Kannan V, Darshan VMD, Sivaramakrishnan V. Nucleotides modulate synoviocyte proliferation and osteoclast differentiation in macrophages with

potential implications for rheumatoid arthritis. 3. Biotech. 2021;**11**(12):504

[134] Zhang WJ, Luo HL, Zhu ZM. The role of P2X4 receptors in chronic pain: A potential pharmacological target. Biomedicine & Pharmacotherapy. 2020;**129**:110447

[135] Tsuda M, Kuboyama K, Inoue T, Nagata K, Tozaki-Saitoh H, Inoue K. Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays. Molecular Pain. 2009;**5**:28

[136] Hamoudi C, Zhao C, Abderrazak A, Salem M, Fortin PR, Sévigny J, et al. The purinergic receptor P2X4 promotes Th17 activation and the development of arthritis. Journal of Immunology. 2022;**208**(5):1115-1127

[137] Li F, Guo N, Ma Y, Ning B, Wang Y, Kou L. Inhibition of P2X4 suppresses joint inflammation and damage in collagen-induced arthritis. Inflammation. 2014;**37**(1):146-153

[138] Lopez-Castejon G, Theaker J, Pelegrin P, Clifton AD, Braddock M, Surprenant A. P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases. Journal of Immunology. 2010;**185**(4):2611-2619

[139] Fan Z-D, Zhang Y-Y, Guo Y-H, Huang N, Ma H-H, Huang H, et al. Involvement of P2X7 receptor signaling on regulating the differentiation of Th17 cells and type II collagen-induced arthritis in mice. Scientific Reports. 2016;**6**:35804

[140] Gao F, Li X. P2Y11 receptor antagonist NF340 ameliorates inflammation in human fibroblastlike synoviocytes: An implication in rheumatoid arthritis. IUBMB Life. 2019;**71**(10):1552-1560

[141] Orriss IR, Wang N, Burnstock G, Arnett TR, Gartland A, Robaye B, et al. The P2Y6 receptor stimulates bone resorption by osteoclasts. Endocrinology. 2011;**152**(10):3706-3716

[142] Su X, Floyd DH, Hughes A, Xiang J, Schneider JG, Uluckan O, et al. The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling. The Journal of Clinical Investigation. 2012;**122**(10):3579-3592

[143] Lazarowski ER, Harden TK. UDP-sugars as extracellular signaling molecules: Cellular and physiologic consequences of P2Y14 receptor activation. Molecular Pharmacology. 2015;**88**(1):151-160

[144] Stamp LK, Hazlett J, Roberts RL, Frampton C, Highton J, Hessian PA. Adenosine receptor expression in rheumatoid synovium: A basis for methotrexate action. Arthritis Research & Therapy. 2012;**14**(3):R138

[145] Knowles HJ. The adenosine a(2B) receptor drives osteoclastmediated bone resorption in hypoxic microenvironments. Cell. 2019;**8**(6):624

[146] Moncrieffe H, Ursu S, Pesenacker A, Gordon-Smith S, Zheng D, Wedderburn L. Autoimmune susceptibility gene critically influences CD39 T cell expression and function in modulating human inflammation (P3313). The Journal of Immunology. 2013;**190**(1 Supplement):175.174

[147] da Silva JLG, Passos DF, Bernardes VM, Leal DBR. ATP and adenosine: Role in the immunopathogenesis of rheumatoid arthritis. Immunology Letters. 2019;**214**:55-64

[148] Botta Gordon-Smith S, Ursu S, Eaton S, Moncrieffe H, Wedderburn LR. *Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

Correlation of low CD73 expression on synovial lymphocytes with reduced adenosine generation and higher disease severity in juvenile idiopathic arthritis. Arthritis & Rhematology. 2015;**67**(2):545-554

[149] Sadovnick AD, Gu BJ, Traboulsee AL, Bernales CQ, Encarnacion M, Yee IM, et al. Purinergic receptors P2RX4 and P2RX7 in familial multiple sclerosis. Human Mutation. 2017;**38**(6):736-744

[150] van Olst L, Rodriguez-Mogeda C, Picon C, Kiljan S, James RE, Kamermans A, et al. Meningeal inflammation in multiple sclerosis induces phenotypic changes in cortical microglia that differentially associate with neurodegeneration. Acta Neuropathologica. 2021;**141**(6):881-899

[151] Dziedzic A, Miller E, Saluk-Bijak J, Niwald M, Bijak M. The molecular aspects of disturbed platelet activation through ADP/ P2Y(12) pathway in multiple sclerosis. International Journal of Molecular Sciences. 2021;**22**(12):657

[152] Vincenzi F, Corciulo C, Targa M, Merighi S, Gessi S, Casetta I, et al. Multiple sclerosis lymphocytes upregulate A2A adenosine receptors that are antiinflammatory when stimulated. European Journal of Immunology. 2013;**43**(8):2206-2216

[153] Álvarez-Sánchez N, Cruz-Chamorro I, Díaz-Sánchez M, Lardone PJ, Guerrero JM, Carrillo-Vico A. Peripheral CD39-expressing T regulatory cells are increased and associated with relapsing-remitting multiple sclerosis in relapsing patients. Scientific Reports. 2019;**9**(1):2302

[154] Jakovljevic M, Lavrnja I, Bozic I, Milosevic A, Bjelobaba I, Savic D, et al. Induction of NTPDase1/CD39 by reactive microglia and macrophages is associated with the functional state during EAE. Frontiers in Neuroscience. 2019;**13**:410

[155] Domercq M, Zabala A, Matute C. Purinergic receptors in multiple sclerosis pathogenesis. Brain Research Bulletin. 2019;**151**:38-45

[156] Sidoryk-Węgrzynowicz M, Strużyńska L. Astroglial and microglial purinergic P2X7 receptor as a major contributor to neuroinflammation during the course of multiple sclerosis. International Journal of Molecular Sciences. 2021;**22**(16):8404

[157] Villani AC, Sarkizova S, Hacohen N. Systems immunology: Learning the rules of the immune system. Annual Review of Immunology. 2018;**36**:813-842

[158] Eberhardt N, Bergero G, Mazzocco Mariotta YL, Aoki MP. Purinergic modulation of the immune response to infections. Purinergic Signal. 2022:**18**(1):93-113

[159] Costales MG, Alam MS, Cavanaugh C, Williams KM. Extracellular adenosine produced by ecto-5′ nucleotidase (CD73) regulates macrophage pro-inflammatory responses, nitric oxide production, and favors Salmonella persistence. Nitric Oxide. 2018;**72**:7-15

[160] Raczkowski F, Rissiek A, Ricklefs I, Heiss K, Schumacher V, Wundenberg K, et al. CD39 is upregulated during activation of mouse and human T cells and attenuates the immune response to Listeria monocytogenes. PLoS One. 2018;**13**(5):e0197151

[161] Chiacchio T, Casetti R, Butera O, Vanini V, Carrara S, Girardi E, et al. Characterization of regulatory T cells identified as CD4(+)CD25(high) CD39(+) in patients with active

tuberculosis. Clinical and Experimental Immunology. 2009;**156**(3):463-470

[162] Théâtre E, Frederix K, Guilmain W, Delierneux C, Lecut C, Bettendorff L, et al. Overexpression of CD39 in mouse airways promotes bacteria-induced inflammation. Journal of Immunology. 2012;**189**(4):1966-1974

[163] Savio LEB, de Andrade MP, Figliuolo VR, de Avelar Almeida TF, Santana PT, Oliveira SDS, et al. CD39 limits P2X7 receptor inflammatory signaling and attenuates sepsis-induced liver injury. Journal of Hepatology. 2017;**67**(4):716-726

[164] Amaral EP, Ribeiro SC, Lanes VR, Almeida FM, de Andrade MR, Bomfim CC, et al. Pulmonary infection with hypervirulent mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis. PLoS Pathogens. 2014;**10**(7):e1004188

[165] Li X, Kondo Y, Bao Y, Staudenmaier L, Lee A, Zhang J, et al. Systemic adenosine triphosphate impairs neutrophil chemotaxis and host defense in sepsis. Critical Care Medicine. 2017;**45**(1):e97-e104

[166] Taylor JM, Han Z. Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus. PLoS One. 2010;**5**(12):e15784

[167] Pacheco PA, Faria RX, Ferreira LG, Paixão IC. Putative roles of purinergic signaling in human immunodeficiency virus-1 infection. Biology Direct. 2014;**9**:21

[168] Corrêa G, de ALC, Fernandes-Santos C, Gandini M, Petitinga Paiva F, Coutinho-Silva R, et al. The purinergic receptor P2X7 role in control of dengue virus-2 infection

and cytokine/chemokine production in infected human monocytes. Immunobiology. 2016;**221**(7):794-802

[169] Hasan D, Shono A, van Kalken CK, van der Spek PJ, Krenning EP, Kotani T. A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling. Purinergic Signal. 2022;**18**(1):13-59

[170] Nikolova M, Carriere M, Jenabian MA, Limou S, Younas M, Kök A, et al. CD39/adenosine pathway is involved in AIDS progression. PLoS Pathogens. 2011;**7**(7):e1002110

[171] Schuler PJ, Macatangay BJ, Saze Z, Jackson EK, Riddler SA, Buchanan WG, et al. CD4<sup>+</sup> CD73+ T cells are associated with lower T-cell activation and C reactive protein levels and are depleted in HIV-1 infection regardless of viral suppression. AIDS. 2013;**27**(10):1545-1555

[172] Tóth I, Le AQ, Hartjen P, Thomssen A, Matzat V, Lehmann C, et al. Decreased frequency of CD73+CD8+ T cells of HIV-infected patients correlates with immune activation and T cell exhaustion. Journal of Leukocyte Biology. 2013;**94**(4):551-561

[173] Séror C, Melki MT, Subra F, Raza SQ, Bras M, Saïdi H, et al. Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection. The Journal of Experimental Medicine. 2011;**208**(9):1823-1834

[174] Orellana JA, Velasquez S, Williams DW, Sáez JC, Berman JW, Eugenin EA. Pannexin1 hemichannels are critical for HIV infection of human primary CD4+ T lymphocytes. Journal of Leukocyte Biology. 2013;**94**(3):399-407

[175] Freeman TL, Swartz TH. Purinergic receptors: Elucidating the role of these

*Cross Talk of Purinergic and Immune Signaling: Implication in Inflammatory and Pathogenic… DOI: http://dx.doi.org/10.5772/intechopen.104978*

immune mediators in HIV-1 fusion. Viruses. 2020;**12**(3):290

[176] Giroud C, Marin M, Hammonds J, Spearman P, Melikyan GB. P2X1 receptor antagonists inhibit HIV-1 fusion by blocking virus-coreceptor interactions. Journal of Virology. 2015;**89**(18):9368-9382

[177] Manzoor S, Akhtar U, Naseem S, Khalid M, Mazhar M, Parvaiz F, et al. Ionotropic purinergic receptors P2X4 and P2X7: Proviral or antiviral? An insight into P2X receptor signaling and hepatitis C virus infection. Viral Immunology. 2016;**29**(7):401-408

[178] Tsai CY, Liong KH, Gunalan MG, Li N, Lim DS, Fisher DA, et al. Type I IFNs and IL-18 regulate the antiviral response of primary human γδ T cells against dendritic cells infected with dengue virus. Journal of Immunology. 2015;**194**(8):3890-3900

[179] Zarei M, Sahebi Vaighan N, Ziai SA. Purinergic receptor ligands: The cytokine storm attenuators, potential therapeutic agents for the treatment of COVID-19. Immunopharmacology and Immunotoxicology. 2021;**43**(6):633-643

[180] Leão Batista Simões J, Fornari Basso H, Cristine Kosvoski G, Gavioli J, Marafon F, Elias Assmann C, et al. Targeting purinergic receptors to suppress the cytokine storm induced by SARS-CoV-2 infection in pulmonary tissue. International Immunopharmacology. 2021;**100**:108150

[181] Simões JLB, Bagatini MD. Purinergic signaling of ATP in COVID-19 associated Guillain-Barré syndrome. Journal of Neuroimmune Pharmacology. 2021;**16**(1):48-58

[182] Caillon A, Trimaille A, Favre J, Jesel L, Morel O, Kauffenstein G. Role of neutrophils, platelets, and extracellular vesicles and their interactions in COVID-19-associated thrombopathy. Journal of Thrombosis and Haemostasis. 2022;**20**(1):17-31

[183] Schultz IC, Bertoni APS, Wink MR. Purinergic signaling elements are correlated with coagulation players in peripheral blood and leukocyte samples from COVID-19 patients. Journal of Molecular Medicine (Berlin, Germany). 2022:**100**(4):569-584

[184] Ahmadi P, Hartjen P, Kohsar M, Kummer S, Schmiedel S, Bockmann JH, et al. Defining the CD39/CD73 axis in SARS-CoV-2 infection: The CD73(−) phenotype identifies polyfunctional cytotoxic lymphocytes. Cell. 2020;**9**(8):1750

[185] Ribeiro DE, Oliveira-Giacomelli Á, Glaser T, Arnaud-Sampaio VF, Andrejew R, Dieckmann L, et al. Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology. Molecular Psychiatry. 2021;**26**(4):1044-1059

[186] Di Virgilio F, Tang Y, Sarti AC, Rossato M. A rationale for targeting the P2X7 receptor in coronavirus disease 19. British Journal of Pharmacology. 2020;**177**(21):4990-4994

#### **Chapter 3**

## Purinergic Signaling in Covid-19 Disease

*Hailian Shen*

#### **Abstract**

SARS-CoV-2 virus infection causes the Covid-19 disease pandemic. Purinergic signaling is a form of extracellular signaling. Purinergic signaling plays significant role in the pathology of Covid-19. Purinergic system includes extracellular nucleotides, nucleosides, ectonucleotidases, and purinergic receptors. ATP, ADP, and adenosine are the main nucleotides, nucleosides. CD39 and CD73 are the main ectonucleotidases. There are two classes of purinergic receptors, P1 and P2. Each of them can be further divided, P1 into A1, A2A, A2B, and A3, P2 into P2X, and P2Y. In Covid-19, the purinergic system is disordered. SARS-CoV-2 viruses invading leads to extracellular ATP and ADP accumulation, purinergic receptor abnormally activation, tissue homeostasis balance is broken, which lead to inflammation even hyperinflammation with cytokine storm and thrombosis et al. symptoms. Currently, Covid-19 therapeutic medicine is still in shortage. Target purinergic system components is a promising way to treat Covid-19, which will help inhibit inflammation and prevent thrombosis. Currently, many relevant preclinical and clinical trials are ongoing. Some are very promising.

**Keywords:** purinergic system, purinergic signaling, purinergic receptor, SARS-CoV-2, Covid-19

#### **1. Introduction**

Coronavirus disease 2019 (Covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. The first Covid-19 case was reported at the end of December 2019 in Wuhan, China [2]. From then on, the virus transmission was swiftly spread leading to the Covid-19 pandemic. So far, more than 6 billion people have infected SARS-CoV-2 virus worldwide, and more than 6million people died in the pandemic. Although now there are several kinds of vaccines have been put into use and been proven to be safe, effective and life-saving, the pandemic does not stop. There are several reasons may be responsible for this. The first one, not 100 percent people inoculate with vaccines. Second, like all the other vaccines, the Covid-19 vaccines do not fully protect everyone who is vaccinated, namely the efficacy rates of the vaccines are less than 100%. The SARS-CoV-2 is enveloped positive-chain RNA virus, which is prone to mutate. New mutation strains more easily escape from the vaccine defense. Third, vaccine protection is time-dependent, not lifelong. The SARS-CoV-2 virus transmission is through hand-mouth-eye contact and infected droplets released

by coughing and sneezing. After the virus enters the body, it will combine with the cell surface angiotensin-converting enzyme 2 (ACE2) receptor through its envelope spike protein [3]. With the help of transmembrane serine protease2 (TMPRSS2), the virus will enter successfully into the cell [4]. There are some other cell-surface proteins, like Eph receptors, Neuropilin 1, and CD147 et al. which can also act as SARS-CoV-2 virus cell entry helpers [5, 6]. From asymptomatic to life-threatening acute respiratory distress syndrome (ARDS), the manifestations of the SARS-CoV-2 virus-infected people are quite variant. The most common symptoms found in the clinical presentation are fatigue, anosmia, ageusia, dizziness, headache, obtundation, myalgia, diarrhea, anorexia, fever, cough, pneumonia, and dyspnea [7, 8]. In severe cases, the virus infection will cause hyper inflammation with cytokine storm and also thrombogenesis can occur [9].

Purinergic signaling plays a pivotal role in SARS-CoV-2 virus infection, participates in the regulation of the innate immune system and platelet function, which are highly relevant for hemostasis, inflammatory, and thrombosis processes [10]. The purinergic system may be a possible target for SARS-CoV-2 treatments [11]. In this chapter, the purinergic signaling in Covid-19 disease will be introduced.

#### **2. Purinergic system**

Purinergic signaling is a form of extracellular signaling. In 1972, Geoffrey Burnstock proposed that adenosine triphosphate (ATP) can act as a neurotransmitter, which opened up a new area [12]. Purinergic system is composed of extracellular nucleotides, nucleosides, ectonucleartides, and purinergic receptors.

#### **2.1 Nucleotides and nucleosides**

The nucleotides and nucleosides are mainly ATP, adenosine diphosphate (ADP), and adenosine (Ado). In addition, Uridine diphosphate (UDP), uracil-diphosphatesugar (UDP -sugar), and Nicotinamide adenine dinucleotide (NAD+) can also act as purinergic signal molecules [13]. Besides as a purinergic signal molecule, ATP is the universal currency of energy metabolism. 10% ATP is produced in cytoplasm by glycolysis. In this way, 1 molecular glucose lysis can generate 2 molecular ATPs. While the 90% ATP is synthesized in mitochondria through Krebs cycle. 1 molecular glucose oxidative-phosphorylation can create 30 ATPs. The Krebs cycle needs oxygen. As ACE2 receptors are highly expressed in lung, SARS-CoV-2 virus very easily invades this tissue leading to lower ventilation rates in Covid-19 patients. Hypoxia will cause ATP production inefficiency to lead to low intracellular ATP [14, 15]. The situation will be further intensified by ATP going outside the cell through Pannexin-1 channel (PANX1). PANX1 expression is increasing in Covid 19. Normally, extracellular ATP concentration is very low, commonly the concentration is less than 3 nanomolar, while intracellular one can be high at millimolar (2–8 mM). So, Covid-19 patients' extracellular ATP level is dramatically increased, along with extracellular ADP accumulation. SARS-CoV-2 virus-infected cell lysis is another important reason for high extracellular ATP. Extracellular ATP catalyzed by CD39 (ectonucleoside triphosphate diphosphohydrolase-1, ENTPD1) to dephosphorylated into ADP, further into AMP which is still by CD39. Then CD73 (ecto-5′-nucleotidase, NT5E) will fully convert AMP to Ado. From ATP to Ado, this pathway is called canonical adenosinergic pathway, which was firstly illustrated by Yegutkin et al. Ado can be produced through

a non-canonical alternative pathway, which starts from NAD+. NAD+ first is metabolized into ADP-ribose (ADPR) by CD38, then into AMP by CD203a, and further into Ado by CD73. So, two pathways converge on CD73. The life of Ado is very short. It will soon be deaminized into inosine by adenosine deaminase (ADA). Inosine is much more stable. Ado can also be taken into the cell through equilibrative nucleoside transporter-1 (ENT1) [16].

#### **2.2 Ectonucleartidases**

Ectonucleotidases are nucleotide metabolizing enzymes, which located on cytoplasmic membrane. The main function of Ectonucleotidases is to catalyze nucleotides hydrolyzation to balance nucleotides and neucleosides. Ectonucleotidases can be classed into 4 families: the ectonucleoside triphosphate diphosphohydrolases (NTPDase1-4,8), the ectonucleotide pyrophosphatase phosphodiesterases (NPP1- 3), ecto-5′-nucleotidase and alkaline phosphatase (**Table 1**). CD39 is type 1 ectonucleoside triphosphate diphosphohydrolase, while CD73 is ecto-5′-nucleotidase. CD39 and CD73 not only regulate AMP existing, but also GMP state [17]. CD39 was overexpressed in COVID-19 patients' plasma and some immune cell subsets and related to hypoxemia [18]. Plasma soluble form of CD39 (sCD39) was related to length of hospital stay and independently associated with intensive care unit admission. Soluble Plasma CD39 may be used to predict covid-19 patients' clinical prognosis, which is suggested as a promising biomarker for COVID-19 severity. CD39 is a defined marker of exhausted T cell [19]. T cell exhaustion and dysfunction are hallmarks of severe COVID-19. Both CD4<sup>+</sup> Tim-3<sup>+</sup> CD39<sup>+</sup> T cell and CD8<sup>+</sup> Tim-3<sup>+</sup> CD39<sup>+</sup> T cell significant increase in multiple tissues, like lung, liver, spleen and PBMCs, of critical covid 19 patients [20]. CD39 expression was also found up-regulated in plasmablasts. CD39 higher expression was also reported in the placenta of a 23 year old woman of pregnancy complicated by SARS-CoV-2 virus infection and the accompanying placental complement C4d deposition [21]. Regulatory T (Treg) cells have been shown to play an essential role in immune homeostasis in many diseases and pathological conditions [22]. Some studies have reported that CD4<sup>+</sup> CD25<sup>+</sup> CD39<sup>+</sup> Tregs have more immunosuppressive effects than CD4<sup>+</sup> CD25<sup>+</sup> CD39<sup>−</sup> Tregs [23]. In covid-19, CD39<sup>+</sup> Tregs are decreased in juvenile patients in an age-dependent manner while in adult patients,CD39<sup>+</sup> Tregs increased with disease severity [24]. However, CD73 expression is down-regulated in plasmablasts, CD8<sup>+</sup> T cells and natural killer T cells (NKT)[25]. But there is one paper shows that moderate and severe cases have increased expression of CD39 and CD73 in total leukocytes. CD38, a catalytic case of non-canonical adenosinergic pathway mentioned above, is upregulated.

#### **2.3 Purinergic receptors**

Purinergic receptors can be separated into P1 and P2 [26]. P1 receptor can be further subclassed into A1, A2A, A2B and A3, P2 receptor further into P2X and P2Y. P1 and P2Y are G protein-coupled metabolic receptors, while P2X receptors are fast ligand gated ion channel.

P1 receptor is also known as adenosine receptor as its endogenous ligand is Ado. Caffeine and theophylline are two best known P1 receptor antagonists [27, 28]. Once Ado binding to P1 receptor, the conformation of the receptor will change to activate the coupled G protein. Activated G protein will cause intracellular cyclic adenosine


*\* Intracellular enzymes.*

*Adapt from Seldin and Giebisch, The Kidney Physiological &Pathophysiological, 4th Edition, Oct1, 2007.*

#### **Table 1.**

*Major hydrolysis pathways of Ectonucleotidases.*

monophosphate (cAMP) level change through acting on adenylate-cyclase. 4 subtype P1 receptors in human, encoded by different genes, interact with different G protein subunits. A1 and A3 receptors preferentially bind to inhibitory regulative Gi/o proteins, inhibiting adenylate-cyclase and cyclic AMP production, whereas the receptors of A2 family are generally coupled to stimulative regulative Gs protein that trigger intracellular cAMP accumulation. A1 and A2A are high affinity receptors, while A2B and A3 are low affinity receptors. Mitogen-activated protein kinase signaling pathway has also been reported to be another P1 receptor downstream pathway. In immune system, A1 receptor is mainly expressed on Neutrophils and immature dendritic cells, A2A on most immune cells, A2B on macrophages and dendritic cells, while A3 on neutrophils and mast cells. Ado binding to A1 receptor will produce chemotaxis function. On the contrary, A3 receptor activation will reduce neutrophil and stimulate mast cells degranulation. A2A and A2B receptors evoke immune suppress. The well-known anti-inflammatory effects of Ado are mediated by these two receptors [29]. A2B expression is upregulated in Covid 19.

P2 receptor can be sub-grouped into P2X and P2Y. P2X receptors belong to a larger family of receptors known as the ENaC/P2X superfamily. They are homologs. Structurally, P2X receptors and ENaC are very similar. PX2 receptors have a wide tissue distribution, which being expressed in nervous system, the pulmonary and digestive systems, muscle, bone, and immune system et al. Blood cells, like red cells, lymphocytes and macrophages and platelets, can be traced to have P2X receptors' expression. P2X receptor family contains 7 members, P2X1 to P2X7 respectively, which are heterotrimers or homotrimers. Another name of P2X7 receptor is P2Z. ATP is the full agonist of

#### *Purinergic Signaling in Covid-19 Disease DOI: http://dx.doi.org/10.5772/intechopen.105008*

P2X receptors, which can activate all P2X family receptors. NAD+ is also an activator of P2X receptors. However, there are some nucleotide-specific variations between these two ligands. For example, among the P2X receptor subtypes, the P2X7 receptor is unique in facilitating the induction of nonselective pores that allow entry of organic cations and dye molecules. Upon stimulation with ATP. As little as 100 μM ATP was sufficient to activate the nonselective pore, whereas NAD+ at concentrations up to 2 mM had no effect. The affinities between ATP and different P2X subgroups are also different. ADP and AMP, when purified, are inactive at P2X receptors. Activation of P2X receptors leads to influx of cations such as sodium and calcium, and further to depolarize the excitable cells. Among the 7 P2X receptors, P2X7 which mainly expressed on Macrophages, mast cells, microglia, pancreas, skin, and endocrine organs, is most studied and plays a pivotal role in SARS-CoV-2 virus infection associated inflammation, which is a promising target to treat Covid-19 disease [30]. Beside to P2X7, P2X1, P2X4, and P2X5 have been detected increasing expression in Covid 19 patients either.

P2Y receptors are seven-transmembrane proteins belonging to the class A family of G protein-coupled receptors (GPCRs), which are the δ group of rhodopsinlike GPCRs [31]. Structurally, P2Y receptors are characterized by extracellular N-terminal, which followed by seven hydrophobic transmembrane (7-TM) α-helices (TM-1 to TM-7) connected by three extracellular loops (ECL) and three intracellular loops, and ending in an intracellular C-terminus. An ECL serves to bind the receptor ligand(s), while intracellular regions mediate G protein activation and participate in P2Y receptor regulation. P2Y receptors can form both homodimers and heterodimers to further increase the biochemical and pharmacological spectrum of P2YRs. P2Y receptor's family consists of 8 subunits, P2Y [4, 5, 14, 18, 28, 30, 32, 33]. The gaps of the subunit numbers are because of the fact that the assignment of numbers to certain putative P2Y receptors was later shown to be premature, with some of the previously designated sequences being P2Y species homologs and others being other types of receptors. P2Y receptors are present in almost all human tissues, where they exert various biological functions based on their G-protein coupling. Different from P2X receptors which have only ATP and NAD+ two native nucleotide agonists, P2Y receptors respond not only to nucleotides (ATP, ADP, UTP, UDP, NAD+ ) but also to nucleotide sugars such as UDP-glucose. According to the G-protein coupling difference, P2Y can be classed into Gq-coupled, P2Y1-like receptors (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) and Gi -coupled, P2Y12-like receptors (P2Y12, P2Y13 and P2Y14) (**Table 2**). As P1 receptors, when ligands combine to P2Y receptors, the conformations of the receptors will change to transfer the signal to the coupled G-proteins. Heteromeric G-proteins (Gαβγ) will dissociate into Gα subunits and Gβγ complexes, which activate or regulate downstream effector pathways. P2Y11 is the only P2Y member which can activate cAMP pathway. P2Y receptors also play very important roles in in SARS-CoV-2 virus infection related inflammation and can be served as therapeutic targets. P2RY1 and P2RY12 have been shown to be elevated in Covid-19.

#### **2.4 Roles of purinergic signaling in the inflammation of Covid-19**

The inflammation of Covid-19 is the most important biological response of the body tissue to SARS-CoV-2 invasion [34]. Usually inflammation is the innate immune protective response involving immune cells, blood vessels, and molecular mediators. However, in severe covid-19 patients, it can develop into hyperinflammation, which can be life-threatening. Hyperinflammation is thought to be the base to develop into severe Covid 19. About hyperinflammation, currently there is no clear-cut definition.


#### **Table 2.**

*Human P2Y receptors.*

The criteria of hyperinflammation are not consistent. Most people think the condition of hyperinflammation as a form of very severe inflammation with cytokine storm which is out of tissue homeostatic control to lead to ARDS or other organs failure. Purinergic signal system is in the pivotal position of pro-inflammation and antiinflammation axis. Once the balance is broken, pro-inflammation factors being far more than the ones of anti-inflammation, hyperinflammation will happen.

SARS-CoV-2 invasion leads to extracellular ATP and ADP accumulation. ATP will bind to P2X7 receptor. Though P2X7 receptor expressed on almost all type human and mouse cells, the levels of the ones on monocyte and macrophage are much higher. ATP binding to P2X7 receptor leads to pore forming on the cell surface to cause K+ efflux. Intracellular K<sup>+</sup> depletion and extracellular K+ concentration increase is necessary and sufficient to activate and assembly the NLR family pyrin domain containing 3 (NLRP3) inflammasome to promote proteolytic cleavage, maturation and secretion of pro-inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18)[30]. So far there is no evidence to show that SARS-CoV-2 can directly activate NLRP3 inflammasome. Flowing P2X7 receptor activation other cytokines and chemokines, for example, IL-6, TNF-α, CCL2, IL-8, CCL3 and CXCL2, of pro-fibrotic factors such as TGF-β, and extracellular matrix remodeling factors, for example, metalloproteinase-9 and tissue inhibitor of metalloproteinase (TIMP)-1 will also be released. In mild Covid 19, the extracellular ATP concentration lower than 100uM, after proinflammation process starts the anti-inflammation response will be triggered either. First reaction is CD39 / CD73 will convert ATP into Ado. Ado will activate P1 receptor. As above mentioned, A2A and A2B receptors activation will launch immune suppress. At the same time, activation of A2A receptor will promote the differentiation of naïve T-cells towards regulatory T-cells (Tregs). Treg will secrete immune suppressive factors, like IL-10 and TGF-β, to restrict immune reaction. However, when extracellular ATP concentration over 100uM, the situation will become worse, dramatic immune

#### *Purinergic Signaling in Covid-19 Disease DOI: http://dx.doi.org/10.5772/intechopen.105008*

response will lead to sever inflammation. If extracellular ATP concentration is over 1 mM, hyperinflammation will be inevitable in most cases. High amount extracellular ATP accumulation leads to prolong P2X7 receptor activation. P2x7 receptor overactivation leads to macropore formation and cytolysis with uncontrolled ATP outgoing and cytokines release. What making the situation even worse is the anti-inflammation process being out of control. P2X7 receptor activation inhibits he suppressive potential and stability of Tregs. Tregs clonal proliferation and mature are suppressed, Treg death increasing. Treg depletion leads to IL10 et al. immune suppressive factors drop. In severe covid-19 patient, the expression of forkhead box protein P3 (FoxP3), a marker of Treg, was monitored lower than that in health control. On the other side, CD73 express is down-regulated, which blocks the production of Ado, which cause P1 receptors desensitization [35]. So, though A2B is detected to have higher expression in Covid-19, it is less activated. The homeostatic out-control at last results in the hyperinflammation exploding. Not only P2X7 receptor play a role in Covid-19 inflammation, other P2 receptors also have functions in the proinflammation. For ex, ATP-P2X4, ADP-P2Y6, ADP-P2Y12, and UDP-sugure-P2Y14 et al. mediated signaling all can stimulate inflammation via actions on innate immune cells, especially dendritic cells and macrophages.

#### **2.5 Roles of purinergic signaling in the thrombosis of Covid-19**

In addition to inflammation, many Covid-19 patients also have microvascular thrombosis, which have been confirmed by autopsy. Clinical detection has also provided very solid evidence. Covid-19 patients have high level of circulating D-dimeris (a fibrin/fibrinogen degradation product), prolonged prothrombin time, upregulated expression of tissue factor (TF, encoded by F3 gene) et al [32]. The Covid-19 related stroke incidence was reported increase, either. Purinergic system not only participate in inflammation but also involved in thrombosis, it is like a bridge to connect the two processes. The complex interplay between the two processes is described as thromoinflammation [36, 37].

Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. In Covid-19 disease the balance of coagulation and fibrinolysis is broken, which leads to thrombosis happen [38]. Blood coagulation can be divided into two pathways: intrinsic pathway and extrinsic pathway. Intrinsic cascade starts from blood contacts the damaged blood vessel surface or other high molecular surface with negative charges to induce factor XII activation, which following by factor XI and activation. On the phospholipid surface of the activated platelet, factor IX together with factor VIII (vW factor) and Ca2+ will activate factor X. Extrinsic pathway, which also called tissue factor pathway, is beginning from factor VII being activated by tissue factor. Activated factor VII can directly activate factor X. So, two coagulation pathways converge on factor X activation. Activated factor collaborated with factor V and Ca2+catalyzes prothrombin to become thrombin. Also, the phospholipid surface of the activated platelet is necessary for this reaction. Thrombin will continue to catalyze fibrinogen to convert into fibrin. Purinergic system can promote coagulation from several aspects [33]. Activated platelet plays very important roles in blood coagulation. ADP can directly activate platelet. As above mentioned, Covid-19 patients have extracellular ADP accumulation. The accumulated ADP can bind to P2Y12 receptor located on the surface of the platelet to activate it. The activated platelet will secrete more ADP and vW factor et al. ADP can also activate platelet through combine to P2Y1 receptor. ATP is also a platelet activator. ATP can interact with platelet P2X1 receptor. ATP binds to macrophage P2X7 receptor enhance tissue factor expression and release to trigger extrinsic pathway [39]. The third way is that purinergic signal can stimulate neutrophils activation to produce reactive oxygen (ROS). The overwhelming production of ROS can result the release of neutrophil extracellular traps (NETs), which is web-like structures composed of chromatin containing neutrophil granule proteins [40]. NETs can further activate factor XII to activate the intrinsic pathway [19].

#### **3. Therapeutic targets**

Regarding the important roles purinergic signal plays in Covid-19 disease, the members of purinergic system have been used as therapeutic targets to reduce morbidity and mortality.

#### **3.1 Adenosine and Ado metabolism enzymes**

As above described, Ado can exert anti-inflammation effects through active P1 receptor [41, 42]. Clinically, Ado is used in cardiac diseases diagnosis and treatment. Preclinically, Ado administration was demonstrated to be able to attenuate lung injury [43]. Ado being reported can also be applied in Covid-19 patient treatment. A patient suffering from SARS-CoV-2-related ARDS on routine therapies who did not show clinical improvements, inhaled adenosine in a mixture of 21% oxygen was applied. After 5 days, the SARS-CoV-2 test was negative and a rapid improvement in clinical condition as well as radiological pictures were shown. The main concern about Ado used in disease treatment is its short half-life in vivo. In the future more stable Ado analogs may be developed. Ado metabolism enzymes blocking methods is another way to elevate extracellular Ado. Pentostatin (2′ deoxycoformycin) and EHNA (erytho-9-(2-hydroxy-3-nonyl) adenine hydrochloride) are two ADA inhibitors. Clinically, pentostatin is used in Hairy Cell Leukemia treatment. It is suggested pentostatin might be beneficial in late-stage ARDS. Not like pentostatin which only inhibits ADA enzyme activity, EHNA can also bind to P1 receptors and adenosine deaminase complexing protein 2(CD26). EHNA potentially has anticancer effects, but so far has been used clinically. EHNA is also suggested to be potentially used in Covid-19 therapy. Dipyridamole (DIP) is a ENT1 inhibitor, which can prevent extracellular Ado uptake [44]. DIP is an approved antiplatelet drug, clinically being used to prevent stroke, and being proved to have high safety [45]. The bleeding risk of DIP is similar to that of aspirin. Currently, three clinical trials evaluating efficacy of dipyridamole for the treatment of COVID-19 have been registered (identifiers: NCT04424901; NCT04391179; NCT04410328) [46, 47]. Apart from anticoagulant and anti-inflammatory effects, it is speculated that DIP can also blunt SARS-CoV-2 replication.

#### **3.2 CD39**

As CD39 plays very important roles in extracellular ATP and ADP hydrolysis, its expression and activity closely related to inflammation and thrombosis [48]. Several approaches have been attempted to target CD39. One of them is using soluble CD39 to antithrombosis [48]. However, this method easily causes bleeding. To overcome this side effect, new strategies is worked out. The core of these new strategies is to link the recombinant soluble CD39 to other molecules, like PSGL-1, the receptor for P selectin on leukocyte surface, and single chain antibody (scFV) specific against GPIIb/IIIa, the platelet fibrinogen receptor, and glycoprotein VI (GPVI) Fc fusion protein et al.

#### **3.3 P1 receptors**

P1 receptor family contain1 A1, A2A, A2B, A3. These 4 receptors have different functions [49]. Activated A2A and A2B can suppress immune response. As mentioned above that caffeine and theophylline are two best known P1 receptors antagonists. Both are non-selective antagonists except for A3 receptor, they can inhibit the other 3 P1 receptors, namely A1, A2A, and A2B, at therapeutic concentrations. Theophylline is more potent. As these receptors have different functions in inflammation, inhibit these three receptors will have different effects. For example, theophylline has been shown to have both proinflammatory and anti-inflammatory effects [50]. The latter one might be stronger. Recently, shown by preclinical data that theophylline can potentially amplify the anti-inflammatory effect of corticosteroids and reduce corticosteroid resistance. Now one clinical trial, which theophylline is designed to be nasally administrated to treat the Covid-19 patients who have been received intranasal and oral corticosteroids, is on-going (Identifier: NCT04789499). However, there is one report sharing that theophylline treatment induced sinus bradycardia in two cases of Covid-19 patients [51]. Pentoxifylline (PTX) can active A2A receptor. When PTX binds to A2A receptor, it will stimulate to secrete IL-10 et al. immune suppressive molecules to inhibit inflammation. PTX treatment is also shown to help reduce IL-6 serum concentration, as well as diminish IL-1b level. PTX has been recommended to be applied in Covid-19 therapy.

#### **3.4 X2P7 receptor**

X2P7 receptor is the most important pro- inflammation purinergic receptor. X2P7 receptor block is predicated to be able to ameliorate inflammation. X2P7 receptor activation can also potentially induce of VEGF release. P2X7 receptor blockade can inhibit VEGF-dependent increase in vascular permeability, and therefor prevent lung oedema. Several X2P7 receptor antagonists are suggested to be used in Covid-19 therapy [27, 52]. Colchicine is one of such inhibitors. Colchicine is a tricyclic lipid-soluble alkaloid extracted from Colchicum autumnalle and *gloriosa superba*. Colchicine is a well-known of microtubule polymerization inhibitor, which in the early time was found to be able to block cell mitosis. Hereafter, its anti-inflammation effects was revealed. Colchicine has been clinically used as an anti-inflammatory agent for longterm treatment of Behçet's disease and also used to treat many other diseases, like pericarditis, pulmonary fibrosis, biliary cirrhosis, various vasculitides, pseudogout, spondyloarthropathies, calcinosis, scleroderma, and amyloidosis et al. Colchicine not only can inhibit X2P7 receptor, but also can block X2P2 receptor pathway. Several clinical trials have shown that colchicine can limit the production of some cytokines, like IL-1b, IL-18, and IL-6 et al., of Covid-19 patients. NIH has included colchicine in Covid-19 treatment guideline.

Lidocaine is another P2X7 receptor antagonist, which routinely used as local anesthesia in clinic. It's readily available and affordable. Recently, a clinical trial (Identifier:NCT04609865) is carrying out in a French group, in which lidocaine is intravenously administrated to treat Covid-19 disease [6]. However, the halfmaximal effective concentration (IC50) for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. A Peru group modified the protocol. 28 (three mild, 21 moderate and four severe) COVID-19 patients were treated with 0.5% lidocaine HCL solution with an intravenous dose of 1 mg/kg once a day for 2 days and 2% lidocaine HCL solution with a subcutaneous dose of 1 mg/kg once a day for 2 days, which results in the improvement in pain, cough, respiratory rate and oxygen saturation. Another group directly carried out subdermal administration of lidocaine in 6 critical ill Covid-19 induced ARDS patients. The author claimed that although all six patients appeared to respond positively to the treatment and no severe adverse effects were observed, no final conclusions could be made on the efficacy of lidocaine in critically ill COVID-19 patients.

#### **3.5 P2Y12 receptor**

P2Y12 receptor is the main purinergic receptor responsible for SARS-CoV-2 virus related thrombosis, Theoretically, P2Y12 receptor blocking can confine thrombosis of Covid-19, and also can curb the inflammation. Several P2Y12 antagonists (clopidorgel, prasugrel, ticagrelor and cangrelor) have been clinically used to prevent thrombosis in patients at risk of heart attack for about 20 years [27, 53]. Recently, these antagonists have been reevaluated for its effects in Covid-19 related thrombosis treatment. Several clinical trials (Identifiers are NCT04505774; NCT04409834, and NCT04333407.) are on the way. One of them, titled "accelerating Covid-19 therapeutic and vaccines 4 acute (ACTIV-4A, Identifier: NCT04505774)" has been finished and reported [54]. This is a randomized clinical trial, which aims to test if P2Y12receptor antagonists can enhance heparin therapeutic effects in mild Covid-19 patients. The answer is no. The results demonstrated that among non- critical ill hospitalized Covid-19 patients, the use of a P2Y12 receptor antagonist in addition to a therapeutic dose of heparin, compared with a therapeutic dose of heparin only, did not result in an increased odds of improvement in organ support– free days within 21 days during hospitalization. However, as the author mentioned that this trial tested only the combination of a P2Y12 inhibitor with anticoagulant therapy, it remains possible that use of a P2Y12 inhibitor as a sole antithrombotic agent may improve outcomes in patients with COVID-19. In addition, the potential for benefit with a longer treatment duration or at an earlier stage of illness (before hospitalization) cannot be ruled out.

### **4. Conclusion**

Purinergic signal is involved in SARS-CoV-2 viruses causing Covid-19, which plays a pivotal role in the pathology of Covid-19 disease. Purinergic signal participates in the regulation of the innate immune system and platelet function et al., which are highly relevant for hemostasis, inflammatory and thrombosis processes. SARS-CoV-2 virus infection will lead to the abnormality of purinergic system to break the body homeostasis further to inflammation and thrombosis. Purinergic system components have been suggested to be Covid-19 therapeutic targets. Currently many preclinical and clinical trials have been in progress to test this hypothesis. Promising data have brought new hope to the patients.

*Purinergic Signaling in Covid-19 Disease DOI: http://dx.doi.org/10.5772/intechopen.105008*

#### **Author details**

Hailian Shen University of Texas Health Science Center at San Antonio, San Antonio, USA

\*Address all correspondence to: shenh4@uthscsa.edu

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Rat P, Olivier E, Dutot M. SARS-CoV-2 vs. SARS-CoV-1 management: Antibiotics and inflammasome modulators potential. European Review for Medical and Pharmacological Sciences. 2020;**24**(14):7880-7885

[2] Dos Anjos F, Simões JLB, Assmann CE, Carvalho FB, Bagatini MD. Potential therapeutic role of purinergic receptors in cardiovascular disease mediated by SARS-CoV-2. Journal of Immunology Research. 2020:8632048

[3] Doğan HO, Şenol O, Bolat S, Yıldız ŞN, Büyüktuna SA, Sarıismailoğlu R, et al. Understanding the pathophysiological changes via untargeted metabolomics in COVID-19 patients. Journal of Medical Virology. 2021;**93**(4):2340-2349

[4] Dietl P, Frick M. Channels and transporters of the pulmonary lamellar body in health and disease. Cell. 2021;**11**(1):45

[5] Zalpoor H, Akbari A, Samei A, Forghaniesfidvajani R, Kamali M, Afzalnia A, et al. The roles of Eph receptors, neuropilin-1, P2X7, and CD147 in COVID-19-associated neurodegenerative diseases: Inflammasome and JaK inhibitors as potential promising therapies. Cellular & Molecular Biology Letters. 2022;**27**(1):10

[6] Ribeiro DE, Oliveira-Giacomelli Á, Glaser T, Arnaud-Sampaio VF, Andrejew R, Dieckmann L, et al. Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology. Molecular Psychiatry. 2021;**26**(4): 1044-1059

[7] Edwards C, Klekot O, Halugan L, Korchev Y. Follow your nose: A key clue to understanding and treating

COVID-19. Frontiers in Endocrinolology. 2021;**12**:747744

[8] Simões JLB, Bagatini MD. Purinergic signaling of ATP in COVID-19 associated Guillain-Barre Syndrome. Journal of Neuroimmune Pharmacology. 2021;**16**(1):48-58

[9] Hasan D, Shono A, van Kalken CK, van der Spek PJ, Krenning EP, Kotani T. A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling. Purinergic Signal. 2022;**18**(1):13-59

[10] Franciosi MLM, Lima MDM, Schetinger MRC, Cardoso AM. Possible role of purinergic signaling in COVID-19. Molecular and Cellular Biochemistry. 2021;**476**(8):2891-2898

[11] Pacheco PAF, Faria RX. The potential involvement of P2X7 receptor in COVID-19 pathogenesis: A new therapeutic target? Scandinavian Journal of Immunology. 2021;**93**(2):e12960

[12] Franco R, Rivas-Santisteban R, Lillo J, Camps J, Navarro G, Reyes-Resina I. 5-Hydroxytryptamine, glutamate, and ATP: Much more than neurotransmitters. Frontiers in Cell and Development Biology. 2021;**9**:667815

[13] Grahnert A, Klein C, Hauschildt S. Involvement of P2X receptors in the NAD(+)-induced rise in [Ca (2+)] (i) in human monocytes. Purinergic Signal. 2009;**5**(3):309-319

[14] Liu W, Zhu X, Mozneb M, Nagahara L, Hu TY, Li CZ. Lighting up ATP in cells and tissues using a simple aptamer-based fluorescent probe. Mikrochimica Acta. 2021;**188**(10):352

*Purinergic Signaling in Covid-19 Disease DOI: http://dx.doi.org/10.5772/intechopen.105008*

[15] Abraham EH, Guidotti G, Rapaport E, Bower D, Brown J, Griffin RJ, et al. Cystic fibrosis improves COVID-19 survival and provides clues for treatment of SARS-CoV-2. Purinergic Signal. 2021;**17**(3):399-410

[16] Schultz IC, Bertoni APS, Wink MR. Purinergic signaling elements are correlated with coagulation players in peripheral blood and leukocyte samples from COVID-19 patients. Journal of Molecular Medicine. 2022;**29**:1

[17] Wu D, Shu T, Yang X, Song JX, Zhang M, Yao C, et al. Plasma metabolomic and lipidomic alterations associated with COVID-19. National Science Review. 2020;**7**(7):1157-1168

[18] Díaz-García E, García-Tovar S, Alfaro E, Zamarrón E, Mangas A, Galera R, et al. Role of CD39 in COVID-19 Severity: Dysregulation of purinergic signaling and thromboinflammation. Frontiers in Immunology. 2022;**13**:847894

[19] Wang N, Vuerich M, Kalbasi A, Graham JJ, Csizmadia E, Manickas-Hill ZJ, et al. Limited TCR repertoire and ENTPD1 dysregulation mark late-stage COVID-19. iScience. 2021;**24**(10):103205

[20] Shahbazi M, Moulana Z, Sepidarkish M, Bagherzadeh M, Rezanejad M, Mirzakhani M, et al. Pronounce expression of Tim-3 and CD39 but not PD1 defines CD8 T cells in critical Covid-19 patients. Microbial Pathogenesis. 2021;**153**:104779

[21] Shimao Y, Yamauchi A, Ohtsuka T, Terao K, Kodama Y, Yamada N, et al. C4d deposition and CD39 downregulation in the placental infection by SARS-CoV-2. Pathology International. 2022

[22] Setoguchi R, Hori S, Takahashi T, Sakaguchi S. Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization. The Journal of Experimental Medicine. 2005;**201**(5):723-735. DOI: 10.1084/ jem.20041982

[23] Bastid J, Cottalorda-Regairaz A, Alberici G, Bonnefoy N, Eliaou JF, Bensussan A. ENTPD1/CD39 is a promising therapeutic target in oncology. Oncogene. 2013;**32**(14):1743-1751. DOI: 10.1038/onc.2012.269

[24] Simsek A, Kizmaz MA, Cagan E, Dombaz F, Tezcan G, Asan A, et al. Assessment of CD39 expression in regulatory T-cell subsets by disease severity in adult and juvenile COVID-19 cases. Journal of Medical Virology. 2022

[25] Wildner NH, Ahmadi P, Schulte S, Brauneck F, Kohsar M, Lütgehetmann M, et al. B cell analysis in SARS-CoV-2 versus malaria: Increased frequencies of plasmablasts and atypical memory B cells in COVID-19. Journal of Leukocyte Biology. 2021;**109**(1):77-90

[26] Alves VS, Leite-Aguiar R, Silva JPD, Coutinho-Silva R, Savio LEB. Purinergic signaling in infectious diseases of the central nervous system. Brain, Behavior, and Immunity. 2020;**89**:480-490

[27] Zarei M, Sahebi Vaighan N, Ziai SA. Purinergic receptor ligands: The cytokine storm attenuators, potential therapeutic agents for the treatment of COVID-19. Immunopharmacology and Immunotoxicology. 2021 Dec;**43**(6):633-643

[28] Simões JLB, de Araújo JB, Bagatini MD. Anti-inflammatory therapy by cholinergic and purinergic modulation in multiple sclerosis associated with SARS-CoV-2 infection. Molecular Neurobiology. 2021;**58**(10):5090-5111

[29] Gonzales JN, Gorshkov B, Varn MN, Zemskova MA, Zemskov EA, Sridhar S, et al. Protective effect of adenosine receptors against lipopolysaccharide-induced acute lung injury. American Journal of Physiology. Lung Cellular and Molecular Physiology. 2014;**306**(6):L497-L507

[30] Leão Batista Simões J, Fornari Basso H, Cristine Kosvoski G, Gavioli J, Marafon F, Elias Assmann C, et al. Targeting purinergic receptors to suppress the cytokine storm induced by SARS-CoV-2 infection in pulmonary tissue. International Immunopharmacology. 2021;**100**:108150

[31] Klaver D, Thurnher M. Control of Macrophage Inflammation by P2Y Purinergic Receptors. Cell. 2021;**10**(5):1098

[32] Brun JF, Varlet-Marie E, Myzia J, Raynaud de Mauverger E, Pretorius E. Metabolic influences modulating erythrocyte deformability and eryptosis. Metabolites. 2021;**12**(1):4

[33] Ziegler O, Sriram N, Gelev V, Radeva D, Todorov K, Feng J, et al. The cardiac molecular setting of metabolic syndrome in pigs reveals disease susceptibility and suggests mechanisms that exacerbate COVID-19 outcomes in patients. Scientific Reports. 2021;**11**(1):19752

[34] Wauters E, Van Mol P, Garg AD, Jansen S, Van Herck Y, Vanderbeke L, et al. Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages. Cell Research. 2021;**31**(3):272-290

[35] Ahmadi P, Hartjen P, Kohsar M, Kummer S, Schmiedel S, Bockmann JH, et al. Defining the CD39/CD73 axis in SARS-CoV-2 infection: The CD73(-)

phenotype identifies polyfunctional cytotoxic lymphocytes. Cells. 2020;**9**(8):1750

[36] Lintzmaier Petiz L, Glaser T, Scharfstein J, Ratajczak MZ, Ulrich H. P2Y14 receptor as a target for neutrophilia attenuation in severe COVID-19 cases: From hematopoietic stem cell recruitment and chemotaxis to thrombo-inflammation. Stem Cell Reviews and Reports. 2021;**17**(1):241-252

[37] Sriram K, Insel PA. Inflammation and thrombosis in COVID-19 pathophysiology: Proteinaseactivated and purinergic receptors as drivers and candidate therapeutic targets. Physiological Reviews. 2021;**101**(2):545-567

[38] Edwards C. New horizons: Does mineralocorticoid receptor activation by cortisol cause ATP release and COVID-19 complications? The Journal of Clinical Endocrinology and Metabolism. 2021;**106**(3):622-635

[39] Furlan-Freguia C, Marchese P, Gruber A, Ruggeri ZM, Ruf W. P2X7 receptor signaling contributes to tissue factor-dependent thrombosis in mice. The Journal of Clinical Investigation. 2011;**121**(7):2932-2944

[40] Caillon A, Trimaille A, Favre J, Jesel L, Morel O, Kauffenstein G. Role of neutrophils, platelets, and extracellular vesicles and their interactions in COVID-19-associated thrombopathy. Journal of Thrombosis and Haemostasis. 2022 Jan;**20**(1):17-31

[41] Garcia-Dorado D, Garcíadel-Blanco B, Otaegui I, Rodríguez-Palomares J, Pineda V, Gimeno F, et al. Intracoronary injection of adenosine before reperfusion in patients with ST-segment elevation myocardial infarction: A randomized controlled

*Purinergic Signaling in Covid-19 Disease DOI: http://dx.doi.org/10.5772/intechopen.105008*

clinical trial. International Journal of Cardiology. 2014;**177**(3):935-941

[42] Jin Z, Duan W, Chen M, Yu S, Zhang H, Feng G, et al. The myocardial protective effects of adenosine pretreatment in children undergoing cardiac surgery: A randomized controlled clinical trial. European Journal of Cardiothoracic Surgery. 2011;**39**(5):e90-e96

[43] Lu Q, Harrington EO, Newton J, Casserly B, Radin G, Warburton R, et al. Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement. American Journal of Physiology. Lung Cellular and Molecular Physiology. 2010;**298**(6):L755-L767

[44] Kanthi Y, Knight JS, Zuo Y, Pinsky DJ. New (re)purpose for an old drug: Purinergic modulation may extinguish the COVID-19 thromboinflammatory firestorm. JCI Insight. 2020;**5**(14):e140971

[45] Liu X, Li Z, Liu S, Sun J, Chen Z, Jiang M, et al. Potential therapeutic effects of dipyridamole in the severely ill patients with COVID-19. Acta Pharmaceutica Sinica B. 2020;**10**(7):1205-1215

[46] Li X, Berg NK, Mills T, Zhang K, Eltzschig HK, Yuan X. Adenosine at the Interphase of Hypoxia and Inflammation in Lung Injury. Frontiers in Immunology. 2021;**11**:604944

[47] Cardoso AM. COVID-19 and purinergic signaling: The need for investigation. Purinergic Signal. 2020;**16**(3):451-452

[48] Morello S, Caiazzo E, Turiello R, Cicala C. Thrombo-inflammation: A focus on NTPDase1/CD39. Cell. 2021;**10**(9):2223

[49] Abouelkhair MA. Targeting adenosinergic pathway and adenosine A(2A) receptor signaling for the treatment of COVID-19: A hypothesis. Medical Hypotheses. 2020;**144**:110012

[50] Sawalha K, Habash FJ, Vallurupalli S, Paydak H. Theophylline in Treatment of COVID-19 Induced Sinus Bradycardia. Clinical Practice. 2021;**11**(2):332-336

[51] Di Nicolantonio JJ, Barroso-Aranda J. Harnessing adenosine A2A receptors as a strategy for suppressing the lung inflammation and thrombotic complications of COVID-19: Potential of pentoxifylline and dipyridamole. Medical Hypotheses. 2020;**143**:110051

[52] Di Virgilio F, Tang Y, Sarti AC, Rossato M. A rationale for targeting the P2X7 receptor in Coronavirus disease 19. British Journal of Pharmacology. 2020;**177**(21):4990-4994

[53] Pereira NL, Avram R, So DY, Iturriaga E, Byrne J, Lennon RJ, et al. Rationale and design of the TAILOR-PCI digital study: Transitioning a randomized controlled trial to a digital registry. American Heart Journal. 2021;**232**: 84-93

[54] Berger JS, Kornblith LZ, Gong MN, Reynolds HR, Cushman M, Cheng Y, et al. ACTIV-4a Investigators. Effect of P2Y12 inhibitors on survival free of organ support among non-critically ill hospitalized patients with COVID-19: A Randomized Clinical Trial. JAMA. 2022;**327**(3):227

Section 2
