Macrophages in Viral Infections

#### **Chapter 14**

## Interaction of Ebola Virus with the Innate Immune System

*Parastoo Yousefi and Alireza Tabibzadeh*

#### **Abstract**

The Ebola viruses (EBOVs) are known as one the most lethal viruses. EBOV systemic infection can cause damage to vital organs and lead to death. The immune responses of the innate immune system and inflammatory cascade are critical elements in the EBOV pathogenesis and mortality. The primary innate immune system response can shape the adaptive immune responses. The innate immune response, due to the pattern-recognition receptors (PRRs), can induce interferons (IFN). IFN is a critical element in the antiviral response. The EBOV can evade the IFN and innate immunity using different mechanisms, whereas a well-controlled and sufficient innate immune response is vital for limiting the EBOV infection. In this regard, a hyperactive inflammation response may lead to cytokine storms and death. In this chapter, we have tried to provide a perspective on the pathogenesis and molecular mechanisms of the innate immune system and its interaction with EBOV infection.

**Keywords:** Ebola virus, immunity, innate immune responses, macrophages, VP35

#### **1. Introduction**

The Ebola virus (EBOV) genus and Marburg viruses are classified in the *Filoviridae* family and *Mononegavirales* order [1]. The EBOV contains a linear single-stranded RNA genome of 18.9-kilobase length and a membrane glycoprotein [2, 3]. Over the past 40 years, there have been 34 episodes of EBOV outbreaks in 11 different Sub-Saharan African countries. The first outbreaks occurred in 1971 in the Democratic Republic of the Congo (DRC) and Sudan [3]. These outbreaks amassed a total of more than 34000 cases and led to 14000 deaths [3]. EBOV Sudan, Bundibugyo, Zaire, and Reston are important species of the EBOV infection in humans [4]. The basic reproductive number (R0) of the EBOV is estimated to be in the range of 1.51–2.53 [4].

Some therapeutic and vaccination strategies have been introduced against the EBOV so far. The remdesivir, monoclonal antibodies [5], and rVSV-ZEBOV (vesicular stomatitis virus-based vaccine which is expressing the glycoprotein of a Zaire Ebola virus) [6] are considered ongoing advances in this area for EBOV treatment and transmission control.

Innate immunity and interferons (IFN) production are one of the most important immune responses to viral infections. Interaction between the Ebola virus and innate immunity with a well-regulated inflammation response can be life-saving in patients with the Ebola virus disease (EBD) [7]. The interaction of the EBOV with the innate immune system is a multifactorial condition, and it is largely associated with cytokines, chemokines, inflammation mediators, the NK cell receptors, and pathogen-associated molecular patterns (PAMPs), such as killer immunoglobulinlike receptor (KIR) and Toll-like receptors (TLRs) [8, 9]. The signaling pathways in the innate immune system are triggered after PAMPs detected by pattern-recognition receptors (PRRs) [10]. Based on protein domain homology, the PRRs are classified into six groups TLRs, a retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), absent in melanoma-2 (AIM2)-like receptors (ALRs), and cyclic GMP-AMP synthase and stimulator of interferon genes (cGAS-STING pathway) [11, 12]. PRRs are also able to recognize molecules released by damaged cells (The damage-associated molecular pattern-DAMPs) and activate natural immunity [13, 14]. The detection of PAMPs or DAMPs by PRRs increases the transcription of genes encoding cytokine and chemokine, IFN type 1, and antimicrobial proteins (AMPs) [15]. Furthermore, most of the TLRs recognize double-stranded RNA (dsRNA) whereas TLR7 and TLR8 bind single-stranded RNA (ssRNA) in endosomes [16]. After recognition, TLRs recruit several adaptor proteins, including Myeloid Differentiation primary response 88 (MyD88), TIR-domain-containing adaptor protein (TIRAP), TIR-domain-containing adapter-inducing interferon-β (TRIF), and TRIF-related adapter molecule (TRAM) [17]. This initiates downstream signaling cascades that lead to the activation of transcription factors, such as transcription factors NF-κB, interferon regulatory factor 3/7 (IRF3/7), and activator protein-1 (AP-1). These factors stimulate the transcription of genes in the cell nucleus and increase the secretion of pro-inflammatory cytokines and IFN [15, 18]. RIG-1 and melanoma differentiation-associated gene 5 (MDA5), recognize viral ss/dsRNA molecule leads to the translocation of NF-kB, mitogen-activated protein kinases (MAPKs), as well as interferon regulatory factors IRF3, IRF7 [19]. RIG-1 signaling pathway plays a crucial role in the antiviral innate immune response. Activated RIG-1 can interact with signaling adaptor protein mitochondrial antiviral signaling protein (MAVS), also known as IFN-β promoter stimulator 1 (IPS-1), to induction of NF-κB signaling or interferon pathways. It was revealed that upon viral infection, MAVs form high-molecular-weight aggregates downstream of RIG-1 and MDA5 signaling [20]. Activated MAVS–RIG-I signaling ultimately results in the activation of the antiviral IFN-I pathway [21]. The binding of type I and III IFNs to their receptors activates the Jak–STAT pathway, which is responsible for enhancing IFNstimulated gene expression, which includes antiviral genes, such as Viperin, MxA, MxB, IFITMs, OAS, and PKR [22, 23]. One of the most important complexes, which participate in host defense by sensing viral infection and promoting innate immune system response, is the inflammasome, first described by Martinon in 2002 [24]. The best-studied inflammasome sensor is NOD-like receptor pyrin domain-containing protein 3 (NLRP3), which consists of a sensor molecule (NLRP3), the adaptor protein ASC, also called PYCARD, and an effector pro-caspase-1 [25]. Activated caspase is required for the cleavage of the inactivated interleukin-1 family (IL-1), such as pro-IL-18 and pro-IL-1β to the mature forms to initiate inflammasome [26].

Dendritic cells (DCs) and macrophages are one of the main axes for filoviruses infections due to the activation of the adaptive immune [27, 28]. In addition, the macrophage's infection and attachment by EBOV result in downstream signaling for alteration of the expression profile in these cells. This alteration leads to the pro-inflammatory cytokine release and virus spreading, which both are important elements in the disease progression and outcome [29, 30]. In this regard, in the current chapter, we tried to provide a perspective on the pathophysiology and molecular mechanisms of the innate immune system and its interaction with EBOV infection.

#### **2. Ebola virus: history and epidemiology**

Over the past 40 years, there are 34 episodes of the EBOV outbreak in more than 10 different Sub-Saharan African countries leading to numerous cases and deaths [31]. Different species of the EBOV have been reported thus far. Three important species are known as Sudan, Bundibugyo, and Reston [32]. The responsible species for the 2014 outbreak of EBOV was identified as the Zaire ebolavirus [32]. The EBOV disease is a zoonotic disease in nature and the major route for transmission is contact with the infected animal especially chimpanzees, fruit bats, and antelope [2, 33]. The virus genome could be detectable for 10 weeks postmortem [34]. Any contact with bodily fluids is a major route for transmission of EBOV [35]. Even after the survival from the disease the viral genome can be detected in semen for 179 days and leads to sexual transmission in some cases [36]. Fever, fatigue, diarrhea, and tachycardia are the most common symptoms and based on the infected strain and the patient's age, 43% of mortality is reported over the 8 days following the infection [37]. In convalesce or a long time after the remission, some rare complications and long-term sequels, such as uveitis or blurred vision, sleeping problems, and meningoencephalitis are reported [38–40].

#### **3. EBOVE virology and pathogenicity**

#### **3.1 Virology and classification**

The Ebola virus (EBOV) genus in *Filoviridae* is a member of the great order of *Mononegavirales* [1]. There are seven important genes in EBOV. The nucleoprotein (NP) is curial for replication and virus formation. Also, viral protein 35 (VP35) is important in the IFN synthesis blocking and virus replication while VP40 acts as an element in virus formation and intracellular trafficking. The glycoprotein (GP) is a major element for viral entry and induces lymphocyte apoptosis by soluble GPs. EBOV VP30 has been assumed to function as a transcription activation factor, which is essential for viral replication. VP24 is necessary for the formation of nucleocapsids (NC) and nucleocapsid-like structures, and Ebola virus L proteins act as subunits of RNA-dependent RNA polymerase, which along with VP35, is necessary for viral replication and transcription [41–43]. A summary of the function of genes is provided in **Table 1**.

The VP35 is a key viral protein in the EBOV virulence. This unique protein inhibits the induction of IFN type I in the infected cell. The IFN type I has long been declared a key element in the host's innate antiviral response [45]. In this regard, the VP35 is considered the most important for virus immune evasion [46]. The IFN blocking strategy in EBOV is not limited to VP35. Other EBOV protein VP24 can actively bind to the karyopherin alpha (KPNA) and interfere with the IFN STAT1 downstream signaling [47]. Furthermore, VP24 seems to be essential for the EBOV replication in macrophages of the guinea pig as an animal model [48].


#### **Table 1.**

*A summary of the important functions of the Ebola virus genes [41, 44].*

#### **3.2 Pathogenicity and treatment**

The EBOV represents an affinity to a wide range of the cellular receptors for virus attachment to the cells. Following the systemic virus infection, viral cytopathology and immune-mediated cell damage are two essential steps of the EBOV pathogenesis and tissue damage [49]. One of the suggested pathogenic mechanisms of the EBOV is the antibody-dependent augmentation of the infection through the attachment of the antibodies to the virus and the C1q-C1q receptor of the complement in the cell surfaces [50].

One of the most important therapeutic agents for EBOV is the remdesivir. The remdesivir acts as an inhibitor of the virus RNA polymerase and leads to chain termination [51]. Efforts for the treatment of the EBOV also lead to some monoclonal antibodies against this virus; for instance, old and newer versions of these monoclonal antibodies are ZMapp, MAb114, and REGN-EB3 [5].

#### **4. Innate immune system and EBOV**

#### **4.1 Primary innate immune response and inflammation**

Interaction between the Ebola virus and innate immunity suggested that a fast and well-regulated inflammation response could be life-saving in patients with the Ebola virus disease (EBD). While a massive monocyte/macrophage activation could be lethal [7]. The mononuclear phagocytes are import element in the EBD pathology [52].

Biomarkers are suggestive elements for disease prognosis and pathogenesis. Some markers such as apoptosis antigen-Fas, IFN-β, IL-29, IL-5, TNFR-II, and FAS ligand levels are associated with the moderate disease while the D-dimer, Granzyme B, IL-10, IL-6, IL-8, TNFR-I, vWF (von Willebrand factor), monocyte chemoattractant proteins and thrombomodulin are associate with severe disease [53]. Furthermore, up-regulation of the IL-1β and IL-6 can suggest a non-fatal infection while the increase in TNF-α, IFN-ɤ, IL-10, IL-1 receptor antagonist, neopterin, IL-8, IL-15, and IL-16 is associated with the lethal outcome [7, 54–56]. By considering all these markers, it has been suggested that in a general view an increase in cytokines and cytokine storm, which it is, represents a hyperactive of the innate immune responses and in the

#### *Interaction of Ebola Virus with the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.104843*

other way, suppression of the adaptive immune responses and lymphocyte apoptosis is the main pathogenesis feature of the EBD and lethal infections [55].

The lymphocyte apoptosis leads to lymphocyte depletion. This apoptotic feature is not due to the replication and infection of the lymphocytes but it mediates through viral and immune system stimulations [57]. sGP, a viral protein produced during EBOV infection and accumulates at high concentrations in the serum, serves as a decoy to prevent the immune system from fighting the infection by binding EBOV-neutralizing antibodies [58]. It is assumed that glycosylation of transmembrane GP may affect neutralizing antibody binding [59]. Virally infected cells, release inflammation mediators that induce Fas and TNF-associated apoptosis-inducing ligands (TRAIL) pathways that can result in lymphocyte apoptosis and lack of an effective adaptive immune response [60–62]. In EBOV infection, lymphocytes are not directly infected, but apoptosis of lymphocytes is a pathological feature of infection. It is hypothesized that the factors (such as TRAIL, TNF-a, and Fas ligand) secreted by macrophages and dendritic cells infected in EBOV, cause lymphocyte apoptosis [52, 60].

Investigation of the EBOV infection in asymptomatic people suggested strong activation of the innate immune system and inflammation, which leads to adaptive immune activation and cytotoxic T cell responses. The strong activation of the innate immune system, inflammation and adaptive immune activation are considered as the optimum immune response to EBOV [63]. The main concept of the current section is summarized in **Figure 1**. The figure represents the EBOV infection in lethal and non-lethal scenarios and the role of the innate immune and inflammation in this process. In addition, some important interactions of the EBOV proteins are noted, for instance, the role of the VP35 and VP24 in type I interferon blocking or the role of the soluble glycoproteins in lymphocyte apoptosis.

#### **Figure 1.**

*A summary of the EBOV infection in lethal and non-lethal scenarios and the role of the innate immune and inflammation. After the EBOV, infection such as any other viral infection innate immunity induces inflammation and IFN. The VP35 and VP24 of the EBOV block the IFN production. A well-controlled and sufficient innate immune response, while it leads to the adaptive immune response, is assumed main cause of asymptomatic or non-lethal infections.*

#### **4.2 Innate immunity receptors and EBOV**

The interaction of the EBOV and the innate immune system is not only limited to the cytokines, chemokines, or inflammation mediators. In this regard, the NK cell and T cell receptors, which are known as killer immunoglobulin-like receptors (KIR), are critical. The role of the NK cells in response to the EBOV viral-like particles highlighted the importance of these cells in the innate immune response to the EBOV [64]. The KIRs are important elements in the host response to infectious diseases. KIR2DS1 and KIR2DS3 of repertoire genotypes of the KIR represent more susceptibility to fatal EBOV [8].

In the EBOV infection, the interaction of the innate and adaptive immune responses is critical for the disease outcome. The Toll-like receptor 4 (TLR4) is a vital element in response to the EBOV glycoprotein and activation of the antigen-presenting cells and T cells. By considering this, TLR4 responses represent a vital role in the regulation of innate and adaptive immunity [9]. The interaction of the TLR4 and monocytes (as antigen-presenting cells) highlighted the importance of the monocytes in the regulation of the immune response. The evidence supports the alteration of the transcriptional patterns in monocytes in EBD [65]. Furthermore, one of the major elements in inflammation and IFN stimulation is NLRP3 [66]. The EBOV infection could increase the IL-1β and IL-18 by the NLRP3 inflammasome activation [67]. This factor highlights the importance of the NLRP3 in pro-inflammatory cytokine production and innate immune system responses.

#### **5. Macrophages and other important innate immune cells in Ebola virus innate immune responses**

The DCs and macrophages infection with the EBOV is critical for adaptive immune activation. The EBOV infection leads to macrophage activation (by increasing the CD163 marker) and decreases T cell activation (by reducing in CD25 marker) in severe cases [68]. The macrophage's infection leads to reductions in the co-stimulatory molecules in these cells, which are known as the main adaptive immune activation and the axis of the antigen presentation [27, 28]. The macrophage infection also leads to alteration in pro-inflammatory cytokine releases, such as IL-1β, TNF-α, and IL-6, which could dysregulate the inflammation [69]. Furthermore, monocytes infection with EBOV is a toll on the virus spreading all around the body [69]. The EBOV uses a mimicking of the apoptosis process for attachment and entry macrophages due to the TAM receptor tyrosine kinases and integrin αV [70]. However, it seems that other cell surface receptors such as DC-SIGN and DC-SIGNR or macrophage galactose-type calcium-type lectin (MGL) are critical for the EBOV infection in DC and macrophages [71, 72]. The attachment of the virus to macrophages regardless of the virus entry or macrophage infection affects the macrophage's expression profile. This alteration is led macrophages to produce high levels of pro-inflammatory and pro-apoptotic signals [30].

Macrophages and dendritic cells are important cell targets of EBOV and the main innate immune cells that secrete cytokines, and chemokines following infection [29, 69]. Although macrophages and DCs are still able to initiate coagulation and inflammation, they are not able to stop the spreading of the Ebola virus systemically due to their impaired ability [28, 73]. These dysfunctions have a major impact on the innate and adaptive immune systems [74]. Macrophages and DCs are the

essential cells of innate immunity and provide a bridge between innate and adaptive immunity [75]. It will highlight the importance of these cells in the disease outcome (**Figure 1**). Furthermore, VP24 and VP35 block latent lymphocyte stimulation through the IFN response [73]. All these clues are critical to combine and work as chains for limiting the infection through sufficient and well-controlled innate immune response activation, which leads to adaptive immune responses.

#### **6. Conclusions**

In this chapter, we tried to provide a perspective on the EBOV infection and innate immune responses. In a glimpse, it is worth mentioning that innate immune responses are critical in the EBOV infection. Sufficient and well-controlled innate immune responses may lead to optimum cytokine release and adaptive immune activation. In contrast, an overreacted innate immunity could affect and hyper-inflammation response.

The macrophages and DCs are also key elements in EBOV infection due to proinflammatory response and virus spreading. The EBOV uses different strategies to dysregulate and evade innate immune responses.

VP35 and VP24 of the virus inhibit the IFN type I stimulation in infected cells. Furthermore, the soluble GPs of the EBOV can induce apoptosis in T cells. The interaction of the EBOV with innate immunity is the most fundamental feature of the infection and determines the disease outcome.

#### **Acknowledgements**

We sincerely acknowledge the IntechOpen publication editorial office and Jelena Vrdoljak the Author Service Manager in IntechOpen for her incredible efforts.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Parastoo Yousefi\* and Alireza Tabibzadeh\* Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

\*Address all correspondence to: parastoo\_y@yahoo.com and alireza.tabibzadeh@outlook.com; tabibzadeh.a@iums.ac.ir

© 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] ICTV. https://talk.ictvonline.org/ taxonomy/. visit 2 March 2022

[2] Pourrut X et al. The natural history of Ebola virus in Africa. Microbes and Infection. 2005;**7**(7-8):1005-1014

[3] Li Y, Chen S. Evolutionary history of Ebola virus. Epidemiology & Infection. 2014;**142**(6):1138-1145

[4] Althaus CL. Estimating the reproduction number of Ebola virus (EBOV) during the 2014 outbreak in West Africa. PLoS Currents. 2014;**2014**:6

[5] Mulangu S et al. A randomized, controlled trial of Ebola virus disease therapeutics. New England Journal of Medicine. 2019;**381**(24):2293-2303

[6] Lévy Y et al. Prevention of Ebola virus disease through vaccination: Where we are in 2018. The Lancet. 2018;**392**(10149):787-790

[7] Baize S et al. Inflammatory responses in Ebola virus-infected patients. Clinical & Experimental Immunology. 2002;**128**(1):163-168

[8] Wauquier N et al. Association of KIR2DS1 and KIR2DS3 with fatal outcome in Ebola virus infection. Immunogenetics. 2010;**62**(11):767-771

[9] Lai C-Y et al. Ebola virus glycoprotein induces an innate immune response in vivo via TLR4. Frontiers in Microbiology. 2017;**8**:1571

[10] Iwasaki A, Medzhitov R. Tolllike receptor control of the adaptive immune responses. Nature Immunology. 2004;**5**(10):987-995

[11] Abe T, Marutani Y, Shoji I. Cytosolic DNA-sensing immune response and

viral infection. Microbiology and Immunology. 2019;**63**(2):51-64

[12] He S et al. Research advancement of innate immunity and pattern recognition receptors. Chinese Journal of Animal Nutrition. 2017;**29**(11):3844-3851

[13] Amarante-Mendes GP et al. Pattern recognition receptors and the host cell death molecular machinery. Frontiers in Immunology. 2018:2379

[14] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell. 2006;**124**(4):783-801

[15] Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010;**140**(6):805-820

[16] Dalpke AH, Helm M. RNA mediated Toll-like receptor stimulation in health and disease. RNA Biology. 2012;**9**(6):828-842

[17] Jang T-h, Park HH. Crystal structure of TIR domain of TLR6 reveals novel dimeric interface of TIR–TIR interaction for toll-like receptor signaling pathway. Journal of Molecular Biology. 2014;**426**(19):3305-3313

[18] Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: Update on Tolllike receptors. Nature Immunology. 2010;**11**(5):373-384

[19] Jensen S, Thomsen AR. Sensing of RNA viruses: A review of innate immune receptors involved in recognizing RNA virus invasion. Journal of Virology. 2012;**86**(6):2900-2910

[20] Hou F et al. MAVS forms functional prion-like aggregates to activate and

*Interaction of Ebola Virus with the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.104843*

propagate antiviral innate immune response. Cell. 2011;**146**(3):448-461

[21] Servant MJ, Grandvaux N, Hiscott J. Multiple signaling pathways leading to the activation of interferon regulatory factor 3. Biochemical Pharmacology. 2002;**64**(5-6):985-992

[22] Schoggins JW. Interferon-stimulated genes: Roles in viral pathogenesis. Current Opinion in Virology. 2014;**6**:40-46

[23] He F et al. Filovirus VP24 proteins differentially regulate RIG-I and MDA5 dependent Type I and III interferon promoter activation. Frontiers in Immunology. Jan 5, 2022;**12**:694105

[24] Martinon F, Burns K, Tschopp J. The inflammasome: A molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Molecular Cell. 2002;**10**(2):417-426

[25] Menu P, Vince J. The NLRP3 inflammasome in health and disease: The good, the bad and the ugly. Clinical & Experimental Immunology. 2011;**166**(1):1-15

[26] Lamkanfi M, Dixit VM. Mechanisms and functions of inflammasomes. Cell. 2014;**157**(5):1013-1022

[27] Mahanty S et al. Cutting edge: Impairment of dendritic cells and adaptive immunity by Ebola and Lassa viruses. The Journal of Immunology. 2003;**170**(6):2797-2801

[28] Bosio CM et al. Ebola and Marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation. The Journal of Infectious Diseases. 2003;**188**(11):1630-1638

[29] Gupta M et al. Monocyte-derived human macrophages and peripheral

blood mononuclear cells infected with Ebola virus secrete MIP-1α and TNF-α and inhibit poly-IC-induced IFN-α in vitro. Virology. 2001;**284**(1):20-25

[30] Wahl-Jensen V et al. Ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression. PLoS Neglected Tropical Diseases. 2011;**5**(10):e1359

[31] Rugarabamu S et al. Forty-two years of responding to Ebola virus outbreaks in Sub-Saharan Africa: A review. BMJ Global Health. 2020;**5**(3):e001955

[32] Baize S et al. Emergence of Zaire Ebola virus disease in Guinea. New England Journal of Medicine. 2014;**371**(15):1418-1425

[33] Rewar S, Mirdha D. Transmission of Ebola virus disease: An overview. Annals of Global Health. 2014;**80**(6):444-451

[34] Prescott J et al. Postmortem stability of Ebola virus. Emerging Infectious Diseases. 2015;**21**(5):856

[35] Judson S, Prescott J, Munster V. Understanding ebola virus transmission. Viruses. 2015;**7**(2):511-521

[36] Mate SE et al. Molecular evidence of sexual transmission of Ebola virus. New England Journal of Medicine. 2015;**373**(25):2448-2454

[37] Bah EI et al. Clinical presentation of patients with Ebola virus disease in Conakry, Guinea. New England Journal of Medicine. 2015;**372**(1):40-47

[38] Varkey JB et al. Persistence of Ebola virus in ocular fluid during convalescence. New England Journal of Medicine. 2015;**372**(25):2423-2427

[39] Jacobs M et al. Late Ebola virus relapse causing meningoencephalitis: A case report. The Lancet. 2016; **388**(10043):498-503

[40] Clark DV et al. Long-term sequelae after Ebola virus disease in Bundibugyo, Uganda: A retrospective cohort study. The Lancet Infectious Diseases. 2015;**15**(8):905-912

[41] Jain S et al. Structural and functional aspects of Ebola virus proteins. Pathogens. 2021;**10**(10):1330

[42] Watt A et al. A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity. Journal of Virology. 2014;**88**(18):10511-10524

[43] Boehmann Y et al. A reconstituted replication and transcription system for Ebola virus Reston and comparison with Ebola virus Zaire. Virology. 2005;**332**(1):406-417

[44] He FB, Melén K, Kakkola L, Julkunen I. Interaction of ebola virus with the innate immune system. In: Okware SI, editor. Emerging Challenges in Filovirus Infections [Internet]. London: IntechOpen; 2019 [cited 2022 May 02]. Available from: https://www. intechopen.com/chapters/67614. DOI: 10.5772/intechopen.86749

[45] Basler CF et al. The Ebola virus VP35 protein functions as a type I IFN antagonist. Proceedings of the National Academy of Sciences. 2000;**97**(22):12289-12294

[46] Woolsey C et al. A VP35 mutant Ebola virus lacks virulence but can elicit protective immunity to wildtype virus challenge. Cell Reports. 2019;**28**(12):3032-3046

[47] Xu W et al. Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with

nuclear import of phosphorylated STAT1. Cell Host & Microbe. 2014;**16**(2):187-200

[48] Mateo M et al. VP24 is a molecular determinant of Ebola virus virulence in guinea pigs. The Journal of Infectious Diseases. 2011;**204**(suppl\_3):S1011-S1020

[49] Baseler L et al. The pathogenesis of Ebola virus disease. Annual Review of Pathology: Mechanisms of Disease. 2017;**12**:387-418

[50] Takada A et al. Antibodydependent enhancement of Ebola virus infection. Journal of Virology. 2003;**77**(13):7539-7544

[51] Tchesnokov EP et al. Mechanism of inhibition of Ebola virus RNA-dependent RNA polymerase by remdesivir. Viruses. 2019;**11**(4):326

[52] Hensley LE et al. Proinflammatory response during Ebola virus infection of primate models: Possible involvement of the tumor necrosis factor receptor superfamily. Immunology Letters. 2002;**80**(3):169-179

[53] McElroy AK et al. Kinetic analysis of biomarkers in a cohort of US patients with Ebola virus disease. Reviews of Infectious Diseases. 2016;**63**(4): 460-467

[54] Villinger F et al. Markedly elevated levels of interferon (IFN)-γ, IFN-α, interleukin (IL)-2, IL-10, and tumor necrosis factor-α associated with fatal Ebola virus infection. The Journal of Infectious Diseases. 1999;**179**(Suppl\_1):S188-S191

[55] Wauquier N et al. Human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis. PLoS Neglected Tropical Diseases. 2010;**4**(10):e837

*Interaction of Ebola Virus with the Innate Immune System DOI: http://dx.doi.org/10.5772/intechopen.104843*

[56] Hutchinson KL, Rollin PE. Cytokine and chemokine expression in humans infected with Sudan Ebola virus. The Journal of Infectious Diseases. 2007;**196**(Suppl\_2):S357-S363

[57] Geisbert TW et al. Apoptosis induced in vitro and in vivo during infection by Ebola and Marburg viruses. Laboratory Investigation. 2000;**80**(2):171-186

[58] Ito H et al. Ebola virus glycoprotein: Proteolytic processing, acylation, cell tropism, and detection of neutralizing antibodies. Journal of Virology. 2001;**75**(3):1576-1580

[59] Francica JR et al. Steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein. PLoS Pathogens. 2010;**6**(9):e1001098

[60] Bradfute SB et al. Mechanisms and consequences of ebolavirus-induced lymphocyte apoptosis. The Journal of Immunology. 2010;**184**(1):327-335

[61] Zaki S, Goldsmith C. Pathologic features of filovirus infections in humans. Current Topics in Microbiology and Immunology. 1999;**235**:97-116

[62] Prescott JB et al. Immunobiology of Ebola and Lassa virus infections. Nature Reviews Immunology. 2017;**17**(3):195-207

[63] Leroy E et al. Early immune responses accompanying human asymptomatic Ebola infections. Clinical & Experimental Immunology. 2001;**124**(3):453-460

[64] Warfield KL et al. Role of natural killer cells in innate protection against lethal ebola virus infection. The Journal of Experimental Medicine. 2004;**200**(2):169-179

[65] Menicucci AR et al. Transcriptome analysis of circulating immune cell subsets highlight the role of monocytes in Zaire Ebola virus Makona pathogenesis. Frontiers in Immunology. 2017;**8**:1372

[66] Chong WC et al. The complex interplay between endoplasmic reticulum stress and the NLRP3 inflammasome: A potential therapeutic target for inflammatory disorders. Clinical & Translational Immunology. 2021;**10**(2):e1247

[67] Halfmann P, Hill-Batorski L, Kawaoka Y. The induction of IL-1β secretion through the NLRP3 inflammasome during Ebola virus infection. The Journal of Infectious Diseases. 2018;**218**(suppl\_5): S504-S507

[68] McElroy AK et al. Macrophage activation marker soluble CD163 associated with fatal and severe Ebola virus disease in humans. Emerging Infectious Diseases. 2019;**25**(2):290

[69] Ströher U et al. Infection and activation of monocytes by Marburg and Ebola viruses. Journal of Virology. 2001;**75**(22):11025-11033

[70] Dahlmann F et al. Analysis of Ebola virus entry into macrophages. The Journal of Infectious Diseases. 2015;**212**(suppl\_2):S247-S257

[71] Simmons G et al. DC-SIGN and DC-SIGNR bind ebola glycoproteins and enhance infection of macrophages and endothelial cells. Virology. 2003;**305**(1):115-123

[72] Usami K et al. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin. Biochemical and Biophysical Research Communications. 2011;**407**(1):74-78

[73] Bray M, Geisbert TW. Ebola virus: The role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever. The International Journal of Biochemistry & Cell Biology. 2005;**37**(8):1560-1566

[74] Mohamadzadeh M, Chen L, Schmaljohn AL. How Ebola and Marburg viruses battle the immune system. Nature Reviews Immunology. 2007;**7**(7):556-567

[75] Olukitibi TA et al. Dendritic cells/ macrophages-targeting feature of Ebola glycoprotein and its potential as immunological facilitator for antiviral vaccine approach. Microorganisms. 2019;**7**(10):402

#### **Chapter 15**

## Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality

*Perla Abou Atmeh, Soraya Mezouar and Jean-Louis Mège*

#### **Abstract**

The role of macrophages in viral infections is well documented. Their activation status also called macrophage polarization categorized by the dichotomy of M1 and M2 phenotype remained poorly investigated. Recent studies have shown the complexity of macrophage polarization in response to viral infection and the limits of its use in infected individuals. The aim of this chapter is to reappraise the concept of macrophage polarization in viral infectious diseases, which are more complicated than the models of macrophage-virus interaction. If this concept has been largely used to describe activation status of myeloid cells in experimental conditions, it has to be assessed in light of high-throughput technologies at molecular and phenotypic levels. We update knowledge on macrophage polarization in viral infectious diseases with a special attention for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection leading to coronavirus disease (COVID-19). Hence, we propose an overview of the concept of macrophages as targets for therapeutic intervention in viral infectious disease. Finally, we tempted to focus our approach on patient investigation restricting the use of *in vitro* experiments and animal models to mechanistic questions.

**Keywords:** macrophages, myeloid cells, polarization, viral diseases

#### **1. Introduction**

First described in 1882 by Ilya Mechnikov, macrophages or "phagocytes" are distributed widely in the body where they acquire specific tissue identities and functions [1]. Macrophages are key effectors of tissue homeostasis contributing to wound healing and tissue repair. When tissue homeostasis is altered, monocytes are recruited from blood to tissues where they differentiate into macrophages. These latter are critical components of innate and adaptive immunity and contribute to inflammation and host defense.

During infection, macrophages represent the first cells on the battle front. They can act as scavengers by engulfing and destroying pathogens or altered host cells, alert the immune system through the secretion of lipid mediators, cytokines, and chemokine; or present antigens to T lymphocytes [2]. The expression of lectins, scavenger receptors, and immunoglobulin receptors enables macrophage phagocytosis, antibody-dependent cell phagocytosis (ADCP) and cytotoxicity (ADCC) [3].

**Figure 1.**

*In vitro and in vivo macrophage polarization markers and protein secretion.*

They are also equipped with pattern recognition receptors (PRRs) that following their stimulation lead to the activation of transcription factors, the release of toxic mediators (reactive oxygen intermediates, proteases, inflammatory molecules) [3]. Because of their ability to promote adaptive immune response to virus via antibody release and CD8 T cell activation, macrophages can contribute to the cure of viral infections.

According to their function in pathological conditions, macrophages are considered as activated or alternatively activated also referred to as M1 and M2 polarization phenotype, respectively. The polarization of macrophages is a concept introduced to describe the features of myeloid cell activation and to classify them in functional categories (**Figure 1**), according to initially reported polarization of immune response into Th1 and Th2 types [4–6]. The M1 macrophages are induced by Th1 cytokines such as interferon (IFN)-γ and/or lipopolysaccharide (LPS) and present a proinflammatory phenotype. They are characterized by the secretion of inflammatory cytokines including tumor necrosis factor (TNF), interleukin (IL)-1α, IL-1β, IL-6, IL-12, IL-23, and also the expression of several markers including CD80, CD86, and CD68. The M2 macrophages are likely more heterogeneous: they have been classified into four categories, M2a, M2b, M2c, and M2d, depending on the stimulus [7, 8]. As described in **Table 1**, macrophages stimulated by IL-4 or IL-13 lead to an M2a profile associating the expression of CD206, IL-1 receptor type 2 (IL1-R2) and arginase and the secretion of transforming growth factor (TGF)-β and IL-10. M2b macrophages are induced by immune complexes, Toll-like receptor (TLR) ligands, or IL-1β. This profile is associated with the secretion of inflammatory cytokines (TNF, IL-1β, IL-6), IL-10, and the chemokine CCL1. M2c macrophages are induced by IL-10, TGF-β, or glucocorticoids; they expressed CD163 and CD206 and exhibit anti-inflammatory activity through the secretion of IL-10 and TGF-β. Finally, M2d polarization is initiated by TLRs (TLR2, TLR4, TLR7, and TLR9) or adenosine receptor ligands (A2A) with the secretion of vascular endothelial growth factor (VEGF) supporting proangiogenic and pro-tumoral functions [9–11]. It is becoming evident that these categories of activation states are an over-simplification of the diversity of macrophage activation modes. We have tried to reappraise the concept of macrophage polarization by associating the type of polarization with the agonist [12]. This approach allows to propose several activation profiles with LPS, IFN-γ, or their combination instead


*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

*IFN: interferon, LPS: polysaccharide; GM-CSF: granulocyte macrophage colony stimulating factor; TLR: Toll like receptor; MHC: major histocompatibility complex; iNOS: inducible nitric oxide; Arg: arginase; TNF: tumor necrosis factor; IL: interleukin; TGF: transforming growth factor; VEGF: vascular endothelial growth factor.*

#### **Table 1.**

*In vitro macrophages polarization sub-types.*

of a unique M1 activation state [13, 14]. In clinical situations, it has been shown that tumor-associated macrophages (TAMs) are clearly specialized cell populations in which polarization and functions are related: M1-like and M2-like TAMs have antitumoral and pro-tumoral activities, respectively [15]. In other clinical situations including infectious diseases, such functional dichotomy is rarely observed [7], and the functional role of macrophage polarization remains an exception.

The mechanisms of macrophage polarization have been the object of a broad literature. It appears now that the metabolism of macrophages is different according to their polarization. Hence, M1 macrophages exhibit increased glycolysis and broken tricarboxylic acid (TCA) cycle, leading to the accumulation of succinate and citrate. In contrast, M2 macrophages have intact TCA cycle leading to the generation of adenosine triphosphate (ATP) [16, 17]. It is becoming evident that the polarization of macrophages is determined by transcriptional changes, as shown by using new technologies [4]. In aortic macrophages studied *in vivo* by scRNA-seq, Chang et al. identified three classes of macrophages: resident-like, inflammatory, and a final group with strong expression of triggering receptor expressed on myeloid cells 2 (TREM2) [18]. Interestingly, M2 markers are found in the inflammatory macrophage population, suggesting that the traditional classification of macrophage polarization does not fully reflect the diversity of the *in vivo* macrophage populations (**Figure 1**). Quantitative mass spectrometry imaging has also been proposed to investigate macrophage polarization *in situ.* It enabled the cartography of functional macrophage population and the visualization of their distribution in normal and pathological tissues [19–22]*.* In addition, macrophage polarization requires dynamic and reversible epigenomic marks at enhancers and promoters of signal responsive genes [4]. The epigenetic mechanisms of M2 polarization reveal the role of histone methylation and acetylation. Hence, the overexpression of DNA methyltransferase 3B or the loss of histone deacetylase-3 (HDAC3) promotes M2 phenotype. The histone demethylase JMJD3 (lysine demethylase 6B, KDM6B) is activated by IL-4 and binds M2 genes, leading to repress M1 inflammatory program. In contrast, IFN-γ increases chromatin accessibility [4, 23]. Finally, polarization and functional responses of macrophages are influenced by differential expression of microRNAs: the literature has reported miRs specialized in M1 polarization and miRs increasing M2-like responses [24].

Is the specialization of M1/M2-like macrophages evolutionary conserved? Two ancient molecular mechanisms, inducible nitric oxide synthase 2 (iNOS2) and arginase (Arg), characterize macrophage polarization. If the ability to produce large amounts of nitric oxide in response to microbial agonists, a hallmark of M1 macrophages, has emerged with vertebrates, the Arg pathway is described in both prokaryotes and eukaryotes [25]. The cytosolic and mitochondrial Args are encoded by two genes, and it has been shown that the duplication of Arg gene occurred after the separation of vertebrates and invertebrates [26]. Hence, it is likely that macrophage polarization occurs during early vertebrate evolution [27].

The aim of this chapter is to update the knowledge about macrophage polarization in viral infections with a special focus on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. We are deciding to analyze papers reporting infections in humans and to restrict animal models for specific questions. The analysis of the literature reveals the heterogeneous definition of macrophage polarization and the frequently inappropriate use of this concept. It is the reason why the final paragraph will describe how macrophage polarization can be studied in patients (**Table 2**) and to propose some recommendations for investigating infected patients.


#### **Table 2.**

*In vivo polarization of macrophage sub-types.*

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

#### **2. Viral infectious diseases in humans**

As obligate intracellular pathogens, viruses require host cells to replicate. Innate defenses are essentials to block or inhibit cell infection, to eliminate virus from infected cells, and to alert cells the adaptive immunity. This crucial stage is regulated by myeloid cells including, dendritic cells, and mainly by macrophages. The early response is based on [1] the recognition of pathogen-associated molecular patterns (PAMPs) by PRRs, [2] the elimination of foreign agents [3] and the activation of type I and II IFNs, and interferon-stimulated gene (ISG), which have broad-spectrum antiviral activity [28, 29]. Although this defense is effective, a complete cure of the infection requires mounting adaptive immune response that is controlled to prevent immune pathogenicity. Macrophage-mediated immune response can be circumvented or hijacked by the virus to allow its replication and persistence in the host.

The first studies highlighting the interaction between myeloid cells and viruses date back to the end of 1970s [30, 31]. In a general point of view, macrophages polarize into M1 following a contact with viruses, which induces a pro-inflammatory response while the phenotype M2 is often found at late stages of infection [32]. It has been reported that virulent strains of viruses will "bias" the M1 polarization profile of macrophages into M2. In contrast, attenuated viruses lead to an M2 polarization profile M2 [33]. M1-activated macrophages play a key role in the elimination of viruses through several strategies, including the secretion of reactive species [34], secretion of antiviral cytokines [35], or activation of other immune cells such as T cells and natural killers [36, 37]. Hence, some viruses are able to counteract M1 macrophages in order to obtain an environment favorable to their replication. Indeed, some viruses are able to inhibit production of nitric oxide [38–41] or pro-inflammatory cytokines [42–44], suppress antigen presentation [45–48], or simply modulate the signaling pathways associated with polarization by inducing M2 phenotype [8, 49–51]. We previously addressed the role of macrophage polarization in bacterial infections [7, 52]; the aim of the present talk is to assess the role of macrophage polarization in viral infections by selecting typical viral infections.

#### **2.1 Hepatitis viruses**

Five viruses, known as hepatitis A, B (HBV), C (HCV), D, and E, are responsible for the majority of acute and chronic hepatitis. HBV infection is responsible of acute self-resolving disease in adults but not in early life. Hepatic macrophages whatever their origin are involved in host response to HBV. HBV antigen and nucleic acids are detected in monocytes and macrophages from patients with hepatitis. The interaction of HBV with macrophages including hepatic macrophages may affect macrophage polarization by suppressing M1 polarization and promoting M2 polarization [53]. M1-associated cytokines are likely protective as shown by higher risk of HBV reactivation and hepatotoxicity in patients with anti-TNF treatment given for inflammatory diseases [54]. During the progression of HBV-related disease (from mild chronic hepatitis B to decompensated cirrhosis), M2-type monocytes expressing CD163 and CD206 are increased, whereas the frequency of cells expressing M1 markers decreases. Monocytes and Kupffer cells expressing an M2 profile are predicting a poor clinical outcome [55]. The non-protective effect of M2-type polarization is illustrated by the observation that IL-10 gene promoter polymorphisms are associated with HBV progression [44]. The measurement of soluble CD163 and soluble mannose receptor may be a pertinent approach of follow-up of patients with chronic hepatitis. Both

markers are released during liver damage and are associated with M2 polarization and fibrosis; their levels are reduced after antiviral treatment [56]. The relationship between chronic evolution of hepatitis B, fibrosis, and infiltration of liver by M2-like macrophages has been demonstrated in a humanized mouse model of infection [57].

HCV is transmitted between adults and is responsible for a high percentage of chronic infections with a major risk of liver cirrhosis and hepatocellular carcinoma [58]. The co-culture of monocyte-derived macrophages with HCV-infected hepatocytes induces M2 surface markers. TGF-β produced by these polarized macrophages activates hepatic stellate cells, leading to fibrosis. However, monocytes and macrophages do not seem completely polarized at the cytokine level [59]. Cell-free virus or exosome-packaged HCV induces the differentiation of monocytes into macrophages with M2 phenotype and non-polarized cytokine production under the control of TLR7/8. Interestingly, TLR7/8 is overexpressed in pro-fibrotic monocytes from chronic HCV-infected patients [60]. It has been also shown that HCV core protein inhibits phagocytosis activity of M1 and M2 macrophages and CD4<sup>+</sup> T cell activation induced by M1 macrophages but promotes that induced by M2 macrophages [61]. M1 and M2 macrophages generated from chronic HCV patients lose their phenotypic characteristics, suggesting that chronic HCV infection is rather associated with an impaired polarization than a reprogramming of macrophages [62]. Finally, in biopsies of HCV-infected patients, it has been shown that non-infected cells such as Kupffer cells are a source of IFNs, demonstrating the interplay between hepatic cells [63]. Taken together, these data suggest that HBV and HCV share the ability to interfere with M1-type macrophage polarization, thus accounting for viral persistence, hepatic fibrosis, and evolution to carcinoma.

#### **2.2 Human immunodeficiency virus (HIV)**

The permissivity of monocytes and macrophages to HIV depends on their differentiation stage, polarization status, and tissue location [64]. The M1/M2 polarization of macrophages impacts the steps of viral cycle including entry, reverse transcription, transcription, and posttranscription. As the level of inflammatory cytokines is high in the early stage of HIV-1 infection [65, 66], it is likely that M1-like macrophages play a role in HIV infection. The entry of R5 and R5/X4 HIV in M1-like macrophages is decreased because of cytokine-mediated downregulation of CD4 and CCR5. In contrast, M1 cytokines such as TNF increase viral transcription. In M2 macrophages elicited by IL-4/IL-13 and/or IL-10-mediated deactivated macrophages, both entry and replication of HIV-1 are decreased [64]. The implication of TNF in M1 polarization of HIV-infected macrophages is debated [67]. Hence, it is likely that M1 macrophages enable the formation of viral reservoirs early in the disease. At later stages, an M2 shift of macrophages is observed. At the onset of acquired immunodeficiency syndrome (AIDS), deactivated macrophages predominate via enhanced clearance of apoptotic cells, which is known to promote M2-like macrophages [68]. Severe evolution of HIV infection is associated with elevated IL-10 levels, but not IL-4 levels, suggesting that AIDS is characterized by IL-10-mediated M2-like phenotype [69]. These findings have been confirmed in acute and chronic HIV and SIV infections. Hence, at the beginning of the infection, the central nervous system, heart, and blood vessels exhibit M1-like macrophages, whereas M2-like macrophages are observed in later responses [32]. It is likely that CD163<sup>+</sup> M2 macrophages play a protective role in SIV-infected macaques through their anti-inflammatory functions [70]. It has been proposed that the persistence of initial activation in patients with chronic infection

and successful antiviral therapy is correlated to non-AIDS complications such neurocognitive disorder and cardiovascular dysfunctions [71]. Unfortunately, few markers have been investigated in these studies, and it is likely that more precise data will be necessary to reanalyze the polarization of macrophages in HIV infection [71, 72].

#### **2.3 Flavivirus**

Flavivirus is responsible for infections essentially dominated by dengue virus (DENV) and Zika virus (ZIKV). First, DENV infection presents a large spectrum of clinical presentations from moderate symptoms to classical dengue and hemorrhagic dengue. Monocytes and macrophages are involved in the infection pathogenesis. Macrophage-colony-stimulating factor (M-CSF)-differentiated macrophages (M2-like macrophages) are poorly sensitive to DENV infection. In contrast, granulocyte macrophage CSF (GM-CSF)-differentiated macrophages (M1-like macrophages) are highly susceptible to DENV infection with high release of cytokines and activation of NLRP3 inflammasome [73]. Nevertheless, some biomarkers such as soluble CD163, known to be associated with M2 polarization of macrophages, seem predictive of severe dengue [74]. In pediatric dengue patients compared with healthy individuals, the number of M2-like macrophages is increased with decreased number of M1-like macrophages. In dengue patients with bleeding trend, both macrophage subsets are decreased and are associated with decreased platelet count [48]. Second, ZIKV is associated with numerous cases of microcephaly and/or central nervous system malformations. ZIKV infects myeloid cells and has a tropism for placenta including maternal and fetal tissues. It has been shown that ZIKV replicates in both placenta macrophages, also named Hofbauer cells, and trophoblasts [75]. Two lineages of ZIKV, African and Asian, have been described, and it has been shown that they exhibit differences in pathogenicity despite close sequence homology [76, 77]. While the Asian strain of ZIKV elicits an expansion of non-classical monocytes from healthy donors and M2-skewed immunosuppressive program, African strain promotes a M1 program [78].

#### **2.4 Cytomegalovirus (CMV)**

Human CMV (hCMV) uses TLR2 and intracytosoplasmic sensors to invade monocytes and macrophages [79]. In monocytes, hCMV stimulates a transcriptomic program in which M1 genes are enriched [80]. On the other hand, a product of hCMV genome, *UL111A* gene, encodes functional homologs of human IL-10 during both productive and latent phases of CMV infection [81]. In CD14<sup>+</sup> monocytes, the viral IL-10 induces M2c phenotype associating increased expression of CD163 and CD14 and downregulation of HLA-DR. The viral IL-10 also upregulates heme oxygenase 1 (HO-1), a driver of phenotype shift to M2 macrophages [82] known to also down-modulate M1-associated cytokines and poorly stimulate CD4<sup>+</sup> T cells [83]. We hypothesize that CMV triggers an M1 program in monocytes and that the release of viral or human IL-10 leads bystander monocytes to be reprogrammed toward an M2 phenotype. The polarization of monocytes in response to hCMV is likely necessary for their differentiation into macrophages. Recently, it has been shown that hCMV stimulates the expression of M1 and M2 markers in monocytes and activates PI3K-Akt axis, leading to caspase 3 activation [84]. CMV susceptibility is dependent on polarization of myeloid cells. M1-like macrophages are more resistant to CMV than M2-like macrophages, and it is likely that this resistance is related to the ability of M1-like

macrophages to induce IFN-γ production by natural killer (NK) cells [85]. This hypothesis is supported by two other studies. Although hCMV susceptibility is higher in M2-like macrophages, productive and persistent viral infection is observed in both M1- and M2-like macrophages. Infected M1- and M2-like macrophages are efficient in stimulating proliferation of autologous T cells from hCMV-seropositive donors [86]. The susceptibility of M2 macrophages is optimal in the early phase of hCMV infection, whereas, in the late phase, macrophage activation necessary for viral replication is dependent on the activation of mammalian target of rapamycin (mTORC)1 complex, as confirmed with experiments including rapamycin [87].

#### **2.5 Influenza virus**

Four *influenza* virus genera (A, B, C, and D) belonging to *orthomyxoviridae* are responsible for flu, a seasonal respiratory epidemics, or pandemics, such as the "Great Influenza" pandemics of 1918 [88]. The severity of influenza pneumonia depends on host susceptibility and strain diversity and can lead to acute respiratory distress syndrome and lethality. These latter complications are associated with uncontrolled inflammatory response. Numerous evidences show that macrophages including alveolar macrophages are involved in the pathophysiology of influenza virus infections via a direct viral infection or the overproduction of cytokines. This is emphasized by the observation that over-pathogenic strains of *influenza* virus productively infect monocytes [89]. Animal models demonstrate that macrophage reprogramming is critical in outcome of influenza virus infections. Hence, GM-CSF protects from mortality and morbidity and redirects responses of alveolar macrophages from M1-like to M-2-like activation. This finding was unexpected because GM-CSF is known as an M1 inducer and depresses arginase, a canonical marker of M2-like status [90]. The inactivation of NOS (nitric oxide synthase) 2 and IFN-γ favors M2 reprogramming and improves outcome of viral infection [91]. In contrast, the treatment of macrophages with baicalin that possesses antiviral properties stimulated an M1 phenotype shift associated with activation of IFN pathway and inhibition of influenza virus replication [92].

The evidences of macrophage polarization in humans infected with influenza virus are scarce. In one study of patients from the 2009 to 2010 pandemics, monocytes have been reported as a marker of severity independently of viral load [93]. Monocytes from patients with severe infection exhibit increased expression of M1 markers and TNF production and a down-modulation of CD163, an M2 marker. Murine models of influenza virus infection also show high proportion of recruited M1 monocytes and decreased number of resident M2 alveolar macrophages, confirming that the severity of influenza virus infection is associated with macrophage reprogramming toward an M1 phenotype [93].

#### **2.6 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)**

SARS-CoV-2, a strain of the coronavirus family, causes coronavirus disease-2019 (COVID-19) characterized in the most severe cases by an increased production of cytokines including IL-1α, IL-1β, IL-6, IL-7, TNF, type I and II IFN, CCL2, CCL3, and CXCL10 [94, 95]. This cytokine storm was initially observed in influenza syndrome that occurs after systemic infection and immunotherapy [96] before it was extended to describe immune response in COVID-19 patients [97]. To better understand the pathophysiology of this emergent disease, the researchers focused on the response of myeloid cells such as macrophages or dendritic cells. Macrophages are permissive

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

to SARS-CoV-2 infection but no viral replication is observed *in vitro* [98, 99]. Interestingly, the production of inflammatory cytokines and chemokines is observed after SARS-CoV-2 infection of macrophages but not after infection of monocytederived dendritic cells [98, 100]. The release of TNF, IL-1β, IL-10, IFN-α/β, and IL-6 by infected-macrophages leads to type I IFN-immune response, suggesting a protective role against viral infection [100].

The polarization of macrophages in COVID-19 has also been investigated. Alveolar macrophages respond differently to infection depending on their polarization status. After SARS-CoV-2 infection, M1 macrophages are associated with viral spreading, whereas M2 polarization induces virus degradation and infection limitation. Indeed, macrophages from human ACE2 transgenic mice present an increased infection rate after *in vitro* treatment by IFN-γ or LPS compared with IL-4 treatment [101]. These results are controversial because the SARS-CoV-2 infection of M1 and M2 macrophages from the THP-1 cell line is similar [99]. The gene expression study of lung alveoli from COVID-19 patients reveals different macrophage patterns depending on their polarization profile. The gene expression study of lung alveoli from COVID-19 patients shows that highly inflammatory macrophages are mostly found in patients with severe COVID-19 [102, 103]. Thus, the polarization of macrophages during infection by SARS-CoV-2 is suggested as determining the severity of the disease, even if involved molecular mechanisms remain unexplored to date.

#### **3. Macrophage polarization and treatment of viral infections**

The role of deregulated immune response in pathogenesis of viral infections such as COVID-19 pandemics justifies the use of drugs that target host response and exhibit anti-infective properties. Hence, chloroquine and hydroxychloroquine are known for their antiviral and immunomodulatory effects, which lead to propose these molecules in the treatment of SARS-Cov2 infection [104]. Beyond the debate about the efficiency of chloroquine and hydroxychloroquine, their immunomodulatory properties affect the macrophage polarization. It is established that treatment with chloroquine can reverse the polarization of TAMs from M2 to M1 phenotype in tumor models [105]. Similarly, chloroquine and hydroxychloroquine interfere with LPS-mediated M1 polarization of macrophages [106]. The combination of hydroxychloroquine and azithromycin is interesting since this latter molecule is known to induce M2 macrophage polarization [106, 107]. Ivermectin, a macrocyclic lactone known for its antiparasitic effect, has an anti-inflammatory effect promoting M2 polarization of macrophages without effect on viral load; it has been proposed to limit the inflammation of respiratory tract and to improve COVID-19 outcome [108]. Remdesivir, an adenosine analog, reduces inflammatory gene expression and has been largely used in COVID-19 treatment [109].

As mentioned above, M1 polarization of macrophages is a determining phenotype against viral infections. The artificial induction of macrophage M1 polarization may be an interesting adjuvant to antiviral treatment for non-COVID19 infectious diseases. Baicaline has been proposed to limit influenza virus infection via the M1 polarization of macrophages, thus activating their antiviral function via the IFN signaling pathway [92, 110]. It is important to note that viral infections can lead to hyperactivation of macrophages, leading to an excessive inflammatory response known as macrophage activation syndrome. In this context, anti-inflammatory molecules such as tofacitinib, anti-IL1R, or IL-6R have been clinically tested, particularly for COVID-19 [111, 112].

#### **4. Conclusion and perspectives**

As the first line of defense, macrophages represent key immune cells against viral infections. In general, the antiviral response is mediated by a pro-inflammatory response of polarized M1 macrophages. Some viruses are able to counteract the antiviral response of macrophages by modulating their polarization by switching the M1 phenotype to M2 phenotype. It should also be noted that viruses such as influenza and SARS-CoV-2 are capable of modulating an M1 over-polarization of macrophages responsible for severe diseases. Interestingly, modulation of macrophage polarization has been investigated as a therapeutic strategy. However, the *in vivo* polarization profile remains more intricate compared with *in vitro* situations. This can be explained by the techniques classically used for investigating macrophage polarization (gene or protein expression) whose limitation is that the observed signals result from a mixture of diverse cells. Hopes for quantitative mass spectrometry imaging as a tissue-level investigative tool remain unanswered to date. Thus, clarifying the tools for investigating macrophage polarization in clinical settings and the associated molecular mechanisms are key steps in the development of therapies in viral infections.

#### **Acknowledgements**

Soraya Mezouar was supported by a "Fondation pour la Recherche Médicale" postdoctoral fellowship (reference: SPF20151234951). This work was supported by the French Government under the "Investissements d'avenir" (Investments for the future) program managed by the "Agence Nationale de la Recherche" (reference: 10-IAHU-03).

#### **Author contributions**

P.A.A, S.M., and J.L.M. conceived and wrote the manuscript.

#### **Declaration of interest**

The authors declare no competing interests.

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

#### **Author details**

Perla Abou Atmeh1,2† , Soraya Mezouar1,2† and Jean-Louis Mège1,2,3\* †

1 Aix-Marseille Univ, IRD, APHM, MEPHI, Marseille, France

2 IHU-Méditerranée Infection, Marseille, France

3 Aix-Marseille Univ, APHM, Hôpital de la Conception, Laboratoire d'Immunologie, Marseille, France

\*Address all correspondence to: jean-louis.mege@univ-amu.fr

† Contributed equally.

© 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] Lavin Y, Mortha A, Rahman A, Merad M. Regulation of macrophage development and function in peripheral tissues. Nature Reviews. Immunology. 2015;**15**(12):731-744

[2] Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nature Reviews. Immunology. 2011;**11**(11):723-737

[3] Kumar V. Phagocytosis: Phenotypically simple yet a mechanistically complex process: Phagocytosis is a very complex but crucial process playing a pivotal role in embryonic development and host defense to maintain immune homeostasis. International Reviews of Immunology. 2020;**39**(3):118-150

[4] Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity. Annual Review of Pathology. 2020;**15**:123-147

[5] Martinez FO, Gordon S. The M1 and M2 paradigm of macrophage activation: Time for reassessment. F1000prime Reports. 2014;**6**:13

[6] Mills CD, Ley K. M1 and M2 macrophages: The chicken and the egg of immunity. Journal of Innate Immunity. 2014;**6**(6):716-726

[7] Benoit M, Desnues B, Mege JL. Macrophage polarization in bacterial infections. Journal of Immunology. 2008;**181**(6):3733-3739

[8] Wang L, Zhang S, Wu H, Rong X, Guo J. M2b macrophage polarization and its roles in diseases. Journal of Leukocyte Biology. 2019;**106**(2):345-358

[9] Nikovics K, Favier AL. Macrophage identification in situ. Biomedicines. 2021;**9**(10):1393

[10] Pinhal-Enfield G, Ramanathan M, Hasko G, Vogel SN, Salzman AL, Boons GJ, et al. An Angiogenic switch in macrophages involving synergy between toll-like receptors 2, 4, 7, and 9 and adenosine A2A receptors. The American Journal of Pathology. 2003;**163**(2):711-721

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

[12] Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: Nomenclature and experimental guidelines. Immunity. 2014;**41**(1):14-20

[13] Murray PJ. Macrophage polarization. Annual Review of Physiology. 2017;**79**(1):541-566

[14] Ouedraogo R, Daumas A, Capo C, Mege JL, Textoris J. Whole-cell MALDI-TOF mass spectrometry is an accurate and rapid method to analyze different modes of macrophage activation. Journal of Visualized Experiments. 2013;**82**:50926

[15] Wu K, Lin K, Li X, Yuan X, Xu P, Ni P, et al. Redefining tumor-associated macrophage subpopulations and functions in the tumor microenvironment. Frontiers in Immunology. 2020;**11**:1731

[16] O'Neill LAJ. A broken Krebs cycle in macrophages. Immunity. 2015;**42**(3):393-394

[17] O'Neill LAJ, Kishton RJ, Rathmell J. A guide to

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

immunometabolism for immunologists. Nature Reviews. Immunology. 2016;**16**(9):553-565

[18] Chang Z, Wang Y, Liu C, Smith W, Kong L. Natural products for regulating macrophages M2 polarization. Current Stem Cell Research & Therapy. 2020;**15**(7):559-569

[19] Unsihuay D, Mesa Sanchez D, Laskin J. Quantitative mass spectrometry imaging of biological systems. Annual Review of Physical Chemistry. 2021;**72**:307-329

[20] Holzlechner M, Strasser K, Zareva E, Steinhäuser L, Birnleitner H, Beer A, et al. In situ characterization of tissue-resident immune cells by MALDI mass spectrometry imaging. Journal of Proteome Research. 2017;**16**(1):65-76

[21] Roussel M, Bartkowiak T, Irish JM. Picturing polarized myeloid phagocytes and regulatory cells by mass cytometry. Methods in Molecular Biology (Clifton, N.J.). 2019; **1989**:217-226

[22] Porta Siegel T, Hamm G, Bunch J, Cappell J, Fletcher JS, Schwamborn K. Mass spectrometry imaging and integration with other imaging modalities for greater molecular understanding of biological tissues. Molecular Imaging and Biology. 2018;**20**(6):888-901

[23] Foster SL, Hargreaves DC, Medzhitov R. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature. 2007;**447**(7147):972-978

[24] Zhong Y, Yi C. MicroRNA-720 suppresses M2 macrophage polarization by targeting GATA3. Bioscience Reports. 2016;**36**(4):e00363

[25] Edholm ES, Rhoo KH, Robert J. Evolutionary aspects of macrophages polarization. Results and Problems in Cell Differentiation. 2017;**62**:3-22

[26] Dzik JM. Evolutionary roots of arginase expression and regulation. Frontiers in Immunology. 2014;**5**:544

[27] Hodgkinson J, Grayfer L, Belosevic M. Biology of bony fish macrophages. Biology. 2015;**4**(4):881-906

[28] Thompson MR, Kaminski JJ, Kurt-Jones EA, Fitzgerald KA. Pattern recognition receptors and the innate immune response to viral infection. Viruses. 2011;**3**(6):920-940

[29] Feng J, Wickenhagen A, Turnbull ML, Rezelj VV, Kreher F, Tilston-Lunel NL, et al. Interferonstimulated gene (ISG)-expression screening reveals the specific antibunyaviral activity of ISG20. Jung JU, editor. Journal of Virology. 2018;**92**(13):e02140-e02117

[30] Wardley RC, Wilkinson PJ. The growth of virulent African swine fever virus in pig monocytes and macrophages. The Journal of General Virology. 1978;**38**(1):183-186

[31] Gazzolo L, Moscovici C, Moscovici MG. Persistence of avian oncoviruses in chicken macrophages. Infection and Immunity. 1979;**23**(2):294-297

[32] Burdo TH, Walker J, Williams KC. Macrophage polarization in AIDS: Dynamic Interface between anti-viral and anti-inflammatory macrophages during acute and chronic infection. Journal of Clinical and Cellular Immunology. 2015;**6**(3):333

[33] Ferrer MF, Thomas P, López Ortiz AO, Errasti AE, Charo N, Romanowski V, et al.

Junin virus triggers macrophage activation and modulates polarization according to viral strain pathogenicity. Frontiers in Immunology. 2019;**10**:2499

[34] Camini FC, da Silva Caetano CC, Almeida LT, de Brito Magalhães CL. Implications of oxidative stress on viral pathogenesis. Archives of Virology. 2017;**162**(4):907-917

[35] Arango Duque G, Descoteaux A. Macrophage cytokines: Involvement in immunity and infectious diseases. Frontiers in Immunology. 2014;**5**(491):491. Available from: http:// journal.frontiersin.org/article/10.3389/ fimmu.2014.00491/abstract

[36] Guerriero JL. Macrophages. In: International Review of Cell and Molecular Biology [Internet]. Elsevier; 2019. pp. 73-93. Available from: https:// linkinghub.elsevier.com/retrieve/pii/ S1937644818300698

[37] Zwirner NW, Domaica CI, Fuertes MB. Regulatory functions of NK cells during infections and cancer. Journal of Leukocyte Biology. 2021;**109**(1):185-194

[38] Kao YJ, Piedra PA, Larsen GL, Colasurdo GN. Induction and regulation of nitric oxide synthase in airway epithelial cells by respiratory syncytial virus. American Journal of Respiratory and Critical Care Medicine. 2001;**163**(2):532-539

[39] Santiago-Olivares C, Rivera-Toledo E, Gómez B. Nitric oxide production is downregulated during respiratory syncytial virus persistence by constitutive expression of arginase 1. Archives of Virology. 2019;**164**(9):2231-2241

[40] Granja AG, Sabina P, Salas ML, Fresno M, Revilla Y. Regulation of

inducible nitric oxide synthase expression by viral A238L-mediated inhibition of p65/RelA acetylation and p300 transactivation. Journal of Virology. 2006;**80**(21):10487-10496

[41] Odkhuu E, Komatsu T, Naiki Y, Koide N, Yokochi T. Sendai virus C protein inhibits lipopolysaccharideinduced nitric oxide production through impairing interferon-β signaling. International Immunopharmacology. 2014;**23**(1):267-272

[42] Bradley JH, Harrison A, Corey A, Gentry N, Gregg RK. Ebola virus secreted glycoprotein decreases the anti-viral immunity of macrophages in early inflammatory responses. Cellular Immunology. 2018;**324**:24-32

[43] Zhu W, Banadyga L, Emeterio K, Wong G, Qiu X. The roles of Ebola virus soluble glycoprotein in replication, pathogenesis, and countermeasure development. Viruses. 2019;**11**(11):999

[44] McElroy AK, Nichol ST. Rift Valley fever virus inhibits a pro-inflammatory response in experimentally infected human monocyte derived macrophages and a pro-inflammatory cytokine response may be associated with patient survival during natural infection. Virology. 2012;**422**(1):6-12

[45] Hurtado C, Bustos MJ, Granja AG, de León P, Sabina P, López-Viñas E, et al. The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens. Archives of Virology. 2011;**156**(2):219-234

[46] Rehm KE, Connor RF, Jones GJB, Yimbu K, Roper RL. Vaccinia virus A35R inhibits MHC class II antigen presentation. Virology. 2010;**397**(1):176-186

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

[47] Rehm KE, Connor RF, Jones GJB, Yimbu K, Mannie MD, Roper RL. Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II. Immunology. 2009;**128**(3):381-392

[48] Schaefer MR, Wonderlich ER, Roeth JF, Leonard JA, Collins KL. HIV-1 Nef targets MHC-I and CD4 for degradation via a final common β-COP–dependent pathway in T cells. Hope TJ, editor. PLoS Pathogens. 2008;**4**(8):e1000131

[49] Wilson HM. SOCS proteins in macrophage polarization and function. Frontiers in Immunology. 2014;**5**:357. Available from: http://journal.frontiersin. org/article/10.3389/fimmu.2014.00357/ abstract

[50] Kwon Y, Meyer K, Peng G, Chatterjee S, Hoft DF, Ray R. Hepatitis C virus E2 envelope glycoprotein induces an Immunoregulatory phenotype in macrophages. Hepatology. 2019;**69**(5):1873-1884

[51] Ouyang P, Rakus K, van Beurden SJ, Westphal AH, Davison AJ, Gatherer D, et al. IL-10 encoded by viruses: A remarkable example of independent acquisition of a cellular gene by viruses and its subsequent evolution in the viral genome. The Journal of General Virology. 2014;**95**(2):245-262

[52] Mezouar S, Mege JL. New tools for studying macrophage polarization: Application to bacterial infections. In: Prakash H, editor. Macrophages [Internet]. London, UK: IntechOpen; 2021. Available from: https://www. intechopen.com/books/macrophages/ new-tools-for-studying-macrophagepolarization-application-to-bacterialinfections

[53] Faure-Dupuy S, Delphin M, Aillot L, Dimier L, Lebossé F, Fresquet J, et al. Hepatitis B virus-induced modulation of liver macrophage function promotes hepatocyte infection. Journal of Hepatology. 2019;**71**(6):1086-1098

[54] Temel T, Cansu DÜ, Korkmaz C, Kaşifoğlu T, Özakyol A. The long-term effects of anti-TNF-α agents on patients with chronic viral hepatitis C and B infections. International Journal of Rheumatic Diseases. 2015;**18**(1):40-45

[55] Liang J, Long Z, Zhang Y, Wang J, Chen X, Liu X, et al. Chloride intercellular channel 3 (CLIC-3) suppression-mediated macrophage polarization: A potential indicator of poor prognosis of hepatitis B virus-related acute-on-chronic liver failure. Immunology and Cell Biology. 2022:imcb.12542

[56] Laursen TL, Wong GLH, Kazankov K, Sandahl T, Møller HJ, Hamilton-Dutoit S, et al. Soluble CD163 and mannose receptor associate with chronic hepatitis B activity and fibrosis and decline with treatment: sCD163 and sMR in chronic hepatitis B. Journal of Gastroenterology and Hepatology. 2018;**33**(2):484-491

[57] Bility MT, Cheng L, Zhang Z, Luan Y, Li F, Chi L, et al. Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: Induction of human-specific liver fibrosis and M2-like macrophages. Walker CM, editor. PLoS Pathogens. 2014;**10**(3):e1004032

[58] Scheel TKH, Rice CM. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nature Medicine. 2013;**19**(7):837-849

[59] Saha B, Kodys K, Szabo G. Hepatitis C virus-induced monocyte differentiation into polarized M2 macrophages promotes stellate cell activation via TGF-β. Cellular and Molecular Gastroenterology and Hepatology. 2016;**2**(3):302-316.e8

[60] Saha B, Kodys K, Adejumo A, Szabo G. Circulating and exosomepackaged hepatitis C singlestranded RNA induce monocyte differentiation via TLR7/8 to polarized macrophages and fibrocytes. Journal of immunology (Baltimore, Md. : 1950). 2017;**198**(5):1974-1984

[61] Hou Z, Zhang J, Han Q, Su C, Qu J, Xu D, et al. Hepatitis B virus inhibits intrinsic RIG-I and RIG-G immune signaling via inducing miR146a. Scientific Reports. 2016;**6**(1):26150

[62] Ahmed F, Ibrahim A, Cooper CL, Kumar A, Crawley AM. Chronic hepatitis C virus infection impairs M1 macrophage differentiation and contributes to CD8+ T-cell dysfunction. Cell. 2019;**8**(4):E374

[63] Park SH, Rehermann B. Immune responses to HCV and other hepatitis viruses. Immunity. 2014;**40**(1):13-24

[64] Herbein G, Varin A. The macrophage in HIV-1 infection: From activation to deactivation? Retrovirology. 2010;**7**(1):33

[65] Lafeuillade A, Tamalet C, Pellegrino P, Tourres C, Yahi N, Vignoli C, et al. High viral burden in lymph nodes during early stages of HIV-1 infection. AIDS (London, England). 1993;**7**(11):1527-1528

[66] Emilie D, Peuchmaur M, Maillot MC, Crevon MC, Brousse N, Delfraissy JF, et al. Production of interleukins in human immunodeficiency virus-1-replicating lymph nodes. The Journal of Clinical Investigation. 1990;**86**(1):148-159

[67] Porcheray F, Samah B, Léone C, Dereuddre-Bosquet N, Gras G. Macrophage activation and human immunodeficiency virus infection: HIV replication directs macrophages towards a pro-inflammatory phenotype while previous activation modulates macrophage susceptibility to infection and viral production. Virology. 2006;**349**(1):112-120

[68] Ogden CA, Pound JD, Batth BK, Owens S, Johannessen I, Wood K, et al. Enhanced apoptotic cell clearance capacity and B cell survival factor production by IL-10-activated macrophages: Implications for Burkitt's lymphoma. Journal of Immunology (Baltimore, Md.: 1950). 2005;**174**(5):3015-3023

[69] Sandanger Ø, Ryan L, Bohnhorst J, Iversen AC, Husebye H, Halaas Ø, et al. IL-10 enhances MD-2 and CD14 expression in monocytes and the proteins are increased and correlated in HIV-infected patients. Journal of Immunology (Baltimore, Md.: 1950). 2009;**182**(1):588-595

[70] Yearley JH, Pearson C, Shannon RP, Mansfield KG. Phenotypic variation in myocardial macrophage populations suggests a role for macrophage activation in SIV-associated cardiac disease. AIDS Research and Human Retroviruses. 2007;**23**(4):515-524

[71] Akiyama H, Jalloh S, Park S, Lei M, Mostoslavsky G, Gummuluru S. Expression of HIV-1 intron-containing RNA in microglia induces inflammatory responses. Journal of Virology. 2020;**95**(5):e01386-20

[72] Lv T, Cao W, Li T. HIV-related immune activation and inflammation: Current understanding and strategies. Journal of Immunology Research. 2021;**2021**:7316456

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

[73] Wu MF, Chen ST, Yang AH, Lin WW, Lin YL, Chen NJ, et al. CLEC5A is critical for dengue virus-induced inflammasome activation in human macrophages. Blood. 2013;**121**(1):95-106

[74] Rahman K, Desai C, Iyer SS, Thorn NE, Kumar P, Liu Y, et al. Loss of junctional adhesion molecule a promotes severe steatohepatitis in mice on a diet high in saturated fat, fructose, and cholesterol. Gastroenterology. 2016;**151**(4):733-746.e12

[75] Quicke KM, Bowen JR, Johnson EL, McDonald CE, Ma H, O'Neal JT, et al. Zika virus infects human placental macrophages. Cell Host & Microbe. 2016;**20**(1):83-90

[76] Wang L, Valderramos SG, Wu A, Ouyang S, Li C, Brasil P, et al. From mosquitos to humans: Genetic evolution of Zika virus. Cell Host & Microbe. 2016;**19**(5):561-565

[77] Miner JJ, Diamond MS. Zika virus pathogenesis and tissue tropism. Cell Host & Microbe. 2017;**21**(2):134-142

[78] Foo SS, Chen W, Chan Y, Bowman JW, Chang LC, Choi Y, et al. Asian Zika virus strains target CD14+ blood monocytes and induce M2-skewed immunosuppression during pregnancy. Nature Microbiology. 2017;**2**(11):1558-1570

[79] Baasch S, Ruzsics Z, Henneke P. Cytomegaloviruses and macrophagesfriends and foes from early on? Frontiers in Immunology. 2020;**11**:793

[80] Chan G, Bivins-Smith ER, Smith MS, Smith PM, Yurochko AD. Transcriptome analysis reveals human cytomegalovirus reprograms monocyte differentiation toward an M1 macrophage. Journal of Immunology (Baltimore, Md.: 1950). 2008;**181**(1):698-711

[81] Jenkins C, Abendroth A, Slobedman B. A novel viral transcript with homology to human interleukin-10 is expressed during latent human cytomegalovirus infection. Journal of Virology. 2004;**78**(3):1440-1447

[82] Naito Y, Takagi T, Higashimura Y. Heme oxygenase-1 and antiinflammatory M2 macrophages. Archives of Biochemistry and Biophysics. 2014;**564**:83-88

[83] Avdic S, Cao JZ, McSharry BP, Clancy LE, Brown R, Steain M, et al. Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses. Journal of Virology. 2013;**87**(18):10273-10282

[84] Cojohari O, Mahmud J, Altman AM, Peppenelli MA, Miller MJ, Chan GC. Human cytomegalovirus mediates unique monocyte-to-macrophage differentiation through the PI3K/ SHIP1/Akt signaling network. Viruses. 2020;**12**(6):E652

[85] Romo N, Magri G, Muntasell A, Heredia G, Baía D, Angulo A, et al. Natural killer cell-mediated response to human cytomegalovirus-infected macrophages is modulated by their functional polarization. Journal of Leukocyte Biology. 2011;**90**(4):717-726

[86] Bayer C, Varani S, Wang L, Walther P, Zhou S, Straschewski S, et al. Human cytomegalovirus infection of M1 and M2 macrophages triggers inflammation and autologous T-cell proliferation. Journal of Virology. 2013;**87**(1):67-79

[87] Poglitsch M, Weichhart T, Hecking M, Werzowa J, Katholnig K, Antlanger M, et al. CMV late phaseinduced mTOR activation is essential for efficient virus replication in

polarized human macrophages. American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2012;**12**(6): 1458-1468

[88] Vemula S, Zhao J, Liu J, Wang X, Biswas S, Hewlett I. Current approaches for diagnosis of influenza virus infections in humans. Viruses. 2016;**8**(4):96

[89] Roberts JMK, Simbiken N, Dale C, Armstrong J, Anderson DL. Tolerance of honey bees to Varroa mite in the absence of deformed wing virus. Viruses. 2020;**12**(5):575

[90] Halstead ES, Umstead TM, Davies ML, Kawasawa YI, Silveyra P, Howyrlak J, et al. GM-CSF overexpression after influenza a virus infection prevents mortality and moderates M1-like airway monocyte/ macrophage polarization. Respiratory Research. 2018;**19**(1):3

[91] Sun K, Metzger DW. Inhibition of pulmonary antibacterial defense by interferon-γ during recovery from influenza infection. Nature Medicine. 2008;**14**(5):558-564

[92] Geng P, Zhu H, Zhou W, Su C, Chen M, Huang C, et al. Baicalin inhibits influenza a virus infection via promotion of M1 macrophage polarization. Frontiers in Pharmacology. 2020;**11**:01298

[93] Cole SL, Dunning J, Kok WL, Benam KH, Benlahrech A, Repapi E, et al. M1-like monocytes are a major immunological determinant of severity in previously healthy adults with lifethreatening influenza. JCI Insight. 2017;**2**(7):e91868

[94] 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**(7821):463-469

[95] Arunachalam PS, Wimmers F, Mok CKP, Perera RAPM, Scott M, Hagan T, et al. Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans. Science. 2020;**369**(6508):1210-1220

[96] Fajgenbaum DC, June CH. Cytokine Storm. The New England Journal of Medicine. 2020;**383**(23):2255-2273

[97] Mehta P, McAuley DF, Brown M, Sanchez E, Tattersall RS, Manson JJ, et al. COVID-19: Consider cytokine storm syndromes and immunosuppression. Lancet (London, England). 2020; **395**(10229):1033-1034

[98] Yang D, Chu H, Hou Y, Chai Y, Shuai H, Lee ACY, et al. Attenuated interferon and proinflammatory response in SARS-CoV-2-infected human dendritic cells is associated with viral antagonism of STAT1 phosphorylation. The Journal of Infectious Diseases. 2020;**222**(5):734-745

[99] Boumaza A, Gay L, Mezouar S, Bestion E, Diallo AB, Michel M, et al. Monocytes and macrophages, targets of severe acute respiratory syndrome coronavirus 2: The clue for coronavirus disease 2019 immunoparalysis. The Journal of Infectious Diseases. 2021;**224**(3):395-406

[100] Zheng J, Wang Y, Li K, Meyerholz DK, Allamargot C, Perlman S. Severe acute respiratory syndrome coronavirus 2-induced immune activation and death of monocyte-derived human macrophages and dendritic cells. The Journal of Infectious Diseases. 2021;**223**(5): 785-795

*Macrophage Polarization in Viral Infectious Diseases: Confrontation with the Reality DOI: http://dx.doi.org/10.5772/intechopen.106083*

[101] Lv J, Wang Z, Qu Y, Zhu H, Zhu Q, Tong W, et al. Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages. Cell Discovery. 2021;**7**(1):24

[102] Liao M, Liu Y, Yuan J, Wen Y, Xu G, Zhao J, et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nature Medicine. 2020;**26**(6):842-844

[103] Channappanavar R, Fehr AR, Vijay R, Mack M, Zhao J, Meyerholz DK, et al. Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoV-infected mice. Cell Host & Microbe. 2016;**19**(2):181-193

[104] Meyerowitz EA, Vannier AGL, Friesen MGN, Schoenfeld S, Gelfand JA, Callahan MV, et al. Rethinking the role of hydroxychloroquine in the treatment of COVID-19. The FASEB Journal. 2020;**34**(5):6027-6037

[105] Chen D, Xie J, Fiskesund R, Dong W, Liang X, Lv J, et al. Chloroquine modulates antitumor immune response by resetting tumor-associated macrophages toward M1 phenotype. Nature Communications. 2018;**9**(1):873

[106] Vitte J, Michel M, Mezouar S, Diallo AB, Boumaza A, Mege JL, et al. Immune modulation as a therapeutic option during the SARS-CoV-2 outbreak: The case for antimalarial aminoquinolines. Frontiers in Immunology. 2020;**11**:2159

[107] Haydar D, Cory TJ, Birket SE, Murphy BS, Pennypacker KR, Sinai AP, et al. Azithromycin polarizes macrophages to an M2 phenotype via inhibition of the STAT1 and NF-κB signaling pathways. Journal of Immunology. 2019;**203**(4):1021-1030

[108] Melo GD, Lazarini F, Larrous F, Feige L, Kornobis E, Levallois S, et al. Attenuation of clinical and immunological outcomes during SARS-CoV-2 infection by ivermectin. EMBO Molecular Medicine. 2021;**13**(8). Available from: https:// onlinelibrary.wiley.com/doi/10.15252/ emmm.202114122

[109] Yin L, Zhao H, Zhang H, Li Y, Dong Y, Ju H, et al. Remdesivir alleviates acute kidney injury by inhibiting the activation of NLRP3 inflammasome. Frontiers in Immunology. 2021;**12**:652446

[110] Li R, Wang L. Baicalin inhibits influenza virus a replication via activation of type-I IFN signaling by reducing miR-146a. Molecular Medicine Reports. 2019. Available from: http://www.spandidos-publications. com/10.3892/mmr.2019.10743

[111] Napoli C, Benincasa G, Criscuolo C, Faenza M, Liberato C, Rusciano M. Immune reactivity during COVID-19: Implications for treatment. Immunology Letters. 2021;**231**:28-34

[112] Mahmudpour M, Roozbeh J, Keshavarz M, Farrokhi S, Nabipour I. COVID-19 cytokine storm: The anger of inflammation. Cytokine. 2020;**133**:155151

#### **Chapter 16**
