**2.1 Complement activation**

Inoculated *Leishmania* promastigotes rapidly interact with the host's complement system. All three complement pathways (alternative, classical and lectin) are involved to varying degrees in *Leishmania* parasite killing and result in the activation of complement (C) protein C3 convertase cleaving C3 to generate C3b (**Figure 2**; reviewed in [17]). C3b facilitates the deposition of the C5b-C9 membrane attack complex (MAC) onto the surface of culture-derived stationary phase *Leishmania* promastigotes (a stage predominately found in the sandfly midgut), resulting in lysis of the parasite and subsequent uptake by phagocytic cells [17, 18]. C3b also acts as an opsonin, promoting direct phagocytosis and destruction by immune cells. *In vitro* experiments demonstrated killing of up to 90% of culture-derived procyclics *Leishmania* promastigotes (including *L*. *donovani*, *L. amazonensis*, *L. infantum* and *L*. *major* species) via complement-mediated lysis within the first few minutes of serum contact [19]. The remaining resistant parasites used the surface bound C3b to enter immune cells and cause infection. Contrary to culture-derived procyclics promastigotes, metacyclic promastigotes (the infective stage that is deposited into the skin by blood-feeding phlebotomine sandflies) are able to subvert phagocytosis to promote their survival and mediate host pathogenesis [17–19]. The glycocalyx component, known as lipophosphoglycan (LPG), and metalloproteinase glycoprotein 63 (GP63), is distinct to the surface of the infective metacyclic promastigotes, preventing the formation of MAC and complement lysis by cleaving the C3b into an inactive form of C3b (iC3b) [18, 20, 21], thereby subverting the complement system. The MAC can also be physically inhibited by elongated LPG on the surface of metacyclic promastigotes [17]. Moreover, iC3b serves as an opsonin that facilitates the parasite's uptake by binding to complement receptor 1 (CR1) and CR3 on macrophages and neutrophils. Binding via CR3 inhibits the production of interleukin 12 (IL-12) and oxidative burst, which provides safe parasite entry into macrophages [22].

#### **2.2 Pattern recognition receptors on innate immune cells**

Pathogen recognition receptors (PRRs) expressed on innate immune cells are critical for recognizing invading pathogens via pathogen-associated molecular patterns (PAMPs) and initiating the host immune response (**Figure 3**). Toll-like receptors (TLRs) and Nod-like receptors (NLRs) are the most studied PRRs in leishmaniasis and play a dual role in promoting protection or resistance depending on the infecting *Leishmania* species, which receptor the parasite interacts with first and the model used [23–25].

Both TLR2 and TLR4 (extracellular receptors) are found on the surface of host macrophages and neutrophils and recognize *Leishmania* promastigote LPG and GP63 *Protective and Pathogenic Immune Responses to Cutaneous Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.101160*

#### **Figure 2.**

*Activation of complement by Leishmania parasites. (A) All three complement pathways are activated by the Leishmania parasite. (B) The alternative pathway is activated directly by the Leishmania parasite and is considered to be the main complement pathway involved in Leishmania clearance. The classical pathway is antibody-driven, while the lectin pathway is activated by the binding of mannose-binding lectin and ficolin on the parasite [16]. (C) Following activation of all pathways, the complement protein C3 convertase cleaves C3 to generate C3b. C3b facilitates the deposition of the C5b-C9 membrane attack complex (MAC) onto the surface of the Leishmania parasite, (D) ultimately resulting in uptake by neutrophils and macrophages following lysis of the parasite. (E) However, the lipophosphoglycan (LPG) metalloproteinase glycoprotein (GP63) on the parasite's surface inhibits MAC formation through its virulence factor, such as activating protein kinase and inducing interleukin-12 (IL-12) [16]. LPG and GP63 resist complement lysis by cleaving the C3b into inactive C3b (iC3b) to inhibit MAC convertase leading to safe entry into host cells and protection from complementmediated attack. Created with Biorender.*

[23–25] and *Leishmania* amastigote LPG (*L. major* specific) and proteophosphoglycan (PPG), which are expressed on the amastigote and promastigote surface [26, 27].

TLRs are activated and use the adaptor protein myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon-β (TRIF) for signal transduction. The MyD88 adapter was shown to be required for the clearance of *L. major* infection in C57BL/6 mice, with MyD88-null C57BL/6 mice showing a greater susceptibility to infection than WT mice [23]. Furthermore knocking out TLR2 in C57BL/6 mice [TLR2−/−] resulted in mice displaying higher resistance to *Leishmania* (*V*.) *braziliensis* infection compared to WT and this resistance was associated with increased enhanced IFN-γ production [24]. Similarly, C57BL/6 TLR2−/− mice infected with *Leishmania* (*L*.) *amazonensis* showed a reduced parasite burden compared to infected WT C57BL/6 mice [25]. It has been proposed that LPG on the surface of *Leishmania* promastigotes may explain why TLRs promote

#### **Figure 3.**

*Macrophage recognition of Leishmania parasites. Toll-like receptors (TLR) are categorized as extracellular receptors (TLR2 and TLR 4) and intracellular receptors (TLR3, TLR7, TLR8 and TLR9). TLRs are activated and use the adaptor proteins (myeloid differentiation primary response 88 (MyD88) or TIR-domaincontaining adapter-inducing interferon-*β *(TRIF)) for signal transduction, which is important for Leishmania clearance. TLR2, TLR4, TLR7, TLR8 and TLR9 use MyD88, TLR3 uses TRIF and TLR4 uses both MyD88 and TRIF. (A) On the macrophage surface, TLR2 and TLR4 recognize lipophosphoglycan (LPG) molecules found on the surface of Leishmania promastigotes and amastigotes (L. major). (B) Upon recognition of Leishmania, macrophages release cytokines and nitric oxide (NO) that promote either parasite death or survival. (C) TLR2 activation by LPG can also induce the release of suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3, which inhibits TLR4 signaling. (D) Complement receptors 1 (CR1) and CR3 are also categorized as extracellular receptors and can recognize LPG and metalloproteinase glycoprotein 63 (GP63) both expressed on the promastigote surface. (E) Fc receptors, located on the extracellular surface of macrophages, can also recognize immunoglobulin G (IgG) on the surface of amastigotes. (F) Intracellular TLRs recognize Leishmania RNA (TLR3, TLR7 and TLR8) and DNA (TLR 9). In the cytoplasm, (G) the NLRP3 inflammasome activates caspase-1, which cleaves pro-interleukin-1*β *(IL-1*β*) and pro-IL-18 to generate mature IL-1*β *and IL-18. Created with Biorender.com.*

both protection and resistance, as the density and diversity of surface polysaccharide extensions to the LPG molecules varies between *Leishmania* species and between their morphological stages [24]. Similarly, TLR4 has a dual role that depends on the time of stimulation [28]. When TLR4 on mouse macrophages is primed *in vitro* with interferon-γ (IFNγ) prior to *L. major* infection, host protective TNF-α and NO are induced, promoting parasite killing. However, when IFNγ is added at the time of infection without sufficient priming time, macrophages increase IL-10 production, favoring parasite persistence [28, 29]. Interestingly, *ex vivo* studies using human monocytes from CL patients revealed that infection with *L. braziliensis* up-regulated TLR2 and TLR4 expression on inflammatory monocytes subsets [30, 31]. Moreover, a correlation with detrimental outcomes of CL was linked to the TLR up-regulation and production of TNF-α and IL-10 in infected monocytes [31]. These results using monocytes from human CL patients infected with *L. braziliensis* suggest that TLR2 and TLR4 expression triggers an inflammatory response and pathology.

TLR3, TLR7 and TLR9 are intracellular receptors recognizing *Leishmania* parasites in the endosomes of macrophages and are activated by *Leishmania* nucleic acids [17]. TLR9 is the most studied intracellular receptor and is associated with disease

*Protective and Pathogenic Immune Responses to Cutaneous Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.101160*

outcome having an important role in the early events of lesion development and parasite burden. A direct correlation was seen between TLR9 expression and lesion size in mice infected with *L. braziliensis* [32, 33]. Similarly *ex vivo* human monocytes from CL patients presenting with larger lesion size, were found to express higher levels of TLR9 [33]. Little is still known about the role of TLR3 in CL. TLR3 promotes immune protection against *L*. (*Leishmania*) *donovani* (visceral *Leishmania* species) through the production of TNF-α and NO [34]. Recent studies identified TLR7 as having an essential role in early *L. major* infection control by neutrophils. In TLR7− / − C57BL/6 mice infection with *L. major* leads to long-term exacerbation of CL [35].

In contrast to TLRs, NLRs are cytoplasmic pattern recognition receptors. The NLRP3 inflammasome is a major regulator of IL-1β and IL-18 in *Leishmania* infection [36]. Similar to TLRs, the involvement and role of NLRs is dependent on the infecting *Leishmania* species. In murine models, activation of the inflammasome and IL-1β production have been shown to be associated with a protective role in parasite control during infection with *L. amazonensis* and *L. braziliensis* [37–39]. In contrast, they have no involvement in resistance to *L. major* infection. Moreover, the NLRP3 inflammasome promotes the development of TH2 cells resulting in nonhealing lesions during *L. major* infection in BALB/c mice [40].

### **2.3 Innate cellular immunity**

The recruitment and activation of innate immune cells are critical for the killing of invading pathogens by phagocytosis. However, these cells can also facilitate the survival of *Leishmania* parasites (**Figure 1**). *Leishmania* has evolved mechanisms to subvert host killing by modulating the response of specific immune cells. Macrophages and monocytes are the primary host cell for *Leishmania* parasites; however, a variety of immune cells are recruited to the inoculation site and play critical roles in determining the course of infection and disease outcome.

### *2.3.1 Neutrophils*

Neutrophils are the first phagocytic cells to arrive at the site of the phlebotomine sandfly bite [41]. These cells are capable of clearing *Leishmania* parasites early in infection through phagocytosis and via the production of an array of microbicidal factors that target *Leishmania* parasites (recently reviewed in [42]). Neutrophils release neutrophil extracellular traps (NETs) to capture and kill *Leishmania* promastigotes through a cell death mechanism (NETosis) [43]. Infected neutrophils degranulate and secrete inflammatory mediators, such as the chemokine macrophage inflammatory protein 1β (MIP-1β) and CC-chemokine ligend-3 (CCL3), aiding in the migration of macrophages, and recruitment of monocytes and dendritic cells [44, 45]. Under normal circumstances, compromised neutrophils undergo spontaneous apoptosis, however prevention of neutrophil apoptosis is an important mechanism that *Leishmania* uses to subvert death [41, 44]. For example, infected apoptotic neutrophils can act as silent vectors by providing a safe entry for *Leishmania* promastigotes into macrophages without triggering mechanisms to kill *Leishmania* [44, 46]. This silent entry into macrophages has been likened to the Trojan horse scenario [41, 47], as the promastigotes suppress neutrophil apoptosis until macrophages arrive at the site of infection and then downregulate the microbicidal responses (ROS and NO) [44, 48]. Infected neutrophils are engulfed by macrophages allowing promastigotes to transform into amastigotes and proliferate. *L. major* is able to delay neutrophil apoptosis for up to two days by inducing the secretion of the anti-apoptotic cytokines IL-8 and granulocyte macrophage colonystimulating factor (GM-CSF) [48]. Infected neutrophils undergoing apoptosis have

also been reported to release higher levels of MIP-1β to attract macrophages to the site of infection thereby ensuring a safe entry for the parasite [44].

The ability of neutrophils to promote parasite killing or parasite survival [35, 49] appears to be *Leishmania* species-specific, impacted by the route of infection [35, 50], and influenced by the genetic background of the host [41, 44, 49, 51–53]. Studies investigating the role of neutrophils in the development of CL utilized two mouse models namely the susceptible (BALB/c) and resistant (C57BL/6) mice and found differences in the number of neutrophils recruited at the site of *L. major* inoculation. Interestingly, only lesions of susceptible mice demonstrated a sustained presence of neutrophils and this was associated with early IL-4 activation and the development of aTH2 response [51]. These observations suggest that in susceptible BALB/c mice the early events of the immune response are important in initiating a subsequent TH differentiation following infection with *L. major*.

*In vitro* studies with human neutrophils suggest that they play either protective or pathogenic roles depending on the infecting *Leishmania* species. A study comparing neutrophils from CL and healthy subjects, which were then infected with *L. braziliensis ex vivo*, observed that neutrophils from CL patients produced more ROS and higher levels of the chemokines CXCL8 and CXCL9 which are both associated with the recruitment of neutrophils and TH1-type cells [54]. Neutrophils from both groups were equally competent to phagocytose *L. braziliensis*, however the cells from CL patients exhibited a pro-inflammatory profile necessary for parasite clearance [54]. The protective role of neutrophils depends on the infecting *Leishmania* species. *In vitro* infection of human neutrophils with *L. amazonensis* resulted in neutrophil production of ROS and leukotriene B4 (an inflammatory mediator) leading to neutrophil degranulation and the killing of *L. amazonensis* [55, 56]. In contrast, human neutrophils infected with *L. major* have been shown to contribute to pathogenesis through the secretion of high levels of MIP-1β, which attracts macrophages to the site of infection. These macrophages then engulf apoptotic infected-neutrophils, thereby providing a silent and safe parasite transmission into macrophages [44].

#### *2.3.2 Macrophages and monocytes*

Macrophages and monocytes are recruited to the inoculation site by degranulating, infected neutrophils releasing inflammatory mediators, such as MIP-1β and CCL2 [44, 57]. These cells become infected either by phagocytosing apoptotic *Leishmania*infected neutrophils, by free *Leishmania* promastigotes that have escaped neutrophils, or by amastigotes that have previously ruptured their host cell [41]. Cells of the monocyte lineage are the main host cells of *Leishmania* parasites and once inside, *Leishmania* promastigotes differentiate into amastigotes, where they survive and replicate.

Both macrophages and monocytes are efficient in controlling *Leishmania* in the early stages of infection (reviewed in [3]). During phagocytosis, these cells release ROS, through a mechanism known as the respiratory burst, which kills *Leishmania* rapidly leading to early parasite control [30]. These cells also produce NO, which is generated by inducible NO synthase (iNOS) [58]. NO diffuses across cell membranes to initiate parasite killing within both the NO-producing cells and bystander cells [58]. For macrophages to release ROS that is sufficient in parasite killing, the cells need to first be activated by IFNγ and TNF-α, which enhance the respiratory burst [59]. Though nonactivated macrophages will still release ROS through the respiratory burst following infection, it is insufficient to kill *Leishmania*. In a mouse model, the respiratory burst and subsequent release of ROS that occurs in *Leishmania*-infected macrophages were found to be insufficient to kill the parasites if the host cell was not previously activated by IFNγ [59]. During infection, the main producers of IFNγ are CD4+ TH1 cells. Prior to the differentiation and activation of CD4+ TH1 cells, natural killer (NK) cells are the

#### *Protective and Pathogenic Immune Responses to Cutaneous Leishmaniasis DOI: http://dx.doi.org/10.5772/intechopen.101160*

primary producers of IFNγ [60]. In contrast, *in vitro studies* with human and mouse monocytes infected with *Leishmania* species showed competence in parasite killing through the secretion of ROS and without the need for prior activation [30, 47, 59].

The majority of studies investigating the role of NO have used rodent models, where NO is considered necessary to control *Leishmania* [58, 61, 62], however it is not yet clear if NO is required for *Leishmania* control in humans as activated human macrophages have not been shown to produce NO upon *Leishmania* infection [59, 63], It has been suggested that inhibiting NO promotes *Leishmania* infection in phagocytes [63]. Similar, the exact role of ROS in human *Leishmania* infection is yet to be elucidated, although it is believed that the production of ROS is an important mechanism in eradicating *Leishmania* parasites throughout the course of disease [59].
