Paradoxical immune response in leishmaniasis: The role of toll‐like receptors in disease progression

Abstract Toll‐like receptors (TLRs), members of pattern recognition receptors, are expressed on many cells of the innate immune system, and their engagements with antigens regulate specific immune responses. TLRs signalling influences species‐specific immune responses during Leishmania infection; thus, TLRs play a decisive role towards elimination or exacerbation of Leishmania infection. To date, there is no single therapeutic or prophylactic approach that is fully effective against leishmaniasis. An in‐depth understanding of the mechanisms by which Leishmania species evade, or exploit host immune machinery could lead to the development of novel therapeutic approaches for the prevention and management of leishmaniasis. In this review, the role of TLRs in the induction of a paradoxical immune response in leishmaniasis was discussed. This review focuses on highlighting the novel interplay of TLR2‐ /TLR9‐driven resistance or susceptibility to 5 clinically important Leishmania species in human. The activation of TLR2/TLR9 can induce diverse anti‐Leishmania activities depending on the species of infecting Leishmania parasite. Infection with L. infantum and L. mexicana initiates TLR2/9 activation leading to host protective immune response, while infection with L. major, L. donovani and L. amazonensis trigger either a TLR2‐ /9‐related protective or non‐protective immune responses. These findings suggest that TLR2 and TLR9 are targets worth pursuing either for modulation or blockage to trigger host protective immune response towards leishmaniasis.

fourth most prevalent tropical infections and ranked second by mortality rate. 6 The disease is endemic to 98 countries affecting about 12 million people. A total of 350 million people are at the risk of infection with an approximate annual incidence of 2 million. 3,6,7 The functional role of the human immune system is to orchestrate a quick and effective response to danger or infection induced by a pathogen, including bacteria, fungi, parasites and viruses. 8 The innate immune system constitutes a non-specific response to pathogens, while the adaptive immune cells provide late but highly specific response to antigens. 9 Among the cells of the innate immune system involved in Leishmania infection are macrophages, neutrophils, dendritic cells, mast cells, basophils, eosinophils and natural killer cells, while the adaptive immune system is made up of T and B lymphocytes. Neutrophils, macrophages and dendritic cells are the most important functional cells of the innate immunity producing ranges of cytokines such as IFNγ, IL-12 and TNFα. 10,11 Leishmaniasis progression depends on efficient proliferation of the parasites intracellularly in the mammalian host. This proliferation is determined by the type and potency of immune responses which can either interfere with or enhance the establishment of leishmaniasis. Thus, the Leishmania-host interactions present a complex paradoxical relationship. The innate immune system uses pattern recognition receptors (PRR) such as toll-like receptors (TLRs), macrophage mannose receptors (MMR), NOD-like receptors (NLR) expressed on antigen-presenting cells (APC) for initial recognition of parasites pathogen-associated molecular patterns (PAMP). 12 Of these PRR, TLRs are first receptors to recognize Leishmaniaassociated PAMPs. 12 PRR signalling initiates several innate immune responses such as the activation of complement cascades, inducing phagocytosis, as well as the production of pro-inflammatory cytokines. 12 In response to host protective immune response, pathogens have developed numerous strategies to conquer the immune machinery. 13 Evasion of innate immunity by Leishmania parasites is a critical step in their survival. The ability to avoid or suppress anti-microbicidal factors produced by innate immune cells is a major evasion strategy employed by Leishmania. Further, Leishmania intracellular promastigotes adopt an adaptive lifestyle that helps them survive in host cells by remodelling the phagosomal compartment and interfering with signalling pathways that mediate parasitic clearance. 13 Additionally, Leishmania parasites survive in host cells by interfering with toll-like receptors signalling pathways which either disrupts immune homeostasis or renders immune cells inactive. 14 Therefore, this review summarizes the paradoxical interaction that exists between host innate immune machinery and

| Neutrophil-leishmania interaction
Neutrophils constitute the first line of immune cells to be deployed within the first few hours to the site of Leishmania infection. 15 Their early recruitment is pivotal to early containment of infection. 15 Neutrophils facilitate immune response through the modulation of several activities, including the engulfing of Leishmania promastigotes, the production of several arrays of antimicrobial factors such as neutrophil extracellular traps (NETs), lytic enzymes, 16 reactive oxygen species (ROS) 17 and differential cytokine production. 18,19 Orchestrated neutrophil immune responses to leishmania infection are modulated by TLRs. Studies have shown that TLRs mediate the early/appropriate recruitment of neutrophils to the site of infection, as well as neutrophils activation and their apoptosis. 20,21 It is worth noting that neutrophil involvement is not limited to the promastigote-mediated phase of infection but extends well into the later phase of infection. 22 Second-wave deployment of neutrophils in L. major-infected C57BL/6 (resistant) mice has been observed 7 days post-infection. 23  amazonensis, the internalization of promastigotes is comparatively more efficient, resulting in TNFα-mediated parasite clearance accompanied by the production of pro-inflammatory cytokines such as IL-12. 15,22 . Nevertheless, besides the protective role of neutrophils against leishmania infection, neutrophils can serve as a Trojan horse transiently spreading infective promastigotes or amastigotes to macrophages. 25 This is achieved by the recruitment of neutrophils to site of infection without activating their lethal antimicrobial factors, uptake of apoptotic cells and hijacking the tendency of early death of neutrophils for recruitment of macrophages. 25 For instance, L.
infantum activated neutrophils migration as well as intracellular effector mechanisms, thereby inducing uptake of promastigotes.
However, minimal release of neutrophil extracellular traps allows the survival of some intracellular promastigotes with active proliferative capacity. 26 Similarly, L. mexicana amastigotes were rapidly internalized by neutrophils; nevertheless, parasitic uptake was relatively silent resulting in death of few parasites. This occurred because L. mexicana amastigote did not trigger ROS production but induces high expression of CD62L which inactivates neutrophilic immune response. 27 This hypothesized the role of neutrophils as a Trojan horse which has been observed in several experimental models involving different species of Leishmania parasite (reviewed in Table 1).

| Macrophage-leishmania interaction
Upon successful infiltration of neutrophils by Leishmania parasite, macrophages provide the next line of defence for the host, by inducing secretion of pro-inflammatory cytokines (IL-1, IL-6, IL-12 and TNF) and nitric oxides. 33 Once they are recruited, free parasites and infected PMNs are phagocytosed; hence, macrophages become the decisive host cells for parasitic persistence and infection establishment as majorly of leishmania parasites differentiate into intracellular infective form (amastigotes) in macrophages. 34 Further, ingestion of promastigotes by macrophages is a process mediated by several receptors including toll-like receptors (TLR), complement receptors (CR), kinases and transcription factors. 33,35 Many of these mediators might negatively impact innate immunity  44 While by its ability to impair the recruitment of synaptotagmin V, an endosomal protein crucial to phagocytosis, LPG reduces phagocytic capacity of host membrane. 45 Nevertheless, Leishmania host surface receptors are recognized by pathogen recognition receptors, especially toll-like receptors to induce innate immune response. For example, toll-like receptors on macrophages recognize LPG of L. infantum and L. braziliensis, thereby inducing the production of nitric oxide (NO). 46 From the above evidences, it is safe to conclude that macrophage-Leishmania interaction also presents a paradoxical interaction.
Hence, the ability of macrophages to elicit either protective or non-protective host immune response to Leishmania infection depends on the signalling cascade expressed during the active stage of infection. Table 2 below gives a summary of experimental reports of some signalling cascade involved in Leishmaniamacrophages interactions.

| Dendritic cell-leishmania interaction
Activation and maturation of DC are triggered after recognition of danger signals called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRR) such as toll-like receptor on DC (TLRs), C-type lectin, simultaneously, concomitantly TA B L E 1 Neutrophil Trojan horse mechanisms during Leishmania Infection Interaction Outcome

Leishmania major
Parasite's promastigotes mimic apoptotic cells by expressing phosphatidylserine This leads to intracellular survival of parasites via PMN inducing the production of TGFβ while downregulating the production of TNFα 28 Upregulating the release of leukotriene B4 and decreasing the production of lipoxin A4 by neutrophils Modulating recruitment of anti-inflammatory lipid mediators such as leukotriene B4 (LTB 4 ) and lipoxin A4 LXA 4 )favouring parasite persistence 29

Leishmania mexicana
Early recruitment of neutrophils to site of infection in infected C57BL/6 mice Ingestion of parasites and formation of NETs; however L. mexicana exploits the early recruitment to block the induction of a protective immune response by impairing recruitment of monocytes and dendritic cells using neutrophils as a safe transient shelter. This contributes to the development of chronic lesions 30 Amastigotes internalization with silenced parasitic uptake by neutrophils Minimal killing of parasite resulting in persistence replication of amastigotes 27

Leishmania amazonensis
Hydrolysis of NETs DNA framework by parasitic enzyme 3'NT/NU Evasion of NETs favours progression of infection 31

Leishmania donovani
Ingestion of promastigotes by lysosome-independent compartment of neutrophils   55 maturation, consequently hindering the production of IL-12, TNFα and γ, thereby presenting Leishmania parasites an escape mechanism dependently or independently of IL-10 production. 60,61 Also, L. amazonensis impairs the activation and maturation of DC through the activation of adenosine A 2B , increasing the production of cAMP and phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2). 61  The observed reduced pathology of leishmanization in these mice models was due to efficient activation of DC and macrophages along with a significant production of pro-inflammatory cytokines.
Resistant to infection confers on conventional susceptible BALB/c mice illustrates the importance of TLR2 in effective clearance of L.
major parasite clearance.
TLR2 and TLR4 are crucial receptors to initiation host defences against L. major infection; however, TLR2 is more expressed on the macrophages of patients with self-healing lesion than those with non-healing lesion when compared to TLR4 expression. 68 Since TLR2 signalling is dependent on MyD88 adaptor protein, MyD88-/-mice were found to be more susceptible to L. major infection marked with larger lesions when compared to WT mice. 69 However, the mechanism of susceptibility is dependent on parasite strains, while MyD88-/-C57BL/6 mice infected with L. major IR75 strains show an increased susceptibility to infection as a consequence of non-protective TH2 response. MyD88-/-C57BL/6 infected with L.
major LV39 strains susceptible to infection is due to impaired Th1 response. 70 It is worthy to note that, despite the ability of TLR2 to form functional heterodimers with other TLRs, TLR2 plays the functional role against L. major independently of TLR1 and TLR6 (potential dimers) ( Figure 1). Halliday et al. 66 observed that TLR1 and TLR6 deficiency have no effect on disease kinetics of L. major infection. 66 In total, evidences from the experimental studies described

| Role of TLR2 in L. mexicana infection
Evidence has surfaced that TLR2-/-mice not TLR1-/-and TLR6-/mice are more vulnerable to L. mexicana infection, a vulnerability which is due to elevated production of anti-inflammatory cytokines such as IL-4, IL-10 and IL-13 by leukocytes in draining regional lymph nodes. 66

| Role of TLR2 in L. infantum infection
The extracellular expression of TLR2 for the production of adequate and efficient cytokines required for clearance L. infantum in canine monocyte-derived macrophages is non-negligible. 77 In the study of Similarly, TLR2 expression was found to be upregulated in blood samples of L. infantum-infected dogs as compared to healthy ones.
And a consequential reduction in the expression of TLR2 in the blood of the sample dogs after treatment with anti-leishmanial agent. 78  It has been elucidated that L. infantum SIR2RP1 (silent information regulator protein 1) protein modulation of B cells and induction of DC maturation to produce TNFα and IL-12 is dependent on TLR2 signalling. 80 However, emerging evidence implicates an association between TLR2 and TLR4 in coordinating innate immune response against visceral leishmaniasis. TLR2 and TLR4 are highly expressed on lymphocytes and monocytes of all patients with active VL in the study of Gatto et al. 81 The expression of these receptors correlates with high production of TNFα, IL-10 and NO before treatment with anti-leishmanial drugs. Furthermore, TLR2-4 expression persists after successful treatment with anti-leishmanial drugs, this expression is accompanied by production of TNFα and NO.
Observations and TH2 response homeostasis. 83 No wonder, pre-treatment of L.
donovani infected macrophages with Arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-dependent immunoprophylactic shifts the TH1/TH2 imbalance response towards protective TH1 via the upregulation of IL-12 production and reduction in IL-10 production. 84 Ara-LAM has also restored impaired splenic CD8 + T cells proliferation in L. donovani infected BALB/c mice and improved IFNγ responsiveness to infection. 85 Similarly, the study of Chowdhury et al. 86  It can be argued that the contrasting reports from the studies above are due to differences in experimental set-up of these studies.
Moreover, the study of Guerra et al. 90  which plays role in initiating protective anti-parasite responses. 93,94 In subsequent subsection, the crosstalk between TLR9 and different Leishmania species will be highlighted.

| Role of TLR9 in L. major infection
To investigate the role of TLR9 in innate immune response against L. major infection, Liese et al. 94  susceptible to this infection. This is due to aberrant TH2 response resulting in low production of IL-12 while increasing the production of IL-10 at draining LN as opposed to TH1 immune response in WT mice. 95 Further, in vitro stimulation of BMDCs of TLR9-/-mice with L. major did not upregulate CD40 and CD80 resulting in failed generation IL-12, IL-6 and IFNβ. This report agrees with that of Liese et al. 94 ; however, they observed TLR9 deficiency did not prevent ultimate resolution of infection. This suggests that, although TLR9 signalling contributes to the maturation of dendritic cells, activation of NK cells and production of pro-inflammatory cytokines for early parasitic clearance, its role is dispensable for a protective T cell response. Similarly, DC activation leading to the production of IL-12 in response to L. infantum infection requires TLR9 signalling. 94 The authors also reported that NK cell activation for the production of IFNγ is dependent on TLR9 signalling. Their hypothesis was confirmed by a comparative study on infected mice models. Leishmanization of WT mice was rapidly followed by NK cell activation in the spleen with induction of IFNγ production. In

| Role of TLR9 in L. amazonensis infection
There is no significant difference in the infectivity of L. amazonensis