Leishmania infection in humans is usually subclinical and parasites may persist for life-time of the host through several escape mechanisms (14). For example, Leishmania blocks the maturation of complement system and C₅–C₉ membrane attacking complex formation, reduces the expression of B7 and CD40 that are required for T-cell anti-parasitic activity, promote overexpression of the iron transporters, modifies the toll-like receptor (TLR)-2/TLR-4 signaling and inhibits Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway in macrophages (MΦs) thereby turnoff the cytokine cascade, and alters the expression profile of cytokines and chemokines etc. It is clear that Leishmania parasites manipulate several key aspects of host defense for their survival. Consequently, targeting immune components is a reliable method to combat the disease. In addition, host innate immune signatures that are specific to Leishmania infection could help early prediction of the disease outcome. These include aspects of innate immune response, such as front-line defense led by the natural killer (NK) cells, mononuclear and polymorphonuclear phagocytes (15). In general, Leishmania parasite resists their uptake by phagocytic dendritic cells (DCs) and MΦs (16, 17) by inhibiting reactive oxygen species (ROS) production that delays phagolysosome formation (18) and blocks lysosomal proteolytic degradation (19). The complement protein C3b, a potent immune opsonin accelerates phagocytosis of Leishmania (17) by interacting with the parasite surface glycoprotein gp63 (20). MΦs and DCs that engulfed Leishmania activate their TLR-9 signaling and produce interleukin (IL)-12, which stimulates NK cells to produce interferon (IFN)-γ, a key cytokine that is responsible for skweing Th1 response (16, 21) and stimulate the MΦs to produce ROS and nitric oxide (NO) for oxidative killing of intracellular amastigotes thereby protects the host (22–25). To establish an early infection, L. major inhibits the NK cell proliferation and IFN-γ production (26) and L. donovani evades inducible nitric oxide synthase (iNOS)-dependent killing of intracellular amastigotes in MΦs via downregulation of iNOS mRNA expression (27, 28) and induction of arginase expression (29) as the arginine is a common substrate for both iNOS and arginase enzymes. Thus, MΦs play a complex role in Leishmania pathogenesis and are associated with both survival and death of the parasites (30). Further, the induction of FasL-mediated apoptosis in Leishmania infected MΦs is a part of host defense mechanisms in innate immunity (31). Similarly, other immune cells also play a key part in the early host defense against leishmaniasis. For example, a drastic reduction in IL-8 and eotaxin secretion from neutrophils and eosinophils, respectively (32), and an elevated number of IL-4⁺ neutrophils and IL-10⁺ eosinophils and reduced number of IFN-γ⁺ and IL-12⁺ eosinophils are observed in active VL patients (33).

While the host innate immune response against leishmaniasis is important, it is now clear that the T-cell mediated immunity and the cytokines produced from various immune cells play a crucial role in determining the disease outcome (shown in Figure 1). However, the cytokines function in autocrine (locally) and paracrine (at a distance from the site of synthesis) fashion to regulate the immune response (34). A longitudinal study on Leishmania pathogenesis and disease recovery highlighted the role of helper T (Th)-cell responses (35). Therefore, immune cells and their cytokines have been recognized as potential targets for immunotherapy to modulate the activity of factors that are crucial in the immune system for healing. In this context, the phenomenon called “Th1-Th2 dichotomy” became popular based on the role of the cytokines produced by these cells in disease progression and/or host protection. Mosmann et al. reported for the first time that the cloned murine Th-cells are in two functional subsets namely Th1 and Th2 based on the production of IFN-γ and IL-4, respectively (36). Thereafter, several studies demonstrated the key role of major cytokines [e.g., IL-10, Transforming growth factor (TGF)-β, IL-4, IL-6, IL-12, and IFN-γ] that implicated the role of Th1/Th2 balance in disease progression or host protection. In general, Th1 type response mediates host resistance and Th2 type response associates with disease progression (37). In resistant mouse strains, the abundance of Th1 type cytokines; IFN-γ, IL-2, and lymphotoxin spontaneously cleared the L. major infection, whereas, in susceptible mouse strains, infection led to the fatal disease by the action of Th2 type cytokines; IL-4, IL-5 and IL-10 (38). IL-4 and IL-10 associated with the visceralization of cutaneous L. major infection (39, 40). However, the Th1-Th2 dichotomy is more complex than previously recognized, which is more evident in certain cases of leishmaniasis (41), such as L. donovani infection where the susceptibility of mouse strains is variable (42). Unlike in cutaneous leishmaniasis (CL), T-cells with Th2 phenotype are difficult to demonstrate in the mouse model of VL (37, 40–43). Similarly, the association between Th1 response and disease resistance to VL is complex in humans (44, 45). Occasionally, individuals respond to the exposure of Leishmania antigens via T-cells even they have no prior exposure to the parasite; this is possible due to the cross-activity by other microorganisms (46).

Immune response in VL patients is characterized by abundant anti-leishmanial antibody titers and low or absence of Leishmania-specific T-cell proliferation and IL-2 and IFN-γ production. Recovery from VL is mostly dependent on the induction of T-cell immunity; preferably Th1 response, which is primed by IL-12⁺ DCs and MΦs (47, 48). IFN-γ produced from IL-12 primed T-cells induce NO-mediated killing of the parasites (49, 50). In contrast, VL progression in humans is associated with abundant production of Th2 type cytokines IL-10, TGF-β, and IL-4 or presence of IL-10⁺ regulatory T cells (Tregs), which diminish the anti-parasitic activity of M1-type MΦs and Th1 response (51–53). However, the presence of abundant IL-10 is crucial rather than a lack of IFN-γ in the VL clinical disease progression (54). IL-10 partially inhibits IFN-γ production but strongly resists IFN-γ mediated activation of MΦs while killing the intracellular parasites (55–57). Likewise, the lack of IFN-γ may result in relatively higher levels of IL-10 in human leishmaniasis resulting in MΦ deactivation (58) and parasite proliferation (59). Murine model of VL demonstrates higher disease susceptibility due to the presence of high IL-10 levels during initial phase of infection (60). The splenic infection of L. donovani causes a constitutive expression of chemokine ligand 2 (CCL2) or monocyte chemoattractant protein 1 (MCP-1), which triggers IL-4 secretion from Th2 cells that activates the MΦs in alternative manner. These M2-type MΦs express arginase in abundant quantity and help in the biosynthesis of polyamines, which favor the survival and growth of the parasite (61). During chronic VL, the high expression of programmeded death protien-1 (PD-1) or Cytotoxic T-lymphocyte Antigen 4 (CTLA-4) causes unresponsiveness in CD4⁺ T-cell, which produce TGF-β in abundant levels and helps in persistence of infection (62). Taken together, there is mounting evidence suggesting that Th1-Th2 imbalance and T-cell unresponsiveness are critical issues in VL pathogenesis.

Role of a whole host cytokines in the resistance and disease progression during VL is increasingly being uncovered. Till date targeting either Th1 or Th2 cytokines produced promising results for leishmaniasis cure. Th1/Th2 balance is not the only determinant of the outcome of leishmaniasis as previously thought because a range of other cytokines have recently been implicated in both disease progression and host protection (Figure 2). Hence, there is a need for in-depth analysis of the role of cytokines and Leishmania pathogenesis to get a comprehensive view of the complex interplay of Leishmania parasite and their hosts. This review aims to summarize and critically analyze the state-of-the-art knowledge relating to cytokines and VL pathogenesis. Special emphasis has been made for the identification of potential cytokine targets that could be used for the development of novel diagnostic assays and immunotherapies for the detection and treatment of VL.

IL-10 is an 18 kDa pleiotropic cytokine, primarily produced by alternatively activated MΦs, DCs, and lymphocytes. As an immunoregulatory cytokine, IL-10 exerts multiple biological effects on different cell types (205). IL-10 is the product of Th2 subset, also known as cytokine synthesis inhibitory factor (CSIF) since it suppresses IFN-γ production from Th1 cells (112). IL-10 is known to inhibit production of cytokines like IL-1, IL-6, IL-12, and tumor necrosis factor (TNF)-α. In addition, Il-10 also inhibits MΦ mediated activation of T-cell through the reduced expression of class II major histocompatibility complex (MHC) and co-stimulatory molecules on the surface of MΦ and results in the inhibition of both innate and T-cell mediated immunity (188). The suppressive role of IL-10 in human VL results in the drastic fall in accumulation of monocyte derived macrophages, which is regulated by migration inhibitory factor (MIF). Further, IL-10 plays a substantial role in the pathogenesis of leishmaniasis by causing the downregulation of Th1 response, MΦ activation (114) and antigen presentation by DCs (58). Furthermore, IL-10 inhibits the leishmanicidal functions of MΦ (206) by diminishing the production of reactive nitrogen intermediates by MΦ, IFN-γ by T and natural killer (NK) cells (115), and IL-12 mediated activation of MΦ (48). High levels of IL-10 during the initial phases of infection due to decreased multifunctional CD4 T cells results in higher susceptibility to VL. Despite elevated levels of IFN-γ during the steady state of infection, parasite burden is not reduced due to higher levels of IL-10 (60). The unfavorable clinical outcome in localized CL was correlated with IL-10 but not with inadequate Th1 response (207). High levels of serum IL-10 is associated with symptomatic VL but absent in asymptomatic individuals. A key function of IL-10 is to protect the tissues from collateral damage due to excessive inflammation (116). However, in the face of parasitic infection an acute inflammatory response is necessary to control the parasite proliferation, hence, the anti-inflammatory role of IL-10 may help the disease progression (117). During active VL, CD8⁺ T-cells could also play an important role in disease progression via abundant production of IL-10 (208). However, the role of IL-10 in VL appears to be species-specific as it was suggested that IL-10 may not be a regulatory cytokine in canine VL. In experimental CL, a group of Treg cells namely, CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25⁻Foxp3⁻ are possible source of IL-10 (209). In contrast, IL-10 in human VL is not produced from thymic Foxp3 Tregs; rather they are produced from IFN-γ co-producing CD4⁺ T cells which are called type 1 regulatory (Tr1) cells (143). The role of Tregs was elucidated in modulating both Th1 (210, 211) and Th2 (210, 212) activity during murine L. major infection.

TGF-β is a 28 kDa homodimer and a potent anti-inflammatory cytokine produced by antigen-activated T-cells and mononuclear phagocytes (75). TGF-β has potent immunosuppressive effects in infectious and autoimmune diseases (118), which include inhibition of T-cell proliferation and MΦ activation. TGF-β inhibits the functions of TNF-α and IFN-γ and controls the expression of inducible nitric oxide synthase (iNOS) and the development of Th1 and Th2 response. Unlike IL-10, the impact of TGF-β on parasite burden and IFN-γ dependent host resistance is marginal during L. donovani infection (119). Locally activated TGF-β favors the parasite growth by modulating innate and adaptive immune responses (120) and enhancing arginase expression (121, 122). In animal models, TGF-β secreted by Leishmania infected lymphocytes diverts the arginine pool from iNOS to arginase for the production of polyamines, which helps in the growth of the parasite (123). Both pro and anti-inflammatory roles of TGF-β have been demonstrated (62, 124). L. chagasi infection induces TGF-β secretion by human MΦs through activation of latent TGF-β itself. L. chagasi infection also induces the expression of TGF-β in spleen and liver tissues of both symptomatic and asymptomatic dogs (213). TGF-β exposure delays the killing of Leishmania parasite and TGF-β overexpression impairs the rate of cure in murine leishmaniasis models (125, 126). In human VL, the elevated levels of IL-10 and TGF-β postively correlate with the parasite load and with increased absolute numbers of FoxP3 Treg cells suggesting the role of Tregs in secretion of these cytokines. There is a significant correlation between the parasite load and circulating antigen specific TGF-β levels in VL patients suggesting its role in parasite multiplication and disease progression in humans (214).

IL-4 is a 20 kDa Th2 subset cytokine that plays a critical role in the regulation of mast cell or eosinophil-mediated immune responses, B-cell mediated IgE class-switching and antibody production. It functions as a growth factor for mast cells and naive CD4⁺ Th2 cells which produce anti-inflammatory cytokines IL-5, IL-10 and IL-13. Both IL-4 and IL-13 inhibit IFN-γ-producing CD4⁺ T-cells and suppress protective Th1 immune response (150) and trigger MΦs to undergo alternative activation resulting in parasite survival and persistence of infection (151, 152). The similarities in IL-4 and IL-13 biological activities are due to a common receptor γ-chain that they both share, which is involved in the signal transduction via signal transducer and activator of transcription (STAT)-6 (215). Studies on murine model established the pathogenic role of IL-4 in leishmaniasis (39). IL-4 inhibits the oxidative burst by inducing low level production of reactive oxygen intermediates and NO in MΦs (153, 154). L. major infected Langerhans cells show increased IL-4R expression and decreased IL-12 production in susceptible mice but not in resistant mice (155).

IL-4 modulated antigen-uptake, endosomal processing, and humoral response are suggested to promote the disease development in Leishmania infection in humans (156). In murine L. donovani infection, IL-4 induces the host protective response (216) and vaccine mediated protection by IFN-γ secretion from CD8⁺ T-cells (157). The peripheral blood mononuclear cells (PBMCs) harvested from cured VL patients produced IFN-γ or IL-4 in response to stimulation by L. donovani promastigote or amastigote crude antigen. In response to purified gp63 antigen, the proliferation capability of the same PBMCs was weak and produced IFN-γ or IL-4 (158). Cytokine analysis in VL unveils the induced expression of IL-10 and/or IL-4 mRNA in tissues and abundant levels of IL-4 in circulation of patients with progressive disease (217). Likewise, the conflicting role of IL-4 in VL is described, though it has a leading role in pathogenesis of VL as it is belonging to Th2 phenotype and anti-inflammatory cytokine subset. Recent studies on human splenic aspirates suggest that blockade of IFN-γ and TNF-α results in increased production of IL-4 which does not contribute to parasite replication and IL-10 production. The biological role of IL-4 in target organ of human VL still remains an outstanding question (218).

IL-13 is a 12-kDa cytokine that is expressed by Th2 and is important in host protection against Leishmania infection. For example, IL-13 knock-out mice infected with L. donovani show retarded hepatic granuloma formation and maturation, depleted IFN-γ secretion and enhanced production of IL-4 and IL-10 (159). IL-13 protects rats from L. major infection through the production of IL-1β and IL-12 (160), which is in contrast to the earlier studies that showed pathogenic role of IL-13 (16, 219). However, studies with BALB/c mice infected by L. mexicana and L. amazonensis have established the pathogenic impact of IL-4 and IL-13 (161). The susceptibility to L. (V.) panamensis infection is predominantly associated with IL-13 but not IL-4. The parasite species and the host genetic background may also influence the dual role of IL-13 and it may not be a potential target for immunotherapy (162).

IL-6 is a 26 kDa pleiotropic cytokine produced by a number of cell types, including monocytes, endothelial cells and T-lymphocytes (220). The main biological activities associated with IL-6 are the induction of acute-phase protein synthesis in hepatocytes, terminal differentiation of B-cells and activation of T-cells (221). It also induces the production of anti-inflammatory proteins, such as IL-1 receptor antagonist (IL-1rα) and soluble TNF receptor (222). IL-6 plays a major role in the switching of monocytes from DC to MΦs (130). IL-6 favors the development of Th2 response, which suppresses the activation and antimicrobial effect of MΦs (75). Contradicting roles of IL-6 have been demonstrated in experimental CL and VL models (223–225). IL-6 has been shown to either promote (226, 227), suppress (228), or do not change (229) the intracellular host defense to leishmaniasis. Function of endogenous IL-6 as a host suppressive cytokine in case of L. donovani infection has outshined its potential pro-host defense effect.

During adoptive transfer of testing splenic DCs, IL-6 induces leishmanistatic effect but not host suppressive effect (226). IL-6 inhibits the IFN-γ mediated gene expression (131) and absence of IL-6 receptor signaling in L. donovani liver infection contributes to enhanced Th1-type response, accelerated tissue inflammation, and rapid parasite killing (132). IL-6 induces the secretion of IL-27 which in turn induces IL-10 production (133–135) in the L. donovani infected mouse model (54). However, L. donovani-infected IL-6⁻/⁻ mice do not show effect on the IL-10 expression (226). This observation raises a potential possibility to target endogenous IL-6 as an anti-leishmanial therapeutic strategy (230). Expansion of CD25⁻FoxP3⁻IL-10⁺CD4⁺ T-cells in vivo and therapeutic efficacy of adoptively transferred DCs against L. donovani infection are regulated by DC-derived IL-6 (226). IL-6 is produced by dogs with active leishmaniasis and is a key player in the pathogenesis of canine leishmaniasis (144, 231). The presence of TNF-α and IL-6 transcripts was found in both Leishmania antigen stimulated and unstimulated cells of asymptomatic infected and uninfected dogs (232). Further, increased anti-leishmanial antibody titers (hypergammaglobulinaemia) in canine VL are usually associated with high levels of IL-6 (224). Contrary to murine VL, the role of IL-6 in human VL is associated with disease severity and death, which is due to the inhibition of TNF-α in the early phase of infection and later by inhibiting the Th1 responses (190, 233).

IL-27 is a member of the IL-6/IL-12 cytokine family and a heterodimer composed of EBI3 and p28. The main cellular source of IL-27 is CD14⁺ spleen cells (140) in particular MΦs and DCs. The anti-inflammatory properties of IL-27 have been demonstrated in various models of infectious diseases and autoimmunity (234). IL-27 mediates anti-inflammatory response by suppressing Th17 cells (138, 139) and inducing IL-10 secretion from activated CD4⁺ T-cells via autocrine action of IL-21 (140). IL-27 plays a multifaceted role characterized by the induction of T-bet (141) in turn inhibition of parasite driven Th2 and Th17 development and Th1 polarization via IL-10 mediated feedback mechanism. IL-27 plays a critical role in the induction of IFN-γ and IL-10 from CD4⁺ T-cells, and suppression of inappropriate Th17 development to achieve immune-balance during intracellular parasite infections. In L. major infection, early burst of IL-4 suppresses IL-27 mediated development of normal Th1 by inducing IL-6 and TGF-β and promote the development of Th17 cells (142). In contrast, IL-27 is not required for the normal development of Th1 response to L. donovani infection (140) but induces IL-10 production (143, 145).

Absence of IL-27 receptor signaling in L. donovani liver infection contributes to the accelerated Th1-type response, tissue inflammation, and rapid parasite killing with reduced parasite burdens in spleen and liver (71, 146). Blocking IL-27 evoke different responses in different mice models. For example, blocking IL-27 results in reduced parasite loads in BALB/c mice and augmented parasite burden is seen in C57BL/6. This dichotomy in the production of IL-27 could be due to the consequence of host immune modulation by the parasite to establish infection (235). IL-27 levels were elevated in human plasma with active VL and splenic mRNA levels of IL-27 and IL-21 were higher in pre-treated biopsies compared with post-treatment samples (140). During VL caused by L. infantum, the sequential pathway of TLR3 and TLR9 recruitment, production of type I IFN and activation of IRF1 in macrophages is induced by IL-27. The secretion of IL-27 increases the Th1 response but also dampens the production of IL-17 which directly impacts the reduced recruitment of neutrophils to target organs (236). Inhibition of IL-27 could be targeted for design of anti-VL treatment in the future.

IL-5 is a glycosylated homodimeric 45–60 kDa protein, functions as an anti-inflammatory cytokine and is produced by Th2 cells, mast cells, and eosinophils. IL-5 stimulates the B-cell growth and promotes the production of cytotoxic T-cells from thymocytes; however, the key function of IL-5 is in the activation, maturation, and survival of eosinophils. Eosinophils activated by IL-5 expel antibody bound parasites while releasing proteins associated with cytotoxic granules. In the case of CL and MCL, PBMCs induce secretion of both IL-4 and IL-5 at the site of the lesion (127, 128), which results in declined Th1 polarization. Several studies have reported that IL-4, IL-5, IL-10, and IL-13 provide favorable atmosphere for intracellular parasite growth and dissemination (129). Patients with chronic lesions produce abundant levels of IL-5 and IL-13, which further inhibits parasite killing by an additive effect of IL-13. IL-5 plays a minor role in the susceptibility to L. major infection in BALB/c mice (237).

IL-9 is a 14 kDa pleiotropic cytokine produced by Th-cells, primarily identified as a mouse T-cell growth factor (237) and mast cell differentiation factor. Erythroid precursors, B-lymphocytes, eosinophils, bronchial epithelial cells and neuronal precursors are the secondary targets of IL-9 (238). It is a Th2-type cytokine produced via both IL-4 dependent (239) and IL-4 independent (240) pathways and involved in the physiological regulation of Th1-Th2 balance in vivo. Very little is known about the role of IL-9 in leishmaniasis. In L. major infection, IL-9 is transiently expressed in susceptible BALB/c as well as in resistant C57BL/6 and DBA mice during early days of infection, but 4-weeks onwards, its expression was only seen in susceptible mice but not in resistant mice (136). In vivo neutralization of IL-9 delays disease progression in BALB/c mice by inducing protective Th1 response suggesting, IL-9 promotes susceptibility to L. major infection. Further, IL-9 induces classical MΦ activity and production of IFN-γ in L. major infected BALB/c mice, which serves as a model system to study the role of IL-9 in human diseases (137).

IL-33 is a member of IL-1 family, which includes IL-1 and IL-18. IL-33 is a crucial player in the defense against nematode infections and allergic reactions, since it causes Th2-type immune response via inducing the production of IL-5 and IL-13 by T-cells, mast cells, basophils, and eosinophils. In addition, IL-33 also induces non-Th2-type inflammation, suggesting its pro-inflammatory role like IL-1 and IL-18. Schmitz et al. first reported that IL-33 functions through ST2 (IL-1R4) orphan receptor present on different immune cell types (241). During L. major infection in BALB/c mice, ST2-expressing CD4⁺ T-cells accumulated in local lesions (147). However, administration of polyclonal anti-ST2 antiserum depleted ST2-expressing cells as well as Th2 cells/cytokines and induced Th1 cytokine production, which in turn reduced the lesion development (148). Rostan et al. reported that the serum IL-33 levels were higher in VL patients besides the presence of IL33⁺ cells in liver biopsy of a patient. Similar results were observed in BALB/c mice infected with L. donovani, additionally, ST2⁺ cells were also observed in mouse liver. ST2 deficient BALB/c mice had shown strong Th1-type immune response via IFN-γ and IL-12 that controlled the hepatic parasite load and hepatomegaly. Recombinant IL-33 treatment of L. donovani infected BALB/c mice dramatically reduced the Th1 immunity and infiltration of polymorphonuclear neutrophils (PMNs) and monocytes in liver. In summary, IL-33 could be a very useful cytokine to determine the host susceptibility and disease prognosis of VL (149).

IFN-γ is a homodimeric glycoprotein consisting of two subunits each about 21 to 24 kDa. It is the most potent type II interferon that helps in MΦ activation to the leishmanicidal state (63). The main cellular sources of IFN-γ production are activated CD4⁺ and CD8⁺ T-cells, and NK cells in response to IL-12 signaling (242). Of the several anti-leishmanial cytokines (23), IFN-γ is the most significant cytokine in host protection, which plays a prominent role in MΦ priming (64) to produce leishmanicidal molecules (243). IFN-γ acts as monocyte-activating factor (65) and enhances release of oxygen radicals, secretion of pro-inflammatory cytokines (TNF-α, IL-l, and IL-6) (66), expression of MHC class-II, and antigen-presentation. In addition, IFN-γ blocks the production of IL-10, which decreases all the above functions of monocytes (67). Several studies demonstrated that the leishmanicidal activity of MΦs can be induced by a variety of cytokines, either alone or in combination. For instance, lipopolysaccharide (LPS) is required to induce the MΦ leishmanicidal activity in vitro (244). In human VL, the response is predominantly Th2-type with absence of PBMCs derived IFN-γ (47). But drug treatment induces a shift in the response so that individuals cured of VL often respond to Leishmania antigen by the production of both IFN-γ and IL-4 (158). In CL patients, however, the response is mainly dominated by IFN-γ and IL-4 is rarely detected (245), indicating that the immunological response to Leishmania in these individuals does not polarize as observed in inbred mouse strains. In vitro studies with T-cell clones (246) and in vivo studies using models of CL (40, 43) have demonstrated that IFN-γ can inhibit the expansion of CD4⁺ Th2-cells, resulting in the preferential expression of Th1 cell-mediated response. Reciprocal regulation is provided by the action of IL-10 on the IFN-γ producing capacity of Th1-type CD4⁺ T-cells (16). A recent study showed that the variation in single nucleotide polymorphisms (SNPs) of IFN-γ gene at the position +874 (A/T) influences the susceptibility to VL such that individuals in southwest of Iran with an AT genotype are susceptible and those with a TT genotype are resistant to VL (247).

IL-12 is a heterodimer consisting of two subunits (35 and 40 kDa) linked by a disulfide bond, mainly produced by activated MΦs and DCs. It is a proinflammatory cytokine that plays a key role in bridging innate and adaptive immune responses (248). Protective immunity against leishmaniasis is typically associated with the production of IL-12 (16, 219). IL-12 drives Th1 response and induces IFN-γ production from both NK cells and T-cells (68), and mediates the leishmanicidal activity by inducing NOS2 expression and NO production (146). In addition, IL-12 also mediates T-cell proliferation and lymphokines production (71, 72). The presence of IL-12 reduces the ability of CD4⁺ T-cells to produce IL-4 and increases the ability to produce IFN-γ. Thus, IL-12 and NK cells seem to play important role in determining the development of Th1 response (249). In vivo studies showed that IL-12 produced in infected mice (219, 250) is important to control Th2 expansion and to promote Th1 type response (69, 70). Neutralization of IL-12 leads to disease exacerbation in L. major and L. donovani infections (49, 69, 77). In contrast, the addition of IL-12 to lymphocyte cultures from VL patients restored IFN-γ production and increased cytotoxic activity of NK cells (48).

TNF-α is a 51 kDa homodimeric cytokine, mainly secreted by the activated MΦs, T-cells, NK cells and mast cells. TNF-α is important in mediating both innate and adaptive inflammatory responses. The regulation of TNF-α production appears to be important because it has potential role in the formation and maintenance of granuloma (76). Antiparasitic activity of TNF-α is mediated through activation of infected MΦs for the destruction of intracellular amastigotes (73). TNF-α production is absent in susceptible mice but present in L. major infected resistant mice. Protective role of TNF-α in L. major infection is characterized by MΦ activation, NO production and parasite clearance or suppression of visceralization (74). Protective T-cell response induced by TNF-α in L. major infected mice is due to the induced production of parasite-specific IgG1 and IgG2a. Acute infection with L. braziliensis resulted in the lack of production of parasite-specific IgG1 and IgG2a antibodies (251). The role of TNF-α in L. braziliensis infection is attributed to controlling the parasite numbers in the skin, lymph nodes and spleen and wound healing process (75). Brazilian patients with MCL had increased levels of TNF-α in both sera (252) and tissue lesions (253).

Treatment with TNF inhibitors, such as pentoxifylline in combination with anti-leishmanial pentavalent antimony, pentoxifylline promotes the re-epithelialization of mucosal tissues (254). However, infection of TNF⁻/⁻ mice with L. major shows some delay but no defect in antigen-dependent T-cell activation (74). IFN-γ independent anti-leishmanial mechanism mediated by endogenous TNF-α was described in IFN-γ knockout (GKO)-1 mouse infected with L. donovani (77, 199). The L. donovani infection provoked endogenous TNF-α level are enough to offer initial resistance to the parasite invasion and critical for the resolution of visceral infection. This is contrasting with the effect of exogenous TNF-α, which has no protective role in established infection and its continuous administration leads to impaired anti-leishmanial activity (255). The polymorphism and upregulation of TNF2 promoter transcription could be involved in enhancing clinical VL infection (256). TNF-α cannot be considered as a good marker of active disease in both human VL and canine VL due to its labile nature (224). The production of TNF-α follows biphasic kinetics due to its effect on target cells mediated by membrane-bound receptors (117). The high expression of IL-32 (especially γ-isoform) in CL and mucosal lesions is associated with endogenous TNF-α production but not with IL-10, suggesting the inflammatory role of IL-32 in host defense against Leishmania infection (257). Absence of IL-32 leads to high infection index but its overexpression opposed the parasite growth via NO cathelicidin and β-defensin 2 syntheses (258). In response to excessive inflammation, increase in the levels of TNF-α might promote the generation of IL-10 producing T-cells as a homeostatic response (78).

IL-2 is a 15 kDa cytokine, produced by activated T-cells and was initially identified as a T-cell growth factor. IL-2 stimulates the proliferation and differentiation of B-cells, NK cells, monocyte/MΦs, oligodendrocytes and lymphocyte activated killer (LAK) cells. IL-2 does not directly stimulate the intracellular antimicrobial activity of MΦs (259) but exerts a range of immunoregulatory effects on T-cells and NK cells and induces the production of IFN-γ (79, 80). IL-2 may act as a susceptibility factor in leishmaniasis (250, 260) by inducing the production of IL-4 from CD4⁺ T-cells (81). But in IL-4 deficiency, the inhibition of disease progression is attributed to IL-13 and IL-2. Interestingly, in CL, both IL-2 and IL-15 are attributed in host protection, while in MCL IL-2 is only protective but not IL-15 (200). Rapid production of IL-2 was observed after successful treatment or acquisition of resistance against L. donovani infection but not during acute phase (261, 262). The endogenous IL-2 could act as a defensive cytokine, only when the mice subsequently challenged after a prior infection with the parasite (82). In contrast, exogenous IL-2 exerts the anti-leishmanial action using L3T4⁺ and Lyt-2⁺ T-cells in acutely infected euthymic mice. IL-2 exerts leishmanicidal activity in splenocytes in vitro even in the absence of IFN-γ (83).

IL-15 is a 14–15 kDa cytokine with four α-helix bundles and plays a central role in the innate and adaptive immune responses to infections (86). The main source of IL-15 is activated peripheral monocytes (263). Due to the common receptor β-chain, immunological functions of IL-15 are similar to IL-2 (84), which includes the induction of T-cell proliferation (87), inhibition of T-cell apoptosis and preservation of memory T-cells (88), B-cell maturation and isotype switching (264). IL-15 also stimulates the proliferation and activation of NK cell (265) and induces the production of IFN-γ and TNF-α, synergistically with IL-12 (85). Nevertheless, IL-15 also shows distinct biological functions from IL-2 due to a different α-chain (266, 267). The possible pleiotropic role of IL-15 is reflected by its action on both Th1 and Th2 subtypes and the ability to induce the activity of IFN-γ and IL-12 (89) as well as IL-5 and IL-13 (90) in various experimental models. Endogenous IL-15 stimulates protective Th1 response by inducing the downregulation of IL-4⁺ Th2 cells (86). IL-15 could be a potential therapeutic agent in acute VL since it upregulates IL-12 production and Th1 development (91). In contrast, other studies have demonstrated that endogenous IL-15 is not necessary for basal expression of IL-12 and MΦ activation and is not able to influence the IL-12 activity and Th1 development in acute VL (268). IL-15 in combination with IFN-γ and/or IL-12 may increase the efficacy of conventional antimonial therapy for VL, because of low toxicity in vivo (201).

IL-17 is a 35 kDa proinflammatory cytokine, primarily produced by activated T-cells (CD4⁺ > CD8⁺) (269) and also by other subsets of T-cells including NKT cells and Th17 cells (96). The development of Th17 subset from naïve T-cells happens in the presence of IL-6 and TGF-β⁺ Tregs (270). IL-17 stimulates different immune cells to produce inflammatory molecules including TNF-α, IL-1, and chemokines (163). At the site of inflammation, IL-17 affects the neutrophil function, reduces the apoptosis, and promotes the secretion of pro-inflammatory cytokines as well as tissue damaging molecules (164, 165). IL-17 induces the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) and G-CSF, which increase the production of neutrophils, monocytes, and chemoattractants for neutrophils (CXCL8, CXCL1, and CXCL6) as well as Th1 cells (CXCL10) (96, 97). IL-17 induces the production of IL-6, which mediates both proinflammatory and regulatory functions (96). IL-17 of Th17 subset and Th1 subset play a complementary role in the host protection from L. donovani infection. Contrastingly, the susceptibility for L. major infection is not only associated with uncontrolled Th2 immunity (271) but also with excessive IL-17 secretion, which mediates neutrophil recruitment (166). Other studies have also demonstrated that IL-17 dependent neutrophil recruitment is essential only during the late stages but not early stages of L. major infection (272). Mucosal disease caused by L. (V.) braziliensis is also associated with elevated levels of IL-17 response (167). Treating VL using curdlan, a b-glucan immunomodulatory molecule induces Th1 cytokines with IL-12, IL-22 and IL-23 (273), while another immunomodulator Astrakurkurone is effective against experimental VL by inducing IL-17 along with IFN-γ (274). While these reports are suggestive of a protective role of IL-17 in VL, other reports suggested the involvement of IL-17 in exacerbating experimental VL in murine model (275) raising questions about its precise role in VL pathogenesis.

IL-18 is a 22 kDa pleiotropic cytokine produced by activated MΦs and Kupffer cells of liver (276). IL-18 promotes Th1 and NK cell development (168), induces IFN-γ production by lymphocytes and NK cells and synergizes with IL-12 (169, 170). IL-18R is expressed on Th1 cells but not on Th2 cells. IL-18 induced Th1 subset produces IFN-γ which indirectly regulates the expansion of Th2 cell (171). IL-18 promotes NK cell activity due to a constitutive expression of IL-18R on NK cells (277) and stimulates TNF-α secretion by human PBMCs (172). IL-18 also induces the activation of memory cells and in combination with IL-12 it prevents the reinfection of BALB/c mice with L. major (173). IL-18⁻/⁻ mice are highly susceptible to L. donovani infection when compared to the wild-type mice. However, endogenous IL-18 induces an initial IFN-γ independent anti-leishmanial effect in L. donovani infection (174). Paradoxically, IL-18 can also stimulate Th2 cytokines production, such as IL-4 from basophils (175) and CD4⁺ T-cells (176) and IL-13 from mast cells. While IL-18 protects BALB/c mice from L. donovani infection, it increases the susceptibility of BALB/c mice to L. major infection. Notably, the resistance and susceptibility of BALB/c mice to L. mexicana infection are not mediated by IL-18 and are influenced by different genetic and immunoregulatory controls (278). IL-18⁻/⁻ BALB/c mice are highly resistant to L. mexicana infection due to increased IFN-γ production and antigen-specific IgG2a, reduced splenic IL-4, antigen-specific IgG1 and total IgE (177). IL-18 plays a critical role in the regulation of Th1 and Th2 balance in vivo, which frequently determines the outcome of many important infectious and autoimmune diseases (168).

IL-22 is a 16.7 kDa cytokine, primarily produced by Th17 cells and to a lesser extent by Th1 and NK cells (96). Immunological functions of IL-22 are associated with the epithelium and mucosal surfaces (92), which include promoting inflammatory response and tissue repair (93, 279). IL-22 stimulates the production of pro-inflammatory molecules, such as S-100A proteins and CXCL5 (93). IL-17 and IL-22 act synergistically on epithelial cells to produce an antimicrobial peptide called β-defensin (95). IL-22 is also involved in protecting the liver (94) during chronic infections. During L. donovani infection, both IL-17 and IL-22 are produced by PBMCs and may exert complementary function along with Th1 cytokines (96, 97). The production of IL-22 requires IL-6 but not TGF-β (98).

IL-1 is synthesized as ~35 kDa precursor, from which two functional agonistic proteins (IL-1-α and IL-1-β each 17 kDa M.W.) and IL-1Ra, receptor antagonist of IL-1R1, are produced. IL-1 is a potent proinflammatory “alarm cytokine” that synergizes the functions of TNF-α and is produced by MΦs. IL-1 builds inflammation by inducing the expression of adhesion molecules on endothelial cells and leukocytes (280, 281). IL-1β, along with other proinflammatory cytokines, is released into the periphery during infection and coordinates immune-to-brain communication (180). IL-1 mediated inflammation is coordinated by adaptive T-cell response and controls the parasite dissemination (178, 179). IL-1 is responsible for regulating the delicate balance between inflammation and immunity which decides the fate of the disease progression in leishmaniasis. In L. major infection, the acute levels of IL-1α, IL-1β, and IL-1Ra are adequately downregulated unlike in L. amazonensis infection (282). Disease progression is inhibited with IL-1α treatment in L. major susceptible BALB/c mice during T-cell differentiation. IL-1β enhances activation of DCs and T-cell priming but do not affect the cytokine profile of DCs and pathogenic Th-cells (178). Contrastingly, Voronov et al. reported that BALB/c mice deficient in IL-1 family genes showed delayed disease progression with L. major infection due to apparent Th1 response even at late stages of the disease. IL-1α deficient mice were slightly more resistant to L. major infection than IL-1β KO mice (181). In L. amazonensis infection, IL-1β treatment induced DCs and cytokine production remains lower than that of L. major infection. IL-1 therapy in murine CL results in a wide range of outcomes depending on the course of treatment and parasite species involved. In this context, IL-1-based treatment may be effective for L. major but not L. amazonensis infection. The decreased production of IL-1 has been associated with L. donovani infection of murine peritoneal MΦs in vitro and human circulatory monocyte population (182, 183). Similarly, human PBMCs failed to produce IL-1 in response to Leishmania-antigen stimulation in vitro, during acute VL. However, following anti-leishmanial therapy, IL-1 and TNF-α levels are usually recovered, which correlates with clinical cure (184). Recombinant IL-1α induces mature granuloma formation in liver and IFN-γ production from spleen cells but is not able to clear the parasite (185).

IL-3 is a 28 kDa glycoprotein derived from T-cells and supports the viability and differentiation of hematopoietic progenitor cells (283, 284) and monocytes (283). With the combination of GM-CSF, M-CSF, and IFN-γ, IL-3 shows an additive effect on human MΦs in the induction of oxidative burst and TNF-α secretion to inhibit the replication and growth of Leishmania parasite. In contrast, IL-3 promotes the infection in murine model of CL highlighting the species-specific differences in the role of IL-3 in leishmaniasis (186). In combination with M-CSF, IL-3 induces superoxide ions production to kill the parasite and may involve in myelopoiesis during acute VL.

IL-7 is a 17 kDa glycoprotein derived from bone marrow stromal cells (285) and regulates a wide variety of functions including multiple effects on B-cells and proliferation of thymocytes (99–101), NK cells (102) and mature T-cells (103). IL-7 induces the production of cytotoxic T-cells with alloreactive, antitumor, and antiviral activities (104). It was reported that IL-7 shows potential effects on monocytic lineages (105, 286). IL-7 enhances the synthesis and secretion of various inflammatory cytokines, such as IL-6, TNF-α, IL-1α, IL-1β, and MΦ inflammatory protein (MIP) 113 by human circulatory monocytes. IL-7 is not as effective as IFN-γ but shows an additive effect with the combination of suboptimal concentrations of IFN-γ in killing the Leishmania amastigotes by inducing the production of TNF-α (105) and NO.

IL-8 is a non-glycosylated proinflammatory cytokine with a M.W. of 8 kDa, which is primarily produced from neutrophils and from other cell types including epithelial cells, keratinocytes, fibroblasts and endothelial cells. In mouse system, IL-8 has two functional homologs like MIP-2 (CXCL2/Groβ) and KC (CXCL1/Groα). During L. major infection in humans, IL-8 promotes the recruitment of neutrophils at lesion sites (106). In mice infected with L. major, IL-8 causes transient production of KC mRNA in the skin, which may associate with granulocyte recruitment (107) which is yet to be demonstrated in vivo (272). Notably, a reduced neutrophil count during active VL is associated with lower IL-8 levels in serum (32). It was identified that polymorphisms at IL-8 −251 position are associated with impaired IL-8 activity and the development of active VL in Iranian individuals (108) but such a correlation was not observed in Brazilian VL patients (287).

IL-23 is a pleiotropic cytokine produced by MΦs and DCs which acts on receptors expressed by T-cells, NK cells and NKT cells (288). During L. donovani infection in BALB/c mice, the IL-23p19 mRNA expression in the liver tissue was comparable to that of wild-type. IL-12 independent protection in visceral infection (76, 109, 110) was mediated by IL-18 and probably by IL-23 also, since the p40 subunit of IL-12 shared by both IL-12p70 and IL-23 (174). IL-23p19 may pair with IL-12p40 to become biologically active (111), which are crucial for host protection. Therefore, IL-23 alone or in combination with other cytokines may be a possible option in immunotherapy of VL.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.