Interleukin-27 Functional Duality Balances Leishmania Infectivity and Pathogenesis

IL-27 is a cytokine that exerts diverse effects on the cells of innate and adaptive immune systems. Chiefly expressed in macrophages and dendritic cells during the early phase of Leishmania infection, IL-27 contributes to the protection against L. major infection but suppresses the protective Th1 response against L. donovani, L. infantum, L. amazonensis and L. braziliensis infections, suggesting its functional duality. During the late stage of Leishmania infection, IL-27 limits the immunopathogenic reactions and tissue damages. Herein, we analyze the mechanism of the functional duality of IL-27 in the resistance or susceptibility to Leishmania infection, prompting IL-27 for anti-Leishmanial therapy.

IL-27 inhibits DC functions, as WSX-1-deficient DCs are hyper-reactive to LPS and promote NK cells and T cells to produce higher amounts of IFN-γ than that induced by wild-type DCs (24). IL-27 pre-treatment of DCs reduces the LPS-stimulated expression of the MHC-II and costimulatory molecules-CD40, CD86-perhaps due to IL-27-induced elevated CD39 expression (24,25). B7-H1 (PD-L1), which provides suppressive signals to T cells, is upregulated by IL-27 in DCs (10,26,27). Consistent with these observations, IL-27 reduces HLA-restricted antigen presentation and inhibits proliferation and cytokine production of allogeneic T cells (28). Through STAT-1 activation (29), IL-27 inhibits proliferation and cytokine production by type-2 Innate lymphoid cells (ILC2)a specialized cell-type that lacks antigen-specific receptors but produces high levels of helper T-cell cytokines and lipid mediators in response to antigen-independent stimuli. ILC2 express GATA3 and partake in airway inflammation via the production of type-2 cytokines. A summary of the effects of IL-27 on innate immune cells is shown in Figure 3A.

Effects of IL-27 on the Adaptive Immune System
Naïve CD4 + T cells abundantly express IL-27Rα or WSX-1, but IL-2 activated and differentiated CD4 + T cells show reduced IL-27Rα expression (30). IL-27 induces T-bet and IL-12Rβ2 expression in CD4 + T cells imparting IL-12 responsiveness during the early phases of Th1 polarization (Figure 2) (31). By contrast, IL-27 inhibits IL-2 production from CD4 + Th cells through SOCS3 induction, limiting T cell responses, and as IL-2 is responsible for the proliferation and survival of Th1 cells, IL-27 mediates suppression of Th1 cell-related immunity (32). Suppression of Th1 cell response by IL-27 also can be explained by the induction of IL-10 an anti-inflammatory cytokine (33). IL-27 inhibits Th2 cell development and production of Th2 cytokines in already polarized Th2 cells by downregulation of GATA3 expression and upregulation of T-bet expression (34). IL-6 and TGF-β trigger naïve CD4 + T cells differentiation to Th17 cells by inducing RORγt expression (35). IL-27 inhibits the Th17 cell differentiation through interference with IL-6 signaling and suppression of the expression RORγt, induction of the IL-10 production and autocrine IL-10-mediated inhibition, and induction of programmed death-ligand 1 (PD-L1) on naïve T cells (36,37).
Two subsets of Treg cells are generated from precursor cells in the thymus including natural Treg (nTreg) and inducible Treg (iTreg) cells, or antigen-induced naive CD4 + T cells in   culminate in the induction of IL-27p28 synthesis are-TLR3/TLR4-TRIF-IRF3/IRF7; IFN-γ/MyD88/IRF8 and IFN-α/IFN-β-STAT1/STAT2-IRF1/IRF9. (C) IFN-γ can induce IL-27  the presence of TGF-β and IL-2, in the peripheral tissues, respectively. Treg cells express the transcription factor FoxP3 and are characterized by the secretion of immunosuppressive cytokines TGF-β, IL-10, and IL-35 (38). The iTReg were divided into two subgroups, including Tr1-and Th3-cells, which are characterized by the production of the large amounts of IL-10 and TGF-β, respectively (39). Indeed, IL-27 supports the development of immune-regulatory Tr1 CD4 + T cells (40). IL-27 also promotes the development of IL-10, producing T-bet + CXCR3 + Treg cells (41). IL-4 and TGF-β elicit naïve CD4 + T cells differentiation to Th9 cells, which express the transcription factor PU.1 and secrete large amounts of IL-9 that contribute to mast cell activation, induction of allergic reactions, and immune responses to intestinal helminth infections (42). IL-27 inhibits the development of Th9 cells (43). IL-6 and IL-21 elicit the T follicular helper (Tfh) cell differentiation from naïve CD4 + T cells. Tfh cells express Bcl6-a transcription factor-and provide CD40L, ICOS, and IL-21 signals required for B cell proliferation and differentiation (44). IL-27 promotes Tfh cell polarization and induces IL-21 (45), which promotes B cell expression of Bcl-6 and Blimp-1 that are critical for plasma cell differentiation and B cell function. Although IL-27 induces B cell proliferation and antibody production, it does not seem to promote the formation of memory B cells (46). CD8 + T cells recognize viral-and tumor-derived antigenic peptides presented by MHC-I molecules and play a fundamental role in the killing of virally infected and cancer cells. IL-27 enhances the proliferation of naïve CD8 + T cells and the production of IFN-γ and granzyme B (47) (Figure 2). IL-27 thus differentially affects the functions of immune cells in both innate and adaptive immune systems. The effects of IL-27 on adaptive immune cells is summarized in Figure 3B.

THE ROLE OF IL-27 IN LEISHMANIA INFECTION
Leishmania is a protozoan trypanosomatid parasite and is transmitted by the sand fly vectors. Leishmania exists as extracellular, flagellated motile promastigotes in sand flies and as sessile, aflagellate, amastigotes within the mammalian macrophages in skin, liver, spleen, bone marrow, and mucosal tissues. It causes a complex of diseases called Leishmaniases to affect 12 million people worldwide and one billion at risk (48,49). Depending on the invading specie of Leishmania, tissue tropism of the parasite, the site and the route of infection and pathophysiology, Leishmaniases are classified into cutaneous (CL), mucocutaneous (MCL), diffuse cutaneous (DCL), visceral (VL), and post-kala-azar dermal Leishmaniasis (PKDL) (49). The CL is the most prevalent form of the disease (70-75%), caused by L. major and L. tropica in the Middle East and North Africa, while L. mexicana and L. braziliensis are predominant in South America (49,50). L. amazonensis causes DCL and CL with disseminated lesions. L. donovani and L. infantum cause VL that disseminates to spleen, liver, and bone marrow (51). Thus, Leishmania species differ in their growth kinetics, the tissue of infection, and clinical outcomes. Although macrophages serve as the host for all these Leishmania sp. the immunopathology of Leishmania is greatly affected by tissue-specific non-immune cells. IL-27 plays a complex role in the immuno-pathology of Leishmaniasis (52).

Do Leishmania Salivary Gland Proteins (SGPs) Affect IL-27 Secretion?
Most species of Leishmania are transferred through the phlebotomine sand-fly vector. The metacyclic promastigotes are embedded in proteophosphoglycan-rich promastigote secretory gel (PSG) secreted by themselves in the anterior midgut of sandflies. PSG must be regurgitated along with infective promastigotes before sand flies can begin hematophagy on host skin. In turn, this event leads to the accumulation of PSG, Leishmania along with sand-fly saliva containing SGPs on host skin. Since the first description of sand-fly SGPs diseaseexacerbating function in CL (53), these are shown to induce cytokines and chemokines with potential contrasting functions (54). Many proteins including LJM19, a novel 11-kDa protein, are identified as anti-Leishmanial vaccine candidates (55,56). Although IL-27-inducing SGPs are not reported, some probable pathways may be proposed. First, as the sand-fly bite is rich in immunomodulatory components like CD39 family ecto-apyrases converting ATP and ADP to AMP and Pi (57), the adenosine receptors on the infected DC and macrophages may modulate IL-27 secretion (58). Second, as platelet activation is associated with the chemotactic migration of effector monocytes, a potent source of IL-27, to the sites of L. major infection (59) an axis of SGPsplatelets activation-monocyte recruitment may modulate IL-27 production affecting the outcome of Leishmania infection. Third, salivary-homogenates from Lutzomiya longipalpis suppress the costimulatory molecule expression and CD40L-induced DCs maturation (60), which may lower available IL-27 pools as observed with IL-10 and TNF-α. A limitation with such studies is that these do not highlight the exact specific composition of sand-fly salivary gland extracts, which may be composed of more than 35 secreted proteins (61). Fourth, although a vasoactive peptide (maxadilan) from the L. longipalpis saliva is shown to indeed increased production of prostaglandin E2 (PGE 2 )   (62) and reciprocally modulate TNF-α and IL-6 production from BALB/c macrophages in a PGE 2 -dependent manner (62), the regulation of IL-27 by maxadilan and a correlation between PGE 2 and IL-27 in experimental Leishmaniasis require further investigation. Similarly, SP15-like protein, D7-related proteins, lufaxin (reversible inhibitor of factor Xa of coagulation cascade), palmitoyl-hydrolase, and YRP proteins (57) may modulate the host IL-27 production by previously unexplored  (Figure 4). Therefore, the infected DCs are the major producers of IL-27 during the early phase of CL.

IL-27 Treatment Suppresses Th2
Responses and Protects the Host From L. major Infection The IL-27 signaling via WSX-1 contributes to protection against L. major infection (69)(70)(71). Upon IL-27 treatment, IL-27 transgenic mice are protected against L. major infection (34). The daily IL-27 administration during the first seven days after L. major infection of BALB/c mice reduces the parasite load and increases the survival rate compared with control mice (34). The lymph node cells from L. major-infected IL-27-treated BALB/c mice secrete higher quantities of IFN-γ and produce lower amounts of IL-4 in comparison with the cells from control mice (34). Therefore, the exogenous IL-27 protects L. majorinfected BALB/c mice from CL by direct suppression of the Th2 cell responses accompanied by the induction of host-protective Th1 cell responses, perhaps due to its ability to augment IFN-γ production from NK and CD4 + T cells (70,71). The L. majorinfected macrophages from patients with healing forms of lesion produce a higher amount of IL-27 and IL-23 as compared with patients having non-healing forms of the lesion, suggesting that IL-27 and IL-23 may play synergistic roles with Th1 cytokines in protection against Leishmaniasis (72). L. major lysate-stimulated lymphocytes from EBI3 −/− mice secrete fewer IFN-γ and high IL-4, IL-10, and IL-13 as compared to the lymphocytes from the control mice on the second and third week post-infection. Thus, at early time points after infection, the Th1/Th2 cell balance in EBI3 −/− mice is diverted toward Th2 cells (69).

IL-27 Deficiency Impairs Host-Protective Th1
Responses and Induces Susceptibility to L. major Infection In WSX-1 −/− mice both IFN-γ production and resistance to L. major are impaired (69,71). EBI3 −/− C57BL/6 mice also display greater susceptibility to L. major infection compared to C57BL/6 wild-type mice, with the maximum differences in the parasites loads and lesion sizes at the sixth-week postinfection; the lesions and parasite load are reduced from the eighth week, and lesions are entirely resolved till 14th week in EBI3 −/− mice (69). Rechallenge of healed EBI3 −/− mice with L. major does not lead to the elicitation of the pathological symptoms, indicating that the expansion and persistence of the protective immunologic memory are not impaired in EBI3 −/− mice (69). These observations suggest the importance of EBI3 for early control of parasite replication (Figure 4). Similarly, when compared with wild-type mice, the lymph node cells from WSX-1 −/− mice produce fewer IFN-γ initially but makes up the deficiency in the later stages of the infection. However, both WSX-1 −/− and wild-type mice successfully control the parasite and resolve the cutaneous lesions (71). Susceptibility to L. major infection in WSX-1 −/− mice is restricted to the initial stages of infection when a considerable level of IL-4 is produced (71). The anti-IL-4 mAb administration during the early stages of infection improves the IFN-γ-dependent host resistance in Leishmania infected WSX-1 −/− mice. The treatment of the WSX-1 −/− mice with blocking anti-IL-4 antibody also reduces the T. cruzi parasite burden and also increases the number of IL-17 + CD4 + cells, indicating that IL-4 inhibits the Th17 cell development in parasitic infections (73,74). The initial IL-4 production during L. major infection determines the necessity for IL-27 in the induction of Th1 cell responses to limit the CL (Figure 4). However, IL-27 is not required to maintain an extended Th1 response during the later stages of L. major infection. Although WSX-1 −/− mice show significant lesions at an early phase of infection, at later stages of infection it ultimately limit parasite loads and resolve the infection (70,71). Accordingly, the increased susceptibility of WSX-1 −/− mice to L. major is accompanied by the Leishmania-induced IL-4 production initially; therefore, blockade of IL-4 during the initial phase of infection abolishes the early requirement for IL-27 for the development of effective anti-parasitic responses. At 6-8 weeks post-infection, IFN-γ production is increased, and the IFN-γ/IL-4 ratio shifts toward Th1 cells in WSX-1 −/− and wild type mice, which were accompanied by parasite killing and lesion resolution (69). The early EBI3 expression is vital for the quick generation of effective Th1 cell-mediated immunity, but healing and immunologic memory develop in an IL-27-independent manner (69).

IL-27 Contributes to Early Development and
Maintenance of Th1 Cells and Mediates Immunity Against L. major It is proposed that IL-27 is initially produced by the parasitestimulated phagocytes and skews Th1 response before IL-12 production is upregulated (75). Indeed, the IL-27-stimulated T cells express IL-12R and are then differentiated to Th1 cells in the presence of IL-12 in vivo (34). Early production of IL-27 may result in Th1 cell differentiation, and later on, maintained by IL-12 produced from L. major-infected DCs (76). Therefore, L. major-infected DCs sequentially produce IL-27 and IL-12 to establish and maintain protective Th1 cellmediated protective immunity against this pathogen. Delayed IL-12 production during L. major infection may cause a delay in the initiation of Th1 responses (77). Indeed, WSX-1 expression diminishes in differentiated Th1 cells; contrastingly, IL-12R is still abundantly expressed in activated Th1 cells. Th1 cells from WSX1 −/− mice display normal IFN-γ production in response to IL-12. Therefore, IL-27 is necessary for the early development of Th1 cells, and IL-12 is necessary for early differentiation, establishment, and full activation of Th1 cells. IL-27 causes Th1 cell differentiation through the induction of p38MAPK that upregulates T-bet. Moreover, it promotes the expression of ICAM-1/LFA-1 adhesion molecules to induce ERK1/2-related signaling pathways. Induction of both signaling pathways causes IL-12Rβ2 expression, which promotes the Th1 differentiation and IFN-γ production in the presence of IL-12 (2). WSX-1-related signaling also induces STAT-1dependent expression of T-bet and trigger IFN-γ production (78), and WSX-1 −/− mice display defects in IFN-γ expression (71). IL-27R-deficient mice exhibit enhanced Th2 cell-dependent asthmatic manifestations (79). Consequently, IL-27 determines the suppression of Th2 cell response. IL-27 inhibits Th2 cell differentiation and associated cytokine-production from fully activated Th2 cells (12). IL-27 also directly inhibits GATA-3 expression in a STAT-1-dependent pathway (80). IL-27 reduces GATA-3 expression while inducing T-bet expression even in polarized Th2 cells (34).

Regulatory Effects of IL-27 During L. major Infection
Severe lesions are reported in L. major-infected WSX-1 −/− mice, which were associated with the appearance of the IL-17 + CD4 + cells, indicating a role for IL-27 in preventing the improper  (Figure 5). Although Th1 polarization is key to the protection from L. major infection, the protection against L. donovani-mediated visceral Leishmaniasis is associated with mixed Th1-and Th2-type cytokine responses; as a result, the cured VL patients display a mixed Th response (93). Although IL-4 is a vulnerability factor in mouse L. major infection, it is necessary for efficient vaccine-induced protection against L. donovani infection (93). WSX-1 −/− mice display severe liver pathology after L. donovani infection. WSX-1 −/− mice exhibit severe liver pathology with large granulomas associated with diffuse foci of inflammation and necrosis, as well as more deposition of collagen and reticulin fiber on days 15 and 30 after infection compared with wild-type mice (92). Therefore, IL-27 plays a critical role in suppressing hepatic inflammation during the acute phase of VL. As mentioned, L. donovani-infected WSX-1 −/− mice produce greater levels of IL-12, IFN-γ, and TNFα during the early phase of infection in comparison to wildtype mice. The depletion of CD4 + T cells or neutralization of both TNF-α and IFN-γ also reduces the severity of liver pathology in WSX-1 −/− mice but renders them susceptible to L. donovani (92). Hence, the IL-27-mediated pathways suppress the tissue inflammation associated with VL by modulating the CD4 + T-cell function ( Figure 5). However, blockade of IFN-γ or TNF-α alone is not sufficient to block liver inflammation or compromise host resistance in WSX-1 −/− mice (92). Therefore, IFN-γ or TNF-α production alone may not be adequate to induce immunopathology in the liver. These findings demonstrate that the IL-27-related pathways, in contrast to its protective role in L. major infection, are involved in the pathogenesis of L. donovani infection.

Immunomodulatory Effects of IL-27 During L. Donovani Infection
It has been indicated that the IL-10 + IL-27 + DCs can promote IL-10 production by Th1 cells in vivo (94). In L. donovani infected C57BL/6 mice, the frequency of splenic IFN-γ + IL-10 + T-bet + CD4 + T cells is increased on day 28 post-infection (94). IL-27 directly alters methylation patterns in CD4 + T cells at the IL-10 gene promoter, thus causing higher IL-10 expression (95) (Figure 5). IL-27 supports IL-10 production by IFN-γ-producing Th1 cells through STAT1, STAT4, and Notch signaling molecules via an alternate pathway (96,97). The optimal generation of CD4 + IL-10 + T cells by IL-27 requires c-Maf, ICOS, and IL-21 expression (98,99). Further, IL-27 and IL-21 together enhance the expansion and differentiation of IL-10-secreting Tr1 cells during VL (40,51). IL-21 also reinforces the IL-10 secretion by IL-27-induced Tr1 cells (100). Therefore, IL-27 controls the severity of VL-associated inflammation by inducing antiinflammatory cytokine IL-10. Recently, in a mouse model of L. donovani infection using BALB/c mice, it has been reported that the administration of the neutralizing anti-EBI3 antibody (but not anti-p28 antibody) to pathogen-infected mice reduce the parasite load in the spleen and liver, and increase the TNFα-and IFN-γ-secreting cells (101). As EBI3 is shared by IL-35 and IL-27, it has been concluded that IL-35 may have more immunomodulatory effects during L. donovani infection (101). These regulatory effects of IL-27 during L. donovani infection are summarized in Figure 5.  (103). The in-vitro experiments indicated that the bone-marrow-derived macrophages (BMDMs) and BMDCs from C57BL/6 mice can produce IL-27 when infected with L. infantum (103). The Leishmania-derived nucleic acids (DNA and RNA) are recognized by intracellular TLRs like TLR3, TLR7, TLR8, and TLR9 (63,104). The BMDMs are deficient in TLR3 and TLR9 or adaptor molecules MyD88 and TRIF are not able to produce IL-27 after L. infantum infection, indicating that nucleic acids from L. infantum activate these pathways to trigger the IL-27 production. L. infantum infection induces IL-27 production in a consecutive process that includes the events, engagement of TLR3 and TLR9, the IFN-β induction, and activation of IRF1 that lead to IL-27 production in macrophages infected with L. infantum. The soluble CD40L also increases the production of several cytokines (IL-12p70, IL-23, IL-27, IL-15, and IL-1β) by L. infantum-infected human macrophages in vitro (105). A negative correlation was observed between the levels of these cytokines in the supernatants of cultured macrophages with the number of infected macrophages as well as amastigotes. Leishmania may also trigger sCD14 and initiate the IL-27, IL-10, and IL-6 production, which subsequently modulate macrophage microbicidal activity, facilitating Leishmania proliferation (106).

IL-27 Is a Regulatory Cytokine That Dictates Susceptibility to L. infantum Infection
For experimental VL both C57BL/6 and BALB/c strains are considered as susceptible models, but only BALB/c mice, not C57BL/6 mice displayed the augmented serum IL-27 levels during an early stage of L. infantum infection. The phenomenon was attributed to the upregulation of p28 expression by splenic DCs and higher parasite burdens in BALB/c mice (102). High expression of TLR2 by DCs from BALB/c mice may support the early IL-27 expression after infection, which contributes to the attenuating of inflammation and promoting infection (102). It has been reported that the L. infantuminfected BMDCs from BALB/c mice produce higher IL-27 levels than C57BL/6 mice. Whereas, the LPS-stimulated BMDCs from C57BL/6 mice produce higher amounts of IL-27 than BALB/c mice (102), indicating that parasite vigorously upregulates the secretion of IL-27 in BMDCs derived from BALB/c, but not from C57BL/6 mice. In both cutaneous and visceral forms of human Leishmaniasis, the IL-27 production is increased when the disease is in the active phase (91,106). When compared with healthy subjects and delayed-type hypersensitivity (DTH) positive individuals, the sera from VL patients before treatment showed high levels of cytokines-IFN-γ, IL-10, IL-6, IL-27, and TNF-α. Besides, the serum levels of these cytokines decrease after treatment (106). B cell activation is also promoted by IL-27, exacerbating the hypergammaglobulinemia in VL patients (106). In a mouse model of VL, IL-27 plays a role in the suppression of the protective immune response during L. infantum infection, which can lead to disease exacerbation (103). IL-27 suppresses the immune response in VL through the induction of IL-10 production in vivo (91). The IL-27 restricts the Th1 cell polarization via inducing the IL-10-producing DCs. In fact, in the p28 −/− mouse model restricted to DCs, CD4 + T cellrelated IFN-γ response is exacerbated (107). The collective production of both IL-27 and IL-10 by L. donovani-infected DCs is essential for IL-10 production by Th1 cells, resulting in parasite persistence (94).

IL-27 Modulates IL-17 to Affect Neutrophil Infiltration in the Spleen of L. infantum Infected Mice
The administration of rIL-27 to in C57BL/6 increases the IL-10 production, while decreasing IFN-γ and IL-12p70 in the peritoneal cavity, and prevents the infiltration of neutrophils in the spleen 24 h after the treatment (102). IL-27 neutralization in acutely infected BALB/c mice reduces parasite burdens and reduces IL-10 levels, while leads to a transient increase in the splenic IFN-γ-producing CD4 + and CD8 + T cells (102). The investigations using EBI3 −/− mice indicate a role for IL-27 in modulating the IL-17 production and neutrophil infiltration during the chronic phase of L. infantum infection (102,103). The EBI3 −/− mice produce considerably higher IL-17A levels than wild-type C57BL/6 mice in both spleen and liver. Restimulation of EBI3 −/− mice-derived splenocyte with L. infantum lysate leads to significant IL-17A production. IL-17A is the critical mediator of neutrophil infiltration in the spleen of infected mice (Figure 6), which induces chemoattractant-CXCL1 expression (103). Higher CXCL1 expression is observed in the spleen of EBI3 −/− mice, which causes elevated neutrophil migration at the fourth week post-infection in the spleen and liver.

IL-27 Leads to Host Vulnerability to L. infantum by Reducing the Neutrophil Influx
Neutrophils have diverse mechanisms for combating pathogens, some of which include the production of cytokines and chemokines that lead to inflammatory cell recruitment as well as the release of proteolytic-enzymes/cathepsin/neutrophilelastase and neutrophil extracellular traps (NETs) mediatedpathogen killing (108,109). Host resistance through neutrophils in response to Leishmania depends on both the host's genetic background as well as the invading Leishmania species (63). For example, L. amazonensis promastigotes are eliminated by NETs, while L. major may survive within neutrophils (110). The host's genetic factors also affect neutrophil performance during Leishmaniasis. The neutrophil-defective C57BL/6 mice display a common L. major infection, while neutrophil-defective BALB/c mice exhibit a weak harmful Th2 cell response (111). The neutrophil-defective Genista mice are resistant to L. mexicana that causes non-healing lesions. During VL, neutrophils play a protective role. In C57BL/6 mice during L. infantum infection, IL-17A levels are reduced in the infected organs (103). The downregulation of IL-17A is mediated by IL-27, as EBI3 deficiency leads to intensified IL-17A secretion. Upon IL-17A neutralization, the resistant EBI3 −/− mice become as susceptible as the control wild-type mice, representing the ability of IL-27 to prevent the IL-17A production directly. Moreover, neutrophil infiltration is also reduced after anti-IL-17A treatment, satisfying the role of IL-17A in neutrophil recruitment to infected organs in parallel to the induction of NO production by macrophages (103). The inhibition of IL-17A by IL-27 during L. infantum infection leads to host vulnerability by reducing the neutrophil influx. Although the EBI3-deficiency impairs the Th1 responses in L. infantum-infected mice, other arms of the inflammatory response such as IL-17A and neutrophils limit the parasite load even in the absence of efficient Th1 cell response (103). Based on the discussion above, the regulatory effects of IL-27 during L. infantum infection are shown in Figure 6.
IL-27 and its receptor are expressed on the infected macrophages and DCs during the early phase of L. infantum infection. TLR3-and TLR9-, and IFN-β related signaling pathways play a prominent role in IL-27 production during L. infantum infection (Figure 6). In addition to the mouse strains, other molecules such as soluble CD40L and sCD14 also influence IL-27 production (112). In L. infantum-infected humans with VL, the IL-27 levels are increased in the active phase, and coming back to basal amounts after treatment, represent that this cytokine may be considered as a marker of VL severity or prognosis. IL-27 is a regulatory cytokine that plays a role in susceptibility to L. infantum infection. As Th1-and Th17 cell-related responses confer protection against L. infantum infection (113), IL-27 increases the susceptibility to parasite by interfering with both protective Th1 and Th17 responses.

Role of IL-27 in L. amazonensis Infection
The C57BL/6 mice are vulnerable to L. mexicana or L. amazonensis infection due to compromised Th1 cell responses (114). This inability has been attributed to elevated IL-10 production, which causes insufficient DC activation and IL-12 production. Although Th1 cell-mediated immunity limits the Leishmania infection, the Th1 cell-released cytokines like TNF-α and IFN-γ involve in the immunopathologic reactions. Indeed, elevated TNF-α and IFN-γ quantities were detected in severe CL and ML patients. L. amazonensis causes anergic DCL and CL with disseminated lesions (50). A different modulating role has been proposed for Treg cells in L. amazonensis infection. The Treg transfers from Leishmania-infected mice to naive mice shortly before L. amazonensis infection decreases lesion development signifying that the role of Treg, cells in limiting immunopathological responses (115). The results from an invitro analyses indicate that the expression of both IL-27 subunits, including p28 and EBI3, is augmented in L. amazonensis-infected macrophages from C57BL/6 mice measured at 4 hours after infection (116).

IL-27 Is a Susceptibility Factor During L. amazonensis Infection
In L. amazonensis-infected C57BL/6 mice, the injection of the rIL-27 into the infected footpads (on days 2, 4, and 6 prior infections) strongly enhances the lesion sizes and parasite number in the footpads and the draining lymph nodes at weeks 2 and 3 following infection (116). Although IL-27 signaling contributes to the Th1 cell-related protective response in the L. major-infected mice, the same pathway may enhance the susceptibility to L. amazonensis, perhaps  due to the differences in protein kinase R (PKR)-mediated signaling between L. major-and L. amazonensis-infected macrophages. The PKR-mediated signaling supports L. amazonensis but reduces L. major infection (117). L. amazonensis activates PKR in macrophages for their survival. The PKR activation by poly I:C (a ligand for TLR3) causes IL-10 production, and IL-10 neutralization prevents the parasite expansion (118), suggesting a TLR3-PKR-IL-27-IL-10 axis in L. amazonensis infection.

TLR2-PKR-IFN-1 Signaling Contributes to L. amazonensis Survival in an IL-27 Dependent Manner
It is reported that the addition of rIL-27 to human macrophages infected with L. amazonensis promastigotes augments the parasite propagation and percentage of infected cells measured on days 3 and 4 after the cytokine addition. The IL-27 neutralization or IL-10 receptor blockade in the infected cell cultures markedly reduce the parasite multiplication (116). Hence, the IL-27-related enhancing effects on L. amazonensis survival and growth occur mainly through IL-10. Treatment of the L. amazonensis-infected human macrophages with a PKR inhibitor abrogates the IL-27-mediated L. amazonensis replication. PKR is activated by IL-27, which is crucial to IL-27mediated Leishmania expansion (116). TLR2-deficient C57BL/6 mice are also less susceptible to L. amazonensis infection than wild-type counterparts (63). TLR2 −/− macrophages express low quantities of IFN-β and PKR post-L. amazonensis infection (63). Indeed, L. amazonensis infection leads to the activation of the PKR/IFN-1 axis via a TLR2-dependent manner, which promotes parasite replication in macrophages (116). Interestingly, the Leishmania-induced IL-27 expression also depends on the TLR2 and IFN-1-mediated signaling (119). The expression of IL-27 is profoundly diminished in infected-macrophages derived from TLR2 −/− or IFN-1R −/− mice (116,119). TLR2-PKR-IFN-1 signaling contributes to the IL-27 production in L. amazonensisinfected macrophages that lead to the intracellular survival of the parasite. When produced, IL-27 elicits new cycles of PKR activation, which supports parasite expansion (116). These regulatory effects of IL-27 during L. amazonensis infection are depicted in Figure 7.
The L. amazonensis-infected macrophages may be one of the main sources of IL-27 during the infection. IL-27 profoundly increases the vulnerability L. amazonensis infection, and its promoting effects on the parasite survival and growth occur mainly through an IL-10-dependent manner. Indeed, the TLR2-PKR-IFN-1 axis contributes to the IL-27 production during L. amazonensis infection that supports the survival of the parasite. When produced, IL-27 triggers more PKR activation, which stimulates its production by a positive feedback mechanism. Accordingly, the targeting of TLR2, PKR, and IFN-1 may impair the IL-27 production, which eventually diminishes the L. amazonensis replication.

Role of IL-27 in L. braziliensis Infection
The L. braziliensis-related CL is associated with robust Th1 responses (65,120) conferring protection against the parasite. However, excessive uncontrolled inflammation is responsible for the development of skin lesions and tissue damages, as observed in ML and CL patients (118). Reduced IL-10 production along with the heightened IFN-γ and TNF-α production are positively correlated with lesions sizes and tissue damage in CL and ML (118,121). A high number of cytotoxic cells are also observed in lesions of patients with CL caused by L. braziliensis and correlated with explicit inflammatory stress and cutaneous tissue damage (122). Patients treated with anti-inflammatory drugs show alleviated lesions size, supporting the deleterious role of inflammation during the L. braziliensis infection (65). The PBMCs from L. braziliensis-infected individuals with CL pattern produce higher levels of IFN-γ and TNF-α than cells from patients with subclinical (SC) pattern after in-vitro stimulation with SLA (123). The SLA-stimulated PBMCs from patients with SC tend to express higher amounts of IL-17 compared with patients with CL pattern (124). The addition of rIL-27 to SLA-stimulated PBMCs from patients with CL reduces IFN-γ production (81,124). Further, higher IL-10 levels were detected in L. braziliensis-infected individuals with SC pattern when compared with CL patients (125). Therefore, IL-10 and IL-27 may modulate the deleterious Th1 cell responses in L. braziliensis infection. Hence, the proper expression of the IL-10 and IL-27 may cause an asymptomatic SC form, while the low expression of IL-10 and IL-27 may lead to symptomatic CL pattern in braziliensis-infected individuals (Figure 8).

IL-27 in L. braziliensis Infection
IL-10 and TGF-β, but not IL-27, modulate the expression of the TNF-α, IFN-γ, and IL-17 by SLA-stimulated PBMCs from ML and CL patients (118). IL-27 was unable to enhance IL-10 production by PBMCs from CL and ML patients (81). The PBMCs from CL and ML patients are unresponsive to the immunomodulatory effects of IL-27. In skin biopsy from patients with L. braziliensis-related CL, the increased IL-10 and IL-27 expression were also associated with high IFN-γ expression, although a similar pattern of cytokine expression was not observed in ML group (126). A positive association has been indicated between the IL-10 expression and IL-27, IL-21, and IFN-γ expression but not with the FOXP3 expression in lesions of patients with CL (127). A new subgroup of Treg cells, namely Tr1-like cells with CD4 + CD25 − CD127 −/low FOXP3 − phenotype was identified in the peripheral blood of L. braziliensis-infected patients with CL that produce higher levels of IL-10 (127). The secretion of IL-10 from CD4 + CD25 − FOXP3 − cells in the initial phase may suppress the Leishmanicidal mechanisms of macrophages, which supports the establishment of infection (127). However, in chronic stages, when a strong Th1 response is dominant, Tr1-like cells may help in protecting tissues from damage caused by an inflammatory reaction. The possible IL-27 contribution to the development of the Tr1-like cells needs to be further investigated. The regulatory effects of IL-27 during L. braziliensis infection are shown in Figure 8.
Collectively, the excessive uncontrolled Th1 cell-mediated inflammation is involved in the tissue damages in ML and CL L. braziliensis. The IL-27, in parallel with IL-10, attenuates the deleterious Th1 cell response during the late stage of the L. braziliensis infection (124). However, the precise role of the IL-27 during the early stage of L. braziliensis infection needs more clarification.

CONCLUSION
IL-27 is expressed during the early phase of Leishmania infection, and the infected phagocytes and DCs are the major producers of this cytokine. The role of IL-27 in Leishmania infection is species-dependent ( Table 1). The protective role of IL-27 during the initial stage of L. major infection is exerted mainly through inhibition of IL-4-mediated Th2 cell responses. Therefore, evaluation of the local therapeutic potential of IL-27 or its agonists concerning the L. major-mediated CL requires more investigations. In mouse models of OVAinduced asthma, the intranasal IL-27 administration significantly improves the clinical symptoms of the disease, decreases the local eosinophilia in the nasal mucosa, modulates the cytokine production by Th1, Th2, and Treg cells, and decrease the

L. donovani
WT BALB/c 2.5 × 10 7 AM Intravenously • Administration of the neutralizing anti-EBI3 antibody (and not anti-p28 antibody) to pathogen-infected mice reduces the parasite load in the spleen and liver and increase the TNF-α-and IFN-γ-secreting cells.
L. infantum WT C57BL/6 WT BALB/c 1 × 10 8 PM Intravenously • Serum IL-27 levels are increased early (at 4 days after infection) in the BALB/c mice, but not in C57BL/6 mice. • The splenic DCs from BALB/c mice but not from C57BL/6 mice upregulate the expression of IL-27p28 24 h after infection. • IL-27 secretion by BMDCs from BALB/c was higher compared to C57BL/6, whereas the LPS-stimulated BMDCs from C57BL/6 mice produce more elevated amounts of IL-27 than BALB/c mice. • The administration of rIL-27 to in C57BL/6 increase the production of IL-10, while decrease IFN-γ and IL-12p70, and prevent the infiltration of neutrophils in the spleen at 24 h after treatment in comparison with infected non-treated animals. (Continued) Frontiers in Immunology | www.frontiersin.org • Neutralization of IL-27 in acutely infected BALB/c decreases parasite burdens transiently reduces IL-10 and a transient increase in splenic IFN-γ producing CD4 + and CD8 + T cells.
L. infantum WT C57BL/6 1 × 10 7 PM Intravenously • High levels of the P28 subunit of IL-27 are detected in the spleen and liver at weeks 4 and 6 post-infection. • The expression of both IL-27R subunits, including WSX-1 and gp130, are upregulated at week 4 after infection.
• Mice produce higher IL-17A levels in both spleen and liver.
• Higher CXCL1expression was observed in the spleen, which causes a peak in neutrophil migration at week 4 post-infection in the spleen and liver. • When IL-17 was blocked, the mice become as susceptible as the C57BL/6 control. serum levels of specific IgE (128,129). The effects of such intranasal IL-27 administration need to be investigated in various Leishmania sp. infections. Leishmania skews naive T cells toward Th2 cells for its survival in the host. On the other hand, IL-27 has the capability to polarize naïve T cells to Th1 cells, which plays host protective roles by jeopardizing parasite growth in the host. Nonetheless, IL-27 may suppress the protective immune responses against L. donovani, L. infantum, L. amazonensis, and L. braziliensis. Therefore, T cell-dependent IL-27 functions are diverse, speciesspecific, and contradictory. At the same time, the infection and pathology of the Leishmania species are unique and vary with IL-27 dependency. The targeting of IL-27 or its receptor using blocking monoclonal antibodies, small molecule inhibitors, and siRNA, alone or in combination with other therapeutic agents, needs to be evaluated as promising strategies for the treatment of the L. donovani-, L. infantum-, L. amazonensis-, and L. braziliensis-related complications. IL-27 or IL-27 associated signaling molecules may be considered as exciting novel targets for immunomodulation in Leishmaniasis. Further, the clinical values of IL-27 as a marker of Leishmaniasis severity and/or as a monitoring marker of treatment efficacy need more considerations. It may be underlined that IL-27 possesses the potential to be a valuable biomarker for active human VL and for monitoring the effectiveness of treatment modality.

AUTHOR CONTRIBUTIONS
AJ, MN, and IS conceptualized and drafted the manuscript and Table 1. BS and AS scrutinized the scientific-content, language, grammar, and edited the manuscript. AP and PC contributed additionally to writing and reviewed the manuscript. PC conceptualized, designed, and digitalized the figures. All authors contributed to the article and approved the submitted version.