Leishmaniases are classified as neglected tropical diseases (1, 2) that are caused by Leishmania spp; a vector-borne intracellular trypanosomatid protozoan parasite. This disease is characterized by a spectrum of clinical manifestations ranging from self-healing skin lesions of localized cutaneous leishmaniasis, mucocutaneous leishmaniasis, diffuse cutaneous leishmaniasis, and visceral leishmaniasis depending on Leishmania parasite species and host immune response (3, 4). Between 700,000 to 1 million new cases of leishmaniasis have been reported annually, with approximately 65,000 deaths (5). Additionally, the prevalence of leishmaniasis is estimated to be over 4.13 million globally and about 1.7 billion people are at risk of infection in more than 98 countries worldwide distributed in all continents except Antarctica (6–8). Cutaneous leishmaniasis (CL) mostly and widely occurs in tropical regions of Western and Central Asia, the Mediterranean basin, and America (5).

Several drugs currently in use for the treatment of various forms of leishmaniasis are known to cause side effects and require a long period of hospitalization for intravenous administration (9, 10). Despite sustained efforts, no effective human vaccine is yet available (11–14). A promising alternative is the use of chemoimmunotherapeutic approaches, which have the potential to increase efficacy and decrease the toxicity of antileishmanial drugs thereby extending their lifespan in clinical use (15). Central to this approach is the identification of correlates of immune protection in the host organism during active infection. Since the early ‘90s, the canonical T-helper (Th) cytokines, interleukin (IL)-4 and IL-13, have been infamously regarded as mediators of susceptibility to CL in mice. However, a paradigm change to this archetypal role was reported in 2001, where early administration of IL-4 during CL in BALB/c mice promoted Th1 immune response leading to the control of the disease (16). IL-4 and IL-13 share a common receptor, IL-4Rα, whose effects in CL have been widely studied in L. major (17, 18) infection and to some extent during L. mexicana infection (19). The phenotypic difference of IL-4^-/- and IL-4Rα^-/- from wildtype BALB/c mice after infection with L. mexicana indicated that gene knockout mice control the infection with characteristic smaller lesions and decreased parasite burden (19, 20) thereby suggesting a role for IL-4 and IL-13 in susceptibility to L. mexicana.

To better understand the specific cell subsets in the host that render IL-4Rα^-/- mice resistant to CL, recent studies have shown that deficiency of IL-4Rα signaling on CD4⁺ T cells protects mice from L. major infections but the protection is sex-dependent in L. mexicana-infected BALB/c mice; with controlled infection in female mice, but persistent infection in male mice (21, 22). Further studies in our laboratory have shown that BALB/c mice lacking IL-4Rα signaling in dendritic cells (DCs) were hyper susceptible to cutaneous L. major infection in the footpad (17). These mice had upregulated Th2 responses and impaired macrophage killing capacity, pointing towards the critical role of IL-4Rα signaling on DCs for protection against CL caused by L. major. Mechanistically, this pointed to early IL-4 signaling instructing DC-derived IL-12 production early after infection, which contributes to the host to control infection. Given that IL-4Rα^-/- mice are protected from L. mexicana infection, it could be possible that the absence of DC IL-4Rα signaling in IL-4Rα^-/- mice infected with L. mexicana could be the primary contributor to protection. Thus, it is interesting to investigate the role of DCs in mice with IL-4Rα specific deletion in DCs following L. mexicana infection. Moreover, since the differences in the outcome of CL in BALB/c mice have been shown to depend on the infecting Leishmania species (23), we sought to investigate the importance of IL-4Rα signaling on DCs in the control of L. mexicana infection in BALB/c mice. To this end, we utilized BALB/c mice with a specific deletion of the IL-4Rα on DCs (17). Herein, we describe the clinical phenotype, humoral and cellular responses of BALB/c mice with IL-4Rα deficiency on DCs during experimental footpad infection with L. mexicana promastigotes. In summary, we show that CD11c^creIL-4Rα^-/lox mice exhibited non-healing lesions similar to their littermate controls and unchanged parasite load in the footpad (FP) and lymph nodes (LN). Furthermore, we found no sex-related difference associated with L. mexicana infection in the absence of IL-4Rα expression on DCs, in contrast to that shown for CD4⁺ T cells (21). Importantly, similar type 2 and type 1 antibody production were observed between CD11c^creIL-4Rα^-/lox and littermate IL-4Rα^-/lox mice, conveying a sustained susceptible type 2 humoral responses compared to IL-4Rα^-/lox mice. Surprisingly, although Th2 cytokine production was upregulated in CD11c^creIL-4Rα^-/lox mice, this did not translate to heightened susceptibility. There was unchanged infiltration of myeloid cells (cDCs, macrophages, neutrophils, and monocyte-derived DCs) in the draining LN of CD11c^creIL-4Rα^-/lox and littermate IL-4Rα^-/lox mice. Interestingly, we found that IL-4-mediated DC instruction was unaltered in IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs) compared to IL-4Rα⁺ BMDCs. Accordingly, the loss of IL-4Rα signaling on CD11c⁺ cells did not alter the maturation or activation. nor the levels of IL-12p70, IL-10, nitrite and arginine-derived urea compared to IL-4Rα⁺ BMDCs. These findings, together, suggest that IL-4/IL-13-responsive DCs are dispensable in host resistance against L. mexicana infection. Moreover, IL-4-mediated instruction of DCs during infection appears to be pathogen and strain-specific in the context of L. major (24) and L. mexicana-induced cutaneous leishmaniasis in mice.

The IL-4Rα pathway, a common receptor for IL-4 and IL-13 signaling, is essential in generating anti-inflammatory responses and mediating susceptibility to infection by Leishmania parasites causing CL in mice. In global abrogation of IL-4Rα, the progression of cutaneous leishmaniasis (caused by L. major and L. mexicana) is severely impaired resulting in protection from infection and eventually a healing outcome (18, 19). Hence there is need for studies on the abrogation of IL-4Rα on specific cells to investigate the cell responsible for resistance to CL infection. IL-4 and IL-13 signaling has been implicated in the maturation and activation of myeloid cells leading to the production of Th1 and Th2 cytokines important for eliminating some parasites (17, 41, 42). In addition, exogenous IL-4 has been shown to instruct DCs to produce IL-12 hence driving healing Th1 immune responses to L. major infection in BALB/c mice (16). This was further exemplified in vivo, where the abrogation of IL-4Rα on DCs (CD11c^creIL-4Rα^-/lox) led to the host’s susceptibility to L. major infection, hence indicating a protective role for IL-4Rα signaling on DCs in BALB/c mice (17). Following this study, we aimed to investigate whether IL-4Rα signaling on CD11c⁺ DCs was the main signaling cascade mediating host protection to L. mexicana using CD11c^creIL-4Rα^-/lox mice and controls. To achieve this, we infected these mice with L. mexicana promastigotes in the footpad, and at 8 weeks post infection, we evaluated disease progression, humoral and cellular immune responses.

CD11c^creIL-4Rα^-/lox mice exhibited non-healing footpad swelling and parasite loads, similar to the littermate IL-4Rα^-/lox, suggesting that IL-4Rα responsive DCs do not play a role in modulating host-protection to CL disease caused by L. mexicana. Sex hormones have been shown to impact the immune responses against leishmaniasis (43). Since, opposite sexes have been found to differ in the intensity, prevalence, and pathogenesis in various infections including CL (44, 45) and in relation to IL-4Rα deletion (21), we compared male and female mice deficient for the IL-4Rα on DCs. By and large, we found no sex-related difference associated with L. mexicana infection in the absence of IL-4Rα expression on DCs. It is by this basis we narrowed our investigation to evaluate the humoral and cellular responses in female mice infected with L. mexicana parasites bearing in mind that in previous studies, female mice deficient for IL-4Rα on T cells developed a healing phenotype whilst males were unchanged to littermate controls (21).

We next investigated the humoral immune response to explain unaltered disease progression in CD11c^creIL-4Rα^-/lox and controls. We measured the levels of total IgE and Leishmania specific IgG1, IgG2a/b antibodies. The high levels of IgG1 in CD11c^creIL-4Rα^-/lox and littermate controls compared to IL-4Rα^-/- mice may be the causative reason why CD11c^creIL-4Rα^-/lox mice and their littermate controls were unable to control lesion development. In support, higher titres of IgG1 are present in patients with active CL disease (46). Previous studies built on this evidence and reported that during L. mexicana infection, IgG1 preferentially binds to FcγRIII on macrophages thereby suppressing protective immunity, likely through macrophage IL-10 production (47). In addition, other studies have shown that the absence of IgG1 in L. major infected mice results in smaller lesions with controlled parasite proliferation (48). Antibody production is dependent on the differentiation of B cells into plasma cells (49). It is therefore not surprising that B cells in totality also do contribute to the host’s susceptibility and are therefore used as correlates of host susceptibility (50). For instance, we show that L. mexicana resistant mice (IL-4Rα^-/-) have decreased population of B cells whilst susceptible CD11c^creIL-4Rα^-/lox mice and controls have higher numbers. We hypothesize that majority of plasma B cells in CD11c^creIL-4Rα^-/lox and their littermate controls may be tailored towards IgG1 and IgE antibody production and not protective IgG2a/b antibodies. In summary, our data demonstrate that IL-4Rα signaling on DCs does not lead to events that alter the production of IgG1 and IgE antibodies associated with susceptibility.

IL-4 and IL-13 bind to type I/II receptor that is constitutively expressed on B cells in both mice and humans (51) to induce IgE and IgG1 class switching (52). In our study, higher levels of IL-4 and IL-13 by LN cells stimulated with α-CD3 and heat killed L. mexicana promastigotes in CD11c^creIL-4Rα^-/lox mice did not correlate with heightened levels of IgG1 and IgE compared to littermate controls. Surprisingly, however, the increased Th2 cytokines in CD11c^creIL-4Rα^-/lox mice did not tilt IgG1 production over littermate controls. The same applies to total IgE, which was significantly lower in IL-4Rα^-/- mice but increased in CD11c^creIL-4Rα^-/lox mice highlighting its systemic role in susceptibility in L. mexicana infected mice. Regarding the latter, a clinical study conducted on patients from Brazil and Iraq revealed that high levels of IgE antibodies are associated with disease activity in CL (53, 54). The low levels of type 1 antibodies, namely, IgG2a and IgG2b, exhibited in CD11c^creIL-4Rα^-/lox and IL-4Rα^-/lox mice also further demonstrate the systemic inability of these mice to control the infection as type 1 antibodies have been associated with parasite clearance (17, 32). In this context, a previous study showed that interaction of mouse IgG1-amastigote immune complexes with FcγRIII on macrophages is detrimental in L. mexicana infections, but not with interactions involving IgG2a/c (28). Interestingly, IgG2a facilitates the killing of L. amazonensis in macrophages (32). Due to the central role of follicular DCs within the germinal center (GC), we speculate that loss of IL-4Rα signaling on DCs did not alter isotype class-switching during GC formation hence no differences in B cell differentiation to plasma cells that could lead to changes in antibody titres (55–57).

The presence of CD4⁺ T cells is important in the control of CL as shown in L. braziliensis and L. major infection (37). We demonstrated that the loss of IL-4Rα signaling on DCs leads to heightened absolute numbers of CD4⁺ and CD8⁺ T cells. Surprisingly, a negative feedback loop appeared to exist in the activation of T cells and IL-4Rα signaling on CD11c⁺ DCs, with higher levels observed in the absence of IL-4Rα⁺ DCs. Notably, the loss of IL-4Rα signaling on CD4⁺ T cells has been shown to protect BALB/c female mice from L. mexicana infection (21).

Central memory T cells (T_CM), are long-lived cells that can survive without the presence of the parasite and can transform into effector memory cells upon secondary infection (58). Our data reveal an increased absolute T_CM cell number in CD11c^creIL-4Rα^-/lox mice suggesting that these mice have long-term memory hence, they are likely to respond effectively in case of secondary infection with L. mexicana. Deletion of IL-4Rα on DCs had no consequences on the infiltration of T cells with naïve phenotype into LNs cells with no prior encounter with L. mexicana. Naïve T-cells can differentiate into effector memory T cells (T_EM) and T_CM to facilitate their function in immune responses against infections (59). We also suspect that the interaction between CD4⁺ and CD8⁺ T cells is important in disease control vs susceptibility (60). In support, bystander CD8⁺ T cells can also contribute to a harmful role in CL disease progression (61). On the other hand, in L. major infections, CD8⁺ T cells play a vital role in protection (62). In this regard, our results clearly show an increased population of CD8⁺ T cells in LNs of CD11c^creIL-4Rα^-/lox and IL-4Rα^-/lox female mice compared to resistant IL-4Rα^-/- mice, suggesting that CD8⁺ T cells perhaps plays a role in the immunopathogenesis of CL caused by L. mexicana infection. In an alternate hypothesis, expansion of CD8⁺ T cells in CD11c^creIL-4Rα^-/lox mice, and their subsequent activation, could lead to degranulation and release of perforin and cytotoxic granzyme effectors, thereby promoting disease severity and mediating immunopathology, as shown in L. braziliensis-infected mice (63–65). In parallel, it may be possible that perforin-mediated attack of L. mexicana-infected cells prevented hypersusceptibility, contrary to that seen in L. major infection (17). Alternatively, this may be either through cell lysis, leading to parasite dissemination, or onset of apoptosis and when apoptotic cells are not cleared by phagocytes, this could lead to an increase in the inflammatory response (66). CD8⁺ T cell effectors generated during L. mexicana infection may have tilted to cytotoxic T lymphocytes (CTLs) exhibiting a cytolytic activity and/or lysis of infected cells without killing the parasites hence promoting disease severity, perhaps through granzyme B production, perforins, and IL-1β/inflammasome activation (63, 67). This finding suggests that CD8⁺ T cells may play a minimal role in the protection of the host from L. mexicana infections indicating that their presence may result in increased pathology (63). Therefore, an understanding of the mechanisms that drive CD8⁺ T cells to become pathogenic or protective depending on Leishmania species, will provide interesting insights.

IFN-γ is unambiguously a host-protector in CL (33). Indeed, studies have shown evidence that IFN-γ secretion by macrophages and DCs fuel a rapid escalation of nitric oxide (NO) production that induces parasite killing (68, 69). Our data show that loss of IL-4Rα signaling on CD11c⁺ DCs does not lead to altered IFN-γ production by CD8⁺ T cells but slight increases in CD4⁺ T cells were observed in the pLN. This may provide an explanation as to why no differences were observed in the footpad lesion progression between CD11c^creIL-4Rα^-/lox and IL-4Rα^-/lox mice even though levels of IL-4 and IL-13 were heightened. Our data, therefore, suggests that the effector functions of IFN-γ on macrophages and DCs remained unchanged by the presence or absence of IL-4Rα signaling on CD11c⁺ DCs. Indeed, it has been documented that the modulation of Type 2 immune responses in murine models of leishmaniasis is more important in the balance of resistance and susceptibility, rather than the Type 1 immune responses. However, the data herein throws caution to this dogma as the heightened IL-4 and IL-13 in CD11c^creIL-4Rα^-/lox did not equate to hyper-susceptibility as previously reported (17).

Innate cells such as macrophages, neutrophils, and DCs are the first line of defense before adaptive immunity comes into action. Surprisingly, these cells may favor parasite survival hence enhancing disease severity (70, 71). The absence of IL-4Rα signaling on DCs did not affect the infiltration of these innate cells into LN. However, the increased levels in CD11c^creIL-4Rα^-/lox mice, as compared to resistant IL-4Rα^-/-, mice suggest that these cells may play a role in susceptibility to L. mexicana, perhaps as host cells that favor replication of parasites (71), and thus investigation of their effector responses could be important in controlling CL. The effect of IL-4 in neutrophil counts and migration has been highlighted during bacterial infections (72). This study particularly demonstrated that mice treated with IL-4 have reduced neutrophil migration possibly by affecting CXCR2-CXCR4 chemokine signaling. In our study, we have demonstrated that the absence of IL-4Rα signaling on DCs leads to increased IL-4 cytokines following α-CD3 stimulation, but this did not reduce the infiltration of neutrophils into LN from circulation during L. mexicana infection. We thus suspect that the effects of IL-4 on neutrophil migration into the LN may have been counteracted by the presence of the parasite. Transient expression of IL-13Rα2 on DCs may have inhibited IL-4 mediated signaling in littermate controls hence similar myeloid infiltration consequently leading to similar disease phenotype to CD11c^creIL-4Rα^-/lox (73). It would therefore be interesting to investigate IL-13Rα2 levels in these animals in future studies. Neutrophils may have facilitated the silent entry of parasites into macrophages in a “Trojan Horse” model hence enhancing the infection (74). Considering absolute cell numbers, increased DC population in CD11c^creIL-4Rα^-/lox mice appears not to reflect in disease control but susceptibility, which suggests that either the loss of IL-4Rα signaling on CD11c⁺ DCs may lead to events that do not cause polarization of these cells to produce pro-inflammatory mediators or signaling in other myeloid cells may be vital in protection against L. mexicana infection. More importantly, the loss of IL-4Rα signaling on CD11c⁺ DCs did not affect the production of nitrite and urea (in ex vivo infection of BMDCs), which are known substrate for iNOS and arginine-derived metabolite respectively. Specifically, L. mexicana infected DCs appear to impair cellular and immunological mechanisms hence leading to the inability to resolve cutaneous disease (39).

Conventional DCs (cDCs) are specialized in uptake of Leishmania parasites, process, and present parasite-derived antigens to T cells. More importantly, the cDC1 identified by their CD8α expression as opposed to cDC2, cross-present antigens to CD8⁺ T cells (75, 76). In addition, cDC1 are IL-12p70 producers hence they are less permissive to Leishmania infection unlike cDC2 cells that skew CD4 T cell differentiation to Th2 cells (77, 78). Therefore, in our study it is more likely that the cDCs may have majorly been of cDC2 subset, given the increase in IL-4 and IL-13 production by CD4⁺ T cells in the dLN of CD11c^creIL-4Rα^-/lox mice during L. mexicana infection.

Inflammatory DCs, also known as monocyte-derived DCs (mo-DCs) expand during L. major infections and been shown to harbour parasites in the lymph node of DC-IL-4Rα deficient mice (24). Analysis on the Mo-DCs revealed unchanged frequencies regardless of the presence or absence of IL-4Rα signaling on DCs. But the IL-4Rα^-/- mice that controlled L. mexicana infection had decreased frequencies of these cells, demonstrating the vital role they may play in susceptibility to L. mexicana infections. In addition, Mo-DCs may have enhanced the differentiation of memory CD8⁺ T cells in both CD11c^creIL-4Rα^-/lox and their littermate controls compared to global IL-4Rα^-/- mice (79).

We also sought to investigate whether the absence of IL-4 signaling in CD11c^creIL-4Rα^-/lox mice affected IL-4-mediated DC instruction in terms of IL-12p70 and IL-10 cytokine production (16, 36). Surprisingly, the amount of IL-12p70 and IL-10 production by BMDCs was unchanged between the two groups. This observation corroborated with a similar expression of maturation and activation markers such as MHCII and CD80 on CD11c⁺ cells respectively when stimulated with IL-4 (76). Together, we conclude that the loss of IL-4Rα signaling on DCs does not affect their maturation and activation, in addition to unaltered IL-4-DC instruction in IL-12 and IL-10 production. These findings may suggest why we observed similar disease outcome between CD11c^creIL-4Rα^-/lox mice and their littermates during L. mexicana infection.

In summary, our data suggest that IL-4Rα signaling on CD11c⁺ DCs does not have an essential role in the control of CL caused by L. mexicana, irrespective of sex and with regard to lack of differences in both humoral and Th1 immune responses. Interestingly, the route or site of infection has been shown to significantly influence the outcome of CL (80, 81), thus it will be interesting to decipher the phenotype exhibited following an intradermal infection with L. mexicana. Surprisingly, Type 2 immune responses, in the absence of IL-4Rα signaling on DCs, do increase substantially, yet disease outcome remains similar compared to littermate control. This is paradoxical to some extent because, in the same mouse strain, disease severity is enhanced during L. major infection, with a characteristic increase in IL-4 (17). Altogether, this may suggest that the outcome of IL-4-mediated instruction of DCs in vivo may be species-specific with respect to CL-causing species.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Faculty of Health Science, Animal Research Ethics Committee, University of Cape Town.

Conceptualization: RH and FB. Methodology and investigation: BOO, RA, RH, ZC, RM, MO, and MBL. Writing, Review, and Editing: BOO, MBL, RH, and FB. Funding Acquisition: RH and FB. Supervision: RH and FB. GM-CSF cell line: MBL. Cell-specific gene-deficient mice: FB. All authors contributed to the article and approved the submitted version.

This research was supported by funds from the National Research Foundation (grant numbers: 113446 and 120407), University of Cape Town, Research, and Innovation: 2019 Enabling Grant Seeker Excellence Award and Poliomyelitis Research Foundation (grant number: 19/12), under RH. Also, financial support from the National Research Foundation (NRF) of South Africa, Department of Science and Technology (DST), South African Research Chair Initiative, International Center for Genetic Engineering and Biotechnology, and Wellcome CIDRI-Africa (grant number: 203135/Z/16/Z) under FB.

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.

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