T cell intrinsic role for galectin-1 during *L. donovani* infection, we generated another set of BM chimeras that comprised a 90:10 mix of WT (CD45.1) and *Lgals1−/−* cells. This allowed *Lgals1−/−* CD4+ T cell activity to be measured in a predominantly WT immunological background and compared with appropriate WT control CD4+ T cells following infection (**Figure 8A**). We found a small, but significant (*p* < 0.05) difference in the frequency of hepatic IFNγ-producing *Lgals1−/−* CD4+ T cells, compared to WT CD4+ T cells, but no difference in Tr1 cell frequency in the 90:10 chimeras after 14 days of *L. donovani* infection (**Figure 8B**). However, we found an increased frequency of galectin-1-deficient hepatic Treg cells (**Figure 8B**), suggesting that similar galectin-1-mediated regulatory circuits established during acute *T. cruzi* infection (43) were unlikely to explain improved control of parasite growth in *L. donovani*-infected *Lgals1−/−* mice. Of note, despite engrafting mice with a 90:10 mix of WT and *Lgals1−/−* BM, respectively, we consistently found that *Lgals1−/−* leukocytes comprised 20–25% of the immune cell compartment (**Figure 8A**), although the ratio of CD4+ T cells in WT and *Lgals1−/−* compartments remained the same (approximately 30%). Together, these results indicate the enhanced CD4+ T cell IFNγ response observed in the liver of *Lgals1−/−* animals involved direct galectin-1 signaling to CD4+ T cells. Furthermore, the lack of effect on hepatic CD4+ T cell IFNγ production in mice with galectin-1-deficient DC (i.e., DT-treated CD11c.DOG:*Lgals1−/−* chimeras), compared to controls (**Figure 7C**), indicated that direct galectin-1 signaling by DCs was not responsible for the increased hepatic CD4+ T cell IFNγ production observed in *Lgals1−/−* mice (**Figure 5C**).

#### DISCUSSION

A substantial amount of work on the immunoregulatory activity of galectin-1, and indeed the other members of the galectin family, has been conducted in the context of cancer and associated illnesses (20). As a result, the anti-inflammatory properties of galectin-1 have been well described (28, 29, 52). Thus, the primary focus of this study was to outline the contribution of galectin-1 signaling during experimental VL, with specific focus on the involvement of galectin-1 in CD4+ T cell IFNγ and IL-10 production. Our studies centered on CD4+ T cells because we and others have previously identified CD4+ T cells, but not CD8+ T cells, to be critical for control of *L. donovani* growth during primary infection (47, 53). CD8+ T cells are important for protection against re-infection the liver (53), and protection in some vaccination strategies (54, 55). Here, we report that galectin-1 plays a limited role in promoting IL-10 production by CD4+ T cells during *L. donovani* infection. However, we found galectin-1-mediated suppression of effector cytokine production by hepatic Th1 cells following infection. This finding was at odds with the proposed immunoregulatory network involving galectin-1 outlined by others both *in vitro* (29), and more recently *in vivo* (43). The latter study reported that *Lgals1−/−* mice had reduced mortality and parasite burden following *T. cruzi* infection, compared to WT controls. However, protection was associated with disruption of an immunoregulatory mechanism involving DC-mediated expansion of Treg cells (43). We found the impact of galectin-1 signaling on Foxp3+ Treg cell IL-10

Mann–Whitney test, relative to naïve (A,B); Mann–Whitney test between IL-10− IFNγ+ and IL-10+ IFNγ+ CD4+ T cells only is shown (C,D)].

production was minimal in experimental VL. Given galectin-1's role in promoting Treg cell suppressive functions (43), our results indicate either a different role for galectin-1 in hepatic parasite clearance during *L. donovani* infection or an IL-10-independent mechanism of galectin-1-mediated Treg cell modulation. Our data from BM chimeric mice showed increased frequencies of Treg cells when either DCs or Treg cells lacked galectin-1 expression. This finding is at odds with what has been reported during acute *T. cruzi* infection (43), where DC galectin-1 expression promoted Treg cell function, and suggests the distinct roles for galectin-1 in different inflammatory settings. In addition, Treg cells have previously been shown to use multiple mechanisms of immune suppression, including TGFβ, IL-35, and CTLA4 (56, 57), and the impact of galectin-1 deficiency on these features of Treg cell functions during experimental VL still needs to be addressed. Another unexplored galectin-1-mediated mechanism of immune regulation in our study was the induction of apoptosis. Again, this needs examination, given the initial reports on the role of galectin-1 in T cell apoptosis (25). Similarly, we have previously reported that γδ T cell-derived IL-17A suppressed early hepatic antiparasitic immunity following *L. donovani* infection (10, 47, 49), and although we have never observed IL-10 production by these cells, we cannot exclude a role for galectin-1 in this response.

Despite the limited impact of galectin-1 deficiency on Tr1 cell development during *L. donovani* infection, Th1 cell numbers in the liver were increased in *Lgals1−/−* mice 14 days after *L. donovani* infection, and this correlated with decreased parasite load during acute hepatic infection. Therefore, the action of galectin-1 may depend on the extent of antigen availability and consequent T cell activation status, rather than the induction of IL-10 during experimental VL. Furthermore, changes in galectin-1 expression by various immune cells were most prominent in the liver during the first 14 days of infection, which may have contributed to the tissue-specific impact on parasite control we observed. Our findings with mixed BM chimeras indicate that galectin-1 influences IFNγ production by CD4+ T cells during the acute

phase of hepatic *L. donovani* infection in a cell intrinsic manner. Previous studies (30, 43) suggest that DCs mediate immunoregulatory effects of galectin-1. This notion was supported by improved parasite clearance early during infection in the liver in *Lgals1−/−* animals, at a time when effector CD4+ T cell responses were being initiated. However, depletion of DC in the presence of either WT or galectin-1-deficient cells did not influence effector T cell function following *L. donovani* infection. Therefore, our data indicate that DC-expressed galectin-1 is unlikely to contribute to the augmented Th1 responses observed in the liver of *Lgals1−/−* animals. However, we cannot exclude the possibility that galectin-1 expressed by other potential antigen-presenting cells, such as macrophages and/or monocytes, might influence T cells responses during experimental VL, an idea supported by the high levels of galectin-1 expression by these cells in the liver following infection. In addition, distinct requirements for DC activity in specific tissue sites by discrete DC subsets in cancer models and *T. cruzi* infection may also help explain lack of requirement for DC-dependent galectin-1-mediated immune regulation in experimental VL.

It is not clear why galectin-1 is expressed on multiple immune cell populations in the liver during *L. donovani* infection, and only influences parasite control in this tissue at the acute stage of infection. One potential explanation is that galectin-1 is preferentially expressed by migrating CD4+ T cell populations that would be expected to be more prominent in the liver during experimental VL. Alternatively, the extent of immune dysregulation in the chronically infected spleen means that abrogation of galectin-1 activity is not, by itself, enough to overcome the *status quo* of impaired cellular responses in this organ. Conversely, the liver, with its delicate balance of pro- and anti-inflammatory responses (4, 50, 51), could be susceptible to more subtle alterations in the immunoregulatory environment. One caveat that must be highlighted, however, is that the flow cytometric analysis of galectin-1 expression in our study does not discern between dimeric and monomeric galectin-1, with obvious implications for interpretation of function. Thus, the mechanism by which galectin-1 impairs parasite clearance remains unclear. Our data indicate a CD4+ T cell intrinsic role for galectin-1 in suppressing IFNγ production. As mentioned previously, monomeric galectin-1 can modulate cell functions by interacting with Ras family proteins (22), and given that suppression of Ras protein activity by farnesyltransferase inhibitors caused reduced IFNγ production by mouse and human CD4+ T cells (58), the manipulation of Ras protein by galectin-1 in CD4+ T cells is one possible mechanism for inhibition of IFNγ production in *L. donovani*infected mice. An important finding in our studies was that *Lgals1−/−* mice exhibit improved parasite clearance prior to the establishment of strong effector T cell activity (day 7 p.i.), thus indicating a broader immunoregulatory potential of galectin-1,

and the extent of which appears to depend on the infected tissue. Both the precise mechanism of galectin-1-mediated suppression of IFNγ production by CD4+ T cells and the broader roles of galectin-1 on other immune cells require further study. One approach could be to block CD45R, as this has previously been shown to be required for galectin-1-mediated functions (25), and might provide novel insights to the mechanism of action of galectin-1 in experimental VL.

In summary, we demonstrate that galectin-1 is an important immunoregulatory molecule in experimental VL. Unexpectedly, galectin-1 did not appear to play a major role in Tr1 cell development or function. The main effect of galectin-1 on anti-parasitic immunity appeared to the suppression of Th1 cells. However, the relatively early effects of galectin-1-deficiency during infection also suggest effects of galectin-1 on innate cell activity, although this is unlikely to be on DCs. Together, these findings provide new insights into the role of galectin-1 in modulating immunity during infectious diseases and highlight both tissue- and diseasespecific roles for this molecule.

#### ETHICS STATEMENT

All animal procedures were conducted with the approval of the QIMR Animal Ethics Committee under the animal ethics number A02-634M and in accordance with the "Australian Code of Practice for the Care and Use of Animals for Scientific Purposes" (Australian NHMRC, Canberra).

# AUTHOR CONTRIBUTIONS

PB, MO, and CRE designed, performed, and analyzed the work, and wrote the paper. FR, RK, CLE, RF, SN, MS, YW, FA, and AH performed the work and analyzed data.

# ACKNOWLEDGMENTS

We thank staff in the QIMR Berghofer flow cytometry laboratory for assistance and staff in the QIMR animal facility for animal husbandry.

# FUNDING

This work was made possible through Queensland State Government funding. The research was supported by grants and fellowships from the National Health and Medical Research Council of Australia, as well as an Australian Post-graduate Awards through Griffith University and the University of Queensland, School of Medicine, and an INSPIRE Fellowship to Rajiv Kumar provided by the Indian government Department of Science and Technology.

# REFERENCES

lymphoproliferative disorders. *Blood* (2011) 117:4315–22. doi:10.1182/ blood-2010-11-320481

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

*Copyright © 2017 Bunn, Montes de Oca, Rivera, Kumar, Edwards, Faleiro, Ng, Sheel, Wang, Amante, Haque and Engwerda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The equivocal Role of Th17 Cells and Neutrophils on immunopathogenesis of Leishmaniasis

*Suênia da C. Gonçalves-de-Albuquerque\*, Rômulo Pessoa-e-Silva, Lays A. M. Trajano-Silva, Tayná Correia de Goes, Rayana C. S. de Morais, Cíntia N. da C. Oliveira, Virgínia M. B. de Lorena and Milena de Paiva-Cavalcanti\**

*Department of Microbiology, Aggeu Magalhães Research Center, Oswaldo Cruz Foundation, Recife, Pernambuco, Brazil*

#### *Edited by:*

*Heinrich Korner, University of Tasmania, Australia*

#### *Reviewed by:*

*Hira Nakhasi, Center for Biologics Evaluation and Research (FDA), United States Julia Walochnik, Medical University of Vienna, Austria*

#### *\*Correspondence:*

*Milena de Paiva-Cavalcanti<EMAIL_ADDRESS>Suênia da C. Gonçalves-de-Albuquerque<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 03 July 2017 Accepted: 16 October 2017 Published: 30 October 2017*

#### *Citation:*

*Gonçalves-de-Albuquerque SdC, Pessoa-e-Silva R, Trajano-Silva LAM, de Goes TC, de Morais RCS, da C. Oliveira CN, de Lorena VMB and de Paiva-Cavalcanti M (2017) The Equivocal Role of Th17 Cells and Neutrophils on Immunopathogenesis of Leishmaniasis. Front. Immunol. 8:1437. doi: 10.3389/fimmu.2017.01437*

Advances in the understanding of leishmaniasis progression indicate that cellular interactions more complex than the Th1/Th2 paradigm define the course of infection. Th17 cells are a crucial modulator of adaptive immunity against *Leishmania* parasites acting mainly on neutrophil recruitment and playing a dual role at the site of infection. This review describes the roles of both these cell types in linking innate defense responses to the establishment of specific immunity. We focus on the Th17–neutrophil interaction as a crucial component of anti-*Leishmania* immunity, and the clinical evolution of cutaneous or visceral leishmaniasis. To date, information obtained through experimental models and patient evaluations suggests that the influence of the presence of interleukin (IL)-17 (the main cytokine produced by Th17 cells) and neutrophils during *Leishmania* infections is strictly dependent on the tissue (skin or liver/spleen) and parasite species. Also, the time at which neutrophils are recruited, and the persistence of IL-17 in the infection microenvironment, may also be significant. A clearer understanding of these interactions will enable better measurement of the influence of IL-17 and its regulators, and contribute to the identification of disease/resistance biomarkers.

Keywords: cutaneous leishmaniasis, visceral leishmaniasis, immunity, T helper 17, interleukin-17, neutrophil, immunopathogenesis

# INTRODUCTION

*Leishmania* parasites are transmitted to mammals by female phlebotomine sandflies and cause a group of diseases with symptoms defined mainly by the parasite species and the host's ability to develop and control immune responses (1). Sickness results from uncontrolled infection, and the wide spectrum of clinical manifestations (from healing cutaneous lesions to fatal visceral infections) is associated with the parasite species involved, among other factors (2). The main clinical forms are cutaneous (e.g., *Leishmania major*, *Leishmania tropica*, *Leishmania mexicana*, and *Leishmania braziliensis*), mucocutaneous (e.g., *L. braziliensis*), and visceral (*Leishmania donovani* and *Leishmania infantum*) leishmaniasis (3). Visceral leishmaniasis (VL) is the most severe form and may compromise important organs and tissues, such as the liver, spleen, bone marrow, and lymph nodes, leading to hepatosplenomegaly, lymphadenopathy, anemia, constant fever, and immunosuppression (4). Cutaneous leishmaniasis (CL) is characterized in most cases by well-delimited ulcerated skin lesions with raised borders (5). Mucocutaneous leishmaniasis (ML) usually compromises mucosal regions of the nose, mouth, and pharynx, and is associated with disfiguring and psychosocial sequelae (6, 7). There are estimated to be 12 million people currently living with leishmaniasis, and the World Health Organization estimates that there are approximately 0.7–1.2 million new cases of CL and 0.2–0.4 million cases of VL per year (6). While CL and ML are associated with social stigma, VL may result in fatality rates of 10–20% if not treated (6).

The parasites have a digenetic lifecycle and two distinct morphologies: the promastigote in the sandfly vector midgut, and the amastigote in mammalian host phagocytes. The motile, flagellated promastigotes exist, multiply, and develop extracellularly in the alimentary tract of blood-sucking female sandflies and are transmitted into the mammalian host during blood meals. Inside the host, they infect macrophages in the reticuloendothelial tissue and differentiate into non-motile amastigotes, and multiply as such in phagolysosomal vacuoles (8).

The interaction of inflammatory and regulatory responses delimited by cell-mediated immune responses drives disease expression and may result in asymptomatic infection, selfhealing, or chronic leishmaniasis (1). Pathogenesis follows a complex set of interactions between factors triggered by the host's innate and acquired immune responses, which are strongly influenced by some aspects such as the host's genetic background (9, 10), infecting species, dose and route of inoculation (1), and sandfly saliva components (8). Only 2–3% of individuals infected with parasites of the *Viannia* subgenus (*L. braziliensis*, *Leishmania guyanensis*, *Leishmania panamensis*, and *Leishmania peruviana*) develop skin lesions (11) and less than 10% of those infected with *L. donovani* or *L. infantum* develop active VL (12). The relationship established between *Leishmania* parasites and the vertebrate host can lead to a self-healing infection or to the clinical manifestations of leishmaniasis with different severity grades (4). The inflammatory responses mediate disease presentation and, in both forms of the disease, the different clinical manifestations are a function of parasite replication and the efficacy of the immune response generated (5). Clinical cure is associated with the activation of macrophages into a leishmanicidal state mediated by some specific proinflammatory cytokines (1). However, there is as yet no consensus on the mechanisms that lead to susceptibility in humans.

Cellular immunity generated by the T helper type 1 profile is considered to be the key mediator of resistance to *Leishmania*. A protective immune response against CL caused by *L. major*, *L. mexicana*, or *Leishmania amazonensis*, as well as VL caused by *L. donovani* or *L. infantum*, depends on the development of the proinflammatory T cell profile (13). However, exacerbated Th1 responses may cause severe tissue damage and are also responsible for the clinical presentation of the disease. High levels of Th1 cytokines in people with localized CL lesions are associated with disease outcome, because individuals infected with *L. braziliensis*, who do not develop the disease, produce less interferon (IFN) γ and tumor necrosis factor (TNF) (14). *L. braziliensis* infections are characterized by excessive production of proinflammatory cytokines such as IFNγ, TNF and interleukin (IL)-6 (12) and have lower levels of Foxp3+ cells (regulatory lineages) and IL-10 than those infected by other *Leishmania* species (15). In fact, the absence of IL-10-mediated regulation of Th1 is more significant in disease development due to *L. braziliensis* infection than the Th2 polarization itself (16). Indeed, high levels of IL-4 are not observed in patients with severe diffuse CL, suggesting Th2 responses may have less influence on disease progression in humans than in animal models (17).

Interleukin-10 is an important regulatory cytokine that inhibits phagocytosis and affects the ability of macrophages to kill intracellular parasites, contributing to the growth and spread of *Leishmania* (18). IL-10 sources have been identified as CD4+/ CD25+ T cells (Th2) (19, 20), CD4+/CD25−/FoxP3+ regulatory T cells (Tregs) (21), and CD4+/CD25−/FoxP3− T cells (Th1) (16), stimulated by the recognition of amastigote antibodies (22). Together with other cytokines from these regulatory populations, IL-10 plays a central role in promoting an alternative kind of macrophage activation, which increases arginase expression and facilitates parasite expansion (13).

A successful cellular immune response, which allows parasite elimination without tissue damage, requires a balance between the set of cytokines secreted in the cellular response, beginning with the recognition of the parasite by antigen-presenting cells (APCs) and antigen presentation, the production of Th1 differentiation cytokines, the activation of IFNγ/TNF-producing Th1 cells, and classical macrophage activation (23), ending with the activation of Th2 cells, which mainly secrete IL-10 and block nitric oxide (NO) production by macrophages (13). However, even in the presence of effector molecules such as IFNγ, TNF, and NO during active disease in humans, parasite multiplication persists in many cases. Ansari et al. (24) suggested that despite high IFNγ levels during *L. donovani* infection, the host fails to control the disease due to an incomplete response to IFNγ. Similarly, high titers of TNF have been observed in sera from patients with active VL and diffuse CL, characterized by high parasite loads (25). The biological network acting for equilibrium between parasite clearance and tissue preservation in humans involves substantial participation from effector T cells other than Th1 and Th2 (26).

Helper T cell responses are now known to include four T cell subsets: Th1, Th2, Th17, and Tregs. Th17 cells have recently emerged as an independent T cell subset that may play an essential role in protecting against certain extracellular pathogens (27). However, this relatively newly discovered (28) T cell population has been demonstrated to influence the balance between inflammatory and anti-inflammatory cytokines, which must be orchestrated in the course of infection to guide a successful effector response to the intracellular *Leishmania* protozoa (13). Particular interest in the Th17 type remains focused on its main cytokine, IL-17. Although it is also produced by other cells including CD8+ T cells and neutrophils, IL-17 is mostly produced by Th17 cells. IL-17 is part of a complex mechanistic web that involves up- and downregulation of anti- and proinflammatory cytokines as well as interferences in the genetic background of the host. Furthermore, IL-17 performs an important function in neutrophil recruitment (29). Since neutrophils are regarded as an important element during *Leishmania* infection (30–33), it would be reasonable to consider that Th17 cells might have a significant role in the complex diseases caused by these parasites. The crosstalk between the neutrophils and APCs present at the site of infection contributes to the type and magnitude of the specific immune response that will develop (30, 34). Nevertheless, at the site of infection, the protecting or damaging role of the neutrophil depends on the parasite species, host, and phase of infection (35). Thus, the effects of Th17/IL-17 are still unclear, as disease-promoting and protective responses have both been attributed to their influence. In this scenario, the Th17 cell activities in the context of the different clinical forms of leishmaniasis, interaction with regulatory cytokines, as well as host particularities, remain to be explored (13, 36).

Although many excellent reviews have discussed the cellular immune responses elicited against the different forms of leishmaniasis (35, 37, 38) or the roles of Th17/IL-17 in infectious and non-infectious diseases (39–42), reviews of the participation of IL-17 and Th17 participation in immunopathogenesis and control of the main clinical forms of leishmaniasis remain scarce (36). In the present review, we identify the Th17–neutrophil interaction as a crucial component of anti-*Leishmania* immunity. A better understanding of immune complexity will contribute to the identification of disease/resistance biomarkers and influence the development of vaccines and immunotherapies for leishmaniasis.

#### Th17 CELLS AND IL-17

The recent recognition of Th17 cells has provided new insights into the mechanisms that are important in autoimmune diseases and antimicrobial host defenses (28, 43). Th17 cells represent a subset of CD4 effector T cells distinct from Th1 and Th2 lineages, and mediate powerful effects on stromal cells; this results in the production of inflammatory cytokines and recruitment of leukocytes, especially neutrophils, creating a link between innate and adaptive immunity (29). *In vivo* studies indicate that IL-17 is an especially potent activator of neutrophils, both through expansion of the lineage and through their recruitment *via* chemokine expression regulation (44). IL-17 cooperates with other cytokines secreted by Th17 (such as IL-17F,1 IL-21, and IL-22) to induce tissue inflammation, leading to different effector functions depending on the pathogen (47).

Th17 cell differentiation in both humans and mice is mediated by the activation of naïve T cells in the presence of a combination of TGF-β, IL-6, IL-1β, and IL-23 (48, 49). IL-6 acts in concert with TGF-β to induce the development of Th17 effector cells. Together, TGF-β and IL-21 upregulate the IL-23 and IL-23 receptors, a decisive step in the full differentiation and maintenance of Th17 cells (50). Although the requirement of TGF-β for the classical generation of Th17 cells *in vitro* and *in vivo* has been demonstrated, as well as the endogenous production of TGFβ for autocrine stimulation (51), it has also been shown that Th17 cells can be generated independently of TGF-β (52), in an alternative manner. Thus, the availability of IL-23 and/or TGF-β has emerged as a determinant of Th17 effector phenotypes; in other words, an abundance of TGF-β with a relative lack of IL-23 favors the generation of "classical" Th17 cells, while the presence of IL-23 alone promotes the generation of "alternative" Th17 cells. Transcription factors that are differentially expressed in "classical" vs. "alternative" Th17 cells will cause differential cytokine expression and effector functions. When "classical" Th17 cells were compared with alternatively induced Th17 cells, the "alternative" Th17 cells were more pathogenic (51, 52) (**Figure 1**).

Th17 differentiation is dependent on the steroid receptortype nuclear receptor (RORγt) (47), as the transduction of naïve T cells with a retroviral vector containing RORγt induces the development of IL-17-producing T cells (53). The IL-23 receptor activates signal transducer and activator of transcription (STAT) 3, which induces RORγt (54). Conversely, in murine models, the generation of Th17 cells is inhibited by IL-4, IL-10, and IFNγ, possibly *via* downregulation of the IL-23 receptor (54, 55). The reciprocity of this antagonism from Th1/Th2 cells remains poorly understood, but as well as Th1 being counterbalanced by Th2 cells (56), Th17 cells are reciprocally related to Foxp3+ Tregs. The presence of TGF-β activates the transcription factor Foxp3 in naïve T cells, whereas the presence of IL-6 suppresses Foxp3, and combined with TGF-β induces RORγt, leading to Th17 differentiation. Thus, the balance between Th17 cells and Foxp3+ Tregs is mediated by the antagonistic interaction of the transcription factors Foxp3 and RORγt [reviewed by Korn et al. (47)].

Th17 is primarily known for its enhancement of host protection against extracellular bacteria and fungi, which requires robust inflammatory infiltrates that are not efficiently cleared by Th1 and Th2 responses (47, 57). IL-17 acts by activating iNOS and inducing the expression of granulocyte macrophage colonystimulating factor, IL-1β, IL-6, IL-8, TNF, and several chemokines, which collaborate to potentiate the inflammatory reaction (58). The induction of these inflammatory mediators suggests that IL-17 plays a role in intracellular parasite infections. Indeed, the differentiation of Th17 subsets has been noticed during infections of *Mycobacterium tuberculosis* (59), *Francisella tularensis*, and *Listeria monocytogenes* (60). The role of IL-17 is also essential for protection against *Trypanosoma cruzi* in the acute phase of Chagas disease (61, 62), suggesting that Th17 cells are important in the successful clearance of kinestoplastid protozoans such as *Leishmania*. However, although Th17 cells and IL-17 have been shown to protect against some intracellular pathogens including *Leishmania* (63, 64), many recent findings point to them as being responsible for excessive inflammation and pathology (65, 66). Currently available data from VL and CL studies are discussed below.

#### IL-17 in VL

Active VL is associated with the upregulation of Th2 cytokines and disease progression. Neutrophil depletion during experimental VL results in enhanced parasite loads (67). The effector role of IL-17A is mediated by the accumulation of neutrophils, the enhancement of IFNγ secretion, and the increased production of antimicrobial peptides, acute phase proteins, mucins,

1 IL-17 is the founding member of the IL-17 family of cytokines, which comprises IL-17A (commonly known as IL-17), IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F (45). The most widely investigated and understood cytokines of this family are IL-17A and IL-17F, which play an essential role in host defenses against microbial infections and are implicated in various inflammatory conditions such as autoimmune diseases, metabolic disorders, and cancer (46).

*Leishmania* infection through alternative macrophage activation, favoring parasite persistence. IL-10 is an important regulatory cytokine that controls Th17 differentiation and regulates NO production. The influx of neutrophils plays a positive role by participating in parasite killing and is limited by the presence of IL-10.

and matrix metalloproteases (45). As IL-17 is associated with neutrophilic inflammation and enhancement of the proinflammatory response, the positive role of this cytokine in VL has been investigated. It is already known that *L. donovani* and *L. infantum* stimulate the differentiation of Th17 cells in peripheral blood mononuclear cells from healthy subjects, as well as the production of IL-6 and IL-23 required for Th17 maintenance (68). A cohort study of individuals with VL showed that IL-17 is strongly associated with protection. This protective role is associated with the increase of CXCL chemokines, which serve as potent chemoattractants for neutrophils and Th1 cells. Thus, naturally resistant subjects, showing greater IL-17 responses, would react more rapidly to *L. donovani*, not only by attracting innate immunity responses, but also by recruiting Th1 cells to tissues (68). In BALB/c mouse models of *L. donovani* infection, parasite clearance was associated with the secretion of Th1 cytokines along with IL-17 and IL-23. The blocking of stimuli by anti-IL-17 and anti-IL-23 led to increased parasite loads in the liver and spleen. Subsequent administration of recombinant IL-17 generated higher levels of IFNγ and NO than recombinant IL-23 did, leading to a stronger association of IL-17 with parasite clearance (69). These data corroborated previous findings (68) that Th17 potentiates the Th1 response. Depending on the infectant parasite load, the speed of Th1 recruitment and the quantity of NO generation may determine disease progression or natural control.

mRNA analysis revealed greater IL-17A production in VL patients than in healthy controls, in a study performed by Nascimento et al. (55). In the same study, the highest production of IL-17A coincided with the parasitic peak in infected C57BL/6 mice. These data demonstrate the stimulation of IL-17 production by *L. infantum*, promoting the control of parasite replication by strengthening T helper type 1 responses and NO production. The authors also demonstrate that the absence of IL-17 signaling leads to the generation of a Treg/IL-10-dominated response and an impaired Th1 profile, resulting in parasite growth. However, although IL-17 receptor *knockout* mice were more susceptible to infection and also exhibited reduced inflammatory infiltration, the high levels of this cytokine observed in humans was not sufficient to generate IFNγ/NO levels that lead to natural recovery from VL (55). Thus, although the presence of this cytokine has been demonstrated, the significance of its participation in the effector response seems to be relative to the host.

Neutrophil influx mediated by IL-17A was revealed by Quirino et al. to be an important mediator of parasite clearance during VL (4). In this work, the authors indicate that IL-27 is an important regulator of Th1 and Th17 profiles in a VL mouse model. Composed of two subunits (IL-27p28 and Ebi3) that bind to IL-27 receptor, leading to STAT-1 (Th1) and -3 (Th17) activation, IL-27 plays a role in the regulation of the immune response (4). The authors observed that Ebi3−/− mice present a peak in neutrophil migration at 4 weeks post-infection, both in the spleen and in the liver. These data reinforce the previous findings of Lopez Kostka et al. (70) that suggest that the IL-17–neutrophil axis has more of an influence in the recovery from VL infection after its establishment (when the antigens are already recognized and presented by DCs and macrophages) than in the prevention of infection. Neutrophils recruited by DCs in a toll-like receptor 2 (TLR2)-dependent manner are efficient producers of NO and provide a prototypal Th1 and Th17 environment, leading to a decrease in parasite number in C57BL/6 mice infected with *L. infantum.* Furthermore, TLR2−/− mice are unable to recruit neutrophils in this way and had higher levels of IL-10, indicating that a non-responsive neutrophil state is associated with parasite persistence (71). The presence of neutrophil infiltrate, however, may cause damage to the tissue, and seems to be most significant in skin, since the Th17 markers IL-23, IL-17, and RORγt were highly expressed during post kala-azar dermal leishmaniasis (PKDL) compared with controls (72).

The cell source of IL-17A expression is another variant to be studied. IL-17A-knockout C57BL/6 mice are highly resistant to VL infection, showing fewer parasites in the liver and spleen. This phenotype was associated with enhanced IFNγ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in fewer granulomas. The source of IL-17 was mainly γλ T cells, which commonly express markers for CD3, but not CD4 or CD8 (67), and are differentiated by IL-1β

and IL-23, suggesting a different mode of activation to the conventional TGF-β and IL-6 that promote CD4+IL-17+ cells. IL-17 produced by γλ T cells suppresses NO-mediated parasite control in macrophages in the liver within 7 days of infection, contributing to susceptibility in C57BL/6 mice (73). The mechanisms that lead to counter-regulation between IFNγ and IL-17 secreted by γλ T cells remain to be elucidated.

#### IL-17 in CL

*Leishmania* infection strongly stimulates IL-17 production. Elevated levels of IL-17 have been measured in patients with CL and ML, showing that Th17 activation is generated by *L. major* (74, 75), *L. braziliensis* (64), *L. tropica* (26), *L. panamensis* (64), *L. guyanensis*, *L. amazonensis*, and *Leishmania naiffi* (66) infections, acting on macrophage activation and neutrophil recruitment. However, clinical studies indicate differences in immune responses during CL between the subgenera *Viannia* and *Leishmania* (76). For example, higher levels of Th17 were found in the sera of patients infected with *L. (Viannia) guyanensis* than in those infected *L. (Leishmania) amazonensis* (66). Nevertheless, the mechanisms of immune evasion used by *L. amazonensis* are known to block cellular immune responses (37) and may also occur by decreasing Th17 activation. Thus, the influence of Th17/ IL-17 on disease depends on the species. As studies on IL-17 are relatively recent, most performed to date have evaluated *L. major* models or *L. braziliensis* infections in patients. Comparing IL-17 participation in other *Leishmania* species seems, therefore, to be an interesting avenue of investigation.

 However, in the case of CL and ML, the immune response itself contributes to lesion progress. In both these forms, patients develop intense inflammatory responses that damage tissues, despite the low number of parasites in lesions (25), so the disease is mostly associated with a failure of anti-inflammatory cytokine secretion, especially IL-10 (77). IFNγ and IL-17 levels are substantially elevated in mice lacking the capacity to respond to IL-10. According to Gonzalez-Lombana et al. (2), in these mice, IFNγ does not seem to contribute to pathology as much as IL-17, since the former promotes mostly monocyte infiltration, while the latter recruits neutrophils, constituting a nonspecific response. Interestingly, blocking IFNγ increases IL-17 level and pathology, supporting the hypothesis that, in C57BL/6 mice, IFNγ may be critical for downregulating the responses of IL-17 as well as IL-10 (2). In another study, self-healing of the infection in mice correlated with the expansion of IFNγ and IL-17-producing CD4 cells, suggesting the existence of other active mechanisms to regulate local inflammation (7).

In experimental models, lesion progression is related to IL-17-mediated neutrophil recruitment. During the course of infection with *L. major*, BALB/c (a susceptible strain) CD4+ cells and neutrophils produced more IL-17 than did cells from (resistant) C57BL/6 mice. IL-17-deficient mice had decreased neutrophil infiltration and smaller lesions than controls (70). Anderson et al. (74) found an association between severe pathology and the presence of IL-17+CD4+ cells, which was also linked to an increase in cellular infiltrate expressing macrophage and neutrophil markers. However, despite contributing to lesion exacerbation, no beneficial effect of controlling parasite replication was observed. Conversely, neutrophils have been thought to contribute to parasite clearance in the early stages of experimental *Leishmania* infection (30), and their expression is closely, but not uniquely, related to IL-17 secretion (78). Thus, neutrophil participation must be better studied and linked to IL-17 associations with leishmaniasis. In resistant strains of mice, the number of polymorphonuclear leukocytes decreases to 2% within 72 h of infection, when the specific immune response is defined. In contrast, insusceptible BALB/c mice, neutrophils continue being recruited and have been detected in large numbers at the site of infection more than 10 days after parasite inoculation (30). Pedraza-Zamora et al. (79) showed that in BALB/c mice, Th17 cells and neutrophils both increased up to 90 days postinfection with *L. mexicana*. This suggests that chronic inflammation throughout *L. mexicana* infection is a consequence of neutrophil recruitment together with Th17 cell differentiation throughout the late phase of the infection. C57BL/6 mice show a more controlled response of these cells and a more resistant infection profile. Together, these data suggest that not only the level of IL-17 but also its persistence during infection resolution, is a decisive factor in disease progression. The excessive activation and maintenance of Th17 by the presence of neutrophils and Th1 may cause a damaging block of regulatory populations and IL-10 secretion, which is necessary to stop inflammation. For example, the study performed by Lopes Kostka et al. (70) suggested that DC-derived IL-23 maintains IL-17 production, thus being responsible for disease progression.

mRNA expression of RORγt and IL-17 is observed in patients with ML, and the synergy of IL-17 and IFNγ is suggested by a positive correlation between IL-17 and IFNγ mRNA expression in ML lesions caused by *L. major* (75), contrasting with the antagonism observed in C57BL/6 mice (2) mentioned earlier. Enzyme-linked immunosorbent assays showed that IL-17 secretion by peripheral blood mononuclear cells was elevated in patients with CL and ML compared with uninfected individuals, and downmodulated by IL-10 and TGF-β (65, 80) similarly to that seen with Th1/ IFNγ, providing further evidence for the existence of IL-17/IFNγ synergy in humans. de Assis Souza et al. (63) showed that levels of IL-17 were significantly higher in patients infected by *L. braziliensis* during active disease than in those who recovered without intervention. Interestingly, the authors associated this decreased expression during healing to a suppressive role exerted by high NO levels. During treatment, NO levels decrease and allow more IL-17 secretion (63). In addition, the number of IL-17+CD4+ cells at lesion sites in patients with CL and ML directly correlates with the number of inflammatory cells, supporting their participation in neutrophil infiltration at lesion sites (65).

Although these important studies have linked the presence of IL-17 to more severe clinical presentations of disease, neutrophils are the first line of defense and are also capable of producing NO and killing parasites (32). Therefore, the presence of neutrophils in this inflammatory context can indicate either protection or injury. The reversal of immunodeficiency in *L. panamensis-*infected mice leads to an increase in IFNγ

and IL-17, allowing parasite control and elimination (64). This suggests that in the absence of anti-inflammatory cytokines (such as IL-10, IL-13, and IL-4), Th1/Th17 cells proliferate simultaneously and are responsible for parasite elimination, although neutrophil migration, and its role in this situation (3.5 weeks post-infection), was not assessed. This observation reinforces the differences in immunomodulation between parasite species. Interestingly, Novoa et al. (81) also found a trend toward increased levels of IL-17 in human subclinical *L. braziliensis* infections compared with symptomatic CL, suggesting no pathological role of IL-17 in CL or ML. Although those authors associated spontaneous cure to a weak type 1 immune response (due to quick parasite control by innate immune responses), IL-17 levels were measured in the study only 96 h after soluble antigen stimuli, when the cellular immune response was probably being established. The expression of IL-27 (which can act as regulator of IL-17) was also higher in patients with CL caused by *L. braziliensis* than in subclinical individuals (81), reinforcing the idea that the Th17 profile contributed to cure in this study, and supporting a role of IL-17 in parasite depletion. Further investigations encompassing the absence of other IL-17 regulatory mechanisms (such as IL-13) in this situation are necessary.

#### NEUTROPHILS IN INNATE AND ADAPTIVE IMMUNITY TO *Leishmania*

Neutrophils are cells that operate in innate immunity as the first line of defense, and play an important role in the elimination of pathogens (82). Toxic granule release from the site of infection, secretion of cytokines, phagocytosis, and formation of fibrous structures, known as NETs (neutrophil extracellular traps) are some of the mechanisms used by these cells to eliminate microorganisms (82, 83). In addition to their role in the elimination of pathogens, neutrophils can also participate in immune regulation as a source of cytokines, including IL-2, IL-10, IFNγ, and TNF, thereby establishing a link between innate and adaptive immunity during infection, and even participating in the presentation of antigens to regulate T cell proliferation and antibody production by B cells (84). In fact, murine *L. (L.) amazonensis*infected macrophages cocultured with neutrophils were better able to destroy the intracellular amastigotes (34). Since no significant difference in *L. (L.) amazonensis* parasite destruction was observed between co-cultures from susceptible BALB/c and resistant C3H/HePas mice, this leishmanicidal activity seemed not to depend on adaptive immunity, which determines an individual's resistance profile (34). However, due to the effector functions in the inflammatory process, neutrophils also need to be regulated because their excessive activity can lead to tissue destruction and even chronic inflammation (82).

Recent studies have addressed the role of neutrophils in *Leishmania* spp. infection. These cells may behave protectively or as enhancers of infection, depending on the parasite species, host, and stage of infection (32, 34). *In vitro* studies demonstrated that several *Leishmania* species are able to induce the formation of NETs, which are basically DNA structures coated with antimicrobial molecules, released by neutrophils (85). These NETs are involved in the sequestration, immobilization, and killing of parasites [reviewed by Bardoel et al. (86)]. However, the efficacy of this immune strategy varies according to the *Leishmania* species involved. For instance, promastigotes of *L. amazonensis* showed susceptibility to NETs, while *L. donovani*, *L. infantum*, and *L. mexicana* were able to escape from this "trap" using different mechanisms (82). Furthermore, the same promastigote surface lipophosphoglycan that is involved in the activation of NETs (as for *L. amazonensis*) may also be associated with parasite resistance to killing through NETs, as occurs with *L. donovani* (87).

There is no consensus on the activity of neutrophils during infection by *Leishmania* species, as besides the complexity of species involved, there is heterogeneity between studies, which explore different host species, host strains, and tissue types, all factors that may interfere directly with the type of response to the infection (32). In an example of how the host strain (genetic background) may change its response, Tacchini-Cottier et al. (33) demonstrated that neutrophil-depleted C57BL/6 mice exhibited a normal course of infection caused by *L. major*, whereas neutrophil depletion in BALB/c resulted in blockage of the early characteristic IL-4 response, thus leading to an inhibition of Th2 type response. Currently, a specific neutropenic mouse strain (Genista) is being used; Hurrell et al. (82) demonstrated that Genista mice, as well as mice with transient neutrophil depletion, are able to control the parasite load and lesion size when infected with *L. mexicana*, indicating that parasite sequestration by neutrophils is associated with disease progression in mice.

Neutrophils that are recruited and that participate in the early stages of infection behave very differently to those associated with the late immune response against *Leishmania* parasites. In the initial phase of infection, neutrophils are recruited as a response to chemotactic substances released from the tissue lesion or from the sandfly's saliva, and also by stimuli from the parasites (32). DCs and neutrophils have been shown by microscopy studies to infiltrate the site of parasite inoculation within a few hours after sandfly bite, where they engulf the parasites. By 48 h after infection, most parasites at the inoculation site are found within macrophages (88). DCs that were recovered from the skin 1 day after infection appeared to have been infected by these parasitized neutrophils (32). *In vitro* and *in vivo* kinetics studies showed that interactions with early infected or not infected neutrophils (from the acute infiltrate), or even with their apoptotic corpses, may impair the innate response of DCs, thus delaying the activation of a specific immune response *via* CD4+ T cells (32). This response may be crucial for parasite depletion before symptom presentation, resulting in subclinical progression. The parasites may enter directly into the macrophages through receptor recognition, or may be transferred silently *via* neutrophils (89). Parasites may be transferred from neutrophils to macrophages in the following two ways: the first involves the release of the parasite from an infected apoptotic neutrophil, in which the free parasite is phagocytosed by the macrophage; the second suggests that the macrophage engulfs live infected neutrophils, referred to as "Trojan horses" (90, 91). Mollinedo et al. (92) have demonstrated viable *L. major*

and *L. donovani* promastigotes inside human neutrophils, and they suggest that this is linked to inhibition of the oxidative burst, as well as prevention of early fusion of lysosomes with parasite-containing phagosomes. The interaction and previous uptake of these early neutrophils by macrophages can drive the development of IL-10-high and IL-12-low macrophages, which are especially permissive to the survival and proliferation of *Leishmania* parasites (32).

Antigen presentation by neutrophils is also inefficient in eliciting adequate T cell differentiation. Recent work revealed that human leukocyte antigen–antigen D related (HLA-DR) positive neutrophils from subjects with VL did not stimulate T-cell proliferation, but they did show higher expression of programmed cell death ligand-1 than other neutrophils, and lymphocytes of the same subjects showed high expression of programmed cell death protein-1, reinforcing the mechanisms of apoptosis that favor *Leishmania* (93). This population of neutrophils also showed elevated expression of IL-10 mRNA and protein, and transcripts encoding arginase-1, which is involved in the suppression of T cell responses (94). Conversely, after *in vitro* infection with *L. braziliensis*, neutrophils from patients with CL produce more reactive oxygen species (ROS) and higher levels of chemokines associated with recruitment of neutrophils and Th1-type cells than neutrophils from healthy control subjects (95), highlighting that the mechanisms used by parasites to explore neutrophils in their favor vary by species.

After the first acute neutrophilic influx, a second wave of neutrophil recruitment to the site of infection occurs approximately 1 week after the first contact with parasites; this is especially mediated by IL-17. Studies using BALB/c IL-17-deficient mice suggest that this cytokine is not the main recruitment factor involved in early stages of infection, but acts beside IL-8 and granulocytecolony-stimulating factor. However, after 4 weeks of infection, low levels of IL-17 lead to a low number of neutrophils (70). Thus, the role of IL-17 in both leishmaniasis forms (cutaneous and visceral) may be strictly associated with neutrophil function, infection stage, and antioxidant capacity, as well as IL-10 regulation. When the neutrophil is engulfed by an infected macrophage, parasite death is increased *via* an elevation in ROS production. However, this second response is also associated with lesion progression, especially in the mucosal form of leishmaniasis (2, 30). According to Gonzalez-Lombana et al. (2), the key point in the healing of a lesion in ML is the downregulation of IL-17 by IL-10 and IFNγ. The huge concentration of granules or products from neutrophils recruited by IL-17 in the site of infection promotes tissue lesioning. In this context, the authors suggest that the IL-17 pathway is an important therapeutic target for the treatment of severe leishmaniasis in patients in whom IL-10 regulatory function is compromised.

In experiments performed in BALB/c mice, Rousseau et al. (96) demonstrated that neutrophils are involved in the early phase of VL by controlling the parasite growth in spleen but not in liver. The authors also found that these cells appear to have no significant effect in late infection in either of these organs. Finally, Almeida et al. (97) found an association between the severity of canine VL, superoxide production and neutrophil apoptosis, observing that neutrophil function alters according to disease stage. Initially, there is a high level of ROS production, ensuring resistance to infection. However, in the late phase of the disease, ROS production overcomes the antioxidant capacity of the cell, leading to oxidative stress followed by neutrophil apoptosis. The increase in apoptosis and the consequent decrease in oxidative activity results in a predisposition to coinfections, commonly seen in severe cases of canine VL (98). In support of this hypothesis, treatments based on antioxidants have generated good results in patients with leishmaniasis (98). In short, like in CL, neutrophils are part of the complex web that governs the establishment and development of VL in different hosts.

Further studies are needed to clarify the role of neutrophils in the control or proliferation of different species of *Leishmania*. Because the infection phase is crucial, time-dependent investigations of ROS production should be carried out, and the regulatory/inflammatory profiles of Th17 cell participation should be explored in different hosts, considering tissue peculiarities.

#### Th17 AND NEUTROPHILS CAN CONTRIBUTE TO PARASITE CLEARANCE OR DISEASE

Recent studies have advanced our knowledge about the influences of each parasite/host factor in the pathogenesis of human leishmaniasis. The characterization of the CD4+ T helper cell population Th17 has further added to the complexity of host–pathogen interactions, previously thought to be determined by Th1 or Th2 polarization. Currently, participation of innate immune responses and complicated interactions between cytokines secreted by different T cell profiles, as well as the diversity of *Leishmania* species and hosts, are known to be relevant to disease outcome.

Neutrophils can potently protect against or enhance infections and can likewise behave as Trojan horses, supporting the invasion of macrophages by *Leishmania*. In addition to DCs and other components of the innate defense system, neutrophils also regulate T cell polarization, through cytokine secretion, thus establishing a link between innate and adaptive immunity during parasitic infection. Although neutrophil presence has been mostly associated with parasite depletion and cure in *L. donovani* and *L. infantum* infections, the infiltration of these cells is suggested to cause more severe disease presentations in tegumentary forms of leishmaniasis. The reviewed data show that regulatory cytokines such as IL-10 and IL-4 are needed to promote the equilibrium in ROS production, which is enhanced by the presence of neutrophils in chronic infections. In the liver and spleen, *L. infantum/ donovani* elicits anti-inflammatory cytokines and stimulates

#### REFERENCES

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Current understanding of the role of Th17 cells in the progression of pathogenesis or contribution to host-protective immunity is evolving rapidly. We now know that *Leishmania* infections trigger CD4+/IL-17+ T cell differentiation in mice and humans. The IL-17 function in neutrophil recruitment has been widely demonstrated in experimental models of leishmaniasis, in which strong inflammatory infiltrates are observed. Although mice are more dependent than humans on IL-17 to combat *L. infantum*/*L. donovani* infections, IL-17 has also been associated with improved recovery from VL in humans. Conversely, higher levels of IL-17 during commitment in skin in patients with PKDL, CL, and ML than in healthy controls suggest that in this tissue, IL-17 contributes less to healing than in other tissues. Most of the current data link the IL-17–neutrophil axis to disease development or lesion exacerbation in CL. Nevertheless, differences in immunomodulation between parasite species, and contradictory results in humans, point to IL-17 stimulatory (IL-23) or regulatory (e.g., IL-27, IL-10, and IL-13) cytokines, which may be important in determining how long IL-17 remains present. Similarly to the role of IL-12 in the context of Th1 generation, identifying the role of these cytokines in the development or maintenance of Th17 is essential for understanding the influence of its persistence at lesion sites.

#### AUTHOR CONTRIBUTIONS

SG-d-A and MP-C concept and drafted the paper. SG-d-A, RS, and LT-S acquired, interpreted data, and wrote the paper. SG-d-A, RS, VL, RM, and MP-C revised critically for intellectual content. All the authors approved the final version to be published. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

#### FUNDING

This study was supported by the Education Ministry of Brazil through the Coordination for the Improvement of Higher Level Personnel (CAPES) and by the National Counsel of Technological and Scientific Development (CNPq), Universal Grant number APQ: 441584/2014-4.

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caused by *Leishmania braziliensis*. *PLoS Negl Trop Dis* (2016) 10(5):e0004715. doi:10.1371/journal.pntd.0004715

**Conflict of Interest Statement:** 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.

*Copyright © 2017 Gonçalves-de-Albuquerque, Pessoa-e-Silva, Trajano-Silva, de Goes, de Morais, da C. Oliveira, de Lorena and de Paiva-Cavalcanti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Rajiv Kumar1 \*, Shashi Bhushan Chauhan2 , Susanna S. Ng3 , Shyam Sundar 2 and Christian R. Engwerda3 \**

*1Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, India, 2Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, 3QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia*

#### *Edited by:*

*Heinrich Korner, University of Tasmania, Australia*

#### *Reviewed by:*

*Gerald Spaeth, Institut Pasteur, France Fabienne Tacchini-Cottier, University of Lausanne, Switzerland*

*\*Correspondence:*

*Rajiv Kumar<EMAIL_ADDRESS>Christian R. Engwerda<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 08 September 2017 Accepted: 23 October 2017 Published: 08 November 2017*

#### *Citation:*

*Kumar R, Chauhan SB, Ng SS, Sundar S and Engwerda CR (2017) Immune Checkpoint Targets for Host-Directed Therapy to Prevent and Treat Leishmaniasis. Front. Immunol. 8:1492. doi: 10.3389/fimmu.2017.01492*

Leishmaniasis encompasses a group of diseases caused by protozoan parasites belonging to the genus *Leishmania*. These diseases range from life threatening visceral forms to self-healing cutaneous lesions, and each disease manifestations can progress to complications involving dissemination of parasites to skin or mucosal tissue. A feature of leishmaniasis is the key role host immune responses play in disease outcome. T cells are critical for controlling parasite growth. However, they can also contribute to disease onset and progression. For example, potent regulatory T cell responses can develop that suppress antiparasitic immunity. Alternatively, hyperactivated CD4+ or CD8+ T cells can be generated that cause damage to host tissues. There is no licensed human vaccine and drug treatment options are often limited and problematic. Hence, there is an urgent need for new strategies to improve the efficacy of current vaccine candidates and/or enhance both antiparasitic drug effectiveness and subsequent immunity in treated individuals. Here, we describe our current understanding about host immune responses contributing to disease protection and progression in the various forms of leishmaniasis. We also discuss how this knowledge may be used to develop new strategies for host-directed immune therapy to prevent or treat leishmaniasis. Given the major advances made in immune therapy in the cancer and autoimmune fields in recent years, there are significant opportunities to ride on the back of these successes in the infectious disease domain. Conversely, the rapid progress in our understanding about host immune responses during leishmaniasis is also providing opportunities to develop novel immunotherapy strategies that could have broad applications in diseases characterized by inflammation or immune dysfunction.

Keywords: leishmaniasis, immunoregulation, immunotherapy, pathology, cell-mediated immunity

#### LEISHMANIASIS

Leishmaniasis describes a collection of neglected tropical diseases caused by protozoan parasites of the genus *Leishmania* that are transmitted by female *Phlebotomine* sand flies (1). It largely affects the poorest populations living in developing countries and is prevalent throughout the tropical and subtropical regions of Africa, Asia, the Mediterranean, Southern Europe, and South

**53**

and Central America. Globally, 350 million people are at risk of developing leishmaniasis and 1.5–2 million new cases occur annually (2). The clinical spectrum of leishmaniasis ranges from the life-threatening visceral form to self-healing cutaneous lesions or a more serious mucosal manifestation.

Visceral leishmaniasis (VL), also known as kala-azar, typically involves long-term, low-grade fever, enlarged spleen and liver, weight loss, pancytopenia, and hypergammaglobulinemia [reviewed in Ref. (3)]. Untreated VL cases are almost always fatal, and more than 90% of cases occur in India, Bangladesh, Nepal, Sudan, Ethiopia, and Brazil with an estimated incidence of at least 500,000 new cases and 50,000 deaths each year (2–4). Of note, the state of Bihar in north east India has been the focus of most VL cases for many years (5), but recent efforts toward elimination, and civil unrest in Southern Sudan, have now made the latter region the source of most cases (6). Post kala-azar dermal leishmaniasis (PKDL), which can be a late cutaneous manifestation of VL, either following drug treatment or sometimes independent of VL development, is confined to the Indian subcontinent [India, Nepal and Bangladesh, and East Africa (Sudan)]. It presents as an accumulation of heavily infected macrophages in the skin (7–9), which appear as nodules, papules, or hypopigmented macules. PKDL can appear from 6 months to years after apparent VL cure in the Indian subcontinent, but can also occur earlier (within 6 months) or along with VL in the Sudan, where the incidence of this disease is higher. PKDL heals spontaneously in a proportion of cases in Africa, but rarely in Indian VL patients and requires prolonged treatment. Since PKDL patients harbor increased parasite numbers in their skin, they are thought to act as parasite reservoir and play an important role in disease transmission in endemic regions. As such, they may be an important population to target with effective host-directed immunotherapies.

Cutaneous leishmaniasis (CL) is characterized by the development of ulcerative skin lesions containing parasites and is the most common form of disease occurring mainly in Afghanistan, Algeria, Brazil, Colombia, the Islamic Republic of Iran, Pakistan, Peru, Saudi Arabia, and Syria (2, 10). Cutaneous lesions are generally localized and may persist for months to years or heal spontaneously within weeks. The development of disfiguring scars at the affected skin areas following healing is a major concern.

Mucocutaneous leishmaniasis (MCL) is prevalent in Bolivia, Brazil, Peru, and Ethiopia (WHO, 2014) and is caused by *L. baziliensis*, *L. panamensis*, and *L. aethiopica*. These species metastasize to mucosal tissue in the mouth and upper respiratory tract, leading to localized tissue destruction. MCL can present from several months to years after the development of a cutaneous lesion. Diffused cutaneous leishmaniasis (DCL), which is more common in central and South America, is thought to occur in immunosuppressed individuals, where parasites can readily disseminate to subcutaneous tissue. As both MCL and DCL are associated with strong and weak host inflammatory responses, respectively, which appear to contribute to disease pathology, they also have potential for improved treatment involving hostdirected immune therapy.

# IMMUNOLOGICAL CHARACTERISTICS OF DISEASE

#### Visceral Leishmaniasis

Studies in experimental VL in mice, caused by infection with the human parasites *L. donovani* or *L. infantum*, show the development of antiparasitic IFNγ-producing, Tbet+ CD4+ T (Th1) cells is critical for resistance against infection (11). Many VL patients fail to generate potent cell-mediated immune responses against parasite antigens and this is thought to be an underlying cause of disease. However, enhanced IFNγ mRNA expression in the spleen and bone marrow, as well as increased circulatory plasma IFNγ, TNF and IL-12 in VL patients, suggests that they do not lack a protective Th1 response, but instead, immunosuppressive mechanism are established to prevent parasite killing (12–15). Importantly, antigen-specific responses in whole blood assays indicate that VL patient CD4+ T cells have the capacity to produce IFNγ in response to parasite antigen (16–18). Therefore, attention has now focused on regulatory mechanisms that prevent Th1 cell-mediated control of parasite growth.

CD4+ T cell IL-10 production has emerged as an important mechanism to dampen T cell activation in parasitic infections, including in humans with VL (15, 19, 20). Importantly, most T cell-derived IL-10 is not produced by thymus-derived Foxp3-expressing regulatory T (Treg) cells. Instead, the IL-10 producing CD4+ T cells often co-produce IFNγ and have been designated type 1 regulatory (Tr1) cells (15). They are increasingly recognized as a critical regulatory CD4+ T cell subset that protects tissue from inflammation (21–23). However, Tr1 cells also appear to promote infection by suppressing Th1 cellmediated immunity. The role of IL-10 in immune suppression and disease progression is well documented in both experimental and human VL (15, 24–28). Human VL is associated with enhanced IL-10 plasma levels, increased IL-10 mRNA expression in lymph nodes, bone marrow, and spleen, and readily detected IL-10 produced by whole blood cells from VL patients following parasite antigen stimulation (15, 28). IL-10 dampens major histocompatibility complex (MHC) class II expression on APC and downregulates TNF and nitric oxide (NO) production, leading to reduced parasite clearance and suppressed activation of Th1 cells (29). Neutralization of IL-10 in VL patient sera can suppress *L. donovani* replication in macrophages (15, 30), and IL-10 blockade in splenic aspirate cultures from VL patients can limit parasite replication and enhance Th1 cell cytokine production (24). IL-10 can also modulate immune responses by promoting T cell exhaustion (31, 32).

A number of immune checkpoint molecules have also been identified on CD4+ T cells from experimental VL models and VL patients (**Table 1**). These include CTLA-4 (CD152) and PD-1, which are negative regulators of T cells and are expressed on exhausted or anergic T cells during chronic infection. CTLA-4 binds to the costimulatory ligands B7-1 (CD80) and B7-2 (CD86), with much higher affinity than CD28, while PD-1 interacts with PD-1 ligand 1 (PD-L1; B7.H1) and PD-L2. Activation of CTLA-4 leads to increased levels of TGFβ, as well as apoptosis of CD4+ T cells in murine VL (33). In mice infected with *L. donovani*,

Table 1 | Immune checkpoint molecules tested for therapeutic effects in leishmaniasis.

*Y, indicates immune checkpoint molecule tested in the disease indicated.*

CTLA-4 blockade decreased parasite burden in both liver and spleen, associated with increased frequencies of IFNγ and IL-4 producing cells, and an accelerated hepatic granulomatous response (34, 35). CTLA-4 blockade has also been shown to increase the efficiency of chemotherapy in *L. donovani* infected mice (36, 37). Similarly, blockade of PD-1 or PD-L1 resulted in enhanced parasite clearance and increased pro-inflammatory cytokine production in experimental VL (38–40). Thus, these studies clearly show the therapeutic potential of targeting immune checkpoint molecules for host-directed immune therapy in VL.

Another important regulatory cytokine involved in VL is IL-27, which is composed of the EBI-3 and p28 sub-units. IL-27 belongs to IL-6/12 cytokine family and was originally described as a co-factor for Th1 cell differentiation, along with IL-12 (63, 64). IL-27 promotes T cell IL-10 production in mice, which is further amplified by autocrine IL-21 production (65, 66). IL-27 receptor-deficient mice infected with *L. donovani* developed enhanced Th1 responses, but this was associated with severe liver pathology (67). Patients with active VL also presented with enhanced IL-27 plasma levels, as well as increased mRNA transcripts encoding EBI-3 and p28 in splenic aspirates (28). IL-27 produced by CD14+ cells, along with IL-21 from T cell sources, promoted the differentiation and expansion of Ag-specific, IL-10–producing T cells in VL patients. Importantly, pro-inflammatory cytokines, such as IFNγ, act on macrophages and stimulate IL-27 production, suggesting a feedback mechanism to stimulate IL-10 production to control IFNγ levels and protect host tissue. IL-27 has also been associated with suppression of CD4+ T cell IL-17A and IL-22 secretion (68, 69). Since *L. donovani* antigen-stimulated production of both IL-17A and IL-22 by PBMC in an apparent disease-resistant Sudanese population, these cytokines were proposed to be protective following *L. donovani* infection and, therefore, elevated IL-27 in VL patients might not only promote disease by increasing IL-10 production, but also by regulating IL-17 production (70). Studies with Indian VL patients showed low levels of IL-17A mRNA transcripts, as well as the IL-17 related transcription factor RORγT during active disease (28). However, there was no direct evidence that this Th17 response was suppressed by IL-27. Furthermore, there is evidence from experimental VL that the impact of IL-17A may depend of the stage of infection, whereby this cytokine impedes antiparasitic immunity early (71), but is protective following establishment of infection (72). Hence, immune dysfunction in VL patients appears to involve multiple immune regulatory pathways that differ both spatially and temporally, and identifying which can be safely and effectively targeted for clinical advantage should be a major research priority.

Other immunosuppressive mechanisms established during VL may be mediated through regulatory T cells. Regulatory T cells can be classified as thymus-derived CD4+CD25+FoxP3+ T (Treg) cells and inducible regulatory T cells that include conventional T cells that convert to FoxP3+ regulatory T cells in peripheral tissues, as well as Tr1 cells (73, 74). To maintain immune homeostatic conditions, Treg cells limit the activity of potentially self-reactive T cell responses and prevent immunemediated pathology and autoimmunity (75, 76). However, these same mechanisms may also contribute to impaired pathogen clearance during parasitic infection. Treg cells function by secreting regulatory cytokines such IL-10 and TGFβ, as well as expressing inhibitory molecules such as CTLA-4 and IL-35 (77). Treg cells express high levels of CD25 (IL-2R), thereby allowing them to form the high affinity receptor for IL-2, which allows them to deprive conventional T cells of this important growth factor, thus causing apoptosis (78). However, there is little evidence for the involvement of Treg cells in human or experimental VL. Studies from VL patients in Bihar, India showed no accumulation of Treg cells in the spleen or blood, and the frequency of these cells did not change during the course of infection (15, 79). Moreover, FoxP3− T cells were the major source of IL-10 mRNA in VL patient spleens, and this finding was in accordance with murine VL studies where IL-10 secretion by FoxP3−CD4+ T cells correlated with disease severity (19). However, other studies have reported the accumulation of Treg cells at sites of infection and suggested their possible role in disease pathogenesis in both human and experimental VL. One study from India suggested that Treg cells were a major source of IL-10 in the bone marrow of VL patients and that IL-10 secretion from Treg cells suppressed conventional T cells (80). In a different study with Indian VL patients, production of IL-10 and TGFβ by Treg cells was positively correlated with parasite load (81). Similarly, TGFβ-producing Treg cells were shown to accumulate in infected tissues in a murine model of VL (82). Thus, further investigation is needed to establish whether Treg cells are involved in the pathogenesis of VL and whether they can be modulated for therapeutic advantage. Interestingly, TGFβ is also secreted by macrophages and dendritic cells (DCs) during experimental VL. A cathepsin B-like cysteine protease present in *L. donovani* can activate TGFβ (83), which, in turn, activates arginase-1, leading to enhanced l-ornithine production and reduced NO secretion, thereby promoting parasite survival in infected cells (84, 85). Human VL patients have enhanced TGFβ plasma levels (13) during active disease, suggesting a possible role in pathogenesis. However, more research is needed to better understanding the precise mechanisms of TGFβ-mediated suppression of antiparasitic immunity before it can be considered as an immune therapy target.

The development of regulatory DC subsets following *L. donovani* infection can also have a major impact on T cell responses during VL. These regulatory DCs are capable of producing antiinflammatory cytokines, such as IL-10, TGFβ, and IL-27 (86). In experimental VL, it was shown that IL-10+IL-27+ DCs were able to promote IL-10 production by Th1 cells *in vivo* and identified this cell population as a potential target for immunotherapy (87). Furthermore, CD11cloCD45RB+ DCs in the spleen of *L. donovani*-infected mice had high levels of IL-10 production, compared to CD11chi populations, and displayed features of immature DCs, including low expression of co-stimulatory molecules and intracellular MHC class II (88). These DCs also produced IL-10 when stimulated with lipopolysaccharide and promoted Treg cell IL-10 production capable of inhibiting mixed lymphocyte reactions driven by conventional DCs (89). The inhibitory effects of these regulatory DCs could be reversed by IL-10 signaling blockade, indicating that IL-10 production was a critical regulatory mechanisms employed by this DC subset (88). Therefore, both cognate and soluble cytokine signals between effector CD4+ T cells, DCs, and infected macrophages have key roles in determining whether parasite growth is controlled and/or disease develops, making these interactions promising targets for immune therapy (**Figure 1**).

CD8+ T cells can kill *Leishmania*-infected macrophages by secreting cytolytic enzymes (90, 91). However, studies with human VL blood samples suggest that CD8+ T cells have an anergic or exhausted phenotype, as indicated by high expression of IL-10, CTLA-4, and PD-1, which may hamper the protective efficiency of these cells during active disease (51). A better understanding of role of CD8+ T cells during VL is needed if the antiparasitic potential of these cells through vaccination or immune therapy can be exploited.

#### POST KALA-AZAR DERMAL LEISHMANIASIS

Post kala-azar dermal leishmaniasis often develops as a cutaneous complication of VL in apparently cured patients, but can also develop independent of VL. The pathogenesis of PKDL remains poorly understood. It has been postulated that immune suppression may allow multiplication of latent parasites from the viscera or residing in the skin (8). Similar to clinical VL, elevated IFNγ and TNF levels are found in lesions of PKDL patients, with the concurrent presence of the immunosuppressive cytokines IL-10 and TGFβ (92). Despite the presence of high IFN-γ and TNF in these tissues, there is reduced expression of IFNγ and TNF receptors in Indian PKDL patients (92, 93), while genetic polymorphisms in the IFNγ receptor 1 gene promoter region have been reported and found to be associated with susceptibility to PKDL in Sudanese patients (94, 95). Treg cells have also been associated with PKDL in the Indian subcontinent and elevated FoxP3, CD25, and CTLA-4 mRNA expression has been reported in the skin of patients. Furthermore, Foxp3, CD25, and IL-10 mRNA levels directly correlated with parasite load in these PKDL patients (96). Since PKDL either develops soon after VL or independent of VL in the Sudanese population, but takes longer to develop after VL in the Indian subcontinent, the immunopathology of PKDL is likely to differ in these populations. PKDL patients from the Sudan display immune responses similar to cured VL patients and their PBMC proliferate in response to parasite antigens and CD4+ T cells secrete IFNγ and IL-10 (97, 98). However, PKDL patients from the Indian subcontinent have high numbers of CD8+ T cells in their lesions and circulation, along with increased antigen-induced IL-10 production by circulating CD8+ T cells and impaired antigen-induced proliferation (99, 100). Studies with Indian PKDL patients have also demonstrated enhanced Th17 cell responses by analyzing mRNA and protein expression of Th17-related IL-23, IL-17A, and RORγt (101). Stimulation of PKDL patient PBMCs with

Figure 1 | Potential immunotherapy targets to manipulate CD4+ T cell-dendritic cell (DC) and CD4+ T cell-infected macrophage (MΦ) interactions during leishmaniasis include both cognate and soluble cytokine signals. Primary signals between CD4+ T cells and macrophages or DCs through major histocompatibility complex class II antigen presentation of parasite peptide to the T cell receptor are indicated (red), as are both positive (green), and negative (orange) costimulatory signals. Positive (blue) and negative (purple) soluble cytokine signals are also show. Note that many of these signals are bidirectional, as indicated by the double-ended arrows. In addition, molecules highlighted by an asterisk have been reported to have the opposite effects in different type of *Leishmania* species infections. Amastigotes residing in macrophages are shown in black.

parasite antigens resulted in IL-17A and IL-23 production, while stimulation with recombinant IL-17A enhanced TNF and NO production. Hence, these data suggest that enhanced Th17 responses may have a role in parasite clearance during PKDL. However, it is still not clear whether regulatory cytokines and/ or other mechanisms suppress IL-17-mediated protective responses during active disease. This knowledge is important if we wish to manipulate this immunoregulatory pathway to improve antiparasitic immunity.

#### CUTANEOUS LEISHMANIASIS

Cutaneous leishmaniasis is caused by several *Leishmania* species, including *Leishmania major*, *L. braziliensis*, *L. mexicana*, *and L. amazonensis.* Cell-mediated immune responses at the site of cutaneous lesions are of primary importance in determining the outcome of disease. Furthermore, in murine models of CL caused by *L. major*, the genetic background of mice also determines disease outcome. In C57BL/6 mice, Th1 cell responses promote a self-healing process, while Th2 responses are associated with parasite persistence in the lesions of BALB/c mice [reviewed in Ref. (102, 103)]. In humans infected with *L. major*, cutaneous lesions have been associated with high IFN-γ, IL-10, and IL-12 mRNA accumulation, indicative of a mixed CD4+ T cell response. Several immune checkpoint molecules have also been identified in experimental CL studies that can modify CD4+ T cell responses to favor parasite clearance (**Table 1**), again demonstrating immune checkpoint blockade as a potential approach to improve disease treatments.

Following transmission of *L. major* to mice *via* sand fly bites or needle, neutrophils rapidly infiltrate the bite site and capture injected parasites (104). Neutrophils rapidly express apoptotic markers following *L. major* uptake, which attracts monocytes and DCs to the site of infection for removal of apoptotic cells (105, 106). This allows the uptake of parasites into phagocytic cells without triggering inflammation, and thereby enabling establishment of infection. Infected neutrophils also express chemokines, such as CCL-3, to attract DCs to the site of infection (107). This may help to stimulate Th1 cell responses following activation of DCs through interaction of DC-SIGN on DC and specific glycans on neutrophils (108). In addition, CCL3 can induce IL-12 secretion by macrophages in C57BL/6 mice, but not in BALB/c mice (107). Given these latter pro-inflammatory properties of neutrophils, any modifications of neutrophil functions may have to be directed specifically toward their activity as a "Trojan horse" for establishment of infection. However, it is important to remember that the role of neutrophils is critically dependent on the *Leishmania* species in question, the parasite lifecycle stage and stage of infection, when trying to manipulate neutrophil functions.

Following *L. major* infection, complement-dependent platelet activation, including the release of platelet-derived growth factors, can stimulate the release of CCL2/MCP-1 by leukocytes and mesenchymal cells, leading to recruitment of Ly6C+ inflammatory monocytes, which can capture and kill parasites *via* oxidative burst (109). Importantly, these monocytes can migrate to lymph nodes and differentiate into specialized DC subsets during *L. major* infection. These monocytes-derived DCs secret high levels of IL-12 and stimulate *L. major*-specific Th1 cell responses, suggesting a contribution to protection against disease (110, 111). Monocytes expressing high levels of CCR2 can also capture *L. major* at the site of infection in C57BL/6 mice, then migrate to draining lymph nodes and differentiate into inducible nitric oxide (iNOS)-producing DC that also promote Th1 cell-mediated protection (112). Hence, the promotion of these activities in the context of vaccination or drug treatment may be desirable.

NK cells are also recruited to the site of infection in mice infected with *L. major* and produce IFN-γ, which can amplify DC IL-12 production required for the development of strong Th1 cell responses (113). However, NK cells can also produce IL-10 during *L. donovani* infection (114), suggesting they can play either antiparasitic or immunoregulatory roles during infection. Depending upon the dose of infection, CD8+ T cells also produce IFN-γ in murine models of CL, which can also help shape early adaptive immune responses associated with protection (115–118). However, resolution of infection following *L. major* infection is primarily associated with CD4+ T cell-mediated immunity (119–121). Despite healing of cutaneous lesions, parasites continue to persist at the original site of infection, in part due to IL-10-mediated mechanisms, and these persisting parasites are thought to help maintain effector memory CD4+ T cells (TEM) that protect against re-infection (122, 123). This TEM response is lost if parasites are eliminated, as shown by studies in which mice were manipulated to achieve sterile cure (122). Thus, concomitant immunity is compromised and protection against a secondary challenge can be lost in the absence of persisting parasites (124). However, there is also evidence that a pool of long-lasting central memory CD4+ T cells (TCM) can develop in absence of persisting parasites, and that these can acquire effector functions after re-infection leading to protection (125). These TCM cells require additional IL-12 signals to develop into fully functional Th1 cells, and in absence of this signal, they can convert into IL-4-producing Th2 cells (126). TCM cells appear to be generated early during infection, and not only help in controlling secondary infections, but also contribute to clearance of primary infection (127). Hence, these findings suggest both TEM and TCM cells participate in maintaining immunity to *L. major* infection, but only the TEM require persistent parasite antigen. Therefore, vaccines designed to protect against leishmaniasis should target the expansion of long-lasting TCM cells, rather than short lived TEM cells.

More recently, skin resident memory (TRM) CD8+ T cells have been shown to provide protection against *L. major* infection, independent of circulatory CD4+ T cells, by recruiting inflammatory monocytes, which rapidly control parasite growth *via* reactive oxygen species (ROS) and NO generation (128). Thus, these TRM cells also represent a potential target cell population for vaccination. Treg cells appear to play a role in *L. major* persistence in C57BL/6 mice by suppressing CD4+ T cell effector functions through IL-10-mediated immunosuppressive mechanisms (122). The IL-10 produced by these Treg cells can also promote parasite persistence by modulating APC function and/or inhibiting parasite killing mechanisms in infected macrophages. Thus, the activity of Treg cells at the site of infection can promote concomitant immunity, but also allow parasites to persist. Therefore, although Treg cells could be targeted for immunomodulation, care would have to be taken to ensure that long-term protection was not compromised.

In non-healing CL caused by *L. major* Seidman strain in C57BL/6 mice, Nlrp3 inflammasome-dependent IL-1β activation plays an important role in determining disease outcome (129). The activation of the Nlrp3 inflammasome enhanced IL-1β activation through caspase-1 cleavage, which caused recruitment of neutrophils to the site of infection, and ultimately resulted in the suppression of immunity, which was confirmed by using neutropenic Genista mice (129). Nlrp3 can promote Th2 cell development in non-healing cutaneous lesions caused by *L. major* infection in BALB/c mice (130). Thus, this inflammasome and related cell signaling pathways are potential targets for immune therapy to treat and promote healing of cutaneous lesions in human CL (see also below). However, inflammasome- and caspase-1-dependent IL-1-β production has been shown to provide resistance against *L. amazonensis* infection in mice by triggering NO production (131), thus emphasizing the need for careful consideration in choosing appropriate targets for immune modulation in specific disease settings.

#### MUCOCUTANEOUS LEISHMANIASIS

Although self-cure is often the outcome of CL, some patients infected with *L. braziliensis, L. panamensis*, and *L. aethiopica* can develop MCL after resolution of their primary lesion, characterized by chronic inflammation of the nasal mucosa and by a hyperactive T-cell response (132, 133), associated with high levels of pro-inflammatory cytokines, such as IFN-γ and TNF-α, and decreased levels of IL-10 and TGF-β (132, 134, 135). Thus, a poorly regulated T cell response is an underlying cause of disease pathogenesis in MCL patients. In patients infected with *L*. *braziliensis,* the number of CD8+ T cells recruited to lesions increased as disease progressed, and these cells expressed high levels of granzymes and perforin, indicating they had elevated cytolytic activity (136). In fact, these CD8+ T cells have now been shown to contribute to inflammation and disease pathology *via* perforin-mediated cytotoxicity (137). In mice co-infected with lymphocytic choriomeningitis virus and *L. braziliensis*, it was found that perforin-mediated CD8+ T cell cytotoxicity in the lesion resulted in enhanced recruitment of neutrophils and monocytes, which produced IL-1-β that contributed to immunopathology and disease severity (138). Importantly, pharmacological blockade of Nlrp3 reduced inflammation caused by cytotoxic CD8+ T cells in this mouse model of MCL, thus identifying this inflammasome, as well as CD8+ T cell-mediated cytotoxicity, as potential targets for immunotherapy. This was supported by additional data from the same study, using skin biopsies and PBMCs from CL patients infected with *L. braziliensis*, which IL-1β was highly expressed in skin lesions and blockade of the Nlrp3 inflammasome prevented the IL-1-β secretion from skin biopsies, suggesting a similar pathogenic mechanism might be operating during clinical MCL.

In addition to CD8+ T cell-mediated pathology, Th17 cells have also been associated with pathogenesis in MCL patients (139). MCL lesions were found to have elevated IL-17A mRNA, as well as TGF-β, ROR-γT, and IL-23 mRNA levels, which are associated with Th17 cell differentiation. Interestingly, IL-17 was not only produced by CD4+ T cells but also by CD8+ T cells, CD14+, and CCR6+ Cells. The enhanced production of IL-17 was associated with infiltration and recruitment of neutrophils into the lesion, suggesting that IL-17 may promote inflammatory responses in MCL patients. Thus, IL-17 production could be a therapeutic target in MCL patients to reduce tissue pathology.

#### DIFFUSE CUTANEOUS LEISHMANIASIS

Diffused cutaneous leishmaniasis is a severe manifestation of CL characterized by a defective cellular immune response to *Leishmania* antigens (140). However, this unresponsiveness is restricted to antiparasitic immune responses, as responses to unrelated antigens remain intact (141, 142). DCL patients have high parasite numbers within skin lesions, which has been associated with low levels of IFN-γ and IL-2 mRNA, and concurrent high levels of IL-10, IL-4, and IL-5 mRNA in lesions (135). Therapeutic cure was associated with enhanced IFNγ production, but low IL-10 expression (143), indicating the requirement for a classical Th1 cells response for favorable clinical outcomes. This disease is also associated with high antibody titers and plasma TGF-β [reviewed in Ref. (102)]. IL-10 and TGF-β, along with Treg cells, can antagonize IFN-γ and TNF activities, resulting in impaired microbicidal activities in infected macrophages [reviewed in Ref. (144)]. However, it is not clear in DCL whether high antigen exposure causes T cell unresponsiveness or if impaired T cell responses promote localized parasite growth in the skin [reviewed in Ref. (144)].

Diffused cutaneous leishmaniasis patients respond poorly to conventional drug treatment (145), but some degree of treatment success has been achieved with immune modulation using IFN-γ combined with viable BCG and antimonial drug (146, 147). Since unresponsive T cells often express inhibitory molecules, such as PD-1, CTLA-4, and LAG-3, these may make attractive targets for immune therapy in DCL patients. IL-1-β has also been associated with disease severity in *L. mexicana*-infected DCL patients (148), making it another potential therapeutic target. Again, care will need to be taken to ensure the promotion of antiparasitic immunity in this context is not at the expense of protection against tissue damage.

#### LEISHMANIZATION

The fact that *L. major*-induced cutaneous lesions often heal spontaneously and protect against future infection is the basis for leishmanization, which involves inoculation with live, virulent parasites in an unexposed part of body to produce a controlled lesion. This strategy has been practiced successfully in the former Soviet Union, Middle East, and Israel, and likely provides protection in humans because it mimics a natural infection, including allowing parasite persistence and development of concomitant immunity. The protection provided by leishmanization is essentially T cell-mediated, whereby IFN-γ-producing CD4+ T cells are recruited to dermal sites of infection where they perform effector functions, including the promotion of microbicidal mechanisms in infected macrophages (123). Importantly, the success of leishmanization depends on the viability and infectivity of injected parasite. Parasites that lost virulence stimulated delayed-type hypersensitive reactions, but did not provide protection from natural re-infection (149). Leishmanization was abandoned in most countries because of logistical problems and safety concerns, due to some immunosuppressed individuals developing non-healing lesions (150). Interestingly, leishmanization can provide cross protection against the visceral form of disease, as leishmanized C57BL/6 mice infected with *L. major* were protected from heterologous visceral infection with *L. infantum*, associated with recruitment of IFN-γ-producing Ly6C+CD4+ T cells to both skin and visceral organs (151). Similarly, longitudinal studies in the Sudan indicated that people residing in an *L. major* endemic area were protected against VL caused by *L. donovani* (152, 153). In addition, CL caused by a *L. donovani* strain in Sri-Lanka provided cross protection against visceral disease (154). These findings suggest that leishmanization could be a strategy employed to increase protection against VL. However, a better understanding of the immunoregulatory mechanisms associated with this process is needed to fully exploit the positive aspects of leishmanization with improved safety.

#### STRATEGIES TO IMPROVE VACCINES

Although different *Leishmania* species cause a broad range of clinical symptoms, genetic analysis indicates a large degree of genomic conservation between species. Thus, it may be possible to generate broadly effective vaccines against different clinical diseases. However, despite many efforts, there is no effective, licensed vaccine to prevent human leishmaniasis. There is a major need for more efficacious and less toxic adjuvants and immune therapies for better vaccines for patients suffering from leishmaniasis. Studies in VL patients and experimental models (15, 155) indicate the rapid development of immunoregulatory networks following exposure to parasites, which raises questions about how these regulatory networks might influence subsequent immunity, particularly to vaccines. It is noteworthy that many vaccines tested in disease endemic regions have not performed as well as when tested in healthy volunteers. For example, the RTS,S/AS01 vaccine in children and infants affords 36 and 25% efficacy against clinical malaria, respectively (156), while a recent study showed that the efficacy of the same vaccine in healthy volunteers in CHMI studies was 52% (157). Similarly, BCG-mediated protection against pulmonary tuberculosis varies geographically and appears to be much less effective in areas with high incidence of previous infection with *M. tuberculosis* or sensitization with environmental *Mycobacteria* (158, 159). Although many reasons could account for the reduced efficacy of vaccines in disease endemic areas, these results suggest that the early establishment of potent, pathogen-specific immunoregulatory networks may be an important factor contributing to this problem (160). Treatment in the field of cancer has been revolutionized by immune checkpoint blockade strategies. These take advantage of the patients own immune system to recognize and kill cancer cells. Although many of the molecules being targeted by this approach were discovered in infectious diseases research, this approach has not been applied to reducing the burden of infection. Therefore, incorporating inhibitors of specific immune checkpoints into vaccine formulations may be one way to transiently reduce immune suppression to allow the generation of robust vaccine-mediated, antiparasitic immunity.

Both CTLA-4 and PD-1 blockade have been successfully used individually and in combination to treat cancer patients (161). Given that leishmaniasis is a chronic infection and shares several key immunoregulatory features with cancer, one strategy could be to "piggy back" on the success of immune checkpoint blockade drugs in cancer to either improve drug treatment protocols by making subsequent immunity more potent and long-lasting or enhance vaccine efficacy. However, it will be important to bear in mind that specific types and combinations of immune checkpoint blockade work best for particular types of cancer, and this is also likely to be the case with the spectrum of diseases caused by *Leishmania* species. Thus, careful consideration will need to be given to types of immune checkpoint blockade best suited to VL, CL, MCL, PKDL, or DCL because they are likely to differ in their outcomes.

#### STRATEGIES TO IMPROVE DRUG TREATMENT

Antimonial chemotherapy was the mainstay for VL treatment for many decades (162). However, parasite resistance against these drugs has developed, especially in the Indian subcontinent (163, 164). Therefore, these drugs are now mainly employed to treat VL in Africa, while drugs such as Amphotericin B, Ambisome, Miltefosine, and paromomycin have been introduced to treat VL in areas of antimonial drug resistance (164, 165). However, these drugs are not without problems, such as toxicity, high cost, potential development of parasite drug resistance, and prolonged treatment regimes [reviewed in Ref. (165)]. Recently, a single dose of Ambisome (lipid formulation of Amphotericin B) was found to be sufficient to successfully treat VL with low toxicity and has now been recommended as a choice of treatment in India subcontinent (166, 167). The oral drug miltefosin has also been used in combination with Amphotericin B. However, based on studies in preclinical models of leishmaniasis, there are concerns that even with combination therapy, drug resistance will develop (168). Further, these drugs do not cause sterile cure and parasites persist in the infected individuals after drug treatment (168–170). This is concerning because these persisting parasites may help promote transmission, with people living in the same household being most at risk of infection (171, 172). Thus, not only should successful cure of disease be a goal of treatment, but lowering the burden of persisting parasites as far as possible is also desired if parasite transmission is to be minimized. However, when considering these goals, it will be important to remember that persistent parasites are also required to maintain concomitant immunity (170), so sterile cure of infected individuals may not necessarily be beneficial. Instead, it may be necessary to establish the level of parasite burden that is low enough to prevent parasite transmission, but at the same time, maintain concomitant immunity, and then try and achieve this through a combination of antiparasitic drug and immunomodulatory strategies.

Drug treatment works most effectively in association with the host immune system, and in particular, cell-mediated immune responses (164). Hence, understanding immunological changes during the course of infection and how these might be modulated to work best with drug is important. The use of biological molecules to stimulate cell-mediated immunity to help achieve therapeutic success has been tested in both preclinical and clinical studies on leishmaniasis (173). In an experimental model of leishmaniasis, treatment with recombinant IL-12 or anti-IL-10 receptor monoclonal antibody, along with pentavalent antimony (Sbv ), resulted in improved clearance of *L. donovani* parasites, compared with animals treated with drug alone (27, 174). In addition, human recombinant IFN-γ has been successfully used to accelerate antiparasitic and clinical responses when used with antimony treatment, and importantly, treat seriously ill VL patients with refractory disease (175–178). Many drugs used to treat VL not only kill parasites, but also promote host immunity. For example, antimonial drugs stimulate the generation of ROS and NO, while miltefosine and Ambisome induce the secretion of IFN-γ, TNF, IL-12, IL-6, and IL-1β from immune cells with a simultaneous decrease in anti-inflammatory cytokine production (179–182). Thus, combining immune-based therapy with conventional antiparasitic drugs is an obvious strategy to improve current treatment protocols.

Rather than supplement or block immune effector molecules, another approach to use host-directed therapy to improve drug treatment is to target intracellular signal transduction pathways. For example, it has been shown that *L. infantum* infection rapidly induces activation of phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase1/2 in bone marrowderived dendritic cells (BMDDC), thereby limiting their maturation and pro-inflammatory cytokine secretion. The blockade of this pathway with wortmannin resulted in reduced infection rates of BMDDC (183). Similarly, the rapid activation of protein tyrosine phosphatase, such as SHP-1, by *Leishmania* is another important parasite evasion strategy, and administration of the SHP-1 inhibitor bpV-phen to mice infected with *L. major* and *L. donovani* promoted control of infection *via* induction of reactive nitrogen intermediates that would otherwise be repressed by parasite-activated SHP-1 (184, 185). Hence, small molecule inhibitors of key cell signaling pathways is another potential approach that could be used with current antiparasitic drug treatment protocols.

# DEVELOPING STRATEGIES FOR RESOURCE POOR SETTING

Leishmaniasis is generally a disease associated with poverty (186) and as such, diagnosis, treatment, and hospitalization costs are an important consideration in disease control programs. In addition, drug development programs for leishmaniasis are often not a high priority for pharmaceutical companies. Furthermore, even if effective, high-cost drugs or vaccines are available, they are unlikely to be used without significant government or philanthropic subsidization. Hence, a practical challenge is to supply relatively cheap drugs in resource poor settings, and this will require the participation of regulatory bodies, as well as public and private sector partnerships. A successful example of this was the implementation of the wider use of Ambisome for VL treatment. This is normally a high-cost drug, but has been substantially reduced in cost for the distribution through the public sector agencies in developing countries, by an agreement negotiated between WHO and the manufacturer (187). However, other issues can complicate such arrangements. For example, the requirement of a reliable cold chain for Ambisome implementation can result in a failure to provide the drug or the use of drug stored under conditions not consistent with storage advice. Therefore, the introduction of new treatment regimes for diseases such as VL will need to consider multiple aspects of drug development, formulation, storage, and delivery. One approach for reducing development costs is to repurpose drugs already licensed for other indications, as was the case with Ambisome, which was first licensed as an anti-fungal drug. Another way of reducing cost is to develop cheaper small molecules rather than more expensive biologics to target parasites or host responses. In addition, as well as usual safety considerations, the stability of drugs in areas of unreliable cold-chain must be considered, as well as ease of manufacturing. Therefore, when considering strategies to promote host directed therapy, regardless whether this be targeting specific immune check points or stimulating microbicidal mechanisms, small molecules are likely to be more cost-effective than antibodies.

# CONCLUDING REMARKS

Leishmaniasis has clear, unmet medical needs. These differ, depending on the disease in question. However, the host immune response to infection is a central component of each of these diseases, whether it is immune dysfunction in the case of VL and DCL or immune-mediated tissue pathology in the case of severe CL and MCL. Therefore, targeting these host responses, as is increasingly occurring in other chronic diseases, such as cancer and autoimmunity, offers promising new opportunities to either improve the efficacy of vaccine candidates or drug treatment protocols. Given the long time lines for vaccine development, the latter approach may have a greater impact in the short term. Furthermore, combining host-directed therapy with antiparasitic drug, offers the added advantage of further reducing parasite loads in treated individuals and improving longterm protective immunity. These outcomes will greatly benefit current disease elimination programs.

#### AUTHOR CONTRIBUTIONS

RK, SC, SN, SS, and CE wrote the paper. SN and SC prepared the figures and table.

#### ACKNOWLEDGMENTS

We thank staff in the Immunology and Infection laboratory at QIMR Berghofer and in Professor Shyam Sundar's research group at Banaras Hindu University for helpful discussions.

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#### FUNDING

This work was made possible through Queensland State Government funding. The research was supported by grants and fellowships from the National Health and Medical Research Council of Australia, a NIAID Tropical Medicine Research Centre grant (2U19AI074321), as well as an Australian Postgraduate Awards through Griffith University and an INSPIRE Fellowship to Rajiv Kumar provided by the Indian government Department of Science and Technology.

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

*Copyright © 2017 Kumar, Chauhan, Ng, Sundar and Engwerda. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

*Ramona Hurdayal1,2,3 and Frank Brombacher1,2\**

*1 Faculty of Health Sciences, Division of Immunology and South African Medical Research Council (SAMRC) Immunology of Infectious Diseases, Institute of Infectious Diseases and Molecular Medicine (IDM), University of Cape Town, Cape Town, South Africa, 2 International Center for Genetic Engineering and Biotechnology (ICGEB), Cape Town, South Africa, 3Department of Molecular and Cell Biology Faculty of Science, University of Cape Town, Cape Town, South Africa*

#### *Edited by:*

*Uwe Ritter, University of Regensburg, Germany*

#### *Reviewed by:*

*Tiago W. P. Mineo, Federal University of Uberlandia, Brazil Ricardo Silvestre, Instituto de Pesquisa em Ciências da Vida e da Saúde (ICVS), Portugal*

> *\*Correspondence: Frank Brombacher<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology*

*Received: 21 July 2017 Accepted: 03 October 2017 Published: 10 November 2017*

#### *Citation:*

*Hurdayal R and Brombacher F (2017) Interleukin-4 Receptor Alpha: From Innate to Adaptive Immunity in Murine Models of Cutaneous Leishmaniasis. Front. Immunol. 8:1354. doi: 10.3389/fimmu.2017.01354*

The interleukin (IL)-4 receptor alpha (IL-4Rα), ubiquitously expressed on both innate and adaptive immune cells, controls the signaling of archetypal type 2 immune regulators; IL-4 and IL-13, which elicit their signaling action by the type 1 IL-4Rα/gamma common and/ or the type 2 IL-4Rα/IL-13Rα complexes. Global gene-deficient mouse models targeting IL-4, IL-13, or the IL-4Rα chain, followed by the development of conditional mice and generation of important cell-type-specific IL-4Rα-deficient mouse models, were indeed critical to gaining in-depth understanding of detrimental T helper (Th) 2 mechanisms in type 1-controlled diseases. A primary example being cutaneous leishmaniasis, which is caused by the protozoan parasite *Leishmania major*, among others. The disease is characterized by localized self-healing cutaneous lesions and necrosis for which, currently, not a single vaccine has made it to a stage that can be considered effective. The spectrum of human leishmaniasis belongs to the top 10 infectious diseases according to the World Health Organization. As such, 350 million humans are at risk of infection and disease, with an incidence of 1.5–2 million new cases being reported annually. A major aim of our research is to identify correlates of host protection and evasion, which may aid in vaccine design and therapeutic interventions. In this review, we focus on the immune-regulatory role of the IL-4Rα chain from innate immune responses to the development of beneficial type 1 and detrimental type 2 adaptive immune responses during cutaneous *Leishmania* infection. We discuss the cell-specific requirements of the IL-4Rα chain on crucial innate immune cells during *L. major* infection, including, IL-4Rα-responsive skin keratinocytes, macrophages, and neutrophils, as well as dendritic cells (DCs). The latter, contributing to one of the paradigm shifts with respect to the role of IL-4 instructing DCs *in vivo*, to promote Th1 responses against *L. major*. Finally, we extend these innate responses and mechanisms to control of adaptive immunity and the effect of IL-4Rα-responsiveness on T and B lymphocytes orchestrating the development of CD4+ Th1/Th2 and B effector 1/B effector 2 B cells in response to *L. major* infection in the murine host.

Keywords: interleukin-4 receptor alpha, interleukin-4/interleukin-13, murine cutaneous leishmaniasis, innate cells, adaptive cells

# INTRODUCTION

Human leishmaniasis, ranging from localized ulcerating lesions (cutaneous) to disseminated (mucocutaneous) and fatal infection (visceral), presents a global health concern, with over 12 million people currently infected and an additional 350 million humans at risk of infection and disease (1, 2). It is therefore not surprising that infection rates surpass 1.5 million new cases annually (3). Despite a concerted effort to develop a vaccine against the parasite, not a single candidate has been proven effective, and current therapeutic approaches are unable to achieve a sterile cure (2, 3). A thorough understanding of the mechanisms by which *Leishmania* spp. evade or exploit host immune mechanisms, to persist and establish disease, is paramount to identifying new and improved strategies for effective management of the disease. To address this, experimental models of cutaneous leishmaniasis (CL), which is the most common form of the disease, was established. In this model, disease is induced by infecting mice subcutaneously with *Leishmania major*. The *L. major* mouse model provided an excellent system for investigating the mechanisms underlying T helper (Th) 1 and Th2 cell differentiation relating to resistance and susceptibility to intracellular infection (4–6). This model, in global gene-deficient mice, established that the archetypal Th2 cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13), are susceptibility factors during *L. major* infection in BALB/c mice, counterregulating a protective Th1 response, and induce their biological functions through a common receptor, the interleukin-4 receptor alpha (IL-4Rα) chain (7–9). However, IL-4Rα-deficient BALB/c mice remain susceptible to *L. major* infection in chronic stages (9, 10), indicating that IL-4/IL-13 may induce protective responses depending on which cell/s the IL-4/IL-13 ligand/s interact with during disease (11). Given the ubiquitous expression of the IL-4Rα signaling receptor on both innate and adaptive immune cells (12), cell-type-specific IL-4Rα-deficient mice were introduced to dissect the cell-specific roles of IL-4/IL-13 in CL. While these studies exemplified the role of IL-4Rα signaling on specific immune cells (10, 11, 13), it also questioned whether the Th1/Th2 paradigm of resistance/susceptibility to infection was in fact still relevant, considering that in certain disease settings, IL-4/IL-13 signaling essentially instructed a beneficial Th1 response (5, 11). Collectively, these reports highlight that the interplay between resistance and susceptibility to murine *L. major* infection involves a complex, dynamic interaction between the IL-4Rα chain and various innate and adaptive immune cells, with different clinical and immunological outcomes. In this review, we focus on the cellspecific requirements of the IL-4Rα chain signaling on crucial cells mediating innate and adaptive immunity to CL, relating primarily to *L. major* infection in mouse models.

#### THE IL-4R**α** CHAIN: COMMON RECEPTOR FOR IL-4 AND IL-13 SIGNALING

#### Interleukin-4

Interleukin-4 plays a critical role in initiating and regulating Th2-type immune responses (14). In mice, IL-4 is a 14–19 kDa glycoprotein localized on chromosome 11, together with the genes for IL-5 and IL-13. During the innate immune response, evidence suggests that early IL-4-producers include basophils (15, 16), mast cells (17), eosinophils (18), natural killer (NK) T cells (19, 20), and innate-like skin keratinocytes (21). T and B lymphocytes orchestrating adaptive immunity, specifically CD4+ Th2 cells (22), B effector 2 (Be2) B cells (23, 24), and γ/δ T cells (25), also secrete IL-4. Apart from regulating the differentiation of Th2 cells, IL-4 also controls immunoglobulin class switching in activated B cells, specifying human B cells to switch to the expression of IgE and IgG4 (26), while in mice, to IgE and IgG1, with the concomitant suppression of IgM, IgG2a, and IgG2b (27, 28). Moreover, alternatively activated macrophages are activated by IL-4 signaling through the IL-4Rα chain (29). Importantly, IL-4 inhibits inducible nitric oxide synthase (iNOS) expression thereby inhibiting IFN-γ-induced classically activated macrophages and induction of a type 1 response. As a whole, IL-4 counter-regulates the expression of IFN-γ (12) and increases the expression of MHC II molecules (30), co-stimulatory molecules CD80 and CD86 (31), and the IL-4 receptor (32). Reports have also indicated that dendritic cells (DCs) can respond to IL-4 *in vivo* and *in vitro* and become alternatively activated, in a manner similar to that described for alternatively activated macrophages, by upregulating multiple alternative activation markers such as mannose receptor and RELM-α (33). Moreover, although IL-4 has been shown to be the primary inducer of Th2 responses, studies have reported IL-4-independent Th2 differentiation, Th2 cytokine production, IL-4Rα signaling, and STAT6 regulation (34–41).

#### Interleukin-13

Murine IL-13 is an immunoregulatory cytokine with a molecular weight of 10–14 kDa (42), also localized on chromosome 11, together with the genes for IL-4 and IL-5. Similar to IL-4, murine IL-13 also promotes upregulation of MHC II antigens, co-stimulatory (CD80/CD86) and adhesion molecules. However, unlike IL-4, murine IL-13 has been shown not to affect Th2 differentiation, B cell switching or upregulation of the low-affinity IgE receptor (CD23), likely due to the absence of a functional IL-13 receptor on those cells in mice. By contrast, human B lymphocytes do respond to IL-13 (34). Innate mast cells, basophils, DCs, NK cells, activated CD4+ Th2 cells, Be2 B cells, and NK T cells are IL-13-producers (34, 43–45). In fact, IL-13 is responsible for activating mast cells, modulating eosinophil function (42), and alternatively activating macrophages in conjunction with IL-4 (29). IL-13 also has immunosuppressive and anti-inflammatory effects on macrophages and monocytes, including suppression of pro-inflammatory cytokines and chemokines. In addition, nitric oxide (NO) production, along with antibody-mediated cytotoxicity, is inhibited by IL-13.

#### The IL-4 and IL-13 Receptor Complexes

The overlapping biological functions of IL-4 and IL-13 on certain cell-types could be partly attributed to the shared IL-4Rα component of natively distinct receptors (34). This theory was initially demonstrated in competitive studies in which treatment of mice with IL-4 antagonists or anti-IL-4Rα antibodies inhibited both IL-4- and IL-13-mediated responses (46–48). The IL-4Rα chain (CD124) is a 140 kDa heterodimeric complex, serving as a common monomer in both the type 1 and type 2 receptor complexes (**Figure 1**). It is ubiquitously expressed in fairly low numbers on hematopoietic and non-hematopoietic cells (12). IL-4Rα interacts with the gamma common (γc) chain to form the type 1 IL-4 receptor and interacts with the 65–70 kDa IL-13-binding receptor alpha 1 (IL-13Rα1) chain to form the type 2 IL-4/IL-13 receptor (**Figure 1**) (12). The former is also shared by the receptors for IL-2, IL-7, IL-9, and IL-15. IL-4 binds the IL-4Rα chain with high affinity. By contrast, IL-13 binds the IL-13Rα1 chain with low affinity. However, when paired with the IL-4Rα chain, IL-13 binds the IL-13Rα1 chain with high affinity forming an active signaling unit (49). Expression of IL-13Rα1 is absent on human or murine T cells but constitutively expressed on B cells, epithelial cells and monocytes in both mice and humans (50, 51). In comparison, IL-13 shows a higher binding affinity for the α2 chain of the IL-13 receptor (IL-13Rα2), which is a 55–60 kDa protein (**Figure 1**). IL-13Rα2 was originally considered a decoy receptor for IL-13, devoid of signal transduction, since its short cytoplasmic domain was not reported to contain any binding motifs for signaling molecules (52). However, well-designed reports have demonstrated a signaling pathway for IL-13 through the IL-13Rα2 chain, which induces the production of TGF-β1 and mediates fibrosis (53). In addition to cell-surface localization,

soluble forms of both IL-4Rα and IL-13Rα2 exist (**Figure 1**), which are capable of binding IL-4 and IL-13 with high affinity as non-signaling monomers. In doing so, the soluble receptors can act as competitive inhibitors of both IL-4 and IL-13 and modulate their effector responses (54, 55).

# Mechanisms of IL-4 and IL-13 Signaling through the IL-4R**α** Chain

Both IL-4 and IL-13 signal transduction *via* the IL-4Rα chain involves activation of the Janus-family kinases (JAK). JAK1 interacts with the IL-4Rα chain whereas JAK3 interacts with the γc chain and JAK2 with the IL-13Rα1 (**Figure 1**) (12, 57, 58). IL-4 engagement with the IL-4Rα chain results in tyrosine phosphorylation of the IL-4Rα chain itself as well as phosphorylation of STAT6 and insulin receptor substrate 2 (IRS-2) by JAKs, which then associates with the phosphoinositol-3 kinase (PI-3) (**Figure 1**) (12, 34). STAT6-deficient mice presented impaired IL-13-mediated functions, which confirmed that IL-13 also uses the JAK/STAT6 pathway for signal transduction (**Figure 1**) (34, 59). IL-13 signaling through the IL-13Rα2 requires initial engagement of the IL-13Rα1/IL-4Rα complexes in conjunction with TNF-α signaling, which increases surface expression of IL-13Rα2. IL-13 then binds to the IL-13Rα2 and, through

Figure 1 | The interleukin-4 (IL-4) and interleukin-13 (IL-13) receptor complexes. IL-4 interacts with the interleukin-4 receptor alpha (IL-4Rα) chain in combination with either the gamma common (γc) chain to form the type 1 receptor or with the IL-13-binding receptor alpha 1 (IL-13Rα1) to form the type 2 receptor complex. The IL-4Rα chain signals *via* the JAK1/STAT6 pathway. Definitively, JAK3 associates with the γc chain and JAK2 with the IL-13Rα1. IL-13 interacts with the type II receptor complex (through IL-13Rα1) or with the α2 chain of the IL-13 receptor (IL-13Rα2). A signaling pathway for IL-13 *via* IL-13Rα2 has recently been identified. TNF-α induces upregulation of IL-13Rα2 expression. IL-13 then binds to the IL-13Rα2, which activates AP-1 to induce gene expression and secretion of soluble TGF-β. Illustration adapted and redrawn from previous publications (12, 34, 42, 52, 56).

TGF-β (**Figure 1**) (52, 53).

CUTANEOUS LEISHMANISIS

#### to *L. major*

The *Leishmania* parasite has a complex digenetic life cycle alternating between two distinct stages; the promastigote form found in the female *Phlebotomus* sandfly vector and the amastigote form replicating within phagolysosomes of host macrophages and DCs. Ironically, macrophages (and DCs) being the primary immune cells involved in the eradication of *Leishmania* in a mammalian host, are the preferred host cells of the parasite (3, 11, 60, 61). Early pioneering studies focused on the modulation of cytokine signaling as a means to alter immune cell activation and Th cell differentiation. These studies were conducted in an attempt

activation of the transcription factor AP-1, drives secretion of

to understand the mechanisms by which *Leishmania* are able to survive and flourish in these hostile environments while also capitalizing on host defense mechanisms to favor the establishment of disease. These mouse models, involving experimental infection with *L. major*, established the Th1/Th2 paradigm of resistance/ susceptibility to intracellular infection (5, 6). Upon infection with *L. major*, genetically resistant C57BL/6 mice develop a healing phenotype associated with an IL-12-driven protective Th1/type 1 immune response, upregulation of IFN-γ and classical activation of macrophages for subsequent killing of intracellular parasites by effector NO production (**Figure 2**) (13, 62–66). IL-18 augments IL-12 activity, both of which interact with the IL-12 receptor β2 chain (IL-12Rβ2) in activated Th1 cells (67) and protects against *L. major* infections (68). CD8+ T cells and NK cells enhance this protective response by secreting IFN-γ that activates macrophages for parasite clearance. By contrast, the lack of healing in genetically susceptible BALB/c mice is associated with a Th2/ type 2 response characterized by the secretion of IL-4, IL-5, IL-9,

Together, a dominant Th1 response is generated by the production of IFN-γ and TNF-α. These cytokines upregulate the expression of inducible nitric oxide synthase (iNOS) and activate infected macrophages for intracellular killing. CD8+ T cells and natural killer (NK) cells control infection by producing IFN-γ and effector killing by macrophages leading to parasite control as observed in C57BL/6 mice. (B) By contrast, parasite persistence in BALB/c mice is due to the failure of an IL-12 dependent redirection of the early Th2 response. This may be due to continued expression of early interleukin-4 (IL-4) from Vβ4+Vα8+ CD4+ T cells, mast cells, and eosinophils at the inoculation site and draining lymph nodes. This delays secretion of IL-12, or loss of responsiveness to IL-12 due to reduced expression of the IL-12Rβ2 chain on activated Th2 cells. IL-4 and IL-13 suppress Th1 responses and inhibit intracellular killing by macrophages. This leads to arginase production by macrophages that favor parasite growth. Sustained neutrophil recruitment to the site of infection and regulatory T cells may promote susceptibility and inhibit Th1 responses by the production of IL-10 and TGF-β that inhibit activation of macrophages. Solid lines represent activation, and broken lines represent inhibition. Illustration drawn from previous publications (6, 75, 76).

IL-10, and IL-13, high type 2 anti-*Leishmania* antibody titers, and alternative activation of macrophages, which altogether promotes parasite survival and growth (**Figure 2**) (7, 8, 69–71). BALB/c mice present with lower levels of IL-12. Evidence suggests that this might be due to sustained IL-4 expression by Vβ4+Vα8+ CD4+ T cells, mast cells and eosinophils at the inoculation site and draining lymph node (LN) (**Figure 2**), which delays secretion of IL-12. Alternatively, reduced expression of the IL-12Rβ2 chain on activated Th2 cells lead to a loss in responsiveness to IL-12. In addition, maintenance of inflammatory neutrophils at the site of parasite inoculation (72) and regulatory T cells (Tregs), constituting a source of IL-10, favors the persistence of parasites in leishmanial skin lesions (**Figure 2**) (73, 74).