Interleukin-17 mediated immunity during infections with Trypanosoma cruzi and other protozoans

Abstract

Host resistance during infection with Trypanosoma cruzi, and other protozoans, is dependent on a balanced immune response. Robust immunity against these pathogens requires of the concerted action of many innate and adaptive cell populations including macrophages, neutrophils, dendritic cells, CD4⁺, and CD8⁺ T cells and B cells among others. Indeed, during most protozoan infections only a balanced production of inflammatory (T_H1) and anti-inflammatory (T_H2/regulatory) cytokines will allow the control of parasite spreading without compromising host tissue integrity. The description of T_H17 cells, a novel effector helper T cell lineage that produced IL-17 as signature cytokine, prompted the revision of our knowledge about the mechanisms that mediate protection and immunopathology during protozoan infections. In this manuscript we discuss the general features of IL-17 mediated immune responses as well as the cellular sources, effector mechanisms and overall role of IL-17 in the immune response to T. cruzi and other protozoan infections.

1. Introduction

Host resistance during infection with Trypanosoma cruzi, and other protozoans, is dependent on a balanced immune response. Robust immunity against these pathogens requires of the concerted action of many innate and adaptive cell populations including macrophages, neutrophils, dendritic cells, CD4⁺, and CD8⁺ T cells and B cells among others [1]. In addition, cytokines play a critical role by participating in the orchestration of such concerted response as well as by exerting direct effector mechanisms. Early evidences supported that cytokines of the T_H1 type such as IFNγ and TNF play a major protective role against T. cruzi infection by activating macrophages to destroy ingested parasites and to release proinflammatory cytokines [2–5]. Further research highlighted that despite being required for parasite clearance, high levels of IFNγ and TNF sustain pathogenesis during chronic Chagas disease [6–9]. Indeed, deficient signaling of regulatory cytokines such as IL-10 correlated with reduced parasite burden but increased mortality during experimental T. cruzi infection, due to overwhelming inflammatory responses mediated by type 1 cytokines [10, 11]. Furthermore, different cytokine expression profiles dictate susceptibility to experimental T. cruzi infection by a mechanism independent on parasite loads [12]. Altogether, current knowledge supports the notion that during T. cruzi infection only a balanced production of inflammatory (T_H1) and anti-inflammatory (T_H2/regulatory) cytokines will allow the control of parasite spreading without compromising host tissue integrity. Similar requirements of balanced effector/regulatory cytokine production have been reported for other protozoan infections including Toxoplasma gondii, Plasmodium spp., Leishmania spp., etc. (reviewed in [13]).

During the last years, new discoveries have challenged our notion about the immune effector pathways that mediate protection against different pathogens. In this regard, the description of T_H17 cells, a novel effector helper T cell lineage that produced IL-17 as signature cytokine [14], lead to the revision of the classical T_H1/T_H2 dichotomy used to explain effector immune response since its enunciation by Mossman et al in 1986 [15]. Consequently, in the last years, many research efforts have been oriented to understand the contribution of IL-17- and T_H17 cell-mediated immune pathways during different pathological conditions, including infections. In this manuscript we discuss the general features of IL-17 mediated immune responses as well as the role of these effector mechanisms in the immune response to T. cruzi and other protozoan infections.

2. Fundamental knowledge about IL-17 and IL-17 producing cell populations

IL-17A, also known as IL17, is the founding member of IL-17 cytokine family and was initially described in 1995 by Yao and colleagues [16]. Since its description, many efforts have been oriented to identify the structure, ligands, receptors, and biological function of this family particularly after the description of T_H17 cells. The current available data in this regard have been recently reviewed [17, 18] and the most important aspects are depicted in Figure 1 and summarized below.

Figure 1. Fundamental knowledge on IL-17 biology.

A) Classical IL-17 producing subsets: IL-17⁺ innate immune cells in steady state are γδ T cells, type three innate lymphoid cells (ILC3) cells (including Lymphoid tissue inducer cells, lymphoid tissue inducer like cells and CCR6⁺ cells) and invariant NK T cells (iNKT) cells. Adaptive immune cells that include CD4⁺ T cells (T_H17) and CD8⁺ T cells (Tc17) produce IL-17 upon engagement with their cognate antigen under inflammatory conditions. Antigen independent CD4⁺ IL-17⁺ cells have also been described and are known as “natural occurring” T_H17 or nT_H17. All these innate and adaptive immune cell subsets are dependent on the expression of the transcription factor RORγt for the stable production of IL-17. B) IL-17 cytokines and subunit receptors family: IL-17 family members share different percentage of sequence homology and are named from A to F. They signal through protein complexes formed by two or more IL-17 receptor subunits named from IL-17RA to IL-17RE. The particular affinity of each cytokine for its receptors and the different IL-17R expression patterns define overlapping or unique effector function for each IL-17 family member depending on the tissue/cell type. C) Intracellular IL-17R signaling and effector mechanisms. Upon IL-17RA binding, IL-17 induces the consecutive recruitment of ACT1 to the SEFIR intracellular domain, and TRAF6 to the ACT1-SEFIR. This process is critical for NF-κB activation and induction of pro-inflammatory cytokines. Another pathway activated by IL-17 signaling is MAPK pathway (ERK, JNK and p38) and stimulation of C/EBP β,δ. Also, one of the most important effects of IL-17 signaling is the stabilization of mRNA transcripts encoding IL-17 target genes through TRAF2/5 molecular adaptors. IL-17 target genes include antimicrobial peptides, chemokines, proinflammatory cytokines and matrix metalloproteinases that drive host immunity against extracellular and intracellular bacteria, fungi, parasites and viruses. Deregulation of these proinflammatory mediators may give rise to chronic inflammation and autoimmunity. Created with Biorender.com

2.1. IL-17 family: cytokines and receptors.

Analysis of the amino acid sequence of IL-17A using protein sequence data banks quickly evidenced that there were other cytokines similar to IL-17. Nowadays, the IL-17 cytokine family is formed by six members, named from IL-17A to IL-17F [18] (Figure 1). As a family, they share four conserved cysteine residues and different percentages of sequence homology in the C-terminal portion. The N-terminal portion of the different cytokine family shows a heterogeneity that is supposed to give the specificity among IL-17 receptors. Of note, IL-17F has 55% sequence homology with IL-17A, and it can bind IL-17A to form the heterodimer IL-17A/F. Furthermore, IL-17F and IL-17A are co-expressed on linked genes and are usually coproduced by the same cells types, showing similar effector functions. Other members such as IL-17B, IL-17C and IL-17D share a homology between 25—30% with IL-17A, while IL-17E show the lowest degree of sequence conservation (16-20%).

IL-17 cytokines signal through receptors formed by homo- or hetero-complexes of two or three subunits of the IL-17 receptor (IL-17R) family. The IL-17R family is composed of 5 members named from IL-17RA to IL-17RE. These subunits share a fibronectin III like domain in the extracellular region and a shared cytoplasmic motif termed a “SEFIR” (SEF/IL-17 receptor), which has a distant relationship with the Toll-IL-1 receptor (TIR) domain. IL-17RA was first discovered as the receptor for IL-17A and IL-17F but it was later reported to also bind to IL-17E and IL-17C; so, it is now considered a common subunit of the receptor family. The subunit that combines with IL-17RA determines the specificity towards a given ligand. The complex formed by IL-17RC and IL-17RA is considered the canonical receptor that enables responsiveness to IL-17A and IL-17F [19]. However, IL-17RA could also complex with IL-17RD to allow IL-17A signaling [20, 21]. Regarding the other cytokine/receptor pairs, it has been reported that IL-17RB and IL-17RE form complexes with IL-17RA to bind to IL-17E and IL-17C, respectively. Differently, a homodimeric receptor formed by IL17RB is required for IL-17B signaling, while the receptor for IL-17D has not been yet identified [18].

Considering that IL-17A and IL-17F are concomitantly produced by immune cell populations and play critical roles in many immunological processes, in the following sections we will focus on the biology of these two cytokines.

2.2. IL-17-producing cell subsets

Specialized CD4⁺ T cells, called T_H17 cells, are the major source of IL-17A and IL-17F in many conditions. T_H17 cells differentiate upon antigenic stimulation in the presence of specific polarizing cytokines that include TFGβ and the inflammatory cytokines IL-6 and IL-1β [22], IL-23, a cytokine of the IL-12 family, further reinforces T_H17 commitment and promotes T_H17 cell survival. The T_H17 lineage developmental program is dictated by the transcription factor RORγt, considered the master regulator of this cell subset, and at a minor extent by other transcription factors such STAT3, RORα and AhR (Aryl hydrocarbon receptor). Of note, a particular population of CD4⁺ T cells that produce both IL-17 and IFNγ has been described in several settings. These cells express T-bet in addition to RORγt and have been particularly associated to pathogenicity in autoimmune diseases and infections, therefore they are named “pathogenic” T_H17 cells [22]. Within other adaptive immune cells, cytotoxic CD8⁺ T cells have been also shown to secrete IL-17. These Tc17 cells follow a transcriptional program that shows marked similarities with that of T_H17 cells [23].

The discovery that RAG deficient mice are able to produce IL-17 demonstrated that different innate cells represent an important source of this cytokine. Among these innate subsets are γδT cells, natural killer (NK) cells, invariant natural killer T (iNKT) cells, TCRβ⁺ natural Th17 (nTh17) cells, and type three innate lymphoid cells (ILC3) including lymphoid-tissue inducer-like cells. Most of these IL-17-producing subsets share some developmental commonalities such as dependence on IL-1β, IL-23 and RORγt, and expression of the chemokine receptor CCR6. Furthermore, these early sources of IL-17 have a central role in the initiation of T_H17 cell program, even before naïve CD4⁺ T cells recognize their antigen. The barrier location of innate IL-17 producing cells positions them as guards of the immune system [24]. In addition to these lymphoid subsets, some reports indicate that myeloid-lineage cells, including neutrophils and microglia, also produce IL-17, though this process is less understood and remains somewhat controversial [25–27].

Within novel innate sources, we reported that B cells are a major source of IL-17 during infection with T. cruzi [28] (see section 3 for further details). Other conditions in which IL-17 producing B cells have been reported include a helminthic infection [29] and autoimmune diseases [30]. It remains to be determined whether B17 cells correspond to a specific B cell lineage or it evidences a particular activation status.

2.3. IL-17 receptor expression and signaling

The complex IL-17RA/IL-17RC is pre-associated, facilitating rapid and specific response to its ligands: IL-17A, IL-17A/F, and IL-17F. As they use the same receptor complex, these three cytokines trigger qualitatively similar signaling pathways; nevertheless, the potency of the signal is the highest for IL-17A homodimers, intermediate for IL-17A/F and low for IL-17F homodimers. These differences are thought to be related to the differential affinity of each receptor subunit for IL-17A and IL-17F. Thus, IL-17RA shows higher affinity for IL-17A than for IL-17F while IL-17RC preferentially binds to IL-17F [18].

On top of the different affinities between ligands and receptor subunits, responsiveness to IL-17A or related cytokines in different cell types or tissues is affected by the profile of expression of IL-17RA and IL-17RC. In this regard, data in the human protein Atlas that systematizes current knowledge about protein expression in healthy human tissue [31] evidence that IL-17RA is ubiquitous expressed. IL-17RA is highly expressed in hematopoietic cells, thyroid, pancreas and tonsil tissue and present at low levels in muscle, kidney, skin, liver and gallbladder. IL-17RC has a more restricted pattern of expression, with low expression in myeloid and lymphoid cells and medium or high expression in non-hematopoietic cells. Remarkably, the IL-17RD protein is expressed at high levels in most of the tissues analyzed but shows a pattern that opposes that of IL-17RA. Besides, it has been shown that IL-17RD signaling diminishes the IL-17A mediated induction of target genes [32]. Altogether, these data support the notion that the combination of particular pattern of IL-17R expression together with different cytokine/receptor affinities may dictate the responsiveness to IL-17 cytokine family, having an impact in the signaling pathways and effector function that is particular to each cell type/tissue.

Given its pro-inflammatory nature, IL-17 signaling pathway activates the NF-κB pathway in a manner that differs from other classic NF-κB activators such as IL-1R and TLR ligands. In fact, the IL-17RA signaling cascade starts after IL-17A binding with recruitment of the adaptor protein ACT1 to the SEFIR region. ACT1 binds to TRAF6, which is indispensable for NF-κB activation. In addition, the IL-17RA signaling drives the activation of the MAPK pathway which includes p38, extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) and leads to the activation of AP1 transcription factor. C/EBPβ and C/EBPδ (CCAAT-enhancer-binding proteins) are also IL-17 targets that have redundant as well as non-redundant functions. Finally, IL-17 signaling also engages a pathway of post-transcriptional mRNA stabilization that is dependent on TRAF2 and TRAF5 [18].

2.4. IL-17-mediated effector mechanisms.

Although initially associated to pathogenic pro-inflammatory responses that sustain autoimmunity, it is currently accepted that IL-17A and IL-17F evolved to protect from infection. These cytokines orchestrate several protective mechanisms that provide resistance against infections at systemic level as well as at mucosal and epithelial surfaces (i.e. intestine, skin, lung, and oral cavity). Their central role in mediating protective immunity against pathogens relies on the induction of molecules collectively known as antimicrobial peptides by different stromal cells such as fibroblasts and endothelial and epithelial cells. The most studies antimicrobial peptides induced downstream IL-17RA/IL-17RC signaling are β-defensins, S100 proteins, serum amyloids and lipocalins. In addition, IL-17A and IL17F promote the enhanced production of cytokines (i.e. TNF, IL-6), inflammatory compounds (i.e. COX2 and iNOS), and matrix metalloproteinases (MMPs) that sustain pro-inflammatory environments. Thus, IL-17A and IL-17F indirectly mediate the activation and recruitment of immune cells to the infection site, promoting a potent immune response against the invading microbe. One of the hallmarks of the inflammatory responses mediated by IL-17 is the accumulation of polymorphonuclear (PMN) cells. Indeed, IL-17 regulates neutrophil production by triggering the induction of growth factors like G-CSF and GM-CSF and promotes neutrophil chemotaxis into tissue by stimulating secretion of chemokines such as CXCL1, CXCL5, and CCL2. In addition, IL-17 induces other chemokines such as CCL20, which recruits CCR6-expressing cells such as T_H17 and ILC3s, as well as CXCL10, which recruits CXCR3⁺ cells like Th1, CD8⁺ and NK cells [17].

Altogether, this plethora of mechanisms allow IL-17A (and to a lesser extent IL-17F) to coordinate the action of stromal, innate, and adaptive immune cells in order to efficiently combat different types of pathogens. The central role IL-17-mediated mechanisms in protective immunity against infections is highlighted by the increased susceptibility of mice deficient for IL-17RA, IL-17A and/or IL-17F to infection with fungi, extracellular and intracellular bacteria, viruses and parasites [33]. Within fungal infections, recent data indicate that IL-17 promotes resistance against not only Candida albicans [34], but also Aspergillus fumigatus [35], Cryptococcus neoformans [36] and dermatophytes such as Microsporum canis [37]. Similarly, immunity mediated by IL-17 is crucial for protection against Staphylococcus aureus in skin [38]. Citrobacter rodentium [39]in intestine and Klebsiella pneumonia in lungs and also collaborate with clearance of different intracellular bacteria [40]. In addition, IL-17 mediated immunity hinders viral replication and virus-induced immunopathology during particular viral infections while it plays the opposite role in others [41]. In the same direction and as discussed in the next sections, IL-17 participates in immune responses against parasites (particularly protozoa) with different outcomes. It is important to mention that, besides their impact in host protection, IL-17-mediated inflammation have been linked in preclinical and clinical settings to many experimental and human chronic diseases like psoriasis, Crohn’s disease, multiple sclerosis, rheumatoid arthritis, and others [42].

3. IL-17 producing subsets in T. cruzi and other protozoan infections.

Infections with different protozoans can trigger production of IL-17 by different subsets of immune cells. Below, we describe these subsets classifying the source of IL-17 as innate or adaptive (Figure 2).

Figure 2. IL-17 producing immune cell subsets during different protozoan infections.

Innate and adaptive immune cells produce IL-17 after in vivo infection or In vitro stimulation with complete protozoans or parasite-derived antigens. Cytokines involved in the induction of IL-17 production have been identified in some of these settings (see section 3).

3.1. Innate and innate-like production of IL-17.

It has been reported that some innate cell types respond early to infections with protozoans by producing IL-17. Particularly in the context of experimental T. cruzi infection, δγT cells and NK cells have been described as relevant sources of IL-17 in infected Balb/c and C57BL/6 mice [43–45] while no studies have evaluated the involvement of ILC3. Nevertheless, during T. cruzi infection there is a great activation of the mucosa and affection of the gut [46], hence, it is expected that ILC3s may play an important role by protecting these compartments.

In addition, we reported that Gr-1⁺ cells from spleen and liver of T. cruzi infected C57BL/6 mice produce IL-17 when stimulated with TLR2 agonists [45] and mature B cells serve as innate-like source of IL-17 in the same setting [28]. In fact, polyclonally activated B cells with an extrafollicular-plasmablast phenotype are the major subset of IL-17 producing cells during T. cruzi infection in C57BL/6 mice. Interestingly, engagement of TLRs and BCR are dispensable for the production of IL-17 by B cells. Signals through IL-6 and IL-23 and the transcription factor RORγt are also not required for the induction of B17 cells. Instead, T.cruzi trans-sialidase, an enzyme that transfers sialic acid to surface proteins, induces IL-17 production by remodeling the glycosylation of CD45 at the B cell membrane.

Lamina propria RORγt⁺ ILC3 cells play a critical role in promoting intestinal barrier function and limiting cellular memory responses to gut-resident commensal bacteria [47]. During oral infection with T. gondii, mucosal ILC3 are reduced in numbers but become activated and limit parasite-specific and commensal-specific CD4⁺ T cell activation and IFN-γ production, preventing excessive intestinal pathology [48]. Strikingly, ILC3 cells exert this regulatory mechanism in steady-state as well as during T. gondii infection independently on IL-17 or IL-22 production but dependent on MHC class II expression and antigen presentation [47, 48]. Also T. gondii infection triggers an early innate IL-17 production, but in this case a major source seems to be splenic NK cells that are activated by IL-6, IL-23 and TGFβ [49].

To date, production of IL-17 by typical innate cells during Plasmodium spp. infection has been scarcely studied. However, one report has recently described a lymphoid progenitor population, defined as LSK⁻ (Lineage⁻ Sca-1⁺ c-Kit⁻) cells, that become the major IL-17 producing population in the spleen of mice (C57BL/6) infected with Plasmodium Yoelii Y17 [50]. Interestingly, LSK⁻ cells upregulate AhR and start to express RORγt after P. yoelii infection. The other IL-17-secreting cell subsets during this infection include γδT and NK cells as well as adaptive immune populations [51]. Of note, IL-17 mediates the differentiation of the lymphoid progenitor LSK⁻ population into mature B cells (see section 4.3. for details).

The sources of IL-17 production during visceral leishmaniasis were investigated using IL-17 reporter mice. Upon infection with Leishmania donovani, B cells and NK cells produce negligible levels of IL-17 while γδ T cells become the major IL-17 producing population, followed by T_H17 cells [51]. Also during experimental cutaneous Leishmania major infection in C57BL/6 there is a strong induction of IL-17 secretion by neutrophils as well as by T_H17 cells [52]. Interestingly, neutrophil IL-17 production is antigen-independent and possibly related to the cytokine environment generated during Leishmania infection.

3.2. Adaptive immune cell sources of IL-17.

Both CD4⁺ and CD8⁺ T cells are the adaptive subsets that have been shown to produce IL-17 during protozoan infections. However, there is incomplete information about the specificity of these cells as well as about the signals that promote and inhibit IL-17 secretion. The available data is discussed below and highlight a strong dependence on the type of parasite, target tissues as well as host genetic background.

During T. cruzi infection, CD4⁺ T cells arise as an important source of IL-17 in spleen, liver and blood, although also CD8⁺ T cells are able to produce this cytokine [43–45]. Whether these cells are specific for T. cruzi antigens or are activated in a by-stander fashion has not been evaluated in experimental setting. In humans with chronic Chagas’ disease, IL-17 is produced mainly by parasite-specific CD4⁺ T cells that are increased in patients with mild clinical disease but reduced in patients with more severe cardiac compromise [53]. Otherwise, it was recently reported that Chagas’ disease patients also present a population of CD4⁺IL-17⁺IFNg⁺ cells whose higher frequency is directly correlated with worsen cardiac disease [54].

Despite that the main effector cells during T. gondii infection are Th1 cells; it has been shown that antigen-experienced T cells produce IL-17 in the context of genetic ablation of IL-27 production [55]. Thus, IL-27 inhibits IL-17 secretion by brain infiltrating CD4⁺ and CD8⁺ T cells and consequently, it restrains the development of chronic central nervous system pathology during T. gondii infection. The inhibitory effect of IL-27 in T_H17 differentiation is independent of the suppressor of cytokine signaling 3 (SOCS3) protein or T-bet but dependent on STAT1 activation.

Analysis of IL-17 secretion during Plasmodium chabaudi infection using reporter mice showed that both CD4⁺T cell and CD8⁺ T cells are able to produce this cytokine [56]. Instead, when studied by intracellular staining, IL-17⁺ cells were only detected within the CD8⁺ T cell pool in liver. In humans, patients with uncomplicated acute vivax malaria have significantly elevated absolute numbers of circulating IL-17-producing CD4⁺ cells in comparison with uninfected controls [57]. The rise of this subset occurs together with an increase in the frequency of CD4⁺ T cells producing IFNγ. Nevertheless, it was not determined whether this concomitant increase may be due to pathogenic IFNγ⁺ T_H17cells.

In experimental cutaneous leishmaniasis caused by L. major infection, CD4⁺ T cells contribute the most to IL-17 production in an antigen-dependent manner [52]. Remarkably, IL-17 secreting ability is higher in T_H17cells from infected C57BL/6 mice compared with counterparts from Balb/c mice. Furthermore, CD8⁺ T cells, γδT cells and neutrophils from infected Balb/c mice produce low levels of this cytokine while neutrophils from C57BL/6 secrete abundant IL-17 even without the requirement of further stimulation [52]. Showing some differences with the cutaneous disease, during experimental visceral disease due to L. donovani infection, most IL-17-producing cells from spleen and liver are from innate origin; though, CD4⁺ T cells are the second largest source of this cytokine [51]. In a similar line, it has been reported that human peripheral blood CD4⁺ T cells from healthy donors produce IL-17 after in vitro L donovani stimulation with approximately half of these induced T_H17 cells coproducing IFNγ [58]. Although not formally tested, induction of the IL-17 producing CD4⁺ T cells by L donovani could be related to the secretion of IL-1β, IL-6 and IL-23 by parasite-stimulated monocytes.

4. IL-17-mediated protective mechanisms during T. cruzi and other protozoan infections

As described in the previous sections, IL-17 signaling triggers several effector pathways that sustain robust immunity against extracellular as well as intracellular pathogens [59]. In this regard, emerging evidences support that IL-17 is induced upon infection with T. cruzi and other protozoans and critically modulates host-parasite interaction. A wealth of the available data supports a protective role of IL-17-mediated effector pathways in protozoan infections by mechanisms that are starting to be delineated, as discussed below.

4.1. Modulation of recruitment and function of innate immune cells

Current knowledge indicates that IL-17 effector pathways are intimately associated to the modulation of neutrophil development, recruitment and function. Therefore, it is not surprising that different studies dissecting the protective mechanisms mediated by this cytokine during protozoan infection involved PMN responses. Of note, some of these findings helped to further understand how neutrophils mediate resistance to certain parasites while others underscored for the first time the protective role of the IL-17/PMN cell axis in that particular infection. Thus, it was early established that IL-17RA knockout (KO) C57BL/6 mice, which fail to reduce parasite burden after oral T. gondii infection, produce low amounts of chemokines such MIP-2 and, consequently, have impaired recruitment of PMN cells into tissues [60]. However, a link between reduced neutrophil influx and reduced parasite control was not formally addressed in this study. More recently, we determined that lack of IL-17RA expression compromised neutrophil recruitment in the context of T. cruzi infection [45]. In this regard, histological evaluation of the liver (target organ of acute T. cruzi infection) demonstrated that lack of IL-17RA signaling influenced the quality of the inflammatory infiltrate that is dominated by mononuclear cells in infected IL-17RA KO mice and by PMN cells in WT counterparts. Further quantitative studies determined that T. cruzi infected IL-17RA KO mice have reduced numbers of CD11b⁺Gr1⁺ neutrophils in the spleen and liver that correlated with a reduced tissue expression of chemokines that participate in neutrophil recruitment such as CXCL1 (KC) and CXCL2 (MIP-2). While these findings suggested that lack of IL-17RA signaling compromise neutrophil recruitment into tissues during T. cruzi infection, a role of this pathway in neutrophil development was ruled out by the conserved G-CSF expression in spleen and liver as well as the normal CD11b⁺Gr1⁺ cell numbers in bone marrow and blood of infected IL-17RA KO mice. Final evidences in this regard were obtained by adoptive transfer experiments that showed that lack of IL-17RA in host cells/tissues, but not in the migrating neutrophils themselves, are required for proper neutrophil recruitment into tissues during T. cruzi infection. Remarkably, we determined that the role of neutrophils in host resistance against T. cruzi is related to not only a possible direct control of parasite replication but also the regulation of exuberant inflammatory responses (see section 4.2.).

While neutrophils are considered the predominant effector cell of IL-17 mediated responses, monocytes/macrophages also respond to this cytokine and activate particular antimicrobial pathways. Thus, it has been reported that IL-17 potentiates the intracellular parasiticidal ability of bone marrow derived and peritoneal macrophages infected with T. cruzi to similar levels than IFNγ [61]. Furthermore, IL-17 stimulation increases the engulfment of parasites selectively in macrophages but not in PMN. Remarkably, IL-17 stimulation in macrophages promotes the actin-mediated uptake of T. cruzi by a phagocytic-like process and also alters intracellular trafficking, restraining parasite escape from the lysosomes to the cytosol. These effects, associated to increased parasite engulfment and prolonged exposure to acidic environment, are further boosted by the up-regulation of iNOS (inducible nitric oxide synthase) expression and activity. Altogether these effector mechanisms underlie the robust trypanocidal profile acquired by macrophages upon IL-17 signaling. In a related In vitro setting, IL-17 was shown to act synergistically with IFNγ to precondition bone-marrow derived macrophages to produce higher Nitric Oxide (NO) levels upon subsequent infection with Leishmania infantum [62]. This increase in NO production induced by IL-17 stimulation has a significant impact on the leishmanicidal ability of the macrophages as the combination of this cytokine with IFNγ significantly reduces the percentage of infected cells. In a different way, the presence of IL-17 alone or together with IFNγ was not able to up-regulate inflammatory mediators or enhance the intracellular parasite killing in peritoneal macrophages with an established infection with Leishmania donovani [63]. Altogether, these results evidence that, depending on the kinetics of the stimulation, IL-17-mediated effector pathways may or may not influence the macrophage activation profiles to enhance the intracellular protozoan elimination.

4.2. Regulation of type 1 inflammation and induction of antimicrobial response

Besides a role in the recruitment and activation of particular innate cell subsets, IL-17 has been reported to mediate host resistance against protozoans by influencing the characteristic of the global inflammatory response. This mechanism seems to be operating in the IL-17 mediated protection during experimental T. cruzi infection, as first reported in 2010 by two research groups. Da Matta Guedes et al. [44] established that neutralization of IL-17 in Balb/c mice infected with T. cruzi (Y strain) by injection of specific antibodies (Abs) before and during the acute phase resulted in increased mortality despite conserved parasitemia and reduced cardiac parasite loads. Remarkably, enhanced susceptibility to T. cruzi in absence of IL-17 correlated with increased inflammatory infiltrate in hearts and elevated concentrations of type 1 inflammatory cytokines such as IL-12, IFNγ and/or TNF in sera and cardiac tissue. These authors finally demonstrated that, in the context of T. cruzi infection, IL-17 controls Th1 differentiation and the expression of chemokines and chemokine receptors in the hearts. Miyazaki and colleagues [43] also reported that IL-17 mediates host protection against T. cruzi as evidenced by the fact that IL-17 KO C57BL/6 mice show increased mortality during acute infection (Tulahuen strain). Mortality is also accompanied by an aggravated multiple organ failure but, in contrast to data from the previous study, infected IL-17 KO mice exhibit augmented parasite burden together with reduced production of inflammatory cytokines by leukocytes from spleen, mesenteric lymph nodes (mLN) and liver. The dissimilarities between these initial studies regarding the possible mechanisms underlying the IL-17 protective effect could be attributed to the differences in mouse and parasite strains that are known to influence the immune mechanisms and the outcome of the infection [64]. Also, alternative approaches utilized to avoid IL-17 signaling (transient neutralization versus permanent genetic deletion) may trigger different compensatory mechanisms such as secretion of other IL-17 family members, leading to alternative phenotypes.

Within this context, our group was aimed to deeply evaluate the role of IL-17 family in the immunity against to T. cruzi unraveling the cellular and molecular mechanisms involved. Thus, to partially overcome the limitations of previous studies, we investigated the progression of T. cruzi infection in mice that lack of the expression of IL-17RA (IL-17RA KO mice) [65], the receptor subunit required for the signaling of not only IL-17 but also IL-17F, IL-17C and IL-17E. In agreement with the initial studies described above, we determined that IL-17RA KO mice exhibit increased susceptibility to T. cruzi infection (Tulahuen strain), corroborating that IL-17 signaling is required for resistance against this parasite [45]. Of note, the phenotype of infected IL-17RA KO mice show a combination of features described for infected anti-IL-17 treated and IL-17 KO mice. Indeed, lack of IL-17RA expression results in conserved control of parasite in blood but increased parasite load in tissues such as liver, together with increased immunopathology as determined by histology and levels of serum markers of hepatic damage. We further determined that infected IL-17RA KO mice present an amplified type 1 inflammatory response with increased serum concentrations of IFNγ and TNF that underlie the hepatic damage and increased host susceptibility. Interestingly, absence of IL-17RA-mediated signaling during T. cruzi infection unleashes inflammation mediated pathology by limiting the recruitment into tissues of IL-10 producing regulatory neutrophils (see previous section).

Further confirmation about the effect of IL-17 in keeping in check type 1 inflammation during T. cruzi infection was obtained by our group with a completely different experimental approach [28]. Indeed, we established that infected muMT mice, which lack mature B cells and therefore lack a major subset able to produce IL-17 upon T. cruzi infection (see section 3.1.), exhibit reduced host resistance. Increased susceptibility of muMT mice to T. cruzi is related to a weakened parasite control together with increased production of IFNγ and TNF that, in turn, sustain immunopathology. Remarkably, adoptive transfer of WT but not IL-17 deficient B cells diminishes type 1 inflammation and restores a resistant phenotype in B cell deficient mice. Altogether, data from our laboratory, interpreted in the context of initial studies from other groups, indicate that the regulation of inflammation mediated by exacerbated secretion of type 1 cytokines is one protective mechanism by which IL-17 participates in host resistance to T. cruzi.

A similar protective immunoregulatory mechanism seems to be operating in the context of T. gondii infection, in which IL-17 deficiency compromise survival upon experimental infection despite conserved control of parasite burden in mLN and ileum [66]. Actually, T. gondii infected IL-17 KO mice have increased frequency of IFNγ producing Th1 cells in mLN and higher amounts of transcript encoding IFNγ, IL-4 and Toxoplasma gondii Heat Shock Protein 70 (T.g.HSP70) in the ileum. Remarkably, the high levels of IFNγ and T.g.HSP70, which have been reported to trigger lethal anaphylactic reactions in infected hosts, were associated with a lower neutrophil recruitment into the ileum of infected IL-17KO mice. Differently, it has been reported that a balanced T_H1/T_H17 response is required for protection against Leishmania infantum infection [62]. IL-17RA KO mice exhibit increased mortality during L. infantum infection as result of an impaired control of parasite load regardless of the degree of immunopathology. Deficiency in IL-17RA promoted immunoregulatory mechanisms associated to regulatory T cells and IL-10 production that limited the induction of an appropriate Th1 response and, consequently, compromised parasite killing as both IL-17 and IFNγ are required for efficient macrophage activation as described before. Furthermore, treatment with neutralizing anti-IL-17 Abs in a model of infection with Trypanosoma congolense resulted in reduced production of IFNγ and IL-10, increased parasitemia and exacerbated tissue damage [67].

Altogether, the available data support the notion that IL-17 plays a protective role during many protozoan infections that is associated to a cross-regulation between type 17 and type 1 responses. This cross-regulation may be antagonist or synergistic likely as consequence of parasite and host genetics. In certain contexts, IL-17 production is required to regulate exuberant secretion of IFNγ (and TNF) and to prevent excessive inflammatory damage to host tissues while in others IL-17 sustain the secretion of type 1 effector cytokines required for proper parasite control. Besides its effect on the cross-regulation of Th1 responses, IL-17 can mediate host protection and impact in the outcome of certain protozoan infections through the induction of innate immunity mediators such as antimicrobial proteins. This mechanism has been mainly studied in infections with the intestinal protozoa Giardia spp. Thus, G. muris infection induces a strong IL-17 production in the intestine that correlates with the production of antimicrobial peptides such as S100A8 and S100A9 [68]. Furthermore, IL-17A and IL-17RA KO mice showed increased susceptibility to G. muris linked to a compromised transit of IgA to the intestinal lumen together with reduced gene expression of molecules known to participate in antimicrobial defense (i.e. α and β defensins, serum amyloids and complement-activating C-type lectins, among others) [69, 70].

4.3. Modulation of recruitment and function adaptive immune cells.

Most of the well-established effector mechanisms mediated by IL-17 involve innate immune pathways. However, as discussed above, the IL-17/IL-17RA axis has also been shown to modulate maturation, recruitment and/or activation of adaptive immune cells during different settings. Within the context of protozoan infections, a couple of recent studies have demonstrated that IL-17 mediated signaling is required to achieve robust humoral and cellular parasite-specific immunity. Thus, Gosh and colleagues reported that infection with P. yoelii induces the expansion of IL-17-producing lymphoid progenitor LSK⁻ cells [50]. Further evaluation of the role of IL-17 in progression of P. yoelii infection took advantage of IL-17RA KO mice that resolve the infection similar than WT mice but exhibit higher parasite burden particularly during the late acute infection. The temporal reduction in the ability of infected IL-17RA KO mice to control parasite replication correlate with a significant decline in the level of parasite-specific Abs. This defect was attributed to the fact that lack of IL-17RA expression during P. yoelii infection compromise not only the development of B cells, as evidenced by reduced numbers of immature and newly formed B cells in the spleen, but also their differentiation into germinal center B cells and antibody secreting cells. In particular, IL-17 was shown to promote the differentiation of LSK⁻ cells into mature B cells in the spleen by an indirect process that relies on the IL-17RA-dependant induction of chemoattractants (i.e. CXCL12), growth factors (i.e. BAFF), and adhesion proteins by stromal cells. Altogether, these results suggest that IL-17 supports B cell responses during P. yoelii infection by instructing splenic stromal cells to create a microenvironment able to support extramedullar lymphopoiesis.

More recently, we reported that IL-17 is critical to sustain CD8⁺ T cell immunity against T. cruzi [71]. Therefore, infected IL-17RA KO mice show an abortive CD8⁺ T cell response as consequence of a premature contraction of parasite-specific CD8⁺ T cells rather than defective induction or expansion of these cells. Remarkably, IL-17 signaling is required once the parasite-specific CD8⁺ T cell response is establish to promote survival of effector cells. Accordingly, IL-17A, but not IL-17F, IL-17C, or IL-17E, is able to stimulate in vitro activated CD8⁺ T cells in a direct fashion, downregulating the pro-apoptotic protein BAD and promoting cell survival. Furthermore, effector CD8⁺ T cells elicited by T. cruzi infection in absence of IL-17RA exhibit a phenotypic, functional and transcriptomic profile compatible with cell exhaustion. In agreement with their deficient CD8⁺ T cell response, infected IL-17RA KO mice show poor control of the parasite in target tissues such as spleen, liver, and heart that can be partially reverted by inhibiting the PD-1/PD-L1 inhibitory pathway [71]. Altogether, our results underscore that during T. cruzi infection, cell populations that produce IL-17, which include T_H17, Tc17, NK cells and B cells [28, 45], may sustain and potentiate parasite-specific CD8⁺ T cell immunity. Accordingly, we determined that depletion of B cells, the major IL-17 producing population during T. cruzi infection, with anti-CD20 Abs affects the magnitude and quality of the parasite-specific CD8⁺ T cell response. This effect is associated with a reduction in the frequency of IL-17A producing cells that affected not only the B cells that are depleted but also non-B cell populations. As reported for infected IL-17RA KO mice, parasite-specific CD8⁺ T cells from B cell depleted infected mice exhibit increased apoptosis and poor effector function. Furthermore, B cell depletion partially arrested CD8⁺ T cell expansion, leading to a premature contraction of the response. Of note, treatment with rIL-17A partially restored CD8⁺ T cell immunity and the control of parasite replication in T. cruzi infected anti-CD20-treated mice[72]. Altogether, these complementary studies from our laboratory support the notion that IL-17 is a key cytokine for the sustenance of CD8⁺ T cell immunity during T. cruzi infection. It remains to be established whether this mechanism of IL-17 mediated effector function is also operating during other protozoan infections where IL-17 mediates control of parasite loads in tissues.

4.4. Evidences of IL-17 mediated protective effects during protozoan infections in humans

Although restricted by the inherent limitations of studies in humans, the investigation of the IL-17-mediated pathways in protozoan-infected patients also suggest a protective function for this cytokine. In this regard, most of the available data suggest that low production of IL-17 and IL-10 together with high production of IFNγ and TNF by peripheral mononuclear cells from patients with chronic Chagas’ disease correlates with increased severity of the cardiac disease [53]. Accordingly, high frequency of IL-17 producing cells are found in patients with Chagas’ disease that show milder disease while high IL-17 expression correlates with conserved cardiac function [73, 74]. Remarkably, polymorphisms in IL17A genes are differentially associated to morbidity in chronic Chagas’ disease, although it is not clear how these polymorphisms impact on IL-17 production [75]. Genetic variations in the IL-17 family have been also connected to cerebral malaria (CM), a neurological complication of the infection with Plasmodium falciparum in humans [76]. In fact, single nucleotide polymorphisms in Il17f and Il17ra but not in Il17a genes show significant association with CM. Remarkably, one of the aggravating Il17f polymorphisms leads to reduced IL-17F production, suggesting a protective role of this cytokine during this infection. Besides its apparent protective roles during Chagas’ disease and Malaria, high level of IL-17 (and IL-22) production is also strongly associated with protection against the most severe visceral disease form or Kala Azar in patients infected with L. donovani [58]. Similarly, secretion of IL-17 and other proinflammatory mediators such as IFNγ and CXCL11 was shown to be higher in patients showing a healing cutaneous leishmaniasis in comparison to the non-healing group [77]. Moreover, T_H17 cells seem to be an important component of the effector memory response in humans infected with Giardia lamblia and increased memory T_H17 frequencies were associated to an earlier control of this intestinal infection [78].

5. IL-17-mediated pathogenic mechanisms during protozoan infections

As discussed above, many studies support the concept that adequate IL-17 responses are required for host protection against certain protozoan parasites. However, there are also reports that described that IL-17 production may play deleterious roles during infections with T. cruzi, T. gondii and Leishmania spp. in a context-dependent manner. Although apparently contradictory with the data reviewed in the previous section, IL-17 seems to be pathogenic particularly in settings with compromised regulatory mechanisms that unleash uncontrollable IL-17 production. This leads to unbalanced effector responses and exacerbated tissue immunopathology.

In this regard, several signaling pathways have been reported to regulate T_H17 induction and therefore modulate host resistance to protozoan infections. Particularly, IL-27 was linked to T_H17 suppression by in vitro experiments that show that this type 1 cytokine counteracts the development T_H17 cells induced by IL-6 and TGF-β [79]. Accordingly, IL-27 receptor-deficient mice chronically infected with T. gondii develop a prominent T_H17 inflammatory response that is associated with a severe neuroinflammation [55].

In a similar direction, mice deficient in the transcription factor BATF2 and infected with T. cruzi exhibit an enhanced T_H17 response that results in severe multiorgan damage due to a greater inflammation in liver and heart despite reduced parasite burden and mortality [80]. BATF2 is an IFNγ induced transcription factor that was shown to act as a negative regulator of IL-23 secretion by innate immune cells and therefore, IL-23 production is highly increased in T. cruzi-infected BATF2 KO mice. Consequently, CD4⁺ T cells from spleen and liver of T. cruzi–infected BATF2 KO mice show remarkably increased levels of IL-17, but not IFN-γ, production in comparison to counterparts from WT mice. This exacerbated T. cruzi-induced T_H17 response and the consequent immunopathology are abrogated in Batf2−/−Il23a−/−mice indicating that IFNγ produced during T. cruzi infection induces BATF that, in turn, balance the IL-23/T_H17 axis to avoid immunopathology. In a similar direction, the severity of cardiac compromise in children with chronic T. cruzi infection has been associated to high levels of T_H17 related cytokines including IL-17 and IL-6, together with increased expression of IFNγ and IL-2 linked to T_H1 responses and pro-fibrotic factors like IL-13 [81]. These data are somehow contradictory with those discussed in the previous section that established a link between IL-17 and conserved cardiac function. These results could be reconciled by possible differences in effector response and disease progression between adults and children that have been reported to rapidly develop cardiomyopathy. Adding a new layer of complexity related to the cellular source of IL-17, a recent study revealed that CD4⁺IL-17⁺IFNγ⁻ (non-pathogenic T_H17) and CD4⁺IL-17⁺IFN-γ⁺ (pathogenic T_H17) as well as CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25^highFoxP3⁺ cells are more frequent in patients with severe cardiac disease [54]. Furthermore, while there is a positive correlation between CD4⁺CD25⁺FoxP3⁺ and CD4⁺IL-17⁺IFN-γ⁺ cells in indeterminate patients, this relationship is not observed in cardiac patients. Remarkably, these authors found that IL-17 expression by conventional and pathogenic T_H17 cells and B cells correlates with worse global cardiac function. The reasons of the difference between this study and those discussed in the previous section remain obscure.

Contradictory roles of IL-17 in resistance and susceptibility to infection with Leishmania spp. are also reported. Indeed, meanwhile IL-17A production during human visceral leishmaniasis has been correlated with protection [58] and IL-17RA expression is required for protection in experimental infection with L. infantum [62]. IL-17A KO mice are highly resistant to visceral leishmaniasis caused by L. donovani, showing decreased parasites in liver and spleen [51]. The unexpected phenotype is associated with enhanced IFNγ production by T cells and decreased accumulation of neutrophils and monocytes, resulting in reduced number of granulomas. Co-infection with Leishmania major, the parasite responsible of cutaneous leishmaniasis, and Staphylococcus aureus produces skin lesions that were two-fold larger than in single infections, regardless of having similar parasite and temporal increased S. aureus burdens [82]. Throughout the first 4 weeks of co-infection, inflammatory lesions in the ears had more neutrophils than lesions induced by single infection. However, most neutrophils were apoptotic. Neutralization studies showed that IL-17A worsen cutaneous disease during co-infection of L. major with S. aureus by exacerbating the inflammatory response as consequence of increased neutrophil recruitment and apoptosis that delays inflammation resolution. These results are in agreement with an early descriptive study that reported that neutrophils are regularly detected in necrotic and perinecrotic areas of patients with mucosal leishmaniasis. Furthermore, neutrophil presence is associated to IL-17 expression in the mucosal lesions, suggesting that this cytokine is involved in mucosal leishmaniasis pathogenesis [83].

Additional data support the idea that the overall biological role of IL-17 during different forms of human and experimental leishmaniasis seems to be also modulated by the crosstalk with regulatory mechanisms. Indeed, in an exhaustive study Gonzalez Lombana et al. [84] demonstrated that mice deficient in the regulatory cytokine IL-10 and infected with L. major developed larger lesions that were not associated to a compromised parasite killing but rather to an exacerbated IL-17- but not IFNγ- mediated inflammation. Furthermore, it was recently described that expressions of Foxp3, IL-10 and IL-17A are significantly higher in lupoid cutaneous leishmaniasis patients than non-lupoid group and in healthy volunteers suggesting that these mediators play important roles in the immunopathogenesis of cutaneous leishmaniasis and that these roles differ depending on the causal leishmania species and the particular body compartment affected [85].

6. Conclusions

During the last decade, several studies have evidenced that IL-17-mediated effector mechanisms play critical roles during host protection against several microbes. Although the relevance of these effector mechanisms seems to be maximal to combat fungi and extracellular bacteria, IL-17A and IL-17F also mediates resistance against intracellular pathogens such as protozoans. In this regard, available data clearly indicate that IL-17 is produced in the context of most protozoan infections (i.e. T. cruzi, T. gondii, Plasmodium spp., Leishmania spp., G. lamblia), both in experimental laboratory animals as well as in humans. Although many different immune subsets are able to produce IL-17 in response to protozoans, the particular contribution of each population will vary according to the type of parasite, the host as well as the kinetics and the site of the infection. Thus, innate cells produce IL-17 at earlier time points as consequence of inflammatory cytokines triggered soon after pathogen recognition while, adaptive immune populations become the major source of this cytokine at later time points once parasite-specific T cell responses are activated. Remarkably, most of the existing data in experimental infections as well as in patients with Chagas’ disease underscore that IL-17 promotes host resistance against T. cruzi favoring the control of parasite burden and regulating immunopathology. Similarly, IL-17 induced pathways favor the control of parasite replication in infection with G. lamblia, T. gondii, and certain Plasmodium and Leishmania species. However, this cytokine has been also linked with the immunopathology generated by these protozoan infections in different settings. In these particular scenarios in which IL-17 responses are often exacerbated, there is an enhancement on inflammatory pathways (i.e. neutrophil recruitment) that promotes tissue damage and increases susceptibility to the pathogen. Beyond apparent inconsistencies, these data may be interpreted as the evidence that the balance between different effector and regulatory pathways rather than a single mechanism modulates inflammation and therefore, clinical progression during protozoan infections. In this context, it is likely that the usage of comprehensive “omics” methodologies will serve to evaluate multiple parameters of immune response against the different protozans that affects humans, shedding light into the integral resistant or susceptible profiles.

Figure 3. IL-17 mediated mechanisms that promote host protection against protozoan infections.

Infection with different protozoans such as T. cruzi, T. gondii, Leishmania spp., Plasmodium spp. and Giardia spp. induces the production of IL-17 by several cell sources including T_H17, Tc17, B17, NK, and γδ T cells and LSK− progenitors. Once produced, IL-17 acts on different cell populations from the stroma and the immune system to trigger several protective mechanisms as follow. A) Recruitment and activation of innate cells: IL-17 produced during T. cruzi and T. gondii infection promotes the secretion of chemokines by stromal cells. These chemokines induce the migration of polymorphonuclear (PMN) into tissues to control parasite spreading. Also, IL-17 in combination or not with IFNγ favors the activation of macrophages (MΘ) to produce more nitric oxide (NO), increase engulfment and enhance intracellular killing of T. cruzi and Leishmania spp.. B) Antimicrobial response: particularly established for infection with Giardia spp., IL-17 provides signals to intestinal epithelial cells to facilitate transit of IgA into the lumen and to produce several antimicrobial peptides. Altogether these downstream effector pathways enhance parasite clearance. C) Modulation of inflammation: IL-17 plays a critical role in the cross-regulation of type 1 responses to achieve a balanced effector immunity able to control parasite replication without excessive tissue damage an immunopathology. Thus, recruitment of IL-10-producing neutrophils during T. cruzi infection controls IFNγ secretion by CD4⁺ and CD8⁺T cells. Similarly, IL-17 triggered by T. gondii reduces production of IFNγ and T.g.HSP70, preventing anaphylactic reactions. Remarkably, IL-17 limits regulatory responses during L. infantum infection favoring IFNγ production to levels required for efficient parasite control. D) Differentiation of adaptive immune cells: IL-17 can also promote adaptive humoral and cellular responses against P. yoelii and T. cruzi, respectively. Thus, IL-17 production by LSK ⁻ progenitors induces splenic stromal cells to produce growth factors that promote LSK⁻ cells differentiation into mature B cells and plasma cells that produce parasite-specific Abs. In addition, IL-17 favors survival of effector CD8⁺ T cells and prevents their exhaustion sustaining prolonged specific cytotoxic responses. Created with Biorender.com

Highlights.

• Protozoan infections trigger IL-17 production by different immune cells.

• IL-17 modulates host-parasite interaction during protozoan infections.

• IL-17-mediated protection requires recruitment and activation of innate and adaptive cells.

• IL-17 may play deleterious roles during protozoan infections in a context-dependent manner.