Cytokine crowdsourcing: multicellular production of T_H17-associated cytokines

Review of critical non-TH17 sources of IL-17A and associated cytokines during inflammation in mucosal tissues, with a focus on leukocytes of the gastrointestinal tract.

Abstract

In the 2 decades since its discovery, IL-17A has become appreciated for mounting robust, protective responses against bacterial and fungal pathogens. When improperly regulated, however, IL-17A can play a profoundly pathogenic role in perpetuating inflammation and has been linked to a wide variety of debilitating diseases. IL-17A is often present in a composite milieu that includes cytokines produced by T_H17 cells (i.e., IL-17F, IL-21, IL-22, and IL-26) or associated with other T cell lineages (e.g., IFN-γ). These combinatorial effects add mechanistic complexity and more importantly, contribute differentially to disease outcome. Whereas T_H17 cells are among the best-understood cell types that secrete IL-17A, they are frequently neither the earliest nor dominant producers. Indeed, non-T_H17 cell sources of IL-17A can dramatically alter the course and severity of inflammatory episodes. The dissection of the temporal regulation of T_H17-associated cytokines and the resulting net signaling outcomes will be critical toward understanding the increasingly intricate role of IL-17A and T_H17-associated cytokines in disease, informing our therapeutic decisions. Herein, we discuss important non-T_H17 cell sources of IL-17A and other T_H17-associated cytokines relevant to inflammatory events in mucosal tissues.

Introduction

The discovery of IL-17A and its role in disease marked a paradigm shift in our understanding of how inflammation is regulated. Originally remarkable for its high homology to Herpesvirus saimiri gene 13 [1], it was evident early on that IL-17A is multifunctional, with wide-ranging effects, including the modulation of NF-κB activation and T cell proliferation [2]. Studies describing the proinflammatory nature of IL-17A soon followed, and the role of IL-17A in the pathology associated with diseases, such as arthritis [3, 4] and psoriasis [5, 6], is now well established. However, recent studies to understand the role of IL-17A in complex diseases of the mucosa, such as colitis, have demonstrated that IL-17A can also serve in a host-protective capacity [7]. Therefore, like many other cytokines, the function of IL-17A appears to be more complex and context dependent than originally appreciated.

IL-17A is primarily produced by immune cells at mucosal sites during inflammatory events. As such, IL-17A is often only 1 component of a complex cytokine microenvironment. There are several cytokines that are often secreted together with IL-17A, including TNF-α, IFN-γ, and T_H17-associated IL-17F, IL-21, and IL-22 and in humans, IL-26. Moreover, recent evidence suggests that some populations of IL-17A-expressing cells that coexpress IFN-γ are primarily pathogenic in nature [8]. Whereas cytokine functions are often deduced via reductionist methods, a movement toward understanding the role of IL-17A and its associated cytokines within the framework of the complete cytokine milieu appears to be the logical next step toward understanding the role of T_H17-associated cytokines in inflammation.

T_H17-ASSOCIATED CYTOKINES

IL-17A and IL-17F

The IL-17 family has 6 members, enumerated IL-17A–IL-17F [9]. Human IL-17A and IL-17F share 40–50% homology at the amino acid level [10, 11], the highest homology between individual members of the IL-17 family, and are adjacently located on chromosome 6 in a head-to-tail fashion [10]. IL-17A and IL-17F can individually homodimerize or form heterodimers of ∼35 kDa [12], both of which bind to a heterodimeric complex containing IL-17RA and IL-17RC [13]. IL-17RA is nearly ubiquitously expressed on a variety of cells within the mucosa, and context-dependent regulation of IL-17RC provides additional signaling capabilities.

Currently, several antibodies targeting IL-17A or IL-17RA are under development, including Ixekizumab (Eli Lilly, Indianapolis, IN, USA), Brodalumab (Amgen, Thousand Oaks, CA, USA), and Secukinumab (Novartis Pharma AG, Basel, Switzerland). All 3 aforementioned drugs are currently showing promise in the treatment of psoriasis [14, 15]. Notably, Secukinumab recently underwent a clinical trial for the treatment of moderate-to-severe Crohn’s disease. Beyond failing to improve Crohn’s disease, treatment with Secukinumab exacerbated the disease in a subset of patients [16]; moreover, Secukinumab was associated with increased infections, including those caused by fungal pathogens [16]. These findings are consistent with the tissue-protective function of IL-17A observed in mouse models of colitis by several groups, including our own [7, 17, 18]. As IL-17A-mediated contributions to intestinal disease have also been observed in mice [19, 20], taken together, these data suggest that the role of IL-17A is complex and perhaps context dependent.

The elimination of IL-17F was shown to be protective in a murine model of colitis by use of dextran sodium sulfate, suggesting that it plays a pathogenic role in intestinal inflammation [18], and the elimination of IL-17A and IL-17F signaling was protective in a T cell-mediated model of colitis [7, 21]. These data suggest that targeting IL-17F may prove more efficacious than targeting IL-17A for the treatment of some mucosal inflammatory disorders, yet therapeutics selectively targeting IL-17F are currently unavailable. New studies determining the utility of targeting multiple IL-17 family members during human disease are needed. Given the complexity in IL-17A/F signal integration, additional microenvironment-specific factors will likely contribute to the efficacy of strategies blocking IL-17 family proteins in patient subpopulations. To appreciate the complexity of the inflammatory milieu in which IL-17A et al. function, going forward, we will consider the roles and cellular producers of disease-relevant, T_H17-associated cytokines.

IL-21

IL-21 is a member of the IL-2 family of cytokines and is located near IL-2 on chromosome 4 in humans [22]. Originally identified as a factor produced by activated T cells that could drive NK cell proliferation and maturation [23], IL-21 appears to be secreted predominantly by T_H17 cells, T_FH cells, and NKT cells (reviewed in ref. [24]). IL-21R is composed of an IL-21-specific component paired with the common γ-chain [23, 25, 26]. This receptor is found on cells of lymphoid origin, including T cells, where IL-21 signaling may act in an autocrine or paracrine fashion to promote T_H17 responses [27]. IL-21 signaling in mucosal diseases appears to be largely pathogenic, especially within the intestinal tract; for example, IL-21^−/− mice are more resistant to chemically induced colitis [28] and the development of CRC [29]. Given the significant association of IL-21 to mucosal disease pathogenesis, therapeutics designed to target the IL-21 signaling pathway are of great clinical interest [30].

IL-22

With 25% homology to IL-10 in humans, IL-22 falls within the larger IL-10 cytokine family [31]. IL-22 is located on chromosome 12 in humans (chromosome 10 in mice), in close proximity to the genes for IFN-γ and IL-26 [32]. One subunit of the IL-22R , the common β-chain receptor IL-10R2, is shared among IL-10, IL-22, and IL-26. The second subunit of the IL-22R, IL-22RA1, also comprises part of the receptors for IL-20 and IL-24 [33]. An agonist of IL-22, termed IL-22BP or IL-22RA2, is a soluble receptor, structurally related to IL-22RA1, that inhibits binding of IL-22 to the membrane-bound IL-22R [34, 35]. IL-22 was discovered originally as a cytokine produced by T cells [36, 37]; T cells and NK1.1⁺ cells are the primary cell types in mucosal tissues implicated in IL-22 production [38]. IL-22-responding cells, however, are of nonhematopoietic lineage and include epithelial cells in mucosal tissues (reviewed in ref. [33]). In addition to stimulating the production of antimicrobial peptides, IL-22 promotes epithelial cell proliferation, thereby maintaining epithelial barrier function (reviewed in ref. [39]); indeed, IL-22 can be protective in murine models of intestinal inflammation [38, 40] and influenza [41, 42]. Consistent with its role in epithelial cell proliferation, IL-22 levels are positively correlated with tumor development, such as in colon cancer [43, 44]. IL-22 can also be proinflammatory at the mucosa, where it contributes to lung pathology following fungal infection [45], chemically induced lung injury [46], or colitis in select models [47]. The proinflammatory roles of IL-22 are not well understood but may be linked to the induction of chemokines [48, 49].

IL-26

Like IL-22, IL-26 is part of the IL-10 cytokine family. Originally coined AK155, IL-26 is located on human chromosome 12 [50] and although expressed in teleosts [51], is not found in rodents [52, 53]. IL-26R is composed of the common β−chain receptor IL-10R2 paired with the α-chain receptor for IL-26, IL-20R1 [54, 55]. In epithelial cells, including intestinal epithelial cells [56], signaling through IL-26R leads to activation of STAT1, STAT3 [54, 55], ERK, stress-activated protein kinase /JNK, and Akt [56]. In addition to being expressed by T_H17 cells, IL-26 is expressed by ILCs. IL-26 has been linked to intestinal inflammation, including Crohn’s disease and ulcerative colitis [56, 57], where it appears to be antiproliferative and to induce proinflammatory cytokine production [56].

NON-T_H17 CELL SOURCES OF T_H17-ASSOCIATED CYTOKINES

During maturation, helper CD4⁺ T cells differentiate into 1 of several effector types, defined primarily by their cytokine and transcription factor expression profiles. Aside from T_H17 populations, the best-characterized lineages are T_H1, T_H2, and T_regs (discussed below); T_H1 cells are defined by their expression of the T-box transcription factor 21 (T-bet) and secretion of IFN-γ, whereas T_H2 cells are defined by expression of GATA-3 and secretion of IL-4, IL-5, and IL-13. More recently, exciting new studies have defined additional subsets, including T_H9 and T_H22 cells, which produce IL-9 and IL-22, respectively [58, 59].

The T_H1/T_H2 paradigm in disease, especially regarding the T_H1-driven pathology, was altered dramatically with the elucidation of IL-23 function [60] and the discovery of T_H17 cells [61, 62]. T_H17 cells are CD4⁺ T cells that express the transcription factors RORγt and RORα [63] and can secrete IL-17A alone or in combination with IL-17F, IL-21, and IL-22 and in humans, IL-26 [64–66]. Antigen stimulation, in the presence of select cytokines, can induce naïve CD4⁺ cells in peripheral lymphoid organs to become T_H17 cells, currently termed iT_H17 cells. Interestingly, a recently identified, thymically derived population of T_H17 cells, coined nT_H17 cells [67], may differ in select developmental requirements and behave more like an innate cell population [66]. The role of T_H17 cells during disease is complex; T_H17 cells can be pathogenic (i.e., rheumatoid arthritis [68]) or protective (i.e., bacterial/fungal infection or in models of colitis [7]), and their contribution to disease pathogenesis may be overt (i.e., psoriasis [69]) or nuanced (i.e., uveitis [70]). Ongoing research to define molecular differences between nT_H17 and iT_H17 cells may clarify differing data on the precise role of T_H17 cells in disease but is unlikely to obviate the clear importance that T_H17 cells play in the immune response.

Whereas T_H17 cells may be one of the best understood IL-17A-secreting cell populations, there are many additional cell types that produce 1 or more T_H17-associated cytokines (Table 1). Remarkably, IL-17 producers are not solely of the T cell lineage, and examination of these cell populations suggests additional important functions for IL-17A. For instance, several secretory cell types, such as neutrophils and Paneth cells, have been reported to produce IL-17A, which hints at a vital role for IL-17A early in the immune/inflammatory response (Fig. 1). More broadly, the wide array of cell populations involved in the secretion of T_H17-associated cytokines speaks to the overall importance of these cytokines in the progression of the immune response. A number of recent developments in the elucidation of cellular sources of T_H17-associated cytokines warrant attention and careful consideration. Toward that end, we will next review important findings that describe and evaluate cellular sources of T_H17-associated cytokines in the mucosa.

TABLE 1.

T_H17-associated cytokine production by non-T_H17 cells and effects at the mucosa

Cytokine	Producers	Receptor	Effects
IL-17A	rTH17, Tregs, CD8+ T cell, γδ T cell, TFH, NKT cell, ILC, neutrophil, Paneth cell, macrophage	IL-17RA/IL-17RC	Neutrophil recruitment [71, 72] Antimicrobial peptide production (e.g., S100A8, S100A9 [73]) Cytokine and chemokine production (e.g., G-CSF, IL-6, CXCL1 [74, 75]) Inhibits TH1 polarization [7]
IL-17F	γδ T cell	IL-17RA/IL-17RC	Neutrophil recruitment [76] Cytokine and chemokine production (e.g., G-CSF, IL-6, CXCL1 [74, 75]) Lymphocyte and macrophage recruitment [18] Mucus hyperplasia in the lung [18]
IL-21	CD8+ T cell, TFH, NKT cell, neutrophil	IL-21R/γc	Cell proliferation, especially NK cells [22] Enhances TH17 responses [27] B cell class-switching [77] Regulatory B cell function [78]
IL-22	rTH17, CD8+ T cell,γδ T cell, NKT cell, ILC, neutrophil, macrophage	IL-22RA1/IL-10R2	Antimicrobial peptide production (e.g., S100A8, S100A9, Reg3γ [73, 79]) Epithelial cell proliferation/transformation [43] Enhances mucosal barrier function [79] Inhibits epithelial apoptosis [46]
IL-26	ILCs	IL-20R1/IL-10R2	Cytokine production (e.g., IL-8, IL-10 [55, 56]) Decreased epithelial proliferation [56]

Reg3γ, Regenerating islet-derived protein 3 γ.

Figure 1. IL-17A production in the small intestine. The healthy small intestinal mucosa serves the dual function of nutrient exchange and immune surveillance. Early in an immune/inflammatory response, IL-17 appears to be generated by several leukocyte and stromal populations. Prolonged inflammation leads to the breakdown of the epithelial barrier, allowing luminal content to enter the lamina propria. The ensuing inflammatory response leads to recruitment of additional immune cells and "late" IL-17A production. IL-17A binds the heterodimeric receptor IL-17RA/IL-17RC that is ubiquitously expressed on many cells within the mucosa, including epithelial cells. IL-17A production by T_FH cells may also support IgA production by neighboring B cells within lymphoid structures, such as Peyer’s patches, leading to more efficient transport of IgA into the lumen of the gut by the polymeric IgR.

CD4⁺ T_regs

Foxp3⁺ and Foxp3⁻ T_regs have been shown to produce T_H17-associated cytokines. Foxp3⁺IL-17A-producing T cells have been observed in vitro and in vivo [80–83], including in IBD and CRC [84–86]. Accordingly, in vitro experiments and in vivo transfer experiments suggest that Foxp3⁺ T_regs can up-regulate RORγt and IL-17A under inflammatory conditions. Further investigation into the stability of T_reg/T_H17 subsets is necessary; remarkable findings from Zhou et al. [87] showed that Foxp3⁺ T_regs can be converted into effector T cells, whereas Rubtsov et al. [88] reported that Foxp3⁺ T_regs are stable, even under inflammatory conditions. This is in line with subsequent data showing that Foxp3 can also be transiently up-regulated by activated T cells [89], which might explain the different outcomes of these studies.

The origin of Foxp3⁺IL-17A-producing T cells under physiologic conditions is still a matter of debate. There are few studies analyzing the function of Foxp3⁺IL-17A-producing T cells in vivo. One study suggests that IL-17A-producing Foxp3⁺ T cells might initiate tumorigenesis, which is, in part, a result of IL-17A production [84]. Foxp3⁺IL-17A⁺ T cells seem to be suppressive, at least in vitro [90], although their immunosuppressive function might be partially impaired by the expression of RORγt [86]. Clearly, further studies will be essential to clarify the function of Foxp3⁺IL-17A-producing T cells in vivo.

Another subset of T_regs has been described as Foxp3⁻IL-10⁺IL-17A⁺ T_H cells and are currently referred to as rT_H17 cells, which have been found in vitro and in vivo [80, 91, 92] and are generated in the small intestine. In contrast to T_H17 cells, rT_H17 cells have anti-inflammatory properties in mouse models of colitis and multiple sclerosis, which is, at least partially, dependent on IL-10 [80]. rT_H17 cells produce IL-22 and IL-17A in comparable amounts with conventional T_H17 cells, suggesting that rT_H17 cells might play an important role during the termination of an immune response and in tissue repair. However, it is currently unclear if this cell type is a stable T_H cell subset or an intermediate step between T_H17 and Foxp3⁻IL-10⁺ T_regs (referred to as a type 1 T_reg cell), which can modify IL-10 and IL-17A production, based on the environment. Further studies are essential to clarify the function, origin, and stability of rT_H17 cells.

CD8⁺ T cells

CD8⁺ T cells can produce T_H17-related cytokines in the mucosa, where they can mediate pathogenic and nonpathogenic effects. Within the lung, CD8⁺ T cells can secrete IL-17A, IL-21, and IL-22 following challenge with influenza [93], and IL-17F-producing CD8⁺ cells have been detected in patients with COPD [94]. Moreover, increased numbers of IL-17A-producing CD8⁺ T cells have been observed in patients with asthma [95] and following lung transplantation [96], supporting the conclusions drawn from murine studies.

Both T_C17 and T_NC17 cells have been described. In vitro, T_C17 cells can be differentiated with TGF-β and IL-6, similar to T_H17 cells; moreover, generation of IL-17A-producing CD8⁺ T cells may require T_H17 cells [97]. It has been suggested that IL-23 is required for the generation of T_C17 but not T_NC17 cells and also for subsequent IL-22 production. Functionally, T_C17 cells have been linked to several diseases of the mucosa. In one model of spontaneous colitis, T_C17 cells appear to mediate pathogenesis through production of IL-17A and IFN-γ in an IL-6-dependent fashion [98]. T_C17 cells also confer host protection; in an influenza model, the adoptive transfer of T_C17 cells promotes survival [93], and T_C17 cells are vital for the vaccine immune response against fungal pathogens [99].

CD8⁺ T cells, in the context of antitumor responses, is a fascinating field as a result of their potential use as an immunotherapeutic [100]. However, whether IL-17A secretion by cytotoxic CD8⁺ T cells leads to more efficient tumor clearance may depend on the presence of other cells of the innate and adaptive immune systems, such as T_H17 cells or MDSCs [101]. In a lung model of melanoma metastasis, IL-17A, produced directly by CD8⁺ T cells [102] or by T_H17 cells that indirectly recruited and activated CD8⁺ T cells [103], led to more efficient tumor clearance. Conversely, the number of T_C17 cells in patients with gastric cancer positively correlates with tumor progression and decreased survival, possibly as a result of recruitment of MDSCs that down-regulate the tumor-suppressive capacity of the T_C17 cells [104]. Thus, additional studies aimed at understanding how IL-17A-producing CD8⁺ T cells function as intratumoral lymphocytes will be invaluable in moving forward with CD8⁺ T cells as therapeutics.

A subset of CD8⁺ cells found in the lung and intestine are MAITs. As the name suggests, MAITs are an innate T cell population that expresses an essentially invariant TCR-α chain [105]. MAITs recognize vitamin B metabolites [106] presented by MR1, a class I-related MHC molecule found on APCs, such as B cells. Given that mammals cannot produce vitamin B and that MAITs require commensals for development [107], it appears likely that MAITs are important during early microbial recognition in the mucosa (reviewed in ref. [108]). Human CD8⁺ MAITs have been shown to produce IL-17A and IL-22 [109] and express CD161, a transmembrane protein suggested to be present on all IL-17A-expressing cells [110]. Moreover, the number of CD8⁺ MAITs is enhanced greatly during colonic inflammation [111].

γδ T cells

An immediate producer of IL-17A during an immune response, γδ T cells can be vital to host protection [112]. Predominantly found at mucosal sites, such as the intestinal epithelia, γδ T cells constitute the vast majority of the population of local T cells [113]. In addition to expressing CCR6, RORγt, IL-1R [114], and IL-23R [115], γδ T cells may be CD8⁺ in the mucosa or lack CD4 and CD8 molecules altogether. Following exposure to IL-23, in combination with IL-1β or IL-18, γδ T cells can be stimulated to produce IL-17A in a TCR-independent mechanism [116]. The role of γδ T cells in early IL-17A production has now been well documented (recently reviewed in ref. [117]), and recent evidence suggests that γδ T cells may additionally produce IL-17F [118].

Murine studies have demonstrated that γδ T cells in the lung contribute to protection from Staphylococcus aureus-mediated pneumonia [119] and appear to be critical for the resolution of allergic lung inflammation [120, 121], among other diseases. In response to Mycobacterium tuberculosis, γδ T cells in the lung produce IL-17A [122], which leads to granuloma formation [123]. The role of IL-17A-producing γδ T cells in murine lung inflammation appears to recapitulate closely that observed during human disease, as there is a significant increase in IL-17A-secreting γδ T cells in patients with active tuberculosis [124], and this correlates with granuloma formation [125].

Within the intestinal tract, γδ T cells are found within the epithelial layer (intraepithelial lymphocytes) and the lamina propria [126], where they can produce IL-17A in combination with other cytokines [126, 127]. Intestinal γδ T cells can be regulated by internal factors, such as the transcription factor PU.1 [128], or by external factors, such as T_regs [129, 130]. The role of γδ T cells in inflammatory conditions of the intestine is an area of active research; given their proximity to gut microbiota and ability to produce IL-17A rapidly, it is not surprising that γδ T cells have been linked to dysregulation of intestinal homeostasis. Intriguingly, a recent report revealed a positive association between tumor-infiltrating, IL-17A-producing γδ T cells and tumor progression in patients with CRC, potentially through the recruitment of immunosuppressive MDSCs [131]. In addition to the direct effects following immediate production of IL-17A [130, 132], IL-17A-secreting γδ T cells may contribute to enhancing T_H17 responses in the colon [118].

Following exposure to select cytokines, γδ T cells can also produce IL-21 and IL-22, as in the case of IL-1 and IL-23 stimulation in a model of experimental autoimmune encephalomyelitis [133]. In a study of pulmonary fibrosis, γδ T cells were shown to be protective through the production of IL-22 [134]. However, the role of γδ T cells in production of IL-21 and IL-22 at mucosal surfaces has not yet been studied extensively. γδ T cells may require the AHR for production of IL-22 [115] and in contrast to αβ T cells, may not require IRF4 for IL-17A or IL-22 production [135].

T_FH cells

T_FH is a subset of T cells that is important in mediating B cell activation in germinal center reactions. Found in secondary lymphoid structures, such as lymph nodes and Peyer’s patches, T_FH cells express CXCL5, allowing them to home to the B cell-rich follicles. Engagement of programmed death 1 (PD-1) on T_FH cells in the presence of IL-12 induces the expression of IL-21 in T_FH [136, 137]. In combination with TGF-β1, IL-21 production by T_FH drives B cell class-switching to an IgA-expressing plasmablast [77]. Moreover, a recent report suggests that T_FH may also secrete IL-17A to assist B cells in IgA production in Peyer’s patches; notably, these cells appear to be derived from T_H17 cells that homed to Peyer’s patches and subsequently differentiated [138].

NKT cells

NKT cells bridge the boundary between the innate and adaptive immune system. NKT cells express an αβ TCR that can recognize the lipid-restricted CD1 family of presentation molecules (recently reviewed in ref. [139]) and also express the NK cell marker NK1.1. One subtype of the NKT cell is termed iNKT cells as a result of their expression of select TCR-αβ chains. Of these, a subset can also express RORγt and secrete T_H17-associated cytokines and is sometimes referred to as NKT17 cells. These T_H17-like iNKT cells appear to require TGF-β [140], IL-15 [141], and E4BP [141] for development but do not require IRF4 [135]. The transcription factor T_H-inducing POZ-Kruppel factor (TH-POK) has been shown to repress formation of this lineage [142, 143]. iNKT cells can be stimulated to produce IL-17A following IL-1β and IL-23 exposure [116]. Originally discovered in the lung, NKT17 cells have been shown to contribute significantly to neutrophilia during Rous sarcoma virus [141] or Klebsiella infection [144], demonstrating that IL-17A, produced by NKT17 cells, can promote lung inflammation. Consistent with the role of NKT cells in producing IL-17A in the murine lung, there is an increase in IL-17A-producing, NKT-like cells in human lung transplant recipients [145] and in patients with cystic fibrosis [146]. Interestingly, a recent study investigating environmental antigens suggested that NKT cells can produce IL-17A following activation by house dust extracts, increasing the number of known antigens that can stimulate NKT cells [147].

NKT cells have been demonstrated to secrete IL-21 following stimulation with anti-CD3 and anti-CD28 or bacillus Calmette-Guérin [148, 149]. A recently described subset of iNKT cells, termed NKT_FH cells, expresses Bcl-6 and secretes IL-21 in the germinal center. This enables NKT_FH cells to help B cells, leading to affinity maturation but not a memory response [150, 151]. Similarly to IL-21, a role for iNKT cell-generated IL-22 has been established in splenocytes [152]; moreover, following influenza infection, IL-22 secreted by iNKT cells is protective against epithelial damage [153], which is consistent with the known role of IL-22 in tissue protection [33]. As previous studies describing NKT cell-generated IL-21 and IL-22 predominantly analyzed splenic cells, interesting questions regarding the presence or absence of similar populations in mucosal tissues remain.

ILCs

A significant portion of T_H17-associated cytokines generated in response to danger signals is thought to come from large numbers of ILCs that populate tissues at the host-pathogen interface [154]. Broadly, ILCs are defined by their resemblance to cells arising from a lymphoid lineage and the absence of lineage markers or antigen receptors resulting from rearranged genes (recently reviewed in ref. [155]). All ILCs require Id2 for development [156–158] but can be subdivided based on their transcriptional and cytokine profiles, as in the recently coined ILC3 populations, which are categorized by their resemblance to the cytokine expression profiles of the T_H subsets [159]. ILC3s, which include LTi cells and NCR⁺ ILC3, require IL-7 for development [157, 160], express RORγt [161, 162], and can produce the T_H17-associated cytokines IL-17A and/or IL-22. More surprisingly, the transcription factor associated with development and homeostasis of T_H2 and ILC2 cells, GATA-3, was demonstrated recently also to be important in the development of ILC3s [163]. Whereas the significance of ILCs, including LTi cells and NCR⁺ ILCs in the innate immune response, is well documented, the exact nature of the different cell types, their lineage, and their individual roles in disease are highly active areas of research.

LTi cells were the 1st ILC3 member identified [164] and need the AHR [165], thymocyte selection-associated HMG box factor (TOX) [166], and receptor activator for NF-κB ligand [167], among others, for development. Capable of producing IL-17A and IL-22 [168], LTi cells may be important in driving T_H17-associated innate immune responses. LTi cells can be subdivided based on expression of CD4 [154], although only CD4⁺ LTi cells appear to be important for protection against the murine enteric pathogen Citrobacter rodentium [169]. Beyond being critical for innate immune responses following challenge, LTi cells are also important in the induction and development of postnatal but not prenatal lymphoid tissue [165].

Besides expressing RORγt, a subset of the ILC3s displays the NCR NKp46 and therefore is called NCR⁺ ILC3s (alternatively NKp46 ILCs, NK22, NCR22, NKR-LTi, and ILC22 [159]). In addition to the previously appreciated requirement for AHR in development of NCR⁺ ILCs, a T-bet and Notch signaling-dependent mechanism has been identified recently for NCR⁺ ILCs [170]. NCR⁺ ILC3s can be derived from CD4⁻ LTi cells in vitro [170], which suggests that ILC3s may be highly plastic in nature. NCR⁺ ILC3s have been found in the skin and intestinal lamina propria of the mouse [171] and in human mucosa and lymphoid tissue [172, 173]. NCR⁺ ILC3s in the intestine can be divided further into NK1.1_lo versus NK1.1⁺ populations, with NK1.1_lo cells as potent producers of IL-22 [171]. IL-22-secreting NCR⁺ ILCs appear to play an important role in protection against intestinal pathogens, such as C. rodentium [170]. Notably, IL-22 secretion does not require NCR [174], which is consistent with the secretion of IL-22 by non-NCR-expressing cells.

In addition to CD4⁺LTi cells and NCR⁺ ILC3s, a population of CD4⁻NCR⁻ ILC3s appears to accumulate during colonic inflammation. These cells express RORγt and can produce IL-17A, IL-22, and IFN-γ following exposure to IL-23 [175]. Ultimately, colonic inflammation can lead to the initiation of CRC, and ILC3-mediated production of IL-22 and to a lesser extent, IL-17A [176] can drive this progression [177]. However, the precise definition of CD4⁻NCR⁻ ILC3s remains unclear.

In humans, CD56⁺NCR⁺ ILC3s within mucosal-associated lymphoid tissue can concomitantly express IL-22 and IL-26 [57]. IL-26 has been linked to IBD [178]; CD3⁻CD56⁺ ILCs, in the lamina propria of humans with ulcerative colitis or Crohn’s disease, secrete IL-26, an effect that can be enhanced with ex vivo IL-23 stimulation [179]. Whether IL-26 production is ultimately tissue protective or a significant source of tissue damage during intestinal inflammation remains unclear.

Neutrophils

Neutrophils constitute a major cell type involved in mediating mucosal T_H17 responses. Surprisingly, human neutrophils express IL-17RA [71, 180]; however, they do not constitutively express IL-17RC and thus, may not bind IL-17A or IL-17F directly [181]. Moreover, neutrophils likely do not express IL-21R [182] or IL-22R. T_H17 cells secrete chemokines (e.g., CXCL8, G-CSF [71, 181, 183]) that attract neutrophils to sites of inflammation. Thus, the recruitment of neutrophils in a T_H17-like immune response is often conceptualized in terms of chemokine gradients [181, 184].

In murine models of inflammation, neutrophils have been implicated in IL-17A generation through direct production, as in the recent, elegant studies out of the Pearlman lab [185], or by promoting the recruitment of IL-17A-expressing cells, such as T_H17 cells [181, 186]. In the lung, murine neutrophils can produce IL-17A mRNA and/or protein following LPS challenge [187] or during infection with inhalational anthrax [188], Cryptococcus infection [186], and Aspergillus fumigatus [189]. Importantly, neutrophilic production of IL-17A in the anthrax and Aspergillus models contributes significantly toward survival to an otherwise lethal challenge. It is currently unclear whether murine model systems fully recapitulate human disease, however. Whereas IL-17A production is associated with neutrophilic influx, as in the case of COPD [190], it is more difficult to define accurately the salient producers in human tissues. Thus, future work to measure directly neutrophilic production of IL-17A will be of key interest to the field.

Data implicating neutrophils in production of IL-21 during mucosal inflammation are limited but have been demonstrated during pediatric celiac disease [191]. Notably, a recently discovered population of neutrophils in the spleen, termed B cell helper neutrophils, secrete IL-21 to induce B cell diversification [192], suggesting that neutrophil production of IL-21 at mucosal surfaces is simply awaiting discovery. Finally, neutrophils can be important producers of IL-22 following colonic injury in an IL-23-dependent fashion and therefore, may be crucial for re-establishing the epithelial barrier [193].

Paneth cells

A specialized epithelial cell found in crypt bases of the small intestine, Paneth cells are typically appreciated for their role in production of antimicrobial peptides and degradative enzymes, such as lysozyme. However, there is growing evidence that Paneth cells may be an important, innate source of IL-17A. First detected in a model of TNF-induced shock, Paneth cell expression of IL-17A was associated with acute inflammation of the small bowel and correlated with increased IL-6 levels in the serum [194]. More recently, ablation of Paneth cells in a model of intestinal ischemia-reperfusion injury drastically reduced levels of serum and intestinal IL-17A and associated pathology. Notably, IL-17A appears to be constitutively present in Paneth cell granules [194], and IL-17A was found at the base of small intestine crypts concomitant with Paneth cell degranulation, 3 h after oral administration of the TLR9 ligand CpG-oligodeoxynucleotide [195]. Therefore, the presence of IL-17A in Paneth cell granules and subsequent Paneth cell degranulation during an inflammatory response may constitute an underappreciated, immediate, and rapid source of IL-17A. How IL-17A present in the intestinal lumen contributes to T_H17-like immune or inflammatory responses remains to be seen.

Macrophages

Although not primary producers of T_H17-associated cytokines, macrophages appear capable of producing IL-17A and IL-22 at the mucosa under select circumstances. In 1 study, following LPS exposure, macrophages could produce IL-17A and IL-22 but only in the absence of IL-10/IL-10R signaling [196]. Moreover, in a model of pulmonary infection during obesity, interstitial but not alveolar macrophages could produce IL-17A [197]. Macrophages could also produce IL-17A in response to infection with Mycobacterium avium [198] and in patients with chronic rhinosinusitis with nasal polyps [199]. Finally, alveolar macrophages can produce IL-22 in response to a model of pulmonary inflammation [200]. Future work, examining the role of macrophages in the generation of T_H17-associated cytokines, may be especially interesting in mucosal sites, such as the lung, given the different macrophage populations that are resident within or that can home to the lung during inflammation.

CONCLUDING REMARKS

Whereas IL-17A is sometimes viewed as synonymous with T_H17 cells, it is a pleiotropic cytokine that can be generated by many different cell types, including cells of the innate immune system that can respond quickly to microbial challenge. Beyond IL-17A, the other T_H17-associated cytokines (i.e., IL-17F, IL-21, IL-22, and IL-26) have important individual contributions to disease severity and resolution that require a complete understanding of the cytokine milieu present in specific conditions. Moreover, there are likely additional cell populations involved in production of T_H17-associated cytokines at the mucosa that have currently only been well characterized at nonmucosal sites (i.e., mast cell-mediated production of IL-17A in inflamed joints [201]). Considering the extended time course during which T_H17-associated cytokines can be produced (from disease initiation to resolution), the understanding of the cellular sources of T_H17-associated cytokines will be useful toward the generation of elegantly designed therapies to modulate inflammation.

Given the relatively recent discoveries of the T_H17-associated family of cytokines, it is not surprising that this is an area brimming with cutting-edge research. However, many questions still remain. How do rT_H17 cells arise, and are they a separate lineage or represent an intermediate cell type? Can we modulate IL-17A production in CD8⁺ T cells to tailor their cytotoxic behavior in the context of an anti-tumor response? How does early production of IL-17A from innate-like immune cells alter the clinical course of mucosal disease compared with late production of IL-17A by the adaptive response? Determining the answers to these questions may provide additional tools for a broad spectrum of diseases ranging from allergic inflammation to tumor progression.

Glossary

AHR

aryl hydrocarbon receptor

COPD

chronic obstructive pulmonary disease

CRC

colorectal cancer

Foxp3

forkhead box p3

IBD

inflammatory bowel disease

ILC2/3

innate lymphoid cell groups 2/3

iNKT

invariant NKT cell

IRF

IFN regulatory factor

iT_H17

induced T_H17

LTi

lymphoid tissue-inducer cell

MAIT

mucosal-associated invariant T cell

MDSC

myeloid-derived suppressor cell

NCR

natural cytotoxicity receptor

NKT_FH

follicular helper NKT cell

nT_H17

natural T_H17

ROR

retinoic acid-related orphan receptor

rT_H17

regulatory T_H17 cell

T_C17

cytotoxic IL-17A-producing CD8⁺ T cell

T_FH

follicular helper T cell

T_NC17

noncytotoxic IL-17A-producing CD8⁺ T cell

T_reg

regulatory T cell

AUTHORSHIP

K.O.B-S., T.W., S.H., and W.O. wrote and edited the review. T.W., K.O.B-S., and W.O. generated the illustration.

DISCLOSURES

The authors declare no conflict of interest.