Interluekin-17A (IL17A)

Abstract

The discovery of the key roles of interleukin-17A (IL-17A) and IL-17A producing cells in inflammation, autoimmune diseases and host defense has led to the experimental targeting of the IL-17A pathway in animal models of diseases as well as in clinical trials in humans. These therapeutic agents include biological products that target IL-17A and IL-23, an upstream regulator of IL-17A production. IL-17A producing T helper cells (Th17 cells) are a distinct lineage from the Th1 and Th2 CD4+ lineages and have been suggested to represent a good drug target in certain inflammatory conditions. Targeting IL-17A has been proven to be a good approach as anti-IL-17A is FDA approved for the treatment of psoriasis in 2015. In host defense, IL-17A has been shown to be mostly beneficial against infection caused by extracellular bacteria and fungi. This review will overview the discovery of IL-17A, the receptors used by this cytokine and its role in mucosal immunity and inflammation.

Introduction

IL-17A is the most widely studied member of the IL-17 family, a group of proteins that have a highly conserved C-terminus containing a cysteine-knot fold structure (Weaver et al., 2007). IL-17A plays a critical role in host defense against various microbial pathogens as well as tissue inflammation. IL-17A and its closest relative IL-17F, signal through the same receptor complex (IL-17R) composed of the subunits IL-17RA and IL-17RC. Recently, IL-17A and IL-17A producing cells have become important targets for drug discovery for the treatment of various forms of autoimmune and inflammatory diseases. Anti-IL-17A is FDA approved for the treatment of psoriasis and this pathway has also been studied in asthma, rheumatoid arthritis, multiple sclerosis, transplant rejection, and inflammatory bowel disease.

Discovery and cellular sources of IL-17A

IL-17A, often time referred to as IL-17, was originally discovered at transcriptional level by Rouvier et al. in 1993 from a rodent T-cell hybridoma, derived from the fusion of a mouse cytotoxic T cell clone and a rat T cell lymphoma (Rouvier et al., 1993). Recent studies now suggest that what was thought to be a mouse sequence was actually derived from the rat lymphoma (Kennedy et al., 1996). Human and mouse IL-17A were cloned a few years later by Yao and Kennedy (Kennedy et al., 1996; Yao et al., 1995b). Yao demonstrated that the IL-17A transcript was expressed in CD4+ T-cells and the protein could induce IL-6 in fibroblasts. Early studies also suggested that IL-17A may be a major vehicle by which T cells communicate with the hematopoietic system (Fossiez et al., 1996; Schwarzenberger et al., 1998). Fossiez demonstrated that IL-17A could signal to bone marrow stromal cells in vitro to induce G-CSF that supported the differentiation of hematopoietic cells to the granulocyte lineage. Schwarzenberger demonstrated that IL-17A over expression in vivo also led to extramedullary granulopoiesis (Schwarzenberger et al., 1998) and this was due to in vivo induction of G-CSF and stem cell factor (Schwarzenberger et al., 2000). Subsequent studies showed that lymphocytes including CD4+, CD8+, gamma-delta T (γδ-T), invariant NKT and innate lymphoid cells (ILCs) are primary sources of IL-17A (Cua and Tato, 2010). Non-T cells, such as neutrophils, have also been reported to produce IL-17A under certain circumstances (Taylor et al., 2014). A critical advance in the field came in 2005 when several groups showed that Th17 cells could be derived from naïve CD4+ T-cells under the control of TGFβ, IL-6 and Th17 lineage commitment was independent of STAT4, STAT6 (Bettelli et al., 2006; Harrington et al., 2005; Park et al., 2005). Subsequent studies demonstrated a critical role of STAT3 (Mathur et al., 2007) and RORC (Ivanov et al., 2006) in Th17 lineage commitment in mice and humans. Accumulating data also suggest that IL-23 is essential for the maturation and maintenance of the Th17 lineage (Gaffen et al., 2014).

Discovery of IL-17A Receptors

A receptor for IL-17A was first isolated and cloned from mouse EL4 thymoma cells and the bioactivity of IL-17A was confirmed by stimulating the transcriptional factor NF-kappa B activity and interleukin-6 (IL-6) secretion in fibroblasts (Yao et al., 1995a). This receptor, IL-17RA, was the founding member of the IL-17 receptor family which includes IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE. IL-17RA appears to be a common receptor chain shared with other IL-17R family members. For example IL-17RA pairs with IL-17RC to allow binding and signaling of IL-17A and IL-17F (Gaffen, 2009) and the IL-17RA/IL-17RC pair was discovered using flow cytometry binding assays as well as Biacore analysis (Kuestner et al., 2007)..

Role of IL-17A in autoimmune diseases

IL-17A producing CD4+ T helper cells, also called Th17 cells, have been studied extensively in the past decade and have been shown to be potent inducers of tissue inflammation and have been associated with the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions. Substantial evidence suggests that IL-17A producing cells including Th17 cells are involved in human psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, and asthma (Korn et al., 2009). The aberrant IL17A expression in affected tissues and the reported cellular sources of IL-17A are summarized in Table 1 and each individual disease is discussed below. Although many studies have reported non-lymphoid cells such as mast cells and neutrophils may produce IL-17A, most of these studies drew these conclusions based on immunohistochemistry staining which does not discriminate between IL-17A producing cells and cells that have bounded IL-17A due the ubiquitous expression of IL-17RA. Regardless of the cellular sources of IL-17A, it is considered to act on the structural cells to initiate tissue inflammation. A cartoon version of IL-17A mediated inflammation in various diseases is illustrated in Figure 1.

Table 1.

IL-17A detection in human diseases.

Disease	Affected tissue or organ	Detection sites of elevated IL-17A	Cellular sources of IL-17A	Reference
IBD	Intestine	Inflamed mucosa and serum	CD4+, CD8+ and ILCs.	(Fujino et al., 2003; Geremia et al., 2011; Holtta et al., 2008)
Psoriasis	Skin	Psoriatic skin lesions and serum	CD3+, neutrophils, mast cells	(de Oliveira et al., 2015; Johansen et al., 2009; Keijsers et al., 2014; Plee et al., 2015)
RA	Joint	Synovial fluid and serum	CD4+, NK, mast cells and B cells	(Church et al., 2010; Hueber et al., 2010a; Metawi et al., 2011; Schlegel et al., 2013)
MS	Brain and spinal cord	Blood and cerebrospinal fluid	CD4+ and CD8+	(Balasa et al., 2013; Durelli et al., 2009; Tzartos et al., 2008)
Asthma	Lung	Bronchoalveolar lavage fluid and blood	CD4+, NK, γδ-T, ILC3, neutrophils and B cells	Reviewed in ref (Chesne et al., 2014; Nembrini et al., 2009)
COPD	Lung	Sputum and bronchial biopsy	CD3+ and mast cells	(Barczyk et al., 2003; Barnes, 2015; Roos et al., 2015)

Figure 1. Schematic illustration of signaling pathway that involves IL-17A in inflammatory diseases.

Naïve CD4 T cells differentiate into IL-17 producing T cells under the cytokine environment including TGF-β, IL-6, IL-1β and IL-23. Th17 differentiation is controlled by transcription factors including RORA, RORC and STAT3. Type 3 innate lymphoid cell (ILC3) and γδ-T cell can also produce IL-17 in respond to IL-1β and IL-23 stimulation. IL-17A acts on structural cells such as epithelial cells, fibroblasts and keratinocytes in various tissue including skin, gut as well as lung. Structural cells that express IL-17 receptor produce inflammatory cytokines such as G-CSF and IL-6 as well as chemokines to attract neutrophils and macrophage to the inflamed tissues. These inflammatory cells can both clear the infection and initiate pathogenic inflammation.

Multiple sclerosis (MS) is a neurological disease caused by immune cells, which attack and destroy the myelin sheath that insulates neurons in the brain and spinal cord. This disease and its animal model experimental autoimmune encephalomyelitis (EAE) have historically been associated with the discovery of Th17 cells (Harrington et al., 2005; Park et al., 2005). However, elevated expression of IL-17A in multiple sclerosis (MS) lesions as well as peripheral blood has been documented before the identification of Th17 cells (Lock et al., 2002; Matusevicius et al., 1999). Human TH17 cells have been shown to efficiently transmigrate across the blood-brain barrier in multiple sclerosis lesions, promoting central nervous system inflammation (Kebir et al., 2007). Recent publications also suggest that GM-CSF instead of IL-17A produced by Th17 cells is a critical pathogenic factor in EAE (Codarri et al., 2011; El-Behi et al., 2011). Nonetheless, IL-17A may still be able to serve as a predictive or surrogate biomarker in MS based on the data from preclinical models.

Psoriasis is an auto-inflammatory skin disease characterized by circumscribed, crimson red, silver-scaled, plaque-like inflammatory lesions. Many types of immune cells can be found in these lesions such as infiltrating T cells (mainly CD4+ cells) and dendritic cells in the dermis as well as cytotoxic T cells and neutrophils in the epidermis (Lowes et al., 2007). These immune cells trigger rapid keratinocyte proliferation, abnormal keratinocyte differentiation, and dermal angiogenesis (Lowes et al., 2007). Initially, psoriasis was considered to be a Th1-mediated disease since elevated levels of IFN-γ, TNF-α, and IL-12 was found in the serum and lesions of psoriasis patients (Di Cesare et al., 2009). However, the finding of IL-17-producing cells as well as IL17A transcripts in the lesions of psoriatic patients suggested that Th17 cells may synergize with Th1 cells in driving the pathology in psoriasis (Cai et al., 2011; Harper et al., 2009). Furthermore, Th17 cells have also been found to be localized in the dermis in atopic dermatitis, with a higher prevalence present in acute compared to chronic lesions (Koga et al., 2008). Thus, IL-17 may be a target in inflammatory skin disorders beyond psoriasis. Secukinumab (anti-IL-17A) has been evaluated in psoriasis and the first report showing Secukinumab is effective when compared with placebo was published in 2010 (Hueber et al., 2010b). In 2015, the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved anti-IL-17 for the treatment of psoriasis (Beringer et al., 2016).

Another autoimmune disease strongly associated with Th17 cells is rheumatoid arthritis (RA), a chronic disorder with symptoms include chronic joint inflammation, autoantibody production, which lead to the destruction of cartilage and bone (McInnes and Schett, 2011). Interestingly, the levels of IL-17A in the synovium correlate with tissue damage, whereas levels of IFN-γ correlate with protection (Kirkham et al., 2006). Direct clinical significance of IL-17A in RA comes from recent clinical trials which found that two anti-IL-17A antibodies, namely Secukinumab and Ixekizumab significantly benefit these patients (Genovese et al., 2014; Genovese et al., 2010). The first trial on Secukinumab in RA showed improved clinical response (Hueber et al., 2010b) but in another phase II trial, the same efficacy was not achieved (Genovese et al., 2013).

Th17 cells and IL-17 have also been linked to Crohn’s disease (CD) and ulcerative colitis (UC), the two main forms of inflammatory bowel diseases (IBD) in man. Th17 cells infiltrate massively to the inflamed tissue of IBD patients and both in vitro and in vivo studies have shown that Th17-related cytokines may initiate and amplify multiple pro-inflammatory pathways (Monteleone et al., 2012). Elevated IL-17A levels in IBD have been reported by several groups (Fujino et al., 2003; Rovedatti et al., 2009). Nonetheless, Th17 signature cytokines, such as IL-17A and IL-22, may target gut epithelial cells and promote the activation of regulatory pathways and confer protection in the gastrointestinal tract (Li et al., 2014; Sarra et al., 2010). To this end, recent clinical trials targeting IL-17A in IBD were negative and actually showed increased adverse events in the treatment arm (Hueber et al., 2012). This data raised the question regarding the role of IL-17A in IBD pathogenesis and suggested that the elevated IL-17A might be beneficial for IBD patients.

Systemic lupus erythematosus, commonly referred as SLE or lupus, is a complex immune disorder affects the skin, joints, kidneys, and brain. Although the exact cause of lupus is not fully known, it has been reported that IL-17 and Th17 cells are involved in disease pathogenesis (Garrett-Sinha et al., 2008). It has been reported that serum IL-17 levels are also elevated in SLE patients compared to controls (Vincent et al., 2013; Wong et al., 2008) and the Th17 pathway has been shown to drive autoimmune responses in pre-clinical mouse models of lupus (Hsu et al., 2008; Jacob et al., 2009). More importantly, IL-17 and IL-17 producing cells are also been detected in kidney tissue and skin biopsies from SLE patients (Crispin et al., 2008; Oh et al., 2011; Yang et al., 2009), suggesting a possible role for IL-17 in the pathophysiology of SLE and a potential therapeutic option of using anti-IL-17A in treating patients with SLE.

IL-17A in Lung Diseases

Asthma

Asthma is a major chronic disease characterized by airway inflammation and bronchial hyperresponsiveness, leading to recurrent chest wheezing, cough, and shortness of breath. Elevated levels of IL-17A have been found in the sputum and in bronchoalveolar lavage fluid of patients with asthma (Molet et al., 2001) and a positive correlation between IL-17A production and asthma severity has been established (Chesne et al., 2014). In murine models, treatment with dexamethasone inhibits the release of Th2-related cytokines but does not affect IL-17A production (McKinley et al., 2008). Furthermore, Th17 cell-mediated airway inflammation and airway hyperresponsiveness are steroid resistant, indicating a potential role for Th17 cells in steroid-resistant asthma (McKinley et al., 2008). Heterogeneous expression of IL-13 (Th2 signature cytokine) and IL-17 (Th17 signature cytokine) related genes have been observed in both patients and animal models, suggesting combination therapy targeting both pathways could be effective in treating asthma (Choy et al., 2015). However, a recent trial using anti-IL-17RA did not show efficacy in subjects with asthma (Busse et al., 2013). Further studies in stratifying and phenotyping patients are needed to determine the contributions of IL-17A in asthma.

COPD

Chronic obstructive pulmonary disease (COPD) is a pulmonary disease marked by progressive emphysematic changes in the lung. Recent studies have suggested the involvement of immunological mechanisms in COPD (Alcorn et al., 2010). An increase in Th17 cells was observed in patients with COPD compared with current smokers without COPD and healthy subjects, and inverse correlations were found between Th17 cells with lung function (Vargas-Rojas et al., 2011). Increased production of IL-17A in the lungs were also found by different research groups (Cazzola and Matera, 2012). Gene expression profiling of bronchial brushings obtained from COPD patients also linked lung function to several Th17 signature genes such as SAA1, SAA2, SLC26A4 and LCN2 (Steiling et al., 2013). Animal studies have shown that cigarette smoke promotes pathogenic Th17 differentiation and induces emphysema (Chen et al., 2011b), while blocking IL-17A using neutralizing antibody significantly decreased neutrophil recruitment and the pathological score of airway inflammation in tobacco-smoke-exposed mice (Cazzola and Matera, 2012; Shen et al., 2011).

Role of IL-17A in host defense

The primary function of Th17 cells appears to be control of the gut microbiota (Ivanov et al., 2009; Kumar et al., 2016) as well as the clearance of extracellular bacteria and fungi. IL-17A and IL-17 receptor signaling has been shown to be play a protective role in host defenses against many bacterial and fungal pathogens including Klebsiella pneumoniae, Mycoplasma pneumonia, Candida albicans, Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis (Chen and Kolls, 2013). However, IL-17A seems to be detrimental in viral infection such as influenza through promoting neutrophilic inflammation (Crowe et al., 2009).

The requirements of IL-17A and IL-17 receptor signaling in host defense were well documented and appreciated before the identification of Th17 cells as an independent T helper cell lineage (Ye et al., 2001). In experimental pneumonia models, IL-17A or IL-17RA knock mice have increased susceptibility to various Gram-negative bacteria, such as Klebsiella pneumoniae (Ye et al., 2001) and Mycoplasma pneumonia (Wu et al., 2007). Upon K. pneumoniae infection, IL-17RA knockout mice showed reduced G-CSF production and neutrophil recruitment in the lungs and, as a consequence, the IL-17RA knockout mice had higher bacterial burdens in the lung as well as increased systemic dissemination to the spleen when compare to their littermate controls (Ye et al., 2001). Early IL-17A production, principally from γδ T-cells, in response to K. pneumonia requires TLR4 signaling and IL-23 production (Happel et al., 2003). Mice lacking IL-23p19, a subunit that is IL-23 specific, have decreased levels of IL-17A and exhibit similar defects as the IL-17RA knockout mice with reduced production of proinflammatroy cytokines and chemokines such as G-CSF and CXCL1, 2, and 5 (Happel et al., 2005). In IL-23p19 knockout mice, mucosal administration of recombinant IL-17A rescued the chemokine production and neutrophil infiltration to the lungs and resulted in decreased bacterial burden as well as systemic dissemination (Happel et al., 2005). Similar results have also been observed in M. pneumoniae infection (Wu et al., 2007). In contrast, data suggest that IL-23 and IL-17A are not required for protection against primary infection by the intracellular bacteria Mycobacterium tuberculosis. Both the IL-17RA knock out mice and the IL-23p19 knock out mice cleared primary infection with M. tuberculosis (Khader et al., 2005; Sun and Metzger, 2008). However, M. tuberculosis infection induces Il23a expression in the lungs and overexpression of IL-23p19 in M. tuberculosis vaccination significantly boosted antigen-specific Th1 responses (Khader et al., 2007; Wozniak et al., 2006a; Wozniak et al., 2006b). Interestingly, IL-17A is required for protection against primary infection with a different intracellular bacteria, Francisella tularensis. It has been shown in the mouse model that IL-17A is required for the production of IL-12p70 after infection and the Th1 responses promoted by IL-17p70 is responsible for the ultimate clearance of the infection (Lin et al., 2009).

Mouse model studies using the IL-17RA knock out mice and the IL-17A knock out mice with the murine adapted influenza strain (PR8) (Crowe et al., 2009) as well as the 2009 pandemic H1N1 stain (Li et al., 2012) both support that IL-17A plays a detrimental role in mediating the acute lung injury, suggesting that therapeutic anti-IL-17A treatment may be useful in an influenza pandemic.

While the essential role of the IL-17A in protection against primary infection caused by a variety of extracellular bacteria and fungi has been documented for many years, the role of adaptive immune responses mediated by antigen specific Th17 has been investigated more recently. The development of antigen specific memory Th17 cells could benefit the host by mounting a more robust pathogen specific recall response upon re-countering the same pathogen. Thus, Th17 cells may have an advantage as a source of IL-17A as innate sources of IL-17A such as γδ-T and ILC3 cells do not mount typical immunological memory responses. In addition, antigen specific Th17 cells were also shown to recognize conserved protein antigens among different K. pneumoniae strains and provide broad-spectrum serotype-independent protection (Chen et al., 2011a). This concept has also been extended to fungal infection where an IL-17 producing clone with a TCR specific for calnexin from Blastomyces dermatitidis confers protection with evolutionary related fungal species including Histoplasma spp (Wuthrich et al., 2015). In another pneumonia mouse model, it has been shown that antigen specific CD4 T cells limit nasopharyngeal colonization of S. pneumoniae (Trzciński et al., 2008). More importantly, CD4 T cell-derived IL-17A, but not IFNγ or IL-4, was required for the clearance of Pneumococcal colonization (Lu et al., 2008). Furthermore, immunization with pneumococcal whole cell antigen and several derivatives provided IL-17-mediated, but not antibody dependent, protection against S. pneumoniae challenge (Malley et al., 2006; Moffitt et al., 2012). These studies demonstrated an essential role of antigen specific Th17 in host defense in the lungs. Antigen specific Th17 responses are also observed in other mucosal site such as the gastrointestinal tract. It has been shown that segmented filamentous bacterium (SFB) is sufficient to induce the Th17 development in the small intestine of mice (Ivanov et al., 2009) and these Th17 cells have been shown to be SFB antigen specific (Yang et al., 2014). These cells appear to constrain SFB growth in the intestine as loss of IL-17R specifically in the gut epithelium leads to SFB expansion (Kumar et al., 2016).

Role of IL-17A in Cancer

The IL-17A response, while constituting a protective arm defending the body against various infections, also functions as a double-edged sword constituting a risk factor that mediates the development of autoimmune diseases. The two sides of IL-17A can be even observed in one disease such as cancer. Recently, a pathogenic role of IL-17A in cancer is suggested (Chang et al., 2014; Coffelt et al., 2015; Wu et al., 2014). In contrast, anti-tumor effect of IL-17 producing cells was also observed in animal models (Martin-Orozco et al., 2009). With the prevalence of cancer increasing year to year, it is of importance to find new pathogenic pathways as well as new therapeutics to combat this disease.

In tumorigenesis, IL-17A has been shown to recruit myeloid derived suppressor cells (MDSCs) to dampen anti-tumor immunity (Chang et al., 2014; He et al., 2010). IL-17A can also enhance tumor growth in vivo through the induction of IL-6, which in turn activates oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) and upregulates pro-survival and pro-angiogenic genes in tumors (Wang et al., 2009). The exact role of IL-17A in angiogenesis has yet to be determined and current data suggest that IL-17A can promote or suppress tumor development (Houghton, 2013). IL-17A seemed to facilitate development of colorectal carcinoma by fostering angiogenesis via promote VEGF production from cancer cells (Liu et al., 2011) and it has been show that IL-17A also mediates tumor resistance to anti-VEGF therapy through the recruitment of MDSCs (Maniati and Hagemann, 2013). In a murine lung cancer model, Th17 cells have been shown to recruit myeloid cells and promote tumor proliferation (Chang et al., 2014) and in a murine model of pancreatic cancer, IL-17 also accelerates pancreatic intraepithelial neoplasia initiation and progression (McAllister et al., 2014). However IL-17A KO mice were more susceptible to developing metastatic lung melanoma (Martin-Orozco et al., 2009), suggesting that IL-17A can possibly promote the production of the potent antitumor cytokine IFN-γ, produced by cytotoxic T cells. Indeed, data from ovarian cancer suggest that Th17 cells are positively correlated with NK cell–mediated immunity and anti-tumor CD8 responses (Kryczek et al., 2009). A novel immune evasion mechanism by the tumors through the inhibition of Th17 cell development is proposed (Kryczek et al., 2009).

Targeting IL-17A in diseases

IL-17A producing T cell subsets are attractive targets for pharmaceutical intervention. In contrast, the generation of pathogen-specific T cell responses may be desired for advances in vaccine development. Other than the monoclonal antibodies that have been discussed above, highly specific and potent inhibitors targeting Th17 specific transcription factor RORγt have been identified and found to be highly effective (Huh and Littman, 2012). These small molecules will also lead us to the better understanding of how IL-17A functions both in normal development and disease settings. Vitamin D, a potent immunomodulator, has also been shown to suppress Th17 cell differentiation and function by several research groups (Hayes et al., 2015). Since IL-17A signals through the receptor complex composed of the subunits IL-17RA and IL-17RC, both can serve as targets for inhibiting IL-17A signaling (Gaffen, 2009). IL-17RA is expressed in nearly every cell type of the body, including epithelial cells, endothelial cells, fibroblasts and myeloid cells. Although IL-17RC is also expressed on epithelial cells and fibroblasts, its expression on myeloid cells is lower (Ge and You, 2008). Based on this more restricted expression of IL-17RC, it is thought that fibroblasts, epithelial cells and endothelial cells are a major target of IL-17A and IL-17F and targeting IL-17RC could be beneficial to the patients with a better specificity and selectivity.

Concluding remarks

Recent studies on IL-17A and Th17 subset provide new evidence of the contribution of T helper cells (other than Th1 and Th2) to chronic inflammation and tissue destruction. An increasing numbers of diseases have been linked to IL-17A and Th17 cells and their specific contribution to disease pathogenesis is being revealed. Both pre-clinical research and clinical trials are underway to test these concepts. Knowledge of the molecular pathways responsible for the regulation of the Th17 lineage and the production of IL-17A is expanding rapidly. This information will undeniably be essential for the development of novel therapeutic strategies for the treatments of an array of inflammatory diseases in humans.

Highlights.

• Overview of the discovery, cloning and identification of the cellular sources of IL-17A

• Summary of the roles of IL-17A in mucosal immunity and autoimmune diseases

• Summary of IL-17A targeted drug development