Th17 deficiency in human disease

The IL-17 Cytokine Family

The IL-17 cytokine family consists of six members (IL-17A-F). Among IL-17 family members, IL-17A and IL-17F share the highest amino acid sequence homology (50%)¹⁰. The receptors for IL-17 include a family of five receptors (IL-17RA-E). Heterodimers of IL-17RA and IL-17RC constitute the receptor for IL-17A and IL-17F. IL-17A and IL-17F are preferentially produced by Th17 cells (along with IL-21 and IL-22). IL-17A and IL-17F form homo-and heterodimers and induce expression of pro-inflammatory cytokines (IL-1, IL-6, TNFα, G-CSF, GM-CSF), chemokines (CXCL1, CXCL5, IL-8, CCL2, CCL7), antimicrobial peptides(AMPs) (defensins, S100), and matrix metalloproteinases (MMP1, MMP3, MP13) from the endothelium, epithelia, and fibroblasts⁹. As a result, Th17 cells can promote granulopoesis and recruitment of neutrophils to sites of infection and provide host defense against extracellular bacteria and fungi. Interestingly, a variety of other T cell types, including CD8+ T cells (Tc17), γδ T cells (γδ-17), and NKT cells (NKT-17) can produce IL-17A and IL-17F (reviewed in¹¹). Further, innate immune cells including neutrophils, monocytes, NK cells, and lymphoid tissue inducer (LTi)-like cells can produce IL-17A and IL-17F. Finally, IL-17A has been shown to be produced by intestinal Paneth cells¹², whereas colonic epithelial cells have been shown to express IL-17F mRNA, suggesting that a variety of cell types may play roles in host defense against fungi and extracellular bacteria⁸. Less is known about the regulation of IL-17 from these cell types.

AD-HIES due to mutations in STAT3

AD-HIES due to mutations in STAT3 is a multisystem immune deficiency characterized by highly elevated serum levels of IgE, local and invasive infections with S. aureus that result in cold abscesses and recurrent pneumonia with pneumatocele formation, skeletal abnormalities, and coarse facial features^26–28. Additionally, development of CMC is a common complication in these patients. The cytokines IL-6 and IL-21, which activate STAT3, play significant roles in the development of human Th17 T cells²⁹, as discussed above. As previously mentioned, STAT3 activation induces the expression of RORγt, a transcription factor required for the development of Th17 T cells and the subsequent expression of IL-17, IL-21, IL-22, and IL23R, which play essential roles in Th17 function. Since patients with AD-HIES due to STAT3 mutations are susceptible to CMC disease and STAT3 is essential for Th17 cell development and Th17 cells play an important role in mucosal immunity against Candida, Ma et al²³ evaluated these patients for potential defects in Th17 development. Analysis of T cell blasts generated from AD-HIES total CD4+ T cells revealed significant reductions in IL-17+ T cells and subsequent stimulation of these CD4+ T cells resulted in no production of IL-17. Furthermore, production of IL-22, a Th17-derived cytokine involved in epithelial and mucosal immunity, was absent from AD-HIES CD4+ T cells. Consistent with the essential role of STAT3 in RORγt expression, upregulation of RORγt expression was reduced in stimulated AD-HIES CD4+ T cells compared to healthy controls. Furthermore, sorted naïve CD4+ T cells from AD-HIES patients failed to differentiate into Th17 T cells when stimulated with anti-CD2 and anti-CD3 plus anti-CD28 in the presence of IL-1β plus IL-6 or IL-23, confirming the requirement for STAT3 in Th17 differentiation. Consistent with these observations, Milner et al³⁰ found that blood cells obtained from AD-HIES patients that were stimulated overnight with S. aureus or Candida failed to generate memory CD4+IL-17+ T cells. Thus, analysis of T cells from patients with STAT3 mutations has clearly demonstrated an essential role for STAT3 in Th17 differentiation and mucosal and epithelial immunity against Candida and S aureus ^{23, 30, 31}(Fig. 1). As mentioned above, Tc17, γδ-17, and NKT-17 are also sources of Th17 cytokines¹¹; however, they either do not produce Th17 cytokines or they produce inadequate quantities of Th17 cytokines in AD-HIES patients to prevent CMC disease. This suggests that STAT3 is likely to be involved in Th17 cytokine production in Tc17 cells, γδ-17 cells, and NKT-17 cells.

DOCK8

AR-HIES due to mutations in DOCK8 is characterized by highly elevated serum IgE levels, hypereosinophilia, recurrent sinopulmonary infections, unusual susceptibility to herpesvirus infections, candidal dermatitis, and atopy^32–34. In addition, DOCK8 deficient patients tend to be T cell lymphopenic with poor T cell proliferation after activation with anti-CD3 and anti-CD28. The molecular basis of cutaneous candidal infections in these patients is incompletely understood at present. Previous analysis of a cohort of AR-HIES patient, some of whom were later confirmed to have DOCK8 deficiency, revealed that these patients have defects in Th17 differentiation²⁴. RORγt expression, which is critical for Th17 differentiation, was markedly reduced in peripheral T cells from patients with AD-HIES due to STAT3 mutations, as well as in AR-HIES patients. Interestingly, in vitro induction of RORγt expression by naïve T cells was intact in the AR-HIES, but not AD-HIES patients. These data suggest that the initial steps of Th17 differentiation are intact in the AR-HIES patients, but subsequent steps of differentiation are impaired (Fig. 1). IL-17 production was impaired in both groups of HIES patients, although the impairment was more severe in the patients with STAT3 mutations. Thus, defective Th17 differentiation occurs by a different mechanism in AR-HIES versus AD-HIES due to STAT3 mutations. It is likely that impaired Th17 differentiation and IL-17 production contributes to the susceptibility of AR-HIES patients with DOCK8 mutations to candidal dermatitis. It is also possible that a more global impairment in T cell function in DOCK8 deficient patients, as demonstrated clinically by their susceptibility to viral infections, also contributes to candidal infections. A clearer understanding of the cause(s) of impaired Th17 differentiation in DOCK8 patients may help to further elucidate the complex process of Th17 differentiation.

Gain of function mutations in STAT1

In a search for genetic causes of AD-CMC disease, two groups of investigators independently identified heterozygous mutations in STAT1 as a novel cause of deficiency of Th17 differentiation and AD-CMC. In one study, van de Veerdonk et al³⁵ analyzed five families that had severe CMC disease. Other documented infections included pneumonia and one case of Pneumocystis jirovecii pneumonia with CMV infection. Additionally, several patients suffered from autoimmune disease (autoimmune hepatitis, autoimmune hemolysis, hypothyroidism) and a few developed cancers (esophageal cancer, squamous cell carcinoma). Functional analysis of blood cells revealed defects in Candida-stimulated production of interferon-γ, IL-17, and IL-22. Further functional analyses demonstrated defective functioning of IL-12 and IL-23 signaling pathways. These studies were followed by array-based sequence capture followed by next generation sequencing of 100 candidates genes relevant to IL-12 and IL-23 signaling and Th1 and Th17 responses. This analysis identified heterozygous mutations within highly conserved residues in exon 10 of STAT1, which lies within coiled-coil domain.

Simultaneously, Liu et al³⁶ analyzed additional genetic causes of CMC disease by applying a genome-wide strategy based upon whole-exome sequencing. All patients suffered CMC disease and several of these patients suffered autoimmune disorders (thyroid autoimmunity, systemic lupus erythematosis), cerebral aneurysms, or squamous cell carcinoma. Candidate variations were selected by filtering out known polymorphisms obtained from several databases. This analysis identified heterozygous variations in the STAT1 gene all within the coiled-coil domain. Functional analysis of one of the STAT1 mutant alleles (STAT1R274Q) revealed enhanced activation of a gamma activated sequence reporter construct in response to IFNα, IFNγ, and IL-27. The increased transcriptional activation by the STAT1R274Q allele was associated with impaired nuclear dephosphorylation. Thus, STAT1 mutations within the coiled-coil domain appeared to be gain of function mutations. Stimulation of EBV-immortalized B cells and fibroblasts from patients with gain of function mutations in STAT1 resulted in augmented responses to IFNα, IFNγ, and IL-27, cytokines known to be antagonistic to Th17 development^{37, 38}. Interestingly, cellular responses to predominantly STAT3 activating cytokines that promote Th17 differentiation, like IL-6 and IL-21, also resulted in increased STAT1 phosphorylation in EBV B cells from patients with gain of function STAT1 mutations compared to healthy controls. The enhanced STAT1 activation may be significant since STAT1 activation has been shown to be antagonistic to Th17 development¹³(Fig. 1). Finally, percentages of IL17A+, IL17F+, and IL-22+ T cells, as well as production of IL-17A and IL-22 in patients with gain of function STAT1 mutations were significantly lower than healthy controls and patients with loss of function STAT1 mutations, providing the molecular basis for CMC disease in these patients. Interestingly, enhanced cellular responses to type 1 interferon in these patients may underlie a predisposition to autoimmune disorders observed in several of these patients, since autoimmune disorders have been associated with an increased type 1 interferon signature^{39, 40}. A fascinating aspect of the role of STAT1 in human immune deficiencies is that mutations in one gene can lead to three very different phenotypes: CMC disease³⁶, mycobacterial disease⁴¹, or susceptibility to viral disease⁴².

APS1/APECED and Thymoma

The syndrome of autoimmune polyendocrine syndrome type 1 (APS-1), or autoimmune polyendocrinopathy, candidiasis, and ectodermal dystrophy (APECED) is the result of autosomal recessive mutations in autoimmune regulator (AIRE) gene⁴³. AIRE is a transcription factor expressed by medullary thymic epithelial cells, which is responsible for the expression of a wide variety of tissue specific antigens within the thymus. Expression of these tissue specific antigens in the thymus leads to deletional self tolerance in T cells maturing within the thymus. However, the lack of functional AIRE in APECED patients results in impaired central T cell tolerance, leading to the generation of autoantibodies. The clinical presentation of APECED is variable, but the majority of patients develop CMC disease, hypoparathyroidism, and Addison’s disease²⁵. CMC is the sole immune deficiency in APECED patients and is one of the earliest manifestations of the syndrome. Neutralizing antibodies against cytokines, in particular type 1 interferons and Th17 cytokines, are the most prevalent autoantibodies in APECED patients (reviewed in²⁵). This high prevalence of autoantibodies against type 1 interferons and IL-22 is virtually diagnostic for APECED. Autoantibodies against IL-17A, IL-17F, and IL-22 appear to be more prevalent in APECED patients with CMC than those without CMC, suggesting a causal role in CMC (Fig. 1). Kisand et al⁴⁴ also observed that IL-17 and IL-22 production was markedly decreased from Candida-stimulated blood cells derived from APECED patients with CMC disease, perhaps due to autoimmune destruction of IL-17+ and IL-22+ T cells. Interestingly, Pedroza et al⁴⁵ observed that the AIRE protein can also function as a signaling effector downstream of Dectin-1 in blood cells and a monocytic cell line (Fig. 1). These observations suggest that AIRE mutations in APECED may contribute to impaired innate responses to Candida, which could subsequently impair IL-17 and IL-22 production, although this remains to be formally evaluated.

Thymomas are tumors that originate from thymic epithelial cells. The majority of these tumors fail to express AIRE (Fig. 1). Thymomas are associated with autoimmune manifestations, most notably with antibodies against muscle acetylcholine receptors that cause myasthenia gravis. Autoantibodies against type 1 interferons are also common in thymoma patients; however, autoantibodies against Th17 cytokines are rare⁴⁴. As a result, CMC is only rarely associated with thymoma. For unknown reasons, APECED and thymoma patients, despite having high titers of neutralizing antibodies against IFNα and IFNω, do not have increased susceptibility to viral infections. Possible explanations are that IFNβ and IFNλ may compensate or local concentrations of type 1 interferons may be adequate to activate biological responses. The rare occurrence of CMC disease in thymoma patients with anti-IL-17 cytokine antibodies provides additional circumstantial evidence of the importance of IL-17 cytokines in CMC disease.

Inborn errors of IL-17RA and IL-17F

Recently, two genetic causes of pure CMC disease have been identified in humans (Table 1). Puel et al⁴⁶ studied individuals and families with infectious susceptibility limited to CMC for inborn errors of IL-17, IL-17R, and IL-22. Other known causes of CMC disease were excluded clinically and genetically. Evaluation of an infant who was the product of a consanguineous union that presented with candidal dermatitis and subsequent Staphylococcus aureus dermatitis resulted in the first description of autosomal recessive CMC disease due to a homozygous nonsense mutation in the IL-17RA gene (Q248X/Q248X). The Q248X/Q248X mutation resulted in absent expression of IL-17RA on the patient’s blood cells and fibroblasts and a complete absence of responsiveness to IL-17A/F. Responsiveness to IL-17 cytokines was restored to the patient’s fibroblasts by transfection with wild type IL-17RA, confirming the lack of IL-17RA expression as the cause the patient’s CMC disease.

Additionally, Puel⁴⁶ et al evaluated a multiplex family with autosomal dominant inheritance of CMC disease, identifying a heterozygous missense mutation in the IL-17F gene. The mutation resulted in the substitution of the serine residue at position 65 of the protein with a leucine residue (S65L). The S65 residue is conserved across all mammalian species and is believed to be involved in ligand-receptor interaction. The production of inflammatory cytokines by normal fibroblasts and blood cells elicited by mutant IL-17F(S65L) homodimers was severely impaired. In addition, stimulation of normal fibroblasts and keratinocytes with heterodimers consisting of IL-17F(S65L)/IL-17FWT or IL-17F(S65L)/IL-17A resulted in severely impaired production of inflammatory cytokines, demonstrating a dominant negative effect of IL-17F(S65L). Interestingly, the IL-17F(S65L) mutation was also found in two apparently healthy members of the family members, suggesting incomplete clinical penetrance. At present, although experience is very limited with patients having genetic defects in IL-17F or IL-17RA, these observations support a role for Th17 cells and Th17 cytokines that is limited to mucosal immunity against Candida, and possibly S aureus (Fig. 1).