TLR2 Supports γδ T cell IL-17A Response to ocular surface commensals by Metabolic Reprogramming.

The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival γδ T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival Vγ4 T cells through a combination of γδ TCR ligation and IL-1 signaling. Here, we identify Vγ6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that Vγ6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, IκBζ, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal γδ T cells.

Too Much of a Good Thing: Extended Duration of Gut Microbiota Depletion Reverses Protection From Experimental Autoimmune Uveitis.

Purpose: Using the model of experimental autoimmune uveitis (EAU) induced by immunization with a retinal antigen, two studies have reported contradictory results on disease development following oral antibiotic treatment (ABX). We showed that long-term ABX did not affect EAU, but another study showed that short-term ABX was protective. We therefore studied the effects of ABX on EAU, gut microbiota, and host immune responses as a function of treatment duration. Methods: EAU-susceptible mice were treated orally with broad-spectrum antibiotics starting at least 10 weeks (long-term) or 1 week (short-term) before immunization until termination of the experiment. Gut microbiota were characterized by 16S amplicon sequencing, and host gut immune elements were studied phenotypically and functionally. Results: Long-term ABX had no effect, whereas short-term ABX delayed EAU, as previously reported by us and others, respectively. Microbial sequencing revealed progressive reduction of gut microbiota that showed some differences in the two ABX groups. Interestingly, duration of ABX was associated with a gradual disappearance of the CD4+ and CD4+CD8+ subset of gut intraepithelial lymphocytes (IELs). This IEL subset is microbiota dependent and is absent in germ-free mice. Relative abundance of Lactobacillus reuteri correlated with the frequencies of CD4+CD8+ IELs. IELs suppressed antigen-specific activation of autoreactive T cells in culture. Conclusions: Gut microbiota may play dual roles in uveitis development: They promote EAU development but also help maintain IEL populations that have regulatory function against autoreactive T cells. We propose that the progressive loss of this population during long-term ABX reverses the EAU-ameliorating effects of microbiota depletion.

T cell-intrinsic role for Nod2 in protection against Th17-mediated uveitis.

Mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) cause Blau syndrome, an inflammatory disorder characterized by uveitis. The antimicrobial functions of Nod2 are well-established, yet the cellular mechanisms by which dysregulated Nod2 causes uveitis remain unknown. Here, we report a non-conventional, T cell-intrinsic function for Nod2 in suppression of Th17 immunity and experimental uveitis. Reconstitution of lymphopenic hosts with Nod2-/- CD4+ T cells or retina-specific autoreactive CD4+ T cells lacking Nod2 reveals a T cell-autonomous, Rip2-independent mechanism for Nod2 in uveitis. In naive animals, Nod2 operates downstream of TCR ligation to suppress activation of memory CD4+ T cells that associate with an autoreactive-like profile involving IL-17 and Ccr7. Interestingly, CD4+ T cells from two Blau syndrome patients show elevated IL-17 and increased CCR7. Our data define Nod2 as a T cell-intrinsic rheostat of Th17 immunity, and open new avenues for T cell-based therapies for Nod2-associated disorders such as Blau syndrome.

The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24.

Dysregulated Th17 cell responses underlie multiple inflammatory and autoimmune diseases, including autoimmune uveitis and its animal model, EAU. However, clinical trials targeting IL-17A in uveitis were not successful. Here, we report that Th17 cells were regulated by their own signature cytokine, IL-17A. Loss of IL-17A in autopathogenic Th17 cells did not reduce their pathogenicity and instead elevated their expression of the Th17 cytokines GM-CSF and IL-17F. Mechanistic in vitro studies revealed a Th17 cell-intrinsic autocrine loop triggered by binding of IL-17A to its receptor, leading to activation of the transcription factor NF-κB and induction of IL-24, which repressed the Th17 cytokine program. In vivo, IL-24 treatment ameliorated Th17-induced EAU, whereas silencing of IL-24 in Th17 cells enhanced disease. This regulatory pathway also operated in human Th17 cells. Thus, IL-17A limits pathogenicity of Th17 cells by inducing IL-24. These findings may explain the disappointing therapeutic effect of targeting IL-17A in uveitis.

Autoimmunity to neuroretina in the concurrent absence of IFN-γ and IL-17A is mediated by a GM-CSF-driven eosinophilic inflammation.

IFN-γ and IL-17A can each elicit ocular autoimmunity independently of the other. Since absence of IFN-γ or IL-17A individually failed to abolish pathology of experimental autoimmune uveitis (EAU), we examined EAU development in the absence of both these cytokines. Ifng-/-Il17a-/- mice were fully susceptible to EAU with a characteristic eosinophilic ocular infiltrate, as opposed to a mononuclear infiltrate in WT mice. Retinal pathology in double-deficient mice was ameliorated when eosinophils were genetically absent or their migration was blocked, supporting a pathogenic role for eosinophils in EAU in the concurrent absence of IFN-γ and IL-17A. In EAU-challenged Ifng-/-Il17a-/- mice, ocular infiltrates contained increased GM-CSF-producing CD4+ T cells, and supernatants of retinal antigen-stimulated splenocytes contained enhanced levels of GM-CSF that contributed to activation and migration of eosinophils in vitro. Systemic or local blockade of GM-CSF ameliorated EAU in Ifng-/-Il17a-/- mice, reduced eosinophil peroxidase levels in the eye and in the serum and decreased eosinophil infiltration to the eye. These results support the interpretation that, in the concurrent absence of IFN-γ and IL-17A, GM-CSF takes on a major role as an inflammatory effector cytokine and drives an eosinophil-dominant pathology. Our findings may impact therapeutic strategies aiming to target IFN-γ and IL-17A in autoimmune uveitis.

A novel role for lipoxin A4 in driving a lymph node-eye axis that controls autoimmunity to the neuroretina.

The eicosanoid lipoxin A4 (LXA4) has emerging roles in lymphocyte-driven diseases. We identified reduced LXA4 levels in posterior segment uveitis patients and investigated the role of LXA4 in the pathogenesis of experimental autoimmune uveitis (EAU). Immunization for EAU with a retinal self-antigen caused selective downregulation of LXA4 in lymph nodes draining the site of immunization, while at the same time amplifying LXA4 in the inflamed target tissue. T cell effector function, migration and glycolytic responses were amplified in LXA4-deficient mice, which correlated with more severe pathology, whereas LXA4 treatment attenuated disease. In vivo deletion or supplementation of LXA4 identified modulation of CC-chemokine receptor 7 (CCR7) and sphingosine 1- phosphate receptor-1 (S1PR1) expression and glucose metabolism in CD4+ T cells as potential mechanisms for LXA4 regulation of T cell effector function and trafficking. Our results demonstrate the intrinsic lymph node LXA4 pathway as a significant checkpoint in the development and severity of adaptive immunity.

Regulated Tristetraprolin Overexpression Dampens the Development and Pathogenesis of Experimental Autoimmune Uveitis.

Non-infectious uveitis, a common cause of blindness in man, is often mediated by autoimmunity, a process in which cytokines play major roles. The biosynthesis and secretion of pro-inflammatory cytokines are regulated in part by tristetraprolin (TTP), an endogenous anti-inflammatory protein that acts by binding directly to specific sequence motifs in the 3'-untranslated regions of target mRNAs, promoting their turnover, and inhibiting synthesis of their encoded proteins. We recently developed a TTP-overexpressing mouse (TTPΔARE) by deleting an AU-rich element (ARE) instability motif from the TTP mRNA, resulting in increased accumulation of TTP mRNA and protein throughout the animal. Here, we show that homozygous TTPΔARE mice are resistant to the induction of experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP), an established model for human autoimmune (noninfectious) uveitis. Lymphocytes from TTPΔARE mice produced lower levels of the pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and TNFα than wild type (WT) mice. TTPΔARE mice also produced lower titers of antibodies against the uveitogenic protein. In contrast, TTPΔARE mice produced higher levels of the anti-inflammatory cytokine IL-10, and had higher frequencies of regulatory T-cells, which, moreover, displayed a moderately higher per-cell regulatory ability. Heterozygous mice developed EAU and associated immunological responses at levels intermediate between homozygous TTPΔARE mice and WT controls. TTPΔARE mice were able, however, to develop EAU following adoptive transfer of activated WT T-cells specific to IRBP peptide 651-670, and naïve T-cells from TTPΔARE mice could be activated by antibodies to CD3/CD28. Importantly, TTPΔARE antigen presenting cells were significantly less efficient compared to WT in priming naïve T cells, suggesting that this feature plays a major role in the dampened immune responses of the TTPΔARE mice. Our observations demonstrate that elevated systemic levels of TTP can inhibit the pathogenic processes involved in EAU, and suggest the possible use of TTP-based treatments in humans with uveitis and other autoimmune conditions.

Microbiota as Drivers and as Therapeutic Targets in Ocular and Tissue Specific Autoimmunity.

Autoimmune uveitis is a major cause of blindness in humans. Activation of retina-specific autoreactive T cells by commensal microbiota has been shown to trigger uveitis in mice. Although a culprit microbe and/or its immunogenic antigen remains to be identified, studies from inducible and spontaneous mouse models suggest the potential of microbiota-modulating therapies for treating ocular autoimmune disease. In this review, we summarize recent findings on the contribution of microbiota to T cell-driven, tissue-specific autoimmunity, with an emphasis on autoimmune uveitis, and analyze microbiota-altering interventions, including antibiotics, probiotics, and microbiota-derived metabolites (e.g., short-chain fatty acids), which have been shown to be effective in other autoimmune diseases. We also discuss the need to explore more translational animal models as well as to integrate various datasets (microbiomic, transcriptomic, proteomic, metabolomic, and other cellular measurements) to gain a better understanding of how microbiota can directly or indirectly modulate the immune system and contribute to the onset of disease. It is hoped that deeper understanding of these interactions may lead to more effective treatment interventions.

AS101 ameliorates experimental autoimmune uveitis by regulating Th1 and Th17 responses and inducing Treg cells.

AS101 is an organotellurium compound with multifaceted immunoregulatory properties that is remarkable for its lack of toxicity. We tested the therapeutic effect of AS101 in experimental autoimmune uveitis (EAU), a model for human autoimmune uveitis. Unexpectedly, treatment with AS101 elicited Treg generation in vivo in otherwise unmanipulated mice. Mice immunized for EAU with the retinal antigen IRBP and treated with AS101 developed attenuated disease, as did AS101-treated recipients of retina-specific T cells activated in vitro. In both settings, eye-infiltrating effector T cells were decreased, whereas regulatory T (Treg) cells in the spleen were increased. Mechanistic studies in vitro revealed that AS101 restricted polarization of retina-specific T cells towards Th1 or Th17 lineage by repressing activation of their respective lineage-specific transcription factors and downstream signals. Retina-specific T cells polarized in vitro towards Th1 or Th17 in the presence of AS101 had impaired ability to induce EAU in naïve recipients. Finally, AS101 promoted differentiation of retina-specific T cells to Tregs in vitro independently of TGF-ß. We conclude that AS101 modulates autoimmune T cells by inhibiting acquisition and expression of effector function and by promoting Treg generation, and suggest that AS101 could be useful as a therapeutic approach for autoimmune uveitis.

Microbiome and Autoimmune Uveitis.

Commensal microbes affect all aspects of immune development and homeostasis in health and disease. Increasing evidence points to the notion that the gut commensals impact not only intestinal diseases but also diseases in tissues distant from the gut. Autoimmune or non-infectious uveitis is a sight-threatening intraocular inflammation that affects the neuroretina. It is strongly T cell driven, but the precise causative mechanisms are not fully understood. We and others observed that depletion of gut microbiota in animal models of uveitis attenuated disease. Using a spontaneous model of the disease, we questioned how retina-specific uveitogenic T cells are primed when their cognate antigens are sequestered within the immune privileged eye. The data suggested that gut commensals provide a signal directly through the retina-specific T cell receptor and cause these autoreactive T cells to trigger uveitis. This activation of retina-specific T cells in the gut appears to be independent of the endogenous retinal antigen. Rather, the findings point to the notion that gut microbiota may mimic retinal antigen(s), however, the actual mimic has not yet been identified. Microbiota may also serve as an "adjuvant" providing innate signals that amplify and direct the host immune response for development of uveitis. In contrast, spontaneous uveitis that develops in AIRE-/- mice appears to be independent of gut microbiota. To date, available data on human microbiota in association with uveitis are very limited and causative relationships are difficult to establish. This review will summarize the current knowledge on the role of microbiome in uveitis and its underlying mechanisms, and discuss unresolved questions and issues in an attempt to explore the concept of gut-retina axis.

STAT-3-independent production of IL-17 by mouse innate-like αß T cells controls ocular infection.

Appropriate regulation of IL-17 production in the host can mean the difference between effective control of pathogens and uncontrolled inflammation that causes tissue damage. Investigation of conventional CD4+ T cells (Th17 cells) has yielded invaluable insights into IL-17 function and its regulation. More recently, we and others reported production of IL-17 from innate αß+ T cell populations, which was shown to occur primarily via IL-23R signaling through the transcription factor STAT-3. In our current study, we identify promyelocytic leukemia zinc finger (PLZF)-expressing iNKT, CD4-/CD8+, and CD4-/CD8- (DN) αß+T cells, which produce IL-17 in response to TCR and IL-1 receptor ligation independently of STAT-3 signaling. Notably, this noncanonical pathway of IL-17 production may be important in mucosal defense and is by itself sufficient to control pathogenic Staphylococcus aureus infection at the ocular surface.

Gut microbiota as a source of a surrogate antigen that triggers autoimmunity in an immune privileged site.

Recent discoveries on the role of commensal microbiota have significantly changed our understanding of human physiology. The host-microbiota interplay is now an important aspect to take into account to understand immune responses and immunological diseases. Autoimmune uveitis is a sight-threatening disease that arises without a known infectious etiology. It is unknown where and how autoreactive T cells become primed to trigger disease in the eye, which is an immune privileged site. We recently reported data supporting the notion that retina-specific T cells receive a signal in the gut from commensal microbiota-derived cross-reactive antigen(s) and trigger autoimmune uveitis in the R161H mouse model. Here we discuss our published findings, as well as our recent attempts to identify the responsible microbe(s) by using different antibiotic treatments, 16S rDNA sequencing and homology searches for candidate antigenic mimic(s) of the retinal antigen.

Regulation of Autoimmunity by the Microbiome.

Intestinal microbes have profound effects on inflammatory autoimmunity in sites distant from the gut. The mechanisms whereby this happens are only now beginning to be understood and may include such diverse effects as innate stimulation of migrating immune cells and effects of circulating bacterial metabolites. Our studies add to this the demonstration that microbiota may provide a source of cross-reactive antigenic material that activates autoreactive lymphocytes within the gut environment. In a spontaneous model of autoimmune uveitis, T lymphocytes specific to a retinal autoantigen are activated through their specific antigen receptor in the gut and acquire the ability to fuel inflammatory autoimmunity in the eye. In view of the huge diversity of commensals, it is conceivable that they may provide surrogate antigens for activation of autoreactive lymphocytes(s) of other tissue specificities, and might therefore be involved in the etiology of autoimmune diseases more frequently than is currently appreciated.

Preparation of Protein-containing Extracts from Microbiota-rich Intestinal Contents.

The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). Recently, we showed that protein extracts from gut commensal microbiota can activate retina-specific T cells, allowing these autoreactive T cells to then break through the blood-retinal barrier and trigger autoimmune uveitis in the recipient (Horai et al., 2015). The protocol below describes the method to prepare intestinal protein-rich extracts that can be used in various in vitro andin vivo immunological studies.

Tertiary Lymphoid Tissue Forms in Retinas of Mice with Spontaneous Autoimmune Uveitis and Has Consequences on Visual Function.

During chronic inflammation, tertiary lymphoid tissue (TLT) can form within an inflamed organ, including the CNS. However, little is known about TLT formation in the neuroretina. In a novel spontaneous autoimmune mouse model of uveitis (R161H), we identified well-organized lymphoid aggregates in the retina and examined them for TLT characteristics. Presence of immune cells, tissue-specific markers, and gene expression patterns typically associated with germinal centers and T follicular helper cells were examined using immunohistochemistry and gene analysis of laser capture microdissected retina. Our data revealed the retinal lymphoid structures contained CD4(+) T cells and B cells in well-defined zonal areas that expressed classic germinal center markers, peanut lectin (agglutinin) and GL-7. Gene expression analysis showed upregulation of T follicular helper cell markers, most notably CXCR5 and its ligand CXCL13, and immunohistochemical analysis confirmed CXCR5 expression, typically associated with CD4(+) T follicular helper cells. Highly organized stromal cell networks, a hallmark of organized lymphoid tissue, were also present. Positive staining for phospho-Zap70 in retina-specific T cells indicated CD4(+) T cells were being activated within these lymphoid structures. CD138(+)/B220(+) plasma cells were detected, suggesting the retinal lymphoid aggregates give rise to functional germinal centers, which produce Abs. Interestingly, eyes with lymphoid aggregates exhibited lower inflammatory scores by fundus examination and a slower initial rate of loss of visual function by electroretinography, compared with eyes without these structures. Our findings suggest that the lymphoid aggregates in the retina of R161H mice represent organized TLT, which impact the course of chronic uveitis.

Microbiota-Dependent Activation of an Autoreactive T Cell Receptor Provokes Autoimmunity in an Immunologically Privileged Site.

Activated retina-specific T cells that have acquired the ability to break through the blood-retinal barrier are thought to be causally involved in autoimmune uveitis, a major cause of human blindness. It is unclear where these autoreactive T cells first become activated, given that their cognate antigens are sequestered within the immune-privileged eye. We demonstrate in a novel mouse model of spontaneous uveitis that activation of retina-specific T cells is dependent on gut commensal microbiota. Retina-specific T cell activation involved signaling through the autoreactive T cell receptor (TCR) in response to non-cognate antigen in the intestine and was independent of the endogenous retinal autoantigen. Our findings not only have implications for the etiology of human uveitis, but also raise the possibility that activation of autoreactive TCRs by commensal microbes might be a more common trigger of autoimmune diseases than is currently appreciated.

IL-1 receptor type 2 suppresses collagen-induced arthritis by inhibiting IL-1 signal on macrophages.

IL-1α and IL-1ß (in this article referred to as IL-1) play important roles in host defense against infection and inflammatory diseases. IL-1R1 is the receptor for IL-1, and IL-1R2 is suggested to be a decoy receptor, because it lacks the signal-transducing TIR domain in the cytoplasmic part. However, the roles of IL-1R2 in health and disease remain largely unknown. In this study, we generated EGFP-knock-in Il1r2(-/-) mice and showed that they were highly susceptible to collagen-induced arthritis, an animal model for rheumatoid arthritis in which the expression of IL-1R2 is augmented in inflammatory joints. Il1r2 was highly expressed in neutrophils but had only low expression in other cells, including monocytes and macrophages. Ab production and T cell responses against type II collagen were normal in Il1r2(-/-) mice. Despite the high expression in neutrophils, no effects of Il1r2 deficiency were observed; however, we found that production of inflammatory mediators in response to IL-1 was greatly enhanced in Il1r2(-/-) macrophages. These results suggest that IL-1R2 is an important regulator of arthritis by acting specifically on macrophages as a decoy receptor for IL-1.