Draft Reference Genome Sequence of Corynebacterium mastitidis RC, an Ocular Commensal, Isolated from Mouse Conjunctiva.

Here, we report the genome sequence of a protective commensal, Corynebacterium mastitidis RC, isolated from mouse conjunctiva. The C. mastitidis RC genome sequence is 2,153,054 bp in size and 96.95% complete, and we believe that it can contribute to the understanding of the functional immune attributes of the ocular commensal microbiome.

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.

Opportunities to Improve Antibiotic Appropriateness in U.S. ICUs: A Multicenter Evaluation.

OBJECTIVES: To use a standardized tool for a multicenter assessment of antibiotic appropriateness in ICUs and identify local antibiotic stewardship improvement opportunities. DESIGN: Pilot point prevalence conducted on October 5, 2016; point prevalence survey conducted on March 1, 2017. SETTING: ICUs in 12 U.S. acute care hospitals with median bed size 563. PATIENTS: Receiving antibiotics on participating units on March 1, 2017. INTERVENTIONS: The Centers for Disease Control and Prevention tool for the Assessment of Appropriateness of Inpatient Antibiotics was made actionable by an expert antibiotic stewardship panel and implemented across hospitals. Data were collected by antibiotic stewardship program personnel at each hospital, deidentified and submitted in aggregate for benchmarking. hospital personnel identified most salient reasons for inappropriate use by category and agent. MEASUREMENTS AND MAIN RESULTS: Forty-seven ICUs participated. Most hospitals (83%) identified as teaching with median licensed ICU beds of 70. On March 1, 2017, 362 (54%) of 667 ICU patients were on antibiotics (range, 8-81 patients); of these, 112 (31%) were identified as inappropriate and administered greater than 72 hours among all 12 hospitals (range, 9-82%). Prophylactic antibiotic regimens and PICU patients demonstrated a statistically significant risk ratio of 1.76 and 1.90 for inappropriate treatment, respectively. Reasons for inappropriate use included unnecessarily broad spectrum (29%), no infection or nonbacterial syndrome (22%), and duration longer than necessary (21%). Of patients on inappropriate antibiotic therapy in surgical ICUs, a statistically significant risk ratio of 2.59 was calculated for noninfectious or nonbacterial reasons for inappropriate therapy. CONCLUSIONS: In this multicenter point prevalence study, 31% of ICU antibiotic regimens were inappropriate; prophylactic regimens were often inappropriate across different ICU types, particularly in surgical ICUs. Engaging intensivists in antibiotic stewardship program efforts is crucial to sustain the efficacy of antibiotics and quality of infectious diseases care in critical care settings. This study underscores the value of standardized assessment tools and benchmarking to be shared with local leaders for targeted antibiotic stewardship program interventions.

Interleukin 22 ameliorates neuropathology and protects from central nervous system autoimmunity.

IL-22 has opposing effects in different tissues, from pro-inflammatory (skin, joints) to protective (liver, intestine) but little is known about its effects on neuroinflammation. We examined the effect of IL-22 on retinal tissue by using the model of experimental autoimmune uveitis (EAU) in IL-22-/- mice, as well as by intraocular injections of recombinant IL-22 or anti-IL-22 antibodies in wild type animals. During EAU, IL-22 was produced in the eye by CD4+ eye-infiltrating T cells. EAU-challenged IL-22-/- mice, as well as WT mice treated systemically or intraocularly with anti-IL-22 antibodies during the expression phase of disease, developed exacerbated retinal damage. Furthermore, IL-22-/- mice were more susceptible than WT controls to glutamate-induced neurotoxicity, whereas local IL-22 supplementation was protective, suggesting direct or indirect neuroprotective effects. Mechanistic studies revealed that retinal glial Müller cells express IL-22rα1 in vivo, and in vitro IL-22 enhanced their ability to suppress proliferation of effector T cells. Finally, IL-22 injected into the eye concurrently with IL-1, inhibited the (IL-1-induced) expression of multiple proinflammatory and proapoptotic genes in retinal tissue. These findings suggest that IL-22 can function locally within the retina to reduce inflammatory damage and provide neuroprotection by affecting multiple molecular and cellular pathways.

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.

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.

An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells.

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

Mincle Activation and the Syk/Card9 Signaling Axis Are Central to the Development of Autoimmune Disease of the Eye.

Uveitis, which occurs in association with systemic immunological diseases, presents a considerable medical challenge because of incomplete understanding of its pathogenesis. The signals that initiate T cells to target the eye, which may be of infectious or noninfectious origin, are poorly understood. Experimental autoimmune uveoretinitis (EAU) develops in mice immunized with the endogenous retinal protein interphotoreceptor retinoid binding protein in the presence of the adjuvant CFA. EAU manifests as posterior ocular inflammation consisting of vasculitis, granulomas, retinal damage, and invasion of self-reactive T cells, which are key clinical features of human uveitis. Our studies uncover Card9 as a critical genetic determinant for EAU. Card9 was responsible for Th17 polarization and Th17-associated Ag-specific responses, but not Th1-associated responses. Nonetheless, Card9 expression was essential for accumulation of both lineages within the eye. Consistent with its recently identified role as an intracellular signaling mediator for C-type lectin receptors (CLRs), a Card9-dependent transcriptional response in the neuroretina was observed involving genes encoding the CLRs Dectin-1, Dectin-2, and Mincle. Genetic deletion of these individual CLRs revealed an essential role for Mincle. Mincle activation was sufficient to generate the EAU phenotype, and this required activation of both Syk and Card9. In contrast, Dectin-1 contributed minimally and a possible repressive role was shown for Dectin-2. These findings extend our understanding of CLRs in autoimmune uveitis. The newly identified role of Mincle and Syk/Card9-coupled signaling axis in autoimmune uveitis could provide novel targets for treatment of patients with ocular inflammatory disease.

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.

NK-DC crosstalk controls the autopathogenic Th17 response through an innate IFN-γ-IL-27 axis.

IFN-γ is a pathogenic cytokine involved in inflammation. Paradoxically, its deficiency exacerbates experimental autoimmune encephalomyelitis, uveitis, and arthritis. Here, we demonstrate using IFN-γ(-/-) mice repleted with IFN-γ +/+: NK cells that innate production of IFN-γ from NK cells is necessary and sufficient to trigger an endogenous regulatory circuit that limits autoimmunity. After immunization, DCs recruited IFN-γ-producing NK cells to the draining lymph node and interacted with them in a CXCR3-dependent fashion. The interaction caused DCs to produce IL-27, which in turn enhanced IFN-γ production by NK cells, forming a self-amplifying positive feedback loop. IL-10, produced by the interacting cells themselves, was able to limit this process. The NK-DC-dependent IL-27 inhibited development of the adaptive pathogenic IL-17 response and induced IL-10-producing Tr1-like cells, which ameliorated disease in an IL-10-dependent manner. Our data reveal that an early NK-DC interaction controls the adaptive Th17 response and limits tissue-specific autoimmunity through an innate IFN-γ-IL-27 axis.

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.

Characterization of a New Epitope of IRBP That Induces Moderate to Severe Uveoretinitis in Mice With H-2b Haplotype.

PURPOSE: Experimental autoimmune uveitis (EAU) induced in mice using the retinal antigen interphotoreceptor retinoid binding protein (IRBP) is an animal model for posterior uveitis in humans. However, EAU induced by native IRBP protein or its widely used epitope amino acid residues 1 to 20 of human IRBP (hIRBP1-20) is inconsistent, often showing low scores and incidence. We found an urgent need to identify a better pathogenic epitope for the C57BL/6 strain. METHODS: Mice were immunized with uveitogenic peptides or with native bovine IRBP. Clinical and histological disease and associated immunological responses were evaluated. Truncated and substituted peptides, as well as bioinformatic analyses, were used to identify critical major histocompatibility complex (MHC)/T cell receptor (TCR) contact residues and the minimal core epitope. RESULTS: The new uveitogenic epitope of IRBP, amino acid residues 651 to 670 of human IRBP (LAQGAYRTAVDLESLASQLT [hIRBP651-670]) is uveitogenic for mice of the H-2b haplotype and elicits EAU with a higher severity and incidence in C57BL/6 mice than the previously characterized hIRBP1-20 epitope. Using truncated and substituted peptides, as well as bioinformatic analysis, we identified the critical contact residues with MHC/TCR and defined the minimal core epitope. This made it possible to design MHC tetramers and use them to detect epitope-specific T cells in the uveitic eye and in lymphoid organs of hIRBP651-670-immunized mice. CONCLUSIONS: Data suggest that hIRBP651-670 is an epitope naturally processed from a conserved region of native IRBP, potentially explaining its relatively high uveitogenicity. This epitope should be useful for basic and preclinical studies of uveitis in the C57BL/6 model and gives access to genetically engineered mice available on this background.

Retina-specific T regulatory cells bring about resolution and maintain remission of autoimmune uveitis.

Experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal Ag interphotoreceptor retinoid-binding protein (IRBP) is a model of human autoimmune uveitis. We examined whether T regulatory cells (Tregs) found in uveitic eyes are IRBP specific, functionally suppressive, and play a role in natural resolution of disease and in maintenance of remission. Progressive increase of Foxp3(+) Treg to T effector cell (Teff) ratio in uveitic eyes correlated with resolution of disease. At peak disease, up to 20% of Tregs (CD4(+)Foxp3(+)) and up to 60% of Teffs (CD4(+)Foxp3(-)) were IRBP specific, whereas in lymphoid organs retina-specific T cells were undetectable. Tregs isolated from eyes of mice with EAU efficiently suppressed IRBP-specific responses of Teffs from the same eyes. Importantly, systemic depletion of Tregs at peak disease delayed resolution of EAU, and their depletion after resolution triggered a relapse. This could be partially duplicated by depletion of Tregs locally within the eye. Thus, the T cell infiltrate in uveitic eyes of normal mice with a polyclonal T cell repertoire is highly enriched in IRBP-specific Tregs and Teffs. Unlike what has been reported for Tregs in other inflammatory sites, Tregs from uveitic eyes appear unimpaired functionally. Finally, Foxp3(+) Tregs play a role in the natural resolution of uveitis and in the maintenance of remission, which occurs at least in part through an effect that is local to the eye.

IL-27p28 inhibits central nervous system autoimmunity by concurrently antagonizing Th1 and Th17 responses.

Central nervous system (CNS) autoimmunity such as uveitis and multiple sclerosis is accompanied by Th1 and Th17 responses. In their corresponding animal models, experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE), both responses are induced and can drive disease independently. Because immune responses have inherent plasticity, therapeutic targeting of only one pathway could promote the other, without reducing pathology. IL-27p28 antagonizes gp130, required for signaling by IL-27 and IL-6, which respectively promote Th1 and Th17 responses. We therefore examined its ability to protect the CNS by concurrently targeting both effector responses. Overexpression of IL-27p28 in vivo ameliorated EAU as well as EAE pathology and reduced tissue infiltration by Th1 and Th17 cells in a disease prevention, as well as in a disease reversal protocol. Mechanistic studies revealed inhibition of Th1 and Th17 commitment in vitro and decreased lineage stability of pre-formed effectors in vivo, with reduction in expression of gp130-dependent transcription factors and cytokines. Importantly, IL-27p28 inhibited polarization of human T cells to the Th1 and Th17 effector pathways. The ability of IL-27p28 to inhibit generation as well as function of pathogenic Th1 and Th17 effector cells has therapeutic implications for controlling immunologically complex autoimmune diseases.

Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen.

Despite presence of circulating retina-specific T cells in healthy individuals, ocular immune privilege usually averts development of autoimmune uveitis. To study the breakdown of immune privilege and development of disease, we generated transgenic (Tg) mice that express a T cell receptor (TCR) specific for interphotoreceptor retinoid-binding protein (IRBP), which serves as an autoimmune target in uveitis induced by immunization. Three lines of TCR Tg mice, with different levels of expression of the transgenic R161 TCR and different proportions of IRBP-specific CD4⁺ T cells in their peripheral repertoire, were successfully established. Importantly, two of the lines rapidly developed spontaneous uveitis, reaching 100% incidence by 2 and 3 months of age, respectively, whereas the third appeared "poised" and only developed appreciable disease upon immune perturbation. Susceptibility roughly paralleled expression of the R161 TCR. In all three lines, peripheral CD4⁺ T cells displayed a naïve phenotype, but proliferated in vitro in response to IRBP and elicited uveitis upon adoptive transfer. In contrast, CD4⁺ T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells that appeared to have been peripherally converted from conventional CD4⁺ T cells rather than thymically derived. Thus, R161 mice provide a new and valuable model of spontaneous autoimmune disease that circumvents the limitations of active immunization and adjuvants, and allows to study basic mechanisms involved in maintenance and breakdown of immune homeostasis affecting immunologically privileged sites such as the eye.

Rodent models of experimental autoimmune uveitis.

The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproducible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from retinal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study of basic mechanisms as well as in clinically relevant interventions.

The living eye "disarms" uncommitted autoreactive T cells by converting them to Foxp3(+) regulatory cells following local antigen recognition.

Immune privilege is used by the eye, brain, reproductive organs, and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable and, therefore, also the most "privileged" of tissues; paradoxically, it remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using Foxp3-GFP reporter mice expressing a retina-specific TCR, we now show that uncommitted T cells rapidly convert in the living eye to Foxp3(+) Tregs in a process involving retinal Ag recognition, de novo Foxp3 induction, and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye because of its function in the chemistry of vision. Nonconverted T cells showed evidence of priming but appeared restricted from expressing effector function in the eye. Pre-existing ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment and, instead, caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue.

Inhibition of experimental auto-immune uveitis by the A3 adenosine receptor agonist CF101.

Uveitis is an inflammation of the middle layer of the eye with a high risk of blindness. The Gi protein associated A3 adenosine receptor (A3AR) is highly expressed in inflammatory cells whereas low expression is found in normal cells. CF101 is a highly specific agonist at the A3AR known to induce a robust anti-inflammatory effect in different experimental animal models. The CF101 mechanism of action entails down-regulation of the NF-κB-TNF-α signaling pathway, resulting in inhibition of pro-inflammatory cytokine production and apoptosis of inflammatory cells. In this study the effect of CF101 on the development of retinal antigen interphotoreceptor retinoid-binding protein (IRBP)-induced experimental autoimmune uveitis (EAU) was assessed. Oral treatment with CF101 (10 µg/kg, twice daily), initiated upon disease onset, improved uveitis clinical score measured by fundoscopy and ameliorated the pathological manifestations of the disease. Shortly after treatment with CF101 A3AR expression levels were down-regulated in the lymph node and spleen cells pointing towards receptor activation. Downstream events included a decrease in PI3K and STAT-1 and proliferation inhibition of IRPB auto-reactive T cells ex vivo. Inhibition of interleukin-2, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production and up-regulation of interleukin-10 was found in cultured splenocytes derived from CF101-treated animals. Overall, the present study data point towards a marked anti-inflammatory effect of CF101 in EAU and support further exploration of this small molecule drug for the treatment of uveitis.