Induction Of Experimental Autoimmune Uveitis Research Articles

Interleukin-17 receptor C is essential for the pro-inflammatory pathogenicity of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in experimental autoimmune uveitis

Autoimmune uveitis is an inflammatory disorder of the eye triggered by the responses of autoreactive T cells to ocular autoantigens. This study aims to understand the role of granulocyte–macrophage-colony-stimulating factor (GM-CSF)-producing T helper (ThGM) cells in the pathophysiology of mouse experimental autoimmune uveitis (EAU). We established an EAU model by immunizing mice with interphotoreceptor retinoid-binding protein (IRBP) 651–670. Splenic or eye-infiltrating ThGM cells were analyzed and enriched by flow cytometry according to the levels of an array of surface markers, transcription factors, and cytokines. Lentiviral transduction was conducted to silence or overexpress the target gene in differentiated ThGM cells. The adoptive transfer was applied to determine the pathogenicity of ThGM cells in vivo. We found that ThGM cells were present in the spleen and the eye after EAU induction. Both splenic and eye-infiltrating ThGM cells were phenotypically CD4+CCR7-CXCR3-CCR6-CCR10hi. Eye-infiltrating ThGM cells up-regulated interleukin-1β (IL-1β), interleukin-6 (IL-6), and IL-17 receptor C (IL-17RC) relative to splenic ThGM cells. IL-17RC overexpression enabled interleukin-17A (IL-17A)-induced up-regulation of IL-1β and IL-6 production in ThGM cells. Adoptive transfer of IL-17RC overexpressing ThGM cells exacerbated EAU severity, as evidenced by a higher histology score as well as increased pro-inflammatory cytokines and inflammatory cells in the eye. However, IL-17RC-silenced ThGM cells did not impact EAU. Therefore, for the first time, this study unveils the essential pro-inflammatory role of IL-17RC-expressing ThGM cells in EAU pathophysiology. We discovered a novel mechanism underlying the pathophysiology of autoimmune uveitis.

Towards molecular mechanisms for pathogenic Th17 cell differentiation in ocular autoimmunity

Abstract CD4+ T helper (Th) cells play critical roles in coordinating proper immune responses by differentiating into effector subtypes specialized against different types of pathogens, while dysregulated Th cells can drive autoimmune disease by triggering inappropriate immune responses. Pathogenic Th17 (pTh17) cells, which combine behaviors of classic Th1 and Th17 effector cells by producing both interferon-gamma (IFNg) and interleukin-17A (IL17A), have been implicated in autoimmune diseases. Our goal is to map regulatory networks and identify novel regulators underlying pTh17 differentiation and activation using the murine model experimental autoimmune uveitis (EAU), in which ocular autoimmunity is driven by pTh17 cells. To track pTh17 differentiation, we bred a double reporter mouse with IL17A-cre/tdTomato-flox tracer and IFNg-YFP. We sorted both Tomato+YFP+ and Tomato+YFP- CD4+ T cells from eyes and spleens after EAU induction for single-cell RNA-seq. Clustering of single-cell transcriptomes revealed distinct transcriptomes between eyes and spleen. In the eye, we identified clusters similar to pTh17 and non-pathogenic (np)Th17 populations defined in a myelin autoimmunity model. The pTh17 cluster contained both double positive and Tomato single positive cells, suggesting a heterogeneous pathogenic population. In contrast, the npTh17 cluster only contained single positives. Differential gene expression analysis showed that pathogenic and non-pathogenic clusters utilize distinct sets of transcription factors. We are further characterizing epigenetic differences between pTh17 and npTh17 to better understand how distinct regulatory networks shape pTh17 differentiation. Supported by the NEI Intramural Research Program fund (ZIAEY000569-01)

Optimization of determinant factors associated with the efficiency of experimental autoimmune uveitis induction in C57BL/6 mice

Experimental autoimmune uveitis (EAU) is a widely used animal model for uveitis research. The C57BL/6 mouse strain is the most commonly used mouse strain in the research of genetic modification, but C57BL/6 mice are not sufficiently susceptible to EAU induction, partly due to experimental factors. This work aims to optimize relevant factors to improve the efficiency of EAU induction in C57BL/6 mice. To induce EAU, mice were immunized via intraperitoneal injection with pertussis (PTX) and subcutaneous injection with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified with complete Freund's adjuvant (CFA). The severity of inflammation was assessed using several approaches. The relevant experimental factors were evaluated, including methods of emulsification and doses of peptide and PTX. Uveitis occurred at 8-12 days after immunization and reached its peak at 18-20 days, while T helper type 17 (Th17) cells peaked earlier at 14-18 days after immunization. Based on clinical and histological scores, 500 µg of IRBP peptide was the optimal dose required to induce EAU. The PTX dose demonstrated no influence on EAU incidence, but potentially affected the severity of uveitis. A single injection of 1,000 ng of PTX induced the most severe EAU and the highest proportion of Th17 cells. Compared to extruded emulsion, sonicated emulsion produced a higher incidence, higher histological score, and a 2-day-earlier onset of EAU. Electron microscopy showed a significantly different microstructure between the 2 emulsions. This work optimized the protocols of EAU induction and obtained a high and stable induction rate with severe inflammation in the C57BL/6 mouse strain. Our results facilitate future experimental research involving uveitis.

Inhibition of experimental autoimmune uveitis by intravitreal AAV-Equine-IL10 gene therapy.

Equine recurrent uveitis (ERU) is a spontaneous, painful, and vision threatening disease affecting up to 25% of equine populations worldwide. Current treatments of ERU are non-specific and have many side effects which limits them to short-term use. In order to develop an effective therapy for ERU, we investigated the use of adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by equine interleukin-10 (Equine-IL10). The purpose of this study was to evaluate the therapeutic efficacy of a single intravitreal (IVT) dose of AAV8-Equine-IL10 gene therapy for inhibition of experimental autoimmune uveitis (EAU) in rats. Each rat was dosed intravitreally (IVT) in both eyes with either balanced salt solution (BSS) (control; n = 4), AAV8-Equine-IL10 at a low dose (2.4x109 vg; n = 5) or high dose (2.4x1010 vg; n = 5). EAU was induced in all groups of rats 7 days after IVT injections and euthanized 21 days post-injection. Ophthalmic examination and aqueous humor (AH) cell counts were recorded with the observer blinded to the treatment groups. Histopathology and qPCR were performed on selected ocular tissues. Data presented herein demonstrate that AAV8-Equine-IL10 treated rats exhibited a significant decrease in clinical inflammatory scores and AH cell counts compared to BSS-treated EAU eyes on days 10, 12 and 14 post EAU induction at both administered vector doses. Mean cellular histologic infiltrative scores were also significantly less in AAV8-Equine-IL10 dosed rats compared to the BSS group. Intravitreal injection of AAV8-Equine-IL10 resulted in Equine-IL10 cDNA expression in the ciliary body, retina, cornea, and optic nerve in a dose-dependent manner. A single IVT injection of AAV8-Equine-IL10 appeared to be well-tolerated and inhibited EAU even at the lowest administered dose. These results demonstrate safety and efficacy of AAV8-Equine-IL10 to prevent EAU and support continued exploration of AAV gene therapy for the treatment of equine and perhaps human recurrent uveitis.

Melatonin, an endogenous hormone, modulates Th17 cells via the reactive-oxygen species/TXNIP/HIF-1α axis to alleviate autoimmune uveitis

BackgroundMelatonin, an indoleamine produced by the pineal gland, plays a pivotal role in maintaining circadian rhythm homeostasis. Recently, the strong antioxidant and anti-inflammatory properties of melatonin have attracted attention of researchers. We evaluated the therapeutic efficacy of melatonin in experimental autoimmune uveitis (EAU), which is a representative animal model of human autoimmune uveitis.MethodsEAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 1–20 (IRBP1–20). Melatonin was then administered via intraperitoneal injection to induce protection against EAU. With EAU induction for 14 days, clinical and histopathological scores were graded to evaluate the disease progression. T lymphocytes accumulation and the expression of inflammatory cytokines in the retinas were assessed via flow cytometry and RT-PCR, respectively. T helper 1 (Th1), T helper 17 (Th17), and regulatory T (Treg) cells were detected via flow cytometry for both in vivo and in vitro experiments. Reactive-oxygen species (ROS) from CD4 + T cells was tested via flow cytometry. The expression of thioredoxin-interacting protein (TXNIP) and hypoxia-inducible factor 1 alpha (HIF-1α) proteins were quantified via western blot.ResultsMelatonin treatment resulted in notable attenuation of ocular inflammation in EAU mice, evidenced by decreasing optic disc edema, few signs of retinal vasculitis, and minimal retinal and choroidal infiltrates. Mechanistic studies revealed that melatonin restricted the proliferation of peripheral Th1 and Th17 cells by suppressing their transcription factors and potentiated Treg cells. In vitro studies corroborated that melatonin restrained the polarization of retina-specific T cells towards Th17 and Th1 cells in addition to enhancing the proportion of Treg cells. Pretreatment of retina-specific T cells with melatonin failed to induce EAU in naïve recipients. Furthermore, the ROS/ TXNIP/ HIF-1α pathway was shown to mediate the therapeutic effect of melatonin in EAU.ConclusionsMelatonin regulates autoimmune T cells by restraining effector T cells and facilitating Treg generation, indicating that melatonin could be a hopeful treatment alternative for autoimmune uveitis.

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneousinjection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund's Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

CD47 Deficiency Ameliorates Ocular Autoimmune Inflammation.

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. The CD47 is a ubiquitously expressed transmembrane protein which plays multiple roles in fundamental cellular functions including phagocytosis, proliferation, and adhesion. Signal regulatory protein alpha (SIRPα), one of the CD47 ligands, is predominantly expressed in myeloid lineage cells such as dendritic cells (DCs) or macrophages, and CD47-SIRPα signaling pathway is implicated in the development of autoimmune diseases. Our current study demonstrates how CD47 depletion is effective in the prevention of experimental autoimmune uveitis (EAU), an animal model of human autoimmune uveitis, in animals deficient of CD47 (CD47-/-). Systemic suppression of SIRPα+ DCs in animals deficient in CD47 resulted in the inability of autoreactive CD4+ T cells to develop, which is crucial to induction of EAU. Of interest, retinal microglia, the resident immune cell of the retina, express SIRPα, however these cells were not operative in EAU suppression in response to CD47 depletion. These results identify CD47 as a significant regulator in the development of SIRPα+ DCs that is vital to disease induction in EAU.

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.

Longdan Xiegan Decoction alleviates experimental autoimmune uveitis in rats by inhibiting Notch signaling pathway activation and Th17 cell differentiation

This study aimed to investigate the dynamic effects of the traditional Chinese medicine compound Longdan Xiegan Decoction (LXD) on the inhibition of Notch signaling pathway activation and T helper (Th) cell differentiation in rats with experimental autoimmune uveitis (EAU). Based on a network pharmacology strategy, we conducted protein interaction network analysis to construct an active ingredient-disease treatment network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were further used to screen out the possible signaling pathways regulated by LXD in the treatment of uveitis. In the subsequent functional studies, we established an EAU rat model and investigated the regulatory role of LXD in the Notch signaling pathway and Th cell differentiation in rats with EAU. Female Lewis rats were randomly divided into a normal control (NC) group, an EAU group, and an LXD group. After the induction of EAU, the ocular inflammation and pathological changes in the rats in each group were observed; for documentation, a scanning laser ophthalmoscope (SLO) was used to observe fundus inflammation on day 12 after immunization. Additionally, quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of Notch1, DLL4, IL-10 and IL-17A in the spleen, lymph nodes and ocular tissues of each group at 0, 6, 9, 12, 15 and 18 days after immunization. In addition, the dynamic frequencies of the CD4+, CD8+, Th17 and Treg cell subsets in the spleen, lymph nodes and ocular tissues were measured by flow cytometry. We found that the Notch signaling pathway was activated and the Th17 frequency was elevated in rats with EAU, leading to disrupted CD4+/CD8+ and Th17/Treg balance. The expression of Notch1, DLL4 and IL-17 mRNA and proteins in the EAU and LXD groups reached a peak on day 12, and then gradually decreased (all P < 0.05), and the ratios of the CD4+/CD8+ and Th17/Treg also peaked on day 12. However, after treatment with LXD, the expression of Notch1, DLL4 and IL-17 mRNA and proteins was significantly decreased (all P < 0.05), and the CD4+/CD8+ and Th17/Treg ratios significantly gradually returns to balance. LXD can efficiently inhibit Th17 cell differentiation, decrease inflammatory cytokine expression, and restore the CD4+/CD8+ and Th17/Treg balance by inhibiting the activation of the Notch signaling pathway in rats with EAU, thus effectively alleviating eye inflammation, protecting eye tissue structures, and positively regulating the immune state of the whole body and the intraocular microenvironment.

Cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis on murine photoreceptor cells.

To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis (EAU) as well as their secreted interferon (IFN)-γ and interleukin (IL)-17A on murine photoreceptor (661W) cells. An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein (IRBP) emulsion supplemented with complete Freund's adjuvant (CFA) and Mycobacterium tuberculosis (TB). On day 12 after induction of EAU, specific T cells from spleen and lymph node tissues were isolated and cultured for 4d and the levels of IFN-γ and IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). T cells and their supernatants were added to 661W cells to observe the alteration of cell morphology; IFN-γ and IL-17A were separately added to 661W cells to observe the effect of IFN-γ and IL-17A on cell proliferation. The levels of IFN-γ and IL-17A in the T cell supernatants were 1568.64±38.79 pg/mL and 1456.57±46.98 pg/mL, respectively. The supernatants apparently inhibited 661W cell proliferation (P<0.05). T cells could also attach to the surface of 661W cells, and IFN-γ showed a more serious cytotoxic effect on 661W cells than IL-17A, inhibiting cell proliferation (P<0.01). IFN-γ and IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation, and IFN-γ shows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.

Activation of the interleukin-23/interleukin-17 signalling pathway in autoinflammatory and autoimmune uveitis.

Uveitis is a group of diseases characterized by intraocular inflammation, of which some are driven by autoinflammatory or autoimmune responses, such as Vogt-Koyanagi-Harada disease, Behçet's disease, uveitis associated with spondyloarthritis, ocular sarcoidosis, sympathetic ophthalmia and birdshot chorioretinopathy. These entities have various clinical forms, but genetic and biomarker data suggest that they share a common molecular basis, activation of the Interleukin (IL)-23/IL-17 pathway. Multiple factors including genetic predisposition, various cytokine imbalances, infectious agents and gut alterations are found to trigger an aberrant response of this pathway. The enhanced activity of the IL-23/IL-17 pathway is committed to the expansion and pathogenicity of Th17cells. Evidence from animal models demonstrates that the development of pathogenic Th17cells is responsible for the induction of experimental autoimmune uveitis. Further findings indicate that retinal pigment epithelium (RPE) cells may be a target of IL-17. IL-17 triggers downstream inflammatory cascades and causes dysfunction of RPE cells, which may affect retinal barrier function and thereby promote intraocular inflammation. Currently, several emerging drugs blocking the IL-23/IL-17 pathway have been assessed for the treatment of uveitis in pilot studies. The purpose of this is to summarize updated biological knowledge and preliminary clinical data, providing the rationale for further development and evaluation of novel drugs targeting the IL-23/IL-17 pathway in autoinflammatory and autoimmune uveitis. Future studies may focus on translational medicine targeting the IL-23/IL-17 pathway for the improvement of diagnosis and treatment of uveitis. In conclusion, activation of the IL-23/IL-17 pathway is a critical biological event and can be an important target for the treatment of autoinflammatory and autoimmune uveitis.

Suppression of the Reactive Oxygen Response Alleviates Experimental Autoimmune Uveitis in Mice.

Reactive oxygen species (ROS) are produced by host phagocytes and play an important role in antimicrobial actions against various pathogens. Autoimmune uveitis causes blindness and severe visual impairment in humans at all ages worldwide. However, the role of ROS in autoimmune uveitis remains unclear. We used ROS-deficient (Ncf1−/−) mice to investigate the role of ROS in experimental autoimmune uveitis (EAU). Besides, we also used the antioxidant N-acetylcysteine (NAC) treatment to evaluate the effect of suppression of ROS on EAU in mice. The EAU disease scores of Ncf1−/− mice were significantly lower than those of wild-type mice. EAU induction increased the levels of cytokines (interleukin (IL)-1α, IL-1β, IL-4, IL-6, IL-12, IL-17, and tumor necrosis factor (TNF)-α) and chemokines (monocyte chemoattractant protein (MCP)-1) in the retinas of wild-type mice but not in those of Ncf1−/− mice. EAU induction enhanced the level of NF-κB activity in wild-type mice. However, the level of NF-κB activity in Ncf1−/− mice with EAU induction was low. Treatment with the antioxidant NAC also decreased the severity of EAU in mice with reduced levels of oxidative stress, inflammatory mediators, and NF-κB activation in the retina. We successfully revealed a novel role of ROS in the pathogenesis of EAU and suggest a potential antioxidant role for the treatment of autoimmune uveitis in the future.

More roles of IL-4 than being as an antagonist of IFN-γ in experimental autoimmune uveitis

Abstract IL-4 appears to be beneficial for some autoimmune diseases. Despite its role in antagonizing pathogenic Th1 response, our recent work has demonstrated IL-4 possesses functions affecting the regulatory T (Treg) cells. Here we aimed to identify the role of IL-4 in the development of experimental autoimmune uveitis (EAU). EAU was induced in WT and IL-4 KO mice via active interphotoreceptor retinoid binding protein (IRBP) immunization, and the disease development was monitored by funduscopic and histology analysis as well as the IRBP-specific immune responses by flow cytometric analysis and ELISA. It showed that the onset of disease, if the clinical score equals to 1, was day 14 and 20 in IL-4KO and WT mice, respectively. The fundoscopic images demonstrated the more and bigger size of lesions, increased leukocyte infiltration, vasculitis and neovascularization in IL-4KO mice. The histological analysis also confirmed the mononuclear cell infiltration. Moreover, Th1 effector is known as the dominant pathogenic mediators, whereas Treg cells play an important and indispensable role in control of EAU progression and disease remission. The splenic T cells from IL-4 KO EAU animals produced significantly elevated level of IFN-γ in responding to IRBP stimulation. However, there seems no significant but some alternation in the presence of Treg cells (Foxp3+) among CD4+CD25+ T cells in the spleens and lymph nodes of IL-4 KO and WT mice. These results demonstrate the lack of IL-4 disrupting the induction of EAU and leading to an increased disease severity through not only antagonizing IFN-γ but also involved mechanisms of immune-regulation. Therefore, IL-4 might play more roles than being just an antagonist of IFN-γ in autoimmune uveitis.

AAV-mediated expression of HLA-G1/5 reduces severity of experimental autoimmune uveitis

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.

Increased duration of antibiotic treatment progressively alters gut microbiota and revokes protection from ocular autoimmunity

Abstract Autoimmune uveitis, thought to be driven by retina-specific T cells, is a major cause of blindness. Using a model of experimental autoimmune uveitis (EAU), we showed that long-term elimination of commensals by oral antibiotic treatment (ABX) did not affect susceptibility to EAU, but others reported that short-term ABX was protective. To reconcile these contradictory findings, we studied whether duration of ABX differentially affected EAU development, gut microbiota and host-immune responses. B10.RIII mice were treated orally with a broad-spectrum antibiotic cocktail, starting 12-weeks (long-term) or 1-week (short-term) before IRBP immunization for EAU induction, and continuing until the end of the experiment. Short-term ABX delayed the onset of EAU, whereas long-term ABX showed a pattern of EAU kinetics and severity similar to untreated SPF controls. Metagenomic analyses revealed ABX duration-dependent depletion of gut microbial communities. Interestingly, ABX also resulted in progressive depletion of CD4+ and CD4+CD8+ T cells from the gut intraepithelial lymphocyte (IEL) compartment. Notably, these IELs showed some cytotoxic activity in vitro, and are thought to help preserve gut mucosal barrier integrity. We propose that microbiota play dual roles in uveitis development: they provide a stimulus for uveitogenic effector cells, but also maintain a “regulatory” IEL population, whose progressive loss reverses the temporary protection seen with short-term ABX. Further understanding of the role of IELs in the microbiota-gut-eye axis may lead to novel treatments for autoimmunity through manipulation of the commensal microbiome and gut epithelium.

Metronidazole Attenuates the Intensity of Inflammation in Experimental Autoimmune Uveitis

Autoimmune uveitis is a serious sightthreatening disease that in many cases fails to respond to conventional immunosuppressive or biological therapy. Experimental models used in research allow more detailed study of pathogenesis of the autoimmune process and testing new therapeutic strategies. Recent results show that infection can trigger autoimmune diseases, and some commensal microorganisms are essential in causing disease activity. The aim of this work was to assess the effect of broadspectrum antibiotics – combination of metronidazole and ciprofloxacin or metronidazole alone – on the intensity of intraocular inflammation in experimental autoimmune uveitis (EAU). EAU was induced in mouse strain C57BL/6J by interphotoreceptor retinoid- binding protein in complete Freund’s adjuvant and pertussis toxin. The grade of uveitis was assessed clinically and histologically in haematoxylin and eosin- stained tissues. Lymphocytes and macrophages were detected in cryosections using the immunoperoxidase method with antibodies. The therapy was commenced one week before EAU induction and continued throughout the experiment. In addition, metronidazole treatment was also started two weeks before EAU induction. Antibiotics significantly reduced the intensity of uveitis compared to the control group (P &lt; 0.05). The effects of combination of ciprofloxacin and metronidazole and of metronidazole alone were similar when the therapy started one week before EAU induction (P &lt; 0.05). Metronidazole commenced two weeks before EAU induction and throughout the experiment suppressed the intensity of EAU with even higher statistical significance (P &lt; 0.0001). It can be assumed that the high protective effect of metronidazole on EAU intensity may be due not only to its antimicrobial effect, but also to its immunomodulatory activity.

Aryl Hydrocarbon Receptor Regulates Apoptosis and Inflammation in a Murine Model of Experimental Autoimmune Uveitis.

Uveitis is characterized as a common cause of blindness worldwide. Aryl hydrocarbon receptor (AhR), a ligand-activated nuclear receptor, has been implicated to play a role in human uveitis, although the exact mechanisms remain poorly understood. The purpose of this study was to enhance our knowledge concerning the role of AhR during intraocular inflammation. We immunized wild-type and AhR-knockout C57BL/6J mice with IRBP651–670 to induce experimental autoimmune uveitis (EAU). Disease severity was evaluated with both clinical and histopathological grading. Blood–retinal barrier (BRB) integrity was tested by Evans blue and tight junction proteins qualifications. Apoptosis was measured using TdT-mediated dUTP nick end labeling staining. Macrophage/microglia activation and polarization were studied by immunofluorescence and Western blot. Following EAU induction, AhR−/− mice had more severe clinical and histopathological manifestations of uveitis than AhR+/+ mice. Increased vascular permeability and apoptotic cells were observed in AhR−/− EAU mice when compared with AhR+/+ EAU mice. In addition, AhR−/− EAU mice showed evidence of a significantly increased macrophage/microglia cells and a stronger polarization from the M2 to the M1 phenotype as compared to AhR+/+ EAU mice. The levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β were increased in AhR−/− EAU mice, which was associated with the activation of NF-κB and signal transducers and activators of transcription (STAT) pathways. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an agonist of AhR, caused a significant decrease in the clinical and histopathological manifestations, preserved BRB integrity, reduced apoptotic cells, inhibited macrophage/microglia activation, and shifted their polarization from M1 toward M2. Moreover, decreased expression of pro-inflammatory cytokines including TNF-α, IL-6, and IL-1β and inhibition of NF-κB and STAT pathways were found in EAU mice following TCDD treatment. In conclusion, AhR activation with TCDD exhibits an immunomodulatory effect by reducing BRB breakdown, inhibiting retinal cell apoptosis, and reducing pro-inflammatory cytokine expression during EAU. The underlying mechanism may involve the modulation of macrophages/microglia polarization and the downregulation of NF-κB and STAT pathways.

Targeting CD6 for the treatment of experimental autoimmune uveitis

ObjectiveCD6 is emerging as a new target for treating many pathological conditions in which T cells are integrally involved, but even the latest data from studies of CD6 gene engineered mice were still contradictory. To address this issue, we studied experimental autoimmune uveitis (EAU), a model of autoimmune uveitis, in wild-type (WT) and CD6 knockout (KO) mice. MethodsAfter EAU induction in WT and CD6 KO mice, we evaluated ocular inflammation and compared retinal antigen-specific T-cell responses using scanning laser ophthalmoscopy, spectral-domain optical coherence tomography, histopathology, and T cell recall assays. Uveitogenic T cells from WT and CD6 KO mice were adoptively transferred into WT naïve mice to confirm the impact of CD6 on T cells. In addition, we immunized CD6 KO mice with recombinant CD6 protein to develop mouse anti-mouse CD6 monoclonal antibodies (mAbs) in which functional antibodies exhibiting cross-reactivity with human CD6 were screened and identified for treatment studies. ResultsIn CD6 KO mice with EAU, we found significantly decreased retinal inflammation and reduced autoreactive T-cell responses, and confirmed the impaired uveitogenic capacity of T cells from these mice in an adoptive transfer experiment. Notably, one of these cross-reactive mAbs significantly ameliorated retinal inflammation in EAU induced by the adoptive transfer of uveitogenic T cells. ConclusionsTogether, these data strongly suggest that CD6 plays a previously unknown, but pivotal role in autoimmune uveitis, and may be a promising new treatment target for this blinding disease. In addition, the newly developed mouse anti-mouse/human CD6 mAbs could be valuable tools for testing CD6-targeted therapies in other mouse models of human diseases.

Total glucosides of paeony suppresses experimental autoimmune uveitis in association with inhibition of Th1 and Th2 cell function in mice.

Total glucosides of paeony (TGP) are active components extracted from the roots of Paeonia lactiflora Pall. In this study, we investigated the role and mechanisms of TGP in experimental autoimmune uveitis (EAU) model of mice. The C57BL/6 mice were randomly divided into three groups: sham group, EAU-control group, and EAU-TGP group. Clinical score of images of the eye fundus were taken on 7, 14, 21, and 28 days after induction of EAU. The concentrations of proinflammatory cytokines in intraocular fluid were measured at 14 days after EAU induction with the use of a multiplex assay system. Flow cytometry was used to analyze the frequency of CD4+, CD8+, interferon-gamma (IFN-γ), and CD4+/CD8+ ratio in spleen and lymph nodes. Western blotting was used to measure expressions of mitogen-activated protein kinase (MAPK) pathway-related proteins in retina. Clinical scores for uveitis were lower in TGP-treated EAU mice than those without TGP treatment. Importantly, the concentrations of cytokines induced by T-helper 1 (Th1) and T-helper 2 (Th2) cells in intraocular fluid were reduced in EAU mice treated with TGP. Furthermore, the frequency of CD4+, IFN-γ, and CD4+/CD8+ ratio was decreased and the frequency of CD8+ was increased in spleen and lymph nodes of mice treated with TGP. The anti-inflammatory effects of TGP were mediated by inhibiting the MAPK signaling pathways. Our results showed that TGP suppressed uveitis in mice via the inhibition of Th1 and Th2 cell function. Thus, TGP may be a promising therapeutic strategy for uveitis, as well as other ocular inflammatory diseases.

Vitamin A deficiency impacts acquisition, but not expression, of autoimmunity to the neuroretina

Abstract Vitamin A (VitA) and its derivative retinoic acid (RA) are essential for immunological responses. Acquisition of effector responses is impeded in VitA-deficient (VAD) mice, but little is known about maintenance and expression of previously acquired effector function in the VAD environment, or its impact on progression of autoimmune diseases. We examined this using two models of uveitis: experimental autoimmune uveitis (EAU) induced by active immunization and spontaneous uveitis in retina-specific T cell receptor transgenic (R161H) mice, and in the model of experimental autoimmune encephalomyelitis (EAE). VAD was induced by dietary lack of VitA from before birth, or by daily injections of a pan-RA receptor inhibitor BMS493 in adult mice fed with standard diet. VAD mice were essentially resistant to induction of EAU or EAE and displayed impaired effector T cell responses. Defective priming/acquisition of effector function by VAD T cells was also evident in vitro. Interestingly, however, effector T cells primed in a VitA-sufficient environment were able to function in VAD recipients and induced EAU. Furthermore, spontaneously uveitic R161H mice fed with VAD diet, in which priming of pathogenic T cells occurs before onset of full VitA deficiency, appeared to develop unreduced or even exacerbated spontaneous uveitis compared to VitA-sufficient R161H mice. We conclude that although priming of naïve T cells in the VAD environment is defective, effector function acquired under VitA-sufficient conditions is maintained and can be expressed under VAD conditions. Because dietary lack of VitA is rarely profound and may be seasonal, our findings may shed light on immunity and autoimmunity in geographical regions where dietary VitA is limiting.