Polyvinylpyrrolidone-curcumin nanoparticles with immune regulatory and metabolism regulatory effects for the treatment of experimental autoimmune uveitis.

Uveitis comprises a cluster of intraocular inflammatory disorders characterized by uncontrolled autoimmune responses and excessive oxidative stress leading to vision loss worldwide. In the present study, curcumin (CUR) was conjugated with polyvinylpyrrolidone (PVP) to form PVP-CUR nanoparticles with significantly elevated solubility and outstanding multiple radical scavenging abilities. In vitro studies revealed that PVP-CUR nanoparticles markedly mitigated oxidative stress and reduced apoptosis in a H2O2-induced human retinal pigment epithelial cell line (ARPE-19) and promoted phenotypic polarization from M1 to M2 in an LPS-induced human microglial cell line (HMC3). Further in vivo studies demonstrated the prominent therapeutic effects of PVP-CUR nanoparticles on experimental autoimmune uveitis (EAU), which relieved clinical and pathological progression, improved perfusion and tomographic manifestations of retinal vessels, and reduced blood-retinal barrier (BRB) leakage; these effects may be mediated by mitigating oxidative stress and attenuating macrophage/microglia-elicited inflammation. Notably, treatment with PVP-CUR nanoparticles was shown to regulate metabolite alterations in EAU rats, providing novel insights into the underlying mechanisms involved. Additionally, the PVP-CUR nanoparticles showed great biocompatibility in vivo. In summary, our study revealed that PVP-CUR nanoparticles may serve as effective and safe nanodrugs for treating uveitis and other oxidative stress- and inflammation-related diseases.

CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis.

Autoimmune uveitis is a leading cause of severe vision loss, and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq, RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU), revealing that EAU causes broad retinal neuron degeneration and marker downregulation, and that Müller glia may act as antigen-presenting cells. Moreover, EAU immune response is primarily driven by Th1 cells, and results in dramatic upregulation of CC chemokines, especially CCL5, in the EAU retina. Accordingly, overexpression of CCR5, a CCL5 receptor, in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU, by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together, our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.

Small extracellular vesicle-based delivery of interleukin-10 improves treatment of experimental autoimmune uveitis.

Non-infectious uveitis is an intraocular autoimmune disease mainly characterized by immune dysregulation of the eye, which may cause blindness if not well treated. Interleukin 10 (IL-10) is a potent cytokine with multiple immunoregulatory functions. However, it's in vivo activity is unstable owing to its inherent protein instability and short plasma half-life. Therefore, our previous research tried to establish IL-10-overexpressing MSC-sEVs (sEVs-IL10) using lentiviral transfection. While this approach indeed improved drug delivery, it also suffered from tedious procedures and limited loading efficiency. Accordingly, we constructed a novel MSC-sEVs-based delivery system for IL-10 (IL-10@sEVs) by sonication. The obtained formulation (IL-10@sEVs) had relatively higher loading efficiency and exerted a more profound immunomodulatory effect than sEVs-IL10 in vitro. Furthermore, IL-10@sEVs had significant therapeutic effects in a mouse model of experimental autoimmune uveitis (EAU) by decreasing the percentage of Th17 cells, increasing regulatory T cells in the eye, and draining lymph nodes. In summary, sonication outperforms conventional transfection methods for loading IL-10 into MSC-sEVs.

Dimethyl itaconate inhibits antigen-specific Th17 cell responses and autoimmune inflammation via modulating NRF2/STAT3 signaling.

Pathogenic Th17 cells play a crucial role in autoimmune diseases like uveitis and its animal model, experimental autoimmune uveitis (EAU). Dimethyl itaconate (DMI) possesses potent anti-inflammatory effects. However, there is still a lack of knowledge about the role of DMI in regulating pathogenic Th17 cells and EAU. Here, we reported that intraperitoneal administration of DMI significantly inhibited the severity of EAU via selectively suppressing Th17 cell responses. In vitro antigen stimulation studies revealed that DMI dramatically decreased the frequencies and function of antigen-specific Th17, but not Th1, cells. Moreover, DMI hampered the differentiation of naive CD4+ T cells toward pathogenic Th17 cells. DMI-treated DCs produced less IL-1ß, IL-6, and IL-23, and displayed an impaired ability to stimulate antigen-specific Th17 activation. Mechanistically, DMI activated the NRF2/HO-1 pathway and suppressed STAT3 signaling, which subsequently restrains p-STAT3 nuclear translocation, leading to decreased pathogenic Th17 cell responses. Thus, we have identified an important role for DMI in regulating pathogenic Th17 cells, supporting DMI as a promising therapy in Th17 cell-driven autoimmune diseases including uveitis.

Foxo1 Drives the TGFß1-Dependent Dichotomy of Th17 Cell Fates.

T-helper 17 (Th17) cells play a dual role in immunological responses, serving as essential components in tissue homeostasis and host defense against microbial pathogens while also contributing to pro-inflammatory conditions and autoimmunity. While Transforming Growth Factor-beta 1 (TGFß1) is pivotal for the differentiation of non-pathogenic Th17 cells, the role of TGFß3 and Activin in steering Th17 cells toward a pathogenic phenotype has been acknowledged. However, the molecular mechanisms governing this dichotomy remain elusive. In this study, we demonstrate that the transcription factor Foxo1 is upregulated in a TGFß1 dose-dependent manner, serving as a critical regulator that specifically modulates the fate of pathogenic Th17 cells. Analyses in both uveitis patients and an Experimental Autoimmune Uveitis (EAU) mouse model reveal a strong correlation between disease severity and diminished Foxo1 expression levels. Ectopic expression of Foxo1 selectively attenuates IL-17A production under pathogenic Th17-inducing conditions. Moreover, enhanced Foxo1 expression, triggered by TGFß1 signaling, is implicated in fatty acid metabolism pathways that favor non-pathogenic Th17 differentiation. Our drug screening identifies several FDA-approved compounds can upregulate Foxo1. Collectively, our findings offer evidence that Foxo1 serves as a molecular switch to specifically control pathogenic versus non-pathogenic Th17 differentiation in a TGFß1-dependent manner. Suggest that targeting Foxo1 could be a promising therapeutic strategy for autoimmune diseases.

MiR-223-3p attenuates M1 macrophage polarization via suppressing the Notch signaling pathway and NLRP3-mediated pyroptosis in experimental autoimmune uveitis.

Autoimmune uveitis is an intraocular inflammatory disease with a high blindness rate in developed countries such as the United States. It is pressing to comprehend the pathogenesis of autoimmune uveitis and develop novel schemes for its treatment. In the present research, we demonstrated that the Notch signaling pathway was activated, and the level of miR-223-3p was significantly reduced in rats with experimental autoimmune uveitis (EAU) compared with the level of normal rats. To investigate the relationship between miR-223-3p and Notch signaling, EAU rats received miR-223-3p-carrying lentivirus, miR-223-3p vector-carrying lentivirus (miR-223-3p-N), and γ-secretase inhibitor (DAPT), respectively. The results of Q-PCR, immunological experiments, and flow cytometry analysis all support the hypothesis that both miR-223-3p and DAPT, a Notch signaling pathway inhibitor, had similar inhibitory effects on the EAU pathological process. That is to say, they could both inhibit the activation of the Notch signaling pathway via modulating recombination signal binding protein-Jκ (RBPJ) to restore the polarization imbalance of M/M2 macrophages in EAU rats. In addition, miR-223-3p could also inhibit NLRP3 inflammasome activation and inflammasome-induced pyroptosis in ocular tissues. Taken together, our findings indicate that miR-223-3p serves as an important regulator in M1 macrophage polarization and pyroptosis, thereby alleviating the inflammatory response in uveitis.

The Role of Adenosine in γδ T-Cell Regulation of Th17 Responses in Experimental Autoimmune Uveitis.

Autoimmune diseases caused by T cells can arise from either T-helper 1 (Th1) or T-helper 17 (Th17)-type pathogenic T cells. However, it is unclear whether these two T-cell subsets are influenced by distinct pathogenic factors and whether treatments that are effective for Th1 responses also work for Th17 responses. To compare these two pathogenic responses, we conducted a systematic analysis in a mouse model of experimental autoimmune uveitis (EAU) to identify the factors that promote or inhibit each response and to determine their responses to various treatments. Our study found that the two types of pathogenic responses differ significantly in their pathological progressions and susceptibility to treatments. Specifically, we observed that extracellular adenosine is a crucial pathogenic molecule involved in the pathogenicity of inflammation and T-cell reactivity and that reciprocal interaction between adenosine and gamma delta (γδ) T cells plays a significant role in amplifying Th17 responses in the development of autoimmune diseases. The potential effect of targeting adenosine or adenosine receptors is analyzed regarding whether such targeting constitutes an effective approach to modulating both γδ T-cell responses and the pathogenic Th17 responses in autoimmune diseases.

Combined Therapy of Experimental Autoimmune Uveitis by a Dual-Drug Nanocomposite Formulation with Berberine and Dexamethasone.

Purpose: Autoimmune uveitis is a kind of sight-threatening ocular and systemic disorders. Recent treatments on autoimmune uveitis still remain many limitations due to extreme complexity and undetermined pathogenesis. In this study, a novel dual-drug nanocomposite formulation is developed to treat experimental autoimmune uveitis by a combined and sustained therapy method. Methods: The dual-drug nanocomposite formulation is constructed by integrating berberine (BBR)-loaded mesoporous silica nanoparticles (MSNs) into dexamethasone (DEX)-loaded thermogel (BBR@MSN-DEX@Gel). The BBR@MSN-DEX@Gel is characterized by transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectrometer and rheometer. The in vitro drug release profile, cytotoxicity and anti-inflammation effectiveness of BBR@MSN-DEX@Gel on lipopolysaccharide-stimulated human conjunctival epithelial cells are investigated. After the in vivo drug release profile and biosafety of the dual-drug nanocomposite formulation are confirmed, its treatment effectiveness is fully assessed based on the induced experimental autoimmune uveitis (EAU) Lewis rat's model. Results: The dual-drug nanocomposite formulation has good injectability and thermosensitivity, suitable for administration by an intravitreal injection. The BBR@MSN-DEX@Gel has been found to sustainably release both drugs for up to 4 weeks. The carrier materials have minimal in vitro cytotoxicity and high in vivo biosafety. BBR@MSN-DEX@Gel presents obviously anti-inflammatory effectiveness in vitro. After administration of BBR@MSN-DEX@Gel into Lewis rat's eye with EAU by an intravitreal injection, the nanocomposite formulation significantly suppresses inflammatory reaction of autoimmune uveitis via a dual-drug combined and sustained therapy method, compared with the equivalent dose of single-component formulations. Conclusion: BBR@MSN-DEX@Gel serves as a promising dual-drug nanocomposite formulation for future treatment of autoimmune uveitis.

Comprehensive Grading System for Experimental Autoimmune Uveitis in Mice.

Experimental autoimmune uveitis (EAU) is the most commonly used animal model to study the progression of chronic uveitis and to test various therapies to treat the disease. However, to accurately evaluate the effectiveness of such treatments, a grading system that combines the latest imaging techniques with definitive quantitative grading thresholds is required. This study aimed to develop a comprehensive grading system that objectively evaluates EAU progression in C57BL/6J mice. EAU was induced following immunisation with interphotoreceptor retinoid-binding protein (IRBP) and pertussis toxin. Weekly fundus and optical coherence tomography (OCT) images were acquired over 12 weeks using a Micron IV imaging system. Each mouse was graded (between 0 to 4) based on changes seen on both the fundus (optic disc, retinal blood vessels and retinal tissue) and OCT (vitreous and retinal layers) images. A total EAU response (with a maximum score of 48) was calculated for each mouse based on the sum of the individual scores each week. Analysis of the clinical scores depicted a gradual increase in inflammatory signs including optic disc and vascular swelling, leukocyte infiltration in the vitreous, lesions in the retina and formation of granulomas and hyper-reflective foci in the retinal layers in EAU mice, with most signs reaching a plateau towards the end of the study period. Development of these signs into sight-threatening complications such as optic disc atrophy, structural damage to the retina and subretinal oedema were noted in 80-90% of mice suggesting consistent disease induction. Overall, a comprehensive and objective grading system encompassing all pathologies occurring in EAU mice was developed to enhance the preclinical evaluation of novel uveitis treatments.

Regulatory T cell intravitreal delivery using hyaluronan methylcellulose hydrogel improves therapeutic efficacy in experimental autoimmune uveitis.

Autoimmune uveitis refers to several intraocular inflammation conditions, which are mediated by autoreactive T cells. Regulatory T cells (Tregs) are immunosuppressive cells that have shown potential for resolving various autoimmune diseases, including uveitis. However, poor donor cell dispersion distal to the injection site and plasticity of Treg cells in an inflammatory microenvironment can present obstacles for this immunotherapy. We assessed the use of a physical blend of hyaluronan and methylcellulose (HAMC) as immunoprotective and injectable hydrogel cell delivery system to improve the efficacy of Treg-based therapy in treating experimental autoimmune uveitis (EAU). We demonstrated that the Treg-HAMC blend increased both the survival and stability of Tregs under proinflammatory conditions. Furthermore, we found that the intravitreal HAMC delivery system resulted in a two-fold increase in the number of transferred Tregs in the inflamed eye of EAU mice. Treg-HAMC delivery effectively attenuated ocular inflammation and preserved the visual function of EAU mice. It significantly decreased the number of ocular infiltrates, including the uveitogenic IFN-γ+CD4+ and IL-17+CD4+ T cells. In contrast, intravitreal injection of Treg cells without HAMC only achieved marginal therapeutic effects in EAU. Our findings suggest that HAMC may become a promising delivery vehicle for human uveitis Treg therapy.

Glucocorticoids improve the balance of M1/M2 macrophage polarization in experimental autoimmune uveitis through the P38MAPK-MEF2C axis.

Uveitis is a common ocular disease that can induce serious complications and sequelae. It is one of the major causes of blindness. Currently, mounting evidence suggests that glucocorticoids (GCs) can suppress ocular inflammation and promote the healing of damaged ocular tissues, but the underlying mechanism remains unclear. The present study aimed to elucidate the mechanism by which GCs modulate the homeostasis of M1/M2 macrophage polarization in experimental autoimmune uveitis (EAU) through the p38MAPK-MEF2C axis. Female Lewis rats were randomly divided into four groups: a normal control (NC) group, an EAU group, an EAU + glucocorticoid (EAU + GC) group, and an EAU + p38MAPK inhibitor (EAU + SB) group. The EAU model was induced in EAU, EAU + GC, and EAU + SB groups, followed by the treatments of normal saline, GC (predisione), and SB203580, respectively. The findings demonstrated that the rats in GC and SB groups had much less ocular inflammation, and the clinical and pathological scores decreased. Further research revealed that GC and SB treatment could inhibit iNOS and CD86 expression while promoting Arg-1 and CD206 secretion in IRBP-induced uveitis rats. Moreover, we found that the role of GC was similar to the results of SB203580, but the role of GC was masked by the C16-PAF (a p38MAPK activator) treatment. Molecular docking and western blot results confirmed that GC's therapeutic action against EAU is mediated via the p38MAPK-MEF2C axis. It regulates macrophage polarization by encouraging M1 to M2 transition and releasing anti-inflammatory factors.

γδ T cells in autoimmune uveitis pathogenesis: A promising therapeutic target.

Autoimmune uveitis is a non-infectious, inflammatory intraocular disease that affects the uveal and adjacent tissues. It frequently causes varying degrees of visual loss. Evidence for the strong association between activated γδ T cells and the development of autoimmune uveitis is growing. The innate and adaptive immune response are connected in the early phases by the γδ T cells that contain the γ and δ chains. γδ T cells can identify antigens in a manner that is not constrained by the MHC. When activated by various pathways, γδ T cells can not only secrete pro-inflammatory factors early on (such as IL-17), but they can also promote Th17 cells responses, which ultimately exacerbates autoimmune uveitis. Therefore, we review the mechanisms by which γδ T cells affect autoimmune uveitis in different activation and disease states. Moreover, we also prospect for immunotherapies targeting different γδ T cell-related action pathways, providing a reference for exploring new drug for the treatment of autoimmune uveitis.

Single-cell transcriptomics-based multidisease analysis revealing the molecular dynamics of retinal neurovascular units under inflammatory and hypoxic conditions.

The retinal neurovascular unit (NVU) is paramount to maintaining the homeostasis of the retina and determines the progression of various diseases, including diabetic retinopathy (DR), glaucoma, and retinopathy of prematurity (ROP). Although some studies have investigated these diseases, a combined analysis of disease-wide etiology in the NUV at the single-cell level is lacking. Herein, we constructed an atlas of the NVU under inflammatory and hypoxic conditions by integrating single-cell transcriptome data from retinas from wild-type, AireKO, and NdpKO mice. Based on the heterogeneity of the NVU structure and transcriptome diversity under normal and pathological conditions, we discovered two subpopulations of Müller cells: Aqp4hi and Aqp4lo cells. Specifically, Aqp4lo cells expresses phototransduction genes and represent a special type of Müller cell distinct from Aqp4hi cells, classical Müller cells. AireKO mice exhibit experimental autoimmune uveitis (EAU) with severe damage to the NVU structure, mainly degeneration of Aqp4hi cells. NdpKO mice exhibited familial exudative vitreoretinopathy (FEVR), with damage to the endothelial barrier, endothelial cell tight junction destruction and basement membrane thickening, accompanied by the reactive secretion of proangiogenic factors by Aqp4hi cells. In both EAU and FEVR, Aqp4hi cells are a key factor leading to NVU damage, and the mechanism by which they are generated is regulated by different transcription factors. By studying the pattern of immune cell infiltration in AireKO mice, we constructed a regulatory loop of "inflammatory cells/NVU - monocytes - APCs - Ifng+ T cells", providing a new target for blocking the inflammatory cascade. Our elucidation of the cell-specific molecular changes, cell-cell interactions and transcriptional mechanisms of the retinal NVU provides new insights to support the development of multipurpose drugs to block or even reverse NVU damage.

Pertussis toxin-induced inhibition of Wnt/ß-catenin signaling in dendritic cells promotes an autoimmune response in experimental autoimmune uveitis.

BACKGROUND: Previous reports have indicated that disrupting the Wnt/ß-catenin pathway in dendritic cells (DCs) may affect the progression of autoimmune inflammation; however, the factors and timing that regulate Wnt/ß-catenin signaling have not been clearly understood. METHODS: Experimental autoimmune uveitis (EAU) mice and Vogt-Koyanagi-Harada disease (VKH) patient samples were used to detect the expression of Wnt/ß-catenin pathway genes. Western blot, real-time PCR, flow cytometry, and ELISA were performed to examine the expression of components of the Wnt/ß-catenin pathway and inflammatory factors. DC-specific ß-catenin knockout mice and 6-bromoindirubin-3'-oxime (BIO) administered mice were used to observe the effect of disrupting the Wnt pathway on EAU pathogenesis. RESULTS: Wnt/ß-catenin signaling was inhibited in DCs during the induction phase of EAU. The inhibition was mediated by pertussis toxin (PTX), which promoted DC maturation, in turn promoting pathogenic T cell proliferation and differentiation. In vivo experiments confirmed that deleting ß-catenin in DCs enhanced EAU severity, and pre-injection of PTX advanced EAU onset. Administration of a Wnt activator (BIO) limited the effects of PTX, in turn ameliorating EAU. CONCLUSIONS: Our results demonstrate that PTX plays a key role as a virulence factor in initiating autoimmune inflammation via DCs by inhibiting Wnt/ß-catenin signaling in EAU, and highlight the potential mechanism by which infection can trigger apparent autoimmunity.

Prednisone acetate modulates Th1/Th2 and Th17/Treg cell homeostasis in experimental autoimmune uveitis via orchestrating the Notch signaling pathway.

Uveitis is an immune eye disease that can seriously impair vision. Glucocorticoids (GCS) have been extensively used to treat uveitis, though the mechanisms have not been fully elucidated. In this study, we investigated the regulatory effects of prednisone acetate (PA) on the Th1/Th2 and Th17/Treg balance in experimental autoimmune uveitis (EAU) through modulating the Notch signaling pathway. Briefly, Lewis rats were randomly divided into the normal control (NC), EAU, and EAU + PA groups. Rats in EAU and EAU + PA groups were induced EAU, while those in the EAU + PA group were treated with PA. Clinical and histopathological scores were employed to assess the progression of EAU. The expression levels of Notch signaling-related molecules (Notch1, Notch2, Dll3, Dll4, and Rbpj) and Th-associated cytokines (IFN-γ, IL-4, IL-10, and IL-17) were assessed via quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). In addition, the frequencies of Th1, Th2, Th17 and Treg cells were detected by flow cytometry. These experimental results indicated that activation of the Notch signaling pathway occurred in EAU rats and resulted in a severe imbalance of the Th17/Treg and Th1/Th2 ratios. PA treatment significantly alleviated ocular inflammation, inhibited activation of the Notch signaling pathway, and declined Th1, and Th17 cell differentiation, thereby restoring the Th1/Th2 and Th17/Treg balance. Collectively, PA can positively enhance the systemic immune response and improve the intraocular microenvironmental homeostasis by inhibiting activation of the Notch signaling pathway and by restoring Th1/Th2 and Th17/Treg balance, thus achieving the goal of treating uveitis.

microRNA 146a ameliorates retinal damage in experimental autoimmune uveitis.

Introduction: Uveitis and related intraocular inflammations are a major cause of blindness due to retinal damage caused by degeneration and loss of the photoreceptor cells. In mouse experimental autoimmune uveitis (EAU) previously we have shown mitochondrial oxidative stress with marked upregulation of αA crystallin in the inner segments of the photoreceptors. Furthermore, αA crystallin treatment prevented photoreceptor mitochondrial oxidative stress by suppressing innate and adaptive immunity in EAU. Methods: Since these immune processes are modulated by microRNAs, in this study we investigated (a) modulation of microRNAs during development of EAU by αA crystallin administration and (b) microRNA therapeutic intervention. Results: Few microRNAs were significantly upregulated in EAU mice with intravenous injection of αA crystallin and among these, computational bioinformatic analysis revealed that the upregulated microRNA 146a targets the innate and adaptive immune responses. In EAU, intravenous as well as intravitreal administration of this microRNA prevented inflammatory cell infiltration in uvea and retina and preserved photoreceptor cells. Discussion: This protective function suggests that microRNA146a can be a novel therapeutic agent in preventing retinal damage in uveitis.

A new small molecule DHODH-inhibitor [KIO-100 (PP-001)] targeting activated T cells for intraocular treatment of uveitis - A phase I clinical trial.

Uveitis is a T cell-mediated, intraocular inflammatory disease and one of the main causes of blindness in industrialized countries. There is a high unmet need for new immunomodulatory, steroid-sparing therapies, since only ciclosporin A and a single TNF-α-blocker are approved for non-infectious uveitis. A new small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), an enzyme pivotal for de novo synthesis of pyrimidines, has a high potency for suppressing T and B cells and has already proven highly effective for treating uveitis in experimental rat models. Systemic and intraocular application of KIO-100 (PP-001) (previously called PP-001, now KIO-100) could efficiently suppress rat uveitis in a preventive as well as therapeutic mode. Here we describe the outcome of the first clinical phase 1 trial comparing three different doses of a single intraocular injection of KIO-100 (PP-001) in patients with non-infectious posterior segment uveitis. No toxic side effects on intraocular tissues or other adverse events were observed, while intraocular inflammation decreased, and visual acuity significantly improved. Macular edema, a sight-threatening complication in uveitis, showed regression 2 weeks after intraocular KIO-100 (PP-001) injection in some patients, indicating that this novel small molecule has a high potential as a new intraocular therapy for uveitis. Clinical trial registration: [https://www.clinicaltrials.gov/ct2/show/NCT03634475], identifier [NCT03634475].

Bidirectional Effect of IFN-γ on Th17 Responses in Experimental Autoimmune Uveitis.

Pro- and ant-inflammatory effects of IFN-γ have been repeatedly found in various immune responses, including cancer and autoimmune diseases. In a previous study we showed that the timing of treatment determines the effect of adenosine-based immunotherapy. In this study we examined the role of IFN-γ in pathogenic Th17 responses in experimental autoimmune uveitis (EAU). We observed that IFN-γ has a bidirectional effect on Th17 responses, when tested both in vitro and in vivo. Anti-IFN-γ antibody inhibits Th17 responses when applied in the initial phase of the immune response; however, it enhances the Th17 response if administered in a later phase of EAU. In the current study we showed that IFN-γ is an important immunomodulatory molecule in γδ T cell activation, as well as in Th17 responses. These results should advance our understanding of the regulation of Th17 responses in autoimmunity.

Photoreceptor Cells Constitutively Express IL-35 and Promote Ocular Immune Privilege.

Interleukin-27 is constitutively secreted by microglia in the retina or brain, and upregulation of IL-27 during neuroinflammation suppresses encephalomyelitis and autoimmune uveitis. However, while IL-35 is structurally and functionally similar to IL-27, the intrinsic roles of IL-35 in CNS tissues are unknown. Thus, we generated IL-35/YFP-knock-in reporter mice (p35-KI) and demonstrated that photoreceptor neurons constitutively secrete IL-35, which might protect the retina from persistent low-grade inflammation that can impair photoreceptor functions. Furthermore, the p35-KI mouse, which is hemizygous at the il12a locus, develops more severe uveitis because of reduced IL-35 expression. Interestingly, onset and exacerbation of uveitis in p35-KI mice caused by extravasation of proinflammatory Th1/Th17 lymphocytes into the retina were preceded by a dramatic decrease of IL-35, attributable to massive death of photoreceptor cells. Thus, while inflammation-induced death of photoreceptors and loss of protective effects of IL-35 exacerbated uveitis, our data also suggest that constitutive production of IL-35 in the retina might have housekeeping functions that promote sterilization immunity in the neuroretina and maintain ocular immune privilege.

N6-methyladenosine demethylase FTO regulates inflammatory cytokine secretion and tight junctions in retinal pigment epithelium cells.

OBJECTIVE: Uveitis is an intraocular inflammatory disease. Epigenetics has been associated with its pathogenesis. However, the role of N6-methyladenosine (m6A) in uveitis has not been reported. We aimed to examine the role of m6A and its regulatory mechanism in experimental autoimmune uveitis (EAU). METHODS: The mRNA expression of m6A-related methylase and demethylase of retinal pigment epithelium (RPE) between mice with EAU and control mice was detected by RT-qPCR. The overall m6A level of ARPE-19 cells was detected by an m6A quantitative detection kit. Cell proliferation was observed by CCK-8 assays, and ELISA was used to test the secretion of inflammatory factors. The expression of tight junction proteins and the target genes of FTO were examined by western blotting and MeRIP-PCR. RESULTS: A decreased expression of FTO in RPE cells was found in mice with EAU. Increased overall m6A%, proliferation of cells and secretion of IL-6, IL-8 and MCP-1 were found after FTO knockdown in ARPE-19 cells. However, ZO-1 and occludin protein expression was decreased. ATF4 protein expression was decreased in the FTO knockdown (shFTO) group as compared with the control (shNC) group. In contrast, the m6A level of ATF4 was elevated, as shown by MeRIP-PCR. Functional analysis showed that p-STAT3 expression was increased in the shFTO group, and the change in occludin expression was reversed in ATF4 rescue experiment. CONCLUSION: FTO may affect the translation of ATF4 by regulating its m6A level, resulting in the increased expression of p-STAT3 and inflammatory factors, and leading to uveitis.