YY1 Lactylation Aggravates Autoimmune Uveitis by Enhancing Microglial Functions via Inflammatory Genes.

Activated microglia in the retina are essential for the development of autoimmune uveitis. Yin-Yang 1 (YY1) is an important transcription factor that participates in multiple inflammatory and immune-mediated diseases. Here, an increased YY1 lactylation in retinal microglia within in the experimental autoimmune uveitis (EAU) group is observed. YY1 lactylation contributed to boosting microglial activation and promoting their proliferation and migration abilities. Inhibition of lactylation suppressed microglial activation and attenuated inflammation in EAU. Mechanistically, cleavage under targets & tagmentation ï¼ˆCUT&Tag） analysis revealed that YY1 lactylation promoted microglial activation by regulating the transcription of a set of inflammatory genes, including STAT3, CCL5, IRF1, IDO1, and SEMA4D. In addition, p300 is identified as the writer of YY1 lactylation. Inhibition of p300 decreased YY1 lactylation and suppressed microglial inflammation in vivo and in vitro. Collectively, the results showed that YY1 lactylation promoted microglial dysfunction in autoimmune uveitis by upregulating inflammatory cytokine secretion and boosting cell migration and proliferation. Therapeutic effects can be achieved by targeting the lactate/p300/YY1 lactylation/inflammatory genes axis.

Decoding the Key Functional Combined Components Group and Uncovering the Molecular Mechanism of Longdan Xiegan Decoction in Treating Uveitis.

Objective: Longdan Xiegan Decoction (LXD) is a famous herbal formula in China. It has been proved that LXD has been shown to have a significant inhibitory effect on suppresses the inflammatory cells associated with uveitis. However, the key functional combination of component groups and their possible mechanisms remain unclear. Methods: The community detecting model of the network, the functional response space, and reverse prediction model were utilized to decode the key components group (KCG) and possible mechanism of LXD in treating uveitis. Finally, MTT assay, NO assay and ELISA assay were applied to verify the effectiveness of KCG and the accuracy of our strategy. Results: In the components-targets-pathogenic genes-disease (CTP) network, a combination of Huffman coding and random walk algorithm was used and eight foundational acting communities (FACs) were discovered with important functional significance. Verification has shown that FACs can represent the corresponding C-T network for treating uveitis. A novel node importance calculation method was designed to construct the functional response space and pick out 349 effective proteins. A total of 54 components were screened and defined as KCG. The pathway enrichment results showed that KCG and their targets enriched signal pathways of IL-17, Toll-like receptor, and T cell receptor played an important role in the pathogenesis of uveitis. Furthermore, experimental verification results showed that important KCG quercetin and sitosterol markedly inhibited the production of nitric oxide and significantly regulated the level of TNF-α and IFN-γ in Lipopolysaccharide-induced RAW264.7 cells. Discussion: In this research, we decoded the potential mechanism of the multi-components-genes-pathways of LXD's pharmacological action mode against uveitis based on an integrated pharmacology approach. The results provided a new perspective for the future studies of the anti-uveitis mechanism of traditional Chinese medicine.

Systemic and Ocular Anti-Inflammatory Mechanisms of Green Tea Extract on Endotoxin-Induced Ocular Inflammation.

Introduction: Green tea extract (GTE) alleviated ocular inflammations in endotoxin-induced uveitis (EIU) rat model induced by lipopolysaccharide (LPS) but the underlying mechanism is unclear. Objectives: To investigate the systematic and local mechanisms of the alleviation by untargeted metabolomics using liquid chromatography-tandem mass spectrometry. Methods: Sprague-Dawley rats were divided into control group, LPS treatment group, and LPS treatment group treated with GTE two hours after LPS injection. The eyes were monitored by slip lamp and electroretinography examination after 24 hours. The plasma and retina were collected for metabolomics analysis. Results: In LPS treated rats, the iris showed hyperemia. Plasma prostaglandins, arachidonic acids, corticosteroid metabolites, and bile acid metabolites increased. In the retina, histamine antagonists, corticosteroids, membrane phospholipids, free antioxidants, and sugars also increased but fatty acid metabolites, N-acetylglucosamine-6-sulphate, pyrocatechol, and adipic acid decreased. After GTE treatment, the a- and b- waves of electroretinography increased by 13%. Plasma phosphorylcholine lipids increased but plasma prostaglandin E1, cholanic metabolites, and glutarylglycine decreased. In the retina, tetranor-PGAM, pantothenic derivatives, 2-ethylacylcarinitine, and kynuramine levels decreased but anti-oxidative seleno-peptide level increased. Only phospholipids, fatty acids, and arachidonic acid metabolites in plasma and in the retina had significant correlation (p < 0.05, r > 0.4 or r < -0.4). Conclusions: The results showed GTE indirectly induced systemic phosphorylcholine lipids to suppress inflammatory responses, hepatic damage, and respiratory mitochondrial stress in EIU rats induced by LPS. Phospholipids may be a therapeutic target of GTE for anterior chamber inflammation.

The innate immune receptor Nlrp12 suppresses autoimmunity to the retina.

BACKGROUND: Nod-like receptors (NLRs) are critical to innate immune activation and induction of adaptive T cell responses. Yet, their role in autoinflammatory diseases of the central nervous system (CNS) remains incompletely defined. The NLR, Nlrp12, has been reported to both inhibit and promote neuroinflammation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), where its T cell-specific role has been investigated. Uveitis resulting from autoimmunity of the neuroretina, an extension of the CNS, involves a breach in immune privilege and entry of T cells into the eye. Here, we examined the contribution of Nlrp12 in a T cell-mediated model of uveitis, experimental autoimmune uveitis (EAU). METHODS: Mice were immunized with interphotoreceptor retinoid-binding protein peptide 1-20 (IRBP1-20) emulsified in Complete Freund's adjuvant, CFA. Uveitis was evaluated by clinical and histopathological scoring, and comparisons were made in WT vs. Nlrp12-/- mice, lymphopenic Rag1-/- mice reconstituted with WT vs. Nlrp12-/- CD4+ T cells, or among bone marrow (BM) chimeric mice. Antigen-specific Th-effector responses were evaluated by ELISA and intracellular cytokine staining. Cellular composition of uveitic eyes from WT or Nlrp12-/- mice was compared using flow cytometry. Expression of Nlrp12 and of cytokines/chemokines within the neuroretina was evaluated by immunoblotting and quantitative PCR. RESULTS: Nlrp12-/- mice developed exacerbated uveitis characterized by extensive vasculitis, chorioretinal infiltrates and photoreceptor damage. Nlrp12 was dispensable for T cell priming and differentiation of peripheral Th1 or Th17 cells, and uveitis in immunodeficient mice reconstituted with either Nlrp12-/- or WT T cells was similar. Collectively, this ruled out T cells as the source of Nlrp12-mediated protection to EAU. Uveitic Nlrp12-/- eyes had more pronounced myeloid cell accumulation than uveitic WT eyes. Transplantation of Nlrp12-/- BM resulted in increased susceptibility to EAU regardless of host genotype, but interestingly, a non-hematopoietic origin for Nlrp12 function was also observed. Indeed, Nlrp12 was found to be constitutively expressed in the neuroretina, where it suppressed chemokine/cytokine induction. CONCLUSIONS: Our data identify a combinatorial role for Nlrp12 in dampening autoimmunity of the neuroretina. These findings could provide a pathway for development of therapies for uveitis and potentially other autoinflammatory/autoimmune diseases of the CNS.

Preventive effects of cristacarpin on experimentally induced uveitis by targeting NF-κB.

Cristacarpin is a novel prenylated pterocarpan that reportedly exhibits broad anti-cancer activity by enhancing endoplasmic reticulum stress. However, whether and how cristacarpin affects in-flammatory processes remain largely unknown. In the present study, the anti-inflammatory effect of cristacarpin on lipopolysaccharide (LPS)-induced inflammation was investigated using zebrafish embryos, RAW 264.7 macrophages, and mouse uveitis models. In the non-toxic concentration range (from 20 to 100 µM), cristacarpin suppressed pro-inflammatory mediators such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while stimulating anti-inflammatory mediators such as IL-4 and IL-10 in LPS-stimulated RAW 264.7 cells and uveitis mouse models. Cristacarpin decreased cell adhesion of macrophages through downregulation of the expression of Ninjurin1 and matrix metalloproteinases. Furthermore, cristacarpin reduced macrophage migration in zebrafish embryos in vivo. Cristacarpin also increased cytosolic levels of inhibitor of nuclear factor-κB and suppressed the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells. Collectively, our results suggest that cristacarpin is a potential therapeutic candidate for developing ocular anti-inflammatory drugs.

Mesenchymal stem cells moderate experimental autoimmune uveitis by dynamic regulating Th17 and Breg cells response.

Mesenchymal stem cells (MSCs) are adult stem cells from mesoderm with multi potential differentiation, and are being widely studied as a promising treatment for autoimmune diseases. The main inflammatory factors of experimental autoimmune uveitis (EAU) are T helper type 1 (Th1) and Th17. Regulatory B cells (Bregs) are a newly designated B cell subgroup, which has been proved to play a key role in regulating inflammation, autoimmunity and cancer. In this regard, we establish the EAU model by injecting interphotoreceptor retinoid-binding protein combined with complete Freund's adjuvant into the tail vein and bilateral thighs of rats, and inject MSCs or equal volume of phosphate buffer saline intraperitoneally on the day of immunization. Dynamic changes of cell subsets and cytokine expression are tested at different time periods to explore the relationship between MSCs treatment and disease prognosis during EAU course. Our results suggest that compared with the model control group, MSCs treatment can significantly reduce the production of Th1 and Th17 cytokines during EAU, while the production of regulatory B cells (Bregs) cytokines is significantly increased. At the same time, MSCs can reduce the proportion of Th17 in lymphocytes while the proportion of Bregs is elevated, thus inhibiting the differentiation and activity of interleukin in EAU rats. All this results provide more powerful evidence for cell therapy of autoimmune uveitis.

Research Progress on the Mechanism of Natural Product Ingredients in the Treatment of Uveitis.

BACKGROUND: As the spectrum of ophthalmic diseases keeps changing, uveitis has gradually become one of the major blinding eye diseases in the world. In recent years, it has become a research hotspot to select effective components for uveitis treatment from natural drugs. METHODS: We searched PubMed and EMBASE databases for studies written in English as well as Chinese National Knowledge Infrastructure (CNKI), CQVIP, and Wan Fang database for studies written in Chinese (inception through 30 December 2020). RESULTS: Eight kinds of natural product ingredients were included in this article. They were found to not only regulate the expression of cytokines, proliferation, and differentiation of T help cells but also inhibit the damage of cytokines and inflammatory cells to uvea, blood aqueous barrier, and blood retinal barrier. CONCLUSION: Natural product ingredients have their unique advantages in the treatment of uveitis. They have good anti-inflammatory effects without causing serious adverse reactions, which enables them to be promising choices for preventive and therapeutic strategy of uveitis.

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.

Chitosan Oligosaccharides Suppress Nuclear Factor-Kappa B Activation and Ameliorate Experimental Autoimmune Uveoretinitis in Mice.

We investigated the therapeutic potential and mechanism of chitosan oligosaccharides (COS) for experimental autoimmune uveoretinitis (EAU) in mice. EAU was induced in C57/BL6 mice by injection of human interphotoreceptor retinoid-binding protein (IRBP) peptides. At the same time, a high or low dose (20 or 10 mg/kg) of COS or phosphate-buffered saline (PBS) was given to mice daily after EAU induction. We found that mouse EAU is ameliorated by the high-dose COS treatment when compared with PBS treatment. In the retinas of high-dose COS-treated mice, the nuclear translocation of NF-κB subunit (p65) was suppressed, and the expression of several key EAU inflammatory mediators, IFN-γ, TNF-α, IL-1α, IL-4, IL-5, IL-6, IL-10, IL-17 and MCP-1 was lowered. These results suggest that COS may be a potential treatment for posterior uveitis.

Dracocephalum heterophyllum (DH) Exhibits Potent Anti-Proliferative Effects on Autoreactive CD4+ T Cells and Ameliorates the Development of Experimental Autoimmune Uveitis.

Experimental autoimmune uveitis (EAU) is a CD4+ T cell-mediated organ-specific autoimmune disease and has been considered as a model of human autoimmune uveitis. Dracocephalum heterophyllum (DH) is a Chinese herbal medicine used in treating hepatitis. DH suppressed the production of inflammatory cytokines through the recruitment of myeloid-derived suppressor cells (MDSCs) to the liver. However, it remains elusive whether DH can directly regulate CD4+ T cell biology and hence ameliorates the development of CD4+ T cell-mediated autoimmune disease. In the current study, we found that DH extract significantly suppressed the production of pro-inflammatory cytokines by CD4+ T cells. Further study showed that DH didn't affect the activation, differentiation, and apoptosis of CD4+ T cells. Instead, it significantly suppressed the proliferation of conventional CD4+ T cells both in vitro and in vivo. Mechanistic study showed that DH-treated CD4+ T cells were partially arrested at the G2/M phase of the cell cycle because of the enhanced inhibitory phosphorylation of Cdc2 (Tyr15). In addition, we demonstrated that treatment with DH significantly ameliorated EAU in mice through suppressing the proliferation of autoreactive antigen specific CD4+ T cells. Taken together, the current study indicates that DH-mediated suppression of CD4+ T cell proliferation may provide a promising therapeutic strategy for treating CD4+ T cell-mediated diseases.

The role of neuroinflammation in the pathogenesis of glaucoma neurodegeneration.

The chapter is a review enclosed in the volume "Glaucoma: A pancitopatia of the retina and beyond." No cure exists for glaucoma. Knowledge on the molecular and cellular alterations underlying glaucoma neurodegeneration (GL-ND) includes innovative and path-breaking research on neuroinflammation and neuroprotection. A series of events involving immune response (IR), oxidative stress and gene expression are occurring during the glaucoma course. Uveitic glaucoma (UG) is a prevalent acute/chronic complication, in the setting of chronic anterior chamber inflammation. Managing the disease requires a team approach to guarantee better results for eyes and vision. Advances in biomedicine/biotechnology are driving a tremendous revolution in ophthalmology and ophthalmic research. New diagnostic and imaging modalities, constantly refined, enable outstanding criteria for delimiting glaucomatous neurodegeneration. Moreover, biotherapies that may modulate or inhibit the IR must be considered among the first-line for glaucoma neuroprotection. This review offers the readers useful and practical information on the latest updates in this regard.

A cell-permeable peptide inhibitor of p55PIK signaling alleviates ocular inflammation in mouse models of uveitis.

PURPOSE: Previously we developed TAT-N24 as a synthetic cell-permeable peptide inhibitor of p55PIK signaling and demonstrated its anti-inflammatory effects. This study aimed to evaluate the potential of TAT-N24 as a new agent for the treatment of ocular inflammatory diseases. METHODS: The endotoxin-induced uveitis (EIU) model was established by intravitreal injection of lipopolysaccharide (LPS) in BALB/c mice and experimental autoimmune uveitis (EAU) model was established by subcutaneous injection of a peptide spanning amino acid residues 161-180 of interphotoreceptor retinoid binding protein (IRBP161-180) with complete Freund's adjuvant (CFA) in B10.RIII mice. TAT-N24 was topically administered in EIU model and intraperitoneally administered in EAU model. The severity levels of uveitis were assessed by clinical and histopathological scores. The mRNA levels of inflammatory cytokines in iris-ciliary body (ICB) and retina were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein levels of inflammatory factors were determined by ELISA or Western blotting. RESULTS: The results showed that TAT-N24 alleviated clinical signs, decreased inflammatory cell infiltration and the expression of inflammatory cytokines in both EIU and EAU models. Furthermore, protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in aqueous humor and mRNA and protein levels of NF-κB p65 in the ICB significantly decreased in EIU model. In EAU model, TAT-N24 application induced a significant decrease of IFN-gamma (IFN-γ) and interleukin-17 (IL-17) in the retina, which were secreted by Th1 and Th17 cells, respectively. CONCLUSION: In conclusion, TAT-N24 suppressed intraocular inflammation in both EIU and EAU models, and the anti-inflammatory effects were mediated by suppressing the expression of inflammatory cytokines by PI3K/NF-κB signaling pathway. TAT-N24 could be potential candidate for the treatment of ocular inflammatory diseases.

Matairesinol ameliorates experimental autoimmune uveitis by suppression of IRBP-specific Th17 cells.

We investigated the effects of matairesinol (MAT) in the experimental autoimmune uveitis (EAU), a classical animal model of uveitis. We found that treatment with MAT could alleviate intraocular inflammation of EAU. Notably, Th17 cells in eyes of EAU mice could be predominantly restrained by MAT. Furthermore, MAT could inhibit Th17 differentiation in vitro. In addition, MAT inhibited the signaling of MAPK and ROR-γt, a pivotal transcription factor for Th17 cell differentiation in vitro and in vivo. Taken together, these results suggested that MAT had immune-suppressive effects on autoimmune inflammation through Th17 cells.

An Increase of Oxidized Phospholipids and the Role of Macrophages in Intraocular Inflammation.

Purpose: The present study was conducted to examine the profile of oxidized phospholipids (OxPLs) in uveitis using rat model and clinical specimens, and to elucidate the role of macrophages in the metabolism of OxPLs. Methods: Lewis rats were immunized with a bovine interphotoreceptor retinoid- binding protein (bIRBP) peptide with complete Freund's adjuvant (CFA) to induce experimental autoimmune uveitis (EAU). The aqueous humor (AH) was collected 2 weeks after immunization. Fifty-four human AH specimens, among which 21 eyes had a history of chronic uveitis, were collected during their cataract surgery. The profile of OxPLs in the AH specimens were analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). In addition, the involvement of macrophages in the viability of cells treated by OxPLs was investigated through a WST-1 assay using ARPE-19 cells and C57BL/6 mouse alveolar macrophages (AMs). The influence of macrophages in the trend of OxPLs was traced by thin layer chromatography (TLC) using AMs. Results: Six species of OxPLs were detected in the AHs of rats and humans. The content of each OxPL was higher in the uveitis group. Four kinds of OxPLs found in AHs showed cytotoxicity to ARPE-19 cells in a dose-dependent manner. The cytotoxicity was reduced by pretreatment of OxPLs with AMs. When the OxPLs were applied on AMs, a marked reduction of OxPLs in the medium was observed. Conclusions: The OxPLs formed by intraocular inflammation could induce cytotoxicity. The present findings suggest that the phagocytic macrophages emerging in the inflammation site eliminate OxPLs, and prevent intraocular tissue damage following uveitis.

Tetramethylpyrazine attenuates endotoxin-induced retinal inflammation by inhibiting microglial activation via the TLR4/NF-κB signalling pathway.

Ocular inflammation is a common pathological condition of a series of retinal degenerative diseases. Tetramethylpyrazine (TMP), a Chinese herbal extraction, is widely used in the treatment of several ocular diseases in Eastern countries. However, the exact mechanisms correlating the vision protective effects of TMP have not been elucidated. Thus, this study aimed to investigate TMP's molecular targets in anti-inflammatory activity in endotoxin lipopolysaccharide (LPS)-induced retinal inflammation both in vitro and in vivo. The primary cultured retinal microglial cells were pretreated with TMP and then activated by LPS. We found pretreatment with TMP significantly inhibited LPS-induced upregulation of CD68, a marker of mononuclear microglia activation. The morphological changes induced by LPS were also inhibited by the TMP pretreatment. Moreover, Toll like receptor 4 (TLR4), phosphorylation of inhibitor of NF-κB alpha (p-IκB-α) and the translocation of nuclear factor kappa B p65 (NF-κB p65) were significantly downregulated in retinal microglial cells with TMP pretreatment, which indicated that TMP might suppress LPS-induced retinal microglial activation through TLR4/NF-κB signalling pathway. And these results were confirmed in vivo. Pretreatment with TMP inhibited microglial activation, migration and regeneration, especially in ganglion cell layer (GCL). In addition to the inhibition of TLR4, TMP significantly inhibited the translocation of NF-κB p-65 to the nucleus in vivo. The downstream genes of NF-κB, such as the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), were significantly downregulated by TMP pretreatment in the retina. Accordingly, the increased expression of cleaved caspase-3 and the decreased ratio of B-cell lymphoma-2 (Bcl-2) to Bcl-2 associated X Protein (Bax) were significantly attenuated by TMP. TUNEL assay also demonstrated that TMP exerted neuroprotective effects in the retina. Therefore, this study elucidated a novel mechanism that TMP inhibits retinal inflammation by inhibiting microglial activation via a TLR4/NF-κB signalling pathway.

Computational model to analyze and characterize the functional mutations of NOD2 protein causing inflammatory disorder - Blau syndrome.

Blau syndrome (BS), which affects the eyes, skin, and joints, is an autosomal dominant genetic inflammatory disorder. BS is caused by mutations in the NOD2 gene. However, there are no direct treatments, and treatment with conventional anti-inflammatory drugs such as adrenal glucocorticoids, anti-metabolites, and biological agents such as anti-TNF and infliximab have all been attempted with varying degrees of success. In this study, we tried to identify all the reported mutations in the NOD2 protein that cause BS. Collectively, 114 missense mutations were extracted from the UniProt, ClinVar, and HGMD databases. The mutations were further subjected to pathogenic, stability, and conservation analyses. According to these computational analyses, six missense mutations (R334Q, R334W, E383G, E383K, R426H, and T605P) were found to be highly deleterious, destabilizing, and positioned in the conserved position. ADP to ATP conversion plays a crucial role in switching the closed-form of NOD2 protein to the open-form, thus activating the protein. Accordingly, the mutations in the ADP binding sites have received more attention in comparison to the mutations in the non-ADP binding positions. Interestingly, the W490L mutation is positioned in the ADP binding site and exhibits highly deleterious and destabilizing properties. Additionally, W490L was also found to be conserved, with a ConSurf score of 7. Therefore, we further performed homology modeling to determine the 3D structure of native NOD2 and the W490L mutant. Molecular docking analysis was carried out to understand the change in the interaction of ADP with the NOD2 protein. We observed that ADP had a stronger interaction with the native NOD2 protein compared to the W490L mutant. Finally, ADP complexed with native NOD2 and W490L mutant were subjected to molecular dynamics simulations, and the trajectories were analyzed. In the simulations, we observed decreased deviation and fluctuations in native NOD2, whereas decreased compactness and inter- and intramolecular hydrogen bonds were observed in the W490L mutant. This study is expected to serve as a platform for developing targeted drug therapy for BS.

Network pharmacology-based identification of the key mechanism of Qinghuo Rougan Formula acting on uveitis.

BACKGROUND: Qinghuo Rougan Formula (QHRGF) is a traditional Chinese medicine (TCM) that has been widely apllied to treat uveitis for several decades. However, the inhibitory mechanism of QHRGF in uveitis has remained to be an enigma. METHODS: The Chinese herbal medicine pharmacology data and analysis platform wereused to search and screen for the effective components of the QHRGF compound injection and to analyse possible therapeutic targets based on network topology. In addition, various known disease target databases were enraolled, the therapeutic target proteins in uveitis were screened, and a protein-protein interaction (PPI) network was constructed. Enrichment analysis was performed on key nodes. Finally, the inhibitory effect of QHRGF on uveitis was verified by experiments. RESULTS: We identified 259 major candidate targets of QHRGF and successfully constructed a 'QHRGF-compound-target-uveitis' network. Above-mentioned targets revealed by Gene enrichment analysis have played an significant role in the cell cycle, autoimmune disease, apoptosis and related signal pathways. We demonstrated that QHRGF attenuates local inflammation in experimental autoimmune uveoretinitis (EAU) rats by regulating natural killer T (NKT) cells and inhibiting MAPK signal pathways. CONCLUSION: QHRGF may regulate the local immune response and inflammatory factors mainly through the MAPK signal pathway. For autoimmune uveitis, QHRGF may be a promising, long-lasting treatment strategy.

Green tea catechins alleviate autoimmune symptoms and visual impairment in a murine model for human chronic intraocular inflammation by inhibiting Th17-associated pro-inflammatory gene expression.

Autoimmune uveitis is a sight-threatening disease mainly caused by dysregulation of immunity. We investigated the therapeutic effects of green tea extract (GTE) and its major component, epigallocatechin-3-gallate (EGCG), on a murine model of experimental autoimmune uveoretinitis (EAU). Oral administration of GTE, EGCG, dexamethasone, or water, which started 5 days before the induction, was fed every two days to each group. On day 21 post induction, the eyes were examined by confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT), fundus fluorescein angiography (FFA) and electroretinography (ERG) prior to sacrificing the animals for histological assessments and gene expression studies. Retinal-choroidal thicknesses (RCT) and major retinal vessel diameter were measured on OCT sections and FFA images, respectively. Comparing to water-treated EAU animals, GTE attenuated uveitis clinical manifestations, RCT increase (1.100 ± 0.013 times vs 1.005 ± 0.012 times, P < 0.001), retinal vessel dilation (308.9 ± 6.189 units vs 240.8 units, P < 0.001), ERG amplitudes attenuation, histopathological ocular damages, and splenomegaly in EAU mice. The therapeutic effects of GTE were dose dependent and were comparable to dexamethasone. EGCG, a major active constituent of GTE, partially alleviated uveitic phenotypes including recovering visual function. Th-17 associated pro-inflammatory gene [interleukin 1 beta (IL-1ß), IL-6, IL-17A, and tumor necrosis factor alpha (TNF-α)] expressions were down regulated by GTE and EGCG treatments, which showed no detectable morphological defects in liver and kidney in non-induced and EAU mice. Our findings suggest that GTE consumption can serve as a potent therapeutic agent as well as a food supplement for developing alternative treatments against autoimmune uveitis.

Familial Blau syndrome:First molecularly confirmed report from India.

Blau syndrome (BS) is a rare autoinflammatory disorder characterized by the clinical triad of arthritis, uveitis, and dermatitis due to heterozygous gain-of-function mutations in the NOD2 gene. BS can mimic juvenile idiopathic arthritis (JIA)-associated uveitis, rheumatoid arthritis, and ocular tuberculosis. We report a family comprising a mother and her two children, all presenting with uveitis and arthritis. A NOD2 mutation was confirmed in all the three patients - the first such molecularly proven case report of familial BS from India.

Amelioration of Endotoxin-Induced Inflammatory Toxic Response by a Metal Chelator in Rat Eyes.

Purpose: Metal ions play a key role in exacerbating toxicity associated with oxidative stress and inflammation. This study examines the effects of a formulation containing the metal chelator ethylenediaminetetraacetic acid (EDTA) and permeability enhancer methyl sulfonyl methane (MSM) on the early course of inflammation in endotoxin-induced uveitis (EIU). The proprietary MSM/EDTA formulation of Livionex, Inc., which was used for this study, is covered by several patents and pending patent applications. Methods: EIU was induced by using subcutaneous injection of lipopolysaccharide (LPS) into the thighs of Lewis rats. Treatment consisted of topical application to the eyes of either PBS or eye drops designated as ME that contain EDTA and MSM. Clinical signs of uveitis were monitored at 6 and 24 hours postinjection. Oxidative and inflammatory markers were evaluated by ELISA or immunohistochemistry. Results: Rats treated with ME showed fewer clinical signs of uveitis including reduced miosis, fibrinous exudates, and dilated blood vessels. The aqueous humor of treated rats contained fewer leukocytes, lower protein levels, and less PGE2. Formation of protein adducts with the lipid peroxidation end-product, 4-hydroxynonenal, expression of NF-κB, TNF-α, and MMP-9 were all reduced in rats treated with ME. Conclusions: Our results indicate that ME eye drops downregulate the ocular inflammatory response in LPS treated rats, suggesting that induction of EIU involves metal ions and chelation therapy with ME is a potential treatment for uveitis.