Experimental Autoimmune Uveoretinitis Research Articles

Defining mesenchymal stem/stromal cell-induced myeloid-derived suppressor cells using single-cell transcriptomics

Mesenchymal stem/stromal cells (MSCs) modulate the immune response through interactions with innate immune cells. We previously demonstrated that MSCs alleviate ocular autoimmune inflammation by directing BM cell differentiation from pro-inflammatory CD11bhiLy6ChiLy6Glo cells into immunosuppressive CD11bmidLy6CmidLy6Glo cells. Herein, we analyzed MSC-induced CD11bmidLy6Cmid cells using single-cell RNA sequencing and compared them with CD11bhiLy6Chi cells. Our investigation revealed 7 distinct immune cell types including myeloid-derived suppressor cells (MDSCs) in the CD11bmidLy6Cmid cells, while CD11bhiLy6Chi cells included mostly monocytes/macrophages with a small cluster of neutrophils. These MSC-induced MDSCs highly expressed Retnlg, Cxcl3, Cxcl2, Mmp8, Cd14 and Csf1r as well as Arg1. Comparative analyses of CSF-1RhiCD11bmidLy6Cmid and CSF-1RloCD11bmidLy6Cmid cells demonstrated that the former had a homogeneous monocyte morphology and produced elevated levels of IL-10. Functionally, these CSF-1RhiCD11bmidLy6Cmid cells, compared with the CSF-1RloCD11bmidLy6Cmid cells, inhibited CD4+ T cell proliferation and promoted CD4+CD25+Foxp3+ Treg expansion in culture and in a mouse model of experimental autoimmune uveoretinitis. RELM-γ encoded by Retnlg, one of the highly-upregulated genes in MSC-induced MDSCs, had no direct effects on T cell proliferation, Treg expansion or splenocyte activation. Together, our study revealed a distinct transcriptional profile of MSC-induced MDSCs and identified CSF-1R as a key cell-surface marker for detection and therapeutic enrichment of MDSCs.

Brain‐derived neurotrophic factor alleviates clinical symptoms of experimental autoimmune uveoretinitis and supports retinal regeneration

Purpose: Brain‐derived neurotrophic factor (BDNF) is one of the most important neurotrophins that has a fundamental role in the regulation of neuronal growth, proliferation, and regeneration, as well as an important modulatory effect on the immune cells. In addition, BDNF potentiates the neurodifferentiation of Müller cells (MCs), thus supporting retinal regeneration. Therefore, we aimed to investigate the anti‐inflammatory effect of BDNF in experimental autoimmune uveoretinitis (EAU), a severe neuroinflammatory condition, and whether BDNF could facilitate the regenerative capacity of MCs in EAU.Methods: EAU in mice was induced by s.c. administration of retinal antigen IRBP1‐20 emulsified in a complete Freud's adjuvant, followed by i.v. administration of Pertussis toxin. BDNF was administered daily in the form of eye drops from day 8 to day 14 after immunization. On day 14, fundoscopy was performed to assess clinical signs of EAU. Mice were then euthanized, and their eyes and retinas were isolated for further Western blot, RT‐qPCR, flow cytometry, and immunohistochemistry analyses.Results: The treatment with BDNF significantly improved the clinical manifestations of EAU, including optic disc oedema, vasculitis, and inflammatory infiltrates in the retina. BDNF significantly decreased the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ myeloid cells into the retina and also suppressed the expression of several pro‐inflammatory cytokines and chemokines. In addition, BDNF administration significantly increased the expression of markers of neurogenesis in retinal MCs.Conclusions: Together, our results demonstrate that BDNF has important anti‐inflammatory and neurogenic properties and that it may serve as a promising target for developing novel therapeutic strategies for neurodegenerative and neuroinflammatory diseases of the retina.Support.This study was supported by the following grant projects: GAUK 378421, 260 648/SVV/2023, and COOPERATIO Neurosciences.

Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.

Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

Targeted delivery of autoantigen to dendritic cells prevents development of spontaneous uveitis.

Restoration of immunological tolerance to self antigens has been a major drive in understanding the mechanisms of, and developing new treatments for, autoimmune and autoinflammatory disease. Sessile dendritic cells (DC) are considered the main instruments underpinning immunological tolerance particularly the CD205+ (DEC205+) cDC1 subset in contrast to DCIR2+ cDC2 which mediate immunogenicity. Targeting DC using autoantigen peptide-antibody fusion proteins has been a well explored methodology for inducing tolerance. Here we show that subcutaneous (s.c.) inoculation of hen-egg lysozyme (HEL)-DEC205 Ig fusion prevents the development of spontaneous uveoretinitis (experimental autoimmune uveoretinitis, EAU) in a transgenic mouse model generated by crossing interphotoreceptor retinol binding protein (IRBP)-HEL (sTg HEL) with HEL specific TCR (sTg TCR) mice. Prolonged suppression of EAU required injections of HEL-DEC205 Ig once weekly, reflecting the half life of s.c. DC. Interestingly, HEL-DCIR2 Ig also had a suppressive effect on development of EAU but less so than DEC205 Ig while it had minimal effect on preventing the retinal atrophy associated with EAU. In addition, HEL-DEC205 Ig was only effective when administered s.c. rather than systemically and had no effect on EAU induced by adoptive transfer of HEL-activated T cells. These data demonstrate the importance of systemic (lymph node) rather than local (eye) antigen presentation in the development of EAU as well as suggest a potential therapeutic approach to controlling sight-threatening immune-mediated uveitis provided relevant antigen(s) can be identified.

Stimulating the Melanocortin System in Uveitis and Diabetes Preserves the Structure and Anti-Inflammatory Activity of the Retina.

The endogenous neuropeptide α-Melanocyte Stimulating Hormone (α-MSH) is a potent suppressor of inflammation and has an essential role in maintaining the normal anti-inflammatory microenvironment of the retina. While the therapeutic use of α-MSH peptide in uveitis and diabetic retinopathy models has been demonstrated, its short half-life and instability limit its use as a therapeutic drug. A comparable analog, PL-8331, which has a stronger affinity to melanocortin receptors, longer half-life, and, so far, is functionally identical to α-MSH, has the potential to deliver melanocortin-based therapy. We examined the effects of PL-8331 on two mouse models of retinal disease, Experimental Autoimmune Uveoretinitis (EAU) and Diabetic Retinopathy (DR). PL-8331 therapy applied to mice with EAU suppressed EAU and preserved retinal structures. In diabetic mice, PL-8331 enhanced the survival of retinal cells and suppressed VEGF production in the retina. In addition, retinal pigment epithelial cells (RPE) from PL-8331-treated diabetic mice retained normal anti-inflammatory activity. The results demonstrated that the pan-melanocortin receptor agonist PL-8331 is a potent therapeutic drug to suppress inflammation, prevent retinal degeneration, and preserve the normal anti-inflammatory activity of RPE.

Colitis in a transgenic mouse model of autoimmune uveitis may be induced by neoantigen presentation in the bowel

Undifferentiated uveitis (intraocular inflammation, IOI) is an idiopathic sight-threatening, presumed autoimmune disease, accountable for ~ 10% of all blindness in the developed world. We have investigated the association of uveitis with inflammatory bowel disease (IBD) using a mouse model of spontaneous experimental autoimmune uveoretinitis (EAU). Mice expressing the transgene (Tg) hen egg lysozyme (HEL) in the retina crossed with 3A9 mice expressing a transgenic HEL-specific TCR spontaneously develop uveoretinitis at post-partum day (P)20/21. Double transgenic (dTg TCR/HEL) mice also spontaneously develop clinical signs of colitis at ~ P30 with diarrhoea, bowel shortening, oedema and lamina propria (LP) inflammatory cell infiltration. Single (s)Tg TCR (3A9) mice also show increased histological LP cell infiltration but no bowel shortening and diarrhoea. dTg TCR/HEL mice are profoundly lymphopenic at weaning. In addition, dTg TCR/HEL mice contain myeloid cells which express MHC Class II-HEL peptide complexes (MHCII-HEL), not only in the inflamed retina but also in the colon and have the potential for antigen presentation. In this model the lymphopenia and reduction in the absolute Treg numbers in dTg TCR/HEL mice is sufficient to initiate eye disease. We suggest that cell-associated antigen released from the inflamed eye can activate colonic HEL-specific T cells which, in a microbial micro-environment, not only cause colitis but feedback to amplify IOI.

The role of microbiome in uveitis in experimental mice model

AbstractUnlike the individual's genetic makeup, gut microbiota can be influenced, and thus used in the disease therapy or even prevention. We have shown that absence or reduction of microbiota modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). Our further purpose of the project was to test influence of probiotics on intensity of intraocular inflammation in EAU. Uveitis was induced in C57BL/6J mice by interphotoreceptor retinoid binding protein in complete Freund's adjuvant and with pertussis toxin. The grade of uveitis was assessed in vivo by using otoscope connected to camera and post mortem on haematoxylin and eosin stained tissues. Bacteria Escherichia coli were administered orally three times a week and saline solution was administered to control group. Different regimens of administration were tested. Interestingly, mice administered with probiotics reached statistically significant lower grade of uveitis in comparison to the control group in clinical and histological evaluation. This may have valuable implications for the control of sight‐threatening uveitis in patients.Supported by research project AZV MZ CR 17‐31248A.Key words: microbiome, probiotics, Escherichia coli, experimental autoimmune uveitis, EAU.

Genetic Ablation of Nrf2 Exacerbates Neuroinflammation in Ocular Autoimmunity.

Experimental autoimmune uveoretinitis (EAU) is an animal model of non-infectious uveitis and is developed by immunization with retinal antigen, interphotoreceptor retinoid-binding protein (IRBP). Nuclear factor erythroid 2- (NF-E2-) related factor 2 (Nrf2) is responsible for regulating antioxidant and inflammatory responses. In this study, we investigated the role of Nrf2 on the development of EAU. Clinical and pathological examination demonstrated that retinal inflammation was exacerbated in Nrf2 knockout (Nrf2 KO) mice compared to wild type (WT) mice, and the expression of inflammatory cytokines (IFN-γ, IL-6, and IL-17) in the retina was significantly elevated in Nrf2 KO mice. GFAP positive cells (astrocytes) and Iba-1 positive cells (microglia cells) in the retina were more numerous in Nrf2 KO mice compared to WT mice. Furthermore, we examined the suppressive effect of the Nrf2 activator CDDO-Im (2-cyano-3,12 dioxooleana-1,9 dien-28-oyl imidazoline) on the development of EAU. The treatment with CDDO-Im significantly reduced the clinical and pathological score of EAU compared to those of vehicle-treated mice. These findings suggest that Nrf2 plays a regulatory role in the pathogenesis of autoimmune uveoretinitis and the activation of the Nrf2 system may have therapeutic potential for protecting vision from autoimmune neuroinflammation.

MHC Class I Enables MSCs to Evade NK-Cell-Mediated Cytotoxicity and Exert Immunosuppressive Activity.

Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.

PD-1 Targeted Nanoparticles Inhibit Activated T Cells and Alleviate Autoimmunity via Suppression of Cellular Energy Metabolism Mediated by PKM2.

BackgroundEffector T cells, especially T helper 1 (Th1) cells and T helper 17 (Th17) cells, are involved in the pathogenesis of many autoimmune diseases such as uveitis. Under hyperactive immune conditions, these effector T cells pathologically maintain a high expression level of programmed cell death protein 1 (PD-1) receptors and distinctively engage aerobic glycolysis via cellular energy metabolism mediated by pyruvate kinase M2 (PKM2). Therefore, we proposed that the synergy of metabolic inhibition and receptor guidance might target and down-regulate these hyperactive effector T cells to achieve anti-immune effects.MethodsPD-1 antibody and TEPP-46 were integrated by polyethylene glycol (PEG) modified poly (lactic-co-glycolic acid) (PLGA) as a nanoplatform (TPP). Characteristics of TPP were basically detected. The biosafety of TPP was evaluated in vitro and in vivo. The targeting effect of TPP was detected by laser scanning confocal microscopy and flow cytometry (FCM). Interleukin-2 (IL-2)/interleukin-17A (IL-17A)/interferon-gamma (IFN-γ) producing cells were detected by FCM. Experimental autoimmune uveoretinitis (EAU) was induced in C57BL/6J mice as the inflammatory model.ResultsTPP had homogeneous distribution, good stability in vitro, and high biosafety in vitro and in vivo. Encapsulated TEPP-46 showed a sustained release profile with burst, steady and slow release periods. Early activation and proliferation of effector T cells was inhibited by TPP treatment in vitro. Th1 and Th17 cells were suppressed by TPP in vitro and in vivo. EAU was alleviated in mice by systemic administration of TPP.ConclusionThe novel nanoplatform TPP could suppress Th1 and Th17 cells and exhibited an anti-inflammatory effect on EAU, providing an alternative approach to ameliorate autoimmune diseases mediated by these cells.

Depletion of Retinal Dopaminergic Activity in a Mouse Model of Rod Dysfunction Exacerbates Experimental Autoimmune Uveoretinitis: A Role for the Gateway Reflex.

The gateway reflex is a mechanism by which neural inputs regulate chemokine expression at endothelial cell barriers, thereby establishing gateways for the invasion of autoreactive T cells into barrier-protected tissues. In this study, we hypothesized that rod photoreceptor dysfunction causes remodeling of retinal neural activity, which influences the blood–retinal barrier and the development of retinal inflammation. We evaluated this hypothesis using Gnat1rd17 mice, a model of night blindness with late-onset rod-cone dystrophy, and experimental autoimmune uveoretinitis (EAU). Retinal remodeling and its effect on EAU development were investigated by transcriptome profiling, target identification, and functional validation. We showed that Gnat1rd17 mice primarily underwent alterations in their retinal dopaminergic system, triggering the development of an exacerbated EAU, which was counteracted by dopamine replacement with L-DOPA administered either systemically or locally. Remarkably, dopamine acted on retinal endothelial cells to inhibit NF-κB and STAT3 activity and the expression of downstream target genes such as chemokines involved in T cell recruitment. These results suggest that rod-mediated dopamine release functions in a gateway reflex manner in the homeostatic control of immune cell entry into the retina, and the loss of retinal dopaminergic activity in conditions associated with rod dysfunction increases the susceptibility to autoimmune uveitis.

Osteopontin is a biomarker for early autoimmune uveoretinitis.

Osteopontin (OPN) is an extracellular matrix protein with a diverse range of functions, including roles in cell adhesion, migration, and immunomodulation, which are associated with the modulation of neuroinflammation in the central nervous system. The present study was performed to evaluate the involvement of OPN in the eyes of an experimental autoimmune uveoretinitis (EAU) model. The EAU model was developed by immunization of Lewis rats with interphotoreceptor retinoid-binding protein. The results showed the OPN level was remarkably upregulated in the eye of EAU rats on day 9 post-immunization. The level of CD44, a ligand of OPN, was increased in the ciliary body of EAU rats. Furthermore, OPN was also detected in the ciliary body and activated microglia/macrophages in the EAU retina. The results suggest that OPN was significantly upregulated in the eyes of EAU rats, and that it may be useful as an early biomarker of ocular autoimmune diseases. All animal experiments were approved by the Institutional Animal Care and Use Committee of Jeju National University (approval No. 2020-0012) on March 11, 2020.

Beneficial effect of dimethyl fumarate on experimental autoimmune uveitis is dependent of pro-inflammatory markers immunomodulation.

Autoimmune uveitis is an inflammatory disease of the eye and is one of the major causes of blindness worldwide. Experimental autoimmune uveoretinitis (EAU) constitutes an animal disease model of human endogenous uveitis. In our study, we investigated the immunomodulatory effect of dimethyl fumarate (DMF) using bovine retinal extract-induced uveitis in a Female Wistar rats. To evaluate the in vivo efficacy, Female Wistar rats were divided into seven experimental groups: control group (n = 5), consisting of non-immunized animals; Uveoretinitis (n = 5), and DMF/Uveoretinitis groups (n = 15), which received a subcutaneous injection of bovine retinal extract emulsified in complete Freund's adjuvant; MC group (n = 5), treated by daily intragastric administration of methylcellulose 0.08% in tap water; DMF group, consisting of control positive group, rats received daily oral gavage administration of 500μL of dimethyl fumarate at 100mg/Kg dissolved in 0.08% methylcellulose in tap water (n = 5). On day 14 post immunization, the rats were then euthanized and associated indications were investigated to evaluate the therapeutic efficacy. Nitric oxide (NO) and TNF-α were assessed in plasma. Meanwhile, eyes were collected for histological and immunohistochemical studies. The retinal expression of iNOS, CD68, CD20, CD25, CD4, and CD8 was examined. Interestingly, DMF enhanced a significant reduction of NO and TNF-α production in the treated group. This effect was strongly related to the histological structure of eyes improvement. In the same context, a significant decrease of iNOS, CD68, and CD20 expression and CD25 increase expression were reported in retinal tissue of DMF/Uveoretinitis group in comparison to the immunized group. Collectively, our results indicate that DMF treatment has a beneficial effect in experimental autoimmune uveoretinitis and could constitute a good candidate for monitoring an ocular inflammatory diseases.

Macrophage-like iPS-derived Suppressor Cells Reduce Th1-mediated Immune Response to a Retinal Antigen

ABSTRACT Purpose To investigate the immunotherapeutic effects of macrophage-like induced pluripotent stem (iPS) cell-derived suppressor cells (SCs) in ocular immune response and experimental autoimmune uveoretinitis (EAU). Methods The genes of Oct3/4, Sox2, Klf4, and c-Myc were transferred to B cells enriched from the spleen cells of C57BL/6 mice by using retrovirus vectors. Transferred B cells were cultured for 17 days to obtain colonies of iPS cells. Through additional steps, iPS-SCs were induced. An antigen-specific T cell proliferation assay was performed with CD4+ T cells collected from draining lymph nodes of the mice immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide and co-cultured with iPS-SCs. Cytokine concentrations in the culture supernatant were examined. Mice were immunized with hIRBP peptide to induce EAU. The iPS-SCs were administered into the mice one day before the induction of EAU. Results The iPS-SCs decreased hIRBP-specific T cell proliferation depending on the number of cells. Productions of tumor necrosis factor-α and interferon-γ were significantly decreased; however, transforming growth factor-β1, nitric oxide, interleukin (IL)-13, IL-17A, and IL-17 F levels were elevated in the supernatant when the collected T cells were co-cultured with iPS-SCs. The iPS-SCs had immunosuppressant effects even without cell-to-cell contact, and their effects were non-specific to the antigen preloaded on iPS-SCs. EAU was significantly milder in the mice administered iPS-SCs prior to immunization. Conclusions Macrophage-like iPS-SCs reduced Th1 immune response to a retinal antigen and Th1-mediated EAU in mice. These results showed the possibility of the application of iPS technology to the treatment of noninfectious ocular inflammation, endogenous uveitis, in the future.

Dynamics of Cyclooxygenase-1 Positive Microglia/Macrophage in the Retina of Pathological Model Mice as a Biomarker of the Retinal Inflammatory Diseases.

In an intraocular inflammatory state, microglia residing in the retina become active and migrate inside the retina. In this study, we investigated whether cyclooxygenase-1 (COX-1) expressed by retinal microglia/macrophage can be a biomarker for the diagnosis of retinal diseases. COX-1 was immunopositive in microglia/macrophage and neutrophils, while COX-2 was immunopositive in astrocytes and neurons in the inner layer of normal retina. The number of COX-1 positive cells per section of the retinal tissue was 14 ± 2.8 (mean ± standard deviation) in normal mice, which showed significant increase in the lipopolysaccharide (LPS)-administrated model (62 ± 5.0, p = 8.7 × 10−9). In addition to microglia, we found neutrophils that were positive for COX-1. In the early stage of inflammation in the experimental autoimmune uveoretinitis (EAU), COX-1 positive cells, infiltrating from the ciliary body into the retinal outer nuclear layer, were observed. The number of infiltrating COX-1 positive cells correlated with the severity of EAU. Taken together, the increased number of COX-1 positive microglia/macrophage with morphological changes were observed in the retinas of retinal inflammatory disease models. This suggests that COX-1 can be a marker of disease-related activities of microglia/macrophage, which should be useful for the diagnosis of retinal diseases.

The protective function of invariant natural killer T cells in the relapse of experimental autoimmune uveoretinitis

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

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.

Low-dose 2-deoxy glucose stabilises tolerogenic dendritic cells and generates potent in vivo immunosuppressive effects

Cell therapies for autoimmune diseases using tolerogenic dendritic cells (tolDC) have been promisingly explored. A major stumbling block has been generating stable tolDC, with low risk of converting to mature immunogenic DC (mDC), exacerbating disease. mDC induction involves a metabolic shift to lactate production from oxidative phosphorylation (OXPHOS) and β-oxidation, the homeostatic energy source for resting DC. Inhibition of glycolysis through the administration of 2-deoxy glucose (2-DG) has been shown to prevent autoimmune disease experimentally but is not clinically feasible. We show here that treatment of mouse bone marrow-derived tolDC ex vivo with low-dose 2-DG (2.5 mM) (2-DGtolDC) induces a stable tolerogenic phenotype demonstrated by their failure to engage lactate production when challenged with mycobacterial antigen (Mtb). ~ 15% of 2-DGtolDC express low levels of MHC class II and 30% express CD86, while they are negative for CD40. 2-DGtolDC also express increased immune checkpoint molecules PDL-1 and SIRP-1α. Antigen-specific T cell proliferation is reduced in response to 2-DGtolDC in vitro. Mtb-stimulated 2-DGtolDC do not engage aerobic glycolysis but respond to challenge via increased OXPHOS. They also have decreased levels of p65 phosphorylation, with increased phosphorylation of the non-canonical p100 pathway. A stable tolDC phenotype is associated with sustained SIRP-1α phosphorylation and p85-AKT and PI3K signalling inhibition. Further, 2-DGtolDC preferentially secrete IL-10 rather than IL-12 upon Mtb-stimulation. Importantly, a single subcutaneous administration of 2-DGtolDC prevented experimental autoimmune uveoretinitis (EAU) in vivo. Inhibiting glycolysis of autologous tolDC prior to transfer may be a useful approach to providing stable tolDC therapy for autoimmune/immune-mediated diseases.

Anti-CD40 monoclonal antibody ameliorates experimental autoimmune uveoretinitis in mice.

To investigate the effects of CD40 on ocular inflammation in experimental autoimmune uveoretinitis (EAU) in B10.RIII mice. EAU-susceptible B10.RIII mice were subcutaneously immunized with interphotoreceptor retinoid-binding protein (IRBP) 161-180 in complete Freund's adjuvant and evaluated clinically and pathologically on days 7, 14, 21, 28, and 35 postimmunization. Anti-CD40 antibody was intraperitoneally injected into mice every other day from days 7 to 14 postimmunization. Phosphate-buffered saline (PBS)-injected EAU mice were used as the controls. The frequencies of CD11c+ CD40+ dendritic cells (DCs), CD11c+ MHC-II+ DCs, and CD11c+ CD40+ MHC-II+ DCs in splenocytes were evaluated by flow cytometry on days 0, 7, 14, and 21 after immunization. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in CD11c+ DCs was assessed by ELISA. IRBP-specific lymphocyte proliferation was assessed using a modified MTT cell proliferation assay. The number of CD11c+ CD40+ DCs, CD11c+ MHC-II+ DCs, and CD11c+ CD40+ MHC-II+ DCs increased at the onset of EAU, peaked at the height of disease severity, and was sustained at a high level until day 21. Treatment with anti-CD40 antibody significantly alleviated clinical and pathological activities related to EAU. Compared with the control mice, antibody-treated EAU mice showed few CD11c+ CD40+ DC and CD11c+ CD40+ MHC-II+ DC frequencies in splenocytes. The anti-CD40 antibody significantly suppressed IRBP-specific lymphocyte proliferation and TNF-α and IL-6 production by DCs in EAU mice. The increased expression of CD40 and major histocompatibility complex (MHC) class II molecules in the splenocytes of EAU mice were correlated with inflammatory activity. Anti-CD40 treatment can significantly attenuate EAU activity by inhibiting systemic IRBP-specific immune responses.

Mesenchymal stromal cells induce distinct myeloid-derived suppressor cells in inflammation.

Mesenchymal stem/stromal cells (MSCs) regulate immunity through myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of immature myeloid cells with phenotypic and functional diversity. Herein, we identified a distinct subset of MDSCs induced by MSCs in the BM under inflammatory conditions. MSCs directed the differentiation of Ly6Glo BM cells from CD11bhiLy6Chi cells to CD11bmidLy6Cmid cells both in cell contact-independent and -dependent manners upon GM-CSF stimulation in vitro and in mice with experimental autoimmune uveoretinitis (EAU). RNA-Seq indicated that MSC-induced CD11bmidLy6CmidLy6Glo cells had a distinct transcriptome profile from CD11bhiLy6ChiLy6Glo cells. Phenotypic, molecular, and functional analyses showed that CD11bmidLy6CmidLy6Glo cells differed from CD11bhiLy6ChiLy6Glo cells by low expression of MHC class II and costimulatory molecules and proinflammatory cytokines, high production of immunoregulatory molecules, lack of change in response to LPS, and inhibition of T cell proliferation and activation. Consequently, adoptive transfer of MSC-induced CD11bmidLy6CmidLy6Glo cells significantly attenuated the development of EAU in mice. Further mechanistic study revealed that suppression of prostaglandin E2 (PGE2) and HGF secretion in MSCs by siRNA transfection partially reversed the effects of MSCs on MDSC differentiation. Altogether, data demonstrate that MSCs drive the differentiation of BM cells toward CD11bmidLy6CmidLy6Glo MDSCs, in part through HGF and COX-2/PGE2, leading to resolution of ocular autoimmune inflammation.