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

ABSTRACT: 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-?B subunit (p65) was suppressed, and the expression of several key EAU inflammatory mediators, IFN-?, TNF-?, IL-1?, 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.

SUBMITTER: Hsu SM

PROVIDER: S-EPMC7664198 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Hsu Sheng-Min SM Yang Chang-Hao CH Tsai Hsien-Yang HY Lin Chia-Jhen CJ Fang Yi-Hsuan YH Shieh Chi-Chang CC Chen Shun-Hua SH

International journal of molecular sciences 20201106 21

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 tre ...[more]

PMID: 33171990

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Project description:BACKGROUND: Recent reports show that gene therapy may provide a long-term, safe and effective intervention for human diseases. In this study, we investigated the effectiveness of adeno-associated virus 2 (AAV2) based human interferon-alpha (hIFN-?) gene therapy in experimental autoimmune uveoretinitis (EAU), a classic model for human uveitis. METHODOLOGY/PRINCIPAL FINDINGS: An AAV2 vector harboring the hIFN-? gene (AAV2.hIFN-?) was subretinally injected into B10RIII mice at two doses (1.5×10(6) vg, 1.5×10(8) vg). AAV2 vector encoding green fluorescent protein (AAV2.GFP) was used as a control (5×10(8) vg). The expression of hIFN-? in homogenized eyes and serum was detected by ELISA three weeks after injection. The biodistribution of vector DNA in the injected eyes, contralateral eyes and distant organs was determined by PCR. EAU was induced by immunization with IRBP(161-180) three weeks following vector injections, and evaluated clinically and pathologically. IRBP-specific proliferation and IL-17 expression of lymphocytes from the spleen and lymph nodes were assayed to test the influence of the subretinal delivery of AAV2.hIFN-? on the systemic immune response. hIFN-? was effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2.hIFN-? vector. DNA of AAV2.GFP was observed only in the injected eyes, but not in the distant organs or contralateral eyes. Subretinal injection of both doses significantly attenuated EAU activity clinically and histologically. For the lower dose, there was no difference concerning lymphocyte proliferation and IL-17 production among the AAV2.hIFN-?, AAV2.GFP and PBS injected mice. However, the higher dose of AAV2.hIFN-? significantly suppressed lymphocyte proliferation and IL-17 production. CONCLUSIONS/SIGNIFICANCE: Subretinal delivery of AAV2.hIFN-? lead to an effective expression within the eye for at least three months and significantly attenuated EAU activity. AAV2.hIFN-? was shown to inhibit the systemic IRBP-specific immune response.

Project description:BACKGROUND: Advances in gene transfer techniques have provided long-term, safe and stable transduction of retinal cells following subretinal injection with adeno-associated viral (AAV) vectors. In this study we investigated whether subretinal injection of AAV2-murine IL-27p28 vector was effective in inhibiting experimental autoimmune uveoretinitis (EAU) induced in B10RIII mice. METHODOLOGY/PRINCIPAL FINDINGS: An AAV2 vector encoding the murine IL-27p28 gene (rAAV2-CMV-mIL-27p28) was prepared and subretinally injected into B10RIII mice (4.35×10(8) vector genome (v.g.)). AAV2 vector mediating green fluorescent protein (rAAV2-CMV-GFP) served as a control (5×10(8) v.g.). The concentration of mIL-27p28 in homogenized eyes and serum was assayed by enzyme linked immunosorbent assay (ELISA) after subretinal injection. Human IRBP(161-180) peptide and Complete Freund's Adjuvant were injected into mice receiving either the rAAV2-CMV-mIL-27p28 or rAAV2-CMV-GFP vector. EAU was evaluated clinically and pathologically. The level of IL-17 and IL-10 in homogenized eyes was measured on day 12 and day 21 following immunization. Delayed type hypersensitivity (DTH) and IRBP(161-180)-specific proliferation of lymphocytes from the spleen and lymph nodes were assayed to examine the influence of the subretinal delivery of rAAV2-CMV-mIL-27p28 on the systemic immune response. IL-27p28 was detectable by ELISA within the eyes from two weeks following subretinal injection of the rAAV2-CMV-mIL-27p28 vector and showed a sustained high expression from day 14 to 9 months with a highest expression at 5 months. Subretinal injection of the vector significantly attenuated the severity of EAU disease clinically and pathologically in association with a significantly decreased IL-17 expression and an increased IL-10 expression. The IL-27p28 vector did not affect the systemic immune response, as determined by DTH and IRBP(161-180)-specific lymphocyte proliferation. CONCLUSIONS/SIGNIFICANCE: A high and stable expression of IL-27p28 was observed for at least 9 months following subretinal delivery of rAAV2-CMV-mIL-27p28. The amelioration of EAU disease severity was associated with a decreased IL-17 expression and an increased IL-10 expression.

Project description:Despite advances in clinical imaging and grading our understanding of retinal immune responses and their morphological correlates in experimental autoimmune uveoretinitis (EAU), has been hindered by the requirement for post-mortem histology. To date, monitoring changes occurring during EAU disease progression and evaluating the effect of therapeutic intervention in real time has not been possible. We wanted to establish whether optical coherence tomography (OCT) could detect intraretinal changes during inflammation and to determine its utility as a tool for accurate scoring of EAU. EAU was induced in C57BL/6J mice and animals evaluated after 15, 26, 36 and 60 days. At each time-point, contemporaneous Spectralis-OCT scanning, topical endoscopic fundal imaging (TEFI), fundus fluorescein angiography (FFA) and CD45-immunolabelled histology were performed. OCT features were further characterised on retinal flat-mounts using immunohistochemistry and 3D reconstruction. Optic disc swelling and vitreous opacities detected by OCT corresponded to CD45+ cell infiltration on histology. Vasculitis identified by FFA and OCT matched perivascular myeloid and T-cell infiltrates and could be differentiated from unaffected vessels. Evolution of these changes could be followed over time in the same eye. Retinal folds were visible and found to encapsulate mixed populations of activated myeloid cells, T-cells and microglia. Using these features, an OCT-based EAU scoring system was developed, with significant correlation to validated histological (Pearson r(2) = 0.6392, P<0.0001, n = 31 eyes) and TEFI based scoring systems (r(2) = 0.6784, P<0.0001). OCT distinguishes the fundamental features of murine EAU in vivo, permits dynamic assessment of intraretinal changes and can be used to score disease severity. As a result, it allows tissue synchronisation with subsequent cellular and functional assessment and greater efficiency of animal usage. By relating OCT signals with immunohistochemistry in EAU, our findings offer the opportunity to inform the interpretation of OCT changes in human uveitis.

Project description:The melanocortin system plays an essential role in the regulation of immune activity. The anti-inflammatory microenvironment of the eye is dependent on the expression of the melanocortin-neuropeptide alpha-melanocyte stimulating hormone (α-MSH). In addition, the melanocortin system may have a role in retinal development and retinal cell survival under conditions of retinal degeneration. We have found that treating experimental autoimmune uveitis (EAU) with α-MSH suppresses retinal inflammation. Also, this augmentation of the melanocortin system promotes immune tolerance and protection of the retinal structure. The benefit of α-MSH-therapy appears to be dependent on different melanocortin receptors. Therefore, we treated EAU mice with α-MSH-analogs with different melanocortin-receptor targets. This approach demonstrated which melanocortin-receptors suppress inflammation, preserve retinal structure, and induce immune tolerance in uveitis. At the chronic stage of EAU the mice were injected twice 1 day apart with 50 μg of α-MSH or an α-MSH-analog. The α-MSH-analogs were a pan-agonist PL8331, PL8177 (potent MC1r-only agonist), PL5000 (a pan-agonist with no MC5r functional activity), MT-II (same as PL5000) and PG901 (MC5r agonist, but also an antagonist to MC3r, and MC4r). Clinical EAU scores were measured until resolution in the α-MSH-treated mice, when the eyes were collected for histology, and spleen cells collected for retinal-antigen-stimulated cytokine production. Significant suppression of EAU was seen with α-MSH or PL8331 treatment. This was accompanied with significant preservation of retinal structure. A similar effect was seen in EAU-mice that were treated with PL8177, except the suppression of EAU was temporary. In EAU mice treated with PL5000, MTII, or PG901, there was no suppression of EAU with a significant loss in whole retina and outer-nuclear layer thickness. There was significant suppression of IL-17 with induction of IL-10 by retinal-antigen stimulated spleen T cells from EAU mice treated with α-MSH, PL8331, PL8177, or PL5000, but not from EAU mice treated with MT-II, or PG901. Our previous studies show the melanocortin system's importance in maintaining ocular immune privilege and that α-MSH-treatment accelerates recovery and induces retinal-antigen-specific regulatory immunity in EAU. Our current results show that this activity is centered around MC1r and MC5r. In addition, the results suggest that a therapeutic potential to target MC1r and MC5r together to suppress uveitis induces regulatory immunity with potentially maintaining a normal retinal structure.

Dataset Information

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

Publications

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

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