Effect of berberine on spleen transcriptome and gut microbiota composition in experimental autoimmune uveitis.

BACKGROUND: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? METHODS: EAU was induced in B10R.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. RESULTS: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. CONCLUSIONS: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

Lutein and Factor D: two intriguing players in the field of age-related macular degeneration.

Age-related macular degeneration (AMD) is a progressive eye disease that impairs central vision among elderly populations in Western, industrialized countries. In this review we will focus on the role of factor D (FD) and lutein in AMD. FD is a rate-limiting enzyme of the alternative complement activation pathway that may play an important role in the development of AMD. Several independent studies have shown a significant increase in the level of a number of complement factors of the alternative pathway, including factor D in the blood of AMD patients as compared to healthy individuals, which suggests a systemic involvement in the pathogenesis of AMD. FD, also called adipsin, is mainly produced by adipose tissue. Besides playing a role in the activation of the alternative pathway, FD is also known to regulate the immune system. Of interest is our preliminary finding that lutein supplementation of early AMD cases was shown to lower the level of systemic FD. If confirmed, these findings provide further support for the application of anti-factor D intervention as a new approach to control the development of this disease.

Lutein supplementation leads to decreased soluble complement membrane attack complex sC5b-9 plasma levels.

PURPOSE: The purpose of this study was to investigate the effect of lutein on systemic complement activation in elderly individuals. METHODS: Seventy patients with signs of early age-related macular degeneration (AMD) were included in this study. All subjects were randomly assigned to receive a 10 mg daily dose of lutein or a placebo for a time period of 1 year. EDTA blood was collected before and at various time-points during the study (0, 4, 8 and 12 months). The plasma level of the soluble complement membrane attack complex sC5b-9 was measured by ELISA. RESULTS: We found a significant 1.1 ng/ml monthly decrease in the plasma sC5b-9 concentration in the lutein group (p<0.001), resulting in a decrease from 60.3 ng/ml at baseline to 46.3 ng/ml at 12 months. For the placebo group, we found a significant 0.6 ng/ml monthly increase in plasma sC5b-9 concentration (p=0.001), resulting in an increase from 51.6 ng/ml at baseline to 58.4 ng/ml at 12 months. CONCLUSIONS: Lutein supplementation inhibits the systemic activation of the complement system, which provides further functional evidence for the reported beneficial effects of this carotenoid in the management of AMD.

Consuming a buttermilk drink containing lutein-enriched egg yolk daily for 1 year increased plasma lutein but did not affect serum lipid or lipoprotein concentrations in adults with early signs of age-related macular degeneration.

Dietary lutein intake is postulated to interfere with the development of age-related macular degeneration (AMD). Because egg yolk-derived lutein has a high bioavailability, long-term consumption of lutein-enriched eggs might be effective in preventing AMD development, but alternatively might increase cardiovascular disease risk. Here, we report the effect of 1-y daily consumption of a buttermilk drink containing 1.5 lutein-rich egg yolks on serum lipid and lipoprotein and plasma lutein concentrations. Additionally, subgroups that could potentially benefit the most from the intervention were identified. Men and women who had early signs of AMD in at least 1 eye, but were otherwise healthy, participated in a 1-y randomized, placebo-controlled parallel intervention trial. At the start of the study, 101 participants were included: 52 in the experimental (Egg) group and 49 in the control (Con) group. Final analyses were performed with 45 participants in the Egg group and 43 participants in the Con group. As expected, the increase in plasma lutein concentrations in the Egg group was 83% higher than that in the Con group (P < 0.001). Changes in serum total, HDL, and LDL cholesterol, as well as the ratio of total cholesterol to HDL cholesterol, were not different between the 2 groups. Interestingly, participants classified as cholesterol absorbers had higher serum HDL cholesterol concentrations than participants classified as cholesterol synthesizers or participants with average campesterol-to-lathosterol ratios (P < 0.05) at baseline. In addition, cholesterol absorbers had a 229% higher increase in plasma lutein concentrations than participants who were classified as having an average campesterol-to-lathosterol ratio upon consumption of the lutein-enriched egg yolk drink (P < 0.05). Moreover, the change in serum HDL cholesterol upon consumption was significantly different between these 3 groups (P < 0.05). We suggest that cholesterol absorbers particularly might benefit from the lutein-enriched buttermilk drink. This study was registered at clinicaltrials.gov as NCT00902408.

The effect of modified eggs and an egg-yolk based beverage on serum lutein and zeaxanthin concentrations and macular pigment optical density: results from a randomized trial.

UNLABELLED: Increasing evidence suggests a beneficial effect of lutein and zeaxanthin on the progression of age-related macular degeneration. The aim of this study was to investigate the effect of lutein or zeaxanthin enriched eggs or a lutein enriched egg-yolk based buttermilk beverage on serum lutein and zeaxanthin concentrations and macular pigment levels. Naturally enriched eggs were made by increasing the levels of the xanthophylls lutein and zeaxanthin in the feed given to laying hens. One hundred healthy volunteers were recruited and randomized into 5 groups for 90 days. Group one added one normal egg to their daily diet and group two received a lutein enriched egg-yolk based beverage. Group three added one lutein enriched egg and group four one zeaxanthin enriched egg to their diet. Group five was the control group and individuals in this group did not modify their daily diet. Serum lutein and zeaxanthin concentrations and macular pigment densities were obtained at baseline, day 45 and day 90. Macular pigment density was measured by heterochromatic flicker photometry. Serum lutein concentration in the lutein enriched egg and egg yolk-based beverage groups increased significantly (p<0.001, 76% and 77%). A strong increase in the serum zeaxanthin concentration was observed in individuals receiving zeaxanthin enriched eggs (P< 0.001, 430%). No changes were observed in macular pigment density in the various groups tested. The results indicate that daily consumption of lutein or zeaxanthin enriched egg yolks as well as an egg yolk-based beverage show increases in serum lutein and zeaxanthin levels that are comparable with a daily use of 5 mg supplements. TRIAL REGISTRATION: ClinicalTrials.gov NCT00527553.

The effect of lutein supplementation on blood plasma levels of complement factor D, C5a and C3d.

Lutein is selectively taken up by the primate retina and plays an important role as a filter for harmful blue light and as an antioxidant. Recent studies have shown that lutein has systemic anti-inflammatory properties. Dietary lutein has been associated with reduced circulating levels of inflammatory biomarkers such as CRP and sICAM. Whether lutein also affects activation of the complement system has not yet been addressed and was the purpose of the study described here. Seventy-two subjects with signs of early macular degeneration were randomly assigned to receive either a 10 mg lutein supplement or a placebo during one year. EDTA blood samples were collected at 0, 4, 8 and 12 months. Complement factor D (CFD), a rate limiting component of the alternative pathway of complement activation and the complement activation products C5a and C3d were determined in the plasma samples by ELISA. A significant 0.11 µg/ml monthly decrease in plasma CFD concentration was observed in the lutein group (p<0.001), resulting in a 51% decrease from 2.3 µg/ml at baseline to 1.0 µg/ml at 12 months. The C5a concentration showed a significant 0.063ng/ml monthly decrease in the lutein group (p<0.001) resulting in a 36% decrease from 2.2ng/ml at baseline to 1.6ng/ml at 12 months. The C3d concentration showed a significant 0.19µg/ml monthly decrease in the lutein group (p=0.004) that gave rise to a 9% decrease from 15.4µg/ml at baseline to 14.4µg/ml at 12 months. In the placebo group we found a significant 0.04 µg/ml monthly decrease in plasma CFD concentration, whereas no changes were observed for C5a and C3d. Lutein supplementation markedly decreases circulating levels of the complement factors CFD, C5a and C3d levels, which might allow a simple method to control this inflammatory pathway of the innate immune system.

Effect of berberine on proinflammatory cytokine production by ARPE-19 cells following stimulation with tumor necrosis factor-α.

PURPOSE: Berberine (BBR) is a well-known drug used in traditional medicine and has been shown to possess anti-inflammatory properties. Whether it can affect the production of inflammatory cytokines by RPE cells is not yet clear and was therefore the subject of our study. METHODS: ARPE-19 cells were cultured with TNF-α in the presence or absence of BBR to different time points. Concentrations of IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) in the supernatant were measured by using an ELISA. The mRNA expression of these cytokines was measured by real-time PCR. Phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) was measured by Western blot assay. The signal transduction mechanisms involved in cytokine production were evaluated using various inhibitors for p38, ERK1/2, and JNK. RESULTS: TNF-α significantly increased the expression of IL-6, IL-8, and MCP-1 in ARPE-19 cells at both the protein and mRNA levels. It promoted the phosphorylation of p38, ERK1/2, and JNK. Inhibitory experiments showed that IL-6 was modulated by p38, whereas IL-8 and MCP-1 were modulated by p38, ERK1/2, and JNK signal pathways. BBR inhibited the expression of IL-6, IL-8, and MCP-1 remarkably at both protein and mRNA levels and down-regulated the phosphorylation of p38, ERK1/2, and JNK upon stimulation with TNF-α. CONCLUSIONS: The present results suggested that BBR significantly inhibits the expression of inflammatory cytokines in ARPE-19 cells and that the inhibitory effect is mediated by down-regulation of the p38, ERK1/2, and JNK pathways.

Regulatory effects of IFN-ß on the development of experimental autoimmune uveoretinitis in B10RIII mice.

BACKGROUND: Experimental autoimmune uveoretinitis (EAU) serves as a model for human intraocular inflammation. IFN-ß has been used in the treatment of certain autoimmune diseases. Earlier studies showed that it ameliorated EAU; however, the mechanisms involved in this inhibition are still largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: B10RIII mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 161-180 in Complete Freund's adjuvant. Splenocytes from different time points after immunization were used to evaluate the expression of IFN-ß. An increased expression of IFN-ß was observed during EAU and its highest expression was observed on day 16, 3 days after the peak of intraocular inflammation. Splenocytes and draining lymph node cells from mice immunized with IRBP(161-180) on day 13 and control mice were activated with anti-CD3/anti-CD28 antibodies or IRBP(161-180) to evaluate the production of IFN-γ and IL-17. The results showed that IFN-γ and IL-17 were significantly higher in immunized mice as compared to the control mice when exposed to anti-CD3/anti-CD28 antibodies. However, the production of IFN-γ and IL-17 was detected only in immunized mice, but not in the control mice when stimulated with IRBP(161-180). Multiple subcutaneous injections of IFN-ß significantly inhibited EAU activity in association with a down-regulated expression of IFN-γ, IL-17 and an enhanced IL-10 production. In an in vitro system using cells from mice, IFN-ß suppressed IFN-γ production by CD4(+)CD62L(-) T cells, IL-17 production by CD4(+)CD62L(+/-) T cells and proliferation of CD4(+)CD62L(+/-) T cells. IFN-ß inhibited the secretion of IL-6, but promoted the secretion of IL-10 by monocytes. IFN-ß-treated monocytes inhibited IL-17 secretion by CD4(+)CD62L(+/-) T cells, but did not influence IFN-γ expression and T cell proliferation. CONCLUSIONS/SIGNIFICANCE: IFN-ß may exert its inhibitory effect on EAU by inhibiting Th1, Th17 cells and modulating relevant cytokines. IFN-ß may provide a potential treatment for diseases mediated by Th1 and Th17 cells.

Increased Regulatory T Cells in Spleen during Experimental Autoimmune Uveoretinitis.

PURPOSE: To investigate the role of Foxp3-positive regulatory T cells in the development of experimental autoimmune uveoretinitis (EAU). METHODS: B10RIII mice were immunized with 50 microg IRBP(161-180) in complete Freund's adjuvant (CFA) to induce EAU. EAU was evaluated clinically and pathologically on days 0, 7, 14, 21, and 28. Foxp3 mRNA levels were detected using reverse transcription-PCR (RT-PCR) and the frequencies of CD4(+)Foxp3(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells in splenocytes were assessed by flow cytometry at the aforementioned time points. RESULTS: The first clinical signs of EAU were observed on day 8-9, worsened up to day 14, and then gradually resolved. Histopathologic results showed that inflammatory signs occurred on day 7, reached their peak on day 14, and then gradually decreased. The levels of Foxp3 mRNA and the frequencies of CD4(+)Foxp3(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells in splenocytes increased on day 7, reached a peak on day 14, and then maintained at a high level until day 28. CONCLUSION: An upregulation of Foxp3 expression is induced in EAU and paralleled with disease activity, suggesting a role for this lymphocyte subpopulation in the regression of this experimental uveitis model.

Contribution of CD4+CD25+ T cells to the regression phase of experimental autoimmune uveoretinitis.

PURPOSE: To investigate the role of CD4(+)CD25(+) Treg cells in the development of experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in B10RIII mice by immunization with IRBP(161-180) in complete Freund's adjuvant and evaluated clinically and pathologically on days 0, 7, 14, 21, and 28. Lymphocytes from draining lymph nodes (LNs) were subjected to flow cytometry to analyze the frequency of CD4(+)CD25(+) Treg cells. CD4(+)CD25(+) Treg cells and CD4(+)CD25(-) T cells were separated by means of magnetic-assisted cell sorting and cocultured or crossover cultured for 3 days. Proliferation of CD4(+)CD25(-) T cells was measured using a modified MTT assay. The levels of IFN-gamma and IL-17 in the supernatants were determined by enzyme-linked immunosorbent assay. RESULTS: Clinical and histopathologic results showed a severe intraocular inflammation in the immunized mice. The frequency of CD4(+)Foxp3(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells in the draining LN lymphocytes was increased on day 7, reached its peak on day 14, and maintained a high level up to day 42. CD4(+)CD25(+) Treg cells obtained from mice on days 14 and 28 after immunization showed a stronger inhibitory effect on the proliferation of CD4(+)CD25(-) T cells and the production of IFN-gamma by CD4(+)CD25(-) T cells compared with those obtained from control mice. CD4(+)CD25(+) Treg cells did not affect IL-17 production. Transfer of CD4(+)CD25(+) Treg cells obtained from EAU mice was able to suppress EAU induction by IRBP(161-180) that was not observed after transfer of cells from mice that had received CFA alone, suggesting antigen specificity of the Treg response. CONCLUSIONS: A significantly increased frequency and immunoregulatory action of CD4(+)CD25(+) Treg cells is associated with the development and regression of EAU, suggesting that CD4(+)CD25(+) Treg cells are induced during EAU and may be involved in its regression.

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis.

BACKGROUND: T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR V(beta) 8.3, an experimental autoimmune uveoretinitis (EAU)-associated gene, was able to prevent the disease. METHODS: EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA). The clinical and histological appearances were scored. Delayed type hypersensitivity (DTH) and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay (ELISA). Gene expression of TCR V(beta) 8.3 on CD(4)(+) T cells was examined by real time quantitative polymerase chain reaction (PCR). RESULTS: After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin (IL)-2 in aqueous humour, interferon (IFN)-gamma and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR V(beta) 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR V(beta) 8.3 gene was significantly reduced in the group of fourfold inoculations. CONCLUSION: Vaccination with the synthetic TCR V(beta) 8.3 peptide could remarkably inhibit the development of EAU.