Objective To investigate the effect of peptidoglycan (PGN) on the secretion of pro-inflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis. Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs. DCs cultured for six days were randomly divided into two groups: PGNtreated group and control group. The DCs in PGNtreated group were stimulated with PGN and the same volume of phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor alpha; (TNF-alpha;), IL-6,IL-1beta;were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Peptide fragment of interphotoreceptor retinoidbinding protein (IRBP1-20)specific T cells, which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier, were co-cultured with PGN-treated or untreated DCs, respectively. Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor gamma;t (ROR-gamma;t), IL-17, T-box expression in T cells (T-bet), interferon gamma; (IFN-gamma;) mRNA were detected by realtime RT-PCR. On the second, the fifth and the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-gamma;, IL-17 positive cells. Results The real-time RT-PCR results revealed that the level of IL-23, IL-1beta;, IL-6, TNF-alpha; mRNA from PGNstimulated DCs were significantly increased compared to the control group (t=-14.363, -5.627, -3.85, -28.151; P＜0.05). The level of RORgamma;t, IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601, -19.76；P＜0.05). However, the level of T-bet, IFN-gamma; mRNA from the T cells cocultured with PGNstimulated DCs were significantly decreased compared with the control group (t=4.717, 11.207; P＜0.05). Data of flow cytometry showed that at two days, five days, seven days after cocultured with PGN-treated DCs, the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944, -3.03, -4.81; P＜0.05), and the percentages of IFN-gamma; positive T cells had no remarkable change (t=-1.25, -0.18, -2.16; P＞0.05). Conclusion PGN can promote the secretion of Th17-related cytokines by DCs, which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis.
ObjectiveTo observe the expression of interleukin (IL)-17, IL-4 and interferon γ (IFN-γ) in experimental autoimmune uveoretinitis (EAU). MethodsC57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein 1-20 to induce EAU. The inflammatory reaction before and on 7, 14, 21, 28 days after immunization were observed. The level of IL-17, IL-4 and IFN-γ in the serum were measured by enzyme-linked immune sorbent assay (ELISA). mRNA and protein expression of spleen and retina were analysed using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at the same time, respectively. ResultsThe most serious inflammatory reaction occurred at the 14th day after immunization. The highest level of IFN-γ in serum, highest mRNA and protein expression of IFN-γ in spleen and retina of mice occurred at day 7 after being immunized. The highest level of IL-17, IL-4 in serum, highest mRNA and protein expression of IL-17, IL-4 in spleen and retina of mice occurred at day 14 after being immunized. The increase degree of IL-17 was more than IFN-γ and IL-4. At 7, 14 and 21 days after immunization, compared with the pre-immunization, the level of IL-17, IL-4, IFN-γ in serum of mice were significantly increased (F=1 817.346, 268.600, 164.621; P < 0.05). There was no difference in the levels of IL-17, IL-4, IFN-γin serum of mice between pre-and 28 days after immunization (P > 0.05). At 7, 14 and 21 days after immunization, compared with the pre-immunization, the protein expression of IL-17, IL-4, IFN-γ in spleen (F=312.67, 114.250, 216.220) and retina (F=271.504, 85.370, 80.722) of mice were significantly increased (P < 0.05). There was no difference in protein expression of IL-17, IL-4, IFN-γ in spleen and retina of mice between pre-and 28 days after immunization (P > 0.05). ConclusionsThere were IL-17, IL-4 and IFN-γ expression in EAU. IL-17, IL-4 and IFN-γ play a key role in the occurrence and development of the EAU.
ObjectiveTo observe the expression of miR-142-5p and forkhead transcription protein O subgroup 3 (FOXO3) in CD4+ T cells of experimental autoimmune uveitis (EAU) model rats, and preliminarily explore the targeting relationship between the two and the effect on EAU impact.MethodsTen Lewis rats were randomly divided into model group and control group. Rats in the model group wree induced an EAU animal model by adoptive immunization. Twenty days after immunization, CD4+ T cells were extracted from the eyeballs and draining lymph nodes of rats in the control group and model group, and divided into control group, model group, mimic-negative control (NC) group, miR-142-5p-mimic group, and small interference (si)-NC group, si-FOXO3 group for in vitro experiments. The miR-142-5p-mimic group and si-FOXO3 group were transfected with miR-142-5p-mimic and si-FOXO3, respectively. Twenty-five Lewis rats were randomly divided into model group, mimic-NC transfected group, miR-142-5p-mimic transfected group, si-NC transfected group, and si-FOXO3 transfected group. The above-mentioned in vitro experimental groups were injected with cells respectively. Slit lamp microscopy and EAU score were performed on 4, 8, 12, 16, 20 days after immunization; on 20 days after immunization, hematoxylin-eosin staining was performed for histopathological grading. Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression of miR-142-5p and FOXO3 mRNA in CD4+ T cells and eye tissues of rats in each group, and helper T cell 17 (Th17) marker interleukin (IL)-17, IL-22, retinoic acid-related orphan receptor gamma (ROR gamma) relative expression level in the supernatant. Bioinformatics website and dual luciferase was used to predict the targeting relationship between miR-142-5p and FOXO3. One-way analysis of variance or t test was used for comparison between groups.ResultsAll rats in the model group showed symptoms of EAU to varying degrees, and the symptoms became worse with time. Compared with the control group, the relative expression of miR-142-5p mRNA in CD4+ T cells of the model group increased, and the relative expression of FOXO3 mRNA decreased. The differences were statistically significant (t=7.374, 10.423; P=0.002, 0.001). Compared with the mimic-NC group, the relative expression of miR-142-5p mRNA in the CD4+ T cells of the miR-142-5p-mimic group increased, and the difference was statistically significant (t=6.540, P=0.003). Compared with the model group, mimic-NC group, and si-NC group, the relative expression of IL-17, IL-22, and RORγ mRNA in CD4+ T cells in the miR-142-5p-mimic group and si-FOCO3 group increased significantly. The difference was statistically significant (F=26.110, 6.292, 5.269, 55.660, 10.490, 11.430; P＜0.05). Compared with the mimic-NC transfected group, the relative expression of miR-142-5p mRNA in the ocular tissues of the miR-142-5p-mimic transfected rats increased significantly, and the difference was statistically significant (t=6.690, P＜0.05). Compared with the transfected si-NC group, the relative expression of FOXO3 mRNA in the eye tissue of the transfected si-FOXO3 group was significantly decreased, and the difference was statistically significant (t=17.751, P＜0.05). Rats in the mimic-NC transfected group, miR-142-5p-mimic transfected group, si-NC transfected group, and si-FOXO3 transfected group prolonged with time after immunization, and the EAU scores showed an upward trend. The EAU score and histopathological grade of rats in the miR-142-5p-mimic transfected group were higher than those in the mimic-NC transfected group, and the difference was statistically significant (t=5.633, 6.286; P＜0.05). The EAU score and histopathological grade of the rats in the transfected si-FOXO3 group were higher than those in the transfected si-NC group, and the difference was statistically significant (t=6.852, 6.635; P＜0.05). FOXO3 has a targeting relationship with miR-142-5p.ConclusionsIn EAU rat CD4+ T cells, the expression of miR-142-5p is up-regulated, while the expression of FOXO3 is down-regulated. miR-142-5p targets the expression of FOXO3 to promote the development of Th17 cell-related inflammatory factors.
ObjectiveTo investigate the effect of interleukin (IL)-23 receptor (IL-23R) overexpression on the balance of T helper 17 (Th17 cells)/regulatory T cells (Treg cells) in experimental autoimmune uveitis (EAU) mice. MethodsTwelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group, with 6 mice in each group. Two groups of mice were injected with LV-Ctrl and LV-IL-23Rlentiviruses through the tail vein, respectively; 7 days after injection, the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors. Starting from 13 days after immunization, the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed; 30 days after immunization, hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina. The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay; the proportion of Th17 cells and Treg cells was detected by flow cytometry. The relative mRNA expression of IL-23R, IL-17, retinoic acid-related orphan receptor γt (RORγt), IL-10 and forkhead transcripyion factor p3 (Foxp3) were detected by real-time quantitative polymerase chain reaction. Comparisons between groups were performed using repeated measures analysis of variance, independent samples Mann-Whitney U test, and independent samples t test. ResultsCompared with the LV-Ctrlgroup, the retinal inflammatory reaction of the LV-IL-23R group was more severe. At 13 days after immunization, there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group (t=－2.001, P=0.058); 15-29 days after immunization. The fundus inflammation scores of LV-IL-23Rgroup were higher than those of LV-Ctrl group, and the difference was statistically significant (t=－4.429, －6.578, －7.768, －10.183, －6.325, －7.304, －4.841, －6.872; P＜0.001). Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased, the retinal structure was damaged more seriously, and the histopathological score was significantly increased, and the difference was statistically significant (t=－4.339, P=0.001). Compared with the LV-Ctrl group, the relative expression of IL-23RmRNA in the spleen of the LV-IL-23R group was significantly increased, and the difference was statistically significant (Z=2.087, P=0.037). The relative expression of IL-17 and RORγt mRNA increased, while the relative expression of IL-10 and Foxp3 mRNA decreased, and the differences were statistically significant (t=－6.313,－5.922, 4.844, 7.572; P=0.003, 0.004, 0.008, 0.002). Compared with the LV-Ctrl group, the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased, and the difference was statistically significant (t=－5.423, P=0.002); the proportion of Th17 cells in the spleen and lymph nodes was significantly increased, whereas, the proportion of Treg cells was significantly reduced, and the difference was statistically significant (t=－4.290, 3.700; P=0.002, 0.006). ConclusionIL-23R overexpression can promote Th17/Treg imbalance in EAU mice, and aggravate the clinical and pathological manifestations of EAU.
Objective To investigate whether p38 mitogen activated protein kinase (p38MAPK) inhibitor can reduce acute lung injury (ALI) caused by lipopolysaccharide (LPS) by regulating Th17/Treg balance. Methods Balb/c mice were randomly divided into a control group, an ALI group and an intervention group. The mice in the control group were injected with phosphate-buffered saline, the mice in the ALI group were intraperitoneally injected with 40 mg/kg LPS, and the mice in the intervention group were injected with SB203580 (0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg) intraperitoneally 1 h prior to the intraperitoneal injection of LPS. All mice were killed on 12 h later respectively. Hematoxylin-eosinstin staining was used to observe the pathological changes of lung tissue, and cell classification, counting, and total protein levels in bronchoalveolar lavage fluid (BALF) were detected. Transcript expression of forkhead box p3 (Foxp3) and retinoic acid receptor-related orphan receptor-γt (RORγt) was detected by real-time polymerase chain reaction. Interleukin (IL)-6, IL-10, IL-17, IL-23 and transforming growth factor-β (TGF-β) in lung tissue and IL-6, tumor necrosis factor-α (TNF-α) in serum were measured by enzyme-linked immunosorbent assay. The Th17 and Treg subset distribution in spleen was determined by flow cytometry. Results Histopathological examination showed that LPS induced inflammatory cell infiltration in lung tissue, increased cell count and protein levels in BALF (P<0.05), and increased proportion of neutrophils and monocytes in the ALI mice. SB203580 significantly attenuated tissue injury of the lungs in LPS-induced ALI mice. Serum levels of IL-6 and TNF-α in the ALI group were significantly higher than those in the control group, and inflammatory cytokines were decreased after SB203580 intervention. Compared with the ALI group, the production of inflammatory cytokines associate with Th17, including IL-17, IL-23, RORγt was inhibited, and the production of cytokines associate with Treg, such as IL-10 and Foxp3 in lung tissue was increased in the intervention group in a concentration-dependent manner with SB203580. After SB203580 intervention, Th17/Treg ratio was significantly decreased compared with the LPS group (P<0.05). Conclusion p38MAPK inhibitor can reduce LPS-induced ALI by regulating the imbalance of Treg cells and Th17 cells.