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.
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.
ObjectiveTo investigate the role of acetyl CoA carboxylase (ACC)-induced Th17 development in the pathogenesis of asthma.MethodsA total of 24 C57BL/6 mice were randomly assigned to 4 groups (6 in each group), namely a normal group, an asthma group, a DMSO control group and an ACC inhibitor group. The mice in the asthma group, the DMSO control group and the ACC inhibitor group were sensitized and challenged with ovalbumin (OVA) to establish a model of acute asthma, the mice in the normal group were administrated with equal volume of phosphate buffered saline (PBS). The mice in the ACC inhibitor group were intraperitoneally administrated with TOFA, an ACC inhibitor (dissolved in DMSO firstly, then diluted with PBS solution) twice a week, while the DMSO control group were intraperitoneally administrated with equal concentration of DMSO. All mice were sacrificed 24 days later, then lung tissue and serum were collected. The inflammatory cells infiltrated in lung tissue were assessed by the means of HE staining under light microscope. The total level of IgE in serum was detected by ELISA. The percentage of Th17 cells in CD4+ T cells in lung tissue was evaluated by flow cytometry.ResultsThe inflammatory cells infiltration in the asthma group, the DMSO control group and the ACC inhibitor group increased significantly compared with that of the normal group (3.50±0.14, 3.47±0.08, 2.07±0.20vs. 0.50±0.17, allP<0.001), but there were no significant differences between the DMSO control group group and the asthma group (P>0.05), while administration of TOFA could significantly decrease the inflammatory cells infiltration (P<0.001). The total level of serum IgE in the asthma group, the DMSO control group and the ACC inhibitor group was significantly higher than that in the normal group [(5 680.40±831.40) ng/ml, (5 624.79±365.50) ng/ml, (2 028.95±134.60) g/mlvs. (400.52±57.13) ng/ml, allP<0.008], but there were no significant differences between the DMSO control group and the asthma group (P>0.05), while the total level of serum IgE in the ACC inhibitor group was significantly lower than that in the asthma group (P<0.008). The percentage of Th17 cells in CD4+ T cells in lung tissue increased markedly in the asthma group, the DMSO control group and the ACC inhibitor group compared with the normal group [(2.01±0.12)%, (1.95±0.16)%, (0.82±0.04)%vs. (0.59±0.03)%, bothP<0.008], and also there were no significant differences between the DMSO control group and the asthma group (P>0.05), while administration of TOFA could notably reduce the percentage of Th17 cells (P<0.008).ConclusionInhibition of ACC significantly alleviates the airway inflammation of OVA-induced asthma, and reduces the percentage of Th17 cells in lung tissue and the total level of serum IgE. ACC may participate in the pathogenesis of asthma by inducing Th17 development.
Objective To investigate the expression of Th17 cells in peripheral blood of patients with sarcoidosis at different stage. Methods Flow cytometry was used to detect the Th17 cells in peripheral blood of 38 patients with sarcoidosis, including 18 cases of newly diagnosed active patients with obvious symptoms such as cough, fever, fatigue and weight loss, and 20 stable cases who were followed up regularly.15 cases of healthy volunteers were enrolled as control. Serumangiotensin-converting enzyme ( SACE) of the patients with sarcoidosis was detected by ultraviolet spectrophotometry. The cell classification and CD4 + /CD8 + T in the BALF of the newly diagnosed active patients were calculated. Results The expression of Th17 cells in peripheral blood in the patients with active sarcoidosis were higher than that in the sable patients and the controls [ ( 1. 59 ±0. 44) % vs. ( 0. 56 ±0. 32) % and ( 0. 49 ±0. 23) % , all P lt; 0. 05] . Th17 cells in peripheral blood in the patients with stable sarcoidosis and the controls were not different significantly ( P gt;0. 05) . The levels of SACE in the patients with active sarcoidosis were higher than that in the patients with stable sarcoidosis [ ( 56. 6 ±14. 6) IU/L vs. ( 35. 8 ±18. 3) IU/L, P lt; 0. 05) . There was not significant correlation between the Th17 cells in peripheral blood and SACE in the patients with sarcoidosis ( P gt;0. 05) . In the patients with active sarcoidosis, the Th17 cells in peripheral blood were not significantly correlated with lymphocyte percentages in BALF( P gt; 0. 05) , but significantly correlated with CD4 + /CD8 + in BALF ( r=0. 63, P lt;0. 05) .Conclusion In patients with active sarcoidosis, the increased expression of Th17 cells in peripheral blood may correlate with the activity of sarcoidosis.
Objective To investigate the effects of ulinastatin on Treg/Th17 and immune status in patients with severe sepsis.Methods A total of 80 patients with severe sepsis, who were hospitalized in ICU during October 2011 to July 2012, were randomly divided into a routine group and a ulinastatin group. The patients in the ulinastatin group were intravenously administered 30mg ulinastatin three times per day for 5 days in addition to routine bundle treatment. The expression of Treg, Th17 and HLA-DR were detected on the first day in ICU and 5 days after treatment. 20 healthy individuals served as controls. Results Compared with the control group, the severe sepsis group had overexpression of Treg and Th17 ( P lt;0. 01) , higher ratio of Treg/Th17( P lt;0. 01) , and decreased HLA-DR expression of CD14 monocyte ( P lt; 0. 01) . In the severe sepsis patients, ulinastatin injection reduced the abnormal expression of Treg and Th17 ( P lt; 0. 01) , decreased the ratio of Treg/Th17( P lt; 0. 01) , and improved the expression of HLA-DR ( P lt; 0. 01) more effectively compared with the routine treatment. Ulinastatin also lowered 28-day mortality of the patients with sepsis, but the difference between the ulinastatin group and the routine group was not significant. Conclusions In severe sepsis patients, there were abnormal overexpression of Treg and Th17, imbalance of Treg/Th17, and underexpression of HLA-DR which imply an immune suppression. Ulinastatin can decrease the expression of Treg and Th17, inverses the ratio of Treg/Th17, and improve the expression of HLA-DR, so as to improve the prognosis of severe sepsis patients.
ObjectiveTo study the protective effect and mechanism of ophiopogonin D (OP-D) on lipopolysaccharide induced acute lung injury (ALI) in mice.MethodsFifty SPF C57BL/6 mice were randomly divided into five groups, ie. a control group, a sham operation group, a model group, an OP-D group (10 mg·kg–1·d–1), and a dexamethasone group (2 mg·kg–1·d–1), with 10 mice in each group. One day before the establishment of the model, the OP-D group and the dexamethasone group received the corresponding drugs by gavage. The model group, the OP-D group and the dexamethasone group received lipopolysaccharide (2 mg/kg, 30 μL) through the trachea to establish the ALI model. The sham operation group received the same volume of normal saline. The blank control group was not treated. Six hours after the operation, the mice were weighed and then killed for peripheral blood and lung tissue. The weight of lung tissue was measured to evaluate the degree of pulmonary edema; the pathological changes of lung tissue were observed by hematoxylin-eosin staining; the mRNA expressions of interleukin (IL)-6, IL-10, and IL-17 in lung tissue were detected by qPCR; the percentage of Th17 and Treg cells in peripheral blood was detected by flow cytometry.ResultsCompared with the model group, the degree of pulmonary edema in the OP-D group decreased significantly (P<0.05), the lung tissue injury decreased, the mRNA expressions of IL-6 and IL-17 in the lung tissue and the proportion of Th17 cells in the peripheral blood decreased significantly (P<0.05), the proportion of Treg cells in the peripheral blood and the mRNA expression of IL-10 in the lung tissue increased significantly (P<0.05).ConclusionOP-D may have therapeutic effect on LPS induced ALI in mice by regulating the balance of Th17/Treg cells.
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.
ObjectiveTo investigate the effects of smoking on Th17/Treg T cell subsets and cytokines expression in patients with chronic obstructive pulmonary disease (COPD) in stable stage. MethodsFrom February 2012 to June 2013, sixty outpatients with stable COPD (20 cases of non-smokers, 40 cases of smokers) and 15 normal volunteers were recruited in the study in the Traditional Chinese Medicine Hospital affiliated to Xinjiang Medical University. Th17/Treg level in peripheral blood was detected by flow cytometry method. Cytometric bead array system was used to detect TGF-β, IL-10, IL-17A, IFN-γand other inflammatory factors in serum. ResultsThe patients' age, duration of disease, lung function, disease severity, and other related data were comparable between the smoking COPD group and the non-smoking COPD group (P > 0.05). Th17/Treg level was increased in the smoking COPD group compared with the normal group (P < 0.05), and showed an increasing trend from the normal group to the non-smoking COPD group and the smoking COPD group. The level of IL-2 in the smoking and non-smoking COPD groups was lower than that in the normal group. Compared with the normal group, the level of TNF-αwas significantly decreased in the smoking and non-smoking COPD groups(P < 0.05). ConclusionsSystemic inflammatory response continuously exists in patients with COPD even in the stable phase. Smoking can partly enhance the inflammatory reaction in COPD. The Th17/Treg T cell subsets associated cytokine regulation has gradually tended to a balance in the stable phase, and inflammatory factors related recovery speed is not consistent, suggesting that smoking may play a certain role in the recovery of balance.
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.