Effect of mTORC1 on intestinal group 3 innate lymphocytes function / 上海交通大学学报（医学版）

Objective : To investigate the effect of mechanistic target of rapamycin complex 1 (mTORC1) on group 3 innate lymphoid cells (ILC3) function. Methods ¡¤ Intestinal lamina propria leukocytes (LPL) of C57BL/6 wild type mice were stimulated by rapamycin, the specific inhibitor of mTORC1 signaling pathway, in vitro, and then quantity and function of ILC3 were detected by flow cytometry. Next, purified ILC3 from mice intestinal LPL were sorted by flow cytometry. After the activation of ILC3 with IL-23, mRNA expression levels of Rorc (the gene encoding retinoic acid receptor related orphan receptor, i.e. RORγt), Il22 and Rptor (the gene encoding key component protein of mTORC1, i.e. Raptor) were detected by real-time qPCR. For further study, a genetically engineered mouse model specifically knocked out Raptor in ILC3 was constructed. Effects of mTORC1 loss on the quantity and function of ILC3 as well as gut structure were detected by flow cytometry, real-time qPCR and hematoxylin-eosin staining. Results ¡¤ The total ILC3 number had no change, but the secretion of IL-22 by ILC3 reduced after stim-ulation with rapamycin. Il22, Rorc and Rptor mRNA expression levels were upregulated simultaneously in ILC3 after activation with IL-23. In addition, there was no significant difference in the numbers and proportions of total ILC3 and ILC3 subsets as well as gut structure in Rap-tor-deficient mice, but the cytokine IL-22 secretion level of ILC3 significantly decreased in these mice. Conclusion ¡¤ Loss of mTORC1 func-tion inhibits ILC3 from secreting IL-22 but has no effect on the intestinal structure and intestinal ILC3 development, which reveals the positive regulation of mTORC1 signaling on intestinal ILC3 function.

Effects of interleukin-22 on the expression of tazarotene-induced gene 3 in HaCaT cells / 中华皮肤科杂志

Objective To evaluate the effects of interleukin-22(IL-22)on the expression of tazarotene-induced gene 3(TIG3)in HaCaT cells. Methods Cultured HaCaT cells were randomly divided into several groups to be treated with different concentrations (12.5, 25, 50, 100 μg/L)of IL-22 alone, or the combination of 50 μg/L IL-22 with the MAPK-ERK1/2 inhibitor PD98059 or the JAK/STAT inhibitor AG490 for 24 hours. Those HaCaT cells treated with phosphate buffered saline served as the control group. Subsequently, total proteins and mRNAs were extracted from the HaCaT cells. An immunofluorescence assay, Western blot and enzyme-linked immunosorbent assay (ELISA) were performed to determine the protein expression level of TIG3, and real-time fluorescence-based quantitative PCR to quantify the mRNA expression of TIG3 in HaCaT cells. Results The immunofluorescence assay showed that TIG3 protein was mainly expressed in the cytoplasm of HaCaT cells. As Western blot revealed, the protein expression level of TIG3 was 0.743 ± 0.035, 0.678 ± 0.040, 0.582 ± 0.041 and 0.328 ± 0.032 in HaCaT cells treated with IL-22 of 12.5, 25, 50 and 100μg/L, respectively, significantly lower than that in the control group (0.839 ± 0.045, all P<0.05). ELISA also showed a decrease in the protein expression of TIG3 in IL-22-treated HaCaT cells, which was consistent with Western blot results. Further more, the mRNA expression level (2-△△Ct)of TIG3 was significantly weaker in HaCaT cells treated with IL-22 of 12.5, 25, 50 and 100μg/L than in the control group (0.838 ± 0.036, 0.686 ± 0.061, 0.565 ± 0.047 and 0.457 ± 0.033 vs. 1.000, all P< 0.05). The decrease in TIG3 mRNA and protein expressions was significantly attenuated in HaCaT cells treated with the combination of 50 μg/L IL-22 with PD98059 or AG490 compared with those treated with 50 μg/L IL-22 alone. Conclusion IL-22 can dose-dependently inhibit the expression of TIG3 in HaCaT cells, likely through the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways.

Mechanisms underlying interleukin-22-induced expression of heparin-binding epidermal growth factor-like growth factor in HaCaT cells / 中华皮肤科杂志

Objective To investigate the mechanisms underlying intedeukin-22 (IL-22)-induced expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in HaCaT cells.Methods Some HaCaT cells were divided into several inverention groups treated with IL-22 at concentrations of 12.5,25,50,100 μg/L,respectively and a control group treated with phosphate buffer saline (PBS).After 24-hour culture,total proteins were extracted from the HaCaT cells,and Western blot was performed to measure the expression of phosphorylated extracellular signalregulated kinase 1/2 (P-ERK1/2) in the mitogen-activated protein kinase (MAPK)-ERK1/2 pathway,as well as phosphorylated-JAK2 (P-JAK2) and phosphorylated-signal transducer and activator of transcription 3 (P-STAT3) in the JAK2/STAT3 pathway.In a blocking experiment,some HaCaT cells were divided into 4 groups to be treated with PBS,IL-22,PD98059 (an inhibitor of MAPK-ERK1/2) combined with IL-22 (PD98059 group),AG490 (an inhibitor of JAK2/STAT3) combined with IL-22 (AG490 group),respectively.After 24-hour treatment,total proteins and mRNAs were extracted from the HaCaT cells followed by Western blot and real-time quantitative reverse transcription-PCR for the measurement of protein and mRNA expressions of HB-EGF respectively.Statistical analysis was carried out with the software SPSS 16.0 by one-way analysis of variance (ANOVA) for intergroup comparisons and by Bonferroni's test for multiple comparisons.Results After treatment with IL-22 at the above 4 concentrations,the expressions of P-ERK1/2,P-JAK2 and P-STAT3 in HaCaT cells were all increased compared with the control group (all P ＜ 0.05).The protein and mRNA expression levels (expressed as the HB-EGF/β-actin ratio and 2-△△Cr respectively) of HB-EGF were both significantly decreased in the PD98059 group and AG490 group than in the IL-22 group (protein:0.183 ± 0.020 and 0.199 ± 0.011 vs.0.924 ± 0.032,F =37.700,36.400,respectively,both P ＜ 0.05; mRNA:1.034 ± 0.072 and 0.989 ± 0.038 vs.1.844 ± 0.135,F =11.271,13.429,respectively,both P ＜ 0.05).Conclusions IL-22 can activate the MAPK-ERK1/2 and JAK2/STAT3 signaling pathways in HaCaT cells,which may contribute to IL-22-induced expression of HB-EGF in HaCaT cells.

Dynamic expressions of IL-22 and hepatic stellate cells senescence in mice in-fected with Schistosoma japonicum / 中国血吸虫病防治杂志

Objective To investigate the dynamic expressions of interleukin-22(IL-22),Interleukin-22 receptor 1(IL-22R1),and hepatic stellate cells(HSC)senescence in mice with Schistosoma japonicum infection. Methods A murine model of S. japonicum infection was established and the serum samples and liver tissues were collected 4,6,8,12 weeks post-infection. The serum samples were detected for the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST). The pathological changes and proliferation of hepatic collagen fibers in the liver tissue were observed after HE staining and Masson staining. The HSC senescence in fibrotic livers was determined by the detection of senescence-associatedβ-galactosidase(SA-β-Gal). Sandwich ELISA was used to measure the expressions of IL-22,and Real-time PCR was used to test the mRNA levels of IL-22 and IL-22R1. The control group without S. japonicum infection was set up. Results The serum levels of ALT and AST signifi-cantly increased 8 weeks and 12 weeks after the infection(vs. 0 week,all P<0.05). The level of IL-22 increased 4 weeks and 6 weeks after the infection(vs. 0 week,both P<0.05),but reduced 8 weeks post-infection,and was even lower 12 weeks post-in-fection(vs. 4 weeks and 6 weeks,both P<0.01). Being consistent with the dynamic expression of IL-22 protein,the mRNA ex-pression of IL-22 began to increase 4 weeks and reached the peak 6 weeks after the infection(vs. 0 week,both P<0.05),and continuously declined 8 weeks and 12 weeks post-infections(vs. 6 weeks,both P<0.05). The increase of the expression of IL-22R1 mRNA was correlated with the progression of fibrosis,and the peak was in 12 weeks post-infections(vs. 0 week and 6 weeks,both P<0.05). The number of senescence-associated beta-galactosidase-positive HSCs was reduced with the decreasing expression of IL-22 in the advanced liver fibrosis. Conclusion IL-22 and IL-22R1 are involved in the pathogenesis of schistoso-miasis liver fibrosis. As an inflammation factor,IL-22 significantly increases in the early stage of fibrosis. The expression of IL-22 decreases in the late stage of fibrosis,which may contribute to HSC senescence and restrict liver fibrosis.

Expression of interleukin-22 and relative CD4+ T cell subsets in ulcerative colitis / 中华微生物学和免疫学杂志

Objective To detect the expression of interlenkin-22 (IL-22) and relative CD4+ T cell subsets in patients with ulcerative colitis (UC),and to explore their roles in the pathogenesis of UC.Methods Thirty-five adult UC patients were enrolled in this study and 35 healthy subjects were taken as control.Plasma IL-22 level was quantified by ELISA.The percentages of Th1,Th17 and Th22 cells in peripheral blood were determined by flow cytometry.The relationships of these results and disease activity were analyzed.Results Plasma IL-22 levels in UC patients [ (354.12±104.22 ) pg/ml ]were significantly higher than that of healthy controls ( P＜0.05 ),and the levels increased significantly in severely active patients.The percentages of Th17 cells in UC patients [ (2.36±0.94) ％ ]were elevated compared to healthy controls ( P＜0.05 ),and the percentages increased significantly in moderately active and severely active patients.The percentages of Th22 cells in UC patients [ (2.27±0.87 ) ％ ]were elevated compared to healthy controls (P±0.05),and the percentages increased significantly in severely active patients.The percentage of Th1 cells was not significantly different between UC patients and normal controls.ConclusionOur resuits demonstrate elevated IL-22 correlated to Th17 and Th22 cells may play an important role in the immunopathogenesis of UC.