Effect Of Th17 Cells In A Bleomycin-Induced Murine Model Of Systemic Sclerosis-associated Pulmonary Fibrosis

PART IThe expression and significance of Th17 cells in a bleomycin-induced murine model of systemic sclerosis associated pulmonary fibrosisObjective To study the expression of Th17 cells and related factors in a blemycin (BLM)-induced mrurie model of systemic sclerosis (SSc) associated pulmonary fibrosis (PF, SSc-PF). To explore whether Th17 cells are involved the pathogenesis of SSc and SSc-PF.Methods 30 female BALB/c mice were randomly divided into two groups, including control group 10 and BLM-induced SSc model group 20 which are divided into two groups according to a semi-quantitative score of PF(Ashcroft), that group SSc-A (PF<3 level) and group SSc-B (PF>3 level). The pathological changes, inflammation, fibrosis score in murine back skin of the injecting and lungs were observed by HE and Masson's staining and the content of hydroxyproline (HYP) was determined. The percentage of CD4~+IL-17~+(Th17) cells in peripheral blood, skin and lung tissue was detected by Flow Cytometry. The mRNA expression of RORyt, interleukin (IL)-17A and transforming growth factor (TGF)-β1 were deteced by real-time (RT)-PCR. The content of IL-17A and TGF-β1 in serum was determined by ELISA. Analyze the relationship between Thl7 cells and the inflammation and fibrosis lesion of skin and lungs.Results According to the Ashcroft score,9 model mice were less than grade 3 as group SSc-A and 11 model mice were epual or more than grade 3 as group SSc-B. HE and Masson's staining showed that the structure of skin and lung tissue were integrity and hadn't apparent inflammatory cells infiltration and collagen hyperplasia in control group, and there were significantly skin thickening, inflammatory cells infiltration, adnexal atrophy and collagen proliferation in model mice. In group SSc-A, lung structure without obvious damaged and with a small amount fibrous thickening in the alveolar and bronchial wall. In group SSc-B, there were obvious fibrous thickening and inflammatory cells infiltration in interstitial lung, alveolar and bronchial wall, and lung structure was damaged in part of the mice. Dermal and pulmonary inflammation, and PF score in group SSc-B(2.82±0.75,2.36±0.81 and 4.0±1.41), group SSc-A (2.44±0.73,1.33±0.50 and 1.56±0.73) were significantly increased as compared with that in the control group(0.40±0.52,0.40±0.52 and 0.60±0.70), all P<0.05. The content of pulmonary HYP in group SSc-B(0.64±0.08) were significantly increased compared to the SSc-A(0.45±0.08) and control group(0.38±0.16)mg/g, and the content of cutaneous HYP in group SSc-B(3.07±1.66), SSc-A (2.44±0.61) were significantly increased compared to the control group(1.45±0.40)mg/g, all P<0.05. Th17 cells of peripheral blood and skin in group SSc-B [(1.61±0.49)%, (3.48±1.59)%], SSc-A[(1.36±0.51)%, (2.89±1.55)%] were significantly increased than control group[(0.85±0.23)%, (1.28±0.37)%]. The percentage of Th17 cells of lung tissue in group SSc-B(3.83±1.44)%were significantly increased than group SSc-A (2.30±0.98)%and control group(1.32±0.37)%, all P＜0.05. The mRNA levels of IL-17A, RORyt and TGF-β1 of skin in group SSc-B and SSc-A were significantly higher than control group. The mRNA levels in group SSc-B of IL-17A (2.79±2.16), RORyt (30.54±32.97)and TGF-β1 (0.24±0.23)in lung tissue were significantly higher than control group (0.19±0.35,2.04±4.46, 0.03±0.03) (P＜0.01), and IL-17A in group SSc-B more than group SSc-A (P<0.05). The levels of IL-17A and TGF-β1 in serum of group SSc-B [(72.45±5.05)pg/ml], [(1397.02±157.64)pg/ml] and in group SSc-A [(70.83±5.05)pg/ml], [(1382.72±161.77)pg/ml] were obviously increased than control group [(63.89±4.48)pg/ml], [(1165.76±140.44)pg/ml], all P＜0.05. Th17 cells in peripheral blood were positively correlated with inflammation of skin and lungs, HYP content of skin and lungs and the levels of IL-17A in serum (r value is 0.499,0.604,0.387,0.374,0.560,P＜0.05). Th17 cells in lung tissue was positively correlated with pulmonary inflammation, the content of HYP, IL-17A mRNA of lung tissue (r value is 0.683,0.663,0.605, P＜0.01).Th17 cells in skin was positively correlated with inflammation of skin, the content of HYP, IL-17A mRNA of skin(r value is 0.632,0.544,0.406,P＜0.05). The level of IL-17A in serum was positively correlated with inflammation and HYP of skin and lung tissue and was positively correlated with the levels of TGF-β1 (r value is 0.728,0.642,0.652,0.650,0.401,P＜0.05)Conclusion Th17 cells and IL-17A were significantly increased in peripheral blood, skin and lungs of SSc murine model and with a enhanced activity, especially in the mice with obvious PF. These changes had an intimate relationship with inflammation and fibrosis lesion of the skin and lungs. Th17 cells may involve the pathogenesis of SSc and SSc-PF through producing IL-17A and interaction with TGF-β1. PART IIThe chemotaxis of CCR6/CCL20 to Th17 cells in a bleomycin-induced murine model of systemic sclerosis associated pulmonary fibrosisObjective To study the level of CCR6-expressing Th17 cells and its ligand CCL20 and related factors in peripheral blood, skin and lungs of SSc or SSc-PF mice, and to assess the chemotaxis of CCR6/CCL20 to Th17 cells.Methods 30 female BALB/c mice were randomly divided into two groups, including control group, model group (group SSc A and group SSc B, like the first part). The pathological changes, inflammation, fibrosis score in murine back skin of the injecting and lungs were observed by HE and Masson's staining and the content of hydroxyproline (HYP) was determined. The percentage of CD4~+CCR6~+cells, CD4~+CCR6~+IL-17~+cells in peripheral blood, skin and lungs were detected by flow cytometry. The mRNA of CCL20, IL-17A, TGFβ1, IL-6 in skin and lungs were measured by RT-PCR. The content of CCL20, IL-17A, TGFβ1, IL-6 in peripheral blood were detected by ELISA.Results Dermal and pulmonary inflammation, PF score, HYP content of skin in group SSc-B and SSc-A were significantly increased compared to the control group, all P<0.05. The content of pulmonary HYP in group SSc-B were more than SSc-A and control group, P<0.05(With the first part of the same). The percentage of CD4~+CCR6~+cells in peripheral blood, skin and lungs of SSc-B and SSc-A groups was significantly increaseed as compared with control group. The percentage of CD4+CCR6+IL-17+cells of peripheral blood and skin in SSc-B [(0.18±0.07)%, (0.36±0.16)%]and SSc-A [(0.19±0.07)%, (0.33±0.17)%] was significantly increased compared with the control group[(0.11±0.03)%, (0.15±0.05)%]. The percentage of CD4+CCR6+IL-17~+cells in lungs of SSc-B group (0.48±0.32)%was significantly increased compared with SSc-A (0.29±0.24)%and control groups (0.25±0.15)%, all P<0.05. The mRNA of CCL20 in skin and lungs and the content of CCL20 in peripheral blood in SSc-B group was significantly increased compared with SSc-A and control groups, all P<0.05. The frequency of CD4~+CCR6~+IL-17~+cells in peripheral blood had a positive correlation with inflammation of skin and lungs, content of pulmonary HYP, and content of CCL20(r value is 0.409,0.435,0.414,0.494, all P＜0.05). The frequency of CD4~+CCR6~+IL-17~+cells in skin and lungs respectively had positive correlation with inflammation of skin and lungs, content of cutaneous and pulmonary HYP, and the mRNA level of CCL20 (r value is 0.376～0.738, P<0.05). Compared with the control group, the mRNA level in skin and lungs and content in peripheral blood of IL-17A, TGFβ1, IL-6 in SSc-B and SSc-A groups were significantly increased, all P<0.05. The frequency of CD4~+CCR6~+ IL-17~+cells and the expression level of CCL20 in peripheral blood and lungs had a positive correlation with the expression level of IL-17 A, TGFβ1, IL-6, respectively (r value is 0.400～0.781, P＜0.05). The frequency of CD4~+CCR6~+ IL-17~+cells in skin had a positive correlation with the expression level of IL-17A, TGFβ1, IL-6.Conclusions Th17 cells can recruit in the lesion site of skin and lung in SSc-PF mice through the chemotactic axes role of the CCR6/CCL20, and that is one of the reasons result in increasing of Thl7 cells. PART IIIThe expression of IL-21 on CD4+T cells in a m urine model of systemic sclerosis and the effect that IL-21 and TGF-β1 on differentiation of CD4+T cells to Th17 cells in vitroObjective Observing the ratio of CD4~+IL-17~+L-21~+cell and CD4~+IL-21R~+ cell in peripheral blood, skin and lung tissue of SSc mice. To research the effect of IL-21 and TGF-p 1 on the differentiation of CD4+T cells to Thl7 cells, and further clarify the increased reason of Thl7cells and the mechanism in inflammatory and fibrotic lesions of SSc.Methods 30 female BALB/c mice were randomly divided into two groups, including control group, model group (group SSc A and group SSc B, like the first part). The pathological changes, inflammation, fibrosis score in murine back skin of the injecting and lungs were observed by HE and Masson's staining and the content of hydroxyproline (HYP) was determined. The proportion of CD4~+IL-17~+L-21~+cells and CD4~+IL-21R~+T cells in peripheral blood, skin and lungs of SSc mice were detected by flow cytometry; the content of peripheral blood IL-21 were tested by ELISA. CD4+T cells were insolated by magnetic beads from spleencytes of SSc mice, and were cultured in medium with rIL-21 (IL-21 group), TGF-β1 (TGF-β1 group), IL-21 and TGF-β1 (IL-21 TGF-β1 group), and the control group respectively in vitro for 72 hours. The proportion of Th17 cells in each group were determined by flow cytometry. The mRNA levels of RORyt and IL-17A were detected by RT-PCR, and the levels of IL-17A in the culture supernatants were tested by ELISA.Results Dermal and pulmonary inflammation, PF score, HYP content of skin in group SSc-B and SSc-A were significantly increased compared to the control group, all P<0.05. The content of pulmonary HYP in group SSc-B were more than SSc-A and control group, P＜0.05(With the first part of the same). The percentage of CD4~+IL-17~+IL-21~+cells in peripheral blood, lung tissue, skin in group SSc-B [0.11±0.06)%, (0.13±0.05)%, (0.15±0.09)%], group SSc-A [0.10±0.06)%, (0.06±0.03)%, (0.16±0.10)%] were obviously increased compared to the control group [0.02±0.02)%,0.02±0.02)%,0.00±0.01)%], and the group SSc-B in lung was significantly increased than the SSc-A group, all P＜0.05. The percentage of CD4~+IL-21R~+in peripheral blood, lungs and skin in group SSc-B [0.89±0.42)%, (1.56±0.39)%,(2.17±1.02)%], group SSc-A [(0.84±0.24)%,(1.50±0.50),(2.17±0.98)%]were significantly increased compared to the control group [0.51±0.18)%,(1.03±0.40)%,(1.13±0.52)%],all P< 0.05. The content of IL-21 in peripheral blood in group SSc-B [(136.68±40.61 pg/ml] and group SSc-A [(125.42±47.88 pg/ml] were significantly increased compared to the control group [(76.89±26.46 pg/ml], all P< 0.05. The proportion of peripheral blood CD4~+IL-17~+IL-21~+cells and CD4~+IL-21R~+cells were related closely with the inflammation of skin and lungs, PF score and the content of IL-21 (r value is 0.383～0.668, all P< 0.05). The proportion of CD4~+IL-17~+ IL-21~+cells in skin was positive correlated with content of cutaneous HYP (r value is 0.523, P= 0.003). The proportion of CD4~+IL-17~+IL-21~+cells and CD4~+IL-21R~+cells in lungs were positive correlated with pulmonary inflammation and HYP content (r value is 0.444～0.767, all P< 0.05). The content of IL-21 in peripheral blood were closely related with the inflammation of skin and lungs, the pulmonary HYP content, CD4~+IL-17~+IL-21~+cells in peripheral blood (r value is 0.395～0.514, P＜0.05). Compared to the control group, the proportion of Thl7 cells of IL-21 group (3.34±1.38)%and IL-21 TGF-β1 group (5.85±2.45)%in SSc murine splenic CD4~+T cells culture were significantly increased(all P＜0.05). The mRNA level of IL-17 A and ROR yt, and the IL-17 content of cell culture supernatants in IL-21 group and IL-21 TGF-(31 group were significantly increased(all P<0.05), especially in IL-21 TGF-β1 group.Conclusion:The increased proportion of CD4~+IL-17~+IL-21~+and CD4~+ IL-21R~+cells in the peripheral blood, skin and lung tissue of SSc mice were detected. Th17 cells may produce IL-21 to involve skin and lung inflammation and fibrosis lesions of SSc mice through the IL-21/IL-21 R pathway. IL-21 can induce CD4~+T cell differentiate into Th17 cells, and combined with TGF-β1 might have a more stronger effect. PART IVThe influence of IL-17A on fibroblasts proliferation and secretory function in vitroObjective Observing the influence of IL-17A and TGF-β1 on fibroblasts (FB) in the proliferation and secretory function and the interaction between them. To further explore the role of Th17 cells in fibrosis formation of SSc.Method Pulmonary FB of the SSc murine were cultured primarily, and observed the morphology through the inverted microscope, and identified the purity through vimentin immunohistochemistry techniques. Observed the effects of IL-17A and TGF-β1 in different concentrations in 24 hours on proliferation of FB by using MTT method and find the most suitable concentration. FB was cultured respectively with IL-17A, TGF-β1, IL-17A and TGF-β1, and DMEM medium. Detect proliferation activity of FB through Brdu ELISA, and the mRNA level and protein concentration of type I collagen secreted by FB through RT-PCR and Western blot(WB), and the level of IL-6 in cell culture supernatants by ELISA after in the culture of 24,48 and 72 hours. Detect the mRNA level of TGF-β1 and content of TGF-β1 in cell culture supernatants in with IL-17A stimulation group.Results The cultured Lung tissue blocks were observed using by the inverted microscope in black shading area and adherent tightly, and the individual cells swam out about in 4-10d. The cells were spindle mainly and intensive gradually, and can be passaged in 11-20d. The immunohistochemical staining of vimentin in the cells was positive. The MTT result showed IL-17A and TGF-β1 could promote the proliferation of FB in 24h with the increasing of concentration. IL-17A in 25μg/L and TGF-β1 in 12.5μg/L can arrive maximal proliferation activity of FB. Compared with the control group, the proliferation activity of FB increased gradually in 24h,48h,72h after added in IL-17A, TGF-β1. The between-subject factors F value was 773.040, and the within-subject factors F value was 140.739, all P< 0.01. Compared with the control group, the expression of mRNA and protein concentration of type I collagen secreted by FB were increased gradually in 24h,48h,72h after added in IL-17A and/or TGF-β1, and so did IL-6 of cell culture supernatants. The between-subiect factors F value was 55.242,202.817,69.145, and the within-subject factors F value was 80.062,149.71,159.62, respectively, all P< 0.01, expecially in the group of IL-17A combine TGF-β1. Compared with the control group, the mRNA level of TGF-β1 and content of TGF-β1 in cell culture supernatants secreted by FB were increased significantly after added in IL-17A(allP<0.05).Conclusion IL-17A can promote FB to secrete type I collagen and IL-6, with time-dependent, and combined with TGF-β1 might have a more stronger effect. IL-17A could enhance FB to secrete TGF-β1, and they could have synergistic effect in fibrotic lesions of SSc.