The Effect Of IL-27 On Differentiation Of T Lymphocytes And Cytokines In Bleomycin Induced-pulmonary Fibrosis Mice

Objective:To investigate the effect of IL-27 on differentiation of T lymphocytes and cytokines in bleomycin induced- pulmonary fibrosis mice,we perform that mice were given injection of interleukin-27(rmIL-27) and interleukin-27 antibody(Anti IL-27).Methods:1.120 6-8 weeks old 18-22gC57BL/6 mice were randomly divided into normal group (A group), BLM model group (B group), BLM+IL-27 group (C group), BLM+ anti IL-27group (group D)., each group of 30.2. Mice were anesthetized, A group was given a one-time injection of saline 0.05ml in intratracheal. B, C, D group were injected with saline solution diluted bleomycin 0.05ml (5ml/kg) in intratracheal. C group were injected with mouse IL-27 protein 1μg/day for 7 days through abdominal subcutaneous after building model. D group were injected with mouse anti IL-27 protein 200mg through abdominal3.5 mice were sacrificed in B group at 3,7,14,28 days respectively.IL-27 staining of T lymphocytes, B lymphocytes, natural killer cell, dendritic cells, and macrophages in the lung were detected by flow cytometry.4.5 mice from each group were sacrificed on day 7,28 days ① Detection of IL-27 expression in the lung tissue of four groups;② Detection of Thl, Th2, Th17 and Treg cellsin the spleen proportion by now cytometry;③Detection of different subsets of Th cells related cytokines in bronchoalveolar lavage fluid by Elisa.Results:1.3,7,14,28 days with IL-27 staining of T lymphocyte cells in the share ratio was 32.51士 7.37%,39.98±4.19%,70.7±0.54%,43.82±2.28%；B lymphocyte percentage of cells was 0.03±0.05％,0.07±0.09%,4.35±0.46%,7.66±0.91%；percentage proponion of cells by dendritic cells were 4.54±0.71%,15.96±0.84%,20.97±0.81%,16.13± 1.15%；Natural killer cells for cell proportions were 0.58±0.3%,6.63±0.55%,26.69+ 0.82%,0.48+0.49%；macrophage cell ratio was 0.31±0.08%,1.12±0.51%,21.97+ 0.93％,8.69+0.2%,T lymphocyte cells at each time point for the highest proporion(P< 0.05).2.In the 28 days of each group changes of T lymphocyte subsets:Thl cells are the largest proportion of group C(0.93±0.04%),A group(0.32±0.01)Second,group B (0.15± 0.01)followed,D group(0.04±0.01)minimum(P<0.05)；Treg cell ratio C(9.23± 0.85%)higher than gronp A(2.57±0.39%),B(0.77±0.11%),D (0.59±0.05%)group(P <0.05)；Th2 D group(3.8±0.28%)is greater man A(0.12±0.04%),B(1.34±0.22%),C (0.38±0.16%)group(P<0.05)；group B eater than group C(P<0.05)；Th17 cell ratio D group(0.94±0.16%)is greater than A(0.13±0.07%),B(0.54±0.08%),C (0.38± 0.07%)group(P<0.05)；group B higher than group C (P>0.05).3.Changes of these cytokines in alveolar lavage fluid concentration:7,28 days IFN-Y group C (49.67±3.06 pg/ml),(54.11±7.03pg/ml)is significantly higher than group A (37.1± 4.51 pg/ml),(42.98±2.87pg/ml),A group is higher than group B(20.79±8.45 pg/ ml),(16.01±2.98pg/ml),group C is higher than group B,group C is higher than D group(17.01±4.63 pg/ml),(13.85±2.49pg/ml)(P<0.05);IL-4 group B(47.7± 3.06pg/ml),(67.87±16.65pg/ml)is higher than group A(4.98±2.01pg/ml),(6.99± 1.07 pg/ml),C group(28.28±4.62pg/ml),(38.48±5.73pg/ml)is lower mall group B, D group(60.81±5.68pg/ml),(71.41±2.371.41±2.3pg/ml)is higher than group C(P <0.05)；IL-10 group B(156.57+54.71pg/ml),(243.8+58.29pg/ml)is higher than that in group A(89.59+53.01pg/ml),(120.28+32.73pg/ml),C group(129.43+30.13pg/ml), (166.2+45.77pg/ml)is lower than those in group D(226.65+31.87pg/ml),(265.97+ 72.71pg/ml),the difference is statistically significant(P<0.05);B group is higher than C group(P>0.05);IL-17 group B(51.23±5.78pg/ml),(56.05±0.42pg/ml)is higher than A group(30.93±4.78pg/ml),(32.53±3.05pg/ml),D group(60.31±19.69pg/ml), (73.03±18.4pg/ml)is higher than gr4oup C(42.59±14.84pg/ml),(31.82±6.48pg/ml), A group(P<0.05)；group B is higher than group c(P>0.05)；TGF β1 in 7 days,28 days group B(334.67±65.83g/ml),(489.72±104.82pg/ml)is higher than group A(168.29 ±37.61pg/ml),(177.6±33.34pg/ml),D group(432.76±59.22pg/ml),(1025.28± 175.33pg/ml)is higher than group C(255.5±102.05pg/ml),(232.76±67.8pg/ml)(P <0.05);group C(255.5±102.05pg/ml)is lower than group B(489.72±104.82pg/ml) (P<0.05)in 28 days;IL-6 in 7 days group A(168.91±42.5pg/ml)is less than B(212.68 ±73.88pg/ml),C(167.41±112.64pg/ml),D(273.43±118.05pg/ml)group,The cytokine levels of B,C,D groups did not differ(P>0.05);28 days group B(503.47± 90.13pg/ml)is higher than group A (197.12±27.45pg/ml),group B (503.47±90.13pg/ ml)is higher than group C(275.12±36.62pg/ml),D(816.43±118.98pg/ml)is higher man group C(P<0.05).Conclusion:1. IL-27 mainly comes from the T lymphocyte in bleomycin induced-pulmonary fibrosis mice.2.IL-27 might increase the differentiation of Th1.Treg cells and inhibit the production of Th2.Th17 cells in bleomycin induced-pulmonary fibrosis mice.3.IL-27 might increase the level of IFN-Y.and decrease the levels of IL-4.IL-6.IL-10. IL-17and TGF-β in bleomycin induced-pulmonary fibrosis mice.Moreover,this study also suggested that the IL-27 might in hibit the pulmonary fibrosis through the TGF-β1 signaling pathway.