| BackgrandLung interstitial fibrosis is a chronic lung disease characterized by excessiveaccumulation of extracellular matrix (ECM), remodelling of lung architectures and seriouscomplications in a variety of rheumatic autoimmune diseases. It is an inflammatoryinterstitial lung disease caused by a variety of reasons, mainly involving interstitial lunglesions, may also be involved in alveolar epithelial cells and pulmonary vascular. Theproportion of patients suffer from secondary pulmonary fibrosis in systemic sclerosis,rheumatoid arthritis, polymyositis/dermatomyositis, systemic lupus erythematosus,ankylosing spondylitis, and mixed connective tissue disease, clinically very common.Most patients with lung interstitial fibrosis would die of progressive respiratory failurewithin3–8years of the onset of the symptoms [1]. Despite the high mortality, no effectivetherapies are available to halt or reverse the progression of lung interstitial fibrosis. Thus, there is a large and unmet medical need for new therapeutic strategies to treat lunginterstitial fibrosis and other diseases that involve pathological tissue fibrosis.T helper cell17(Th17) has great significance in the defense reaction of the body andautoimmune diseases， which can secrete interleukin-17(IL-17) of the T-cell subsets. Th0cell differentiate to Th17cells stimulated by TGF-β, IL-1β, IL-6and IL-23. Th17cellsplay a very important role in the pathogenesis of a variety of immune-related diseases,such as psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease andasthma. In recent years researches, Th17cells also has a very important role in fibrosis.Mononuclear phagocyte precursors are induced to differentiation by themicroenvironment of certain microbial products and cytokines, and different inducedfactors exhibit phenotypic and functional characteristics. It can be roughly divided intotwo different subtypes, namely M1and M2macrophages, the functional differences of thevarious subtypes. Although at home and abroad have been reported in the literaturemacrophages can promote the differentiation of Th17cells, but the role of each subtype indifferentiation of Th17cells is not very clear.In pulmonary fibrosis, certain cells particularly macrophages and neutrophils expresshigh level of CD147and CD147may promote the process of pulmonary fibrosis. Havebeen reported in the literature, CD147also have a certain relationship with TGF-β, IL-6,so we infer CD147may relate with Th17differentiation. The purpose of this project is tolink interstitial pulmonary fibrosis, macrophage subtype, Th17cells and CD147togetherand to clarify the relationship and the mechanism of M1, M2macrophages and Th17celldifferentiation in interstitial lung fibrosis, and to study CD147in the role.AimTo establish a murine model of pulmonary interstitial fibrosis, and treat with HAb18mAbs, observe the changes of M1, M2macrophages and Th17cells; to co-culture M1, M2macrophages and CD4+T cells isolated from mice lung tissues of the bleomycin group,and observe the differentiation of Th17cells. to clarify the relationship and the mechanismof M1, M2macrophages and Th17cell differentiation in interstitial lung fibrosis, and to study CD147in the role.Methods and resultsCD147/HAb18mAbs treatment of lung interstitial fibrosisInduction of murine models of pulmonary interstitial fibrosisC57BL/6female mice were assigned randomly to the BLM-induced group,24mice, thetreatment group,6mice and treatment control group,6mice. The experimental mice wereinjected intraperitoneally with10mg/kg HAb18G/CD147mAbs (prepared in ourlaboratory) from d1, and one day at a time. Three groups were included in the experiment:normal group treated with sterile saline, bleomycin group and HAb18G/CD147mAbstreatment group.Testing and researchMice were killed at d4,7,14following induction of lung fibrosis and the fibrotic micetreated with HAb18G/CD147mAbs were killed at d14. The top-left lung was surgicallyremoved, stabilize with4%0.1PBS paraformaldenyde for24hours. After routinedehydration and paraffin denvelopment, the3μm laraffin slices were cut from theparaffin-embedded lung samples for HE and Masson staining. Mice lung tissues of thebleomycin group were minced and digested with collagenase and hyaluronidase,respectively. After lysis of RBC, the dissociated cells were underlaid with5ml oflymphocyte separation solution (Mediatech) and were centrifuged at2000rpm for20min.The mononuclear cells in the middle layer were collected for flow cytometer analysis.Total RNA was isolated from purified lymphocytes and macrophages. ComplementaryDNA was prepared by reverse transcription and RNA expression levels were determinedby real-time quantitative PCR.ResultsHistologic examination of lung tissues Hematoxylin-eosin staining showed that the lungs of mice in saline group and normalcontrol group were normal and light pink in color. No inflammatory cell infiltration orobvious changes on alveolar septa were observed under the microscope. In the bleomycingroups, multifocal diffuse changes, including some combinations of thickened alveolarsepta, interstitial hyperplasia, intra-alveolar fibrosis with myofibroblasts, occasional fociof dense fibrosis, increased alveolar macrophages, and some infiltration of inflammatorycells, were increased at all times. The histological examination and masson trichromtaining of the lungs on d14after bleomycin injection displayed intact lung architecture incontrol group. In bleomycin group, collagen fibers increased significantly and a largenumber of fibroblasts gathered around interstitial and small peribronchial, whereascollagen deposition decreased in HAb18G/CD147mAbs-treated group.The changes of M1, M2macrophages and Th17cellsFlow cytometry showed that the quantity of M1, M2macrophages and Th17cellsincreased gradually and reached peak at d14. M1macrophages were higher than M2macrophages at d4and d7, however, at d14, M2macrophages took dominate placeslightly. In HAb18G/CD147mAbs-treated group, the presence of M1and M2macrophages and Th17cells in the fibrotic lungs showed that Th17cells decreasedobviously compared with those in bleomycin group (1.56%±0.45%to3.24%±0.63%),and M1macrophages were lost slightly (1.96%±0.35%to2.83%±0.47%), but noobvious changes were observed in M2macrophages (4.86%±0.67%to4.73%±0.54%).Real-time quantitative PCR analysisReal-time quantitative PCR analysis performed on RNA isolated from fibrotic lungsfor TGF-β, IL-6, IL-1β, IFN-γ and IL-4showed that treatment with HAb18G/CD147mAbs decreased TGF-β, IL-6and IL-1β mRNA evidently, but no obvious effect was seenon IFN-γ and IL-4.Macrophages-induced differentiation of Th17cells Isolation and co-culture of CD4+T cell and macrophage subsetsMice lung tissues of the bleomycin group were minced and digested with collagenase andhyaluronidase, respectively. After lysis of RBC, the dissociated cells were underlaid with5ml of lymphocyte separation solution (Mediatech) and were centrifuged at2000rpm for20min. The mononuclear cells in the middle layer were collected for flow cytometeranalysis. The M1macrophages, defined as CD14+CD16/32+cells, the M2macrophages,defined as CD14+CD206+cells and CD4+T cells were sorted by fluorescence-activatedcell sorter.Testing and researchM1macrophage and M2macrophages incubated in a6-well plate for2h respectively.Then, CD4+T cells with5ug/ml anti-CD3and10ug/ml anti-CD28(purchased from BDPharmingen) were co-cultured respectively with M1macrophages or M2macrophages inthe6-well plate, and3days later, CD4+T cells were collected for flow cytometry.ResultsMacrophages-induced differentiation of Th17cellsCollection CD4+T cells and flow cytometric analysis showed that the expressions ofCCR6, IL-17and RORγt activated by M1macrophages were respectively~5(4.96%±0.46%to1.22%±0.21%),~3(3.87%±0.32%to1.24%±0.23%) and~4(2.54%±0.28%to0.60%±0.15%) folds higher than the expressions activated by M2macrophages.CD147in M1macrophages-induced Th17cellsWith presence of HAb18G/CD147mAbs, the levels of CCR6in HAb18G/CD147mAbs treatment group were lower than those in the normal M1macrophages group(1.84%±0.45%to4.15%±0.68%)(Figure3B) and with the cut-down expression ofCD147using shRNA lentivirus interference, Th17cells in CD147-deficient group had lower activation compared with that in normal M1macrophages group (2.06%±0.48%to4.84%±0.72%)ConclusionIn summary, the findings of this study have shown that M1macrophages have apositive effect on the differentiation of Th17cells, and CD147promotes this process byregulating Th17-promoting cytokines, including TGF-β, IL-6and IL-1β. However, thedifferentiation of Th17cells is a complex process involving different kinds of cells andcytokines, and further experimental investigations are needed to further explore whichsignal path CD147takes to produce the effect. |
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