| Background Dermatomyositis(DM)and polymyositis(PM)are two subtypes of idiopathic inflammatory myopathy(IIM).DM/PM are autoimmune diseases which primarily manifest involved skin in the former,coupled or uncoupled with involved muscle,and primarily manifest involved muscle in the latter.However,there are other organs involved except for skin and muscle,such as vasculitis,arthritis,raynaud's phenomenon,interstitial lung disease(ILD).ILD associated with DM/PM is the most important factor which affects the prognosis of these patients,especially for patients with rapidly progressive interstitial lung disease(RP-ILD).DM/PM-ILD patients have worse outcome,and take heavy economical burden to family and society.There is no effective and specific therapy because of undefined pathogenesis associated with ILD in DM/PM till now.The pathogenesis of myositis-associated ILD is primarily the following hypothesis:excessive inflammation can't be terminated in time,which results in the status of fiber proliferation in lung.No definite etiology has been found for MA-ILD,however,virus infection is regarded as one possible reason.In literature,hepatitis C virus(HCV)is related to MA-ILD and anti-Jo-1-associated ILD,cytomegalovirus(CMV)is related to RP-ILD,and parvovirus B19 is related to the damage of microvessel in ILD patients.In addition,humoral and cellular immunity are related to MA-ILD,more than 75% patients with anti-synthetase antibody accompanied with ILD,and there were more T lymphocytes in BALF(Bronchoalveolar Lavage Fluid),moreover,more CD8+ T cells were activated in lung of DM/PM-ILD patients.However,the action of the factors above mentioned to MA-ILD is only relevant study,and it is not known how these factors work.So,it is urgent to explore the pathogenesis of ILD associated with DM/PM further.There is consistency in clinic,physiology,pathology and image about different ILDs in spite of different reasons,meanwhile,the interface between alveoli and blood capillary is hurted by the same pathway(s)in different ILDs,and they manifest the same reticular and honeycomb shade in lung HRCT.More work has been done in animal and IPF about the pathogenesis of ILD and pulmonary fibrosis,which could be used to study the pathogenesis of DM/PM-ILD.miR-200 c,miR-125 b and ILD.Micro RNAs(miRNAs)are small non-coding RNAs about 18-25 nucleotides in length,which function as regulating the expression of protein-encoding genes by targeting 3'UTR of m RNAs and resulting in inhibiting of translation or promoting degradation of these m RNAs.Up to now,miRNAs have been confirmed to play important roles in diverse diseases,such as tumors,cardiovascular diseases,fibrotic diseases(including lung fibrosis).Mi R-200 c is significantly downregulated in the lungs of mice with experimental lung fibrosis,and in the lungs of patients with IPF.Moreover,miR-200 c has higher expression in alveolar epithelial cells(AECs)than in lung fibroblasts,and introduction of miR-200 c alleviates experimental lung fibrosis by inhibiting epithelial-mesenchymal transition(EMT)in mice.So,miR-200 c was believed to have a protective effect on pulmonary fibrosis by maintaining the epithelial phenotype of AECs and inhibiting EMT.Mi R-125 b has been shown disease-suppressing properties and disease-promoting functions in many different types of diseases,such as solid tumors,hematological malignancies and autoimmune diseases,by targeting different protein-coding genes and influencing the proliferation,differentiation,migration of different types of cells.In the mice of acute lung injury,the count of total cells,neutrophils,proinflomatory cytokines,and chemokines were reduced by miR-125 in BALF,which protected lung from damage.miR-200 c,miR-125 b and T lymphocytes.miR-200 c and miR-125 not only play a crucial role in differentiation,proliferation and migration of AECs and lung fibroblasts,but also show important roles in proliferation,differentiation and apoptosis of lymphocytes.miR-125 b inhibits the differentiation of T lymphocytes from na?ve T-cells to effector T-cells.miR-200 c inhibits T lymphocyte response.T lymphocytes and ILD.T lymphocytes play crucial roles in the development of diverse ILDs(including ILD associated with DM/PM).It was reported that T lymphocyte activation was more significant in BALF of ILD patients than in peripheral blood.There were more CD8+ T lymphocytes in BALF in a patient with amyopathic dermatomyositis associated with fatal rapidly progressive interstitial pneumonitis.Enelow RI,et al reported antigen-specific CD8+ T lymphocytes recognized an alveolar epithelial autoantigen,triggering an inflammatory cascade and cause interstitial pneumonitis in experimental lung disease.mTOR pathway and T lymphocytes.Mammalian target of Rapamycin(mTOR)was reported to promote various kinds of biosynthesis,including protein synthesis,proliferation,growth,differentiation of different cells.It is reported that mTOR pathway participates in the proliferation of eukaryocytes widely.In vivo,mTOR pathway influences the migration of CD8+ T lymphocytes and differentiation of CD8+ memory T lymphocytes.miR-200 c,miR-125 b and mTOR pathway.We predicted that S6 K and 4E-BP1,which are in the downstream of mTOR pathway,cell proliferation pathway,are the target genes of miR-200 c and miR-125 b by bioinformatics.So,miR-200 c and miR-125 b maybe influence the development of ILD associated with DM/PM by controling the proliferation of CD8+ T lymphocytes through regulating the expression of S6 K and 4E-BP1.Basis of the roles of miR-200 c,miR-125-b,and CD8+ T lymphocytes in ILD and the bioinformatics prediction of target genes for those miRNAs,we caught up with a hypothesis: miR-200 c and miR-125 b are related to the development of ILD associated with DM/PM by influencing the proliferation of CD8+ T lymphocytes.We verified our hypothesis by three steps: firstly,we studied the levels of miR-200 c and miR-125 b in PBMCs of DM/PM patients,and analyzed the correlation between miRNAs levels and the degree of lung injury;secondly,we analyzed the association between the expression levels of miR-200 c and miR-125 b and SP-D,TGF-?1,the marks of lung injury in BALF;thirdly,we studied the association between the levels of miR-200 c,miR-125 b and the percentage of CD8+ T lymphocytes in BALF.Objectives1.To explore the relationship between the levels of miR-200 c and miR-125 b and the degree of lung injury in DM/PM-ILD patients.2.To analyze the association between the levels of miR-200 c and miR-125 b and SP-D,TGF-?1,the marks of lung injury.3.To explore the association between the levels of miR-200 c,miR-125 b and the percentage of CD8+ T lymphocytes in BALF to make clear furtherly the role of the miR-200 c and miR-125 b in ILD associated with DM/PM.Methods1.30 consecutive inpatients and outpatients were recruited into current study from Anhui Provincial Hospital.All these patients were arranged for high resolution computerized tomography(HRCT)of chest and pulmonary function test(PFT).According to HRCT,PFT and the diagnosis criteria of ILD from American Thoracic Society /European Respiratory Society(ATS/ERS),these patients were judged with ILD or without ILD,and all these patients were classified into three groups: non or mild ILD group(9 patients),moderate ILD group(10 patients),and severe ILD group(11 patients).23 blood donors were included as healthy control.2.Quantitative reverse transcription-polymerase chain reaction(QRT-PCR): PBMCs were gained from 30 DM/PM patients and 23 healthy control by lymphocyte separating medium,then total RNA was extracted from these PBMCs.QRT-PCR was used to detect the expression levels of miR-200 c and miR-125 b in PBMCs of all patients and BALF cells of DM/PM-ILD patients.The expression levels of the two miRNAs were compared among different degree ILD groups,and between PBMCs and BALF cells.3.Bronchoalveolar lavage: 13 DM/PM-ILD patients were recruited for BAL to gain BALF.The BALF was filtered to collect cells for detecting these two miRNAs by QRT-PCR and to gain supernatant for detecting TGF-?1 and SP-D by enzyme linked immunosorbent assay(ELISA).4.Detecting TGF-?1 and SP-D: TGF-?1 and SP-D in serum and the BALF supernatant of DM/PM and DM/PM-ILD patients were detecting by ELISA.The levels of TGF-?1 and SP-D were compared between plasma and BALF supernatant.The correlations between miR-200 c,miR-125 b and TGF-?1,SP-D in BALF were analyzed.5.Differential counts of bronchoalveolar cells: Total cell count was done in a hematocytometer for BALF samples in 13 DM/PM-ILD patients.Four cytospin specimens were prepared and stained with Wright-Giemsa.These stained specimens were examined in microscope and differentially counted.6.Flow cytometry(FCM): PBMCs of 10 DM/PM-ILD patients and Bronchoalveolar cells of 13 DM/PM-ILD patients were stained with fluorescein isothiocyanate-,phycoerythrin-,allophycocyanin-.or peridinin chlorophyll protein-conjugated monoclonal antibodies to CD3,CD4,CD8 and CD45.Stained cells were analyzed by FCM.The percentages of CD3+,CD4+,and CD8+ T lymphocytes were calculated.The correlations between miR-200 c,miR-125 b,and CD8+ T lymphocyte in BALF were analyzed.Results1.The levels of miR-200 c and miR-125 b in PBMCs from DM/PM patients: The expression levels of miR-200 c and miR-125 b were significantly higher in patients' PBMCs than in control's PBMCs in statistics(P=0.0006,0.034,Mann-Whitney Test).2.The comparison of levels of miR-200 c and miR-125 b in BALF with those in PBMCs: The expression levels of miR-200 c and miR-125 b in BALF were higher than those in PBMCs,and the differences were significant in statistics(P=0.0313,0.0188,Wilcoxon Signed Ranks Test).3.There was positive correlation between the level of miR-200 c in PBMCs and severity degree of ILD associated with DM/PM from without or mild ILD(group 1)to moderate(group 2)and severe ILD(group 3)(group1 VS group 3 and group 2 VS group 3,P=0.0024,0.0083).There was positive correlation between the level of miR-125 b in PBMCs and severity degree of ILD associated with DM/PM from without or mild ILD(group 1)to moderate(group 2)and severe ILD(group 3)(group1 VS group 3 and group 2 VS group 3,P=0.0107,0.0133).4.Comparison of the levels of TGF-?1 and SP-D in BALF supernatant with those in serum from DM/PM-ILD patients: There was significantly higher TGF-?1 in BALF supernatant than in serum(525.79(492.67-651.76)ng/m L VS 35.59(18.02-72.25)ng/m L,P=0.0009,Mann-Whitney Test).The level of SP-D in BALF supernatant was also higher than that in serum,the difference was not significant(P=0.0799,Mann-Whitney Test).5.The correlations between the levels of miR-200 c and miR-125 b in BALF cells and the levels of TGF-?1 and SP-D in BALF supernatant: There were positive correlation between the expression levels of miR-200 c and miR-125 b in BALF cells,and that of SP-D in BALF supernatant(P=0.0083,0.002,Spearman correlation),but there was no significant correlation between miR-200 c,miR-125 b and TGF-?1(P=0.3333,0.3333,Spearman correlation).6.The differential counts of bronchoalveolar cells: There was higher count of total cells in BALF(>10*10^9/L for all 13 specimens).Most of these cells were neutrophils(>40% for 7 patients)and lymphocytes(>40% for 7 patients),which demonstrated lymphocyte predominance and neutrophil predominance(>5% for neutrophils and >15% for lymphocytes in all specimens).7.Comparison of the percentages of T lymphocyte subsets in BALF with those in PBMCs: In PBMCs,the percentages of CD3+,CD8+and CD4+ T lymphocytes were all normal in all 9 specimens and only one patient had lowered percentage of CD4+ T lymphocytes.There were higher percentage of CD3+ and CD8+ T lymphocytes in BALF than those in PBMCs(P=0.0006,P=0.0002,Paired T test).The percentage of CD4+ T lymphocytes was lower in BALF than in PBMCs(P=0.011,Paired T test).The ratio of CD3+ CD4+ / CD3+ CD8+ T lymphocytes was significantly lower in BALF than in PBMCs(P=0.003,Paired T test).8.The correlation between the levels of miR-200 c,miR-125 b and the percentage of CD8+ T lymphocytes in BALF: There were negative correlation between the levels of miR-200 c,miR-125 b and the percentage of CD8+ T lymphocytes(P=0.0172,0.0083,Spearman correlation).There were positive correlation between the levels of miR-200 c,miR-125 b and the ratio of CD4+/CD8+ T lymphocytes(P=0.002,0.0007,Spearman correlation).Conclusions1.There were positive correlations between the levels of these miRNAs and the degree of lung injury in DM/PM-ILD patients,which means that miR-200 c and miR-125 b maybe influence the development of ILD in DM/PM patients.2.There were negative correlations between the levels of miR-200 c,miR-125 b and the enhanced percentage of CD8+ T lymphocytes in BALF,which suggests miR-200 c and miR-125 b maybe protect lung from injury by inhibiting the proliferation of CD8+T lymphocytes in DM/PM patients. |
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