The Role Of Cytokines TWEAK And BAFF In The Pathogenesis Of Polymyositis And

Idiopathic inflammatory myopathies (IIM) are a group of acquired, heterogeneous systemic diseases that mainly affect skeletal muscle. Polymyositis (PM) and dermatomyositis (DM) are two common subsets of IIM characterized by symmetrical proximal muscle weakness, decreased muscle endurance, increased serum creatine kinase, presence of autoantibodies, and inflammatory infiltrates in skeletal muscle tissue. In addition to affecting muscles, IIM also present pulmonary, heart, renal,cutaneous manifestations. PM and DM have now emerged as one of the most serious diseases that threating the health of people in China. However, the pathological mechanism of PM and DM remains unclear. Recent studies suggest that an interplay between adaptive immune, innate immune, and nonimmune mechanisms may be responsible for the damage and dysfunction that occur in myopathic muscle tissue.Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), belonging to the tumor necrosis factor (TNF) superfamily, has emerged as a multifunctional cytokine that regulates multiple cellular responses. TWEAK exerts pleiotropic functions through binding to its receptor Fn14 and activating downstream signaling pathways. Increased levels of TWEAK were found in many types of autoimmune diseases, such as rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus, suggesting a crucial role of TWEAK in autoimmune disorder. However, information about TWEAK-Fnl4 expression in PM and DM is limited. Therefore, in order to further explore the role of TWEAK in the pathogenesis of PM and DM, we investigated the levels of circulating TWEAK in sera and the expression of TWEAK and Fn14 in the muscle tissues of patients with PM and DM at the mRNA and protein levels. In addition, we explored the stimulating effects of TWEAK protein to cultured myoblast, therefore could provide experimental evidence for clarifying the pathogenic role of TWEAK-Fn14 in PM and DM.To date, several serum biomarkers have been identified as possible indicators of disease activity of PM and DM, such as creatine kinase (CK), the anti-Jo-1 antibody, and KL-6. However, these potential biomarkers have their limitations in monitoring general PM and DM disease activity. As a result, a better understanding of biomarkers that are present in the duration of the disease would significantly help to determine disease activity and inspire optimal treatment for each individual patient. B-cell activating factor (BAFF), belonging to the tumor necrosis factor family, plays a crucial role in B-cell maturation and survival. Serum BAFF levels have been found to be elevated in patients with various autoimmune diseases, implying that BAFF could be a potential serological biomarker reflecting disease activity. BAFF concentrations in Chinese patients with PM/DM and the nature of their association with IIM disease activity were still unknown. Therefore, we set out to investigate the serum levels of BAFF in Chinese patients with PM/DM and to systematically clarify their clinical significance. Furthermore, we designed a cross-sectional study and a longitudinal study involving Chinese PM/DM patients to examine the association between serum BAFF levels determined by ELISA and disease activity measured by the Myositis Disease Activity Assessment Visual Analog Scales (MYOACT), therefore clarifying the potential clinical usefulness of serum BAFF levels in the disease activity evaluation of PM and DM.The main findings in the present study are as follows:1. The expression and potential role of TWEAK and Fn14 in PM and DM(1) Serum levels of TWEAK in patients with PM/DM Serum levels of TWEAK were measured in 98 patients with PM/DM, and the results showed that the median value of serum TWEAK concentrations in PM/DM patients (442 pg/ml, range 81-1385 pg/ml) were significantly lower compared to that in healthy controls (516 pg/ml, range 282-797 pg/ml, P<0.001)). In addition, serum levels of TWEAK showed no statistical difference between PM and DM patients (P>0.05). No significant differences were observed when TWEAK levels were compared in patients with PM/DM subgrouped based on autoantibody positivity, serum CK levels, presence of clinical manifestations (interstitial lung disease, oropharyngeal dysphagia, Mechanic’s hands, Raynaud’s phenomenon), (P value all>0.05). No correlation was found between serum TWEAK levels and MYOACT global disease activity scores in 42 PM/DM patients involved in the corss-sectional study. In addntion, for 12 newly diagnosed patients, after treatment, all the 12 patients were considered as on remission stage and their MYOACT global disease scores were significantly decreased (P<0.01). However, no significant difference was observed in the patients’serum TWEAK levels before and after treatment (P>0.05).(2) Increased expression of Fn14 mRNA in muscle tissue of patients with PM/DM The expression of TWEAK and Fn14 mRNA in the muscle tissue of 17 PM,19 DM patients and of 10 healthy controls was analyzed by real time RT-PCR. The results showed that the expression of TWEAK mRNA in the muscle tissue of patients with PM/DM was not significantly different from that of the healthy controls (P>0.05). However, the expression of Fnl4 mRNA was significantly higher in the muscle tissue of PM/DM patients than in the healthy controls (P<0.01). Additionally, the Fn14 mRNA levels showed no significant difference between PM and DM patients (P>0.05). Interestingly, we found that patients with oropharyngeal dysphagia had significantly higher Fn14 mRNA levels than the patients without oropharyngeal dysphagia. In addition, the Fn14 mRNA levels correlated with MYOACT muscle disease activity scores (r=0.512, P<0.01), while did not correlate MYOACT global disease activity score (P>0.05).(3) Highly expressions of TWEAK and Fn14 protein in PM/DM muscle biopsies We analyzed the expressions of TWEAK and Fn14 in the muscle biopsies of 13 patients with DM,10 patients with PM, and seven healthy controls using immunofluorescence staining, and found that strong expressions of TWEAK were observed in PM and DM patients, while none of the healthy controls showed obvious expression of TWEAK and Fn14 in muscle biopsies. Remarkably strong expression of TWEAK were mainly found in the field where abundant infiltrating monocytes existed, and TWEAK immunoreactivity was also observed around the sarcolemma of muscle fibers partially invaded or surrounded by monocytes. as well as in a large number of fibers which was not near inflammatory cells. However, Fn14 were mainly expressed on the sarcolemma of muscle fibers. We therefore could find that TWEAK and Fn14 are frequently coexpressed in the same myofibers on overlay images by double immunofluorescence staining.(4) The stimulating effect of TWEAK on myoblast Recombinant human TWEAK proteins (100ng/ml) were used to stimulate in vitro cultured human myoblast. After 48 hours, the myoblasts were harvested and western blot analyses were applied to examine the expression of MHC-I molecules, and the levels of cell proliferation, apoptosis, autophagy. As a result, we found that the levels of cell proliferation, apoptosis remained no significant changes after TWEAK protein stimulating. However, the expression of MHC-I molecules by myoblast was significantly upregulated after TWEAK protein stimulating. Intriguingly, we found that the expressions of autophagy related proteins Beclinl and LC3B were obviously increased, indicating elevated levels of autophagy in myoblasts under TWEAK protein stimulating.2. BAFF as a serological biomarker for PM and DM(1) Serum levels of BAFF in patients with PM and DM Serum levels of BAFF in PM/DM patients were determined in 41 patients with PM, 51 patients with DM and 25 healthy controls. We found that the median value of serum BAFF concentration in PM patients was 1627.5 pg/ml (range 239.2-19950 pg/ml), and the median value of serum BAFF concentration in DM patients was 1582.5 pg/ml (range 506.5-18360 pg/ml), while the median value of that in healthy controls was 842 pg/ml (range 512-1354 pg/ml). Therefore serum levels of BAFF were significantly higher in PM patients (P<0.001) and DM patients (P<0.001) than those in the control subjects. And there was no significant difference between the serum levels of BAFF in PM patients and those in DM patients (P>0.05).(2) Clinical associations of serum BAFF levels in patients with PM and DM Serum levels of BAFF in PM/DM patients complicated with interstitial lung disease (ILD) (49 PM/DM patients, median value 2472 pg/ml, range 497.5-19950 pg/ml) were statistically higher than that in patients without ILD (43 PM/DM patients, median value 1105 pg/ml, range 239.2-6150pg/ml) (P<0.001). By chai-square analysis, the PM/DM patients with high serum BAFF levels showed significant increasd incidence of ILD compared with patients with normal serum BAFF levels (χ2 value=20.886, P<0.001). No further clinical associations were found between serum BAFF levels and other clinical manifestations including oropharyngeal dysphagia, Mechanic’s hands, Raynaud’s phenomenon, arthritis (P value all>0.05). The results of correlation analyses between serum BAFF levels and laboratory data showed that PM/DM patients with positive antinuclear antibody (ANA) (39 PM/DM patients, median value 1929 pg/ml, range 506.5-19950 pg/ml) had significantly higher serum BAFF levels compared to patients with negative ANA (53 PM/DM patients, median value 1533pg/ml, range239.2-12865 pg/ml) (P<0.05). Additionally, PM/DM patients with positive anti-Jo-1 antibody (21 PM/DM patients, median value 3838pg/ml, range 1385.5-19950 pg/ml) had significantly higher serum BAFF levels compared to patients with negative ANA (71 PM/DM patients, median value 1348.5pg/ml, range 239.2-18360 pg/ml) (P<0.001). Moreover, in patients with positive anti-Jo-1 antibody, serum levels of BAFF positively correlated with serum concerntrations of anti-Jo-1 antibody (r=0.822, P<0.001). Furthermore, serum BAFF levels of PM/DM patients were found to be positively correlated with serum CK levels (r=0.675,0.723, respectively, P value all<0.01). In our cohort,49 patients have detailed records of levels of their peripheral blood lymphocyte subsets. Thus, the correlation between BAFF levels and levels of peripheral blood lymphocyte subsets was analyzed by using Spearman’s correlation analysis. Serum BAFF levels of PM/DM patients was negatively correlated with the following:CD3+ cell count (r=-0.334, P<0.05), CD3+CD4+ cell count (r=-0.302, P<0.05), CD3+CD8+ cell count (r=-0.320, P<0.05), and CD19+CD5- cell count (r=-0.322, P<0.05). However, no significant correlation was found between serum BAFF levels and NK cell count (r=0.047, P>0.05).(3) Correlation of serum BAFF levels with disease activity in PM and DM: cross-sectional studyThe disease activity was evaluated according to the MYOACT tool by two experienced physician at the time when the serum sample was collected. We found significant correlations between serum BAFF levels and global disease activity scores both in PM patients (r=0.481, P<0.01) and in DM patients (r=0.536, P<0.01). Interestingly, a mild correlation between serum BAFF levels and muscle disease activity scores was found in PM patients (r=0.257, P<0.05), as well as in DM patients (r=0.228, P<0.05). In addition, we also found a mild correlation between serum BAFF levels and cutaneous disease activity scores (r=0.354, P<0.05).(4) Correlation of serum BAFF levels with disease activity in PM and DM: longitudinal studyIn the longitudinal study,24 patients were included and followed. Each of them had between two and seven serum samples collected. The time intervals between the first and last follow-up visits ranged from 0.5 to 6 years. Among the 24 patients,20 were followed up for longer than two years and more than three longitudinal serum samples were obtained from each patient. We collected 73 samples from the 24 patients involved in the longitudinal study, and these serial patient samples were analyzed by using Spearman’s correlation analysis. A modest correlation of serum levels of BAFF was found with global disease activity scores (r=0.68, P<0.001) as well as with BAFF levels (P<0.001), as well as in global disease activity scores (P<0.001). In addition, the change in BAFF levels and the change in global disease activity before and after treatment were found modestly correlated (r=0.434, P<0.05).Taken together, our study demonstrated the abberant expression of TWEAK-Fnl4 in PM and DM patients. Especially in muscle tissue, the expression of TWEAK-Fn14 were significantly increased, indicating the possible pathogenic role of TWEAK derived from infiltrating inflammatory lymphocytes in muscle tissue of PM/DM patients. Our study suggested that TWEAK may expressed by infiltrating inflammatory cells and then induce high expression of Fn14 by adjacent muscle cells, consequently activating downstream signal pathways and resulting the skeletal muscle cell damage. By in vitro experiment, we demonstrated that the expression of MHC-I molecules and autophagy related proteins in human myoblast significantly unregulated, indicating the potential pathogenic role of TWEAK-Fn14 in the pathogenesis of PM and DM.Further more, our study also revealed high expression levels of BAFF in the sera of PM/DM patients, as well as significant correlations with the presence of autoantibodies and ILD in PM/DM patients. Cross-sectional assessment and longitudinal study demonstrated that serum BAFF levels significantly correlated with disease activity scores of PM/DM patients, suggesting the potential clinical usefulness of serum BAFF levels in the disease activity evaluation of PM and DM.