TLR9 regulates the autophagy-lysosome pathway to promote dendritic cell maturation and activation by activating the TRAF6-cGAS-STING pathway.

Dysregulated maturation and activation of dendritic cells (DCs) play a significant role in the progression of systemic lupus erythematosus (SLE). The autophagy-lysosome pathway has been identified as a potential mechanism to inhibit DC activation and maturation, but its precise workings remain unclear. We investigated the role and regulatory mechanism of TLR9 in modulating the autophagy-lysosome pathway and DCs activation. The mRNA and protein expressions were assessed using qRT-PCR and/or western blot. NZBW/F1 mice was used to construct a lupus nephritis (LN) model in vivo. Cell apoptosis was analyzed by TUNEL staining. Flow cytometry was adopted to analyze DCs surface markers. Lyso-tracker red staining was employed to analyze lysosome acidification. Levels of anti-dsDNA, cytokines, C3, C4, urine protein and urine creatinine were examined by ELISA. The results showed that TLR9 was markedly increased in SLE patients, and its expression was positively correlated with SLEDAI scores and dsDNA level. Conversely, TLR9 expression showed a negative correlation with C3 and C4 levels. Loss-of function experiments demonstrated that TLR9 depletion exerted a substantial inhibition of renal injury, inflammation, and DCs numbers. Additionally, upregulation of TLR9 promoted DCs maturation and activation through activation of autophagy and lysosome acidification. Further investigation revealed that TLR9 targeted TRAF6 to activate the cGAS-STING pathway. Rescue experiments revealed that inactivation of the cGAS/STING signaling pathway could reverse the promoting effects of TLR9 upregulation on DCs maturation, activation, and autophagy-lysosome pathway. Overall, our findings suggested that TLR9 activated the autophagy-lysosome pathway to promote DCs maturation and activation by activating TRAF6-cGAS-STING pathway, thereby promoting SLE progression.

LncRNA PICSAR promotes cell proliferation, migration and invasion of fibroblast-like synoviocytes by sponging miRNA-4701-5p in rheumatoid arthritis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have drawn increasing attention because they play a pivotal role in various types of autoimmune diseases, including rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), a prominent component of hyperplastic synovial pannus tissue, are the primary effector cells in RA synovial hyperplasia and invasion which can lead to joint destruction. In this study, we investigated whether lncRNAs could act as competing endogenous RNAs to regulate the pathological behaviors of RA-FLSs. METHODS: LncRNA microarray was conducted to establish lncRNA expression profiles in FLSs isolated from RA patients and healthy controls (HCs). Differentially expressed lncRNAs were verified by quantitative real-time PCR (qRT-PCR) on RA-FLSs and synovial fluid. The functional role of lncRNA PICSAR downregulation was evaluated in RA-FLSs. We conducted molecular biological analysis to predict miRNAs which have a potential binding site for PICSAR and further refined the results by qRT-PCR. Luciferase reporter assay was adopted to validate the interaction of lncRNA PICSAR and miR-4701-5p. Western Blot and qPCR were used to identify the target gene and protein. The functional role of miR-4701-5p upregulation was examined in RA-FLSs. FINDINGS: We identified a long intergenic non-protein-coding RNA162 (LINC00162), also known as lncRNA PICSAR (p38 inhibited cutaneous squamous cell carcinoma associated lincRNA), has significantly higher expression in RA-FLSs and RA synovial fluid. The cell proliferation, migration, invasion and proinflammatory cytokines production of RA-FLSs showed significant alterations after the lncRNA PICSAR suppression. Mechanistically, lncRNA PICSAR functioned through sponging miR-4701-5p in RA-FLSs. INTERPRETATION: Our results reveal PICSAR may exert an essential role in promoting synovial invasion and joint destruction by sponging miR-4701-5p in RA and that lncRNA PICSAR may act as a biomarker of RA.

Long Non-Coding RNA GAPLINC Promotes Tumor-Like Biologic Behaviors of Fibroblast-Like Synoviocytes as MicroRNA Sponging in Rheumatoid Arthritis Patients.

Rapidly accumulating evidence has now suggested that the long non-coding RNAs (LncRNAs), a large and diverse class of non-coding transcribed RNA molecules with diverse functional roles and mechanisms, play a major role in the pathogenesis of many human inflammatory diseases. Although some LncRNAs are overexpressed in plasma, T cell, and synovial tissues of patients with rheumatoid arthritis (RA), there is a dearth of knowledge in what role these transcripts play in fibroblast-like synoviocytes (FLSs) of these patients. Here, our studies showed that GAPLINC, a newly identified functional LncRNA in oncology, displayed a greater degree of expression in FLSs from RA than in patients with traumatic injury. GAPLINC suppression in RA-FLS cells revealed significant alterations in cell proliferation, invasion, migration, and proinflammatory cytokines production. Additionally, we performed a preliminary bioinformatics analysis of GAPLINC gene sequence in order to find its target molecules, using miRanda, PITA, RNAhybrid algorithms, Kyoto encyclopedia of genes and genomes, and gene ontology analysis. Since the results predicted that some of microRNAs and mRNA may interact with GAPLINC, we simulated a gene co-action network model based on a competitive endogenous RNA theory. Further verification of this model demonstrated that silencing of GAPLINC increased miR-382-5p and miR-575 expression. The results of this study suggest that GAPLINC may function as a novel microRNAs sponging agent affecting the biological characteristics of RA-FLSs. Additionally, GAPLINC may also promote RA-FLS tumor-like behaviors in a miR-382-5p-dependent and miR-575-dependent manner. Based upon these findings, LncRNA GAPLINC may provide a novel valuable therapeutic target for RA patients.

uPAR promotes tumor-like biologic behaviors of fibroblast-like synoviocytes through PI3K/Akt signaling pathway in patients with rheumatoid arthritis.

Urokinase-type plasminogen activator receptor (uPAR), is a multifunctional receptor on cell surface, widely present in endothelial cells, fibroblasts, and a variety of malignant cells. Current studies have suggested that uPAR overexpressed on synovial tissues or in synovial fluid or plasma in patients with rheumatoid arthritis (RA). However, there are limited researches regarding the role of uPAR on fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLSs) and its underlying mechanisms. Here, our studies show that the expression of uPAR protein was significantly higher in fibroblast-like synoviocytes (FLSs) from RA than those from osteoarthritis or traumatic injury patients. uPAR gene silencing significantly inhibited RA-FLSs cell proliferation, restrained cell transformation from the G0/G1 phase to S phase, aggravated cell apoptosis, interfered with RA-FLSs cell migration and invasion, and reduced activation of the PI3K/Akt signaling pathway, which may be associated with ß1-integrin. Cell supernatants from uPAR gene-silenced RA-FLSs markedly inhibited the migration and tubule formation ability of the HUVECs (a human endothelial cell line). Therefore, we demonstrate that uPAR changes the biological characteristics of RA-FLSs, and affects neoangiogenesis of synovial tissues in patients with RA. All of these may be associated with the ß1-integrin/PI3K/Akt signaling pathway. These results imply that targeting uPAR and its downstream signal pathway may provide therapeutic effects in RA.