RhoA Promotes Synovial Proliferation and Bone Erosion in Rheumatoid Arthritis through Wnt/PCP Pathway.

Ras homolog gene family member A (RhoA) plays a major role in the Wnt/planar cell polarity (PCP) pathway, which is significantly activated in patients with rheumatoid arthritis (RA). The function of RhoA in RA synovitis and bone erosion is still elusive. Here, we not only explored the impact of RhoA on the proliferation and invasion of RA fibroblast-like synoviocytes (FLSs) but also elucidated its effect on mouse osteoclast and a mouse model of collagen-induced arthritis (CIA). Results showed that RhoA was overexpressed in RA and CIA synovial tissues. Lentivirus-mediated silencing of RhoA increased apoptosis, attenuated invasion, and dramatically upregulated osteoprotegerin/receptor activator of nuclear factor-κB ligand (OPG/RANKL) ratio in RA-FLSs. Additionally, the silencing of RhoA inhibited mouse osteoclast differentiation in vitro and alleviated synovial hyperplasia and bone erosion in the CIA mouse model. These effects in RA-FLSs and osteoclasts were all regulated by RhoA/Rho-associated protein kinase 2 (ROCK2) and might interact with Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways.

Exosomal Dvl3 promoted the aggressive phenotypic transformation of RA-FLS via wnt pathway.

OBJECTIVE: This study was performed to explore the function and mechanism of Dvl3 in RA-FLS by exosome intervention. METHODS: The expression pattern of Dvl3 was examined by IHC, WB, and qPCR. Modified exosomes obtained from culturing supernatant of RA-FLS infected with Dvl3 over expression (OE) lentivirus were administrated to the target RA-FLS. The ability of survival, migration, and the production of inflammatory factor influenced by exosomal Dvl3 were detected by CKK8 kits, Tunel, migration test, qPCR, and enzyme-linked immunosorbent assay (ELISA) respectively; Flow cytometry analysis was conducted to explorer the inflammatory moderate role of exosomes on CD4+ T cells. The possible downstream pathways of Dvl3 were screened by qPCR and WB and verified by double luciferase reporter experiment. RESULTS: The expression level of Dvl3 was significantly increased in RA and CIA. Exosomes from the OE group could significantly promote cell proliferation activity, migration/invasion ability. The augment of TNF-α, IL-1ß, IL-17, and IL-21 was observed in exosomal Dvl3-OE group. Th1 and Th17 cells polarisation and cytokines related were both enhanced by Exosomal Dvl3. Over expression of Dvl3 was accompanied by the significant increase of ß-catenin and RhoA activities. CONCLUSION: This study discovered the high expression of Dvl3 of exosomes derived from RA patients which may possessed the ability to promote phenotypic transformation of RA-FLS through Wnt pathway.

The intervention of intestinal Wnt/ß-catenin pathway alters inflammation and disease severity of CIA.

Autoreactive T cell is one of the leading causes of immunological tolerance defects in the chronic inflammatory lesions of rheumatoid arthritis (RA). There have been several extracellular signals and intracellular pathways reported in regulating this process but largely remain unknown yet. In this study, we explored the roles of intestinal Wnt/ß-catenin on disease severity during collagen-induced arthritis model (CIA), an animal model of RA. We first testified the activity pattern Wnt/ß-catenin shifted by intragastric administration of LiCl and DKK-1 in the intestine by real-time PCR and WB analysis. The arthritis scores showing the disease severity in the DKK-1 group was significantly ameliorated compared with the control group at the late stage of the disease, while in the LiCl group, the scores were significantly elevated which was consistent with pathology score analysis of H&E staining. Next, ELISA was performed and showed that TNF-α and IL-17 in the LiCl group were significantly higher than that of the control group. IL-10 in the DKK-1 group was significantly higher than that in the LiCl-1 group and control group, P < 0.05. Flow cytometry of spleen T cells differentiation ratio showed that: Th1 from the DKK-1 and LiCl groups and Th17 from the LiCl group was significantly different from that of the blank model group, P < 0.05. Finally, we explored the effects of intestinal Wnt/ß-catenin on T cell differentiation regulator ROR-γt and TCF1 and found that both transcription factors were up-regulated in the LiCl group. Together, these data suggested the pro-information role of Wnt/ß-catenin pathway from the intestine in the CIA mouse, implying its use as a potential therapeutic target for the treatment of inflammatory diseases such as RA.

MSX3 Switches Microglia Polarization and Protects from Inflammation-Induced Demyelination.

The major challenge for progressive multiple sclerosis therapy is the promotion of remyelination from inflammation-induced demyelination. A switch from an M1- to an M2-dominant polarization of microglia is critical in these repair processes. In this study, we identified the homeobox gene msh-like homeobox-3 (Msx3) as a new pivotal regulator for microglial polarization. MSX3 was induced during microglia M2 polarization and repressed in M1 cells. The expression of MSX3 in microglia was dynamically regulated during experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. The overexpression of MSX3 in microglia promoted M2 but impeded M1 polarization. Interrupting MSX3 expression in microglia accelerated inflammation-induced demyelination and neurodegeneration. The conditioned medium from MSX3-transduced microglia promoted oligodendrocyte progenitor survival, differentiation, and neurite outgrowth. The adoptive transfer of MSX3-transduced microglia suppressed EAE and facilitated remyelination within the murine CNS in EAE and the LPC model. Mechanically, chromatin immunoprecipitation assays also indicated that MSX3 directly regulated three key genes associated with microglia M2 polarization, including Pparg, Stat6, and Jak3. Importantly, we found that overexpression of MSX3 in human-derived microglia represents the M2 phenotype and ameliorated EAE after intraventricular injection. Our findings suggest a new homeobox protein-dependent mechanism for driving microglia M2 polarization and identify MSX3 as an attractive therapeutic approach for preventing inflammation-induced demyelination and promoting remyelination.

Insulin-like growth factor-binding protein 7 is up-regulated during EAE and inhibits the differentiation of oligodendrocyte precursor cells.

Oligodendrocyte precursor cells (OPCs) differentiation failure is one of the leading causes for remyelination defects in the demyelinating lesions of multiple sclerosis (MS). In this study, we explored the roles of insulin-like growth factor-binding proteins 7 (IGFBP-7) on OPCs differentiation during experimental autoimmune encephalomyelitis (EAE). We first investigated the expression pattern of IGFBP-7 by real-time PCR and immunofluorescence staining. It showed that IGFBP-7 was expressed in astrocytes (ACs), oligodendrocytes (OLs) and neurons both in vitro and in vivo. The mRNA and protein level of IGFBP-7 was also increased in the spinal cord from mice at the peak of EAE disease. Next we found that IGFBP-7 acted as a negatively regulator of the OPCs differentiation. Together, these data suggest that IGFBP-7 was up regulated during EAE and inhibit the transition from OPCs to mature OLs, implying its use as a potential therapeutic target for the treatment of inflammatory demyelinating diseases.

[MicroRNAs in microglia polarization and CNS diseases: mechanism and functions].

Microglia are resident macrophages of central nervous system (CNS), and thus act as the crucial stuff of immune response and play very important roles in the progress of various CNS diseases. There are two different polarization statuses of activated microglia, M1 and M2 phenotypes. M1 polarized microglia are important for eradicating bacterial and promoting inflammation, whereas M2 cells are characterized by anti-inflammation and tissue remodeling. Recently, more and more evidence indicated that different polarized microglia showed diverse microRNA (miRNA) expression profiles. MiRNAs regulate microglia polarization, and thus affect the progress of CNS diseases. Fully exploring the polarization status of microglia during CNS diseases and the role of miRNAs in microglia polarization will be very helpful for a deep understanding of the roles of microglia in immunopathologic mechanism of different CNS diseases and offer the theoretical foundation of searching more effective therapies for these disorders.