Abstract
The complement system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Besides driving lectin pathway (LP) activation, the mannan-binding lectin (MBL)-associated serine proteases (MASPs) also play a key role in regulating the alternative pathway (AP). We evaluated the effects of N-acetylgalactosamine (GalNAc)-conjugated MASP-1 and MASP-2 duplexes in vitro and in mice with and without arthritis to examine whether knockdown of MASP-1 and MASP-2 expression affects the development of arthritis. GalNAc-siRNAs for MASP-1 and MASP-2 demonstrated robust silencing of MASP-1 or MASP-2 at pM concentrations in vitro. To evaluate the impact of silencing in arthritic mice, we used the collagen antibody-induced arthritis (CAIA) mouse model of RA. Mice were injected a 10 mg/kg dose of GalNAc-siRNAs 3x s.q. prior to the induction of CAIA. Liver gene expression was examined using qRT-PCR, and protein levels were confirmed in the circulation by sandwich immunoassays and Western blot. At day 10, CAIA mice separately treated with MASP-1 and MASP-2 duplexes had a specific reduction in expression of liver MASP-1 (70–95%, p < 0.05) and MASP-2 (90%, p < 0.05) mRNA, respectively. MASP-1-siRNA treatment resulted in a 95% reduction in levels of MASP-1 protein in circulation with no effect on MASP-2 levels and clinical disease activity (CDA). In mice injected with MASP-2 duplex, there was a significant (p < 0.05) 90% decrease in ex vivo C4b deposition on mannan, with nearly complete elimination of MASP-2 in the circulation. MASP-2 silencing initially significantly decreased CDA by 60% but subsequently changed to a 40% decrease vs. control. Unexpectedly, GalNAc-siRNA-mediated knockdown of MASP-1 and MASP-2 revealed a marked effect of these proteins on the transcription of FD under normal physiological conditions, whereas LPS-induced inflammatory conditions reversed this effect on FD levels. LPS is recognized by Toll-like receptor 4 (TLR4), we found MBL not only binds to TLR4 an interaction with a Kd of 907 nM but also upregulated FD expression in differentiated adipocytes. We show that MASP-2 knockdown impairs the development of RA and that the interrelationship between proteins of the LP and the AP may extend to the transcriptional modulation of the FD gene.
Highlights
Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease of the joints, affecting ∼0.24% of the entire world population [1]
A robust silencing in the small interfering ribonucleic acid (siRNA)-treated groups was observed, i.e., 93% of mannan-binding lectin-associated serine protease-1 (MASP-1) (Figure 2A) and 69% of MBL-associated serine protease-2 (MASP-2) (Figure 2B) expression was silenced after a single subcutaneous administration
Our data confirm that MBL-associated serine proteases (MASPs)-1 and MASP-2 proteases are generated by the liver and there is little extrahepatic generation of these proteases to effect disease phenotype locally in the joints
Summary
Rheumatoid Arthritis (RA) is a chronic inflammatory autoimmune disease of the joints, affecting ∼0.24% of the entire world population [1]. Despite clinical improvement afforded by these therapeutics, the radiological damage in RA can continue over time due to an underlying inflammatory process [6] and treatment with anti-TNF antagonists, despite clinical improvement, has little effect on diagnostic and pathologically relevant markers like anti-citrullinated protein antibodies (ACPAs) [7, 8]. Autoantibodies which develop against these epitopes, designated anti-cyclic citrullinated peptide (CCP) antibodies, can circulate to deposit in damaged joint tissues, which are rich in citrullinated proteins. It has been shown recently that transgenic (Tg) mice expressing IgM with the V region of an anti–CCP mAb cloned from a RA patient, from a naturally generated anti-CCP Ab, failed to develop arthritis [9], a finding consistent with the hypothesis that anti-CCP Abs alone are not the cause of disease. A “second hit” has been postulated to be required for localization of this immune
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