Division of Rheumatology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
Alnylam Pharmaceutical Inc., Boston, MA, United States.
Front Immunol. 2020 Feb 21;11:201. doi: 10.3389/fimmu.2020.00201. eCollection 2020.
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 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 . 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%, < 0.05) and MASP-2 (90%, < 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 ( < 0.05) 90% decrease in 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 K 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.
补体系统在类风湿关节炎（RA）的发病机制中起着重要作用。除了驱动凝集素途径（LP）激活外，甘露聚糖结合凝集素（MBL）相关丝氨酸蛋白酶（MASP）在调节替代途径（AP）方面也起着关键作用。我们评估了 N-乙酰半乳糖胺（GalNAc）缀合的 MASP-1 和 MASP-2 双链体在有和没有关节炎的小鼠中的作用，以检查 MASP-1 和 MASP-2 表达的敲低是否会影响关节炎的发展。MASP-1 和 MASP-2 的 GalNAc-siRNA 在 pM 浓度下对 MASP-1 或 MASP-2 进行了强大的沉默。为了评估在关节炎小鼠中的沉默影响，我们使用胶原抗体诱导的关节炎（CAIA）RA 小鼠模型。在 CAIA 的诱导之前，小鼠接受了 10 mg/kg 剂量的 GalNAc-siRNA 3x s.q.。使用 qRT-PCR 检查肝脏基因表达，并通过夹心免疫测定法和 Western blot 在循环中确认蛋白质水平。在第 10 天，分别用 MASP-1 和 MASP-2 双链体处理的 CAIA 小鼠中，肝脏 MASP-1（70-95%，<0.05）和 MASP-2（90%，<0.05）mRNA 的表达均特异性降低。MASP-1-siRNA 治疗导致循环中 MASP-1 蛋白水平降低 95%，而对 MASP-2 水平和临床疾病活动（CDA）没有影响。在注射 MASP-2 双链体的小鼠中，甘露聚糖上的 C4b 沉积显著减少（<0.05），减少了 90%，而循环中的 MASP-2 几乎完全消除。MASP-2 沉默最初使 CDA 显著降低 60%，但随后与对照组相比降低了 40%。出乎意料的是，GalNAc-siRNA 介导的 MASP-1 和 MASP-2 的敲低在正常生理条件下对 FD 的转录显示出这些蛋白质的明显作用，而 LPS 诱导的炎症条件逆转了对 FD 水平的这种作用。LPS 被 Toll 样受体 4（TLR4）识别，我们发现 MBL 不仅与 TLR4 结合，相互作用的 Kd 为 907 nM，而且还上调了分化脂肪细胞中 FD 的表达。我们表明，MASP-2 敲低会损害 RA 的发展，LP 和 AP 中的蛋白质之间的相互关系可能扩展到 FD 基因的转录调节。
