The complement system is a major component of the immune system and plays a central role in many protective immune processes, including circulating immune complex processing and clearance, recognition of foreign antigens, modulation of humoral and cellular immunity, removal of apoptotic and dead cells, and engagement of injury resolving and tissue regeneration processes. In stark contrast to these beneficial roles, however, inadequately controlled complement activation underlies the pathogenesis of human inflammatory and autoimmune diseases, including rheumatoid arthritis (RA) where the cartilage, bone, and synovium are targeted. Recent studies of this disease have demonstrated that the autoimmune response evolves over time in an asymptomatic preclinical phase that is associated with mucosal inflammation. Notably, experimental models of this disease have demonstrated that each of the three major complement activation pathways plays an important role in recognition of injured joint tissue, although the lectin and amplification pathways exhibit particularly impactful roles in the initiation and amplification of damage. Herein, we review the complement system and focus on its multi-factorial role in human patients with RA and experimental murine models. This understanding will be important to the successful integration of the emerging complement therapeutics pipeline into clinical care for patients with RA.

The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

Rheumatoid arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated Abs or small interfering RNAs (siRNAs) or small molecule inhibitors against C5 and C5aR1 (CD88) that have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. In this study, we test in collagen Ab-induced arthritis (CAIA) a new therapeutic strategy using a novel anti–C5ab-C5 siRNA conjugate. We first demonstrate that although C5aR2 or C5L2 (GPR77) plays no role in CAIA, C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking C5, C5aR1, or the combination decreased clinical disease activity in mice with CAIA by 45%, 51%, and 58%, respectively. Anti-C5 Ab (BB5.1) has only limited efficacy, but significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1 Ab–protamine–C5 siRNA conjugate. To our knowledge, we show for the first time that whereas unconjugated Ab plus siRNAs reduce arthritis by 19%, our anti-C5aR1 Ab–protamine–C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils, and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized.

Rheumatoid arthritis (RA) is a complex autoimmune disease with an etiology that is not yet well understood, disproportionally affects women and also varies in incidence and prevalence by population. The presence of anti-citrullinated protein antibodies (ACPA) is a highly specific biomarker for the diagnosis of clinically apparent RA. ACPA are also present in the serum for an average of 3-5 years prior to the onset of RA during an asymptomatic period characterized by mucosal inflammation and local ACPA production at these sites. We hypothesized that systemic complement activation products might be generated during the pre-clinical initiation of RA and/or provide a second hit that promotes subsequent arthritis development in the joints. In addition, we evaluated which demographic and genetic features and environmental exposures could influence the complement activation process. We analyzed plasma from healthy subjects, subjects at-risk for the development of RA based on serum ACPA positivity in absence of inflammatory arthritis (IA), and ACPA positive RA subjects by Multiplex Assay and ELISA for eighteen complement system components, factors and activation products belonging to the classical, lectin and alternative pathways. By using regression models, associations between complement proteins and various demographic, genetic, and environmental factors previously found to be associated with RA, including sex, smoking, shared epitope, and oral contraceptive use, were examined. We found no evidence of systemic complement activation in ACPA positive subjects without IA, but in contrast found evidence of systemic involvement of the both classical and alternative pathways during the stage of the disease where classified RA is present, (i.e. during joint inflammation and damage). With regard to the demographic, genetic, and environmental variables, females who reported current or past oral contraceptive use and subjects with current tobacco exposure demonstrated alterations of the alternative pathway of complement. Furthermore, RA subjects with established disease who have a body mass index categorized as obese demonstrated higher levels of C2 compared to RA subjects who are not considered obese. In sum, the complement system may be involved in the pathogenesis of RA, with only localized mucosal effects during the preclinical period in those at-risk for RA but in the joint as well as systemically in those who have developed clinically apparent arthritis.

Previous studies using mannose-binding lectin (MBL) and complement C4–deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen Ab–induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases—MASP-1, MASP-2, and MASP-3—and with two MBL-associated proteins designated sMAP and MBL-associated protein of 44kDA (MAp44). Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies, we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming GFP (AdGFP) expression were injected i.p. in C57BL/6 wild type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity (CDA) score by 81% compared with mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA score and histopathologic injury scores. In addition, administration of AdhMAp44 significantly diminished the severity of Ross River virus–induced arthritis, an LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis.