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282 Generation of hydrolyzed complement component C3 is substantially elevated in SLE
  1. Michelle Elvington,
  2. M Kathryn Liszewski,
  3. John P Atkinson and
  4. Alfred H Kim
  1. Washington University School of Medicine


Background Complement activation is a central pathophysiologic event in several autoimmune diseases. A key activation event is the conversion of native C3 to C3(H2O), where a highly reactive thioester bond in C3 is hydrolyzed. C3(H2O) can be utilized to generate C3 convertase, which further drives complement activation via the alternative pathway. C3(H2O) has been elusive to measure, but we have recently developed a novel ELISA-based assay allowing for its accurate measurement. We hypothesized that in autoimmune diseases where complement activation is a central feature, C3(H2O) levels will be elevated reflecting a primed state for triggering inappropriate alternative pathway activation and amplification.

Methods Healthy adult subjects (n=5) and adults with classified SLE (n=6) were enrolled and consented for serum collection at Washington University School of Medicine. Frozen serum was tested either immediately after thawing or after incubation at room temperature or 37°C for 6 or 24 hours. C3(H2O) was measured by an ELISA assay that utilizes a capture antibody to C3b and a detection antibody to C3a. Data was analyzed using Prism version 7.0d (GraphPad Software, Inc.). Statistical significance was determined using 2-way ANOVA with Dunns multiple comparisons test.

Results Substantial elevation of serum C3(H2O) levels were observed from patients with SLE following incubation at RT and 37°C compared to healthy controls. The differences were most pronounced in samples incubated at 37°C for 6 hours, with over a 4-fold increase in C3(H2O) levels in both the SLE samples. Interestingly, sera from both groups (healthy controls and SLE) did not reflect any difference in baseline C3(H2O) levels.

Conclusions Both SLE and RA are associated with elevated C3(H2O) levels following in vitro incubation compared to healthy controls. These data suggest that the potential for C3(H2O) formation in SLE and RA patients are higher compared to healthy controls, which could support additional complement activation or utilization of C3(H2O) in other pathways such as intracellular activation in immune cells. Thus, In addition to a possible diagnostic tool for pathogenic autoimmunity, these data may also suggest novel mechanisms of how complement could drive symptomatic autoimmune disease.

Funding Source(s): NIH R01 GM0099111

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