Complement Activation Signatures in Systemic Lupus Erythematosus: Castle Study

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Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease that causes organ damage, leading to significant morbidity and mortality. SLE is characterized by the generation of autoantibodies, which bind to an individual’s own tissues to induce inflammation and organ damage. An important mechanism for SLE-induced inflammation is activation of the complement cascade. The complement system is an important part of the immune system, which is well-designed eliminate pathogens with the help of antibodies and other immune components. In SLE though, autoantibodies that have bound to patients’ organs activated complement to generate damaging inflammatory responses. Currently, clinicians assess complement activation through the complement components C3 and C4 in serum. As the complement cascade is activated, both C3 and C4 become attached to the surfaces of pathogens and cells and serum C3 and C4 levels decrease as a result. In SLE, since complement activation occurs during flares, C3 and C4 levels should also decrease in flares. During systemic inflammation though, the liver produces both C3 and C4. Thus, during SLE flares, there is both consumption (due to their activation on cell surfaces) and production (due to inflammation-induced liver production) of C3 and C4. This leads to the underdetection of SLE flares. Complement activation generates numerous split products (or fragments) that are found on the surface of cells and interact with numerous complement receptors (both stimulatory and regulatory). Given the central role of complement activation in SLE, identifying the array of complement signatures on immune cells and blood will likely generate important observations regarding SLE pathophysiology. Indeed, the value of complement split products have just started to be realized, as increased erythrocyte and platelet bound C4d can provide utility in the diagnosis of SLE. Nevertheless, a comprehensive, qualitative assessment of the complement fragment deposition on immune cells from patient with various states of SLE has yet to be performed. Here, we hypothesize identifying complement signatures on immune cells using mass cytometry and blood complement split products will provide invaluable insight in the role of complement activation on SLE. We have two aims to test this hypothesis: 1) Evaluate the complement split product iC3b as a dramatically improved biomarker of SLE disease activity, and 2) Fully delineate the complement signatures found on immune cells from patients with SLE. Two recent technological improvements have made evaluating these Aims possible: 1) Development of an investigational medical device that rapidly determines blood iC3b and C3 levels without artefactual elevation of iC3b; and 2) Highly multiplexed phenotyping tools for immune cells such as mass cytometry has emerged as an innovative approach to analyze complex multicellular systems. The device that measures iC3b and C3 does so within 20 minutes, and our preliminary data demonstrate a strong correlation between iC3b and C3 levels to SLE disease activity. Applying MC to the study of complement signatures on immune cells in SLE will provide a level of detail and quantification that has not been possible. Thus, rather than evaluating an incomplete profile of complement proteins, we have the potential to quantify the levels and types of complement fragments, complement receptors, and membrane regulators on multiple cell types at the single cell level. This proposal will help establish the role of iC3b and iC3b/C3 ratios as a promising approach to assessing SLE disease activity, and significantly improve our understanding of how cell surface complement activation signatures on immune cells drive pathophysiology in SLE.