TLR4-Independent Mechanisms of Complement Activation During Endotoxemia

Abstract Almost two million American adults are diagnosed with sepsis annually of which 350,000 succumb to the infection. Toll-like-receptor 4 (TLR4) canonically recognizes lipopolysaccharide (LPS), an outer membrane structure of Gram-negative bacteria. This recognition leads to pro-inflammatory cytokine release and propagation of other immune compartments. Complement is an acellular innate immune recognition system and has three general categories for activation: classical, lectin and alternative pathway. Previous research has found that complement proteins are dysregulated independent of TLR4 in serum during murine endotoxemia. This study investigates mechanisms of complement activation by describing location of complement gene upregulation and protein distribution in WT and TLR4−/− mice. mRNA was extracted from murine organ samples collected 0, 6, 12, and 18 hours post-induction of endotoxemia. RT-qPCR is then performed to measure expression fold changes of complement genes F5, F13a1, C1qb, Cfd, Masp1, and Mbl1. Genes involved in the lectin pathway were observed to be upregulated at the 6-hour timepoint and then downregulated throughout the progression of disease. C3 is a complement protein that is cleaved to create C3a, an anaphylatoxin, contributing to activation of acute phase inflammatory responses. Levels of C3a are analyzed using Western blots from WT and TLR4−/− mice as a marker of activation. The effects of LPS chemical structure on the efficiency of C3 cleavage is also analyzed. Identification of TLR4-independent pathways will allow for more targeted immune therapies for adverse outcomes related to sepsis. Supported by National Institute of Health and Towson University Bridges to Doctorate Program NIH (1T32GM146694-01), Fisher College of Science and Math at Towson University, and an FY2022 grant from the Towson University Faculty Development and Research Committee (FDRC) awarded to Erin Harberts titled “Septic Shock: Defining Alternative Pathways of Activation”.