Abstract 1632 Investigating Transcriptional Regulation of Complement Genes During Endotoxemia Using a qPCR Array

Symptoms of septic shock are caused by dysregulation of the host immune system, leading to an uncontrolled inflammatory response and organ dysfunction. This project aims to identify and define a compensatory immune signaling pathway activated during sepsis when classical activation molecules are not present. Sepsis affects 47-50 million people and causes over 11 million deaths every year. It is the number one cause of nosocomial deaths, accounting for 1 in 5 worldwide. In Gram-negative bacterial sepsis, Toll-like receptor 4 (TLR4) recognizes lipopolysaccharides (LPS), a component of the outer membrane of Gram-negative bacteria and alerts the innate immune system to attack the bacteria. In extreme cases, if the immune system becomes overwhelmed by the infection, cell surface TLR4 receptors are downregulated, and immune cells deplete. Despite the absence of canonical receptors, markers of innate immune activation are found in TLR4-/- samples, indicating the presence of a TLR4 independent compensatory pathway. This project investigates the potential transcriptional regulation of complement pathway genes in an endotoxemia model of septic shock. Individual RT-qPCRs of complement genes C1qb, Masp1, and F5 indicate dysregulation in TLR4-/- and WT samples over the course of endotoxemia. A qPCR array was designed to include genes coding for complement protein initiators (including Mbl2, Masp1, C1q), cascade propagators (including C2, C3), and downstream signaling proteins (including C6, C7, C8b, C9). Transcriptional activation and fold change over course of endotoxemia using ΔΔCT analysis was compared between liver samples collected at 0, 6, and 12 hr timepoints in WT C57BL6 and TLR4-/- (B6.B10ScN-Tlr4lps-del/JthJ) mice. Future experiments will confirm dysregulation of identified genes at the protein level using Western blots. This project supports the development of complement specific therapies for people experiencing secondary infection after septic shock. Funding for this project was provided by a Fisher College of Science and Math grant awarded to Phoebe Calkins titled "Using a qPCR Array to Determine Pathway Regulation Activated During Septic Shock".