Manipulating TLR4 surface expression to investigate its influence on UV-induced cell death 3666

Abstract Description Activation of TLR4 signaling by UV has been shown to cause cell death by apoptosis promoting UV-induced carcinogenesis. Without TLR4, necroptosis, an inflammatory cell death, may occur instead and lead to an immune-driven decrease in UV-carcinogenesis. This study investigates the use of lipopolysaccharide (LPS) to induce decreased TLR4 surface expression and how this effects cell death mechanism following UV exposure. If cell death can be skewed toward necroptosis with addition of LPS to skin cells before UV exposure, the incidence of UV-induced carcinogenesis may decrease. To investigate this hypothesis, thioglycolate-induced mouse peritoneal macrophages cells were stimulated with 0 mJ/cm2, 25 mJ/cm2, and 50 mJ/cm2 of UVB to measure for cell death. The UVB 50 mJ/cm2 dose reliably results in visible cell death via DNA laddering assays and cleavage of caspase-3 as measured by Western blot. Cells were treated with 0.1 ng/mL, 1 ng/mL, or 10 ng/mL LPS for 24 hours and decreased surface expression of TLR4 is confirmed by immunohistochemistry. Cells are then exposed to 50 mJ/cm2 of UVB, 24 hours later evidence of cell death including DNA laddering, cleavage of caspase-3, phosphorylation of necroptotic protein MLKL, and concentration of thymine dimers is investigated. Understanding how innate immune ligands impact epithelial cell fate may provide proof of principal for use of prophylactic low dose LPS application before UV exposure. Funding Sources FCSM Undergraduate Research Grant Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)