Characterizing the PolyIC-induced activation and apoptosis of MuTuDCs by determining the influence of Bim isoforms and Corticosterone

Dendritic cells (DCs) are a critical subset of immune cells responsible for connecting the innate and adaptive immune response. Dendritic cells are activated by exposure to Pathogen Associated Molecular Patterns (PAMPs). Polyinosinic:polycytidylic acid (PolyIC) is synthetic double-stranded RNA that functions as a PAMP. Corticosterone (CORT), a stress hormone, is generally immunosuppressive. The response of murine DCs (MuTuDC) to PolyIC and CORT exposure is being investigated. ELISA demonstrated that PolyIC exposure increased IL-12 production, an indicator of DC activation. Increased cell death was also observed following PolyIC exposure, but pre-treatment with CORT decreased cell death. The literature suggests that DC death post activation is important for regulating the immune response. However, the pathway mediating PolyIC-induced DC death and the influence of CORT on the process is loosely characterized. Literature suggests that apoptosis occurs through the pro-apoptotic protein Bim. It is hypothesized that CORT is altering Bim isoform expression to reduce cell death. Bim has three predominant isoforms, varying in function, that regulate apoptosis. Primers derived from the literature for both murine and human Bim mRNA isoforms were analyzed using the NCBI BLAST program to ensure detection of the three isoforms. However, detection of Bim protein and mRNA isoforms in MuTuDCs was difficult. Therefore, Human-Diploid Fibroblast cells immortalized with T-antigen (HDF+T) were used to establish necessary RT-PCR and Western protocols. After demonstrating Bim isoform(s) presence in HDF+Ts, future studies will involve MuTuDCs pretreatment with CORT and then treatment with PolyIC, or another apoptosis inducer such as 5-fluorouricil (5-FU). Elucidating the PolyIC-mediated cell death pathway and the impact of CORT will provide novel information about factors influencing DC abundance and lifespan, which ultimately influence the immune response