ABSTRACTROLE OF ARYL HYDROCARBON RECEPTOR (AhR) POLYMORPHISMS AND TRANSCRIPTIONAL ACTIVITY IN TCDD-INDUCED SUPPRESSION OF THE B CELL IgM RESPONSEByNatalia KovalovaPrevious studies have demonstrated that most of the intraspecies variation in sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including suppression of antibody responses, in murine models is due to single nucleotide polymorphisms (SNPs) within the aryl hydrocarbon receptor (AhR) gene. The underlying reason for the variation in sensitivity to TCDD-induced suppression of IgM responses among humans is not well understood but is thought, in part, to be a result of various polymorphic forms of the AhR expressed by different individuals. Additionally, it is well known that AhR mediates toxic effects of TCDD by, most likely, deregulating gene expression. However, the exact mechanism, as wells as transcriptional targets of TCDD are not well understood. Therefore, this dissertation research had two primary long-term goals. The first goal was to identify and characterize a core set of evolutionarily conserved gene expression responses across mouse, rat and human primary B cells focusing on the role of AhR in mediating immunotoxic effects of TCDD. The second goal was to comprehensively evaluate the correlation between B cell sensitivity to TCDD-mediated suppression of the IgM response and previously identified SNPs within the human AhR. I investigated the functional properties of six (P517S, R554K, V570I, V570I+P517S, R554K+V570I and P517S+R554K+V570I) human AhR variants stably expressed in the human B cell line, SKW 6.4. I have demonstrated that the R554K human AhR SNP alone altered sensitivity of human B cells to TCDD-mediated induction of Cyp1B1 and Cyp1A2 metabolizing enzymes. By contrast, attenuation of TCDD-induced IgM suppression required a combination of all three SNPs P517S, R554K, and V570I. The second part of my dissertation research focused on identification of the B cell-specific molecular targets of TCDD. Based on preliminary findings, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 544 human, 2527 mouse and 772 rat genes over the 24-h period. Only 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with calcium ion binding, cell adhesion and inflammatory response were overrepresented in all three species. Collectively, these results suggest that species-specific gene expression profiles mediate the species-specific effects of TCDD despite the conservation of the AhR and its signaling mechanism.
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