Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Rosana Schafer

Committee Co-Chair

Stacey Anderson

Committee Member

John Barnett

Committee Member

Barbara J Mean

Committee Member

Paul Siegel

Abstract

Toluene diisocyanate (TDI) is a low molecular weight chemical sensitizer and a major cause of occupational allergic disease and asthma. The roles of immunomodulatory elements such as microRNAs, which alter gene expression, and regulatory T cells (Tregs), which suppress T cell-mediated responses, in chemical sensitization are not fully elucidated. In order to further investigate the functional role of miR-210 and Tregs during TDI sensitization, BALB/c mice were dermally exposed to a single, sensitizing dose of TDI (0.5-4% v/v) and evaluated for immunologic endpoints. dLN T regs were phenotyped during TDI sensitization and a heterogeneous, expanded population was observed. To examine Treg functionality, a carboxyfluorescein succinimidyl ester-based Treg suppression assay was conducted. Tregs isolated from the dLNs of TDI sensitized mice were more suppressive than their control counterparts. The Treg population was then depleted prior to TDI sensitization by in vivo administration of a CD25-targeting antibody. The sensitization response was intensified following Treg depletion, as evidenced by amplified total dLN cellularity, Th2 expansion, and IL-4 mRNA production. This data suggests that Tregs are important negative regulators of the TDI sensitization response.;We have previously shown that miR-210 expression in the draining lymph node (dLN) is augmented during TDI sensitization and several putative mRNA targets identified for this miRNA (foxp3 and runx3 ) were directly related to Treg differentiation and function. Therefore, we hypothesized that miR-210 targets transcription factors central to Treg development and function, antagonizing the development and functional role of this subset during TDI sensitization. In this work, we show that miR-210 expression is increased in the mouse dLN and Treg subsets during TDI sensitization. Putative miR-210-target/key player interactions were examined using an ex vivo stimulation system in which miR-210 mimics were transfected into primary, stimulated dLN cells; decreases in multiple Treg-related genes were observed via qPCR analysis. Alterations in dLN and Treg mRNA and protein expression of foxp3, runx3, ctla4, and cd25 were observed at multiple time points following TDI exposure. These molecules are directly involved in Treg expansion and function; therefore, miR-210 may potentially negatively target Treg differentiation and function during TDI sensitization. In order to examine the effect of miR-210 modulation on Treg functionality we performed a carboxyfluorescein succinimidyl ester-based Treg suppression assay using dLN T regs from naive mice transfected with miR-210 mimic. While the suppressive ability of TDI/mimic Tregs was increased compared to the acetone/mimic counterparts, the TDI/mimic samples had less expression of miR-210 compared to the acetone/mimic controls suggesting other factors, such as additional miRNAs, might be involved in the regulation of the functional capabilities of these cells. These novel findings indicate that Treg s likely play an important role in modulating allergic responses during TDI sensitization and that miR-210 may have an inhibitory role in this process. Because the functional roles of miRNAs and Tregs have not been previously elucidated in a model of chemical sensitization, these data contribute to the understanding of the immunologic mechanisms of chemical induced allergic disease and may aid in the development of preventative strategies.

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