A murine model to study the effect of cold-induced stress (CIS) on Chlamydia muridarum genital infection and immune response has been developed in our laboratory. Previous results in the lab show that CIS increases the intensity of chlamydia genital infection, but little is known about the effects and mechanisms of CIS on the differentiation and activities of CD4+ T cell subpopulations and bone marrow-derived dendritic cells (BMDCs). The factors that regulate the production of T helper 1 (Th1) or T helper 2 (Th2) cytokines are not well defined. In this study, we examined whether CIS modulates the expressions of beta-adrenergic receptor (β-AR), transcription factors, hallmark cytokines of Th1 and Th2, and differentiation of BMDCs during C. muridarum genital infection in the murine model. Our results show that the mRNA level of the beta2-adrenergic receptor (β2-AR) compared to β1-AR and β3-AR was high in the mixed populations of CD4+ T cells and BMDCs. Furthermore, we observed decreased expression of T-bet, low level of Interferon-gamma (IFN-γ) production, increased expression of GATA-3, and Interleukin-4 (IL-4) production in CD4+ T cells of stressed mice. Exposure of BMDCs to Fenoterol, β2-AR agonist, or ICI118,551, β2-AR antagonist, revealed significant β2-AR stimulation or inhibition, respectively, in stressed mice. Moreover, co-culturing of mature BMDCs and naïve CD4+ T cells increased the production of IL-4, IL-10, L-17, and IL-23 cytokines, suggesting that stimulation of β2-AR leads to the increased production of Th2 cytokines. Overall, our results show for the first time that CIS promotes the switching from a Th1 to Th2 cytokine environment. This was evidenced in the murine stress model by the overexpression of GATA-3 concurrent with elevated IL-4 production, reduced T-bet expression, and IFN-γ secretion.

Running Title: Stress and Chlamydia muridarum genital infection ABSTRACTA mouse model to study the effect of cold-induced stress on Chlamydia muridarum genital infection and immune response has been developed in our laboratory. Our previous results show that cold-induced stress increases the intensity of chlamydia genital infection, but little is known about the effect of cold-induced stress on differentiations and activities of T cell subpopulations and bone marrow derived dendritic cells (BMDCs). The factors that regulate the production of T helper 1 (Th1) or T helper 2 (Th2) cytokines is not clear. The objective of this study was to examine whether cold-induced stress modulates the expression of transcription factors and hallmark cytokines of Th1 and Th2 or differentiation of BMDCs during C. muridarum genital infection in mice. Our results show that mRNA level of beta2-adrenergic receptor (2-AR) compared to 1-AR and 3-AR was high in mixed population of CD4+ T cells and BMDCs.Further, decreased expression of T-bet and low level of interferon-gamma (IFN-) production and increased expression of GATA-3 and interleukin-4 (IL-4) production in CD4+ T of stressed mice was observed. Exposure of BMDCs to feroterol (2-AR agonist) or ICI,118551 ( 2-AR antagonist), respectively, revealed significant stimulation or inhibition of 2-AR in stressed mice. Moreover, co-culturing of mature BMDC and naïve CD4+ T cells resulted in increased production of IL-4, IL-10, and IL-17 in culture supernatants, suggesting that stimulation of 2-AR leads to the increased production of Th2 cytokines. Overall, our results show for the first time that cold-induced stress is able to modulate the pattern of Th1 and Th2 cytokine environment, suggesting that it promotes the differentiation to Th2 rather than Th1 by the overexpression of GATA-3 correlated with elevated production of IL-4, IL-10, in contrast to a low expression of T-bet correlated with less IFN- secretion in the mouse model.

Chlamydia caused by Chlamydia trachomatis is the leading cause of bacterial STDs worldwide and the USA. Although there are more clinical and epidemiological studies of chlamydia genital infection, the effect of stress on chlamydia disease is not well explored. Previous results from our lab show that cold-induced stress results in increased intensity of chlamydia genital infection in mice; however, the mechanisms are unknown. Thelper 1(Th1) is known to be protective against chlamydia, whereas Th2 leads to infection.  However, the differentiation of Th1 and Th2 during exposure to cold stress is not well understood. Several studies show GATA-3 and T-bet are associated with Th1 and Th2 cytokine production. The purpose of this study was to evaluate the effect of cold water on the activity off Th1 and Th2 during Chlamydia muridarum genital infection. We hypothesized that cold stress alters the differentiation and variation in gene expression profile of transcription factors and cytokine productions in T cells. T cells isolated from the genital tract were proliferated for 48 h incubated at 37OC and 5% CO2. The mRNA levels and detection of cytokines in culture supernatants was determined by quantitative real time PCR and ELISA, respectively. Our results showed increased IL-4 production, which was associated with upregulated expression of transcription factor GATA-3. In contrast, decreased gamma interferon production was associated with decreased T-bet gene expression in T cells. Based on this data, cold stress modulates the dynamics of signature Th1 and Th2 cytokine productions that are regulated by expression of GATA-3 and T-bet.  This work was supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and NIH Grant P20GM103434 awarded to Bluefield State College.