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.

The beta-2-adrenergic receptor (β2-AR) is one of the β-AR subsets found in most immune cells and is stimulated by norepinephrine (NE) binding. We showed previously that exposure of a mouse model to cold water stress leads to high level production of NE that may result in increased intensity of Chlamydia muridarum genital infection but the mechanism is not well defined. The purpose of this study was to develop and characterize a β2-AR knockout (KO) stress mouse model to investigate its susceptibility to C. muridarum genital infection. We hypothesized that lacking the β2-AR leads to a decreased C. muridarum shedding from the genital tract of stressed mice compared to that of stressed wild type (WT) mice. Stressed and non-stressed C57BL/6J WT and β2-AR KO mice were infected intravaginally with C. muridarum. Swabbing at 3-day intervals during primary infection and C. muridarum isolation was performed by culturing in McCoy tissue culture following standard methods. β2-AR KO stressed mice had a mean of 8540 inclusion forming units/ml (IFU/ml) compared to a mean of 18000 IFU/ml of stressed wild type at day 9 post infection. The overall course of primary infection in the β2-AR KO mouse model showed slightly reduced Chlamydia shedding. In addition, transfer of CD4+ T cells from black J6 WT to β2-AR KO resulted in a significant increase of shedding compared to that of non-stressed WT transfer (6000 versus 1000 IFU/ml). Secretion of interferon gamma was restored in β2-AR KO mice compared to WT. Overall, these results imply that deficiency in β2-AR leads to decreased C. muridarum shedding compared to the WT. However, other members of the alpha- and beta-adrenergic receptor family may compensate for the absence in β2-AR KO mice thus more investigation is needed.

Chlamydia trachomatis genital infection is the most common bacterial sexually transmitted disease worldwide. Previously we reported that cold-induced stress results in immune suppression of mice that subsequently leads to increased intensity of Chlamydia muridarum genital infection. Furthermore, we demonstrated that stressed mice orally fed with active hexose correlated compound (AHCC) have reduced shedding of C. muridarum from the genital tract. However, the mechanism of AHCC on reducing the organ load and changes in the immune response in the stress model is not well known. This study evaluated infection and changes in immunological parameters of stressed AHCC-fed mice with or without C. muridarum genital infection. We hypothesized that AHCC feeding to stressed mice restores the protective immune function and reduces susceptibility to C. muridarum genital infection. Results show that oral feeding of stressed mice with AHCC resulted in decreased shedding of C. muridarum from the genital tract, reduced production of plasma catecholamines, increased expression of T-bet and reduced GATA-3 in CD4+ T cells, increased production of IL-12 and IFN-γ, and reduced production of IL-4 in CD4+ T cells, and enhanced expression of surface markers and co-stimulatory molecules of CD4+ T cells, bone marrow-derived dendritic cells (BMDCs), and natural killer cells. Co-culturing of mature BMDCs with splenic CD4+ T cells led to the increased and decreased production of T-helper 1 and T-helper2 cytokines, respectively. Overall, our results show that AHCC fosters the restoration of Th1 cytokine production while reducing Th2 cytokine production, which would promote C. muridarum clearance in the murine stress model.

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.