Program Director/Principal Investigator (Last, First, Middle): Belay, Tesfaye Project Summary Chlamydia trachomatis genital infection is the most common sexually-transmitted bacterial disease in the world. If left untreated, chlamydia genital infection leads to pelvic inflammatory disease, ectopic pregnancy, and infertility. It is widely known that the immunopathological reactions to this disease, rather than the infection itself, remain a serious public health problem. There is medical interest in understanding how stress may impact resistance or susceptibility to sexually transmitted diseases. Many investigators have examined the immunological responses to C. trachomatis, but the effect of stress on chlamydia genital infection has not yet been explored and is not understood. Our research seeks to understand the relationship between them. To accomplish this, we have developed a chronic-stress model in mice by daily immersing mice in cold water for five minutes daily for 21 days. This cold-induced stress (CIS) mouse model shows the increased intensity of Chlamydia muridarum genital infection, which is associated with the elevation of the stress hormone norepinephrine (NE) during both primary and secondary infections. Our research has demonstrated that CIS promotes increased beta2- adrenergic receptor (β2-AR) expression in CD4 + T cells, as well as T helper 2 (Th2) differentiation, specifically by increasing the expression of GATA-3 and IL-4 secretion. Moreover, we showed that β1/β2-AR KO are less susceptible to C. muridarum genital infection than the WT C57BL/6J. Those observations prompted us to use a β2-AR KO model and explore the immunopathogenesis of chlamydia genital infection. We recently found that the β2-AR KO is more resistant to C. muridarum than the WT C57BL/6J. However, the underlying mechanisms of chronic CIS on the infection and the immunopathogenesis in our mouse model remain unknown. The central objective of this proposal is to define the mechanism(s) by which stress may suppress the immune system and increase the intensity of genital C. muridarum infection and its immunopathologies in mice. We hypothesize that the NE produced by CIS leads to stimulation of the 𝛽𝛽2-AR signaling pathway in mice which, in turn, suppresses the protective immune response against C. muridarum genital infection. To test this hypothesis, we will conduct the following three experiments as part of this project: In Aim #1, we will compare the CD4+ T cell subset immune responses of stressed β2-AR-/- mice and C57BL/6J (β2-AR+/+) mice. We anticipate that the deficiency of β2-AR enhances the restoration of Th1 cytokine production in stressed mice, which may result in the clearance of C. muridarum. In Aim #2, we will explore the downstream signaling pathways from the β2-AR of stressed KO mice compared to stressed C57BL/6J mice. Data obtained may provide insight into the involvement of β2-AR in cAMP-protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) pathways in suppressing the immune system. In Aim#3, we will assess the histopathological changes of stressed β2-AR KO as compared to WT C57BL/6J mice, and we will then evaluate the contributions of cytokines to pathology. Data from this method will demonstrate a reduced oviductal pathology in β2-AR KO mice. The proposed experiments will provide insight into the immunosuppression mechanisms resulting from the interactions of the endocrine and immune systems in the immunopathogenesis of chlamydia. Overall, results obtained from these β2-AR deficient mice, combined with our previous findings and those of others, could reveal more evidence of the effects of chronic stress on the severity of chlamydial disease in humans. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page

Program Director/Principal Investigator (Last, First, Middle): Belay, Tesfaye Narrative The purpose of this proposed project is to investigate the mechanism(s) with which cold water-induced norepinephrine secretion influences intensity of infection and immune cell functions and pathology in a mouse model. We will use stress-hormone-receptor-deficient mice to test whether infection is minimized when compared to control. These experiments may, for the first time, isolate the mechanism(s) through which stress worsens chlamydia disease in mice. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page