Investigating the effect of sex hormones on the immune response to TB and HIV.

Abstract

Background: Global incidence rates for tuberculosis (TB) indicate a gender-bias to the disease, with almost twice as many men actively infected than women (male/female ratio of 1.9). Sex hormones are known to regulate immune function and their role in tuberculosis has been suggested experimentally in animal models. However the role of sex hormones in human TB infection and whether there is any synergistic effect in HIV and TB co-infection is unknown. In addition, there is data to suggest that manipulation of sex hormones with progestin-based injectable contraceptives, particularly DMPA, increases susceptibility to both HIV and TB. Quantitation of sex hormones has relied on antibody based immunoassays, such as ELISA, which suffer both from a lack of specificity and sensitivity. Aims: Consequently, the purpose of this study was to develop a method for quantitation of sex hormones and their contraceptive analogues utilizing a targeted LC-MS/MS approach that has been demonstrated to increase the accuracy of hormone quantitation and to determine the effect of sex hormones and hormonal contraceptives on the immune response to TB and HIV. Method: Sex hormone levels were measured in plasma samples from three separate cohorts, using MRM run in positive ESI mode, on a ABSciEx Q-TRAP 5500 mass spectrometer. The method was validated and DMPA levels were measured in the FRESH cohort (n= 62) and CAPRISA CAP004 study (n= 38) to determine effects on DMPA on increased risk in HIV acquisition. Testosterone, progesterone and DMPA levels were measured in the HIV chronic patients from the Cryptococcal cohort (n = 271) and sex hormone associated changes in phenotypic expression and immunological responses using intracellular cytokine staining were determined by flow cytometry analysis. Results: We validated our assays and were able to identify and quantitate levels of DMPA in two blinded studies. In the FRESH cohort, we were able to correctly identify and quantitate DMPA levels in Depo-Provera users and injectable progestin-based (IPC) contraceptive use was associated with high risk of HIV acquisition (p = 0.0142). IPC users were found to have significant increase in CCR5+CD4+ T cells in the cervix as well as increased CCR5 expression. Preliminary data in the CAP004 study, showed differential expression of mucosal proteins in the cervicovaginal lavage associated with DMPA use. In the Cryptococcal study, we found, as expected, significant differences in testosterone and progesterone levels between male and female patients, p <0.0001 and p=0.0001 respectively. Given that only 3 female patients reported to be using the contraceptive DMPA, we identified 42 of the total number of 172 females to have significant levels of DMPA (greater than LOQ = 0.064ng/ml) and these females had significantly lower progesterone levels than females not using DMPA (p <0.0001). This large under-reporting of contraceptive use indicates the value in direct measurement as opposed to self-reporting. As expected negative correlation was observed between progesterone and DMPA levels (p = 0.0016, r = -0.2445). However, individual response profiles are highly variable and decay rates of DMPA in longitudinal samples vary greatly between individuals. We hypothesis this will impact the immunomodulatory effect of DMPA, again suggesting the need for direct measurement. In this small sample we find no significant differences in activation and exhaustion of CD4 and CD8 T cells (determined using HLA-DR, CD38 and PD1 expression), as well as T regulatory cells (FoxP3 expression) when comparing female patients with high progesterone, low progesterone, and injectable contraceptive DMPA users, as well as males with high testosterone and low testosterone levels. However, females with high progesterone levels generally had higher CD38 expression, though non-significant. Also, we observe no significant differences in cytokine expression (TNFα, IFNγ, and IL-2) as well as markers, CD107a and Mip-1β, upon stimulation with SEB, PPD, pp65 CMV and HIV peptides. Conclusion: We successfully optimized and validated a method for quantitation of sex hormones using LC-MS/MS and were able to detect and quantify levels of testosterone, progesterone and DMPA. This method has the potential in clinical studies, to eliminate the need to rely on self-reported information, as exogenous hormones or contraceptive analogues can be detected with high sensitivity and specificity. Changes in the female genital tract which may be associated with increased risk of HIV were found in injectable progestinbased contraceptive users, particularly DMPA. However, no significant immunological effects of hormone levels on immune response and phenotype expression were found in blood.