# Murine modeling of menstruation identifies immune correlates of protection during Chlamydia muridarum challenge

## TL;DR

A mouse model of menstruation reveals that immune cells called NK cells help protect against chlamydia infection during a specific phase of the cycle.

## Contribution

The study introduces a murine model of menstruation to identify immune correlates of protection against chlamydial infection.

## Key findings

- NK cell abundance and proinflammatory signaling correlate with reduced bacterial burden during the late luteal phase.
- NK cells in the cervicovaginal tissues are transcriptionally distinct and enriched for effector functions.
- Depletion of NK cells during the late luteal phase leads to productive chlamydial infection.

## Abstract

The menstrual cycle influences the risk of acquiring sexually transmitted infections (STIs), including those caused by the pathogen Chlamydia trachomatis (C. trachomatis). However, the underlying immune contributions are poorly defined. A mouse model simulating the repetitive immune-mediated process of menstruation could provide valuable insights into tissue-specific determinants of protection against chlamydial infection within the cervicovaginal and uterine mucosae of the female reproductive tract (FRT). Here, we used the pseudopregnancy approach for inducing menstruation in naïve C57Bl/6 mice and performed vaginal challenge with Chlamydia muridarum (C. muridarum) over the course of decidualization, endometrial tissue remodeling, and menstruation. This strategy identified that a time point over pseudopregnancy corresponding to the late luteal phase of the menstrual cycle correlated with reduced bacterial burden. By evaluating the early infection site following challenge at this time point, we found that a greater abundance of NK cell populations and proinflammatory signaling, including IFNγ, were strongly correlated with protection. FRT immune profiling in uninfected mice over pseudopregnancy or in pig-tailed macaques over the menstrual cycle identified periodic NK cell infiltration into the cervicovaginal tissues and luminal surface occurring over a similar time frame. Notably, these cell populations were transcriptionally distinct and enriched for programs associated with NK cell effector functions. Depletion of FRT NK cells during the late luteal phase time frame resulted in a loss of protection, enabling productive infection following C. muridarum challenge. This study shows that the pseudopregnancy murine menstruation model recapitulates dynamic changes occurring in mucosal immune states throughout the FRT as a result of endometrial remodeling and identifies NK cell localization at the FRT barrier site of pathogen exposure as essential for immune protection against primary C. muridarum infection.

Although the vast majority of women and adolescent girls of reproductive age experience menstruation, we have limited insight into how this tissue remodeling process alters mucosal immune defenses against infection by genitourinary pathogens. In this study, we used a murine model of menstruation to investigate how endometrial shedding and repair alters the FRT immune landscape to influence chlamydial infections. Using this approach, we identified that endometrial remodeling regulates a substantial pro-inflammatory immune response, including periodic NK cell recruitment into the cervicovaginal tissues. Transcriptional profiling showed that these cells were distinct from FRT NK cells in MPA-treated mice and exhibited an immune-activated state. The localization and enrichment of NK cells at the cervicovaginal barrier were determined to be responsible for providing rapid immune protection that reduced C. muridarum burden, as experimental depletion of these cells at this time point led to productive infections. Taken together, this study identifies that murine models of menstruation can be a valuable tool for investigating how the menstrual cycle modulates immune homeostasis and for identifying ways to strengthen mucosal immune defenses against genitourinary pathogens in women.