Sarah C. Vick et al. BioRxiv
Keywords
● Mucosal NK cells
● Vaginal immunology
● Sexually transmitted infections (STIs)
● Epithelial barrier integrity
● Amphiregulin
Main Findings
The vaginal mucosa is a complex microenvironment comprised of diverse immune cell populations, a dynamic microbiome, and a protective epithelial barrier. Several mechanisms work together to protect the vaginal microenvironment against viral and bacterial pathogens, while also preserving epithelial barrier integrity. Although tissue-resident natural killer (NK) cells have been extensively characterized at other mucosal sites such as the gut, their role in the vaginal tract remains poorly understood. In the current study, the authors characterized circulating and vaginal tissue (VT) NK cells in both human and murine samples to better define their functions during homeostasis and infection.
First, the authors first performed single-cell RNA sequencing on paired VT and peripheral blood mononuclear cell (PBMC) samples to characterize the immune cell populations present. They found that immune cells from the VT and PBMCs clustered separately, suggesting that these populations may have distinct gene signatures. Specifically, circulating NK cells had a high expression of cytotoxic and effector-associated genes, whereas VT NK cells were enriched for tissue-repair genes. They further characterized these NK cell populations and identified 313 genes upregulated in the VT NK cells that were associated with tissue residency and immune regulation, which was subsequently validated by flow cytometry.
The authors also examined CD56brightCD16- and CD56dimCD16+ NK cell subsets to assess markers of maturation. They found a higher frequency of CD56brightCD16-NK cells in the VT, which are known to be a less mature and immunoregulatory NK cell population. Conversely, CD56dimCD16+ NK cells were found at a higher frequency in the blood, a subset described as highly cytotoxic and mature.
Notably, the VT NK cells expressed amphiregulin (Areg), a key growth factor involved in tissue protection and repair. Ex vivo stimulation of VT NK cells with the damage-associated alarmins IL-18 and IL-33 significantly increased Areg expression. Furthermore, although the VT NK cells exhibited a predominately immunoregulatory and quiescent phenotype, they rapidly responded to an inflammatory stimulus. Following incubation with interleukin (IL)-12 and IL-18 for 24 hours, there was a significant increase in granzyme B, interferon (IFN)γ and several activation markers.
Subsequently, the authors used a mouse model to characterize NK cell function during an in vivo infection. First, they confirmed their initial findings, identifying distinct NK cell populations in the blood and vaginal tissue of C57BL/6 mice. The mice were then intravaginally inoculated with an Herpes Simplex Virus (HSV)-2 strain and samples were collected from two points; 2 days post-infection (dpi) and 10 dpi. At 2dpi, the VT NK cells produced high amounts of granzyme B and IFNγ, and this returned to baseline by 10dpi. Similarly, there was a peak in Areg expression at 2dpi, which decreased by 10dpi. Finally, the authors investigated whether VT NK cells play a direct role in tissue repair during infection. Using the same model, they depleted NK cells after HSV-2 inoculation and examined vaginal tissue damage. They found a significant increase in vaginal epithelial damage and lymphocyte infiltration compared to control mice, despite no differences in HSV-2 viral load.
In summary, this study identifies a distinct population of vaginal tissue-resident NK cells that exhibit an immunoregulatory phenotype. These mucosal NK cells respond rapidly to an HSV-2 infection and express amphiregulin, a key tissue-protective factor involved in repair processes. Importantly, depletion of vaginal NK cells results in increased tissue damage despite viral clearance, highlighting their important role in maintaining tissue integrity during an infection.
Limitations
· The study would have benefited from a larger sample size for both the human and murine experiments. While the data is compelling, a larger number of samples would’ve increased robustness.
· As the authors discussed, while there were some experiments involving a mouse model of HSV-2 infection, there was no disease state explored for the human VT NK cells. This represents an important gap, as human VT NK cells may behave differently during an infection. Validation can be done in a few ways such as the analysis of biobanked samples from participants with HSV infection to directly assess NK cell function. As this sample type may be difficult to access, the development of in vitro models such as a vaginal epithelial cell infection model supplemented with human NK cells can provide an important system for examining human NK cell responses.
· While IL-18 and IL-33 were identified as activators of Areg expression, there was limited exploration into mechanisms of action. Specifically, the authors did not investigate specific cell populations that are producing these cytokines and directly assessing their contribution to Areg expression in the VT NK cells (which can be explored with a co-culture experiment or depletion experiment). Furthermore, there was no exploration into inhibitory factors, which is critical in understanding how the tissue repair phenotype is regulated. Identifying inhibitors will help define the threshold and/or magnitude of Areg expression in the VT NK cells, and how this subsequently impacts tissue repair.
· It would have been interesting to perform co-culture experiments with other cell types and examine a larger panel of cytokines that may activate or inhibit Areg expression.
· Notably, the authors did not discuss the possible implications of the vaginal microbiome. There is a large body of literature describing the important role the microbiome plays in vaginal immunology; the authors did not explore or acknowledge the possible role microbes may have on VT NK cell function and/or Areg expression.
· It remains unclear whether AREG expression is a part of a repair response that could determine the outcome of infection or increase pathology. Exploring the downstream effects of this repair response will help answer this question.
· The authors did not directly compare the VT NK cells to an NK cell population from a distinct mucosal site (i.e. lung) to determine how unique the tissue repair phenotype is to VT NK cells.
· The authors did not explore activating and inhibitory NK cell receptor-ligand interactions in the vaginal tract. NK cell activity is largely mediated by a balance between activating and inhibitory signals, which is critical for an antiviral response. Herpesviruses such as CMV and HSV have several mechanisms that modulate these signals, including the downregulation of activating ligands and effects on MHC inhibitory signals to evade NK-cell recognition. Consideration of these receptor-ligand interactions is important when examining NK cell function in the vaginal tract during HSV infection.
Significance/Novelty
This study is the first to provide an in-depth characterization of vaginal tissue NK cells, both during homeostasis and a localized HSV-2 infection. Notably, it not only identifies a distinct population of VT NK cells with a unique gene signature, but also describes amphiregulin expression in this population, which has not been previously reported. Using in vitro and in vivo models, the authors effectively demonstrate that the depletion of this NK cell population results in significant epithelial barrier damage, which is novel to the field.
Furthermore, the results of this study have important implications for the field of reproductive health research. While vaginal immunology research is increasing, there is still a significant knowledge gap in how this microenvironment functions, and the dynamic factors involved. This study demonstrates that immune cell populations such as NK cells have a functionally distinct role in the vaginal tract to not only protect against pathogens, but to also maintain epithelial barrier integrity. Moreover, this study identifies an important factor involved in vaginal tissue repair, which may aid in the development of novel therapeutics that protect against sexually transmitted infections such as HIV.
Credit
Reviewed by Rameen Jamil as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute and the University of Toronto.
The author declares no conflict of interests in relation to their involvement in the review.