Single-cell analysis of the aged ovarian immune system reveals a shift towards adaptive immunity and attenuated cell function
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eLife assessment
The study by Ben Yaakov et al. describes a single cell analysis of the mammalian ovary in young, adult and old mice. Based on gene expression profiles, the authors identified cell clusters corresponding to immune cell populations in mouse ovaries and compared their abundance in aged compared to adult animals. In comparison with previous studies that used single cell RNAseq to characterize the heterogeneity of cell types in the ovary, this study focuses only on immune cells resulting in much better coverage to characterize the changes that these cells undergo as a function of age. The combination of single-cell RNA sequencing and flow cytometry used by the authors is a robust and unbiased approach to characterize immune cell alterations in aging ovaries. Overall, the data and analyses presented in this study reveal profound modifications of the immune system in the aging reproductive system in mice. However, while both the data and biology presented are quite interesting, this study is perhaps too wide in breadth such that no individual result is extensively and rigorously explored.
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Abstract
The immune system plays a major role in maintaining many physiological processes in the reproductive system. However, a complete characterization of the immune milieu in the ovary, and particularly how it is affected by female aging, is still lacking. Here, we utilize single-cell RNA sequencing and flow cytometry to construct the complete description of the murine ovarian immune system. We show that the composition of the immune cells undergoes an extensive shift with age towards adaptive immunity. We analyze the effect of aging on gene expression and chemokine and cytokine networks and show an overall decreased expression of inflammatory mediators together with an increased expression of senescent cells recognition receptors. Our results suggest that the fertile female’s ovarian immune aging differs from the suggested female post-menopause inflammaging as it copes with the inflammatory stimulations during repeated cycles and the increasing need for clearance of accumulating atretic follicles.
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eLife assessment
The study by Ben Yaakov et al. describes a single cell analysis of the mammalian ovary in young, adult and old mice. Based on gene expression profiles, the authors identified cell clusters corresponding to immune cell populations in mouse ovaries and compared their abundance in aged compared to adult animals. In comparison with previous studies that used single cell RNAseq to characterize the heterogeneity of cell types in the ovary, this study focuses only on immune cells resulting in much better coverage to characterize the changes that these cells undergo as a function of age. The combination of single-cell RNA sequencing and flow cytometry used by the authors is a robust and unbiased approach to characterize immune cell alterations in aging ovaries. Overall, the data and analyses presented in this study reveal …
eLife assessment
The study by Ben Yaakov et al. describes a single cell analysis of the mammalian ovary in young, adult and old mice. Based on gene expression profiles, the authors identified cell clusters corresponding to immune cell populations in mouse ovaries and compared their abundance in aged compared to adult animals. In comparison with previous studies that used single cell RNAseq to characterize the heterogeneity of cell types in the ovary, this study focuses only on immune cells resulting in much better coverage to characterize the changes that these cells undergo as a function of age. The combination of single-cell RNA sequencing and flow cytometry used by the authors is a robust and unbiased approach to characterize immune cell alterations in aging ovaries. Overall, the data and analyses presented in this study reveal profound modifications of the immune system in the aging reproductive system in mice. However, while both the data and biology presented are quite interesting, this study is perhaps too wide in breadth such that no individual result is extensively and rigorously explored.
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Reviewer #1 (Public Review):
This work leverages single-cell RNA-sequencing to probe changes in various immune functionings within the ovary in aging. The data provided is the most comprehensive of ovarian immune cells at the resolution of single-cell transcriptomics to-date and will be valuable to other researchers. The authors explore four distinct immune functionings:
- The authors identify macrophages and a unique CD3+CD8-CD4- T-cell subpopulation that change in abundance with aging. While these are interesting findings that align with flow cytometry results, the lack of batch correction and application of single-cell differential abundance tools limit the strength of the claims. The authors also do not further probe gene expression changes specific to these populations.
- The authors also analyze changes in global gene expression …
Reviewer #1 (Public Review):
This work leverages single-cell RNA-sequencing to probe changes in various immune functionings within the ovary in aging. The data provided is the most comprehensive of ovarian immune cells at the resolution of single-cell transcriptomics to-date and will be valuable to other researchers. The authors explore four distinct immune functionings:
- The authors identify macrophages and a unique CD3+CD8-CD4- T-cell subpopulation that change in abundance with aging. While these are interesting findings that align with flow cytometry results, the lack of batch correction and application of single-cell differential abundance tools limit the strength of the claims. The authors also do not further probe gene expression changes specific to these populations.
- The authors also analyze changes in global gene expression across cell types using an enrichment analysis; Figure 3B specifically is an excellent visualization summarizing potential global and cell-type specific changes in gene expression programs during aging.
- The authors infer differences in cell-cell communication mediated by various chemokines and cytokines. In this analysis, they claim a decreased inflammatory response due to aging. Here, the global decrease in gene expression in many cell types is not accounted for. Visualizations and quantitative analyses could benefit from existing, specialized in cell-cell communication tools.
- A discussion of changes to the expression of SASP receptors on immune cell types.
While both the data and biology presented are quite interesting, this study is perhaps too wide in breadth such that no individual result is extensively and rigorously explored.
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Reviewer #2 (Public Review):
The paper by Ben Yaakov et al. describe a single cell analysis of the mammalian ovary in young, adult and old mice. In comparison with previous studies that used single cell RNAseq to characterize the heterogeneity of cell types in the ovary, this study focuses only on immune cells resulting in much better coverage to characterize the changes that these cells undergo as a function of age. The paper provides a useful dataset and informative data analysis with interesting findings including the increases in DNT cells in the ovary of old mice. Some discussion on how the presented results might be related to reduced fertility with age would be good to tie the results back to the original questions with which the authors start their paper.
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Reviewer #3 (Public Review):
This work studied age-related alterations in the ovarian immune cells in mice using single-cell RNA sequencing and flow cytometry. Based on gene expression profiles, the authors identified cell clusters corresponding to immune cell populations in mouse ovaries and compared their abundance in aged compared to adult animals. The authors identified two parallel immune processes in aging ovaries: a decrease in proportions of myeloid cells such as macrophages and neutrophils accompanied by an increase in proportions of CD3+ T cells. The latter cell population was increased in abundance due to an expansion of CD3+ cells that do not express CD4 and CD8, referred to as "double-negative T cells." These immune alterations were identified by single-cell RNA sequencing using small numbers of mice, and the authors …
Reviewer #3 (Public Review):
This work studied age-related alterations in the ovarian immune cells in mice using single-cell RNA sequencing and flow cytometry. Based on gene expression profiles, the authors identified cell clusters corresponding to immune cell populations in mouse ovaries and compared their abundance in aged compared to adult animals. The authors identified two parallel immune processes in aging ovaries: a decrease in proportions of myeloid cells such as macrophages and neutrophils accompanied by an increase in proportions of CD3+ T cells. The latter cell population was increased in abundance due to an expansion of CD3+ cells that do not express CD4 and CD8, referred to as "double-negative T cells." These immune alterations were identified by single-cell RNA sequencing using small numbers of mice, and the authors partially validated the data using flow cytometry analysis in larger groups of animals. In addition, based on the gene expression data, they predicted which signaling pathways were altered in the aged immune cells and analyzed putative changes in the chemokine and cytokine networks, pointing at potential crosstalk of immune cell populations with senescent cells in aging ovaries.
The combination of single-cell RNA sequencing and flow cytometry used by the authors is a robust and unbiased approach to characterize immune cell alterations in aging ovaries. Overall, the data and analyses presented in this study reveal profound modifications of the immune system in the aging reproductive system in mice. Additional computational approaches predicting cell-cell communications affected by aging in the ovaries presented in this study can extend our understanding of the aging immune system. However, most of the conclusions from single-cell RNA sequencing results are not confirmed using additional approaches, including a more detailed flow cytometry analysis of ovarian immune cell subsets and functional validations of the predicted biological processes affected by aging.
The presented data do not specify whether the identified changes in the ovarian immune system are specific to aging ovaries or reflect a common alteration of the aging immune system in mice. Recently, several papers unbiasedly identified immune alterations associated with aging in different tissues using single-cell RNA sequencing and flow cytometry techniques (e.g., Almanzar et al., Nature 2019; Kimmel et al., Genome Res 2019; Mogilenko et al., Immunity 2021). This study does not compare the findings with previous single-cell-based results from different tissues and does not clearly state if the immune aging in the ovaries is paralleled by similar alterations in immune cell subsets in other tissues in mice.
The authors show that the CD4- CD8- double-negative T cell subset is profoundly increased in abundance in aging ovaries. However, the population of double-negative T cells is not sufficiently characterized in the study. For example, it is unclear if similar cells can be found in aged tissues other than the ovaries. Moreover, using single-cell RNA sequencing, the authors show that the double-negative T cells co-express Trbc2 (TCRb) and Tcrgc2 (TCRg) genes, but the flow cytometry analysis of TCRg/d expression on these cells is not presented. The authors speculate that the double-negative T cells might have a regulatory function. However, a recent paper identified a population of pro-inflammatory T cells that co-express TCRab and TCRgd in mice and humans (including CD4- CD8- double-negative cells) (Edwards et al., J Ex Med 2020), suggesting that the double-negative T cells might be pro-inflammatory. It remains unclear if the double-negative T cell subset is unique to aging ovaries or phenotypically similar to the previously characterized double-negative TCRab+ and TCRgd+ cells.
The authors identified multiple transcriptional changes in genes encoding cytokines and chemokines, reflecting their decreased expression in aged ovarian immune cells. This observation is interesting because it contradicts the basic assumption of enhanced inflammation in old tissues. However, the presented findings are limited by the single-cell RNA sequencing level of evidence and are not supported or exemplified by an orthogonal analysis showing similar changes at the protein levels.
The authors claim that aging affects the recognition of senescent cells by ovarian immune cells. This exciting statement is based only on the single-cell RNA sequencing data in immune cells. The interaction between the immune cells and senescent cells in the ovaries involving the discussed pathways is not validated at protein levels in this study.
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