Origin and microenvironment contribute to the sexually dimorphic phenotype and function of peritoneal macrophages

Background:

Tissue resident macrophages are key controllers of tissue homeostasis, inflammation, and tissue repair. Recent studies in immunology have clarified the origins and different phenotypes of macrophages in various tissues, and begun to reveal the tissue features that help imprint those phenotypes. While some tissue features are understood, the role of sex in shaping features of tissue resident macrophages, and their turnover and homeostasis, remains severely understudied. In general, the importance of sex in studies of both model organisms and humans has been understudied, with the NIH only highlighting the need for more careful consideration of sex in mouse studies in the last few years (Clayton and Collins, 2014). With autoimmune diseases being among the most sexually dimorphic diseases in humans, clarifying the role of sex in immune biology is of critical importance. This preprint, from Bain et al, takes on issues of both sex difference and tissue resident macrophage biology, and illustrates how differences in peritoneal macrophage function emerge and are controlled by sex differences in mice.

 

Key Findings:

• Peritoneal macrophages in female mice are differentially replenished

The authors used bone marrow chimeras and CD11c fate-mapping mice to measure turnover and replenishment of peritoneal macrophages in male and female mice. Macrophages in male mice turned over more rapidly, as showed by increased donor-derived peritoneal macrophages in chimeric mice and increased labeling in the fate-mapping mice. This was not due to cell-intrinsic differences, as female bone marrow cells in male mice showed higher replenishment. Additionally, this was dependent on sexual maturity, as young female mice did not show lower turnover than young male mice, and ovariectomy increased turnover in female mice. These turnover differences were unique to peritoneal macrophages.

• Peritoneal macrophages from female mice have a unique transcriptional program

Bulk RNA-sequencing and single-cell RNA-sequencing were used to characterize peritoneal macrophages from both female and male mice. Consistent with the higher rate of turnover in male mice, peritoneal macrophages from male mice showed increased expression of cell cycle and proliferation genes. Peritoneal macrophages from female mice expressed higher levels of immune receptors CD209a/b and Tim4, the chemokine CXCL13, and apolipoproteins ApoE and ApoC1. Single-cell sequencing showed that these differences were mostly driven by differences in distribution between shared cell populations, rather than unique cell populations present only in male or female mice.

• Sex differences in peritoneal macrophage phenotypes are driven both by local environment and proliferation

The authors examined sexually dimorphic genes in peritoneal macrophages by flow cytometry, using CCR2 KO mice to prevent replenishment by circulating monocytes. Some macrophage populations changed similarly in both sexes of mice, such as a decrease in Tim4- cells in both male and female mice, suggesting that acquisition of Tim4 is related to time-of-residence in the peritoneum. This suggests that some of the differences in overall expression observed between sexes are due to the different turnover and longevity of peritoneal macrophages, and not programmed by unique features of the environment – consistent with differences in cluster frequency as seen in the single-cell RNA-sequencing, rather than unique populations present in one sex. Other sex differences were independent of replenishment, such as CXCL13 expression (which was not reduced in female CCR2 KO mice) and higher proliferation (Ki67 expression) in male mice, and are therefore likely to be programmed by the unique environments by sex, rather than being related to macrophage longevity.

 

Importance:

This study clearly shows the role of tissue imprinting in sex-specific differences of resident macrophages. This shows two axes of important study in tissue macrophage function, the role of sex and the time of residence in tissue. These data may suggest mechanisms that underlie sexually dimorphic disease susceptibility. In particular, sex differences in both resident cell turnover and renewal as well as tissue environments imprint different features of tissue-resident macrophages. Additionally, this work thoroughly characterizes peritoneal macrophage phenotype across time and sex, providing strong foundations for the future study of the role of peritoneal macrophages in various aspects of immunity, including    the production of “natural” antibodies, as discussed by the authors.

 

Moving Forward / Questions for Authors:

• The sham surgery induced some level of peritoneal macrophage turnover, possibly due to a response to tissue injury. It would be interesting to see alternative experimental setups for the ovariectomy experiment – perhaps surgery before the transfer, so the injury has time to heal, or chemical methods of estrogen depletion (such as leuprolide treatment). Each comes with its own caveats, but different experiments might narrow down the precise cause of the increased turnover seen with ovariectomy.
• It is interesting to note the lipoprotein components of the female peritoneal macrophage gene signature – ApoE, ApoC1, and Saa genes. Are these genes changed with ovariectomy? They did not change with high-fat diet, so it would be interesting to understand the role of these genes in tissue residence and adaptation when it is not due to adiposity.
• This study is a blueprint for future studies of resident macrophages in other tissues. The “female peritoneal” signature (18 genes) is substantially smaller than the overall “female” signature – how uniform these genes are across tissues will be interesting. It will also be critical to consider the differences between bone-marrow derived, but slowly replenishing, tissue macrophages and yolk-sac derived “true” tissue resident macrophages, and the role of extended tissue residency in shaping their similarities and differences. It will be exciting to see this work progress!

 

References:

Clayton JA and Collins FS, 2014, https://www.nature.com/news/policy-nih-to-balance-sex-in-cell-and-animal-studies-1.15195

 

doi: https://doi.org/10.1242/prelights.15590

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