Biological sex, not reproductive cycle, influences peripheral blood immune cell prevalence in mice

Abstract

Key points Traditionally the female sex, compared with the male sex, has been perceived as having greater variability in many physiological traits, including within the immune system. We investigated effects of biological sex and the female reproductive cycle on numbers of circulating leukocytes in C57BL/6J mice. We show that biological sex, but not female reproductive cyclicity, has a significant effect on peripheral blood immune cell prevalence and variability, and that sex differences were not consistent amongst common inbred laboratory mouse strains. We found that male C57BL/6J mice, compared with female mice, have greater variability in peripheral blood immunophenotype, and that this was influenced by body weight. We created summary tables for researchers to facilitate experiment planning and sample size calculations for peripheral immune cells that consider the effects of biological sex. Abstract Immunophenotyping (i.e. quantifying the number and types of circulating leukocytes) is used to characterize immune changes during health and disease, and in response to pharmacological and other interventions. Despite the importance of biological sex in immune function, there is considerable uncertainty amongst researchers as to the extent to which biological sex or the female reproductive cycle influence blood immunophenotype. We quantified circulating leukocytes by multicolour flow cytometry in young C57BL/6J mice and assessed the effects of the reproductive cycle, biological sex, and other experimental and biological factors on data variability. We found that there are no significant effects of the female reproductive cycle on the prevalence of peripheral blood B cells, NK cells, CD4+ T cells, CD8+ T cells, monocytes, or neutrophils. Immunophenotype composition and variability do not significantly change between stages of the female reproductive cycle. There are, however, sex‐specific differences in immune cell prevalence, with fewer monocytes, neutrophils, and NK cells in female mice. Surprisingly, immunophenotype is more variable in male mice, and weight is a significant contributing factor. We provide tools for researchers to perform a priori sample size calculations for two‐group and factorial analyses. We show that immunophenotype varies between inbred mouse strains, and that using equal sample sizes of male and female mice is not always appropriate for within‐sex evaluations of immune cell populations in peripheral blood.

The Effects of Biological Sex on Sepsis Treatments in Animal Models: A Systematic Review and a Narrative Elaboration on Sex- and Gender-Dependent Differences in Sepsis

“…This study highlights how sex-dependent differences in immune function may lead to differential outcomes in sepsis depending on various precipitating factors or other superimposed comorbidities. Interestingly, biological sex, but not female reproductive cyclicity, has been shown to influence the peripheral blood immune cell prevalence in mice ( 56 ). Specifically, it has been observed that female mice have fewer monocytes, neutrophils, and natural killer cells and that males exhibit greater variability in the peripheral blood immunophenotype ( 56 ).…”

“…Interestingly, biological sex, but not female reproductive cyclicity, has been shown to influence the peripheral blood immune cell prevalence in mice ( 56 ). Specifically, it has been observed that female mice have fewer monocytes, neutrophils, and natural killer cells and that males exhibit greater variability in the peripheral blood immunophenotype ( 56 ). The authors also demonstrated that the common practice of using equal numbers of male and female mice is often not appropriate and subsequently provided guidance to facilitate sample size calculations for peripheral immune cells (i.e., that consider the effects of sex) ( 56 ).…”

Effects of Obesity-Associated Chronic Inflammation on Peripheral Blood Immunophenotype Are Not Mediated by TNF in Female C57BL/6J Mice

“…Flow cytometry of bone marrow and peripheral blood leukocytes Blood was collected retro-orbitally in heparinized capillary tubes under isoflurane anesthesia. Peripheral blood leukocytes (monocytes, neutrophils, NK cells, CD4 1 T cells, CD8 1 T cells, and B cells) were analyzed by flow cytometry as previously described (41). Bone marrow leukocytes were flushed from femurs, disrupted with an 18-gauge needle and analyzed by flow cytometry as previously described (49).…”

“…Sexual dimorphism also influences regulation of hematopoietic stem cells and innate immune signaling pathways (8,38,39), which may contribute to findings that male mice with diet-induced obesity have enhanced bone marrow myelopoiesis, as well as enhanced macrophage accumulation within adipose tissue, compared with their female counterparts (6,8). Males generally have higher levels of circulating monocytes under conditions of homeostasis (40,41) and PBMC production of TNF in response to acute inflammation is generally lower in females compared with males (40,42). Together, these data emphasize the sex-specific differences that exist in immunometabolic regulation and highlight the fact that researchers often overlook sex differences when considering mechanisms that contribute to the development and progression of obesity.…”

“…Samples were run on a BD Biosciences LSR II or BD Biosciences Fortessa flow cytometer (BD Biosciences). Data were analyzed using the FlowJo v9 software (Tree Star) using previously published methods(41). Surface marker expression was quantified by measuring geometric mean fluorescence intensity of each fluorescence marker and subtracting geometric mean fluorescence intensity of isotype controls.…”