Aging modulates homeostatic leukocyte trafficking to the peritoneal cavity in a sex-specific manner

Abstract

Aging is associated with exacerbated systemic inflammation (inflammaging) and the progressive loss of immune system function (immunosenescence). Leukocyte migration is necessary for effective immunity; however, dysregulated trafficking of leukocytes into tissue contributes to inflammaging and the development of age-related inflammatory diseases. Aging modulates leukocyte trafficking under inflammatory conditions; however, whether aging modulates leukocyte trafficking under homeostatic conditions remains to be elucidated. Although immune responses are evidently sexually dimorphic, limited studies have investigated the effect of sex on age-related changes to leukocyte trafficking processes. Here, we investigated age-related and sex-specific changes to the leukocyte populations within the peritoneal cavity of young (3-mo), middle-aged (18-mo) and old (21-mo) male and female wild-type mice in the steady state. We found an age-related increase in the number of leukocytes within the peritoneal cavity of female mice, predominantly B cells, which may reflect increased trafficking through this tissue with age. This was accompanied by an increased inflammatory environment within the aged cavity, including increased levels of chemoattractants, including B cell chemoattractants CXCL13 and CCL21, soluble adhesion molecules, and proinflammatory cytokines, which was more pronounced in aged female mice. Intravital microscopy techniques revealed altered vascular structure and increased vascular permeability within the peritoneal membrane of aged female mice, which may support increased leukocyte trafficking to the cavity with age. Together, these data indicate that aging affects homeostatic leukocyte trafficking processes in a sex-specific fashion.

Keywords: T cells; aging; inflammation; leukocyte trafficking; sexual dimorphism.

Fig. 1.

Baseline trafficking of leukocytes into the resting peritoneum is dysregulated by aging in a sex-specific manner. Total number of peritoneal (A) CD45⁺ leukocytes, (B) Ly6G⁺ neutrophils, (C) CD11c⁺ DCs, (D) CD3⁺ T cells, (E) CD3 ⁺ KLRG1⁺ senescent T cells, (F) CD19⁺ B cells, (G) CD19 ⁺ CD93⁻CD43⁻CD21/35⁻CD23⁻ ABCs, and (H) F4/80^hi macrophages from resting 3-, 18-, and 21-mo-old female (♀) and male (♂) C57Bl6 mice were quantified using flow cytometry. Analysis of variance showed a significant effect of age on the number of each leukocyte subset (P < 0.01) and of sex on the number of leukocytes (P < 0.001), macrophages (P < 0.01), and B cells (P < 0.05) cells. Data are presented as mean ± SEM using n = 5, n = 8, and n = 6 for 3-, 18-, and 21-mo-old mice, respectively, from n = 2 independent experiments per age group for each sex. Color-coded data points indicate independent experiments per age group. *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni multiple comparison posttest.

Fig. 2.

Leukocyte trafficking through blood and lymphoid organs are altered with age, displaying sexual dimorphism. (A, C, D, F) CD45⁺ leukocytes and (B, E) CD19 B cells within the (A, B) blood, (C) bone marrow, (D, E) spleen, and (F) iLNs of naïve 3-, 18- and 21-mo-old female (♀) and male (♂) C57Bl6 mice were quantified using flow cytometry. Analysis of variance showed a significant effect of age on the number of leukocytes in the blood, spleen, and iLNs (P < 0.05), and of sex on leukocyte numbers in the blood (P < 0.001). Analysis of variance analysis also showed a significant effect of age and sex on the number of B cells in the blood and spleen (P < 0.01). Data are presented as mean ± SEM using n = 5, n = 8, and n = 6 for 3-, 18-, and 21-mo-old mice, respectively, from n = 2 independent experiments per age group for each sex. Color-coded data points indicate independent experiments per age group. *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni multiple comparison posttest.

Fig. 3.

Aging skews the peritoneal secretome toward a proinflammatory microenvironment. An abundance of proinflammatory mediators in the peritoneal fluid of naïve 3- and 21-mo-old female (♀) and male (♂) C57Bl6 mice were analyzed using a cytokine array. Fluids from 3 mice per age group for each sex were pooled, in which n = 1. Heatmaps depict the fold change in (A) chemokines/chemoattractants, growth/survival factors, and soluble adhesion molecules, with (D) cytokines of aged mice relative to young mice for each sex. Analytes with <3-fold change are represented by white squares. Quantification of (B) CCL21 and (C) CXCL13 in the peritoneal lavage fluid of 3-, 18-, and 21-mo-old female and male wild-type mice by ELISA. Analysis of variance showed a significant effect of age (P < 0.001) and sex (P < 0.001) on the concentration of CCL21 in the peritoneal fluid and of age (P < 0.001), but not sex, on the concentration of CXCL13. Data are presented as mean ± SEM using n = 4 to 5 per age group, from n = 1 independent experiment. *P < 0.05, **P < 0.01, and ***P < 0.001 by Bonferroni multiple comparison posttest.

Fig. 4.

Aging was associated with increased permeability and modulated structure of the peritoneal membrane vasculature. (A) Relative abundance of angiogenic factors in the peritoneal fluid of naïve 3- and 21-mo-old female (♀) and male (♂) C57Bl6 mice were analyzed using a cytokine array. Fluids from 3 mice per age group were pooled, in which n = 1. Heatmaps depict the fold change in cytokines and angiogenic factors of aged mice relative to young mice for each sex group. The vasculature of the peritoneal membrane from (B, D) young (3 mo) and (C, E) aged (21 mo) (B, C) female and (D, E) male mice were imaged using intravital microscopy. Images were quantified to assess (F) the leakage of fluorescent dextran into the extravascular space, expressed as mean number of gray pixels; (I) average number of vessels; (J) total length of vessels; (K) number of nodes; (L) number of meshes; and (M) total mesh area all expressed per mm². Quantification of the concentration of (G) 20 kDa and (H) 70 kDa fluorescent dextran within the peritoneal lavage fluid postimaging. Data are mean ± SEM for n = 5 independent experiments, using 5 mice per age group. *P < 0.05, **P < 0.01, ***P < 0.001 by Bonferroni multiple comparison test. Scale bar = 10 μm.