Sex differences in resident immune cell phenotype underlie more efficient acute inflammatory responses in female mice by Ramona S. Scotland, Melanie J. Stables, Shimona Madalli, Peter Watson, and Derek W. Gilroy Blood Volume 118(22):5918-5927 November 24, 2011 ©2011 by American Society of Hematology

Distinct resident leukocyte population in the female peritoneal cavity. Distinct resident leukocyte population in the female peritoneal cavity. (A) Increased total resident cell number in peritoneal (n = 13 mice; 3 independent groups) and pleural (n = 5 mice) cavities of female compared with male mice. (B) Total cell number and (C) percentage of F4/80+ macrophages, CD3+ T lymphocytes, CD19+ B lymphocytes, and GR1+ granulocytes in peritoneal cavity of male and female mice were determined by flow cytometry (n = 7 mice). (D) Increased total resident CD8+ and CD4+ T lymphocytes but not CD4+/CD25+ T-regulatory or δγ T lymphocytes in female peritoneal cavity (n = 4 mice). All values are expressed as means ± SEM. All comparisons are relative to male. *P < .05; **P < .01; and ***P < .001 by Student t test. Ramona S. Scotland et al. Blood 2011;118:5918-5927 ©2011 by American Society of Hematology

Increased homeostatic leukocyte recruitment into female peritoneal cavity. Increased homeostatic leukocyte recruitment into female peritoneal cavity. (A) Basal mRNA expression of chemokines CX3CL1, CCL2, CCL7, CXCL12, CXCL5, and CCL5 in mesenteric tissue (n = 6 mice). (B) Chemokine receptor mRNA expression in resident peritoneal cells (n = 4-6 mice). Levels of mRNA for each sample are normalized to corresponding mRNA levels of housekeeping gene for small 18S and calculated as the -fold expression relative to the mean value in males, except CX3CR1 (relative to female). All values are expressed as means ± SEM. *P < .05 and **P < .01 by Student t test. ND denotes chemokine expression that was not detected within 35 PCR cycles. Ramona S. Scotland et al. Blood 2011;118:5918-5927 ©2011 by American Society of Hematology

Elevated pathogen-sensing and phagocytosis by female macrophages. Elevated pathogen-sensing and phagocytosis by female macrophages. (A) Basal mRNA expression of TLRs and Myd88 in naive peritoneal cells (n = 5-6 mice) and (B) Flow cytometric analysis of surface TLR2 and TLR4 protein expression on resident F4/80+ peritoneal macrophages (n = 6-8; 2 independent experiments). (C) Phagocytosis of zymosan A (5 × 106 particles/105 cells, 30 minutes) by equivalent numbers of resident peritoneal leukocytes (macrophage and lymphocytes), measured in vitro by a colorimetric assay (n = 5 mice). Basal levels of TLR mRNA in (D) mesenteric tissue and (E) aortae of male and female mice (n = 6 mice). Levels of mRNA for each sample are normalized to corresponding mRNA levels of housekeeping gene for small 18S and calculated as the -fold expression relative to the mean value in females. All results are shown as means ± SEM. *P < .05; **P < .01; and ***P < .001 compared with male by Student t test. Ramona S. Scotland et al. Blood 2011;118:5918-5927 ©2011 by American Society of Hematology

Reduced severity and neutrophil recruitment in peritonitis in females. Reduced severity and neutrophil recruitment in peritonitis in females. (A-C and F) Male and female mice were treated with GBS (30 × 106 bacteria per mouse IP; n = 7 mice) for 3 hours. (A) Total cell number recovered from the peritoneal cavity, (B) sepsis severity score, and (C) whole-blood bacterial count. (D) Phorbol myristate acetate (1 pg/mL)–induced NADPH oxidase activity in male and female resident peritoneal macrophages (105 cells/sample), measured in vitro by Amplex Red for 7 minutes (n = 3 mice). (E) GBS levels after incubation in vitro (104 bacteria/sample) for 1 hour at 37°C with normal mouse plasma (n = 3 samples from 6 mice in each group). (F) Concentration of GBS-induced cytokines in cell-free peritoneal lavage (n = 7 mice). (G-H) Male and female mice were injected with zymosan A (1 mg IP). (G) Total peritoneal cell number (n = 5-10; 2 independent experiments) and (H) number of F4/80+ macrophages, CD3+ T lymphocytes, CD19+ B lymphocytes, and GR1+ granulocytes in peritoneal cavity of male and female mice 3 hours after injection of zymosan A (n = 6 mice). All values (A-H) are expressed as means ± SEM. All comparisons are relative to male. *P < .05; **P < .01; and ***P < .001 by Student t test; §P < .05 by 2-way ANOVA followed by Bonferroni posttest; #P < .001. Ramona S. Scotland et al. Blood 2011;118:5918-5927 ©2011 by American Society of Hematology

T lymphocytes control the severity of innate inflammatory responses. T lymphocytes control the severity of innate inflammatory responses. (A) Cytokine production in vitro by male and female resident peritoneal cells (2 × 105 cells/sample, n = 6 mice) after 3 hours of stimulation by TLR4-specific LPS (0.1 μg/mL) or the TLR2 agonist Pam3CSK4 (Pam3, 0.1μg/mL). (B) TNFα production by isolated resident male peritoneal macrophages (1.5 × 105 cells/sample, n = 3 mice) treated with LPS (0.1μg/mL, 18 hours) in the absence or presence of CD4+ T lymphocytes (1.5 × 105 cells). (C) Zymosan-induced (1 mg IP for 3 hours) recruitment of GR1+ granulocytes into the peritoneal cavity of C57BL/6 (wild-type) and T-lymphocyte–deficient Rag2 knockout (KO) mice (n = 5 mice). All values are expressed as means ± SEM. #P < .05 by 1-way ANOVA compared with male; §P < .05 by 1-way ANOVA relative to macrophages alone; and *P < .05 by Student t test relative to wild-type. Ramona S. Scotland et al. Blood 2011;118:5918-5927 ©2011 by American Society of Hematology

Ovarian sex hormones contribute to sex differences in resident immune cell population. Ovarian sex hormones contribute to sex differences in resident immune cell population. OVX or sham operation was performed on female mice at 4 weeks of age and they were allowed to recover for 4-5 weeks. (A) Total number of resident F4/80+ macrophages, CD3+ or CD8+ T lymphocytes, and CD19+ B lymphocytes in peritoneal cavity (n = 5 mice). Basal mRNA expression of (B) mesenteric tissue chemokines (n = 6-7 mice), (C) chemokine receptors on resident peritoneal leukocytes (n = 6 mice), and (D) peritoneal leukocyte TLR expression (n = 6 mice). (E) Surface expression of TLR2 or TLR4 on F4/80+ resident peritoneal macrophages (n = 3-5 mice). (F) Phagocytosis of zymosan A (5 × 106 particles/105 cells, 30 minutes, n = 4 mice) by resident peritoneal cells in vitro. (G) GBS-induced (30 × 106 bacteria/mouse IP for 3 hours, n = 7 mice) accumulation of leukocytes in peritoneal cavity. All values (A-G) are expressed as means ± SEM. Expression of mRNA for each sample is normalized to corresponding levels of the housekeeping gene for small 18S and is calculated as the -fold expression relative to OVX females. All comparisons are relative to Sham females. *P < .05; **P < .01; and ***P < .001 by Student t test; NS denotes P > .05. Ramona S. Scotland et al. Blood 2011;118:5918-5927 ©2011 by American Society of Hematology