Sex-Differences In Monocyte Recruitment In A Murine Model Of Zymosan-Induced Peritonitis

E Kay, S Nourshargh, JR Whiteford, RS Scotland. William Harvey Research Institute, Queen Mary University of London, London, UK

Background: In response to inflammatory insult, monocytes and neutrophils are known early responders, migrating out of the circulation to the site of infection. Differences between male and female mice in the response of leukocytes to inflammatory stimuli have previously been reported1. This aim of this study was to determine any sex-differences in the monocyte response in a murine model of zymosan peritonitis, and furthermore to dissect the mechanisms behind this dimorphism.

Material & Methods: Male and female CX3CR1+/gfp and C57Bl/6 mice (8-12wks) were treated with zymosan (1mg, ip) to induce peritonitis. Naïve animals served as controls. Animals were killed by Schedule I and leukocytes were isolated from blood (cardiac puncture), femur bone marrow (BM), and peritoneal cavity (6ml lavage) at 1h, 3h, 24h, 48h or 72h. Lavage supernatants were collected, blood erythrocytes were lysed with ammonium-chloride-potassium (ACK) lysis buffer, and all leukocytes were resuspended in cold PBS with 1% goats serum. Leukocytes were counted with a hemocytometer and incubated (20mins, 4°C) with Fc-blocking antibody [anti-CD16/CD32, eBioscience] prior to addition of PE-anti TLR2 [clone T2.5; eBioscience] and TLR4 [clone MTS510; eBioscience], or respective isotype control antibody (1µg/ml/105cells). Classical monocytes were identified as CD115+Gr1+CX3CR1+, and non-classical monocytes as CD115+Gr1-CX3CR1+. Samples were run on LSRFortessa cytometer (BD Biosciences) with data analysed using FlowJo (v7.6). Data are expressed as mean cell number ±SEM with timecourses analysed by 1-way ANOVA followed by Bonferroni’s post-test, and sex-differences were analysed by student’s t-test (GraphPad Prism).

Results: No difference between male and female mice was observed in basal monocyte subset numbers from blood [classical: 16±2.7 x104/ml (male), 12±2.1 x104/ml (female); non-classical: 12±1.2 x104/ml (male), 14±3.3 x104/ml (female) (p>0.05, n=9-11)], peritoneal cavity [classical: 0.4±0.09 x104 (male) 1.4±0.52 x104 (female); non-classical: 11±0.1 x104 (male), 14±2.4 x104 (female) (p>0.05, n=3-6)] or BM [classical: 145±33.0 x104 (male), 133±6.5x104 (female); non-classical: 46±10.3 x104 (male), 58±12.0 x104 (female) (p>0.05, n=3-5)]. In response to zymosan, classical monocytes accumulated in the circulation by 3h, to a greater extent in male mice [male: 122±15.6 x104/ml; female 65±10.3 x104/ml] (p<0.01, n=15-18), and were subsequently recruited to the peritoneum. Accumulated classical monocytes corresponded to a reduction in BM cells at 3h, [males: 55±9.2x104; female: 54±3.9x104 (p<0.05, n=9)] indicating mobilisation, to the same extent in males and females. Monocyte mobilisation and blood accumulation does not appear earlier in females as no sex-difference was evident at 1h post-zymosan [BM: 130±17.7 (male), 105±22.03 (female); blood: 18±10.3 (male), 5±1.4 (female) (n=3)].

Conclusion: Sex-differences are evident in the monocyte response of mice to zymosan peritonitis. Analysis of naive and inflamed peritoneal cytokine environments revealed dimorphism between males and females and may provide explanation for differences in the responses of male and female mice to zymosan peritonitis. Sex-differences in acute inflammation may provide evidence for differences in leukocyte trafficking which may translate to chronic conditions more prevalent in males, such as atherosclerosis.

Acknowledgements: EK is funded by a British Heart Foundation Studentship

1Scotland RS, et al, Blood 118, 5918-27, 2011