TNF-alpha stimulated immune response: are there sex differences?
Authors: Sanchez Sophie , Didier Kaylin , Forsberg Matthew , Shahi Ankita , Wannebo Brett , Capitini Christian , Schrage William 
Published: May 2023

Background: The purpose of this study is to explore potential sex differences of varying immune responses. Immune cells, specifically macrophages, play a role in the development of cardiovascular diseases. Females exhibit greater innate (macrophages) and adaptive immune responses than males in multiple species. In addition, the overall inflammatory response is typically greater in females than males, especially during reproductive ages, yet females exhibit lower prevalence of CVD. Whether inflammatory responses of macrophages are sex-specific remains unclear. We hypothesized monocytes from females would demonstrate a greater expression of inflammatory cytokines at baseline, as well as after exposure to tumor necrosis factor alpha (TNF-α). Methods: 6 young healthy adult subjects were included in this study (28 ± 5 yr, 22.4 ± 0.7 BMI, 3 F) using an institutional review board (IRB)-approved protocol. Women were studied in days 1-5 of their menstrual cycle to minimize sex hormone differences. Peripheral blood samples were collected to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with anti-human CD14 microbeads for 15 minutes at 4°C. After washing the unbound antibody, cell separation was done using an autoMACS Pro Separator. CD14+ immune cells were incubated for 24 hours in culture media. CD14+ immune cells were exposed to 20ng/ml of TNF-α for 30 minutes. The culture media was recovered, centrifuged at 100 × g for 1 minute to remove any floating cell debris and assayed to measure the resulting expression of inflammatory cytokines (IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and INF-γ). Group comparisons were performed using a two-way repeated measures ANOVA with a p<0.05 significance level. Results: Data are presented as mean±SD. Males trended towards elevated baseline levels of INF-γ and IL-12p70 expression (p=0.09). There were no sex differences in the other measured cytokines expressed at baseline between sexes (p>0.13). There was no difference between sexes (Male vs Female, p-value) when stimulated with TNF-α in the expression (pg/mL for all) of IL-1β (7±10 vs. 15±6, p=0.72), IL-2 (5±7 vs. 52±88, p=0.39), IL-4 (0.5±0.8 vs. 0.5±0.6, p=0.87), IL-6 (11±8 vs. 28 ± 15, p=0.65), IL-8 (748±446 vs. 838±688, p=0.87), IL-10 (8±13 vs. 24±38, p=0.31), IL-12p70 (1.2±1.7 vs. 2.3±3.5, p=0.86), IL-13 (12±9 vs. 27±18, p=0.56), and INF-γ (3.3 ±4.2 vs. 18±29, p=0.53). Discussion: These data indicate macrophages from males and females demonstrate similar baseline cytokine levels (except a trend toward higher INF-γ and IL-12p70 expression in males) and a similar lack inflammatory cytokine response to TNF-α stimulation. Future work will explore longer and higher TNF-α exposures to determine a threshold response and further explore the relationship between sex and immunity. NIH R01 HL150361 and Summer Undergraduate Research Fellowship program supported by the American Physiological Society This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.