Accelerated resolution of inflammation underlies sex differences in inflammatory responses in humans

Abstract

Background: Cardiovascular disease occurs at lower incidence in premenopausal females compared with age-matched males. This variation may be linked to sex differences in inflammation. We prospectively investigated whether inflammation and components of the inflammatory response are altered in females compared with males.

Methods: We performed 2 clinical studies in healthy volunteers. In 12 men and 12 women, we assessed systemic inflammatory markers and vascular function using brachial artery flow-mediated dilation (FMD). In a further 8 volunteers of each sex, we assessed FMD response to glyceryl trinitrate (GTN) at baseline and at 8 hours and 32 hours after typhoid vaccine. In a separate study in 16 men and 16 women, we measured inflammatory exudate mediators and cellular recruitment in cantharidin-induced skin blisters at 24 and 72 hours.

Results: Typhoid vaccine induced mild systemic inflammation at 8 hours, reflected by increased white cell count in both sexes. Although neutrophil numbers at baseline and 8 hours were greater in females, the neutrophils were less activated. Systemic inflammation caused a decrease in FMD in males, but an increase in females, at 8 hours. In contrast, GTN response was not altered in either sex after vaccine. At 24 hours, cantharidin formed blisters of similar volume in both sexes; however, at 72 hours, blisters had only resolved in females. Monocyte and leukocyte counts were reduced, and the activation state of all major leukocytes was lower, in blisters of females. This was associated with enhanced levels of the resolving lipids, particularly D-resolvin.

Conclusions: Our findings suggest that female sex protects against systemic inflammation-induced endothelial dysfunction. This effect is likely due to accelerated resolution of inflammation compared with males, specifically via neutrophils, mediated by an elevation of the D-resolvin pathway.

Trial registration: ClinicalTrials.gov NCT01582321 and NRES: City Road and Hampstead Ethics Committee: 11/LO/2038.

Funding: The authors were funded by multiple sources, including the National Institute for Health Research, the British Heart Foundation, and the European Research Council.

Conflict of interest statement

A. Ahluwalia is a director of Heartbeet Ltd.

Figures

Figure 1. Flow charts of clinical studies.

(A) Study 1. (B) Study 2. PWV, pulse wave velocity; PWA, pulse wave analysis; FMD, flow-mediated dilatation.

Figure 2. Low-grade systemic inflammation induced by typhoid vaccination enhances neutrophil activation state in healthy male but not female volunteers.

Changes in expression of (A) CD11b, (B) CD162, and (C) CD62L on neutrophils in male and female healthy volunteers at baseline, 8 hours, and 32 hours following typhoid vaccine administration. Changes in expression of (D) CD11b, (E) CD162, and (F) CD62L from baseline in male and female healthy volunteers at 8 hours and 32 hours following typhoid vaccine administration. Data expressed as mean ± SEM of n = 12 male and female volunteers for all the panels. Statistical significance determined using 2-way ANOVA with Šídák’s post hoc analysis; *P < 0.05 for all panels.

Figure 3. Low-grade systemic inflammation induced by typhoid vaccination does not cause endothelial dysfunction in healthy female volunteers.

The effect of typhoid vaccination on (A) FMD measured at baseline and 8 and 32 hours following typhoid vaccination. (B)The effect of sex on the change in FMD at 8 hours following typhoid vaccination from baseline All data are expressed as mean ± SEM of n = 12 for each sex. Statistical significance determined using (A) 2-way ANOVA with Šídák’s post hoc analysis, **P < 0.01; and (B) Student’s 2-tailed unpaired test. Maximal dilatation of the brachial artery in response glyceryl trinitrate in (C) male and (D) female healthy volunteers measured at baseline and 8 and 32 hours following typhoid vaccination. Data expressed as mean ± SEM of n = 8. Statistical significance determined using 1-way ANOVA with Šídák’s post-hoc analysis.

Figure 4. Reduced inflammatory response to cantharidin in female compared with male healthy volunteers.

The total (A) volume of fluid, (B) cell count, (C) neutrophil count, (D) inflammatory monocyte count, and (E) CD4+ and (F) CD8+ T cell count at 24 hours and 72 hours following application of cantharidin to the volar aspect of the forearm. Data is shown as mean ± SEM of n = 16 of each sex for all the panels. Statistical analysis was determined using 2-way ANOVA followed by Šídák’s post tests; #P < 0.05, ##P < 0.01 for all panels.

Figure 5. Reduced inflammatory cell activation state in cantharidin-induced blister exudates in female compared with male healthy volunteers.

Mean fluorescence intensity (MFI) of the expression molecules CD162, CD62L, and CD11b on (A) neutrophils, (B) inflammatory monocytes, and (C) CD4+ and CD8+ T cells in healthy male (n = 16) and female (n = 16) volunteers. Data are shown as mean ± SEM. Statistical significances determined using 2-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; followed by Šídák’s post tests, #P < 0.05, ##P < 0.01, and ####P < 0.0001 comparing the sexes at each time point for all the panels.

Figure 6. Female blister exudates display a pro-resolving mediator profile.

Lipid mediators from exudates were extracted using C18 solid-phase extraction and profiled using LC-MS/MS–based lipid mediator profiling. (A) Representative multiple reaction monitoring chromatograms for identified lipid mediators and pathway markers from the 3 major bioactive metabolomes (left panel) and MS/MS spectra employed for their identification (right panel). (B) Partial least squares discriminant analysis of exudate lipid mediator profiles: left panel, 2D score plot; right panel, corresponding 2D loading plot. (C–E) Cumulative levels for the lipid mediator from the (C) docosahexaenoic acid, (D) eicosapentaenoic acid, and (E) arachidonic acid SPMs, arachidonic acid–derived LTB4, and ratio of SPM to LTB4. Results shown are mean ± SEM of n = 11 females and n = 13 males for B–E. Statistical significance determined using Students 2-tailed unpaired t test; *P < 0.05 for C–E.