Investigation of the influence of sex on cantharidin‐induced inflammation in healthy volunteers
Authors: Rathod Krishnaraj Sinhji , Siddiqui Umme , Hartley Amy , Khambata Rayomand S , Ahluwalia Amrita 
Published: November 2023

Introduction

Pre‐menopausal females, compared to age‐matched males, have a lower incidence of cardiovascular disease; an effect thought to be mediated by sex hormones (Mass et al. 2010; Kalin et al. 1990). In pre‐clinical models it has been reported that oestrogen modulates vascular inflammation, in particular a recent study involving intravital microscopy of the mesenteric circulation of wild‐type mice observed that leucocyte recruitment in response to inflammatory cytokines was significantly reduced in female compared to age‐matched male mice (Villar et al.2006). We therefore investigated whether sex differences exist in leucocyte recruitment using a cantharidin‐induced blister model of acute inflammation in healthy male and female volunteers.

Methods

This study received ethics approval by the NRES Committee London ‐ City Road & Hampstead (11/LO/2038). 32 healthy, non‐smoking volunteers (male=16, female=16), aged 18–45, were included after gaining informed, written consent. Subjects attended for a total of 3 days. On Day 1, each subject received a blister by placing a 1 cm2 filter paper disc on the ventral aspect of the forearm and impregnating it with 10 μL of 0.1% cantharidin solution, diluted in acetone. The procedure was repeated on Day 3 to produce a second blister <5 cm in distance from the first one. On Day 4, fluid from both blisters were harvested to obtain samples at two time‐points of the inflammatory reaction – 24 hrs (acute phase) and 72 hrs (resolution phase). Total number of cells in each blister fluid sample was determined using the haemocytometer counting method.

The samples were incubated with antibody conjugated‐fluorophores and analysed by flow cytometry to identify and quantify specific leucocyte sub‐populations and level of expression of three key cell adhesion molecules, CD162, CD62L and CD11b, as a marker of their activation state. Data is shown as mean ± S.E.M.

Results

The total number of cells recruited into cantharidin‐induced blisters showed a trend for reduction in females. Whilst the volume of fluid within the blisters at 24 hrs was similar between the sexes, at 72 hrs blister volume was significantly reduced in females compared to males (61.8 μL ± 26.7 μL vs. 271.1 μL ± 57.3 μL, respectively; p=0.003). There was a sex difference in the proportions of leucocyte subset recruitment into blisters, with significantly reduced monocyte count (24 hrs: 0.72 × 105 ± 0.20 × 105 females vs. 1.62 × 105 ± 0.50 × 105 males and 72 hrs: 0.54 × 105 ± 0.28 × 105 females vs. 2.76 × 105 ± 0.62 × 105 males; p=0.0006) and CD8+ lymphocyte count (24 hrs: 0.027 × 105 ± 0.012 × 105 females vs. 0.080 × 105 ± 0.031 × 105 males and 72 hrs: 0.035 × 105 ± 0.021 × 105 females vs. 0.11 × 105 ± 0.027 × 105 males; p=0.011) in females compared to males. In addition, there was a generalised reduction in the activation state of all major leucocytes as demonstrated by reduced fluorescence intensity of activation markers in females compared to males (Figure 1A–C).

Conclusions

Our findings suggest that female sex suppresses inflammatory leucocyte recruitment in inflammation, with reduced numbers of both inflammatory monocytes and lymphocytes and a reduced activation state of all leucocyte subsets. We suggest that the differences in the profile of cell recruitment and activation state in females suggests that a more efficient resolution of inflammation occurs in females.

Support or Funding Information

KSR is supported by a NIHR Doctoral Research Fellowship