Estrogen and progesterone decrease let-7f microRNA expression and increase IL-23/IL-23 receptor signaling and IL-17A production in patients with severe asthma

Abstract

Background: Women have an increased prevalence of severe asthma compared with men. IL-17A is associated with severe asthma and requires IL-23 receptor (IL-23R) signaling, which is negatively regulated by let-7f microRNA.

Objective: We sought to Determine the mechanism by which 17β-estradiol (E2) and progesterone (P4) increase IL-17A production.

Methods: IL-17A production was determined by using flow cytometry in TH17 cells from women (n = 14) and men (n = 15) with severe asthma. Cytokine levels were measured by using ELISA, and IL-23R and let-7f expression was measured by using quantitative PCR in TH17-differentiated cells from healthy women (n = 13) and men (n = 14). In sham-operated or ovariectomized female mice, 17β-E2, P4, 17β-E2+P4, or vehicle pellets were administered for 3 weeks before ex vivo TH17 cell differentiation. Airway neutrophil infiltration and CXCL1 (KC) expression were also determined in ovalbumin (OVA)-challenged wild-type female recipient mice with an adoptive transfer of OVA-specific TH17 cells from female and male mice.

Results: In patients with severe asthma and healthy control subjects, IL-17A production was increased in TH17 cells from women compared with men. IL-23R expression was increased and let-7f expression was decreased in TH17-differentiated cells from women compared with men. In ovariectomized mice IL-17A and IL-23R expression was increased and Let-7f expression was decreased in TH17 cells from mice administered 17β-E2+P4 compared with those administered vehicle. Furthermore, transfer of female OVA-specific TH17 cells increased acute neutrophil infiltration in the lungs of OVA-challenged recipient mice compared with transfer of male OVA-specific TH17 cells.

Conclusions: 17β-E2+P4 increased IL-17A production from TH17 cells, providing a potential mechanism for the increased prevalence of severe asthma in women compared with men.

Keywords: Estrogen; IL-17A; IL-23 signaling; Let-7f; progesterone; severe asthma.

Figure 1

IL-17A+ memory Th17 cells are increased in women compared to men with severe asthma. (A–C) Percent and total number of IL-17A producing CCR6+ memory Th17 cells in healthy controls and severe asthma patients. * p<0.05, Mann-Whitney U test, n=9 healthy individuals and n=29 severe asthma patients (combined women and men). (D–E) Percent and total number of IL-17A+ or IL-4/IL-17A+ memory Th17 cells in women and men with severe asthma. * p<0.05, Mann-Whitney U test, n=14 (women) and 15 (men).

Figure 2

IL-17A protein expression is increased in Th0 and Th17 cells from women compared to men. (A–B) Naive CD4+ T cells were differentiated into Th0 and Th17 cells. IL-17A and IL-17F protein expression was determined in Th0 and Th17 from healthy women (n=13) or men (n=14). * p<0.05, Kruskal-Wallis test with Dunn's post-test analysis. (C) Total number of CD3+ CD4+ Th17 cells. (D–F) Percent, total number, and/or gMFI for CD3 gated, CD4+, IL-17A+ Th17 cells. * p<0.05, Kruskal-Wallis test, n=10 (women) n=12 (men).

Figure 3

IL-23R surface expression is increased and Let-7f is decreased in Th17 cells from women compared to men. (A–B) Dot plot and total IL-23R+ CD3+ CD4+ T cells from women and men. * p<0.05, Kruskal-Wallis test with Dunn's post-test analysis, n=8 (women) n=7 (men). (C) Let-7f miRNA expression normalized to U6B expression in Th0 and Th17 cells from women and men. * p<0.05, Kruskal-Wallis test with Dunn's post-test analysis, n=12 (women and men).

Figure 4

Let-7f decreased IL-23R surface expression and IL-17A levels from Th17 cells. A 10nM Let-7f inhibitor or negative (neg.) control was transfected into naïve T cells that were then differentiated to Th17 cells. (A–B) Dot plots and the total number of IL-17A+ Th17 cells. (C) IL-23R mRNA expression normalized to GAPDH. * p<0.05, Wilcoxon matched pairs test, n=9 (women) and n=9 (men), hashes are means.

Figure 5

IL-17A protein expression is increased in Th0 and Th17 cells from female mice compared to male mice. (A–C) IL-17A and IL-17F protein expression or IL-23R mRNA expression normalized to GAPDH in Th0 and Th17 cells from female and male adult or prepubescent mice. (D). IL-17A protein expression from adult Th17 cells differentiated with rmIL-23 (0-30ng/ml). * p<0.05, ANOVA with Tukey post-test analysis, n=6, 2 experiments.

Figure 6

In vivo administration 17β-E2 and P4 increased IL-17A protein expression and IL-23R mRNA expression from in vitro Th17 cells. Hormones or vehicle pellets (veh.) were implanted into mice for 21 days followed by naïve T cell isolation and Th17 cell differentiation. (A) IL-17A protein expression. (B) IL-23R mRNA expression normalized to GAPDH. (C) Let-7f miRNA expression normalized to U6B expression. * p<0.05, Kruskal-Wallis test with Dunn's post-test analysis; n=Th17 cells from 5–10 mice for each group, 2 experiments.

Figure 7

Adoptive transfer of female D011.10 Th17 cells increases neutrophilic infiltration in the airways of WT female recipient mice. (A) Schematic of experimental protocol. (B–D) Total number of OVA-specific CD3+ CD4+ T cells (panel B) or the percent and total number of IL-17A+ OVA-specific CD3+ CD4+ Th17 cells (panels C-D) in the lungs of recipient mice on day 4. (E) KC protein expression in whole lung homogenates. (F) Total neutrophils in BAL fluid. * p<0.05, ANOVA with Tukey post-test analysis; n=10–12 recipient mice per group administered D011.10 Th17 cells from female or male mice combined from 2 experiments.

Fig. E1

IL-4+ memory T cells are similar in women and men with severe asthma. (A–C). Percent and total number of IL-17A/IL-4+ memory Th17 cells in women and men with severe asthma. (D–E). Percent and totals for IL-4+ CD3+ CD4+ memory T cells in women and men with severe asthma. * p<0.05, Mann-Whitney U test, n=14 (women) and 15 (men). (F). Spearman correlation of total IgE plasma levels and IL-17A producing CCR6+ memory Th17 cells.

Fig. E2

Cytokine expression in human Th17 differentiated cells from women and men. (A–C) Naïve T cells isolated from healthy women (circles) and men (squares) were differentiated into Th17 cells. Cell culture supernatants were harvested and analyzed for protein expression. * p<0.05, Mann Whitney U test, n=13 Th17 cells from women and n=14 Th17 cells from men.

Fig. E3

IL-17A protein expression in Th17 cells from women does not correlate with 17β-E2 and P4 plasma levels. (A–B) Spearman nonparametric correlations of 17β-E2 or P4 plasma concentration and IL-17A protein expression from Th17 cell culture supernatants, r and p values are listed on the graphs. (C) Naïve T cells isolated from healthy women and men were differentiated into Th17 cells in the presence of 17β-E2 or vehicle (ethanol). Four days after differentiation, IL-17A protein expression was determined from cell culture supernatants. Data is representative from n=8 Th17 cells from women and n=5 Th17 cells from men. * p<0.05, ANOVA with Tukey post-test analysis.

Fig. E4

IL-23R surface expression is increased in memory Th17 cells from women with severe asthma compared to men with severe asthma. IL-23R surface expression was determined on CD3+ CD4+ CCR6+ memory Th17 cells from women and men with severe asthma. * p<0.05, Mann-Whitney U test, n=8 (women) and 7 (men).

Fig. E5

Let-7f miRNA expression is decreased in Th17 cells transfected with Let-7f inhibitor. Let-7f inhibitor (10nM) or negative control (10nM) were transfected into naïve T cells isolated from healthy women and men. Cells were differentiated to Th17 cells and Let-7f miRNA expression was determined by qPCR and normalized to U6B expression. * p<0.05, Wilcoxon matched paired test, n=9 Th17 cells from women and n=9 Th17 cells from men.

Fig. E6

IL-13 and IFN-γ protein expression in Th17 cells. Hormones or vehicle pellets (veh.) were implanted into mice for 21 days. Naïve T cells were isolated from the spleens of mice and Th17 cells were differentiated. As positive controls, Th1 and Th2 cells were differentiated from sham-operated female mice administered vehicle pellets. IFN-γ and IL-13 protein expression was determined by ELISA. * p<0.05 compared to Th17 cells from sham-operated female mice administered vehicle pellets, Kruskal-Wallis test with Dunn's post-test analysis; n=5–10 mice per group combined from 2 experiments.

Fig. E7

Role of 17β-E2 and P4 in IL-17A protein expression from Th17 cells. Schematic proposing how increased 17β-E2 and P4 concentrations in women impact IL-17A protein expression from Th17 cells.