The role of sex hormones in the development of Th2 immunity in a gender-biased model of Trichuris muris infection

Abstract

Trichuris muris infection is an ideal model for defining T-cell-driven immunity, and also provides essential insights that may impact on potential helminth therapies currently in development. Conflicting host variables determine the efficiency of such treatments and we have identified host-derived sex steroid hormones as key factors in the development of immunity. The female-associated hormone 17-beta estradiol (E2) significantly enhanced the generation of a Th2 response in vitro; however, this stimulatory effect was found to be dispensable for the generation of immunity to Trichuris in the gender-biased IL-4KO mouse model. In contrast, the male-associated hormone dihydrotestosterone significantly inhibited the T-cell stimulatory capacity of DC and directly suppressed the immune response of male IL-4KO mice, with worm expulsion restored following castration. This finding was associated with dramatically reduced IL-18 mRNA expression suggesting androgens may act via this cytokine to suppress Th2 immunity to Trichuris. This study has critical implications for the development and efficacy of potential helminth therapeutics and identifies host gender - specifically sex hormones - as important factors in the development of Th2 immunity in susceptible and immunocompromised mice.

Figure 1

Female mice, but not male litter mates show enhanced Th2-associated immunity in susceptible or slow responder strains. (A) AKR and IL-4KO BALB/c mice were infected with T. muris eggs and adult worm burdens assessed at day 14, 21 or 28 and 35 p.i. (B) MLNC from AKR mice were restimulated with T. muris E/S antigen after 40 days p.i. and cytokine production assessed via CBA. (C) Caecal histology of infected male and female AKR mice was compared at day 40 p.i. Data represent two independent experiments of n=10±SEM. ^*p≤0.05, ^**p≤0.01, ^***p≤0.001, Kruskal Wallis ANOVA. Average naïve levels indicated by red lines.

Figure 2

Treatment of BMDC with E2 during T. muris E/S pulsing enhances their Th2 priming capacity. (A) BMDC were pulsed with T. muris E/S(C, thick black line) and in combination with 10⁻¹⁰ M E2 (grey fill) or with E2 and 10⁻⁸ M ICI 182 780 (ICI, dotted black line). The percent and MFI of CD11c⁺ DC expressing CD80 and CD86 and MHC class II are indicated. Isotype controls are indicated by thin black lines. (B) DC were co-cultured with purified CD4⁺ T cells and following stimulation cytokine production in the co-culture supernatants was assayed via CBA. All treatments represent triplicate samples±SEM and the data are representative of three independent experiments. ^*p≤0.05, ^**p≤0.01, ^***p≤0.001, Mann–Whitney t-test.

Figure 3

OVX of female IL-4KO mice does not prevent delayed expulsion of T. muris. (A) Adult worm burdens in the caecum and proximal colon of intact (C) and OVX WT control and IL-4KO BALB/c mice were assessed at day 18 and 28 p.i. alongside. (B) Levels of pro-inflammatory cytokines from E/S restimulated MLNC. (C) T. muris-specific antibody isotype levels in the sera. (D) Caecal histology was assessed in control and OVX female IL-4KO mice. Data represent two independent experiments of n=5±SEM. ^*p≤0.05, ^**p≤0.01, ^***p≤0.001, Kruskal–Wallis ANOVA. Average naïve levels indicated by red lines.

Figure 4

DHT treatment of BMDC inhibits stimulation of T-cell cytokine production. (A) DC were treated with 10⁻⁷ M DHT during pulsing with T. muris E/S and (B) co-cultured with purified CD4⁺ T cells. Cytokine levels in coculture supernatants measured via CBA. All treatments represent triplicate samples±SEM and the data is representative of two independent experiments. ^*p≤0.05, ^**p≤0.01, ^***p≤0.001, Mann–Whitney t-test.

Figure 5

CSX of male IL-4KO mice restores T. muris expulsion. (A) Adult worm burdens of intact (C) and CSX male IL-4KO BALB/c mice infected with T. muris were assessed at day 18 and 28 p.i. (B) Th2 and pro-inflammatory cytokine production from E/S restimulated MLNC at day 28 p.i. (C) Total number and representative percentages of CD4⁺CD25⁺Foxp3⁺ Treg in the draining LN in CSX and control mice were measured via flow cytometry. (D) Relative expression of IL-18 mRNA in the MLN of control and CSX mice assayed during T. muris infection. Data represent two independent experiments of n=5±SEM. ^*p≤0.05, ^**p≤0.01, ^***p≤0.001, Kruskal–Wallis ANOVA. Average naïve levels indicated by grey line.