Characterising the mucosal and systemic immune responses to experimental human hookworm infection

Abstract

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases.

Figure 1. Trial design.

For Trial 1, 20 patients were chosen according to selection criteria and divided into two groups of 10, placebo control and hookworm infected (HW). All 20 patients completed the trial. For Trial 2, seven of the 10 patients from Group 1 in Trial 1 (placebo) chose to participate in the next trial and received hookworm infection and gluten challenge in an identical manner to that described for Group 2 in Trial 1. The only difference was that gut biopsies were taken at week 0 (prior to hookworm infection; denoted in bold font) in addition to biopsies taken at weeks 20 and 21. For the purposes of this study, sample collection ceased at week 20 (prior to gluten administration) and tissues from week 21 were not utilized, except for samples taken from the worm attachment site which were collected at week 21; week 21 tissues were critical for assessment of the effect of hookworm on the anti-gluten response in celiac disease and are therefore highlighted in italicised grey font and have been reported in relevant publications , . ¹Forty millilitres of blood per person were collected at weeks 0, 4, 12 (Trial 1 only), 20 and 21. ²Gut biopsies were taken at weeks 0 (Trial 2 only), 20 (pre-gluten ingestion) and 21 (post-gluten ingestion) using a gastroscope as described elsewhere .

Figure 2. Systemic production of hookworm-specific cytokines.

Peripheral blood mononuclear cells were harvested from subjects in Trial 1 and cultured for 120 h at 37°C in either tissue culture medium (MED) or MED containing 10 µg/ml Necator americanus ES products (NaES). Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) determined using a Cytometric Bead Array. Cytokine levels from PBMCs stimulated with just MED alone were subtracted from those stimulated with NaES. Data were analysed by Kruskal-Wallis non-parametric ANOVA, comparing time points at weeks 4, 12 and 20 to week 0 within each group.

Figure 3. Production of Th2 cytokines in the duodenal mucosa of hookworm infected individuals.

Duodenal biopsies from Trial 1, taken from either the duodenum at week 20 post-infection or from directly adjacent to an adult hookworm attachment site (HW site – determined by endoscopy) at week 21 in the hookworm group only, were cultured for 24 h in tissue culture medium at 37°C with 95% O₂/5% CO₂. Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) were determined using a Cytometric Bead Array. Data were analysed by Mann-Whitney U test.

Figure 4. Ex vivo cytokine gene expression in the duodenal mucosa of hookworm infected individuals.

Duodenal biopsies from Trial 2 were taken before (week 0) and 20 weeks after hookworm infection, and RNA was prepared ex vivo. Levels of IL-4 (A) IL-5 (B), IL-13 (C), IL-9 (D), GATA-3 (E), Foxp3 (F), TGF-β (G), ALDH1A2 (H), IFN-γ (I), IL-15 (J), IL-17A (K), IL-23 (L) and RORγt (M) transcripts were determined by quantitative real time RT-PCR.

Figure 5. Systemic and mucosal hookworm specific immune responses.

Peripheral blood mononuclear cells (PBMCs) from Trial 2 were cultured for 120 h with either tissue culture medium (Med) or Med containing 10 µg/ml Necator americanus ES products (NaES). Duodenal biopsies were also taken at week 20 post-infection, and cultured for 24 h at 37°C, 95% O2/5% CO₂, in either Med alone or 10 µg/ml NaES in Med. Cell-free supernatants were taken from both PBMCs and biopsy cultures and levels of soluble cytokines were determined by cytometric bead array. RNA was also prepared from biopsies after stimulation and quantitative real time RT-PCR was used to determine levels of cytokine gene transcripts. Soluble cytokines released from PBMCs are shown in panels A, D, G, J and M. Biopsy-derived soluble cytokines are shown in panels B, E, H, K and N. Biopsy cytokine transcripts are shown in panels C, F, I, L and O. Cytokine levels determined were IL-4 (A–C), IL-5 (D–F), IL-13 (G–I), IL-9 (J–L) and IL-10 (M–O).

Figure 6. Systemic and mucosal hookworm specific immune responses.

Peripheral blood mononuclear cells (PBMCs) from Trial 2 were cultured for 120 h with either tissue culture medium (Med) or Med containing 10 µg/ml Necator americanus ES products (NaES). Duodenal biopsies were also taken at week 20 post-infection, and cultured for 24 h at 37°C, 95% O2/5% CO2, in either Med alone or 10 µg/ml NaES in Med. Cell-free supernatants were taken from both PBMCs and biopsy cultures and levels of soluble cytokines determined by Cytometric Bead Array. RNA was also prepared from biopsies after stimulation and quantitative real time RT-PCR was used to determine levels of cytokine gene transcripts. Soluble cytokines released from PBMCs are shown in panels A, D and G. Biopsy-derived soluble cytokines are shown in panels B, E and H. Biopsy cytokine transcripts are shown in panels C, F, I, J and K. Cytokine levels determined were IL-2 (A–C), IFN-γ (D–F), IL-17A (G–I), IL-15 (J), TGF-β (K) and IL-22 (L).