Early exposure of infants to GI nematodes induces Th2 dominant immune responses which are unaffected by periodic anthelminthic treatment

Abstract

We have previously shown a reduction in anaemia and wasting malnutrition in infants <3 years old in Pemba Island, Zanzibar, following repeated anthelminthic treatment for the endemic gastrointestinal (GI) nematodes Ascaris lumbricoides, hookworm and Trichuris trichiura. In view of the low intensity of worm infections in this age group, this was unexpected, and it was proposed that immune responses to the worms rather than their direct effects may play a significant role in morbidity in infants and that anthelminthic treatment may alleviate such effects. Therefore, the primary aims of this study were to characterise the immune response to initial/early GI nematode infections in infants and the effects of anthelminthic treatment on such immune responses. The frequency and levels of Th1/Th2 cytokines (IL-5, IL-13, IFN-gamma and IL-10) induced by the worms were evaluated in 666 infants aged 6-24 months using the Whole Blood Assay. Ascaris and hookworm antigens induced predominantly Th2 cytokine responses, and levels of IL-5 and IL-13 were significantly correlated. The frequencies and levels of responses were higher for both Ascaris positive and hookworm positive infants compared with worm negative individuals, but very few infants made Trichuris-specific cytokine responses. Infants treated every 3 months with mebendazole showed a significantly lower prevalence of infection compared with placebo-treated controls at one year following baseline. At follow-up, cytokine responses to Ascaris and hookworm antigens, which remained Th2 biased, were increased compared with baseline but were not significantly affected by treatment. However, blood eosinophil levels, which were elevated in worm-infected children, were significantly lower in treated children. Thus the effect of deworming in this age group on anaemia and wasting malnutrition, which were replicated in this study, could not be explained by modification of cytokine responses but may be related to eosinophil function.

Figure 1. Frequency of IL-5, IL-13, IFN-γ & IL-10 responses in infants by infection status.

Percentage of IL-5, IL-13, IFN-γ and IL-10 responders (cytokine responses >31.24 pg/ml) to:- A) Ascaris antigen in Ascaris positive infants (Asc+, n = 198); Ascaris negative/other worm positive infants (OW+, n = 273); and worm negative infants (W−, n = 195) or B) hookworm antigen in hookworm positive infants (HW+, n = 79); hookworm negative/other worm positive infants (OW+, n = 312); and worm negative infants (W−, n = 164). * P = 0.01–0.05, ** P = 0.001–0.01, *** P<0.001.

Figure 2. Mean level of IL-5, IL-13, IFN-γ and IL-10 responses in infants by infection status.

Mean levels of cytokine responses (+SEM) amongst responders (responses >31.24 pg/ml) to:- A) Ascaris antigen in Ascaris positive (Asc+) infants ( n = 94, 91, 40 and 24, respectively, for IL-5, IL-13, IFN-γ, IL-10); Ascaris negative/other worm positive (OW+) infants (n = 97, 101, 52 and 29, respectively, for IL-5, IL-13, IFN-γ, IL-10); and worm negative (W−) infants (n = 30, 33, 34 and 16, respectively, for IL-5, IL-13, IFN-γ, IL-10) or B) hookworm antigen in hookworm positive (HW+) infants (n = 43, 43, 5 and 6, respectively, for IL-5, IL-13, IFN-γ, IL-10); hookworm negative/other worm positive (OW+) infants (n = 122, 113, 15 and 10, respectively, forIL-5, IL-13, IFN-γ, IL-10) and worm negative (W−) infants n = 29, 28, 6 and 0 respectively for IL-5, IL-13, IFN-γ, IL-10) * P = 0.01–0.05.

Figure 3. Distribution of IL-5, IL-13 responses to Ascaris and hookworm antigens with age by infection status.

The lines describe the estimated linear relationship between cytokine responses and age. A) responses to Ascaris antigen by (i) all Ascaris positive infants, n = 198; Aii) all Ascaris negative/other worm positive infants, n = 273; Aiii) worm negative infants, n = 195 and in Bi) all hookworm positive infants, n = 79; Bii) all hookworm negative/other worm positive infants, n = 312; Biii) worm negative infants, n = 164.

Figure 4. Effects of tri-monthly mebendazole or placebo treatment in infants on parasitology.

A. Prevalence of infection after 4 rounds of tri-monthly mebendazole (n = 296) or placebo (n = 307) treatment on any worm positive infants, Ascaris lumbricoides positive infants (Asc), Trichuris trichuria positive infants (Tric), hookworm positive infants (HW). B. Egg reduction rate after 4 rounds of tri-monthly mebendazole (n = 296) or placebo (n = 307) treatment on Ascaris lumbricoides positive infants (Asc), Trichuris trichuria positive infants (Tric), hookworm positive infants (HW). * P = 0.01–0.05, *** P<0.001.

Figure 5. Comparison of cytokine responses at baseline and follow-up in mebendazole or placebo treated infants.

Left hand graphs show the percentage of responders (responses >31.24 pg/ml), right hand graphs show the mean (+SEM) response of responders to A) Ascaris antigen, B) hookworm antigen C) Trichuris antigens for IL-5, IL-13, IFN-γ, and IL-10 in infants treated with placebo (PL) or mebendazole (MBZ) at baseline and follow-up. * P = 0.01–0.05, ** P = 0.001–0.01, *** P<0.001 significantly different from baseline.

Figure 6. Peripheral blood eosinophil counts at baseline and follow-up and treatment effects.

A) worm egg +ve or −ve infants at baseline; B) worm egg +ve or −ve at follow-up, C) MBZ- vs PL-treated children at follow-up. *** P<0.001.