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Helminth infection promotes colonization resistance via type 2 immunity

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Science  29 Apr 2016:
Vol. 352, Issue 6285, pp. 608-612
DOI: 10.1126/science.aaf3229
  • Fig. 1 Trichuris muris infection reverses intestinal abnormalities in Nod2−/− mice.

    (A and B) SI sections stained with PAS (periodic acid–Schiff)–Alcian blue (A) and quantification of the number of goblet cells displaying normal morphology per villi (B) from uninfected and T. muris–infected WT and Nod2−/− mice (n ≥ 7 per genotype). (C and D) Immunofluorescence (IF) analysis of Reg3β in the small intestine (C) and quantification of the mean fluorescence intensity (D) of the aforementioned mice (n ≥ 8 per genotype). (E) Quantification of the proportion of CD8+ IELs expressing IFN-γ by flow cytometry (n ≥ 11 per genotype). (F to H) Quantification of weight loss (F), SI sections stained with hematoxylin and eosin (G), and quantification of pathology (19) (H) after piroxicam treatment of uninfected and T. muris–infected WT and Nod2−/− mice. The asterisk in (G) denotes an abscess (n ≥ 7 per genotype). *P < 0.05; **P < 0.01; ****P < 0.0001 by analysis of variance (ANOVA), with Holm-Sidak multiple comparisons test for (B), (D) to (F), and (H). ns, not significant. Scale bars represent 50 μm in (A) and 100 μm in (C) and (G). Data are represented in (F) as mean ± SEM (error bars) from at least two independent experiments. In (B), (D), (E), and (H), each data point represents an individual mouse, and each horizontal bar denotes a mean value (at least two independent experiments).

  • Fig. 2 Helminth infection inhibits B. vulgatus colonization through a type 2 immune response.

    (A) Quantification of B. vulgatus colony forming units (cfu) in stool from T. muris–infected WT and Nod2−/− mice (n ≥ 10 per genotype). D represents number of days postinfection. (B) Quantification of pSTAT6 staining in the small intestine of T. muris–infected WT and Nod2−/− mice (n ≥ 3 per genotype). (C) Quantification of B. vulgatus in stool from T. muris–infected WT (Nod2−/− → WT) and Stat6−/− (Nod2−/−Stat6−/−) mice reconstituted with Nod2−/− bone marrow. Both WT and Stat6−/− chimeric mice were gavaged with B. vulgatus to ensure equal colonization before T. muris infection (n ≥ 5 per genotype). (D) Quantification of the total number of SI lamina propria CD4+ T cells expressing IL-13 in uninfected and T. muris–infected Nod2−/− mice (n ≥ 4 per genotype). (E) Fold increase in the number of CD4+ T cells producing IFN-γ, IL-13, or IL-10 in the SI lamina propria of T. muris–infected WT and Nod2−/− mice, normalized to uninfected mice (n ≥ 4 per genotype). (F) Quantification of B. vulgatus associated with SI tissue of uninfected, T. muris–infected, and H. polygyrus–infected Nod2−/− mice (n ≥ 10 per genotype). (G and H) Quantification of goblet cells displaying normal morphology per villi (G) and total number of SI lamina propria CD4+ T cells expressing IL-13 (H) in uninfected and H. polygyrus–infected WT and Nod2−/− mice (n ≥ 3 per genotype). (I and J) Quantification of B. vulgatus in SI tissue (I), and goblet cells displaying normal morphology (J) in H. polygyrus–infected Nod2−/− mice treated with antibody to IL-13 or isotype control (n = 6 per genotype). (K and L) Quantification of goblet cells displaying normal morphology (K) and B. vulgatus in stool (L) in Nod2−/− mice treated with recombinant IL-13 or phosphate-buffered saline (PBS) (n = 8 per genotype). (M) Pathway analysis based on Gene Ontology (GO) terms of genes up-regulated in Nod2−/− mice treated with recombinant IL-13 compared with PBS controls. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ANOVA, with Holm-Sidak multiple comparisons test for (A), (B), (G) and (H) and unpaired t test for (C), (D), (F), and (I) to (L). N.D., not detected. Data in (A), (B), (C), (E), and (L) are represented as mean ± SEM (error bars) from at least two independent experiments. In (D) and (F) to (K), each data point represents an individual mouse, and each bar denotes a mean value [at least two independent experiments, excluding the generation of bone marrow chimeras in (C)].

  • Fig. 3 Inhibition of B. vulgatus is associated with expansion of Clostridiales after helminth infection.

    (A) Quantification of B. vulgatus in stool harvested from uninfected and T. muris–infected Nod2−/− mice cohoused for the duration of the experiment (n ≥ 4). (B) Relative abundance of taxonomic groups in response to T. muris infection in the stool of WT and Nod2−/− mice, as determined by 16S sequencing (n ≥ 5 per genotype). (C) Supervised analysis of 16S sequencing data with LDA effect size (LEfSe), comparing Nod2−/− mice at D0 and D21 postinfection with T. muris using an LDA threshold score of 4 (n ≥ 5). (D) LEfSE analysis to determine alterations to the stool microbiota after recombinant IL-13 treatment of Nod2−/− mice using an LDA threshold score of 4 (n ≥ 5). (E) Quantification of B. vulgatus in stool harvested from Nod2−/− mice gavaged with sterile broth, L. johnsonii, or a mix of 17 Clostridiales and Erysipelotrichales strains (n ≥ 3). (F and G) Quantification of Clostridium species (Clostridiales 28) (F) or B. vulgatus (G) in the presence of varying concentrations of pig intestinal mucin or vehicle in the culture media. ***P < 0.001; ****P < 0.0001 by ANOVA, with Holm-Sidak multiple comparisons test for (E) and (F). Data are represented as mean ± SEM (error bars) from at least two independent experiments.

  • Fig. 4 Helminth colonization in humans is associated with a decrease in Bacteroidales and an increase in Clostridiales levels.

    (A) Beta diversity plots of gut microbiota from urban controls in Kuala Lumpur (red dots) and the Orang Asli (blue dots). (B) Relative abundance of a dominant Bacteroides OTU in the Orang Asli and urban controls. (C to F) Supervised LEfSE analysis (C), relative abundance of Bacteroidales (D) and Clostridiales (E), and alpha diversity as observed OTUs (F) of the Orang Asli stool microbiota before and after treatment with albendazole (n = 19 for urban controls and 55 Orang Asli; n = 53 for deworming experiments). (G) Partial least squares regression biplots examining within-subject variances with repeated measures design to identify bacterial taxa associated with T. trichiura worm burden (illustrated by the intensity of the spots). Red arrows represent Clostridiales taxa; green arrows denote Bacteroidales taxa. (H) Specific OTUs identified to be positively (Dialister) or negatively (Prevotella) associated with changes to T. trichiura egg burdens. (I and J) Microbial network inference demonstrating an antagonistic relationship between Clostridiales and Bacteroidales communities from the Human Microbiome Project (I) and the pediatric IBD RISK cohort (J). The node diameter is proportional to the geometric mean of the OTU’s relative abundance. Numerical values on the edges represent the fraction of edges that are either majority-positive (green) or majority-negative (red). Also see fig. S10. ****P < 0.0001 by unpaired t test in (B) and paired t test in (D) to (F).

Supplementary Materials

  • Helminth infection promotes colonization resistance via type 2 immunity

    Deepshika Ramanan, Rowann Bowcutt, Soo Ching Lee, Mei San Tang, Zachary D. Kurtz, Yi Ding, Kenya Honda, William C. Gause, Martin J. Blaser, Richard A. Bonneau, Yvonne AL Lim P’ng Loke, Ken Cadwell

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S11
    • Tables S1 to S4
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