Type 2 immunity-dependent reduction of segmented filamentous bacteria in mice infected with the helminthic parasite Nippostrongylus brasiliensis

Abstract

Background: Dynamic interactions between the host and gastrointestinal microbiota play an important role for local and systemic immune homeostasis. Helminthic parasites modulate the host immune response, resulting in protection against autoimmune disease but also increased susceptibility to pathogen infection. The underlying mechanisms remain largely unknown.

Results: We showed that the type 2 immune response to enteric Nippostrongylus brasiliensis infection in mice was associated with altered intestinal mucin and AMP expression and shifts in microbiota composition. Most strikingly, infection reduced concentrations of intestinal segmented filamentous bacteria (SFB), known inducers of T helper 17 cells, and IL-17-associated gene expression. Infected mice deficient in IL-13 or STAT6 did not reduce SFB or IL-17, and exogenous IL-25 replicated the effects of parasite infection in wild type mice.

Conclusions: Our data show that parasite infection acts through host type 2 immunity to reduce intestinal SFB and expression of IL-17, providing an example of a microbiota-dependent immune modulation by parasites.

Fig. 1

Infection with N. brasiliensis induces characteristic alterations in intestinal expression of type 2 cytokines, markers for M2 macrophages, and antimicrobial peptides and mucins. N. brasiliensis-infected mice were euthanized at day 11 post-inoculation with infective larvae. qPCR was carried out to examine gene expression of IL-13, Fizz1, and YM-1 in the jejunum (a) and ileum (b), as well as selected AMP and mucins in the ileum (c). The fold change is relative to vehicle after normalization to 18S rRNA. Data shown in bar graphs are the mean ± s.e.m. Two-tailed Student’s t-test was used for comparisons between infected and uninfected mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective vehicle (n = 10 for vehicle group and n = 9 for N. brasiliensis-infected group). Ang4 angiogenin 4, Defa1 defensin alpha 1, Fizz1 found in inflammatory zone 1, Lyz1/2 lysozymes 1/2, Muc2 mucin 2, Muc5Ac mucin 5 AC, Reg3γ regenerating islet-derived protein 3 gamma, Retnlb resistin-like molecule beta, YM-1

Fig. 2

Infection with N. brasiliensis modulates the ileal microbiota in mice. Mice infected with N. brasiliensis were euthanized at day 11 post-inoculation and metagenomic DNA isolated from ileal tissue samples including luminal content and subjected to 16S rRNA gene short amplicon sequencing. a Microbial diversity calculated with the Shannon diversity index and compared with the Mann–Whitney test. b Qualitative (un-weighted UniFrac) and c quantitative (weighted UniFrac) phylogenetic distance calculations. d Taxonomic phyla and e families with differential abundance in N. brasiliensis-infected compared to uninfected mice as calculated with the non-parametric Fisher test as implemented in Metastats. Data shown in bar graphs are the mean ± s.e.m. *P < 0.05 versus vehicle (n = 10 for vehicle group and n = 9 for N. brasiliensis-infected group)

Fig. 3

Infection with N. brasiliensis reduces the abundance of segmented filamentous bacteria (SFB) along the gastrointestinal tract and the expression of Th17 cell-associated genes in the ileum. Metagenomic DNA was extracted from intestinal strips containing the luminal contents or feces. 16S rRNA sequencing was carried out to examine the relative abundance of SFB in the ileum (a). qPCR further confirmed the decrease of SFB abundance in infected mice in ileum (b), jejunum (c), proximal colon (d), and feces (e), relative to vehicle after normalization to total bacteria. Expression of Th17-associated genes was determined by qPCR (f). The fold change in mRNA is relative to vehicle after normalization to 18S rRNA. Data shown in bar graphs are the mean ± s.e.m. Mann–Whitney test (a–e) or two-tailed Student’s t-test (f) was used for comparisons between groups. *P < 0.05, **P < 0.01, and ***P < 0.001 versus respective vehicle (n = 10 for vehicle group and n = 9 for N. brasiliensis-infected group)

Fig. 4

Infection with N. brasiliensis does not affect ileal expression of IL-17-associated genes in SFB-negative mice. Mice from NCI-Frederick (NCI) or Jackson Laboratory (Jackson mice) were infected with N. brasiliensis and metagenomic DNA extracted from ileal strips containing luminal contents or feces. qPCR was carried out to examine the relative abundance of SFB (a). Total RNA was isolated from the ileum and expression of IL-17A (b), SAA1 (c), IL-13 (d), Retnlb (e), or Reg3γ (f) was determined by qPCR. The fold change in mRNA is relative to NCI vehicle after normalization to 18S rRNA. Data shown in bar graphs are the mean ± s.e.m. *P < 0.05 versus respective vehicle; ^ϕ P < 0.05 versus NCI vehicle (n = 6–8 for each group), based on one-way ANOVA followed by Newman-Keuls test

Fig. 5

N. brasiliensis infection-induced alterations in AMP, SFB, and Th17-associated genes depend primarily on IL-13 and STAT6. Mice deficient in IL-13 (a–c) or STAT6 (d–f) were infected with N. brasiliensis and euthanized at day 11 post-inoculation. qPCR was carried out to quantify expression of antimicrobial peptides (a, d), SFB-specific and universal bacterial 16S rRNA (b, e), and Th17-associated genes (c, f). The abundance of SFB is relative to vehicle after normalization to total bacteria based on 16S rRNA qPCR. The fold change in mRNA is relative to vehicle after normalization to 18S rRNA. Data shown in bar graphs are the mean ± s.e.m. Mann–Whitney test (b, e) or two-tailed Student’s t-test (a, c, d, f) was used for comparisons between the two groups. *P < 0.05 versus respective vehicle (n = 7 for vehicle groups and 6 for N. brasiliensis-infected groups; one mouse died after infection)

Fig. 6

Exogenous IL-25 induces type 2 immunity, as well as IL-13-dependent alterations in ileal AMP expression and SFB abundance. WT (a–c) or IL-13^−/− (d–f) mice were injected with IL-25 (i.p., 1 μg per mouse) daily for 3 days and euthanized at day 4. qPCR was carried out to examine expression of antimicrobial peptides (a, d), SFB-specific and universal bacterial 16S rRNA (b, e), and Th17-associated genes (c, f) in the ileum. The abundance of SFB is relative to vehicle after normalization to universal bacterial 16 s rRNA. The fold change is relative to vehicle after normalization to 18S rRNA. Data shown in bar graphs are the mean ± s.e.m. Mann–Whitney test (b, e) or two-tailed Student’s t-test (a, c, d, f) was used for comparisons between the two groups. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective vehicle (n = 7 for BSA groups and WT-IL-25, n = 6 for IL-13^−/−-IL-25)

Fig. 7

Proposed model for the immune regulatory network that connects host, parasitic nematode, and SFB microbiota during N. brasiliensis infection in mice. We showed that SFB-colonized mice reduce SFB and expression of IL-17 upon infection with N. brasiliensis or induction with exogenous IL-25 in a way that is dependent on STAT6 activation by IL-13 and possibly modulation of intestinal AMP and mucin expression. This is consistent with a model in which the host type 2 response to parasite infection leads to SFB depletion with the net result of reduced intestinal IL-17 expression