Commensal-pathogen interactions in the intestinal tract: lactobacilli promote infection with, and are promoted by, helminth parasites

Abstract

The intestinal microbiota are pivotal in determining the developmental, metabolic and immunological status of the mammalian host. However, the intestinal tract may also accommodate pathogenic organisms, including helminth parasites which are highly prevalent in most tropical countries. Both microbes and helminths must evade or manipulate the host immune system to reside in the intestinal environment, yet whether they influence each other's persistence in the host remains unknown. We now show that abundance of Lactobacillus bacteria correlates positively with infection with the mouse intestinal nematode parasite, Heligmosomoides polygyrus, as well as with heightened regulatory T cell (Treg) and Th17 responses. Moreover, H. polygyrus raises Lactobacillus species abundance in the duodenum of C57BL/6 mice, which are highly susceptible to H. polygyrus infection, but not in BALB/c mice, which are relatively resistant. Sequencing of samples at the bacterial gyrB locus identified the principal Lactobacillus species as L. taiwanensis, a previously characterized rodent commensal. Experimental administration of L. taiwanensis to BALB/c mice elevates regulatory T cell frequencies and results in greater helminth establishment, demonstrating a causal relationship in which commensal bacteria promote infection with an intestinal parasite and implicating a bacterially-induced expansion of Tregs as a mechanism of greater helminth susceptibility. The discovery of this tripartite interaction between host, bacteria and parasite has important implications for both antibiotic and anthelmintic use in endemic human populations.

Keywords: Commensal; Duodenum; Lactobacillus; Microbiota; Nematode; Th17; Treg.

Figure 1

Intestinal microbiota composition alters H. polygyrus persistence. (A–F) BALB/c mice were administered untreated water, or water containing 0.5 g/L vancomycin for one week prior to infection with 200 H. polygyrus L3s, and throughout the experiment. Data pooled from two experiments. (A) Experimental protocol. (B) qPCR analysis of fecal 16S rRNA gene expression 28 d post-infection. (C) Intestinal H. polygyrus burden 28 d post-infection. (D) qPCR analysis of fecal Lactobacillus/Lactococcus-specific 16S rRNA gene expression 28 d post-infection. (E) qPCR analysis of fecal Enterobacteriaceae-specific 16S rRNA gene expression 28 d post-infection. (F) qPCR analysis of fecal Eubacterium/Clostridium-specific 16S rRNA gene expression 28 d post-infection. NS denotes no statistical differences; * indicates P = ≤ 0.05; ** indicates P = ≤ 0.01; *** indicates P = ≤ 0.001.

Figure 2

Lactobacillaceae species abundance positively correlates with susceptibility to H polygyrus. Six-week old female mice were left naïve or infected with 200 H. polygyrus L3s. Ten naïve mice and 40 H. polygyrus-infected mice, either C57BL/6 or BALB/c were used in each experiment. Experiments in each strain were performed separately and so data are not intended to be directly comparable between strains. A direct comparison of differential strain resistances to H. polygyrus can be found in ref. 23. (A) Intestinal H. polygyrus burden 28 d post-infection. (B) qPCR analysis of duodenal Lactobacillus/Lactococcus-specific 16S rRNA gene expression 28 d post-infection in C57BL/6 mice. (C) qPCR analysis of duodenal Enterobacteriaceae-specific 16S rRNA gene expression 28 d post-infection in C57BL/6 mice. (D) qPCR analysis of duodenal Lactobacillus/Lactococcus-specific 16S rRNA gene expression 28 d post-infection in BALB/c mice. (E) qPCR analysis of duodenal Enterobacteriaceae-specific 16S rRNA gene expression 28 d post-infection in BALB/c mice. (F) Correlation between intestinal H. polygyrus burden and duodenal Lactobacillus/Lactococcus-specific 16S rRNA gene expression measured by qPCR at day 28 post-infection in BALB/c mice. Statistics shown indicate analysis by Spearman correlation test. * indicates P ≤ 0.05; ** indicates P ≤ 0.01; *** indicates P ≤ 0.001 and r indicates the correlation co-efficient.

Figure 3

Lactobacillaceae species and H. polygyrus abundance both positively correlate with Th17 and Treg phenotypes. Six-week old BALB/c female mice were infected with 200 H. polygyrus L3s. Twenty-eight days post-infection MLN cells were stained for Foxp3 expression, or MLN cells were restimulated with 1 μg HES for 72 h after which IL-17A production was determined by ELISA. (A, B) Correlation between MLN cell HES-specific IL-17A production and (A) duodenal Lactobacillus/Lactococcus-specific 16S rRNA gene expression measured by qPCR, and (B) intestinal H. polygyrus burden 28 d post-infection. (C, D) Correlation between total number of MLN CD4⁺Foxp3⁺ cells 28 d post-infection and (C) duodenal Lactobacillus/Lactococcus-specific 16S rRNA gene expression measured by qPCR, and (D) intestinal H. polygyrus burden 28 d post-infection. at the same time point. Statistics shown indicate analysis by Spearman correlation test. * indicates P ≤ 0.05; ** indicates P ≤ 0.01; and r indicates the correlation co-efficient.

Figure 4

Lactobacillus taiwanensis correlates with Lactobacillus/Lactococcus effects in H. polygyrus infection. (A) Neighbor-joining tree showing relatedness of Lactobacillus species based on the similarity at the region of the gyrB gene shown in Fig. S2. (B) Correlation between duodenal Lactobacillus/Lactococcus-specific 16S rRNA gene expression and duodenal L. taiwanensis-specific gyrB gene expression measured by qPCR 28 d post-infection. (C) Correlation between intestinal H. polygyrus burden 28 d post-infection and duodenal L. taiwanensis-specific gyrB gene expression measured by qPCR at the same time point. (B, C) Statistics shown indicate analysis by Spearman correlation test. * indicates P ≤ 0.05; *** indicates P ≤ 0.001; and r indicates the correlation co-efficient.

Figure 5

Lactobacillus taiwanensis administration enhances H. polygyrus infection. (A) Experimental protocol for (B-D). BALB/c mice were administered untreated drinking water, or water containing 2 × 10⁸ colony forming units (cfu)/ml L. taiwanensis for one week prior to infection with 200 H. polygyrus L3s, and throughout the experiment. (B) Mean H. polygyrus egg output per gram of feces +/− SEM on days 14, 21 and 28 post-infection. (C) Intestinal H. polygyrus burden 28 d post-infection. Data shown are pooled from four independent experiments, each with 6–7 mice per group. (D) Mean intestinal H. polygyrus burden 28 d post-infection. Data shown are paired means from the four independent experiments shown in (C). (E) Experimental protocol for (F-G). BALB/c mice were administered untreated drinking water, or water containing 2 × 10⁸ colony forming units (cfu)/ml L. taiwanensis for one week. (F) % Foxp3⁺ cells among CD4⁺ MLN cells. Data shown are pooled from two independent experiments each with 4 mice per group. (G) % Foxp3⁺ cells among CD4⁺ PP cells. Data shown are pooled from two independent experiments each with 4 mice per group. * indicates P ≤ 0.05; ** indicates P ≤ 0.01.