Effect of Helicobacter felis infection and somatic deletion of NFκB family members on the murine gastric microbiome

Abstract

BackgroundThe development of gastric atrophy in C57BL/6 mice infected with Helicobacter felis is differentially regulated by signalling involving NFκB1 and NFκB2. After infection, more severe atrophy develops in Nfkb1-/- mice than in wild-type (wt) mice, whereas Nfkb2-/- mice are protected from atrophic gastritis. In addition, the development of pathological changes induced by Helicobacter pylori is delayed in INS-Gas mice maintained in germ-free conditons compared with those in conventional animal house conditions. Inflammasome-deficient mice have altered immunological responses that can lead to development of a dominant dysbiosis, which is sufficient to alter the outcome of dextran sodium sulphate induced colitis. We hypothesised that the different phenotypes observed in H felis infected mice lacking specific NFκB proteins could be influenced by altered gastric microbiota. We have therefore quantified the abundance of specific bacterial phyla in Nfkb1-/-, Nfkb2-/-, and C57BL/6 mice with and without H felis infection. MethodsWe used C57BL/6, Nfkb1-/-, and Nfkb2-/- mice aged 6 weeks. Three mice of each type were infected with H felis by gavage, humanely killed at 12 weeks, and gastric antral DNA extracted. Total bacterial load and relative abundance of α-proteobacteria, γ-proteobacteria, Bacteriodetes, Firmicutes, and Actinobacteria were measured by quantitative PCR (qPCR) of 16S rDNA. Colonisation by H felis was assessed with qPCR for FlaA; samples were normalised to murine Gapdh. FindingsUntreated wt mice had 3·4 times and 2·6 times greater universal bacterial transcripts than did Nfkb1-/- and Nfkb2-/- mice, respectively. Actinobacteria abundance was 6·0 times greater in untreated Nfkb1-/- mice and 7·0 times greater in Nfkb2-/- mice than in wt mice. α-proteobacteria were 9·0 times more abundant in untreated Nfkb1-/- mice than in wt mice. H felis infection of wt mice resulted in increases of 5·6 times and 16·7 times in α-proteobacteria and γ-proteobacteria, respectively, compared with uninfected mice. γ-proteobacteria were more abundant in all infected groups, but significantly more so in Nfkb2-/- mice than in others (p<0·05, two-way ANOVA). This finding correlated with a 30-times higher abundance of H felis (phylum γ-Proteobacteria) in infected Nfkb2-/- than in wt mice. Infected Nfkb2-/- mice also had a 3·3-times greater abundance of actinobacteria than did infected wt mice. No statistically significant differences were observed in the abundance of Firmicutes or Bacteroidetes. InterpretationThe constitution of the murine gastric antral microbiome is affected both by H felis infection and by somatic deletion of NFκB family members. Since deletion of NFκB1 and NFκB2 alter susceptibility to H felis induced gastric atrophy, our data support the hypothesis that specific differences in microbiota could cause or signal this altered susceptibility. Further studies are needed to determine whether specific organisms influence the development of gastric disease either individually or within complex communities. FundingWellcome Trust.