The degree of Helicobacter pylori-triggered inflammation is manipulated by preinfection host microbiota

Abstract

Helicobacter pylori infects over 3 billion people worldwide and is the primary risk factor for gastric cancer. Most individuals infected with H. pylori develop only asymptomatic gastritis; however, some develop ulcers or gastric adenocarcinoma. We demonstrate that one previously unappreciated parameter influencing H. pylori disease outcome is variation in the preinfection host microbiota. Utilizing a mouse model, we altered the microbiota by antibiotic treatment and found that these alterations resulted in significantly lowered H. pylori-triggered inflammation. Specifically, antibiotic pretreatment reduced CD4(+) T-helper cells and Ifnγ transcript levels in gastric tissue after H. pylori infection. The bacterial communities in mice with a reduced response to H. pylori displayed many differences from those in untreated mice, including significantly more cluster IV and XIVa Clostridium spp., bacteria known to influence inflammation via regulatory T cell populations. Our findings suggest that microbiota composition, perhaps Clostridium spp., contributes to the variable disease outcome of H. pylori infection by altering the recruitment of CD4(+) T cells to the gastric compartment. Our results suggest that gastric microbiota could be used as a diagnostic tool to determine which patients are at risk for developing severe disease.

Fig 1

Female C57BL/6N mice from different vendors mount different inflammatory responses to H. pylori and possess various amounts of key Lactobacillus spp. (A) Inflammation grade scored on a scale of 0 (no lymphocytic infiltration) to 5 (moderate, widespread and severe multifocal lymphocytic infiltration) as described by Eaton et al. (20). (B) Inflammation grade of the same tissue as in panel A, scored by determining the percentage of fields that contain PMN, gastritis, or metaplasia, as outlined by Eaton et al. (21). Gastric tissue was from mice from either Charles River Laboratories (CRL) or Taconic Farms (TF) infected with wild-type H. pylori strain SS1 (WT) for 6 months or from uninfected (UI) controls. PMN, polymorphonuclear leukocytes (neutrophils). (C) H. pylori colonization levels in CFU counts per gram of stomach tissue at 2 and 6 months postinoculation. (D) Quantitative PCR on the 16S gene for Lactobacillus species ASF360 and ASF361 in 2-month-old uninfected mice from CRL or TF. For all panels, n ≥ 6 mice per group infected with H. pylori, and n = 3 for uninfected groups. For panels B and D, data are presented as averages ± standard errors of the means. *, P < 0.05; **, P < 0.01, Student's t test.

Fig 2

The murine stomach microbiota is refractory to H. pylori-triggered perturbations and similar to the human stomach microbiota. (A) Pie charts depicting phylum and class level distribution of 2,056 taxa that were present in all noninfected TF mice; 74% of the 2,056 taxa are Firmicutes, and, specifically, 44% of the total are members of the class Clostridia. A total of 12,032 taxa were present in at least one of five mouse stomachs. (B) Significant differences in microbiotas are not apparent between H. pylori-infected and noninfected samples, as indicated by nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis distance between samples given the presence/absence of 12,032 taxa present in at least one of nine mouse samples; each dot represents one mouse in the study. H.p., 4-week H. pylori infection; UI, uninfected.

Fig 3

Fewer CD4⁺ T cells infiltrate the stomach in response to H. pylori when mice have been pretreated with antibiotics. (A) Experimental setup for altering the microbiota and infecting with H. pylori. (B) Representative flow cytometry plots showing the percentages of CD4⁺ and CD8⁺ cells of the CD45⁺ CD3⁺ gastric lymphocytes from tissue 4 weeks postinoculation. Numbers in quadrants indicate the percentages of positively stained cells. Quadrants were determined based on isotype controls (data not shown). (C) CD45⁺ cell percentage is presented as the percentage of CD45⁺-stained cells out of 50,000 cells counted (left panel). CD4⁺ and CD8⁺ cell percentages are presented as the percentages of positively stained cells out of 50,000 cells counted (right panel). Data were obtained from two independent experiments with similar results (n ≥ 7 mice for each group). (D to F) Total gastric mRNA expression of Ifnγ, Il17, and Il4. mRNA expression levels were normalized to the level of the housekeeping gene, Gapdh, and are expressed as fold change from mock treatment, using the ΔΔC_T method (19). H.p., H. pylori-infected; Ab, antibiotic treatment; RC, reconstituted with gastric microbiota; WT, wild type; PI, postinfection; wks, weeks. Data in panels C to F are presented as averages ± standard errors of the means. N.S., not significant; *, P < 0.05, by two-way ANOVA and Tukey's range test.

Fig 4

The microbiota of antibiotic-treated mice is significantly different from that of the untreated mice and is dominated by members of the Clostridiales. (A) Significant differences in microbiotas are apparent between antibiotic-treated and untreated H. pylori-infected samples, as indicated by nonmetric multidimensional scaling (NMDS) based on the Bray-Curtis distance between samples given the presence/absence of 12,765 taxa present in at least one of 10 mouse samples; each dot represents one mouse in the study. A total of 4,400 OTUs were different between the antibiotic-treated H. pylori-infected mice and the H. pylori-infected untreated mice. Of those OTUs, 55% were significantly decreased in the antibiotic-treated mice, and 45% were significantly increased. (B and C) Pie charts illustrate phylum and class level distributions between bacterial taxa that either decreased (B) or increased (C) in the antibiotic-treated mice compared to levels in the nontreated mice. The majority of the taxa that were increased in the antibiotic treated mice were members of the Clostridia. (D) Ninety-four percent of the Clostridia OTUs that were increased in the antibiotic-treated mice were in the order Clostridiales; the chart details those members of the Clostridiales at the family and genus level. (E and F) Total gastric mRNA expression of Foxp3 and Il10. mRNA expression levels were normalized to the level of the housekeeping gene, Gapdh, and are expressed as fold change from mock treatment, using the ΔΔC_T method. H.p., H. pylori-infected; Ab, antibiotic treatment; RC, reconstituted with gastric microbiota (n = 5 for each group). For panels E and F, data are displayed as averages ± standard errors of the means. *, P < 0.05, by two-way ANOVA and Tukey's range test.