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. 2022 Jan 26;13(1):525.
doi: 10.1038/s41467-022-28126-w.

V体育ios版 - Defective humoral immunity disrupts bile acid homeostasis which promotes inflammatory disease of the small bowel

Ahmed Dawood Mohammed  1   2 , Zahraa Mohammed  1   3 , Mary M Roland  1 , Ioulia Chatzistamou  1 , Amy Jolly  1 , Lillian M Schoettmer  1 , Mireya Arroyo  1 , Khadija Kakar  1 , Yuan Tian  4 , Andrew Patterson  4 , Mitzi Nagarkatti  1 , Prakash Nagarkatti  1 , Jason L Kubinak  5
Affiliations

Defective humoral immunity disrupts bile acid homeostasis which promotes inflammatory disease of the small bowel

Ahmed Dawood Mohammed et al. Nat Commun. .

VSports手机版 - Abstract

Mucosal antibodies maintain gut homeostasis by promoting spatial segregation between host tissues and luminal microbes. Whether and how mucosal antibody responses influence gut health through modulation of microbiota composition is unclear. Here, we use a CD19-/- mouse model of antibody-deficiency to demonstrate that a relationship exists between dysbiosis, defects in bile acid homeostasis, and gluten-sensitive enteropathy of the small intestine. The gluten-sensitive small intestine enteropathy that develops in CD19-/- mice is associated with alterations to luminal bile acid composition in the SI, marked by significant reductions in the abundance of conjugated bile acids. Manipulation of bile acid availability, adoptive transfer of functional B cells, and ablation of bacterial bile salt hydrolase activity all influence the severity of small intestine enteropathy in CD19-/- mice VSports手机版. Collectively, results from our experiments support a model whereby mucosal humoral immune responses limit inflammatory disease of the small bowel by regulating bacterial BA metabolism. .

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. B cell deficiency is associated with altered BA composition in the gut.
A Fecal BA concentrations of respective cohorts are shown. Tukey’s multiple comparisons test; (WT vs. CD19−/−), **=p = 0.0031, (CD19−/− vs. CD19−/−ciprofloxacin), ***=p = 0.0001, (CD19−/− vs. CD19−/−metronidazole), ***=p = 0.0002. Data representative of two replicate experiments. B Aerobic and anaerobic CFU measurements from ileum and duodenum are shown. Two-tailed unpaired Student’s t-test; ns = non-significant = p = 0.16, *=p = 0.05, **=p = 0.003. Data representative of three replicate experiments. C Ileal BA concentrations are shown. Two-tailed Student’s t-test; ****=p < 0.0001. Data representative of three replicate experiments. D Stacked bar-chart illustrating general shifts in BA abundance as measured by UPLC-MS. Source data are provided as a Source Data file. Data representative of three replicate experiments. E Correlation analysis of relationship between conjugated and unconjugated BA concentrations in the ileum of WT and CD19−/− mice. (upper plot) Two-tailed one-way ANOVA, Fstat(1,31) = 19.93, p < 0.0001. (lower plot) Two-tailed one-way ANOVA, Fstat(1,31) = 1.263, p = 0.27. F The relative abundance of unconjugated and conjugated BAs in WT and CD19−/− mice is shown. Sidak’s multiple comparisons test; ns = >0.99, *=p = 0.04, ***=p = 0.0004. G Comparison of the ratio of unconjugated to conjugated BAs between genotypes is shown. Two-tailed Student’s t-test; **=p = 0.002. H A PcoA plot based on Bray–Curtis distance of ileal microbial community composition is shown. Results of multivariate PERMANOVA hypothesis testing is shown (p = 0.01). I Pie-charts illustrating differential abundance of major bacterial Classes between WT (n = 14) and CD19−/− (n = 15) mice are shown. Source data are provided as a Source Data file. J A heatmap summarizing descriptive statistics (Pearson r-coefficient and r-squared) of relationship between bacterial group abundance shown in (E) with associated ileal BA concentrations from the same mice (left panel). The correlation between Lactobacillaceae abundance and ileal BA concentrations are also shown (right panel). For right panel: two-tailed one-way ANOVA, F(1,16) = 8.041, p = 0.01. K A heatmap summarizing descriptive statistics (Pearson r-coefficient and r-squared) of relationship between Lactobacillaceae abundance and the abundance of specific BAs as measured by UPLS-MS is shown. Right panel: LCA correlation-two-tailed one-way ANOVA, F(1,11) = 7.842, p = 0.02, β-MCA correlation-two-tailed one-way ANOVA, F(1,11) = 6.599, p = 0.03. L Bar-charts representing the top 15 bacterial groups influencing mean accuracy of Random Forest classification of samples by genotype (Blue bars: enriched in WT; Red bars: enriched in CD19−/− mice). M Results of correlation analysis of ASV read abundance of differentially enriched bacterial groups in (L) demonstrating a significant association with ileal BA concentrations. Results of two-tailed one-way ANOVAs are shown in respective plots (each comparison stands alone). J, K Descriptive statistics were generated without adjustment for multiple comparisons.
Fig. 2
Fig. 2. BA abnormalities are not due to host defects in Bile acid synthesis, conjugation, or reabsorption in CD19−/− mice.
A Gene expression of bile acid transporters in the distal ileum of mice. B Volcano plot of major differentially regulated genes in the liver of CD19−/− mice compared to WT controls. The FDR-adjusted significance cutoff (FDR < 0.05) of quasi-likelihood hypothesis testing is illustrated by line along Y-axis. C Gene expression of liver enzymes involved in bile acid synthesis. D Gene expression of liver enzymes associated with taurine biosynthesis and bile acid conjugation. E Gene expression of liver bile acid transporters. A–E Data is representative of one ileal RNAseq and one liver RNAseq experiment each containing five biological replicates for each genotype. Significance determined by quasi-likelihood testing with an FDR significance cutoff of <0.05. None of the genes shown are differentially regulated based on FDR-adjusted p-values. Ileal and liver RNAseq data sets are derived from the same set of 5 female WT and 5 female CD19−/− mice. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. SI enteropathy is associated with increased IEC apoptosis in CD19−/− mice.
A Representative flow cytometry plot demonstrating gating on apoptotic IECs (EpCAM+CD45−PI−AnnV+) and the percentage of apoptotic IECs in WT and CD19−/− mice are shown. Two-tailed unpaired Student’s t-test; **=p = 0.007. Data representative of three replicate experiments. B Representative flow cytometry plot demonstrating gating on live IECs (EpCAM+CD45−ZombieGreen−) and MitoSpy MFI values in live IECs in WT and CD19−/− mice are shown. Two-tailed unpaired Student’s t-test; **=p = 0.006. Data representative of two replicate experiments. C Representative flow cytometry plot demonstrating gating on proliferating IECs (EpCAM+CD45−Ki67+) and the percentage of proliferating IECs in WT and CD19−/− mice are shown. Two-tailed unpaired Student’s t-test; ns=non-significant=p = 0.28. Data representative of three replicate experiments.
Fig. 4
Fig. 4. Manipulation of BA availability alters severity of SI enteropathy.
A Schematic overview of oral TUDCA supplementation experiment. Syringe illustrations in (A) are image elements derived from BioRender.com. B Representative flow cytometry plots and comparison of the percentage of apoptotic IECs in vehicle (PBS) (n = 12) or TUDCA-treated (n = 13) CD19−/− mice are shown. Two-tailed unpaired Mann–Whitney U test; ***=p = 0.0009. C SI enteropathy scoring is shown for vehicle or TUDCA-treated CD19−/− mice. Two-tailed unpaired Mann–Whitney U test; ***=p = 0.0008. D Comparison of individual features of SI enteropathy between vehicle or TUDCA-treated CD19−/− mice are shown. Two-tailed unpaired Mann–Whitney U test; (leukocytosis), n = non-significant =p = 0.0542, (inflammation), ns = non-significant =p = 0.53, **=p = 0.003, ***=p = 0.0008. E Schematic overview of cholestyramine diet exposure experiment. Mouse chow illustrations in (E) are image elements derived from BioRender.com. F Results of SI enteropathy scoring are shown for CD19−/− mice exposed to control versus resin diet. Two-tailed unpaired Mann–Whitney U test; ns = non-significant =p = 0.66. G Comparison of individual features of SI enteropathy between CD19−/− mice exposed to control versus resin diet are shown. Two-tailed unpaired Mann–Whitney U test; (crypt hyperplasia), ns=non-significant=p = 0.0513, (villous blunting), ns = non-significant =p = 0.27, *=p = 0.0.02, ***=p < 0.0009. A–D Data representative of three replicate experiments. E–G Data representative of two replicate experiments.
Fig. 5
Fig. 5. Adoptive transfer of WT B cells rescues CD19−/− mice from SI enteropathy.
A Schematic of adoptive transfer experiment. B Representative histogram plot demonstrating gating on apoptotic IECs and results of flow experiments are shown. Two-tailed unpaired Student’s t-test with Welch’s correction for uneven variance; *=p = 0.01. C Anaerobic CFU titers from ileal contents are shown. Two-tailed unpaired Mann–Whitney U test; ***=p = 0.0006. D Total ileal BA concentrations are shown. Two-tailed unpaired Student’s t-test; *=p = 0.05. E SI enteropathy scoring is shown for CD19−/− mice receiving B cells from WT or CD19−/− mice. Two-tailed unpaired Mann–Whitney U test; ****=p < 0.0001. F Comparison of individual features of SI enteropathy between CD19−/− mice receiving B cells from WT or CD19−/− mice. Two-tailed unpaired Mann–Whitney U test; ns = non-significant =p = 0.21, **=p = 0.004, ***=p = 0.0001 (crypt hyperplasia), ***=p = 0.0001 (villous blunting). G Pie-charts illustrating differential abundance of major bacterial Classes are shown. Source data are provided as a Source Data file. H Heatmaps summarizing descriptive statistics (Pearson r-coefficient and r-squared) of relationship between ileal bacterial group abundance and ileal BA concentrations are shown. I Heatmaps summarizing descriptive statistics (Pearson r-coefficient and r-squared) of relationship between ileal bacterial group abundance and SI Enteropathy severity are shown. B–I Data representative of two replicate experiments. H, I Hatched values indicate bacterial groups that were not found present in more than 75% of samples, and were thus excluded from analysis.
Fig. 6
Fig. 6. Ileal bsh activity is perturbed in CD19−/− mice.
A Total bsh (EC:3.5.1.24) gene abundance in WT and CD19−/− mice is shown. Two-tailed unpaired Student’s t-test; *=p = 0.04. B Correlation between bsh gene abundance and ileal BA concentrations is shown. Two-tailed one-way ANOVA, Fstat(1,20) = 10.66, p = 0.004. C Total 7α-dehydratase (EC:4.2.1.106) gene copies in WT and CD19−/− mice are shown. Two-tailed unpaired Student’s t-test; ns = p = 0.0651. D Correlation between 7α-dehydratase gene abundance and ileal BA concentrations is shown. Two-tailed one-way ANOVA, Fstat(1,20) = 0.13, p = ns. E Correlation between Lactobacillaceae abundance and bsh gene abundance is shown. Two-tailed one-way ANOVA, Fstat(1,23) = 33.48, p < 0.0001. F Heatmaps summarizing descriptive statistics (Pearson r-coefficient and r-squared) of relationship between ileal bacterial bsh gene abundance and specific BA species are shown. Data representative of two replicate experiments. G Correlation between TCA-d4 and CA-d4 abundance (μM) in fecal protein extracts (left panel) and ileal content protein extracts (right panel) from WT and CD19−/− mice. Two-tailed one-way ANOVA (WT fecal protein extracts), Fstat(1,13) = 18.71, p = 0.0008. Two-tailed one-way ANOVA (CD19−/− fecal protein extracts), Fstat(1,13) = 11.04, p = 0.006. Two-tailed one-way ANOVA (WT ileal protein extracts), Fstat(1,7) = 9.186, p = 0.02. Two-tailed one-way ANOVA (CD19−/− ileal protein extracts), Fstat(1,8) = 0.0006, p = 0.98. H Total abundance of deconjugated CA-d4 in fecal and ileal content protein extracts from WT and CD19−/− mice. Two-tailed unpaired Student’s t-test; ns = non-significant =p = 0.63, ***=p = 0.0002. I Total bsh gene copies in feces and ileal contents of CD19−/− mice administered B cells from CD19−/− or WT mice. Two-tailed unpaired Student’s t-test; ns = non-significant =p = 0.68, *=p = 0.04. J Correlation between SI enteropathy and bsh gene copies in feces or ileal contents of CD19−/− mice administered B cells from CD19−/− or WT mice. Two-tailed one-way ANOVA (feces), Fstat(1,13) = 0.13, p = ns. Two-tailed one-way ANOVA (ileal contents), Fstat(1,10) = 7.61, p = 0.01. A–E Data representative of three replicate experiments. G, H Data representative of two replicate experiments. I, J Data representative of two replicate experiments.
Fig. 7
Fig. 7. Microbial bsh activity drives BA defects and contributes to SI enteropathy in CD19−/− mice.
A Schematic overview of B.theta and Δbsh B.theta colonization experiments. B qPCR estimates of the relative abundance of B.theta, Δbsh B.theta, and total Bacteroides are shown. Sidak’s multiple comparisons test; B.theta, ns = p = 0.9972, Eubacteria, ns = p = 0.1483. C Ileal BA concentrations in B.theta-colonized and Δbsh B.theta-colonized CD19−/− mice are shown. Two-tailed unpaired Student’s t-test; *=p = 0.03. D Stacked bar-chart illustrating general shifts in BA abundance as measured by UPLC-MS. Source data are provided as a Source Data file. E The relative abundance of unconjugated and conjugated BAs in B.theta and Δbsh B.theta-colonized CD19−/− mice are shown. Sidak’s multiple comparisons test; ns = p = 0.9998, ***=p = 0.0007, ****=p < 0.0001. F SI enteropathy scoring is shown for CD19−/− mice colonized with WT B.theta (n = 7) or Δbsh B.theta (n = 8). Two-tailed unpaired Mann–Whitney U test; **=p = 0.002. G Comparison of individual features of SI enteropathy between CD19−/− mice colonized with WT B.theta or Δbsh B.theta. Two-tailed unpaired Mann–Whitney U test; ns = non-significant =p = 0.08, *=p = 0.04, **=p = 0.004, ***=p = 0.0008. B–G Data representative of two replicate experiments.
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