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Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation

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Abstract

The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control1,2,3,4,5. In many cases, the microbiota is the presumed cause of the variation. Current solutions to account for phenotypic variability include littermate and maternal controls or defined microbial consortia in gnotobiotic mice6,7. In conventionally raised mice, the microbiome is transmitted from the dam2,8,9. Here we show that microbially driven dichotomous faecal immunoglobulin-A (IgA) levels in wild-type mice within the same facility mimic the effects of chromosomal mutations. We observe in multiple facilities that vertically transmissible bacteria in IgA-low mice dominantly lower faecal IgA levels in IgA-high mice after co-housing or faecal transplantation. In response to injury, IgA-low mice show increased damage that is transferable by faecal transplantation and driven by faecal IgA differences. We find that bacteria from IgA-low mice degrade the secretory component of secretory IgA as well as IgA itself. These data indicate that phenotypic comparisons between mice must take into account the non-chromosomal hereditary variation between different breeders. We propose faecal IgA as one marker of microbial variability and conclude that co-housing and/or faecal transplantation enables analysis of progeny from different dams.

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Figure 1: Low faecal IgA in WT mice is a vertically and horizontally transferable, dominant phenotype driven by ampicillin-sensitive bacteria.
Figure 2: The IgA-low phenotype alters susceptibility to DSS in an IgA-dependent manner.
Figure 3: The IgA-low phenotype correlates with an absence of faecal secretory component and the presence of specific microbial taxa in vivo, which can be anaerobically cultured in vitro.
Figure 4: Culturable anaerobic bacteria present in IgA-low samples degrade secretory component.

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European Nucleotide Archive

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16S rDNA sequencing data have been deposited in the European Nucleotide Archive under accession number PRJEB7854.

Change history

  • 06 May 2015

    The footnote symbols in Fig. 2d and Tukey’s P-value in the Figs 1-4 legends were corrected.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) grants AI08488702 and DK7161907, the Crohn’s & Colitis Foundation of America Genetics Initiative, the Rainin Foundation, and the Helmsley Charitable Trust. C.M. was supported by NIH training grant T32AI007163, and M.T.B. was supported by NIH training grant T32CA009547 and the W.M. Keck Fellowship from Washington University. We thank H. Miyoshi for technical recommendations, D. Kreamalmeyer for animal care and breeding, and members of the Stappenbeck and Virgin laboratories for discussion. Experimental support was provided by the Speed Congenics Facility of the Rheumatic Diseases Core Center (NIH award number P30AR048335) and the Digestive Disease Research Core Center (NIH award number P30DK052574) of Washington University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors

Contributions

M.T.B. and C.M. designed the project, performed experiments, and wrote the paper. T.S.S. and H.W.V. assisted with project design and writing the paper. C.D.B. and M.A.W. assisted with microbial characterization and project design.

Corresponding authors

Correspondence to Herbert W. Virgin or Thaddeus S. Stappenbeck.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 WT mice within two independent facilities exhibit binary faecal IgA levels, and the IgA-low phenotype is transferable between these facilities.

a, Faecal IgA (normalized to faecal weight) from mice housed in either facility 1 (n = 28 IgA-high and n = 22 IgA-low mice) or facility 2 (n = 12 mice per group) was detected by anti-mouse IgA ELISA. b, c, WT IgA-high mice from one mouse facility were transplanted with homogenized faecal material from WT IgA-high or IgA-low mice from the other mouse facility, and faecal IgA was measured 14 days later by anti-mouse IgA ELISA. b, Facility 1 mice pre- (n = 18 mice) and post-faecal transplantation with facility 2 faecal samples (n = 8 post-IgA-high and n = 10 post-IgA-low mice). c, Facility 2 mice pre- (n = 10 mice) and post-faecal transplantation with facility 1 faecal samples (n = 4 post-IgA-high and n = 6 post-IgA-low mice). The dotted lines represent the limit of detection by ELISA. All values are mean ± s.e.m. One-way ANOVA: a, F = 44.59, P < 0.0001; b, F = 20.93, P < 0.0001; c, F = 12.92, P = 0.0004. Means with different footnote symbols are significantly different by Tukey’s multiple comparison test (P < 0.5).

Extended Data Figure 2 IgA-high- and IgA-low-associated microbes can be stably passaged through pIgR−/− recipients, and are vertically transmissible after recolonization.

a, Schematic for repopulation of pIgR−/− microbiota with WT IgA-high/IgA-low samples, followed by faecal transplantation (FT) of pIgR−/− IgA-high or IgA-low samples to WT IgA-high mice. b, Faecal IgA on day 44 depicted in a. Mann–Whitney U-test: P = 0.0006, n = 8 mice per group. c, Experimental schematic of antibiotic treatment and transplant protocol for d and Fig. 1f, g. d, Faecal IgA of post-FT mice on day 30 pre-treated with metronidazole (Metro). One-way ANOVA: F = 6.525, P = 0.0012, n = 13 (pre-Metro), n = 15 (post-Metro), n = 8 (post-IgA-high FT), and n = 5 (post-IgA-low FT). All values are mean ± s.e.m. e, IgA-low mice converted to IgA-high from Fig. 1f were mated, and faecal IgA of their adult progeny was measured. One-way ANOVA: F = 18.29, P = 0.0002, n = 2 breeders, n = 10 progeny from four litters. Different footnote symbols indicate groups significantly different by Tukey's multiple comparison test (P < 0.5). Dotted lines: limit of detection.

Extended Data Figure 3 DSS effects on pIgR−/− mice are dependent on IgA and not microbes.

a, Faecal IgA levels were measured in WT mice from Fig. 2d, e after VNAM treatment and IgA-high/IgA-low faecal transplantation (FT), before the start of DSS treatment. Statistical analysis by Mann–Whitney U-test: P = 0.0006, n = 7 mice per group. b, Representative haematoxylin and eosin-stained histological sections of WT and pIgR−/− mice from Fig. 2d, e after 14 day VNAM treatment + IgA-high/IgA-low faecal transplantation. Representative of n = 3 (WT + IgA-high), n = 6 (WT + IgA-low), n = 8 (pIgR−/− + IgA-high), and n = 10 (pIgR−/− + IgA-low) mice. All values indicated as mean ± s.e.m. Means with different footnote symbols are significantly different by Tukey’s multiple comparison test (P < 0.5). Dotted lines, limit of detection.

Extended Data Figure 4 Plasma cell numbers and pIgR expression are unchanged in the ileum and colon between IgA-high and IgA-low mice.

ad, Ileal and colonic sections from IgA-high and IgA-low mice were stained with anti-IgA (green) and bis-benzamide dye (blue); representative ×20 images are shown of n = 10 (ac) or n = 9 mice (d). Scale bars, 100 μm. e, f, Quantification of ileal plasma cells per villus (e) and colonic plasma cells per ×20 field (area = 1.5 μm × 105 μm) (f) based on IgA staining. All values are mean ± s.e.m. Statistical analysis by Mann–Whitney U-test: e, P = 0.5191, n = 10 mice per group; f, P = 0.3117, n = 10 IgA-high and n = 9 IgA-low mice. gj, Ileal and colonic sections from IgA-high and IgA-low mice were stained with anti-pIgR/secretory component (red) and bis-benzamide dye (blue); representative images are shown (n = 10 mice per group). Scale bars, 100 μm.

Extended Data Figure 5 16S rDNA sequencing identifies biomarkers for IgA-low and IgA-high samples.

a, b, LEfSe analysis31 of 16S rDNA sequencing of IgA-low and IgA-high faecal samples from facilities 1 and 2 identified statistically significant bacterial taxa biomarkers for (a) IgA-low and (b) IgA-high samples. Biomarkers for facility 1 and biomarkers for facility 2 alone were identified by comparison of IgA-high and IgA-low samples within each facility. Biomarkers for facilities 1 and 2 were identified by comparison of all IgA-high and IgA-low samples from both facilities. No IgA-high biomarkers were identified when comparing all IgA-high and IgA-low samples from both facilities. Biomarkers for the indicated groups are plotted as taxonomic trees with GraPhlAn (http://huttenhower.sph.harvard.edu/graphlan); n = 13 (facility 1 IgA-high), n = 14 (facility 1 IgA-low), n = 73 (facility 2 IgA-high), and n = 68 (facility 2 IgA-low) samples. Statistical analysis is shown in Extended Data Table 1.

Extended Data Figure 6 Sutterella is more abundant in IgA-low samples than IgA-high samples in both facilities.

ac, Relative abundance of sequences assigned by QIIME to the bacterial genus Sutterella from 16S rDNA analysis in (a) facility 1 and (b) facility 2. These results are summarized in c. One-way ANOVA: F = 12.85, P < 0.0001. n = 13 (facility 1 IgA-high), n = 14 (facility 1 IgA-low), n = 73 (facility 2 IgA-high), and n = 68 (facility 2 IgA-low) samples. Values in c are indicated as mean ± s.e.m. Means with different footnote symbols are significantly different by Tukey’s multiple comparison test (P < 0.5).

Extended Data Figure 7 IgA-low cultured bacteria can degrade free secretory component in the absence of IgA, and secretory-component-degrading properties of these bacteria are active after freeze/thaw.

Primary intestinal epithelial Transwell monolayers were pre-treated with 10 μM DAPT + 1 μg ml−1 LPS on days 1 and 2 post-seeding to induce differentiation and pIgR expression. Some wells were left untreated as negative controls. On day 3 post-seeding, either 3 μg of normal mouse dimeric IgA or media alone was added to the lower compartment of the Transwells. Different subsets of the DAPT + LPS-treated Transwells were also treated with one of the following conditions in the apical compartment: IgA-high/IgA-low bacterial cultures (pelleted bacterial or supernatant fraction), live or freeze/thawed IgA-high/IgA-low bacterial cultures (pelleted bacterial fraction). Apical Transwell supernatants were collected at 3 h and 6 h, and the amount of secretory component was measured by anti-secretory-component immunoblot. a, Representative anti-pIgR/secretory component and anti-actin immunoblots of intestinal epithelial monolayers at 6 h (one of three experiments). bd, Secretory component degradation in the absence of IgA. (b) Representative anti-secretory-component immunoblot and quantification of undegraded secretory component (denoted by the red brackets) at 3 h (c) and 6 h (d) over four independent experiments by ImageJ. eg, Secretory component degradation by freeze/thawed bacterial cultures. e, Representative anti-secretory-component immunoblot and quantification of undegraded secretory component at 3 h (f) and 6 h (g) over five independent experiments by ImageJ. All values are mean ± s.e.m. One-way ANOVA: c, F = 1.834, P = 0.1831, n = 4 independent experiments with DAPT + LPS, no IgA repeated twice; d, F = 23.96, P = 0.0002, n = 4 independent experiments with DAPT + LPS, no IgA repeated twice; f, F = 7.444, P = 0.0045, n = 5 independent experiments with untreated and DAPT + LPS repeated three times; g, F = 31.53, P < 0.0001, n = 5 independent experiments with untreated and DAPT + LPS repeated three times. Means with different footnote symbols are significantly different by Tukey’s multiple comparison test (P < 0.5).

Extended Data Figure 8 IgA-low cultured bacteria can degrade IgA.

Primary intestinal epithelial cell monolayers were pre-treated with 10 μM DAPT + 1 μg ml−1 LPS on days 1 and 2 post-seeding to induce differentiation and pIgR expression. Some wells were left untreated as negative controls. On day 3 post-seeding, 3 μg of normal mouse IgA was added to the lower compartment of the Transwells. Different subsets of the DAPT + LPS-treated Transwells were also treated with combinations of the following in the apical compartment: live IgA-low bacterial cultures (either the pelleted bacterial or supernatant fraction), freeze/thawed IgA-low bacterial cultures, and a 1× protease inhibitor (PI) cocktail. Apical Transwell supernatants were collected at 3 h (a) and 6 h (b), and the amount of IgA was measured by anti-mouse IgA ELISA. The dotted lines represent the limit of detection by ELISA. All values are mean ± s.e.m. One-way ANOVA: a, F = 26.32, P < 0.0001, n = 8 (untreated), n = 8 (DAPT + LPS), n = 6 (IgA-low culture, pellet), n = 3 (IgA-low culture, supernatant), n = 4 (IgA-low culture, freeze/thawed), and n = 4 (IgA-low culture, +PI); b, F = 35.57, P < 0.0001, n = 8 (untreated), n = 8 (DAPT + LPS), n = 6 (IgA-low culture, pellet), n = 3 (IgA-low culture, sup), n = 3 (IgA-low culture, freeze/thawed), and n = 4 (IgA-low culture, +PI). Means with different footnote symbols are significantly different by Tukey’s multiple comparison test (P < 0.5); ND, not detected.

Extended Data Table 1 16S rDNA analysis of faecal samples from two facilities identified discriminant biomarkers between IgA-high and IgA-low samples
Extended Data Table 2 IgA-low sample cultures enrich for unique bacterial taxa compared with cultures from IgA-high samples

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Moon, C., Baldridge, M., Wallace, M. et al. Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation. Nature 521, 90–93 (2015). https://doi.org/10.1038/nature14139

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