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.
Your institute does not have access to this article
Open Access articles citing this article.
BMC Pharmacology and Toxicology Open Access 21 April 2022
Analysis of gut microbiome profiles in common marmosets (Callithrix jacchus) in health and intestinal disease
Scientific Reports Open Access 15 March 2022
Microbiome Open Access 03 October 2021
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hao, L. Y., Liu, X. & Franchi, L. Inflammasomes in inflammatory bowel disease pathogenesis. Curr. Opin. Gastroenterol. 29, 363–369 (2013)
Ubeda, C. et al. Familial transmission rather than defective innate immunity shapes the distinct intestinal microbiota of TLR-deficient mice. J. Exp. Med. 209, 1445–1456 (2012)
Letran, S. E. et al. TLR5-deficient mice lack basal inflammatory and metabolic defects but exhibit impaired CD4 T cell responses to a flagellated pathogen. J. Immunol. 186, 5406–5412 (2011)
Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009)
Cadwell, K. et al. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell 141, 1135–1145 (2010)
Holmdahl, R. & Malissen, B. The need for littermate controls. Eur. J. Immunol. 42, 45–47 (2012)
Hooper, L. V., Littman, D. R. & Macpherson, A. J. Interactions between the microbiota and the immune system. Science 336, 1268–1273 (2012)
Spor, A., Koren, O. & Ley, R. Unravelling the effects of the environment and host genotype on the gut microbiome. Nature Rev. Microbiol. 9, 279–290 (2011)
Fujiwara, R., Watanabe, J. & Sonoyama, K. Assessing changes in composition of intestinal microbiota in neonatal BALB/c mice through cluster analysis of molecular markers. Br. J. Nutr. 99, 1174–1177 (2008)
Brandtzaeg, P. Secretory IgA: designed for anti-microbial defense. Front. Immunol. 4, 222 (2013)
Johansen, F. E. et al. Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice. J. Exp. Med. 190, 915–922 (1999)
Tsuji, M., Suzuki, K., Kinoshita, K. & Fagarasan, S. Dynamic interactions between bacteria and immune cells leading to intestinal IgA synthesis. Semin. Immunol. 20, 59–66 (2008)
Blutt, S. E. & Conner, M. E. The gastrointestinal frontier: IgA and viruses. Front. Immunol. 4, 402 (2013)
Murthy, A. K., Dubose, C. N., Banas, J. A., Coalson, J. J. & Arulanandam, B. P. Contribution of polymeric immunoglobulin receptor to regulation of intestinal inflammation in dextran sulfate sodium-induced colitis. J. Gastroenterol. Hepatol. 21, 1372–1380 (2006)
Reikvam, D. H. et al. Epithelial-microbial crosstalk in polymeric Ig receptor deficient mice. Eur. J. Immunol. 42, 2959–2970 (2012)
Brandtzaeg, P. & Prydz, H. Direct evidence for an integrated function of J chain and secretory component in epithelial transport of immunoglobulins. Nature 311, 71–73 (1984)
Brown, W. R., Newcomb, R. W. & Ishizaka, K. Proteolytic degradation of exocrine and serum immunoglobulins. J. Clin. Invest. 49, 1374–1380 (1970)
Lindh, E. Increased resistance of immunoglobulin A dimers to proteolytic degradation after binding of secretory component. J. Immunol. 114, 284–286 (1975)
Wexler, H. M. et al. Sutterella wadsworthensis gen. nov., sp. nov., bile-resistant microaerophilic Campylobacter gracilis-like clinical isolates. Int. J. Syst. Bacteriol. 46, 252–258 (1996)
Moon, C., Vandussen, K. L., Miyoshi, H. & Stappenbeck, T. S. Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis. Mucosal Immunol. 7, 818–828 (2014)
Plaut, A. G., Gilbert, J. V., Artenstein, M. S. & Capra, J. D. Neisseria gonorrhoeae and Neisseria meningitidis: extracellular enzyme cleaves human immunoglobulin A. Science 190, 1103–1105 (1975)
Loomes, L. M., Senior, B. W. & Kerr, M. A. A proteolytic enzyme secreted by Proteus mirabilis degrades immunoglobulins of the immunoglobulin A1 (IgA1), IgA2, and IgG isotypes. Infect. Immun. 58, 1979–1985 (1990)
Ichinohe, T. et al. Microbiota regulates immune defense against respiratory tract influenza A virus infection. Proc. Natl Acad. Sci. USA 108, 5354–5359 (2011)
Lee, Y. K., Menezes, J. S., Umesaki, Y. & Mazmanian, S. K. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc. Natl Acad. Sci. USA 108 (suppl. 1). 4615–4622 (2011)
Rakoff-Nahoum, S., Paglino, J., Eslami-Varzaneh, F., Edberg, S. & Medzhitov, R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241 (2004)
Williams, B. L., Hornig, M., Parekh, T. & Lipkin, W. I. Application of novel PCR-based methods for detection, quantitation, and phylogenetic characterization of Sutterella species in intestinal biopsy samples from children with autism and gastrointestinal disturbances. MBio 3, e00261–11 (2012)
Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl Acad. Sci. USA 108, 4516–4522 (2011)
Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7, 335–336 (2010)
Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010)
McDonald, D. et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618 (2012)
Segata, N. et al. Metagenomic biomarker discovery and explanation. Genome Biol. 12, R60 (2011)
Miyoshi, H. & Stappenbeck, T. S. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nature Protocols 8, 2471–2482 (2013)
Miyoshi, H., Ajima, R., Luo, C. T., Yamaguchi, T. P. & Stappenbeck, T. S. Wnt5a potentiates TGF-β signaling to promote colonic crypt regeneration after tissue injury. Science 338, 108–113 (2012)
Kang, S. S. et al. An antibiotic-responsive mouse model of fulminant ulcerative colitis. PLoS Med. 5, e41 (2008)
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671–675 (2012)
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.
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.
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.
a–d, 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 (a–c) 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. g–j, 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.
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.
a–c, 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). b–d, 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. e–g, 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).
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.
About this article
Cite this article
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
BMC Pharmacology and Toxicology (2022)
Analysis of gut microbiome profiles in common marmosets (Callithrix jacchus) in health and intestinal disease
Scientific Reports (2022)
Nature Reviews Immunology (2021)
Monocyte-derived dendritic cells link localized secretory IgA deficiency to adaptive immune activation in COPD
Mucosal Immunology (2021)