1. Immunology and Inflammation
  2. Microbiology and Infectious Disease
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The Crohn’s disease polymorphism, ATG16L1 T300A, alters the gut microbiota and enhances the local Th1/Th17 response

  1. Sydney Lavoie
  2. Kara L Conway
  3. Kara G Lassen
  4. Humberto B Jijon
  5. Hui Pan
  6. Eunyoung Chun
  7. Monia Michaud
  8. Jessica K Lang
  9. Carey Ann Gallini Comeau
  10. Jonathan M Dreyfuss
  11. Jonathan N Glickman
  12. Hera Vlamakis
  13. Ashwin Ananthakrishnan
  14. Aleksander Kostic  Is a corresponding author
  15. Wendy S Garrett  Is a corresponding author
  16. Ramnik J Xavier  Is a corresponding author
  1. Harvard T. H. Chan School of Public Health, United States
  2. Massachusetts General Hospital, United States
  3. Broad Institute of Harvard and MIT, United States
  4. Joslin Diabetes Center, United States
  5. Harvard Medical School, United States
  6. Beth Israel Deaconess Medical Center, United States
  7. Dana-Farber Cancer Institute and Harvard Medical School, United States
Research Article
Cite this article as: eLife 2019;8:e39982 doi: 10.7554/eLife.39982
9 figures, 1 table, 3 data sets and 2 additional files

Figures

Altered microbiota in conventionally-housed WT vs T300A mice.

12 week old WT and T300A mice were conventionally-housed in a specific pathogen free (SPF) facility. Stool samples were collected and analyzed by 16S rRNA gene amplicon sequencing. (a) Population analysis of 16S rRNA gene amplicon sequencing from stool samples from SPF WT vs. T300A mice. (p = phylum, c = class, o = order, f = family, g = genus, s = species) Bacteria Z-scores generated by limma. (b) t-Distributed Stochastic Neighbor Embedding (tSNE) plot analysis of WT vs. T300A SPF mouse stool samples. (c) Box plot analysis of the order Bacteroidales in WT vs. T300A SPF mouse stool samples.

https://doi.org/10.7554/eLife.39982.003
Figure 2 with 1 supplement
Altered microbiota in conventionally-housed WT vs T300A mice with gut inflammation.

12 week old WT and T300A conventionally-housed SPF mice were treated with 2.5% DSS for 7d followed by 7d of regular drinking water. (a) Population analysis of 16S rRNA gene amplicon sequencing from stool samples from WT vs. T300A mice. (p = phylum, c = class, o = order, f = family, g = genus, s = species) Bacteria Z-scores generated by limma. (b) t-Distributed Stochastic Neighbor Embedding (tSNE) plot analysis of WT vs. T300A stool samples post DSS treatment. (c) Box plot analysis of the phylum Bacteroidetes in WT vs. T300A mice post DSS treatment. (d) Box plot analysis of B. ovatus in WT vs. T300A mice post DSS treatment. (e) Percent initial body weight of WT (n = 12) and T300A (n = 15) mice treated with 2.5% DSS for 7d followed by 7d of regular drinking water. Day 8–13 time points, p = < 0.0001. Two-way ANOVA with Tukey‘s post-hoc test.

https://doi.org/10.7554/eLife.39982.004
Figure 2—figure supplement 1
T300A affects the microbiome gene function profile.

Significant alterations in gene function inferred from 16S rRNA gene amplicon sequencing data using PICRUSt from stool samples obtained from 12 wk old WT versus T300A conventionally-housed SPF mice treated with 2.5% DSS followed by 7d regular drinking water.

https://doi.org/10.7554/eLife.39982.005
Distinct gut microbiota composition in mice associated with human stool.

GF mice were associated with human stool samples and analyzed after four wks. (a) tSNE dimensionality reduction analysis based on genus-level abundances of stool samples from human patient stool donor (square) or GF WT (circles) and T300A (triangles) mice four wks post association with 50 mg human stool from patients: healthy control, genotype Het (yellow), inactive CD, genotype WT (blue), inactive CD, genotype T300A (green), active CD, genotype T300A (red), or active UC, genotype T300A (purple). Each symbol represents data from an individual mouse. (b) PCA plot of stool samples from WT vs. T300A mice four wks post association with either active CD (left) or active UC (right) human stool. Each symbol represents data from an individual mouse.

https://doi.org/10.7554/eLife.39982.007
T300A alters gut microbiota composition in GF mice associated with human stool.

Metagenomic population analysis (MetaPhlAn) on stool samples (four wk time point) from WT vs. T300A GF mice associated with human IBD stool samples (a) Box plot of the genus Bacteroides from stool samples from mice associated with active CD or active UC stool samples. (b) Box plot of B. ovatus from stool samples from mice associated with active CD or active UC stool samples. P-values were generated using Benjamini-Hochberg false discovery rate. (a, b) Y-axis is log10 relative abundance.

https://doi.org/10.7554/eLife.39982.008
Figure 5 with 1 supplement
The presence of T300A in humans increases Bacteroides.

16S rRNA gene amplicon data from human stool samples from Jostins et al. 2012 (Jostins et al., 2012) cohort were analyzed for differences in the microbiota associated with the presence (T300A – GG), absence (WT – AA) or heterozygous (Het – AG) genotype. (g = genus, s = species). (a) Relative abundance of Bacteroides. (b) Relative abundance of B. fragilis. (c) Relative abundance of B. caccae.

https://doi.org/10.7554/eLife.39982.009
Figure 5—figure supplement 1
The presence of T300A in humans reduces Clostrida spp. and increases Gammaproteobacteria.

16S rRNA gene amplicon data from human stool samples from Jostins et al. 2012 (Xavier and Podolsky, 2007) cohort were analyzed for differences in the microbiota associated with the presence (T300A – GG), absence (WT – AA) or heterozygous (Het – AG) genotype. (g = genus). (a) Relative abundance of Roseburia. (b) Relative abundance of Blautia. (c) Relative abundance of Oscillospira. (d) Relative abundance of Haemophilus.

https://doi.org/10.7554/eLife.39982.010
Figure 6 with 3 supplements
Alterations in T cell and myeloid cell populations in the gut of T300A mice associated with stool from patients with active Crohn’s disease.

LP flow cytometry of T and myeloid cell populations from GF mice associated with 50 mg of frozen stool from a patient with Crohn’s disease (CD, WT n = 6 and T300A n = 6) or ulcerative colitis (UC, WT n = 7 and T300A n = 5) for four wks. T cells gated as the frequency of RORγt+ (Th17), T-bet+ (Th1), Foxp3+ (Treg) or GATA-3+ (Th2) cells out of CD3+CD4+ cells and myeloid cells gated as the frequency of CD11c+MHCII+ cells out of CD45+ cells (conventional DCs), CD11b-CD103+ cells out of CD11c+MHCII+ cells (tolerogenic DCs), CD11b+GR-1int cells out of CD45+ cells (neutrophils), and CD11b+GR-1hi cells out of CD45+ cells (inflammatory monocytes). CD3+CD4+ and myeloid cell populations were gated on single, live, CD45+ lymphocytes. (a) GF +CD stool colonic LP T cells (top) and myeloid cells (bottom) (b) GF +UC stool colonic LP T cells (top) and myeloid cells (bottom). (c) GF +CD stool ileal LP T cells (top) and myeloid cells (bottom) (d) GF +UC stool ileal LP T cells (top) and myeloid cells (bottom). Data are plotted as the mean ± SEM. ns = not significant. Mann-Whitney U test.

https://doi.org/10.7554/eLife.39982.011
Figure 6—figure supplement 1
Representative T cell flow cytometry gating strategy.

LP cells from either the colon or ileum (distal 10 cm of the small intestine) were gated on single cells, live cells, CD45+, lymphocytes, CD3+CD4+ and finally T-bet+ out of CD3+CD4+ and RORγt+ out of CD3+CD4+.

https://doi.org/10.7554/eLife.39982.012
Figure 6—figure supplement 2
Representative myeloid cell flow cytometry gating strategy.

LP cells from either the colon or ileum (distal 10 cm of the small intestine) were gated on single cells, live cells and CD45+. CD11c+MHCII+ (1 - conventional DCs) were gated out of CD45+ cells and CD11b-CD103+ cells (2 - tolerogenic DCs) were gated out of CD11c+MHCII+ cells. CD11b+GR-1hi (3 - inflammatory monocytes) and CD11b+GF-1int (4 - neutrophils) were gated out of CD45+.

https://doi.org/10.7554/eLife.39982.013
Figure 6—figure supplement 3
Microbiota changes and immune infiltration occur in the absence of disease.

WT and T300A gnotobiotically housed mice were associated with human stool samples from patients with either active CD (WT n = 6, T300A n = 6) or active UC (WT n = 7, T300A n = 5). Histologic colitis/ileitis scores were calculated by grading monocyte infiltration, epithelial hyperplasia, epithelial injury and polymorphonuclear (PMN) cell infiltration, all from 0 to 3 for a maximum total score of 12. Histologic colitis scores from (a) the colon and (b) the ileum from mice associated with stool from patients with active CD or active UC. Data are plotted as mean ± SEM.

https://doi.org/10.7554/eLife.39982.014
No change in immune cell population frequency in the gut of GF or GF mice monoassociated with B. ovatus 8483.

LP flow cytometry of T and myeloid cell populations from GF mice (WT n = 6, T300A n = 6) or GF mice associated with ~109 CFU of B. ovatus 8483 for three wks (WT n = 6, T300A n = 6). T cell populations are gated as the frequency of RORγt+ (Th17), T-bet+ (Th1), Foxp3+ (Treg) or GATA-3+ (Th2) cells out of CD3+CD4+ cells and myeloid cell populations are gated as the frequency of CD11c+MHCII+ cells out of CD45+ cells (conventional DCs), CD11b-CD103+ cells out of CD11c+MHCII+ cells (tolerogentic DCs), CD11b+GR-1int cells out of CD45+ cells (neutrophils), and CD11b+GR-1hi cells out of CD45+ cells (inflammatory monocytes). CD3+CD4+ and myeloid cell populations were gated on single, live, CD45+ lymphocytes. (a) GF colonic LP T cells (top) and myeloid cells (bottom) (b) GF +B. ovatus 8483 colonic LP T cells (top) and myeloid cells (bottom). (c) GF ileal LP T cells (top) and myeloid cells (bottom) (d) GF +B. ovatus 8483 ileal LP T cells (top) and myeloid cells (bottom). Data are plotted as the mean ± SEM. ns = not significant. Mann-Whitney U test.

https://doi.org/10.7554/eLife.39982.016
Figure 8 with 2 supplements
Increased frequency of Th17 cells in the gut of T300A ASF mice associated with B. ovatus 8483.

LP flow cytometry of T cell and myeloid cell populations from ASF mice (WT n = 6 and T300A n = 6) or ASF mice associated with ~109 CFU of B. ovatus 8483 for 3 weeks (WT n = 6 and T300A n = 6). T cells gated as the frequency of RORγt+ (Th17), T-bet+ (Th1), Foxp3+ (Treg) or GATA-3+ (Th2) cells out of CD3+CD4+ cells and myeloid cell gated as the frequency of CD11c+MHCII+ cells out of CD45+ cells (conventional DCs), CD11b-CD103+ cells out of CD11c+MHCII+ cells (tolerogentic DCs), CD11b+GR-1int cells out of CD45+ cells (neutrophils), and CD11b+GR-1hi cells out of CD45+ cells (inflammatory monocytes). CD3+CD4+ cell and myeloid cell populations were gated on single, live, CD45+ lymphocytes. (a) ASF colonic LP T cells (top) and myeloid cells (bottom) (b) ASF +B. ovatus 8483 colonic LP T cells (top) and myeloid cells (bottom). (c) ASF ileal LP T cells (top) and myeloid cells (bottom) (d) ASF +B. ovatus 8483 ileal LP T cells (top) and myeloid cells (bottom). Data are plotted as the mean ± SEM. ns = not significant. Mann-Whitney U test.

https://doi.org/10.7554/eLife.39982.018
Figure 8—figure supplement 1
Immune infiltration in ASF mice associated with B. ovatus occurs in the absence of disease.

WT and T300A ASF mice were associated ~109 CFU of B. ovatus 8483 for 3 weeks (WT n = 6, T300A n = 6). Histologic colitis/ileitis scores were calculated by grading monocyte infiltration, epithelial hyperplasia, epithelial injury and polymorphonuclear (PMN) cell infiltration, all from 0 to 3 for a maximum total score of 12. Histologic colitis/ileitis scores from (a) the colon and (b) the ileum of ASF mice or ASF mice associated with B. ovatus 8483. Data are plotted as mean ± SEM.

https://doi.org/10.7554/eLife.39982.019
Figure 8—figure supplement 2
No differences in colonization levels of B. ovatus 8483 in GF or ASF mice.

GF mice (WT n = 6, T300A n = 6) or ASF mice (n = 6, T300A n = 6) were associated with ~109 CFU of B. ovatus 8483 for three wks. Fecal samples were taken at the three wk timepoint and CFU/g stool was calculated. ns = not significant. Two-way ANOVA with Tukey’s post-hoc test.

https://doi.org/10.7554/eLife.39982.020
Increased expression of Il23p19 in the lamina propria from the colon and ileum of T300A ASF mice associated with B. ovatus 8483.

Expression of Il23p19 by qPCR from colon (a) and ileum (b) LP cells isolated from ASF T300A mice or ASF T300A mice associated with B. ovatus 8483.

https://doi.org/10.7554/eLife.39982.022

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional information
Genetic reagent (M. musculus)T300APMID: 24821797Dr. Ramnik Xavier (Broad/MIT/Harvard)
Chemical compound, drugDextran Sulfate SodiumMP Biomedicals0216011050–50 g
, strain back groundBacteroides ovatusATCC8483
Software, algorithmPicard suitehttps://broadinstitute.github.io/picard/command-line-overview.htmlRRID:SCR_006525
Software, algorithmMetaPhlAnPMID 26418763RRID:SCR_004915
Software, algorithmHUMAnN2http://huttenhower.sph. harvard.edu/humann2RRID:SCR_016280
Software, algorithmRtsnehttps://cran.r-project.org/web/packages/Rtsne/index.htmlRRID:SCR_016342
Software, algorithmPICRUStPMID 23975157RRID: SCR_016856
Software, algorithmHUMAnNPMID 22719234RRID:SCR_014620
Software, algorithmPrism 7.0bhttps://www.graphpad.com/scientific-software/prism/RRID:SCR_002798
Software, algorithmlimmaPMID 25605792RRID:SCR_010943
Software, algorithmQIIMEPMID:
20383131
RRID:SCR_008249
AntibodyRat anti mouse CD16/32 (clone 93)BiolegendCat. #: 101310, RRID:AB_2103871Flow cytometry (FC), FC receptor block 1:100
AntibodyRat anti mouse CD45 (clone 30-F11)BiolegendCat. #: 103114, RRID:AB_312979FC, PE/Cy7 1:100
AntibodyArmenian hamster anti mouse CD3e (clone 145–2 C11)BiolegendCat. #: 100334, RRID:AB_2028475FC, Pacific Blue 1:100
AntibodyRat anti mouse CD4 (clone GK1.5)BiolegendCat. #: 100414, RRID:AB_312981FC, APC/Cy7 1:100
AntibodyMouse anti mouse GATA-3 (clone L50-823)BD BiosciencesCat. #: 560077, RRID:AB_1645303FC, Alexa Fluor 488 1:33
AntibodyMouse anti mouse Foxp3 (clone 150D)BiolegendCat. #: 320007, RRID:AB_492981FC, PE 1:40
AntibodyRat anti mouse RORgt (clone B2D)InvitrogenCat. #: 17-6981-82, RRID:AB_2573254FC, APC 1:40
AntibodyMouse anti mouse T-bet (clone 4B10)BiolegendCat. #: 644806, RRID:AB_1595488FC, PerCP/Cy5 1:33
AntibodyRat anti mouse CD45 (clone 30-F11)BiolegendCat. #: 103126, RRID:AB_493535FC, Pacific Blue 1:100
AntibodyArmenian hamster anti mouse CD11c (clone N418)BiolegendCat. #: 117306, RRID:AB_313775FC, FITC 1:200
AntibodyRat anti mouse CD11b (clone M1/70)BiolegendCat. #: 101228, RRID:AB_893232FC, PerCP/Cy5 1:200
AntibodyRat anti mouse I-A/I-E (MHCII) (clone M5/114.15.2)BiolegendCat. #: 107630, RRID:AB_2069376FC, PE/Cy7 1:200
AntibodyRat anti mouse Ly-6G/Ly-6C (GR-1) (clone RB6-8C5)BiolegendCat. #: 108412, RRID:AB_313377FC, APC 1:200
AntibodyArmenian hamster anti mouse CD103 (clone 2E7)InvitrogenCat. #: 12-1031-82, RRID:AB_465799FC, PE 1:100
CommercialAssay or KitLive/Dead Fixable Yellow Dead Cell StainThermo Fisher ScientificCat. #: L34959FC
Commercial Assay or KitQuant-iT PicoGreen dsDNA Assay KitThermo Fisher ScientificCat. #: P11496DNA quantification
Commercial Assay or KitNextera XT DNA Library Preparation KitIlluminaCat. #: FC-131–1096Sequencing libraries
Commercial Assay or KitAgilent DNA 1000 KitAgilent TechnologiesCat. #: 5067–1504Insert sizes
OtherRNAlaterSigmaCat. #: R0901-500MLSolution for stool sample collection
Commercial Assay or KitQIAamp 96 PowerFecal QIAcube HT Kit (5)QIAGENCat. #: 51531Stool DNA extraction
Commercial Assay or KitAllprep DNA/RNA 96 KitQIAGENCat. #: 80311Stool DNA extraction
OtherIRS solutionQIAGENCat. #: 26000-50-2Solution for stool DNA extraction
Commercial
Assay or Kit
PowerBead Plates, GlassQIAGENCat. #:27500–4-EP-BPStool DNA extraction
OtherBrucella agar sheeps blood/hemin/vitamin K platesHardy DiagnosticsCat. #: A30Bacterial culture plates
Commercial Assay or KitRneasy Mini KitQIAGENCat. #: 74106RNA isolation
Commercial Assay or KitiScript cDNA synthesis kitBio-RadCat. #:1708891cDNA synthesis

Data availability

Human microbiome sequence data are available as noted in Jostins et al. 2012 and requests for access can be made via the NCBI Genotypes and Phenotypes database (additional details here https://dbgap.ncbi.nlm.nih.gov/aa/wga.cgi?page=login). Mouse microbiome data have been submitted for deposit at NCBI sequence read archive SUB4222585.

The following data sets were generated
  1. 1
    NCBI Sequence Read Archive
    1. Kostic Aleksander
    (2018)
    ID 4222585. Illumina HiSeq 2000 sequencing of SAMD00080972.
The following previously published data sets were used
  1. 1
    NCBI Genotypes and Phenotypes database
    1. Cho Judy
    (2008)
    ID phs000130.v1.p1. NIDDK IBDGC Crohn's Disease Genome-Wide Association Study.
  2. 2
    NCBI Genotypes and Phenotypes database
    1. Cho Judy
    (2012)
    ID phs000345.v1.p1. NIDDK IBD Genetics Consortium Ulcerative Colitis Genome-Wide Association Study.

Additional files

Supplementary file 1

Mouse caging and metadata for Figures 18.

Mouse genotype, age (wks), sex, cage, experiment description and figure are listed above. Mice housed together have the same cage number.

https://doi.org/10.7554/eLife.39982.024
Transparent reporting form
https://doi.org/10.7554/eLife.39982.025

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