Research ArticleMUCOSAL IMMUNOLOGY

Microbial antigen encounter during a preweaning interval is critical for tolerance to gut bacteria

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Science Immunology  15 Dec 2017:
Vol. 2, Issue 18, eaao1314
DOI: 10.1126/sciimmunol.aao1314
  • Fig. 1 Bacterial antigen encounter occurs during a specific preweaning interval, is dependent on GCs, and correlates with the presence of colonic GAPs.

    (A) Fold increase in CBir1 T cells after ex vivo culture with LP-MNPs isolated from the SI or colon on DOL7, DOL14, DOL21, or DOL28. (B) Percentage of (CD45.1+) CBir1 T cells of the total CD4+ T cells or (C) percentage of proliferating CBir1 T cells of the total CD45.1+ CBir1 T cells within the SI- or colon-draining MLNs 3 days after transfer into recipient CD45.2 mice on DOL7, DOL17, or DOL27. (D) Percentage of (CD45.1+) DP1 T cells of the CD4+ T cells within the MLNs 3 days after transfer. (E) Percentage of CD45.1 cells of the total CD4+CD3+ T cell population or (F) percentage of Foxp3+ cells of the CD45.1+ cells in the colon LP 7 days after transfer of CD45.1+ CBir1, DP1, or OTII T cells into recipient mice not receiving OVA. (G) Percentage of LP-MNPs staining for luminally administered Ova-647 on DOL7, DOL14, DOL21, or DOL28. (H) FITC-dextran (4 kDa) in serum after gavage in DOL18 GC knockout and littermate control mice. (I) Percentage of LP-MNPs staining for luminally administered Ova-647 in DOL18 GC knockout and littermate control mice. (J) Percentage of CD45.1+ CBir1 cells of CD4+ T cells within the MLNs 3 days after transfer into DOL18 GC knockout and littermate control mice. (K) Ratio of GAPs per GC in the SI and colon from DOL7 to DOL60. *P < 0.001; ns, not significant; n = 4 mice per group. Experiments in (B), (C), (G), (H), and (K) were repeated two independent times.

  • Fig. 2 The microbiota inhibits colonic GAPs and antigen delivery after weaning.

    (A) Expression of TLRs 1 to 9 (labeled by the number), Myd88 (M), and EGFR (E) on fluorescence-activated cell sorting (FACS)–sorted GCs from the SI (blue) or colon (red) of DOL18 or DOL56 mice. (B) Ratio of GAPs per GCs in the colon of DOL18 SPF-housed mice, germ-free (GF)–housed mice, SPF-housed Myd88−/− mice, or SPF-housed mice lacking Myd88 in GCs, with or without luminal heat-killed cecal contents from a DOL56 SPF-housed mice. (C) Ratio of GAPs per GC in the colon of DOL18 mice after luminal LPS with or without inhibition of EGFR (EGFRi) or p42/p44 MAPK (MAPKi) activation. (D) Quantification of 16S ribosomal RNA (rRNA) in the cecal contents from DOL7 to DOL60. (E) Number of 16S rRNA sequences grouped by bacteria class across the first 28 DOLs. Ratio of GAPs per GC in the (F) SI and (G) colon of Myd88f/fMath1PGRCre mice lacking Myd88 in GCs or Cre-negative littermates on DOL8, DOL18, or DOL28. nd, not detected; *P < 0.05; #, not detected; n = 4 mice per group. Experiments in (A) were repeated three independent times, and experiments in (B), (C), (F), and (G) were repeated two independent times.

  • Fig. 3 EGFR activation in GCs inhibits GAP formation and luminal antigen delivery throughout life.

    (A) GAPs per crypt or villus cross section in the SI (blue) or colon (red) of SPF-housed mice in the presence of tropicamide (t) or CCh (c) treatment on DOL8, DOL18, or DOL28. (B) Immunofluorescence staining of phosphorylated EGFR (pEGFR; red) and cytokeratin 18 (CK18; green) in colon sections from DOL8, DOL18, and DOL28 mice; 4′,6-diamidino-2-phenylindole nuclear stain (blue). (C) Amount of phosphorylated EGFR in the colon epithelium of DOL8, DOL18, or DOL28 mice measured by ELISA. (D) GAPs per crypt (colon) or villus (SI) cross section in DOL8, DOL18, or DOL28 SPF-housed mice in the presence or absence of EGFR inhibition (EGFRi). (E) Ratio of GAPs per GC and (F) percentage of LP-MNPs staining with luminal Ova-647 in the SI and colon of DOL7, DOL14, DOL21, or DOL28 mice lacking EGFR in GCs or Cre-negative littermate controls. (G to J) Mice were treated with vehicle or inhibition of EGFR activation (EGFRi) on DOL14 and DOL16 or DOL24 and DOL26 and LP-MNPs isolated or TCR transgenic T cells adoptively transferred on DOL18 or DOL28, respectively. (G) Fold increase in CBir1 transgenic T cells after ex vivo culture with LP-MNPs from the colon isolated on DOL18 or DOL28. (H) Percentage of CD45.1+ CBir1 or DP1 cells of the total CD4+ T cells in the MLNs 3 days after transfer into recipient mice on DOL18 or DOL28. (I) Percentage of naïve (CD62L+) CBir1 or DP1 cells in the MLNs 3 days after transfer into recipient mice on DOL18 or DOL28. (J) Percentage of Foxp3+ CBir1 or DP1 cells in the colon LP 7 days after transfer into recipient mice on DOL18 or DOL28. *P < 0.05; n = 4 mice per group. Experiments in (A) to (D) and (H) were repeated three independent times, and experiments in (E) to (G), (I), and (J) were repeated two independent times.

  • Fig. 4 Luminal EGF inhibits GAPs and antigen delivery before weaning.

    (A) Concentration of EGF in the luminal contents of the stomach, SI, or colon on DOL7, DOL14, or DOL21. (B) Concentration of EGF in the luminal contents, (C) amount of phosphorylated EGFR in the epithelium, or (D) amount of phosphorylated p42/p44 MAPK in the epithelium of SI segments or colon of DOL14 mice measured by ELISA; SI segments: 1 = 0 to 6 cm, 2 = 6 to 12 cm, 3 = 12 to 18 cm, 4 = 18 to 24 cm measured from the pylorus. (E) Ratio of GAPs per GC after incubation with stomach contents (SC) from DOL10 mice or recombinant EGF (EGF) in the presence or absence of EGFR inhibition (EGFRi) or p42/p44MAPK inhibition (MAPKi). (F) Fold increase in OVA-specific OTI T cells after culture with colonic LP-MNPs isolated from DOL18 mice lacking EGFR in GCs or Cre-negative littermate controls given luminal OVA or PBS and treated with intracolonic EGF or tropicamide (Trop; intraperitoneally) or untreated (control). (G) Percentage of CBir1 T cells of the total CD4+ T cells in the MLNs 3 days after cell transfer into DOL18 in mice that received intracolonic EGF (1 μg) or PBS daily from DOL10 to DOL21. *P < 0.05; n = 4 mice per group. Experiments in (A) to (D), (F), and (G) were repeated two independent times. Experiment in (E) was repeated three independent times.

  • Fig. 5 Inhibiting or altering the timing of microbial antigen encounter results in inflammatory T cell responses against gut bacteria.

    (A) Percentage of CD45.1+ CBir1 T cells of the total colon LP CD4+ T cells or percentage of (B) Foxp3+ or (C) interleukin 17–positive (IL-17+), tumor necrosis factor–α–positive (TNFα+), or interferon-γ–positive (IFNγ+) CBir1 T cells among all CBir1 T cells in the colon LP after transfer on DOL16 and analysis on DOL30 in mice treated with intracolonic (ic) vehicle (PBS) or EGF on DOL10 to DOL21. (D) Percentage of CD45.1+ CBir1 T cells of the total colon LP CD4+ T cells or percentage of (E) Foxp3+ or (F) IL-17+, TNFα+, or IFNγ+ CBir1 T cells among all CBir1 T cells in the colon LP after transfer on DOL16 and analysis on DOL30 in mice treated with inhibition of EGFR activation (EGFRi) on DOL14 and DOL16, or on DOL24 and DOL26. *P < 0.05; n = 4 mice per group.

  • Fig. 6 Inhibiting or altering the timing of microbial antigen encounter results in inflammatory T cell responses against gut bacteria and worsened colitis upon epithelial damage.

    (A to F) Mice were given intracolonic PBS or EGF on DOL10 to DOL21 or (G to L) mice were treated with inhibition of EGFR activation (EGFRi) on DOL14 and DOL16 or DOL24 and DOL26, CBir1 T cells were adoptively transferred on DOL16, and treatment groups and control mice were given DSS in drinking water on DOL30 to DOL38. (A and G) Weight loss. (B and H) Histology score. (C and I) Colon length. (D and J) Percentage of CBir1 T cells in the colon LP. Percentage of (E and K) Foxp3+ and (F and L) IL-17+, TNFα+, or IFNγ+ CBir1 T cells after 8 days of DSS treatment. *P < 0.05; n = 4 mice per group.

  • Fig. 7 Deletion of GCs or GAPs during early life results in inflammatory T cell responses against gut bacteria and worsened colitis later in life.

    (A) Enumeration of GCs and GAPs per colonic crypt in DOL18 GC-deficient mice or mice with mAChR4 deleted from GCs and their littermate controls. (A to F) GCs were deleted beginning on DOL12, or (G to K) mAChR4 was deleted from GCs between DOL10 and DOL21, CBir1 T cells were adoptively transferred on DOL16, and mice were placed on DSS from DOL30 to DOL38. (B and G) Weight loss. (C and H) Histology score. (D and I) Colon length. (E and J) Percentage of CBir1 T cells expressing Foxp3. (F and K) Percentage of CBir1 T cells expressing IL-17, TNFα, or IFNγ in the colon LP after 8 days of DSS treatment. *P < 0.05; n = 4 mice per group (A to F) and n = 3 mice per group (G to K).

  • Fig. 8 Summary of time-limited events in early life promoting tolerance to gut bacterial antigens.

    (Top) Events controlling exposure to bacterial antigens that define phases in early life in mice. Left: DOL0 to DOL10 bacterial antigens are largely not encountered by the gut immune system due to the low abundance of gut bacteria and the inhibition of GAPs by high levels of luminal EGF. Middle: DOL11 to DOL20, luminal EGF levels fall, allowing GAPs to form, and bacterial antigens are encountered by the colonic immune system to induce the development of a population of bacterial antigen-specific Tregs. Right: After weaning, the abundant gut microbiota inhibits colonic GAPs, and the gut immune system has limited encounters with bacterial antigens. (Bottom) Left: The stable population of colonic Tregs that develop in early life persist and can limit effector responses to bacterial antigens encountered later in life during inflammation. Middle: Inhibition of bacterial antigen encounter before weaning does not allow bacterial antigen-specific Treg development and does not restrain inflammatory responses upon encounter of bacterial antigens later in life in the setting of inflammation. Right: Extending bacterial antigen encounter beyond weaning by inappropriately inducing colonic GAPs hampers Treg stability and the ability to restrain effector responses to bacterial antigens when encountered later in life in the setting of inflammation.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/18/eaao1314/DC1

    Materials and Methods

    Fig. S1. Representative flow cytometry plots related to Fig. 1.

    Fig. S2. CBir1 epitope–producing bacteria and B. vulgatus in the small intestinal and colonic contents by DOL.

    Fig. S3. M cells are not present on the non–follicle-bearing epithelium in the colon in early life.

    Fig. S4. The extension of TEDs by LP-MNPs is rare in the intestine of preweaning mice.

    Fig. S5. GAP manipulations in early life do not affect antigen-presenting capacity of colonic LP-MNPs.

    Fig. S6. Immunofluorescence staining reveals that dextran-containing epithelial cells express the GC marker cytokeratin.

    Fig. S7. EGF inhibits colonic GAPs in the postneonatal phase of life in a GC intrinsic manner.

    Fig. S8. Inhibition of GAPs in the postneonatal phase of life abrogates T cell responses to gut bacteria.

    Fig. S9. EGFR inhibition after weaning, to inappropriately induce colonic GAPs, but not in the postneonatal phase, when GAPs are present, results in inflammatory cytokine production in the colon-draining MLNs but does not induce overt pathologic changes.

    Fig. S10. Worsened DSS colitis and inflammatory responses to commensal bacteria when encounters with microbial antigens are altered in early life.

    References (7577)

  • Supplementary Materials

    Supplementary Material for:

    Microbial antigen encounter during a preweaning interval is critical for tolerance to gut bacteria

    Kathryn A. Knoop, Jenny K. Gustafsson, Keely G. McDonald, Devesha H. Kulkarni, Paige E. Coughlin, Stephanie McCrate, Dongyeon Kim, Chyi-Song Hsieh, Simon P. Hogan, Charles O. Elson, Phillip I. Tarr, Rodney D. Newberry*

    *Corresponding author. Email: rnewberry{at}wustl.edu

    Published 15 December 2017, Sci. Immunol. 2, eaao1314 (2017)
    DOI: 10.1126/sciimmunol.aao1314

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Representative flow cytometry plots related to Fig. 1.
    • Fig. S2. CBir1 epitope–producing bacteria and B. vulgatus in the small intestinal and colonic contents by DOL.
    • Fig. S3. M cells are not present on the non–follicle-bearing epithelium in the colon in early life.
    • Fig. S4. The extension of TEDs by LP-MNPs is rare in the intestine of preweaning mice.
    • Fig. S5. GAP manipulations in early life do not affect antigen-presenting capacity of colonic LP-MNPs.
    • Fig. S6. Immunofluorescence staining reveals that dextran-containing epithelial cells express the GC marker cytokeratin.
    • Fig. S7. EGF inhibits colonic GAPs in the postneonatal phase of life in a GC intrinsic manner.
    • Fig. S8. Inhibition of GAPs in the postneonatal phase of life abrogates T cell responses to gut bacteria.
    • Fig. S9. EGFR inhibition after weaning, to inappropriately induce colonic GAPs, but not in the postneonatal phase, when GAPs are present, results in inflammatory cytokine production in the colon-draining MLNs but does not induce overt pathologic changes.
    Fig. S10. Worsened DSS colitis and inflammatory responses to commensal
  • bacteria when encounters with microbial antigens are altered in early life.
  • References (75–77).
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