MUCOSAL IMMUNOLOGY Copyright © 2017 Microbial ......development, protection from pathogens, immunity, and metabolism (2–6). To maintain these mutual benefits, interactions between

Knoop et al., Sci. Immunol. 2, eaao1314 (2017) 15 December 2017

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Microbial antigen encounter during a preweaning interval is critical for tolerance to gut bacteriaKathryn A. Knoop,1 Jenny K. Gustafsson,1 Keely G. McDonald,1 Devesha H. Kulkarni,1 Paige E. Coughlin,1 Stephanie McCrate,1 Dongyeon Kim,1 Chyi-Song Hsieh,1 Simon P. Hogan,2 Charles O. Elson,3 Phillip I. Tarr,4 Rodney D. Newberry1*

We have a mutually beneficial relationship with the trillions of microorganisms inhabiting our gastrointestinal tract. However, maintaining this relationship requires recognizing these organisms as affable and restraining inflam-matory responses to these organisms when encountered in hostile settings. How and when the immune system develops tolerance to our gut microbial members is not well understood. We identify a specific preweaning inter-val in which gut microbial antigens are encountered by the immune system to induce antigen-specific tolerance to gut bacteria. For some bacterial taxa, physiologic encounters with the immune system are restricted to this inter-val, despite abundance of these taxa in the gut lumen at later times outside this interval. Antigen-specific tolerance to gut bacteria induced during this preweaning interval is stable and maintained even if these taxa are encountered later in life in an inflammatory setting. However, inhibiting microbial antigen encounter during this interval or ex-tending these encounters beyond the normal interval results in a failure to induce tolerance and robust antigen- specific effector responses to gut bacteria upon reencounter in an inflammatory setting. Thus, we have identified a defined preweaning interval critical for developing tolerance to gut bacteria and maintaining the mutually bene-ficial relationship with our gut microbiota.

INTRODUCTIONThe gastrointestinal tract (GI) is home to trillions of microorganisms (1). The relationship between the host and a healthy gut microbiota is mutually beneficial, with the microorganisms receiving an environ-ment in which to reside and the host receiving benefits related to development, protection from pathogens, immunity, and metabolism (2–6). To maintain these mutual benefits, interactions between the host and the gut microbiota are orchestrated to avoid infection yet main-tain tolerance and prevent inappropriate inflammatory responses. Long- lived effector responses to gut commensal antigens can occur when they are encountered in an inflammatory setting (7). Moreover, loss of tolerance to the gut microbiota, as evidenced by systemic immune responses to gut commensals (8–12), is believed to underlie the patho-genesis of inflammatory bowel disease (IBD), a chronic inflammatory condition of the GI tract. Damage from inappropriate inflammatory responses not only is limited to the host cells but also induces dys-biosis of the gut microbiota, which has been associated with multiple disorders (13) and, in turn, promotes host inflammatory responses (14, 15). Accordingly, how immune tolerance to the microbiota is established and maintained and becomes altered are central topics to understanding multiple facets of health and disease.

Immune tolerance to environmental antigens is largely mediated by peripheral Foxp3+ regulatory T cells (pTregs). Specific gut commensal bacteria taxa promote the induction of pTregs (16–18) via bacterial products (19, 20) or metabolites (21, 22). However, the bacterial anti-gens to which most colonic pTregs respond originate from gut bacteria taxa that are distinct from those identified to promote pTreg induc-

tion (16–18, 23, 24). This suggests that tolerance-promoting bacterial taxa provide an environment that promotes the induction of pTregs directed toward other antigens from other gut bacteria, which may enforce homeostasis and limit inflammatory responses when members of the gut microbiota are reencountered in other settings. Yet, how and when the process of tolerance induction to the larger community of the gut microbiota occurs is largely unknown.

Initial exposure to microbes via the GI tract early in life has been associated with a reduced risk for inflammatory disorders. Observa-tions from multiple studies have suggested the “hygiene hypothesis,” where a decreased susceptibility for immune-mediated diseases later in life is associated with early life exposure to microbes and microbial antigens, which are largely encountered in the GI tract (25, 26). Further, mouse studies have shown that initial exposure to microbes via the GI tract before weaning, but not later in life, reduces susceptibility to colitis later in life (27). These outcomes may be related to encoun-tering microbial antigens in the setting of tolerance-promoting gut bacterial taxa (16–18), establishing immune tolerance to these anti-gens and suppressing inflammatory responses upon future encounters. However, the role for tolerance-inducing gut bacteria is not straight-forward, because these species are most abundant in later childhood and adulthood, when microbe introduction via the GI tract is less effective at reducing the risk of disease. Thus, additional early life and time-limited aspects to the induction of immune tolerance to the gut microbiota exist.

Here, we identify distinct phases in early life in mice: the neonatal phase, days of life (DOLs) 0 to 10, in which luminal antigens are not encountered by the small intestine (SI) or colonic immune system; the postneonatal phase, DOL11 to weaning, in which luminal antigens are encountered almost exclusively by the colonic immune system; and the postweaning phase, in which luminal antigens are encountered almost exclusively by the SI immune system. The inhibition of anti-gen delivery to the SI and colon in the neonatal phase and to the SI in the postneonatal phase was mediated by high levels of luminal

1Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. 2Division of Allergy and Immunology, Department of Pe-diatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA. 3Division of Gastroenterology and Hepatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA. 4Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA.*Corresponding author. Email: [email protected]

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

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epidermal growth factor (EGF) acting on the EGF receptor (EGFR) on goblet cells (GCs) suppressing the formation of GC-associated antigen passages (GAPs). This pattern of antigen delivery allowed the immune system’s encounter with gut microbial antigens and the development of pTregs directed toward members of the gut microbiota at this specific and crucial time in early life. Inhibition of bacterial antigen encounter before weaning or shifting the timing of bacterial antigen encounter by the colonic immune system to the postweaning period abrogated the development of pTregs directed toward members of the gut microbiota and resulted in worse colitis in response to epi-thelial injury and inflammatory responses to gut commensals later in life. These observations identify a specific period in early life that are critical for the induction of tolerance to the gut microbiota, which, once established, serves to limit inflammatory responses upon encounter of gut-resident bacteria in inflammatory settings.

RESULTSLuminal antigen encounter by the gut immune system occurs in phases in early lifeWhereas multiple studies have documented that gut bacteria can affect the outcome of T cell responses, fewer studies have examined how T cells encounter and respond to gut commensal bacterial anti-gens. Results from the few studies examining this topic indicate that T cell responses to gut bacteria are dominated by a relatively small number of taxa that are closely adherent to the epithelium, such as segmented filamentous bacteria (SFB) or Helicobacter, whereas anti gens from other bacteria, such as Bacteroides vulgatus and the Lachnospiraceae bacterium A4, do not elicit responses from antigen- specific T cells in the absence of inflammation (7, 24, 28, 29). A limitation of these studies is that they have examined adult mice or gnotobiotic mice, which may not reflect the physiologic inter-actions of the developing immune system with the microbiota in early life. To overcome this limitation, we isolated lamina propria (LP) CD11c+ MHCII+ populations, referred to as mononuclear phago-cytes (MNPs), from the SI and colon of conventionally housed mice during early life and evaluated their ability to stimulate CBir1 CD4+ T cell receptor (TCR) transgenic T cells specific for a flagellin epi-tope produced by the Lachnospiraceae bacterium, A4, and COE1 (12). We observed that SI LP-MNPs were relatively ineffective at in-ducing CBir1 T cell expansion at all days examined (Fig. 1A). In contrast, colonic LP-MNPs isolated on DOL14 and DOL21, but not earlier on DOL7 or later on DOL28, were able to induce strong pro-liferation of CBir1 T cells, suggesting that these LP-MNPs were loaded with the CBir1 flagellin epitope (Fig. 1A). Adoptive transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled CBir1 T cells recapitulated the temporal and regional pattern of T cell stimulation in the gut in vivo, with effective accumulation and proliferation of CBir1 T cells in the colon-draining mesenteric lymph node (MLN) around DOL21 but not earlier on DOL10 or later on DOL30 and not in the SI-draining MLN on any day examined (Fig. 1, B and C, and fig. S1). Likewise, we observed that adoptively trans-ferred DP1 CD4+ TCR transgenic T cells, specific for an antigen pro-duced by B. vulgatus, localized to the colon-draining MLN around DOL20 but not earlier on DOL10 or later on DOL30 and not to the SI-draining MLN on any day examined (Fig. 1D). CBir1 and DP1 TCR transgenic T cells transferred on DOL18, but not earlier on DOL8 or later on DOL28, and not ovalbumin (OVA)–specific OTII TCR trans-genic T cells in the absence of OVA, expanded in the colon LP, and

expressed Foxp3 7 days later (Fig. 1, E and F, and fig. S1). This sig-nifies that antigen-specific Foxp3 pTreg responses are made toward multiple members of the commensal microbiota selectively during this preweaning interval. The lack of responses in the SI is likely due to the preferential localization of these bacterial taxa in the colon, and the lack of responses in the colon around DOL10 may be ac-counted for by the low levels of these bacteria in the lumen during this time of life (fig. S2). However, the lack of responses in the colon around DOL30 was paradoxical, as the flagellin peptide recognized by CBir1 T cells and the B. vulgatus bacteria recognized by DP1 T cells became more abundant in the colonic luminal contents around

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Fig. 1. Bacterial antigen encounter occurs during a specific preweaning inter-val, 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) per-centage 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) Per-centage 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 lumi-nally 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 inde-pendent times.


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DOL30 (fig. S2), suggesting that LP-MNPs became unable to ac-quire these luminal microbial antigens.

To overcome the inability to assure microbial antigen presence at all time points in early life, we evaluated the ability of SI and colon LP-MNPs to acquire a surrogate antigen OVA. Fluorescent OVA ac-quisition by colonic LP-MNPs occurred in a pattern similar to that of CBir1 antigen (Fig. 1G and fig. S1). In contrast, SI LP-MNPs ac-quired fluorescent OVA around the time of weaning, DOL21, with limited capacity at earlier time points (Fig. 1G). Thus, the inability to stimulate gut bacteria antigen T cell responses before DOL10 might reflect both the limited presence of the antigen and the inability of LP-MNPs to acquire luminal antigens, whereas the inability to stim-ulate gut bacteria antigen T cell response in the colon after weaning is reflective of the inability of colonic LP-MNPs to acquire antigen. Further, the lack of T cell responses to bacterial antigen in the SI is not due to the inability of SI LP-MNPs to acquire luminal antigens after weaning but likely is reflective of the low levels of this bacterial antigen in the SI lumen (fig. S2).

Several processes have been implicated in luminal antigen capture by LP-MNPs; however, these processes have not been investigated in the preweaning intestine. Consistent with observations in the adult intestine, villous M cells, or M cells in the non–follicle-bearing epi-thelium, were absent from the colon before weaning (fig. S3). CD11c+ antigen-presenting cells can extend trans-epithelial dendrites (TEDs) into the lumen of the SI for the purpose of sampling luminal antigens (30–32); however, this has not been observed in the colon of adult mice (33, 34), where most of the antigen is loading onto LP-MNPs before DOL21 (Fig. 1, A and G). In vivo two-photon (2P) imaging of DOL21 CD11cYFP reporter mice did not reveal dendrites protruding past the intestinal epithelium in either the SI or the colon, although we could readily see dendrites in the LP probing the epithelium (fig. S4). Studies evaluating TED formation in adult mice removed luminal contents and mucus before imaging (30–32, 35, 36), a process that in-duces TED formation in the SI but not the colon of adult mice (34). We observed that even after removal of the luminal contents and mucus layer, TEDs did not form in the SI or colon of DOL18 mice (fig. S4). GCs can form GAPs and deliver luminal substances to LP-MNPs in the SI of adult mice. Mouse atonal homolog 1 (Math1) is a transcrip-tion factor required for GC development (37), and inducible deletion of Math1 in intestinal epithelial cells results in GC deletion (38–40). We observed that deletion of GCs in DOL18 Math1f/fvil-Cre-ERT2 mice resulted in an about threefold increase in intestinal permeabil-ity, as evaluated by the presence of 4-kDa fluorescein isothiocyanate (FITC)–dextran in the serum after gavage (Fig. 1H). LP-MNPs from the SI or colon of DOL18 mice lacking GCs could not capture luminal fluorescent OVA (Fig. 1I and fig. S1) yet had a normal number of LP-MNPs and could capture fluorescent OVA when administered system-ically (fig. S5). Moreover, CBir1T cells transferred into DOL18 mice lacking GCs failed to expand in vivo (Fig. 1J and fig. S1), despite the ability of colonic LP-MNPs from mice lacking GCs to expand CBir1 T cells similar to controls when antigen was added to ex vivo cul-tures (fig. S5). Thus, the presence of increased intestinal permeabil-ity did not correlate with the ability of LP-MNPs to capture luminal antigen and to stimulate immune responses in early life and suggest-ed that GCs and GAPs are required for LP-MNPs to acquire luminal antigens in the preweaning period. Therefore, we evaluated the re-gional pattern and timing of GAP formation during early life. GAPs were not seen in the colon or SI before DOL10 (Fig. 1K). Around DOL10, GAPs were present in the colon (fig. S6) at a rate of 0.6 GAPs

per GC and remained at that level until DOL21, that is, the time of weaning, when GAP formation decreased to become rare in the co-lon at DOL24 (Fig. 1K). At DOL18, GAPs became consistently pres-ent in the SI across multiple experiments and became prevalent at 0.6 GAPs per GC on DOL21 and remained at that level throughout adulthood (Fig. 1K). Thus, GCs were required for the delivery of antigens to the LP-MNPs in early life, and the presence of GAPs, but not TED formation, increased intestinal permeability, or villous M cells correlated with the regional pattern and timing of antigen de-livery during early life. We defined three phases of luminal antigen delivery in the gut: the neonatal phase (DOL0 to DOL10), in which antigens are not delivered to the SI or colonic immune system; the post-neonatal phase (DOL11 to weaning), in which antigens are predom-inantly delivered to the colonic immune system; and the postweaning phase, when antigens are delivered predominantly to the SI immune system.

The gut microbiota and breast milk control luminal antigen delivery in early lifeGCs in the colon of adult mice are largely unable to form GAPs due to Myd88-dependent GC intrinsic sensing of the abundant colonic microbes and microbial products (39). GC microbial sensing activates the EGFR and p42/p44 mitogen-activated protein kinase (MAPK), inhibiting GC responses to acetylcholine (ACh), the stimulus- inducing spontaneous GAP formation (39, 41). Like intestinal GCs from adults, GCs from preweaning DOL18 mice expressed Toll-like receptors (TLRs), Myd88, and EGFR (Fig. 2A) (39), suggesting that they would respond similarly to microbial products. Colonic GAPs on DOL18 could be inhibited by heat-killed cecal contents from adult specific pathogen–free (SPF)–housed mice, and this inhibition was dependent on Myd88 in Math1-expressing epithelial lineages, which is largely restricted to GCs in the colon (Fig. 2B). Intraluminal lipo-polysaccharide (LPS) inhibited GAP formation in the DOL18 colon, and this inhibition was dependent on activation of EGFR and MAPK (Fig. 2C), confirming that the pathways downstream of microbial sensing seen in GCs in the adult colon also apply to preweaning co-lonic GCs. The gut microbiota undergoes marked changes in quan-tity and diversity from birth to weaning (42–45). The cecal bacterial load increased little during the neonatal phase but then logarithmi-cally increased during the postneonatal phase and postweaning before plateauing during adulthood (Fig. 2D). This quantitative increase in bacterial load correlated with qualitative changes as seen by 16 s rRNA gene deep sequencing (Fig. 2E), with a switch from gammapro-teobacteria to clostridia, bacilli, and bacteroidia in early life. When Myd88 was deleted from GCs, GAPs formed spontaneously in the postweaning colon, but the density of colonic GAPs in earlier phases of life and in the SI remained unchanged (Fig. 2, F and G), indicating that Myd88-dependent signals suppress GAP formation specifically in the colon in the postweaning period. Therefore, al-though both preweaning and postweaning colonic GCs can respond to microbial signals to inhibit GAP formation, the gut microbiota is permissive for the spontaneous formation of colonic GAPs before weaning.

The lack of GAPs in the neonatal phase of mice lacking Myd88 in GCs could be due to the lack of ACh, the stimulus spontaneously inducing GAPs, or the lack of GC responsiveness to ACh due to other pathways activating the EGFR in GCs. We evaluated the ability of GCs to respond to the ACh analog carbamylcholine (CCh) to form GAPs. SI and colonic GCs in DOL8 mice, the neonatal phase, did


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not form GAPs in response to CCh (Fig. 3A). SI GCs in DOL14 mice, the postneonatal phase, were relatively unresponsive to CCh, whereas colonic GCs were responsive to CCh to form GAPs (Fig. 3A). More-over, spontaneously forming colonic GAPs on DOL14 were suppressed by tropicamide, an antagonist of muscarinic ACh receptor 4 (mAChR4; Fig. 3A), the ACh receptor on GCs inducing GAPs in the adult SI (39). In contrast, GCs in the postweaning phase responded similarly to what has been reported for adults (39), with SI GCs increasing GAPs in response to CCh and spontaneously forming GAPs via mAChR4 signals and colonic GCs being unresponsive to CCh to form GAPs (Fig. 3A). This suppression after weaning was relieved by deletion of Myd88 in GCs, which allowed GAPs to be formed spontaneously in a mAChR4-dependent manner and to be further induced by CCh (fig. S7). This indicates that the pattern of GC responsiveness to ACh to form GAPs changes throughout early life, prompting us to ex-plore whether other ligands/pathways activate EGFR and p42/p44 MAPK at these times of life. Consistent with EGFR activation inhibit-ing GAPs in the preweaning colon, phosphorylation of the EGFR in GCs inversely correlated with the presence of colonic GAPs before weaning (compare Fig. 3, B and C, with Fig. 1K), and inhibition of EGFR phosphorylation with tyrphostin AG1478 (EGFRi), or deletion

of EGFR in GCs, allowed GAP formation in the SI and colon during all phases of early life, independent of changes to the number of GCs (Fig. 3, D and E, and fig. S6).

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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 con-tents 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 experi-ments 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) treat-ment on DOL8, DOL18, or DOL28. (B) Immunofluorescence staining of phosphoryl-ated 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 ab-sence 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 in-dependent times.


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Intraluminal fluorescent Ova-647 was captured by SI and colonic LP-MNPs in all phases of life in mice lacking EGFR in GCs (Fig. 3F), indicating that EGFR activation in GCs was controlling the regional and temporal pattern of antigen delivery in early life. Inhibition of EGFR activation allowed colonic LP-MNPs from postweaning mice to capture microbial antigen to stimulate CBir1 T cells ex vivo (Fig. 3G). The presence of GAPs correlated with MNP recruitment to the co-lonic epithelium and the acquisition of luminal OVA (fig. S5). However, these GAP manipulations did not affect the overall number of MNPs in the colonic LP, the ability of MNPs to capture systemic antigens, or their ability to stimulate bacterial antigen-specific T cell prolifera-tion when antigen was added to ex vivo cultures (fig. S5). This indicates that GAP manipulation specifically affected luminal antigen deliv-ery to MNPs but not their presence within the colonic LP or their antigen presentation capacity. In addition, inhibition of EGFR acti-vation allowed adoptively transferred CBir1 or DP1 cells to expand and become activated in the MLNs of postweaning mice, whereas cells transferred into postweaning vehicle-treated mice, lacking colonic GAPs, remained naïve (Fig. 3, H and I). However, unlike mi-crobial antigen-specific T cells encountering antigen in the post-neonatal phase of life, DOL18, CBir1 and DP1 T cells encountering microbial antigen after weaning, DOL28, had little Foxp3 expression 7 days after transfer (Fig. 3J), indicating that the colon LP of post-neonatal mice is uniquely favorable for the induction of regulatory responses.

Because pathways independent of Myd88 were activating EGFR in GCs to suppress GAPs, we evaluated the pattern of EGFR ligands in the preweaning gut. EGFR ligands, including EGF, amphiregulin, and heparin-binding EGF-like growth factor, are abundant in breast milk after parturition and decrease throughout lactation until wean-ing (46–50). Of these, EGF is the most abundant in breast milk, being about fivefold higher than the others (46). Furthermore, ingested breast milk EGF reaches the offspring’s GI tract in a biologically active form (49, 51, 52). We observed that EGF was present in luminal contents throughout the GI tract of mice before weaning in a con-centration pattern inversely correlating with the presence of GAPs (compare Fig. 4A with Fig. 1K). The concentration of EGF both decreased throughout the GI tract and over time, and correlated with the activation of the EGFR and p42/p44 MAPK in the epitheli-um (Fig. 4, A to D), indicating that the proximal to distal gradient and temporal decrement of luminal EGF could allow GAP forma-tion to occur in a distal to proximal and temporal pattern as we ob-served in the gut during early life. Luminal EGF inhibits GAPs (39), and accordingly, the stomach contents from DOL10 mice, which contain high levels of EGF, inhibited GAPs in the colon of DOL18 mice in a manner dependent on EGFR and p42/p44 MAPK acti-vation (Fig. 4E). Stomach contents also inhibited colonic GAP for-mation in DOL18 Myd88−/− mice (Fig. 4E), confirming that the inhibitory ligands pre sent in the stomach con tents of neonatal mice were not microbial ligands acting via Myd88. Luminal recombinant EGF inhibited GAPs and luminal antigen loading of LP-MNPs in postneonatal colon, in an EGFR- and MAPK- dependent, but not Myd88- independent, manner (Fig. 4, E and F, and fig. S7). More-over, CBir1 cells failed to expand in the colon- draining MLN 3 days after transfer in postneonatal mice given intracolonic recombi-nant EGF (Fig. 4G). These data indicate that breast milk EGF and potentially other EGFR ligands control the temporal and re-gional luminal antigen exposure of the gut immune system be-fore weaning.

Altering the timing of exposure to bacterial antigens results in loss of tolerance to gut bacteria, worsened colitis, and inflammatory responses to gut bacteria after epithelial injuryWe evaluated whether GAP dysregulation or disruption affects the de-velopment of microbial antigen-specific Tregs. CBir1 T cells transferred into mice given intracolonic EGF on DOL10 to DOL21, to inhibit colonic GAPs, had significantly less expansion and expressed significantly less Foxp3 on DOL30 (Fig. 5, A and B, and fig. S8). However, a signifi-cantly larger proportion of the remaining CBir1 T cell population ex-pressed inflammatory cytokines when compared with phosphate- buffered saline (PBS)–treated control mice (Fig. 5C). Conversely, inhibition of EGFR on DOL14 and DOL16, during the postneonatal phase, or on DOL24 and DOL26, after weaning, did not affect the expansion of transferred CBir1 T cells (Fig. 5D). However, unlike control treated mice or mice given EGFR inhibitors on DOL14 and DOL16 during the postneonatal phase, CBir1 T cells in mice given EGFR inhibitors on DOL24 and DOL26 after weaning, to allow encounters with micro-bial antigen outside of the normal window, expressed significantly less Foxp3, and a significantly greater proportion of the population ex-pressed inflammatory cytokines (Fig. 5, E and F). Inhibition of EGFR

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Fig. 4. Luminal EGF inhibits GAPs and antigen delivery before wean-ing. (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 phospho-rylated 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; intra-peritoneally) 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 intra-colonic EGF (1 g) 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.


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during the postneonatal phase on DOL14 and DOL16, when colonic GAPs are already present, resulted in no significant difference in the expression of Foxp3+ or inflammatory cytokines by transferred CBir1 T cells when compared with controls (Fig. 5, E and F). This supports the idea that the effects of EGFR inhibition after weaning on CBir1 T cells are a result of the induction of colonic GAPs and extension of the window of microbial antigen encounter.

Altering the timing of microbial antigen encounter resulted in inflammatory cytokine production in the MLNs 1 week later, but no overt pathology in the colon (fig. S9). We reasoned that the lack of pathology might, in part, be due to the relatively low level of micro-bial antigen encountered by the immune system after weaning, be-cause we observed little or no responses by bacterial antigen-specific T cells adoptively transferred into mice after weaning (Fig. 1, B to D). Therefore, mice were given intracolonic EGF on DOL10 to DOL21 to inhibit microbial antigen encounter and pTreg development, mice were given EGFR inhibitors after weaning on DOL24 and DOL26 to alter the timing of microbial antigen encounter, and control mice were given 3% dextran sulfate sodium (DSS) in drinking water on DOL30 to DOL38 to disrupt the colonic epithelial barrier and allow exposure of the immune system to luminal bacteria. Mice treated with EGF on DOL10 to DOL21, or EGFR inhibition on DOL24 and DOL26, lost significantly more weight (Fig. 6, A and G) and developed significantly worse disease, as evidenced by histology demonstrating increased edema, increased ulceration, increased cellular infiltration, and shortened colons (Fig. 6, B, C, H, and I, and fig. S10). In un-treated mice, mice receiving intrarectal PBS on DOL10 to DOL21, and mice receiving EGFR inhibitors in the postneonatal phase on DOL14 and DOL16 when colonic GAPs are normally present, trans-ferred CBir1 T cells did not expand further in the colon LP (com-

pare Fig. 1E with Fig. 6, D and J) and continued expressing Foxp3 but not inflammatory cytokines after DSS treatment (Fig. 6, E, F, K, and L). However, in mice where microbial antigen encounter was inhibited by EGF, or inappropriately induced after weaning by EGFR inhibition on DOL24 and DOL26, DSS treatment resulted in significant expansion of CBir1 T cells in colon LP (Fig. 6, D and J). The transferred CBir1 T cells had significantly reduced Foxp3 expression and significantly increased inflammatory cytokines (Fig. 6, E, F, K, and L), indicative of effector T cell expansion during inflammation in response to commensal bacteria. Similarly, mice lacking GCs and GAPs beginning on DOL12, or mice with deletion of mAChR4 on GCs between DOL10 and DOL21, which have GCs but are unable to form GAPs during this interval (Fig. 7A), experienced worse DSS- induced colitis (Fig. 7, B to D and G to I). Similar to mice treated with EGF on DOL10 to DOL21 to inhibit GAPs during the postneonatal phase, CBir1 T cells adoptively transferred on DOL16 into mice lack-ing GCs or GAPs had significantly reduced Foxp3 expression and significantly increased inflammatory cytokines after disruption of the epithelial barrier with DSS (compare Fig. 6, F and G, with Fig. 7, G, H, N, and O). These data demonstrate that the timing of gut bacte-rial antigen encounter in early life is a critical com ponent to establish durable tolerance and limit inflammatory responses upon encounter later in life (Fig. 8).

DISCUSSIONHere, we identify a specific preweaning interval in which bacterial anti-gens are encountered by the colonic immune system. In the course

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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––positive (TNF+), or interferon-–positive (IFN+) 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+, TNF+, or IFN+ 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.

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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+, TNF+, or IFN+ CBir1 T cells after 8 days of DSS treatment. *P < 0.05; n = 4 mice per group.


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of normal development, the immune system’s encounter with some bacterial antigens is largely restricted to this interval in the postneo-natal phase of life. However, a limitation of this study is that these events, and their time-limited nature, do not pertain to all gut bac-terial antigens, because some bacteria in the fully developed gut microbiota are not present during this interval, and gut bacteria closely adhering to the epithelium have been observed to generate antigen-specific T cell responses outside of this interval in later life (24, 28, 29). Blocking the encounter of the immune system with bacterial products during this window resulted in failure to develop

pTregs specific for antigens from these bacteria that was not com-pensated for by later encounters, indicating that the timing of these events is critical for developing tolerance to some gut bacteria. Fur-ther, initial encounter of these bacterial antigens outside of this in-terval or extending encounters beyond this interval resulted in the induction of effector T cell responses to gut bacteria. Thus, the en-counter of antigens from some gut bacterial taxa during a specific preweaning interval is critical for the development of tolerance to members of the gut microbiota.

The microbiota drives the development of a long-lived population of pTregs in the preweaning colon, and these pTregs can control effector responses (53–55). Superimposing our findings on these studies suggests that these pTregs can be specific for microbial antigens encountered in the postneonatal phase and that these encounters serve to develop a long-lived pTreg population with the ability to control inflammatory responses. Although a limitation of our study is that it is restricted to mice, it is notable that individuals with Crohn’s disease make systemic immune responses to the CBir1 antigen, and we observed that pTregs specific for CBir1 antigen were largely induced during the postneonatal phase of life in mice. This indicates that the events we identified in preweaning mice have relevance to humans and, combined with ob-servations that antibiotic use in the first year of life is associated with an increased incidence of asthma, allergy, and IBD (56–58), strongly suggests that altering microbial antigen encounters during specific intervals in preweaning children increases the risk of disease.

Effective acquisition of luminal antigens by LP-MNPs was abro-gated in the absence of GCs and correlated with the density of GAPs in all situations, and was not correlated with paracellular leak, villous M cells, or the extension of TEDs by LP-MNPs. Although it is not possible to entirely exclude contributions from these other pathways, and a limitation is that the pharmacologic and genetic manipulations may have other effects beyond altering GAPs, multiple strategies ma-n ipulating GAPs correlated with antigen acquisition by LP-MNPs, strongly implicating that GAPs are major contributors to antigen acquisition in the preweaning intestine. The stratified mucus layer, which separates bacteria from the epithelium, develops in the first week of life (59–61). However, this mucus layer was not sufficient to prevent encounters of the colonic immune system with gut bacterial antigens. This suggests that bacterial antigens, as opposed to whole bacteria, are delivered via GAPs, or alternatively, encounters with live bacteria occur in the proximal colon where the mucus layer is more permeable (62). The need to restrict the encounter of gut bacterial antigens with the immune system to a specific interval is not well under-stood. The need to limit encounters in the neonatal phase might be related to the limited diversity of the gut bacterial population and limited T cell repertoire (63, 64). This might preclude developing a diverse and stable responding T cell population as well as T helper 2 (TH2) skewing from T cells derived from fetal T cell precursors (65, 66), resulting in unbalanced responses to gut bacterial antigens. The need to limit exposure to gut bacterial antigens after weaning, when the immune system is fully competent, may be related to the com-plex environment in which these antigens are encountered, because we observed that inappropriate exposure to gut bacterial antigens after weaning does not result in the induction of pTregs, and previous ob-servations indicate that these encounters result in inflammatory re-sponses (34, 41). In total, these observations indicate that timing of exposure is a critical component to the development of tolerance to some gut bacterial antigens and highlight the unique and time- limited events occurring in the preweaning GI tract.

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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, TNF, or IFN 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).


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The gut microbiota contributes to multiple facets of health, and dysbiosis of the gut microbiota is inferred to contribute to multiple diseases. Interactions between the gut microbiota and the immune system shape each other, resulting in co-development of these two organs, and accordingly these interactions are important to establish a stable relationship that is able to withstand transient perturbations by either member. The unstable relationship between the immune sys-tem and the gut microbiota, characterized by reciprocal inappropri-ate inflammatory responses and dysbiosis, is believed to underlie the pathogenesis of IBD. Here, we demonstrate that the encounters of bacterial antigens with the immune system that establish this rela-tionship are much more complex than previously appreciated and extend beyond the mere presence of specific bacterial taxa in the lumen and responding T cells in the immune compartment. For antigens from some bacterial taxa, these encounters initially occur

in, and are largely limited to, a specific interval in early life, which is critical for developing tolerance to these bacteria and fostering a stable relationship with our gut microbiota.

MATERIALS AND METHODSMiceAll mice were 10 generations or greater on the C57BL/6 background, with the exception of mAChR4f/f mice, which were 6 or more generations on the C57BL/6 background. C57BL/6 mice, OTII TCR transgenic mice (67), CD11cYFP transgenic mice (68), Math1f/f mice (37), Myd88f/f mice (69), and Math1PGRCre mice (70) were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred and main tained in house. EGFRf/f mice (71) were a gift from D. Threadgill (University of North Carolina, Chapel Hill, NC). Myd88−/− mice (72), a gift from S. Akira (Osaka

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Fig. 8. Summary of time-limited events in early life promoting toler-ance 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 bacte-rial antigens are encountered by the colonic immune system to induce the development of a population of bac-terial antigen-specific Tregs. Right: After weaning, the abundant gut mi-crobiota 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 re-sponses to bacterial antigens encoun-tered 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 encoun-ter of bacterial antigens later in life in the setting of inflammation. Right: Extending bacterial antigen encoun-ter beyond weaning by inappropriate-ly inducing colonic GAPs hampers Treg stability and the ability to restrain ef-fector responses to bacterial antigens when encountered later in life in the setting of inflammation.


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University, Osaka, Japan), were bred onto the C57BL/6 background by the Speed Congenics Facility of the Rheumatic Diseases Core Center and maintained in house. mAChR4f/f mice (73) were a gift from J. Wess (National Institutes of Health, Bethesda, MD). CBir1 TCR transgenic mice and DP1 transgenic mice have been previously described (12, 23). Transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the villin promoter (vil-Cre-ERT2) mice (74) were a gift from S. Robine (Institut Curie, Paris, France). To deplete Myd88, EGFR, or mAChR4 in GCs, we bred mice to generate Myd88f/fMath1PGRCre mice, EGFRf/fMath1PGRCre mice, and mAChR4f/fMath1PGRCre mice. These mice and Cre-negative litter-mate controls were injected intra peritoneally with mifepristone (10 mg/kg) every day starting 4 days before use in experiments. Generation of Math1fl/fl Vil-Cre-ERT2 mice and inducible deletion of GCs by treatment with tamoxifen have been previously described (39). Mice used in these experiments were bred in house and weaned at DOL21. Co-housed littermates were used as experimental groups and controls to minimize differences in the gut microbiota. For phar-macologic manipulations, littermates of the same sex were assigned a cage, which contained animals of all treatment groups. For genetic manipulations, cages of weaned mice contained littermates of the same sex and both genotypes. Animal procedures and protocols were carried out in accordance with the institutional review board at Washington University School of Medicine.

Statistical analysisData analysis using Student’s t test or one-way analysis of variance (ANOVA) with Dunnett or Tukey test to correct for multiple com-parisons was performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Biological variables measured were normally distributed. All tests for significance were two-sided. An of P < 0.05 was considered significant.

SUPPLEMENTARY MATERIALSimmunology.sciencemag.org/cgi/content/full/2/18/eaao1314/DC1Materials and MethodsFig. 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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Acknowledgments: We thank M. J. Miller for insightful critiques and assistance with 2P imaging and analysis. The 2P imaging was performed at the In Vivo Imaging Core at Washington University School of Medicine. The Washington University Digestive Diseases Research Center Core (DDRCC) Gnotobiotic Facility, supported by NIH grant P30 DK052574, provided the germ-free mice. The High Speed Cell Sorter Core at the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO, supported in part by National Cancer Institute Cancer Center Support Grant P30 CA91842, provided flow cytometric cell sorting services. The Speed Congenics Facility of the Rheumatic Diseases Core Center, supported by NIH grant P30AR048335, bred the Myd88−/− mice onto the C57BL/6 background. R.D.N., K.G.M., and K.A.K. are inventors on a provisional patent application 62/450,831 held/submitted by Washington University School of Medicine in St. Louis that covers approaches to control dietary and microbial exposure in early life. P.I.T. has equity in, is a member of the scientific advisory board, and is a consultant to MediBeacon Inc., a company that is developing technology to measure gut permeability in humans and is also a co-inventor of a patent that could generate royalty payments. Funding: This study was supported by the following grants: AI131342 (R.D.N. and P.I.T.), DK097317 (R.D.N.), AI1126260 (S.P.H. and R.D.N.), Children’s Discovery Institute grant MD-II-2015-481 (R.D.N. and P.I.T.), P30 DK052574, and DK109006 (K.A.K.). D.H.K. is supported by a Research Fellowship Award from the Crohn’s and Colitis Foundation of America. Author contributions: K.A.K. performed animal colitis models, T cell transfers, enzyme-linked immunosorbent assays (ELISAs), cell isolation, in vitro studies, microarray analysis, flow cytometry, and data analysis. K.A.K., J.K.G., K.G.M., D.H.K., and P.E.C. performed immunofluorescence and data analysis. K.A.K. and K.G.M. performed 2P imaging experiments and data analysis. K.A.K., S.M., and D.K. performed reverse transcription polymerase chain reaction and data analysis. P.I.T., C.-S.H., S.P.H., and C.O.E. provided input on experimental design and data analysis. K.A.K. and R.D.N. conceived the study, directed the experiment design, analyzed the data, and wrote the initial manuscript draft. All authors reviewed and discussed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Data and materials are available upon request from R.D.N.

Submitted 22 June 2017Accepted 6 October 2017Published 15 December 201710.1126/sciimmunol.aao1314

Citation: K. A. Knoop, J. K. Gustafsson, K. G. McDonald, D. H. Kulkarni, P. E. Coughlin, S. McCrate, D. Kim, C.-S. Hsieh, S. P. Hogan, C. O. Elson, P. I. Tarr, R. D. Newberry, Microbial antigen encounter during a preweaning interval is critical for tolerance to gut bacteria. Sci. Immunol. 2, eaao1314 (2017).


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

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

DOI: 10.1126/sciimmunol.aao1314, eaao1314.2Sci. Immunol.

microbes that colonize the colon early in life.lamina propria. Their findings suggest that the development of this particular type of microbial tolerance is restricted to associated antigen passages (GAPs) in the colon, which facilitate transport of bacterial antigens from the lumen to the

−gut develops between 10 and 20 days after birth, and is perfectly synchronized with the formation of goblet cell demonstrate in mice that tolerance to symbionts in the et al.tolerance to gut microbial antigens early in life. Here, Knoop

by competing for essential nutrients. To establish and sustain this symbiotic relationship, it is vital that the host develops Gut microbiota have several important functions, including aiding host metabolism and limiting pathogen invasion

Licensing microbes for symbiosis

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MUCOSAL IMMUNOLOGY Copyright © 2017 Microbial ......development, protection from pathogens, immunity, and metabolism (2–6). To maintain these mutual benefits, interactions between