Innate and Adaptive Immunity Cooperate Flexibly to Maintain Host-Microbiota Mutualism

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Science  31 Jul 2009:
Vol. 325, Issue 5940, pp. 617-620
DOI: 10.1126/science.1172747

Maintaining Mutual Ignorance

Our gut is colonized by trillions of bacteria that do not activate the immune system because of careful compartmentalization. Such compartmentalization means that our immune system is “ignorant” of these microbes and thus it has been proposed that loss of compartmentalization might result in an immune response to the colonizing bacteria. Microorganisms are sensed by cells that express pattern recognition receptors, such as Toll-like receptors, which recognize patterns specific to those microbes. Slack et al. (p. 617) show that Toll-like receptor–dependent signaling is required to maintain compartmentalization of bacteria to the gut of mice. In the absence of Toll-dependent signaling, intestinal bacteria disseminated throughout the body and the mice mounted a high-titer antibody response against them. This antibody response was of great functional importance because, despite the loss of systemic ignorance to intestinal microbes, the mice were tolerant of the bacteria. Thus, in the absence of innate immunity, the adaptive immune system can compensate so that host and bacterial mutualism can be maintained.


Commensal bacteria in the lower intestine of mammals are 10 times as numerous as the body’s cells. We investigated the relative importance of different immune mechanisms in limiting the spread of the intestinal microbiota. Here, we reveal a flexible continuum between innate and adaptive immune function in containing commensal microbes. Mice deficient in critical innate immune functions such as Toll-like receptor signaling or oxidative burst production spontaneously produce high-titer serum antibodies against their commensal microbiota. These antibody responses are functionally essential to maintain host-commensal mutualism in vivo in the face of innate immune deficiency. Spontaneous hyper-activation of adaptive immunity against the intestinal microbiota, secondary to innate immune deficiency, may clarify the underlying mechanisms of inflammatory diseases where immune dysfunction is implicated.

Innate recognition of a few thousand bacteria in the blood and peripheral tissues results in an inflammatory response and, subsequently, induction of both cell-mediated and humoral adaptive immunity (1). Paradoxically, our trillions of intestinal bacteria do not normally induce spontaneous pathological inflammatory responses or high-titer serum antibody responses (2, 3). Many layers of defense compartmentalize commensal bacteria within the gut lumen and mucosal immune compartment (Lamina propria, Peyer’s patches, and mesenteric lymph nodes) (3). Thus, the paradigm emerged that minimizing microbial recognition within the specialized intestinal microenvironment is crucial to establishing mutualism (4), and consequently, it was no surprise that animals with deficient innate immunity successfully contain their microbiota. In contrast, we demonstrate that innate and adaptive immunity can function both sequentially (5), and in a compensatory manner, and the relative functionality of the two systems translates into a set point that permits mutualism.

To study how mammals can adapt to commensal intestinal bacteria in the absence of signaling through Toll-like receptors (TLRs), a major family of innate immune sensors, we maintained mice deficient in the TLR adaptor molecules MyD88 and Ticam1 (also known as TRIF) and control animals with different densities of intestinal commensal bacteria (6). We generated germ-free mice, who can be supposed to have never before encountered a live bacterium, “clean SPF” mice containing a very limited intestinal microbiota free of both Enterococcus faecalis and Escherichia coli, and “conventional SPF” mice reared in standard clean animal house conditions and, thus, free of all monitored mouse pathogens.

To address whether mucosal containment of the microbiota was normal in TLR-deficient mice, we challenged clean SPF Myd88–/– Ticam1–/– mice intragastrically with high doses of a commensal bacterium to which they had never previously been exposed (E. coli K-12). In accordance with previous findings (7), both Myd88–/– Ticam1–/– and control mice had similar loads of culturable E. coli K-12 in the mesenteric lymph nodes 18 hours after challenge (Fig. 1A). In contrast, and in agreement with a recent publication from Vaishnava et al. (8), significantly more live bacteria were recovered from the spleens of Myd88–/– Ticam1–/– mice (Fig. 1A). Similar data were obtained when germ-free Myd88–/– Ticam1–/– mice were naturally colonized by cohousing with conventional SPF mice (fig. S1A), which ruled out any unphysiological complications due to gavage. These data demonstrate a dramatic failure to compartmentalize the intestinal microbiota within the mucosal immune compartment in Myd88–/– Ticam1–/– mice.

Fig. 1

Increased bacterial penetration in Myd88–/– Ticam1–/– mice is not dependent on increased intestinal permeability. (A) Clean SPF Myd88–/– Ticam1–/–, F1 control mice, and F1 mice treated with 7.5 mg indomethacin per kg of body weight 24 hours earlier (F1 + NSAID) were gavaged with 1010 ampicillin-resistant E. coli K-12. After 18 hours, the density of ampicillin-resistant E. coli in the cecal content, mesenteric lymph nodes, and spleen was determined by selective plating (*P < 0.027, **P < 0.002). Data are pooled from three independent experiments. CFU, colony-forming units; mLN, mesenteric lymph node; G, conductance. (B) Ussing chamber measurements of conductance and paracellular permeability, as assessed by serosal 51Cr-EDTA recovery, of jejunum from Myd88–/– Ticam1–/– mice, cohoused C57BL/6 control mice (B6), and positive control (C57BL/6) mice treated with 7.5 mg/kg indomethacin (NSAID). Two or three matched mice were analyzed each day over 5 days. Each data point represents an individual mouse, and all collected data are shown. (C) Enzyme-linked immunosorbent assay (ELISA) for albumin presence in the feces of Myd88–/– Ticam1–/– mice, F1 control mice, NSAID-treated control mice, or DSS-treated control mice. (*P < 0.05). Each data point represents an individual mouse, and all collected data pooled from three independent experiments are shown.

Alterations in epithelial proliferation and increased susceptibility to intestinal pathogens and chemical damage have been reported in Myd88–/– mice (9, 10). We therefore addressed whether altered intestinal barrier function was responsible for loss of mucosal compartmentalization in Myd88–/– Ticam1–/– mice. Treatment of clean SPF control mice with nonsteroidal anti-inflammatory drugs (NSAIDs) to increase small intestine permeability (11), before administering high numbers of E. coli K-12 intragastrically, resulted in increased recovery of live E. coli from mesenteric lymph nodes but not from the spleen (Fig. 1A). This suggested that a nonspecific increase in intestinal permeability was insufficient to cause high numbers of live intestinal bacteria to reach the spleen.

We carried out three additional independent measurements to assess intestinal permeability in Myd88–/– Ticam1–/– mice. We used Ussing chambers to measure permeability in vitro (Fig. 1B and fig. S1B), assessed permeability between the intestinal lumen and the vasculature ex vivo (fig. S1C), and directly measured serum protein loss into the intestinal lumen in vivo (Fig. 1C). In each case, the values were similar in Myd88–/– Ticam1–/– and C57BL/6 mice, but were elevated when permeability was increased by prior treatment of the animals with NSAIDs or dextran sodium sulfate (DSS). Histological examination of intestinal architecture and epithelial cell proliferation also revealed no differences between Myd88–/– Ticam1–/– mice and controls (fig. S1, D and E). Thus, although previous work demonstrated that MyD88 function is important in determining susceptibility to DSS-induced (9) and pathogen-induced (10) inflammation in the intestine, we found no evidence for altered intestinal barrier function in Myd88–/– Ticam1–/– mice with a limited defined intestinal flora. Therefore, our data suggest that loss of mucosal compartmentalization can occur in the presence of normal intestinal barrier function and in the absence of any measurable intestinal pathology.

Bacteria encountered in the blood and spleen induce strong adaptive immune responses (1). We therefore determined the serum titers of commensal bacteria–specific antibodies in conventional SPF Myd88–/– Ticam1–/– mice. To avoid detection of highly cross-reactive epitopes, we determined titers by surface staining of live bacteria and flow cytometric analysis (fig. S2). We found that as expected (7), conventional SPF F1 control mice had no detectable serum antibodies to intestinal commensal bacterial species (Fig. 2A). In contrast, commensal-specific immunoglobulins G and M (IgGs and IgMs) were consistently present in conventional SPF unmanipulated Myd88–/– Ticam1–/– mice (Fig. 2A and S3A), at titers similar to those observed after intravenous vaccinations of control mice with bacteria (fig. S4). Furthermore, commensal-specific antibodies were also observed in clean SPF Myd88–/– Ticam1–/– mice and in an entirely independent colony of Myd88–/– mice (table S1). MyD88 or Ticam1 animals with a single gene deleted (single knockout) maintained under conventional SPF conditions also had spontaneous induction of commensal-specific antibodies, although at lower titers than Myd88–/– Ticam1–/– littermates (fig. S3B and C), which suggested an additive effect of the two genetic lesions.

Fig. 2

Myd88–/– Ticam1–/– mice lose systemic ignorance to their commensal flora. (A and B). The commensal bacteria E. faecalis and Staphylococcus xylosus were stained with whole serum from Myd88–/– Ticam1–/– and control mice, followed by phycoerythrin (PE)–conjugated secondary antibody against a mouse IgG1 and analyzed by flow cytometry. Titrations S. xylosus, E. faecalis, and Salmonella typhimurium IgG1 reactivity from 24-week-old Myd88–/– Ticam1–/– or F1 control mice housed in the indicated conditions. Each line represents an individual mouse. Data are representative of n > 30 mice. (C) Germ-free, 12-week-old Myd88–/– Ticam1–/– and Myd88+/– Ticam1–/– were monocolonized by cohousing with an E. coli K-12 monocolonized sentinel for the indicated amount of time (day 0, day 7, and so on). Serum was taken weekly to follow the development of E. coli K-12 IgG1 responses by bacterial flow cytometry and ELISA. Each line represents an individual mouse. Representative data of two independent experiments are shown. (D) E. coli K-12–specific IgA titers as determined by bacterial surface staining with cleared intestinal lavage from day 28 E. coli–monocolonized mice. Data shown are representative of two experiments.

Commensal bacteria–specific antibodies were shown to be a consequence of colonization with intestinal bacteria, because germ-free Myd88–/– Ticam1–/– mice had no detectable anticommensal IgG (Fig. 2B). Furthermore, this phenomenon occurred independently of opportunistic pathogens in the microbiota, as we detected high titers of E. coli IgG in the serum of Myd88–/– Ticam1–/– mice monocolonized with the nonpathogen (12) E. coli K-12 (Fig. 2C). It was noteworthy that clean SPF Myd88–/– Ticam1–/– mice vaccinated intravenously with peracetic acid–killed bacteria required higher doses of killed bacteria to induce IgG1 antibody responses than control mice (fig. S4), which suggested that Myd88–/– Ticam1–/– mice must experience profoundly elevated systemic commensal bacterial–antigen loads. Intestinal and serum IgA was present at normal concentrations in clean SPF and monocolonized MyD88–/– Ticam1–/– mice (fig. S5) and with normal or elevated titers specific for colonizing organisms (Fig. 2D and fig. S5). This demonstrated that induction of commensal-specific antibodies is microbiota-dependent, but not due to microbial pathogenicity or IgA production defects. Furthermore, although TLR signaling increases the sensitivity of the host for induction of bacteria-specific antibodies (5, 1316), TLR-signaling is not essential for the induction of antibacterial IgG1 responses. This is presumably because of contributions of other innate immune detection pathways, such as the NOD-like receptors, which still operate in the absence of TLRs but with lower sensitivity.

Studies of bacterial infection in MyD88–/– mice imply that there is deficient bacterial clearance (10, 14, 1620), which involves both hematopoietic and/or stromal cells. Indeed, intravenous injection of E. coli K-12 resulted in dramatically higher numbers of culturable bacteria in the spleens of clean SPF MyD88–/– Ticam1–/– mice than in spleens of clean SPF control mice (fig. S6). In addition, unmanipulated phagocyte oxidative burst–deficient mice (21) (Nos2–/– Cybb–/– mice) had serum antibodies specific for their commensal microbiota, whereas cohoused C57BL/6 controls did not (Fig. 3). This suggests that production of commensal-specific IgG in serum is a broad phenomenon associated with innate deficiency, causing impaired bacterial clearance.

Fig. 3

Nos2–/– Cybb–/– mice exhibit serum antibodies directed against their commensal flora. Serum and feces were collected from 8-week-old Nos2–/– Cybb–/– mice and cohoused C57BL/6 controls. Isolates of E. faecalis, Staphylococcus saprophyticus, and Stenotrophomonas maltophilia were obtained by aerobic culture from feces, and pure cultures were stained with serum from Nos2–/– Cybb–/– and control C57BL/6 mice. Antibacterial IgG1 was quantified by flow cytometry. C57BL/6 controls (n = 5) and NOS2Cybb mice (n = 5). Representative data from two independent experiments are shown.

To ask whether spontaneously induced anticommensal immunity was functionally important, we determined whether commensal colonization of Myd88–/– Ticam1–/– mice could correct bacterial clearance defects. Germ-free Myd88–/– Ticam1–/– and C57BL/6 mice were depleted of CD4+ T cells or mock-depleted, and monocolonized with the typical commensal Enterococcus faecalis by cohousing. Four weeks after colonization, Myd88–/– Ticam1–/–, but not C57BL/6 mice, had mounted a robust CD4+ T cell–dependent IgG response to E. faecalis (Fig. 4A and B). As expected, when infected intravenously with mixed E. faecalis and E. coli K-12, germ-free Myd88–/– Ticam1–/– mice had significantly higher splenic counts of both bacterial strains than C57BL/6 germ-free mice (Fig. 4C). In contrast, mock-depleted E. faecalis monocolonized Myd88–/– Ticam1–/– mice still did not clear E. coli, but could now clear E. faecalis from their spleens as efficiently as C57BL/6 mice (Fig. 4C). Monocolonized Myd88–/– Ticam1–/– mice that were depleted of CD4+ T cells (fig. S7A) and had not mounted strong IgG responses remained unprotected (Fig. 4C). Clearance did not require CD4+ T cells per se, as depletion just before challenge did not negatively influence protection (fig. S7, B to D). Further, coating of E. faecalis with serum from 28-day monocolonized Myd88–/– Ticam1–/– mice was sufficient to correct the bacterial clearance defect in Myd88–/– Ticam1–/– mice (fig. S7E). These data imply that adaptive immune responses, particularly high-titer T cell–dependent antibody responses, functionally compensate for the defect in bacterial clearance in Myd88–/– Ticam1–/– mice.

Fig. 4

Serum antibodies successfully protect Myd88–/– Ticam1–/– mice from bacteraemia. Germ-free, 12-week-old Myd88–/– Ticam1–/– and C57BL/6 mice were monocolonized with E. faecalis for 4 weeks by cohousing with monocolonized sentinel mice, with and without continuous CD4+ T cell depletion. (A) Serum antibodies against E. faecalis were quantified by bacterial flow cytometry at days 0 and 28 of colonization. (B) Serum antibodies against E. coli K-12 were quantified by bacterial flow cytometry at day 28 post colonization. (C) Germ-free mice that were monocolonized for 32 days and had been T cell–depleted, or mock-depleted, as indicated, were injected intravenously with a mixture of 107 CFU of nalidixic acid–resistant E. faecalis and 108 CFU of chloramphenicol-resistant E. coli K-12. Spleens were recovered 3 hours post injection and selectively plated (*P < 0.01). Data are representative of two independent experiments. (D) Total live mice of the indicated genotypes found at weaning (male and female mice). (E) Representative mice and weights of female mice at week 4 (7 days after weaning). (F) Protein-losing enteropathy quantified by measuring fecal albumin concentrations at 4 weeks of age. Total mice analyzed N = 97, including n = 2 MyD88–/–JH–/– mice.

In order to confirm that this mechanism is functionally important in unmanipulated mice we bred the Myd88–/– JH–/– double-deficient strain, which lacks antibody production beause of a deletion of the J segments of the immunoglobulin heavy-chain locus. Despite a clean SPF intestinal microbiota, Myd88–/–JH–/– offspring exhibited stunted growth, and only half the expected number survived to weaning (Fig. 4, D and E), whereas Myd88–/–JH+/– control littermates grew and survived normally. MyD88–/–JH–/– mice also displayed evidence of protein-losing enteropathy at weaning (Fig. 4F). These results suggest that antibody-mediated immunity is essential for TLR signaling–deficient mice to mutually coexist with their microbiota.

In this report, we have shown that adaptive immunity is critical for successful mutualism in TLR signaling–deficient mice. We conclude that TLR signaling is required for the normal elimination of low numbers of bacteria that are translocated from the intestinal lumen into the mucosa, but that commensal-specific serum IgG responses, induced in response to “escaped” intestinal bacteria, can restore effective bacterial clearance to wild-type levels. Thus, innate and adaptive immune mechanisms can complement each other to establish and maintain mutualism, as illustrated by the severe phenotype of the MyD88–/–JH–/– mouse. We suggest that there is a flexible set point between innate and adaptive immunity, determined by the functional performance of each system, that acts to protect the host. It is interesting to note that mutations in innate immunity genes are commonly associated with autoinflammatory diseases, the best characterized of which is the association of the non-TLR microbial recognition receptor NOD2 and Crohn’s disease (2224). Our work would clearly suggest that such patients would experience escape of a subset of the intestinal commensal flora, and the subsequent nature of immune responses induced may determine the presence or absence of disease.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S7

Table S1


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank J. Kirundi, J. Jury, and J. Lu for their technical support; A. G. Rolink and A. Strasser for providing reagents; and E. Denou, M. Heikenwälder, C. Reis e Sousa, D. Stetson, J. Danska, and C. Mueller for their helpful comments and editing of the manuscript. Grant support: Canadian Institutes of Health Research, Crohn’s and Colitis Foundation of Canada, Genome Canada, Canada Research Chairs, Canadian Association of Gastroenterology, NIH grant numbers CA105001 and AI56363.
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