Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism

Abstract

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.

Figures

Fig. 1

Increased bacterial penetration in Myd88−/− Ticam1−/− mice is not dependent on increased intestinal permeability. (A) “Clean SPF” Myd88−/− Ticam1−/− (MyD88TRIF), F1 control mice, and F1 mice treated with 7.5 mg/kg Indomethacin 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 3 independent experiments. n.d. Not detectable. (B) Ussing chamber measurements of conductance and paracellular permeability, as assessed by serosal 51Cr-EDTA recovery, of jejunum from Myd88−/− Ticam1−/− (MyD88TRIF) mice, co-housed C57BL/6 control mice (B6), and positive control (C57BL/6) mice treated with 7.5 mg/kg Indomethacin (NSAID). Each data point represents an individual mouse and all collected data is shown. (C) 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 is shown.

Fig. 2

Myd88−/− Ticam1−/− mice lose systemic ignorance to their commensal flora. (A and B). The commensal bacteria Enterococcus faecalis and Staphylococcus xylosus were stained with whole serum from Myd88−/− Ticam1−/− and control mice, followed by PE-anti-mouse IgG1 and analyzed by flow cytometry. Titrations of anti-S. xylosus, anti-E. faecalis and anti-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 is representative of n>30 mice. (C) 12-week old germ-free Myd88−/− Ticam1−/− and Myd88+/− Ticam1−/− mice were monocolonized by co-housing with an E. coli K-12 monocolonized sentinel for the indicated amount of time. Serum was taken weekly to follow the development of E. coli K-12 IgG1 antibody 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.

Fig. 3

Cybb−/− Nos2−/− mice exhibit serum antibodies directed against their commensal flora. Serum and feces were collected from 8-week-old Cybb−/− Nos2−/− mice and co-housed C57BL/6 controls. Isolates of E. faecalis, Staphylococcus saprophyticus and Stentrophomonas maltophilia were obtained by aerobic culture from feces and pure cultures were stained with serum from Cybb−/− Nos2−/− and control C57BL/6 mice. Anti-bacterial IgG1 was quantified by flow cytometry. n=5 C57BL/6 controls and 5 CybbNOS2 mice. Representative data from two independent experiments are shown.

Fig. 4

Serum antibodies successfully protect Myd88−/− Ticam1−/− mice from bacteraemia. 12-week old germ-free Myd88−/− Ticam1−/− and C57BL/6 mice were monocolonized with E. faecalis for four weeks by co-housing with monocolonized sentinel mice, with and without continous 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) 32 day monocolonized and germ-free mice, which had been T cell depleted, or mock-depleted, as indicated, were injected intravenously with 107 CFU of nalidixic acid-resistant E. faecalis and 108 CFU of chloramphenicol-resistant E. coli K-12. Spleens were recovered at three 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 (seven days after weaning). (F) Protein-losing enteropathy quantified by measuring fecal albumin concentrations at four weeks of age. Total mice analysed n=97, including n=3 MyD88−/−JH−/− mice.