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Science 325 (5940): 617-620

Copyright © 2009 by the American Association for the Advancement of Science

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

Emma Slack1,5,*, Siegfried Hapfelmeier1,5, Bärbel Stecher2, Yuliya Velykoredko1, Maaike Stoel1, Melissa A. E. Lawson1, Markus B. Geuking1, Bruce Beutler3, Thomas F. Tedder4, Wolf-Dietrich Hardt2, Premysl Bercik1, Elena F. Verdu1, Kathy D. McCoy1, and Andrew J. Macpherson1,5,*

1 Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, ON L8S 4L8, Canada.
2 Institute of Microbiology, ETH Zürich, 8032 Zürich, Switzerland.
3 Department of Genetics, The Scripps Research Institute, La Jolla, CA 92121, USA.
4 Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.
5 DKF (Department of Clinical Research), Maurice Müller Laboratories, Universitätsklinik für Viszerale Chirurgie und Medizin (UVCM), University of Bern, 3008 Bern, Switzerland.

Figure 1
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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.


Figure 2
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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.


Figure 3
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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.


Figure 4
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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.


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