Supplemental Information. Commensal Fungi Recapitulate. the Protective Benefits of Intestinal Bacteria
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1 Cell Host & Microbe, Volume Supplemental Information Commensal Fungi Recapitulate the Protective enefits of Intestinal acteria Tony T. Jiang, Tzu-Yu Shao, W.X. Gladys Ang, Jeremy M. Kinder, Lucien H. Turner, Giang Pham, Jordan Whitt, Theresa Alenghat, and Sing Sing Way
2 A 1 Villus width (µm) 7 A/PAS Goblet cells (#) 1 1 C Granules (#) 1 1 D Crypt height (µm) 1 A/PAS 1 Goblet cells (#) E 9 Figure S1 (related to Figure 1). C. albicans enteric colonization does not cause intestinal tissue injury (A-C) Representative sections of small intestine stained with hematoxylin and eosin () or alcian blue/periodic acid-schiff (A/PAS), and composite data enumerating villus width (A), goblet cells per villus (), and granules per paneth cell (C), for specific-pathogen free mice administered C. albicans and maintained on antibiotics containing ampicillin, gentamicin, metronidazole, neomycin, vancomycin for 1 days (), compared with antibiotic treated controls without C. albicans inoculation (). (D,E) Representative sections of colon stained with or A/PAS, and composite data enumerating crypt height (D), and number of goblet cells per X field (E) for mice described in panels (A-C). Data are representative of at least two independent experiments. ars, mean ± s.e.m. Original magnification 1X for (A,D), X for (), or X for (C,E).
3 D D17 D D9 Analysis Epithelial ulceration D Total disease score C Edema DSS FITC-dextran (µg/ml) CA 9 Inflammatory cell infiltration Colon length (cm) A Figure S (related to Figure 1). C. albicans intestinal mono-colonization mitigates the severity of DSS induced colitis (A) Analysis after % DSS initiation among specific-pathogen free mice after supplementing the drinking water with an antibiotic cocktail () containing ampicillin, gentamicin, metronidazole, neomycin, vancomycin, or administered C. albicans (CA) three days after initiation (). (-D) Colon length (), serum FITC-dextran concentration four hours after oral gavage (C), representative hematoxylin and eosin () stained colonic sections and composite histological scoring (see Methods) (D) after DSS administration (for six days) for mice described in panel (A). P <.1, P <.1, by unpaired t-test with Welch s correction. Data are representative of at least three independent experiments. ars, mean ± s.e.m. Original magnification 1X for (D).
4 A Fungal CFUs/g (log 1 ) 1 GF GF L.o.D. Survival (%) 1 7 GF (n=1) GF (n=11) DSS 1 1 Days after DSS initiation Figure S (related to Figure 1). Overt susceptibility to DSS induced mortality among germ-free mice is improved following C. albicans intestinal mono-colonization (A) Recoverable fungal CFUs in the feces for germ-free mice 1 days after inoculation with C. albicans (GF), compared to germ-free controls without CA administration (GF). () Percent survival after DSS supplementation in the drinking water (for six days) for mice described in panel (A). P <., by Log-rank (Mantel-Cox) test. ars, mean ± s.e.m. L.o.D., limit of detection.
5 A Intrarectal reconstitution CA Flu A Analysis D D1 D17 Survival (%) 1 7 CA (n=1) Mannan (n=17) PS (n=17) 1 1 Days after Flu A infection K b OVA + CD T cells (#) PS CA Mannan IFN-γ + CD T cells (#) PS CA Mannan Figure S (related to Figure ). Mannan stimulation recapitulates the extra-intestinal benefits of persistent C. albicans mono-colonization (A) Percent survival after influenza A PR-OVA (Flu A) intranasal infection ( x 1 PFUs) among specificpathogen free mice maintained on drinking water supplemented with an antibiotic cocktail () containing ampicillin, gentamicin, metronidazole, neomycin, vancomycin, and administered C. albicans (CA) three days after initiating antibiotic treatment, or intrarectally administered 1 mg mannan or saline (PS) every other day starting one day prior to infection. () Total number of Flu A-specific K b OVA tetramer + CD T cells (left), and IFN-γ + CD T cells after in vitro OVA 7- peptide stimulation (right), from lungs nine days after Flu A infection for mice described in panel (A). P <., P <.1, by Log-rank (Mantel-Cox) test (A) or by Mann Whitney U test (). Data are representative of at least three independent experiments. ars, mean ± s.e.m.
6 Table S1 (related to STAR METHODS). Antibodies used and staining conditions for flow cytometry Protein Fluorophore Clone Vendor Dilution CD FITC GK1. eioscience 1: CDα APC -.7 eioscience 1: CD11b PE-Cy M1/7 eioscience 1: CD11c PE-Cy N1 eioscience 1: F/ PE-Cy M eioscience 1: PE-Cy RA- eioscience 1: IFN-γ efluor XMG1. eioscience 1:1
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DC enriched CD103. CD11b. CD11c. Spleen. DC enriched. CD11c. DC enriched. CD11c MLN
CD CD11 + CD + CD11 d g CD CD11 + CD + CD11 CD + CD11 + Events (% of max) Events (% of max) CD + CD11 Events (% of max) Spleen CLN MLN Irf fl/fl e Irf fl/fl h Irf fl/fl DC enriched CD CD11 Spleen DC enriched
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