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1 SUPPLEMENTARY INFORMATION doi: /nture11225 Numer of OTUs sed on 3% distnce Numer of 16s rrna-sed V2-V4 tg sequences LF MF PUFA Supplementry Figure 1. High-ft diets decrese the richness nd diversity of intestinl microiot in wild type C57BL/6 mice. C57BL/6 mice were fed low-ft (LF), sturted milk ft (MF) or polyunsturted sfflower oil (PUFA) diet for 24 dys. The rrefction curve is sed on 454-sed DNA sequencing of 16S rrna gene lirries from cecl contents nd depicts the numer of unique opertionl txonomic units (OTUs) inned per smple with ech line representing sequences derived from one mouse. The slope of the line is directly relted to the numer of unique OTUs (i.e. incresed diversity) in smple. 1
2 RESEARCH SUPPLEMENTARY INFORMATION. Low-ft Lrd. Firmicutes PUFA MF Bcteroidetes 10 8 δ-proteocteri (Bilophil wdsworthi) Supplementry Figure 2. Lrd-sed sturted ft exerts differentil effects on phyl nd sulfite-reducing cteri compred to milk ft-sed sturted ft., C57BL/6 mice (n=12) were plced on either low-ft (LF), sturted lrd-sed ft, sturted milkft-sed (MF), or polyunsturted sfflower oil-sed (PUFA) diet for 24 dys. Totl ft content of the three high-ft diets ws equl t 37% kcl ut the loom of B. wdsworthi ws only oserved in the MF diet,, nd ws confirmed y qpcr. Attempts to determine which ftty cid component of MF nd Lrd might ccount for the differences in phyl distriution were unsuccessful ecuse of technicl chllenges in soluilizing steric cid- the mjor sturted ftty cid component of MF. 2
3 SUPPLEMENTARY INFORMATION RESEARCH.. *** Colitis score Reltive fold chnge (dsra) LF PUFA MF *** LF PUFA MF d. *** LF PUFA MF LF PUFA MF *** c. Low-ft PUFA Milk-ft ** * LF PUFA MF LF PUFA MF Supplementry Figure 3. Milk ft (MF) increses incidence of colitis nd in cute DSS tretment nd is ssocited with n increse in sulfite-reducing cteri nd TH1 cytokines.. Blinded histologicl colitis scores re shown individully nd represented s mens ± S.E.M., Reltive undnce of SRBs s mesured y qpcr of dsra nd normlized to LF. Presented s mens ± S.E.M. c, H&E stining of distl colon. Scle rs, μm. d, Distl colonic mucosl cytokines determined y ELISA. *** P< , **P<.001, *P<.05 determined y one-wy ANOVA nd Tukey post-hoc. 3
4 RESEARCH SUPPLEMENTARY INFORMATION LF + Bw MF +Bw Supplementry Figure 4. Bilophil wdsworthi cn only e estlished in gnotoiotic mice in the presence of MF. Representtive mice from LF (left) nd MF (right) + monossocition. Note the shrunken cecum in the MF mouse, phenomen oserved with Bw coloniztion. 4
5 SUPPLEMENTARY INFORMATION RESEARCH ** Supplementry Figure 5. B. wdsworthi preferentilly colonized the mucos of germ-free IL10 -/- monossocited mice fed MF. GF IL10 -/- were monossocited with Bw nd fed MF for 24 dys. Quntittive PCR for dsra reveled greter coloniztion of the distl colonic mucos compred to distl colonic luminl contents. dsra vlues were normlized to totl 16s rrna. ** P< determined y student s t-test. 5
6 RESEARCH SUPPLEMENTARY INFORMATION. MF + Bw PUFA + Bw LF + Bw. c c c c c c c c c c c Supplementry Figure 6. B. wdsworthi (Bw) growth in germ-free IL10-/- mice fed low-ft (LF), polyunsturted ft (PUFA, or milk ft (MF) diets., Germ-free IL10-/- were fed LF, PUFA, or MF diets for 7 dys, then monossocited with Bw. Bw surivived only the presence of MF nd this growth induced distl colonc inflmmtion noted y rrows pointing to lymphoid ggregte, which is not typiclly seen in GF mice until colonized with pthogen, nd immune cell infiltrtion. These effects were not oserved in mice consuming LF or PUFA diets. Scle rs, 400µm., Distl colonic mucosl cytokines determined y ELISA. P<.05 determined y one-wy ANOVA nd Tukey post-hoc. 6 W W W. N A T U R E. C O M / N A T U R E
7 SUPPLEMENTARY INFORMATION RESEARCH *** *** *** * Supplementry Figure 7. B. wdsworthi (Bw) growth in IL10 -/- mice supplemented with turocholic cid induces intestinl inflmmtion. SPF IL10 -/- were monossocited with Bw nd then gvged dily with either PBS, glycocholic cid (GC), or turocholic cid (TC) for 21 dys. Bw surivived only the presence of TC. Distl colonic mucosl cytokines determined y ELISA. *** P< , *P<.05 determined y one-wy ANOVA nd Tukey post-hoc. 7
8 RESEARCH SUPPLEMENTARY INFORMATION PBS + Bw. GC + Bw TC + Bw PBS TC GC PBS + Bw GC + Bw TC + Bw. c c c c Supplementry Figure 8. Germ-free IL10-/- mice monossocited with Bw nd gvged with either PBS, glycocholic cid (GC), or turocholic cid (TC)., Distl colon H&E stining shows incresed inflmmtion in monossocited mice gvged with TC s noted y rrows pointing to crypt hyperplsi nd presence of lymphoid ggregte t the se of n re of mucosl ulcertion. These effects were not oserved in the presence of TC gvge lone in the sence of Bw (dt not shown). Scle rs, 400μm., Distl colonic mucosl cytokines determined y ELISA. P<.05 determined y one-wy ANOVA nd Tukey post-hoc. 8 W W W. N A T U R E. C O M / N A T U R E
9 SUPPLEMENTARY INFORMATION RESEARCH Supplementry Tle 1. Experimentl diets nd ftty cid composition Study Diets Low-ft (AIN-93M) Sturted- Milk Ft Sturted- Lrd Ft PUFA (Sfflower oil) Ft (%kcl) -Sturted (%totl) -PUFA Protein Crohydrte Micronutrients/Fier *ND *ND *ND *ND 9
10 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Tle 2. Assignment to the genus level of 324 ner full-length 16s rrna sequences 10
11 SUPPLEMENTARY INFORMATION RESEARCH Supplementry Tle 3. Smple metdt Clone Lirry 454-Pyrosequencing Sequencing depths nd # reds 3525 verge totl sequences 110 sequences/smple 3000 reds/smple Totl Mice C57BL/6 IL10 -/
12 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Tle 4. Correltion etween undnce of B. wdsworthi nd colitis score clculted y Persons correltion coefficient Diet tretment groups Correltion of Bw undnce nd colitis score (R-vlue) SPF IL10 -/- + LF SPF IL10 -/- + PUFA SPF IL10 -/- + MF GF IL10 -/- + LF GF IL10 -/- + PUFA GF IL10 -/- + MF Bile tretment groups SPF IL10 -/- + PBS SPF IL10 -/- + GC SPF IL10 -/- + TC GF IL10 -/- + PBS GF IL10 -/- + GC GF IL10 -/- + TC
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