Supplementary Figure 1. ETBF activate Stat3 in B6 and Min mice colons
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1 Supplementary Figure 1 ETBF activate Stat3 in B6 and Min mice colons a pstat3 controls Pos Neg ETBF b pstat1 pstat2 pstat3 pstat4 pstat5 pstat6 Actin Figure Legend: (a) ETBF induce predominantly pstat3 in colons of C57Bl/6 mice. pstat nuclear proteins were evaluated by western blot on nuclear extracts of colons of 3-4 ETBF-colonized C57Bl/6 mice. Positive and negative controls for each pstat protein are included for comparison. Equal amounts of nuclear protein were loaded in each lane. Actin further serves as a loading control. (b) pstat3 staining in infiltrating leukocytes of the colonic mucosa of an ETBF-colonized Min mouse. An example of ETBFcolonized mucosa in which marked inflammatory infiltrates demonstrated pstat3 staining by IHC.
2 Supplementary Figure 2 Splenic cells display intracellular IFN-γ but minimal IL-17 in ETBF-colonized Min mice. 3.0 CD4 + TCR β + CD8 + TCR β Spleen IFN γ IL 17 Figure Legend: Splenic cells isolated from Min mice colonized with ETBF for 1 wk and activated with PMA, ionomycin and Golgiplug TM for 5 hr were first stained for surface markers followed by intracellular staining for IFN-γ and IL-17.
3 Supplementary Figure 3 CD3 - and CD3 + CD8 + leukocytes are not responsible for IL-17 production in ETBF-colonized Min mice. a IL-17 γδ-tcr 48 αβ-tcr 38 CD3 CD4 CD4 b CD3 + cells IL CD8 Figure Legend: Isolated colonic lymphocytes from Min mice colonized with ETBF for 1 wk and activated 5 hrs in vitro with PMA and ionomycin in the presence of Golgiplug TM. (a) CD3 - leukocytes do not display ICS for IL-17. Two right dot plots show the expression of γδ-tcr, αβ-tcr and CD4 + on the CD3 + IL-17 + lymphocytes. (b) CD3 + CD8 + lymphocytes do not display ICS for IL-17. The dot plot is derived from CD3 + cells. The figure is representative of at least 3 independent experiments.
4 Supplementary Figure 4 Mucosal cytokine expression in NTBF- and ETBF-colonized Min mice P< Cytokine fold-change P= P= P= P= IL-23p19 IL-6 IL-1β IL-17A TGF-β NT ET NT ET NT ET NT ET NT ET Figure Legend: qpcr analysis of Th17 pathway cytokines in the colonic mucosa of Min mice colonized with NTBF or ETBF for 1 wk. The distribution of the cytokine data are shown as box and whisker plots. NT = NTBF-colonized and ET = ETBF-colonized mice. n = per condition except NT, IL-1β (n = 4).
5 Supplementary Figure 5 a Distribution of colon tumor sizes in ETBF-colonized Min mice treated with Th17 pathway neutralizing antibodies. b Number of polyps Polyp size distribution in ETBF Min mice treated with IL-17A and IL-23R blocking antibodies IL23R IL17 ET IgG2a/2b ET Number of polyps Polyp size distribution in ETBF Min mice treated with IL17A blocking antibody IL17A ET IgG2a ET Polyp area (mm 2 ) Polyp area (mm 2 ) Figure Legend: Methylene blue-stained colon tumors were counted at 5 wks after bacterial inoculation and sized by microscopy. In panel (a), ETBF-colonized Min mice were treated with IL-17A and IL-23R neutralizing antibodies or isotype control antibodies. In panel (b), ETBF-colonized Min mice were treated with IL-17A neutralizing antibodies or isotype control antibodies.
6 Supplementary Figure 6 IL-17 blockade does not discernibly modify Stat3 activation by ETBF. Sham IgG + ET IL-17 Ab + ET Min mice pstat3 Actin Sham IgG + ET IL-17 Ab + ET B6 mice pstat3 Actin Figure Legend: Sham and ETBF-colonized Min or C57Bl/6 mice were treated with IL-17 blocking antibody (IL-17 Ab + ET) or isotype control IgG antibody (IgG + ET) for 3 d followed by extraction of mucosal nuclear proteins and western blot for activated Stat3 (pstat3). Actin serves as a loading control.
7 Supplementary Table 1 Mouse colon histology scores 1 wk after NTBF or ETBF colonization in wild-type (WT) or CD4 Stat3-KO C57Bl/6 mice. Median (range) Inflammation Hyperplasia Sham WT (n = 3) 0 (0 0) 0 (0 0) Sham CD4 Stat3-KO (n = 4) 0 (0 1) 0.5 (0 1) NTBF WT (n = 4) 0 (0 0) 0.5 (0 1) NTBF CD4 Stat3-KO (n = 4) 0 (0 1) 0.5 (0 1) ETBF WT (n = 13) 3 (1 4) a,b 2 (2 3) a,d ETBF CD4 Stat3-KO (n = 18) 2 (1 3) c 2 (1 3) e a P vs sham and NTBF WT, independently b P vs ETBF CD4 Stat3-KO c P vs sham and NTBF CD4 Stat3-KO, independently d P 0.03 vs ETBF CD4 Stat3-KO e P vs sham and NTBF CD4 Stat3-KO, independently
8 Supplementary Table 2 Min mouse colon histology scores 4 5 wks after ETBF colonization in mice treated with isotype antibodies or IL-17 and IL-23R neutralizing antibodies or IL-17 neutralizing antibodies alone Median (range) Inflammation Hyperplasia GIN Gross Tumors ETBF (isotype Abs) (n = 24) 2 (1 3) 2 (2 3) 2 (0 6) 10 (2 32) ETBF (IL-17/IL-23R Abs) (n = 14) 1 (0 1) a 2 (1 2) b 0 (0 1) b 5 (1 9) c ETBF (IL-17 Abs) (n = 8) 1 (1 1) d 1 (0 2) b 0.5 (0 2) e 3.5 (1 13) e All comparisons are to ETBF (isotype antibodies). a P < b P < c P < d P = e P < 0.02
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A263 A352 A204. Pan CK. pstat STAT3 pstat3 STAT3 pstat3. Columns Columns 1-6 Positive control. Omentum. Rectosigmoid A195.
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To compare the relative amount of of selected gene expression between sham and
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<20 <20 <20 < <20 <20 <20 <20. Mock
Cross-Lineage Neutralization PRNT 80 Titers Asian Asian West African Indian Ocean Group NHP Strain 181/25 Strain 99659 Strain 37997 Strain LR 142590 80 80 20 40 EILV/CHIKV 150844 640 640 160 320 Mock 150849
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