<i>E</i>. <i>coli</i> LF82_N mice develop severe, transmural colitis following DSS-induced inflammation.

Mice were scored at the time of necropsy for macroscopic parameters of disease including colon length (A) and macroscopic colitis score (as described in Materials and Methods) (B). Additionally, proximal colon was blindly scored for histopathologic parameters of inflammation by a pathologist (JH) (C). Representative photomicrographs of histopathological sections imaged at 40X (scale bar = 100 μm) (D). Shared letters between different groups indicate no statistical significance with an ANOVA or Kruskal-Wallis with multiple group comparisons test. Asterisk indicate statistical significance based on a direct comparison with a Mann-Whitney two-tailed test, *p ≤ 0.05, **p ≤ 0.01. p for colon length data indicates significance based on a linear regression model for 1all groups and between the 2DSS treated groups. A and B: Control n = 17, DSS n = 21, LF82A n = 21, LF82A + DSS n = 28, LF82N n = 24, LF82N + DSS n = 32; C: Control n = 13, DSS n = 12, LF82A n = 16, LF82A + DSS n = 24, LF82N n = 19, LF82N + DSS n = 29.</p

RNAscope^® analysis with HALO^® image analysis software.

RNAscope® analysis with HALO® image analysis software.</p

PCA plots.

PCA plot depicting the clustering of samples originating from control ASF mice and ASF mice colonized with E. coli LF82 in the presence or absence of DSS exposure. A) PCA plot generated by incorporating data based on measurements of colon length, macroscopic lesions, and histological evaluation and B) PCA plot generated by incorporating data based on measurements of colon length, macroscopic, and histologic scores plus tissue cytokine production.</p

Experimental protocol.

Adult gnotobiotic C3H/HeN mice harboring the altered Schaedler flora (ASF) were orally gavaged with E. coli LF82. After three weeks, LF82-infected and ASF-only control mice were paired for breeding. Pups born to E. coli LF82-infected dams were colonized by vertical transmission during the process of microbial succession and were designated as neonatal colonized (LF82N). LF82N and ASF-only pups were weaned after 3 weeks of age. At 6 weeks of age, a subset of ASF-only mice were colonized with E. coli LF82 by oral gavage and designated as adult colonized (LF82A). At 9 weeks of age, mice were treated with filter sterilized 2% dextran sodium sulfate in their drinking water for seven days prior to necropsy.</p

Evaluation of spatial distribution of the microbial community following <i>E</i>. <i>coli</i> LF82 colonization and DSS-induced inflammation.

Proximal colons with intact contents were assessed for overall microbial spatial distribution by fluorescent in situ hybridization (FISH). Spatial distribution of organisms was evaluated using a eubacteria probe (EUB338) to detect bacteria in the lumen, adherent/attaching mucus layer, and translocation into the glands (A). Representative photomicrographs (60X magnification) of E. coli LF82 detected in the microbial community by FISH by labeling the nuclei of mucosal cells with DAPI (blue), total bacteria with a EUB338 probe (FITC, green), and labeling with a E. coli-specific probe (Cy-3, orange) (B). Control n = 5, LF82A + DSS n = 8, LF82N + DSS n = 8.</p

LF82_NH LF82 mice have intermediate colitis scores compared to LF82_N and LF82_A following DSS-induced inflammation.

Mice were horizontally infected as neonates (LF82NH) to assess maternal influence on the effects observed following colonization with E. coli LF82. Mice were evaluated at the time of necropsy for parameters of disease including colon length (A) and macroscopic colitis score (B). Additionally, proximal colon was fixed, sectioned and stained with hematoxylin and eosin and blindly scored by a pathologist for histopathologic parameters of inflammation (C). The cecal load of E. coli LF82 was enumerated by bacteriological culture (D) and 16s rRNA gene amplicon sequencing was performed on DNA recovered from fecal samples to assess relative microbial abundance (E). To mimic the length of time that the neonates were colonized with E. coli LF82, a separate set of adult mice evaluated at 10 weeks post-colonization (10 wk LF82A). Mice were scored at the time of necropsy for parameters of disease including colon length (F) and macroscopic colitis score (G). Shared letters between different groups indicate no statistical significance with an ANOVA or Kruskal-Wallis with multiple group comparisons test; ns indicates no statistical significance based on a direct comparison with a t or Mann-Whitney test. A and B: Control n = 17, DSS n = 21, LF82A n = 21, LF82A + DSS n = 28, LF82NH n = 15, LF82NH + DSS n = 18, LF82N n = 24, LF82N + DSS n = 32; C: Control n = 13, DSS n = 12, LF82A n = 16, LF82A + DSS n = 24, LF82NH n = 5, LF82NH + DSS n = 5, LF82N n = 19, LF82N + DSS n = 29; D and E: LF82NH n = 6, LF82NH + DSS n = 8; A and B: Control n = 17, DSS n = 21, LF82A n = 21, LF82A + DSS n = 28, 10 wk LF82A n = 4, 10 wk LF82A + DSS n = 5, LF82N n = 24, LF82N + DSS n = 32. (TIFF)</p

Inflammation induces differential production of proinflammatory cytokines by colonic explants.

Analysis of IL-17A (A), IFNγ (B), IL-1β (C), and IL-6 (D) released from ex vivo incubated colonic explants. Mice were euthanized and tissues collected on day seven of the DSS exposure. Shared letters between different groups indicate no statistical significance based on an ANOVA with multiple group comparisons test. p indicates significance based on a linear regression statistical model of the IL-17A secretion. Data is presented as the mean ± SEM. A-C: Control n = 11, DSS n = 9, LF82A n = 14, LF82A + DSS n = 19, LF82N n = 15, LF82N + DSS n = 24; D: Control n = 6, DSS n = 7, LF82A n = 6, LF82A + DSS n = 12, LF82N n = 8, LF82N + DSS n = 12.</p

Evaluation of the luminal microbial community following <i>E</i>. <i>coli</i> LF82 colonization and/or DSS-induced inflammation.

At necropsy, the cecal load of E. coli LF82 was enumerated by bacteriological culture (A), and 16s rRNA gene amplicon sequencing was performed on DNA recovered from fecal samples to assess relative microbial abundance (B). A Weighted UniFrac PCoA plot was generated using PhyloSeq to assess changes in beta diversity among the treatment groups (C). Each point represents a single animal, and each color represents a treatment group. The ellipses were generated to visualize the relative variability of each treatment group. Shared letters between different groups indicate no statistical significance with an ANOVA with multiple group comparisons test. A: LF82A n = 11, LF82A + DSS n = 15, LF82N n = 13, LF82N + DSS n = 19; B and C: Control n = 3, DSS n = 3, LF82A n = 4, LF82A + DSS n = 6, LF82N n = 5, LF82N + DSS n = 8.</p

Raw SAS outputs from analyses as described in materials & methods.

(PDF)</p

RNAscope detection of IL-17, IFNγ and CD4 mRNA transcripts in the colon of <i>E</i>. <i>coli</i> LF82 colonized mice treated with DSS.

IL-17, IFNγ, and CD4 mRNA was labeled with a duplex RNAscope® assay. Representative photomicrographs demonstrating dual labeling of CD4- (red) and cytokine-specific (green) mRNA in multiple experimental groups (scale bar = 20 μm). (TIFF)</p