Community Structure Comparison: Libshuff and Parsimony Statistical Analysis of Colon-Associated Bacterial Populations.

<p>Community Structure Comparison: Libshuff and Parsimony Statistical Analysis of Colon-Associated Bacterial Populations.</p

Richness, diversity and taxonomic analysis of the WT and Nod2 KO colon tissue-associated bacterial communities.

<p>Top panel: Incidence of phyla from treatment groups of WT and Nod2 KO littermates. Mean +/− SEM (n = 4–6) for each group is shown. * p≤0.05, ** p<0.01 by 2 way ANOVA with Bonferroni's multiple comparison test. Bottom panels: the Chao and Shannon estimates for richness and diversity were calculated from individual mice from each group as indicated (n = 4–6). The mean +/− SEM are shown. No differences were statistically significant by ANOVA.</p

Comparison of physical and histological parameters and bacterial load of WT and Nod2 KO littermates following DSS damage.

<p><b>A.</b> Timelines and histology assessment for individual mice. No significant difference was observed between the two genotypes for physical parameters (body weight loss, colon length: not shown) nor histological scores between the two groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273.s004" target="_blank">Figure S4</a> for data from 2 additional independent experiments). <b>B.</b> Colon, mesenteric lymph node and spleen tissue-associated bacterial loads assessed by FACS 42 days following DSS damage. ** = p≤0.01 by Anova with Bonferroni's multiple comparison test. <b>C.</b> Residence of commensal bacteria in the muscle layer in Nod2 KO mice. Examples of bacterial staining by EUB338 FISH probes are indicated by closed arrows in these representative images.</p

Phylogenetic tree visualisation of full length 16S rRNA sequences from WT and Nod2 KO mouse colon tissue 42 days post DSS or control as indicated.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#s4" target="_blank">Materials and Methods</a> for parameters.</p

Infiltration of colon by immune cells assessed by immunohistochemistry following DSS damage.

<p>Top panel provides timeline for the experiment with arrows indicating times of histological and immunohistochemical assessment. Representative images for each of the cell markers and from each of the time points are shown. H&E (haematoxylin/eosin histochemical stain), CD3 (T cell marker), F4/80 (macrophage marker) and GR-1/Ly-6G (granulocyte marker).</p

Temporal physical, histological and biomarker analysis of DSS damage.

<p>Mice were administered DSS in their drinking water from day 0 to day 5 and individual parameters outlined in each panel assessed by criteria described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273-Smith1" target="_blank">[41]</a>. * = p≤0.05, ** = p≤0.01, *** = p≤0.001 by 1-way Anova with Bonferroni's multiple comparison test. <b>A.</b> Body weight (physiological parameter for progression of inflammation) expressed as mean +/− SEM, n = 7. <b>B.</b> Fecal Score (combined weighted score of intestinal damage based on the presence of blood in the stool and subjective assessment of diarrhea) as a physiological indicator of colon tissue damage expressed as mean +/− SEM, n = 7. <b>C.</b> Colon length (physiological parameter for progression of inflammation) expressed as mean +/− SEM, n = 7. Statistical comparisons are versus Naive (no DSS-treatment) mice. <b>D.</b> Histology Score (quantitative assessment of microscopic tissue damage identical to that described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030273#pone.0030273-Smith1" target="_blank">[41]</a>) expressed as mean +/− SEM, n = 7. Statistical comparisons are versus Naive (no DSS-treatment) mice. <b>E.</b> Temporal cytokine (as indicated) profiles in colon homogenates from treated mice expressed as mean +/− SEM, n = 7. Statistical comparisons by Anova/Bonferroni. @ = p≤0.05 vs day 5, # = p≤0.05 vs day 8.</p

Proximity and progression of host/commensal interactions following DSS damage.

<p><b>A.</b> Localisation of commensal bacteria by EUB338 FISH probe analysis of FFPE sections from DSS-treated mice. Open arrows highlight examples of luminal or mucosal bacteria, closed arrows indicate bacteria associated with the muscle layers. E, epithelium. M, muscle. Bar = 50 µm. <b>B.</b> Quantification of colon tissue bacteria load in individual mice by FACS following DSS damage. * = p≤0.05, ** = p≤0.01, *** = p≤0.001 vs day 0 (no DSS treatment) by 1-way Anova with Bonferroni's correction.</p