SIHUMI mice with both <i>A. muciniphila</i> and <i>S.</i> Typhimurium display increased MUC2 mRNA levels (A) and reduced numbers of mucin filled goblet cells (B and C).

<p>(A) mRNA was extracted from cecum mucosa of mice belonging to either one of four groups: SIHUMI, SIHUMI-A, SIHUMI-S and SIHUMI-AS. MUC2 mRNA from cecum mucosa was converted to cDNA and expression levels were quantified using real-time PCR (see materials and methods). SIHUMI-A and SIHUMI-AS mice showed significantly higher MUC2 gene expression compared to the other two groups, harboring no <i>A. muciniphila</i>. Data are expressed as mean±standard error. n = 6 per group. <i>*P<0.05, **P<0.01, ***P<0.001.</i> Amuc: <i>A. muciniphila;</i> S. Tm: <i>S.</i> Typhimurium. (B) Formalin fixed cecal tissue sections from SIHUMI, SIHUMI-A, SIHUMI-S and SIHUMI-AS mice were stained with alcian blue (pH-2.5) and haematoxylin. Images are representative of 5 mice per group. Magnification 400-fold. SIHUMI-AS mice display the lowest number of positively stained mucin-filled goblet cells compared to the other three groups. The bar represents 100 µm. (C) Quantitative analysis of the number of acidic mucin-filled goblet cells (blue) enumerated in cecal tissue sections from SIHUMI, SIHUMI-A, SIHUMI-S and SIHUMI-AS mice for a 50 µm stretch of lamina muscularis corresponding to approximately 30 cecal crypts per section. Two sections per mouse were analyzed. The number of cecal mucin filled goblet cells was elevated when <i>A. muciniphila</i> was present (SIHUMI-A) but the concomitant presence of <i>S.</i> Typhimurium (SIHUMI-AS) resulted in the lowest number of mucin filled goblet cells of gnotobiotic SIHUMI mice compared to the other mouse groups. Data are expressed as mean±standard error. n = 5 mice. <i>*P<0.05, **P<0.01, ***P<0.001.</i> Amuc: <i>A. muciniphila;</i> S. Tm: <i>S.</i> Typhimurium.</p

Hypothetical Scheme.

<p>The presence of <i>A. muciniphila,</i> leads to the exacerbation of <i>S.</i> Typhimurium-induced intestinal inflammation. We propose that the presence of <i>A. muciniphila</i> causes changes in mucin composition and production, which in turn facilitates the invasion of <i>S.</i> Typhimurium into the host. Increased inflammatory status was characterized by increased pro-inflammatory cytokines, increased macrophage infiltration and invasion of the pathogen into the lymph nodes, reduced number of mucin-filled goblet cells in SIHUMI-AS mice (B) compared to SIHUMI-S mice (A). Our data suggests that in the presence of both <i>A. muciniphila</i> and <i>S.</i> Typhimurium, mucus sulphation is diminished and this may facilitate the access of <i>S.</i> Typhimurium to sialic acid in mucus. Sialic acid may serve as a substrate and adhesion site for <i>S.</i> Typhimurium in the gut <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone.0074963-Giannasca1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone.0074963-Severi1" target="_blank">[57]</a>. Increased gene expression of IFN-γ and IP-10 indicate an increased NK-cell recruitment. mLN - mesenteric lymph nodes, NK- Natural killer cells. (↑ increased; ↓ decreased; grey dotted line: assumed processes including lectin-sialic acid binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone.0074963-Giannasca1" target="_blank">[56]</a>, M-cells for pathogen transit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone.0074963-McGuckin1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone.0074963-Clark1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone.0074963-Foster1" target="_blank">[59]</a>; black line: supported by data of the present study).</p

<i>S.</i> Typhimurium becomes the dominant species in SIHUMI mice previously associated with <i>A. muciniphila</i>.

<p>Data are expressed as mean±standard error. Different superscripts indicate statistically significant differences (<i>P≤0.05).</i> n = 10 mice per group. DW: dry weight.</p

Presence of both <i>A. muciniphila</i> and <i>S.</i> Typhimurium is accompanied by increased pro-inflammatory cytokines.

<p>(A) Cecal mRNA levels of IFN-γ, IP-10, TNF-α, IL-12, IL-6, IL-17 and IL-18 in gnotobiotic SIHUMI mice were measured. mRNA was extracted from cecum mucosa of mice belonging to either one of four groups: SIHUMI, SIHUMI-A, SIHUMI-S and SIHUMI-AS (see Figure. 1). The mRNA was converted to cDNA for quantitative real-time PCR measurement (see materials and methods). Inoculation of the gnotobiotic SIHUMI mice with <i>A. muciniphila</i> followed by <i>S.</i> Typhimurium infection (SIHUMI-AS) caused an increase in mRNA levels of pro-inflammatory cytokines except IL-18. Data are expressed as mean±standard error. n = 6 per group. Star indicates statistically significant differences (<i>*P<0.05, **P<0.01, ***P<0.001)</i>. AU: Arbitrary units; Amuc: <i>A. muciniphila;</i> S. Tm: <i>S.</i> Typhimurium. (B) Serum protein levels of IFN-γ were increased in SIHUMI-AS mice compared to the other mouse groups. Data are expressed as mean±standard error. n = 10 mice per group. <i>*P<0.05, **P<0.01, ***P<0.001</i>. Amuc: <i>A. muciniphila;</i> S. Tm: <i>S.</i> Typhimurium.</p

Concomitant presence of <i>A. muciniphila</i> and <i>S.</i> Typhimurium results in increased histopathology scores in SIHUMI mice.

<p>(A) Gnotobiotic C3H mice containing 8 defined microbial species (SIHUMI) were subsequently inoculated with <i>A. muciniphila</i> or <i>S.</i> Typhimurium or consecutively with both organisms (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074963#pone-0074963-g001" target="_blank">Figure 1</a>). SIHUMI and SIHUMI-A mice had the lowest histopathology scores (≤4.0) with no signs of inflammation and were therefore taken as baseline (dotted line). Data are expressed as median with range. <i>*P<0.05, **P<0.01, ***P<0.001</i>. n = 10 mice per group. (B) Representative microscopy images of pathological changes observed in cecum tissue sections fixed with formalin and stained with hematoxylin and eosin (4 µm) of the four mouse groups. n = 10 mice per group; Magnification: 1000-fold.</p

Additional file 1 of Dendritic cells under allergic condition enhance the activation of pruritogen-responsive neurons via inducing itch receptors in a co-culture study

Supplementary Material

Scoring for assessment of the disease activity index.

<p>Scoring for assessment of the disease activity index.</p

Local cytokine concentrations in the colon of wild-type, MCL^−/−, and DCIR^−/− mice.

<p>Colons from untreated wild-type mice or from wild-type and MCL^−/− mice (n = 6) (<b>A</b>), or wild-type and DCIR^−/− mice (n = 7 for wild-type and n = 8 for DCIR^−/− mice) (<b>B</b>) treated with 3% DSS for seven consecutive days were homogenized and used for cytokine determination by cytometric bead array. Data are expressed as mean + SEM. Significance is indicated by asterisks (*), ns = no significance.</p

Scoring for the histological evaluation of intestinal lesions.

<p>Scoring for the histological evaluation of intestinal lesions.</p

Histological analysis of colon sections from wild-type and DCIR^−/− mice.

<p>Paraffin sections of the colon from untreated or 3% DSS-treated wild-type and DCIR^−/− mice were prepared at day seven and were stained with hematoxylin and eosin (H&E) for histological evaluation in a blinded manner. (<b>A</b>) Representative images of paraffin-embedded sections of the rectal part of the colon are shown (40x magnification). Arrows indicate a severe ulcer in the colon from DCIR^−/− mice. Each colon was divided into three segments of identical length (oral, middle, rectal) which were separately analyzed. The degree of leukocyte infiltration (<b>B</b>) and mucosal erosion/ulceration (<b>C</b>) was graded from none (score 0) to mild (score 1), moderate (score 3), or severe (score 4). The scores for both, cell infiltration as well as mucosal ulceration in the rectal part of the colon from DCIR^−/− mice were significantly increased compared to wild-type mice. Data are expressed as mean + SEM (n = 5). The <i>p</i>-values were determined using Mann-Whitney’s U test (*<i>p</i><0.05, **<i>p</i><0.01). Significance is indicated by asterisks (*), ns = no significance.</p