Inflammatory cytokines characterize the distal colon of a subgroup of colitic Muc2^-/- mice.

<p>Saponin extracts of proximal, middle and distal colon were prepared and normalized to total protein concentration. Cytokine expression was analyzed by CBA (A, C- F) or ELISA (B, G). Each symbol represents an individual mouse and the solid line indicates the mean of each group. Symbols of the same color in Figs 3 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.s001" target="_blank">S1 Fig</a> represent samples from the same colon segment of the same animal. Mice are segregated into colitic and non-colitic according to neutrophil influx into the colon LP in the respective colon segment. Data are pooled from 5 independent experiments that examined 27 mice. The distal colon panels to the right of the vertical bar show the same distal colon data as to the left of the bar except that the colitic mouse group is segregated into group “A” and “B”. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p

CD103⁺CD11b⁺ Dendritic Cells Induce T_h17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

<div><p>Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells <i>ex vivo</i>. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.</p></div

Altered frequency of iMP populations in the colon of Muc2^-/- mice.

<p>LP cells from proximal, middle and distal colon of Muc2^+/- controls, Muc2^-/-, and colitic Muc2^-/- mice were analyzed by flow cytometry. (A) A representative dot plot of a Muc2^+/- mouse showing gating of viable NK1.1^-TCR^-CD19^-CD11c⁺MHC-II⁺ cells is depicted in the left two dot plots and further analysis of gated CD11c⁺MHC-II⁺ cells for expression of CD103 and CD11b for the indicated genotype of mouse is shown to the right. B-D depict the frequency of CD103⁺CD11b^- "P1" DCs (B), CD103⁺CD11b⁺ "P2" DCs (C), and CD103^-CD11b⁺ "P3" iMPs (D) among viable LP cells in the indicated gates. E-G show the absolute number of viable CD103⁺CD11b^- "P1" DCs (E), CD103⁺CD11b⁺ “P2” DCs (F) and CD103^-CD11b⁺ “P3” iMPs (G). Each symbol represents an individual mouse. For B-G results from the proximal (left column), middle (center column) and distal (right column) colon are shown. The mean of each group is indicated by the horizontal line. Segregating Muc2^-/- mice into colitic and non-colitic is performed according to neutrophil influx in the respective colon segment. Data are from 12 independent experiments that analyzed 14–22 mice per group. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p

Cytokine profiles in the distal colon of colitic mice correlate with increased CD103⁺CD11b⁺ P2 DCs in the same colon segment.

<p>LP cells of the other half of the same distal colon segment of the same mice used for cytokine analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.g003" target="_blank">Fig 3</a> were analyzed by flow cytometry. Cells were gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.g002" target="_blank">Fig 2A</a>. (A-C) The frequency of DCs belonging to subsets P1 (A), P2 (B) or P3 (C) among viable MHCII⁺CD11c^hi cells is shown. (D-E) The absolute number of DCs belonging to subsets P1 (D), P2 (E) or P3 (F) among 10⁶ viable LP cells is depicted. Each symbol represents an individual mouse and the solid line indicates the mean of each group. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p

Colitis influences the ability of intestinal DC subsets to induce CD4⁺ T cell proliferation.

<p>P1, P2 and P3 cells were sorted from pooled MLN from Muc2^+/-, Muc2^-/- or colitic Muc2^-/- and analyzed by flow cytometry. Sorted cells were pulsed with OVA_(323–339) peptide prior to co-incubation with CTV-labeled OT-II cells for 5 days. (A) The gating strategy used to identify P1, P2, and P3 populations from MLN cell suspensions is shown. (B) Dot plots show CTV dilution of OT-II cells co-cultured with the indicated iMP subset pulsed with OVA_(323–339) peptide. (C) Bar graphs show the frequency of proliferating OT-II T cells ± SD from (B).</p

Colitis influences CD4 T cell differentiation induced by intestinal DC subsets.

<p>Cytokine concentrations in the supernatants of the T cell proliferation assays in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.g005" target="_blank">Fig 5</a> were analyzed by Luminex and are shown in (A-C). (D-F) show the absolute number of differentiated CD4 T cells identified by staining for intracellular IFN-γ (left), CD25 and intracellular FoxP3 (middle) or intracellular IL-17A (right). Data are normalized to 10⁴ CD4 T cells in co-cultures with the indicated DC subset after 5 days. Pooled data from 2 independent experiments in duplicates with a total of 6–8 animals/group is shown ± SD. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p

Patient demographics.

<p>*NA; not applicable.</p

Altered abundance of DCs and macrophages characterizes the inflamed colon LP of UC patients.

<p>LP cells from biopsies taken from inflamed areas of UC patients with active inflammation, from UC patients in clinical remission and from non-inflamed controls were analyzed by flow cytometry. (<b>A</b>) The gating strategy used where viable CD3⁻CD19⁻HLADR⁺ cells that were CD11c⁺ (left two plots) were further analyzed to identify CD14⁺ macrophages and CD103⁺ DCs (right two plots). Data from a representative non-inflamed control and an inflamed UC patient are shown. (<b>B</b>) The percent of CD103⁺ DCs and (<b>C</b>) CD14⁺ macrophages among HLADR⁺CD11c⁺ cells is shown. The absolute number of (<b>D</b>) CD103⁺ DCs and (<b>E</b>) CD14⁺ macrophages among 10⁶ viable LP cells is shown. Each symbol represents an individual patient and the median is indicated by a horizontal line. Non-inflamed controls n = 10, UC patients in remission n = 6, UC patients with active inflammation n = 10. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001).</p

Luminal bacteria penetrate colon tissue of Muc2^−/− mice and inflamed UC patients.

<p>qPCR was used to determine the ratio of 23SrRNA to 18SrRNA to assess bacterial presence in mouse colon tissue (<b>A</b>), mouse MLN (<b>B</b>) and biopsies from UC patients with active inflammation, from UC patients in clinical remission and from non-inflamed controls (<b>C</b>). Results were analyzed using the ΔΔCT method with the CT value of 18SrRNA as the endogenous reference gene. (<b>D</b>) Differential gene expression of Relmβ was analyzed using the ΔΔCT method with the CT value of HPRT as the endogenous reference gene. (<b>E</b>) Dot plots show intracellular iNOS expression by gated viable (7AAD⁻) CD11c⁺MHC-II^hiCD11b⁺ cells from the MLN of a representative inflamed Muc2^−/− mouse (left) and a WT mouse (right). (<b>F</b>) Scatter plot show the percent iNOS-expressing cells among total CD11c⁺MHC-II^hiCD11b⁺ cells from the MLN of WT mice (grey circles), Muc2^−/− mice (open circles) and inflamed Muc2^−/− mice (black circles). Mice in the “Muc2^−/− inflamed” group had a higher frequency of colon PMNs relative to other Muc2^−/− mice indicating they were colitic and were analyzed as a separate group; see text for details. Mouse data was obtained from 3 independent experiments with 8–11 mice in each group. Each symbol represents an individual. Mice were between 8–16 weeks of age except 2 WT mice that were 7 weeks old. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001).</p

Histology of colon tissue from Muc2^−/− mice and UC patients.

<p>(<b>A</b>) Carnoy fixed whole colon sections with preserved mucus from WT and Muc2^−/− mice were analyzed by FISH (red) for bacteria localization and counterstained with DAPI. The mucus separating bacteria from the epithelium in WT mice is indicated by a double arrow and bacteria in contact with the epithelium in Muc2^−/− mice are marked by arrows. Scale bars are 100 µm. (<b>B</b>) Sigmoid sections of human biopsies from a control patient and a patient with active UC (Mayo endoscopic score 2) were stained for MUC2 (green) and DAPI. Mucus separating bacteria and the epithelium is indicated by a double arrow and bacteria within the remaining mucus in the UC patient are marked by arrows. Scale bars are 20 µm. (<b>C–D</b>) Representative sections from the proximal, middle or distal colon from (<b>C</b>) Muc2^+/− (18 weeks of age) and (<b>D</b>) inflamed Muc2^−/− (18 weeks of age) are shown. The neutrophil frequency determined by flow cytometry in parallel samples of the displayed tissues is indicated in the inlays. Original magnification is 5x. (<b>E–F</b>) Human rectal tissue from representative sections of (<b>E</b>) an inflamed UC patient at the time of diagnosis and (<b>F</b>) a non-inflamed control are shown. Original magnification is 40x. All tissues were sliced in 5 µm sections and stained with H&E.</p