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

Local accumulation of neutrophils in the colon of Muc2^-/- mice.

<p>LP cells from proximal, middle or distal colon sections of Muc2^-/- and Muc2^+/- littermates were analyzed by flow cytometry. (A) Viable neutrophils were identified as 7AAD^-MHCII^-CD11b⁺Ly6G⁺ cells. The numbers represent the percent of cells in the indicated gate. (B) The frequency of neutrophils among viable LP cells for all mice examined is shown. The dashed line indicates the “cut off” value of 0.36%[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.ref023" target="_blank">23</a>]). (C) The frequency of CD11b⁺MHC^-/low cells among viable LP cells of all mice examined is depicted. Each symbol represents an individual mouse and the horizontal line indicates the mean. Results are from 12 independent experiments with a total of 14–22 mice per group. Statistical significance 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

The composition of colon DCs and macrophages is altered in inflamed Muc2^−/− mice.

<p>LP cells from the colon of Muc2^−/− mice, inflamed Muc2^−/− mice and Muc2^+/− controls were stained for DCs and macrophages and analyzed by flow cytometry. (<b>A</b>) The gating strategy used is shown where viable MHC-II⁺NK1.1⁻TCR⁻CD19⁻ cells (left column) were distinguished by CD11c expression (middle) and CD11c⁺MHC-II⁺ cells were then analyzed for CD103 and CD11b (right). The numbers are the percent of cells in the indicated gates. (<b>B–D</b>) The frequency of CD103⁺CD11b⁻ DCs (<b>B</b>), CD103⁺CD11b⁺ DCs (<b>C</b>), and CD103⁻ macrophages (<b>D</b>) among MHCII⁺CD11c^hi cells in the indicated gates is shown. (<b>E–G</b>) The absolute number of CD103⁺CD11b⁻ (<b>E</b>), CD103⁺CD11b⁺ (<b>F</b>) and CD103⁻ macrophages (<b>G</b>) among 10⁶ viable LP cells is shown. Each symbol represents an individual mouse. The median is indicated by a horizontal line. Data are from 15 independent experiments that analyzed 20–29 mice per group. The mice were between 8–19 weeks of age. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test. Significance is indicated as *p<0.05, **p<0.01, while all other comparisons are non-significant.</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