Faecal secretogranin and chromogranin levels persist over time and are unrelated to disease history and outcome in patients with ulcerative colitis

<p><b>Objectives</b>: Chromogranins (Cg) and secretogranins (Sg) are expressed by endocrine cells and may be important for the pathophysiology of ulcerative colitis (UC). We investigated dynamics of faecal granin expression in patients with UC during a period of 18 months, both during remission and relapse, and association to disease outcome the following 3 years.</p> <p><b>Materials and methods</b>: Secretoneurin (SN), Sg3, CgA and CgB were measured in three to seven serial faecal samples from UC patients who did not (n = 20) or did (n = 20) relapse during study time. All patients were in remission at baseline and disease characteristic were monitored during sampling and 3 years after the final sample.</p> <p><b>Results</b>: Faecal SN, Sg3, CgA and CgB levels showed no association to patient characteristic or disease history at baseline. Faecal granin levels showed low intra-patient variability and levels stayed constant during short and long intervals at remission, did not alter during or after clinical relapse and were not associated to medical therapy. In contrast, high inter-patient variability was detected and multivariate analysis revealed three distinct patient groups, where extensive disease was more common in patients with high levels of SN and CgA as compared to patients with low levels of all granins or patients with high levels of Sg3 and CgB. These patient subgroups did, however, not differ in patient characteristics, disease history or future disease course.</p> <p><b>Conclusions</b>: Faecal granin levels are stable over time but are unrelated to disease history, activity and outcome and are thus not valuable markers for disease activity in UC patients.</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

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

PMNs increase in the colon of Muc2^−/− mice and UC patients.

<p>Colon LP cells from Muc2^−/− mice, Muc2^+/− littermates, UC patients and non-inflamed controls were analyzed by flow cytometry. (<b>A</b>) PMNs in mice were identified as viable (7AAD⁻) MHCII⁻CD11b⁺Ly6G⁺ cells. The numbers represent the percent cells in the indicated gate. The Muc2^−/− mouse shown did not have overt signs of inflammation such as rectal swelling. (<b>B</b>) The frequency of PMNs among viable LP cells for all mice examined is shown. The dashed line indicates the “cut off” value of 0.36% (see text for details). Results are from 17 independent experiments with a total of 19–55 mice per group. (<b>C</b>) PMNs in human tissue were identified as 7AAD⁻MHCII⁻CD15^hiCD66b⁺ cells. The numbers represent the percent cells in the indicated gate. PMN gating was performed using a non-inflamed and inflamed sample from different colon regions from the same UC patient as negative and positive control. (<b>D</b>) The frequency of PMNs among viable LP cells for non-inflamed controls and UC patients with established disease is shown. Statistical significance was assessed using the Mann-Whitney-U-Test; significance is indicated as **p<0.01, ****p<0.0001. Each symbol represents an individual mouse or patient. The mice used were between 8–19 weeks of age.</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

Spontaneous Colitis in Muc2-Deficient Mice Reflects Clinical and Cellular Features of Active Ulcerative Colitis

<div><p>Background</p><p>The colonic mucus layer plays a critical role in intestinal homeostasis by limiting contact between luminal bacteria and the mucosal immune system. A defective mucus barrier in animal models allows bacterial contact with the intestinal epithelium and results in spontaneous colitis. A defective mucus barrier is also a key feature of active ulcerative colitis (UC). Alterations in the immune compartment due to intestinal bacterial breach in mice lacking the colon mucus barrier have not been characterized and correlated to active UC.</p><p>Aims</p><p>To characterize alterations in the immune compartment due to intestinal bacterial breach in Muc2^−/− mice, which lack the colon mucus barrier, and correlate the findings to active UC.</p><p>Methods</p><p>Bacterial contact with colon epithelium and penetration into colon tissue was examined in Muc2^−/− mice and colon biopsies from patients with active UC using fluorescence microscopy and qPCR. Neutrophils, lymphocytes, CD103⁺ dendritic cell subsets and macrophages in colon from Muc2^−/− mice and biopsies from UC patients were quantitated by flow cytometry.</p><p>Results</p><p>Inflamed UC patients and Muc2^−/− mice had bacteria in contact with the colon epithelium. Bacterial rRNA was present in colonic mucosa in humans and Muc2^−/− mice and in the draining lymph nodes of mice. Inflamed Muc2^−/− mice and UC patients had elevated colon neutrophils, T cells and macrophages while a reduced frequency of CD103⁺ DCs was present in the inflamed colon of both mice and humans.</p><p>Conclusions</p><p>The parallel features of the colon immune cell compartment in Muc2^−/− mice and UC patients supports the usefulness of this model to understand the early phase of spontaneous colitis and will provide insight into novel strategies to treat UC.</p></div