A Quantitative Study of the Mechanisms behind Thymic Atrophy in Gαi2-Deficient Mice during Colitis Development

Mice deficient for the G protein subunit Gαi2 spontaneously develop colitis, a chronic inflammatory disease associated with dysregulated T cell responses. We and others have previously demonstrated a thymic involution in these mice and an aberrant thymocyte dynamics. The Gαi2−/− mice have a dramatically reduced fraction of double positive thymocytes and an increased fraction of single positive (SP) thymocytes. In this study, we quantify a number of critical parameters in order to narrow down the underlying mechanisms that cause the dynamical changes of the thymocyte development in the Gαi2−/− mice. Our data suggest that the increased fraction of SP thymocytes results only from a decreased number of DP thymocytes, since the number of SP thymocytes in the Gαi2−/− mice is comparable to the control littermates. By measuring the frequency of T cell receptor excision circles (TRECs) in the thymocytes, we demonstrate that the number of cell divisions the Gαi2−/− SP thymocytes undergo is comparable to SP thymocytes from control littermates. In addition, our data show that the mature SP CD4+ and CD8+ thymocytes divide to the same extent before they egress from the thymus. By estimating the number of peripheral TREC+ T lymphocytes and their death rate, we could calculate the daily egression of thymocytes. Gαi2−/− mice with no/mild and moderate colitis were found to have a slower export rate in comparison to the control littermates. The quantitative measurements in this study suggest a number of dynamical changes in the thymocyte development during the progression of colitis

Reduced IL-37 Production Increases Spontaneous Chemokine Expressions in Colon Epithelial Cells

Microscopic colitis, comprising collagenous colitis and lymphocytic colitis, is a common cause of chronic diarrhea. Previously, we showed enhanced chemokine productions in microscopic colitis patients, indicating dysregulated immune cell chemotaxis in the immunopathogenesis. We also showed decreased mRNA of IL-37, mainly regarded as an anti-inflammatory cytokine, in the colonic mucosa of these patients, potentially an important factor for the chronicity of the colitis. Our aim in this study was to understand the possible role of IL-37 in chemokine production using a cell line model. A colon epithelial cell line, T84, was stimulated with the TLR5 ligand flagellin. IL-37 protein production was reduced 20% using the CRISPR/Cas9 system, and the changes in chemokine mRNA and protein expressions were compared to cells transfected with empty plasmid. The 20% reduction in IL-37 protein levels spontaneously increased CCL5, CXCL8, CXCL10, and CXCL11 mRNA and protein expressions. CCL2 mRNA and protein levels were enhanced upon TLR5 stimulation. CCL3, CCL20, and CX(3)CL1 mRNA expressions were increased either spontaneously or following TLR5 stimulation, whereas CCL4 and CCL22 mRNA expressions were significantly decreased. Even a minor decrease in the ability of colon epithelial cells to produce IL-37 results in altered chemokine expression, mainly an increase in the production of several chemokines. Our results indicate that a decreased IL-37 expression by colon epithelial cells may be an important factor for increasing the recruitment of immune cells and subsequently developing microscopic coliti

Decreased frequencies and numbers of TRECs in peripheral blood CD3⁺ T lymphocytes in colitic Gαi2^−/− mice.

<p>Peripheral blood was collected and the volume recorded from individual mice, whereafter Gαi2^+/− mice were pooled (3 mice/pool) and analysed, while peripheral blood from Gαi2^−/− mice was analysed individually. The control group of wt mice were 4–9 weeks old (n = 11–16, white circles) and the Gαi2^−/− mice were 4–8 weeks old (n = 4–14, gray/black symbols), grouped based on their colitis scores (no colitis = rectangles, mild colitis = triangles). PBMCs were isolated from peripheral blood and the frequencies of CD3⁺ T lymphocytes was analysed by flow cytometry. The number of CD3⁺ (A) and naïve T lymphocytes (B) are presented as total T lymphocytes of the entire blood volume in each mouse, 6–8 ml blood per 100 g body weight <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036726#pone.0036726-Hoff1" target="_blank">[40]</a>. (C) The frequencies of TREC⁺ lymphocytes were analysed by real time-PCR on isolated DNA from flow cytometry sorted peripheral blood CD3⁺ T lymphocytes. (D) Numbers of TREC⁺ CD3⁺ peripheral blood lymphocytes, as calculated from data (A) and (C). Horizontal lines indicate the mean within the group. Statistical analysis was performed using the Mann-Whitney non-parametric test and values of p≤0.05 were considered significant. *p≤0.05; **p≤0.01 and ***p≤0.001 between Gαi2^−/− and wt mice.</p

The frequencies of TRECs in mature thymocytes in Gαi2^−/− mice are independent of the colitis score.

<p>Mature CD4⁺CD8⁻CD62L⁺CD69⁻ and CD8⁺CD4⁻CD62L⁺CD69⁻ thymocytes from wt mice and Gαi2^−/− mice were sorted by flow cytometry. DNA from the sorted thymocytes were analysed for the amount of TRECs specific DNA by real time-PCR. The frequencies of TRECs⁺ cells in CD4⁺CD62L⁺ (white symbols) and CD8⁺CD62L⁺ (gray symbols) thymocytes were analysed in 4–9 weeks old wt mice (n = 28) and 4–8 weeks old Gαi2^−/− mice (n = 5–13), grouped based on colitis scores (rectangles = no colitis, triangles = mild colitis). The mean frequencies within each group are represented as a horizontal line. Statistical analysis was performed using the Mann-Whitney non-parametric test and values of p≤0.05 were considered significant.</p

Elevated fecal peptidase D at onset of colitis in Galphai2-/- mice, a mouse model of IBD.

BACKGROUND:The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms. METHODS:Fecal samples were collected at onset of inflammation in Galphai2-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice. RESULTS:As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2-/- mice at different stages of disease. CONCLUSIONS:These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2

Reduced thymic egression in Gαi2^−/− mice with no/mild and moderate colitis.

<p>A. PBMCs were stained with CD4 and CD62L specific antibodies, as well as Annexin V and Propidium Iodide (PI) to analyse the frequency of naïve peripheral blood CD4⁺ T lymphocytes undergoing apoptosis. The bars depicts the mean frequency ± S.D. of apoptotic CD4⁺ T lymphocytes in wt mice (n = 6, light bar) and Gαi2^−/− mice (n = 5, dark bar (A). The daily egression was calculated by the following equation; , see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036726#s2" target="_blank">Materials and Methods</a>. (B) Four-nine weeks old wt mice (n = 11) and Gαi2^−/− mice (n = 4–11) indicate the individual numbers of thymocytes egressing per day. Gαi2^−/− mice were divided based on their stage of colitis (no colitis = rectangles, mild colitis = triangles). Horizontal lines indicate mean values within each group. Statistical analysis was performed using the Mann-Whitney non-parametric test and values of p≤0.05 were considered significant. *p≤0.05; **p≤0.01 and ***p≤0.001 between Gαi2^−/− and wt mice.</p

Spearman's correlation analysis between age or colitis score and fraction or number of the different thymocyte subpopulations.

a<p>The different age groups were: 4 (n = 9), 5 (n = 7), 6 (n = 5), 7 (n = 6) and 8 weeks (n = 3).</p>b<p>The different colitis score were: 0p (n = 3), 0.5–2p (n = 3), 2.5–4p (n = 14), 4.5–6p (n = 6) and 6.5–8p (n = 4).</p