Progastrin Represses the Alternative Activation of Human Macrophages and Modulates Their Influence on Colon Cancer Epithelial Cells

Macrophage infiltration is a negative prognostic factor for most cancers but gastrointestinal tumors seem to be an exception. The effect of macrophages on cancer progression depends on their phenotype, which may vary between M1 (pro-inflammatory, defensive) to M2 (tolerogenic, pro-tumoral). Gastrointestinal cancers often become an ectopic source of gastrins and macrophages present receptors for these peptides. The aim of the present study is to analyze whether gastrins can affect the pattern of macrophage infiltration in colorectal tumors. We have evaluated the relationship between gastrin expression and the pattern of macrophage infiltration in samples from colorectal cancer and the influence of these peptides on the phenotype of macrophages differentiated from human peripheral monocytes in vitro. The total number of macrophages (CD68+ cells) was similar in tumoral and normal surrounding tissue, but the number of M2 macrophages (CD206+ cells) was significantly higher in the tumor. However, the number of these tumor-associated M2 macrophages correlated negatively with the immunoreactivity for gastrin peptides in tumor epithelial cells. Macrophages differentiated from human peripheral monocytes in the presence of progastrin showed lower levels of M2-markers (CD206, IL10) with normal amounts of M1-markers (CD86, IL12). Progastrin induced similar effects in mature macrophages treated with IL4 to obtain a M2-phenotype or with LPS plus IFNγ to generate M1-macrophages. Macrophages differentiated in the presence of progastrin presented a reduced expression of Wnt ligands and decreased the number and increased cell death of co-cultured colorectal cancer epithelial cells. Our results suggest that progastrin inhibits the acquisition of a M2-phenotype in human macrophages. This effect exerted on tumor associated macrophages may modulate cancer progression and should be taken into account when analyzing the therapeutic value of gastrin immunoneutralization

Succinate activates EMT in intestinal epithelial cells through SUCNR1: a novel protagonist in fistula development

The pathogenesis of Crohn's disease-associated fibrostenosis and fistulas imply the epithelial-to-mesenchymal transition (EMT) process. As succinate and its receptor (SUCNR1) are involved in intestinal inflammation and fibrosis, we investigated their relevance in EMT and Crohn's disease (CD) fistulas. Succinate levels and SUCNR1-expression were analyzed in intestinal resections from non-Inflammatory Bowel Disease (non-IBD) subjects and CD patients with stenosing-B2 or penetrating-B3 complications and in a murine heterotopic-transplant model of intestinal fibrosis. EMT, as increased expression of Snail1, Snail2 and vimentin and reduction in E-cadherin, was analyzed in tissues and succinate-treated HT29 cells. The role played by SUCNR1 was studied by silencing its gene. Succinate levels and SUCNR1 expression are increased in B3-CD patients and correlate with EMT markers. SUCNR1 is detected in transitional cells lining the fistula tract and in surrounding mesenchymal cells. Grafts from wild type (WT) mice present increased succinate levels, SUCNR1 up-regulation and EMT activation, effects not observed in SUCNR1$^{-/-}$ tissues. SUCNR1 activation induces the expression of Wnt ligands, activates WNT signaling and induces a WNT-mediated EMT in HT29 cells. In conclusion, succinate and its receptor are up-regulated around CD-fistulas and activate Wnt signaling and EMT in intestinal epithelial cells. These results point to SUCNR1 as a novel pharmacological target for fistula prevention

IFNγ-Treated Macrophages Induce EMT through the WNT Pathway: Relevance in Crohn’s Disease

Background: Fibrosis is a common complication of Crohn’s disease (CD) in which macrophages play a central role. Epithelial-mesenchymal transition (EMT) and the WNT pathway have been associated with fibrosis. We aim to analyse the relevance of the tissue microenvironment in macrophage phenotype and the EMT process. Methods: Intestinal surgical resections are obtained from control and CD patients with stenotic or penetrating behaviour. Cytokine’s expression, macrophage phenotype, EMT markers and WNT signalling pathway are determined by WB, RT-PCR, ELISA or Cytometry. U937 cells are treated with IFNγ, TNFα, IL1β, IL4 or IL10 and co-cultured with HT29 cells and, in some cases, are treated with XAV939 or miFZD4. The expression of macrophage, EMT and WNT pathway markers in U937 or HT29 cells is analysed by WB or RT-PCR. Results: IFNγ, WNT6, CD16 and CD86 are increased in the intestinal tissue of CD patients. IFNγ-treated U937 activated the EMT process and WNT pathway in HT29 cells, and the EMT process is mediated by FZD4. Conclusions: An IFNγ-rich microenvironment polarises macrophages, which induces EMT through the WNT pathway

Induction of CD36 and Thrombospondin-1 in Macrophages by Hypoxia-Inducible Factor 1 and Its Relevance in the Inflammatory Process

<div><p>Inflammation is part of a complex biological response of vascular tissue to pathogens or damaged cells. First inflammatory cells attempt to remove the injurious stimuli and this is followed by a healing process mediated principally by phagocytosis of senescent cells. Hypoxia and p38-MAPK are associated with inflammation, and hypoxia inducible factor 1 (HIF-1) has been detected in inflamed tissues. We aimed to analyse the role of p38-MAPK and HIF-1 in the transcriptional regulation of CD36, a class B scavenger receptor, and its ligand thrombospondin (TSP-1) in macrophages and to evaluate the involvement of this pathway in phagocytosis of apoptotic neutrophils. We have also assessed HIF-1α, p38-MAPK and CD36 immunostaining in the mucosa of patients with inflammatory bowel disease. Results show that hypoxia increases neutrophil phagocytosis by macrophages and induces the expression of CD36 and TSP-1. Addition of a p38-MAPK inhibitor significantly reduced the increase in CD36 and TSP-1 expression provoked by hypoxia and decreased HIF-1α stabilization in macrophages. Transient transfection of macrophages with a <em>miHIF-1α</em>-targeting vector blocked the increase in mRNA expression of <em>CD36</em> and <em>TSP-1</em> during hypoxia and reduced phagocytosis, thus highlighting a role for the transcriptional activity of HIF-1. CD36 and TSP-1 were necessary for the phagocytosis of neutrophils induced by hypoxic macrophages, since functional blockade of these proteins undermined this process. Immunohistochemical studies revealed CD36, HIF-1α and p38-MAPK expression in the mucosa of patients with inflammatory bowel disease. A positive and significant correlation between HIF-1α and CD36 expression and CD36 and p38-MAPK expression was observed in cells of the lamina propria of the damaged mucosa. Our results demonstrate a HIF-1-dependent up-regulation of CD36 and TSP-1 that mediates the increased phagocytosis of neutrophils by macrophages during hypoxia. Moreover, they suggest that CD36 expression in the damaged mucosa of patients with inflammatory bowel disease depends on p38-MAPK and HIF-1 activity.</p> </div

Diminished Vitamin D Receptor Protein Levels in Crohn’s Disease Fibroblasts: Effects of Vitamin D

Vitamin D (VD) deficiency has been associated to Crohn&rsquo;s disease (CD) pathogenesis, and the exogenous administration of VD improves the course of the disease, but the mechanistic basis of these observations remains unknown. Vitamin D receptor (VDR) mediates most of the biological functions of this hormone, and we aim to analyze here the expression of VDR in intestinal tissue, epithelial cells, and fibroblasts from CD patients. The effects of VD on a fibroblast wound healing assay and murine intestinal fibrosis are also analyzed. Our data show diminished VDR protein levels in surgical resections and epithelial cells from CD patients. In intestinal fibroblasts isolated from damaged tissue of CD patients, we detected enhanced migration and decreased VDR expression compared with both fibroblasts from non-damaged tissue of the same CD patient or control fibroblasts. Treatment with VD increased VDR protein levels, avoided the accelerated migration in CD fibroblasts, and prevented murine intestinal fibrosis induced by the heterotopic transplant model. In conclusion, our study demonstrates diminished VDR protein levels associated with enhanced migration in intestinal fibroblasts from damaged tissue of CD patients. In these cells, VD accumulates VDR and normalizes migration, which supports that CD patients would benefit from the VD anti-fibrotic therapeutic value that we demonstrate in a murine experimental model

HIF-1, p38-MAPK and CD36 correlates in the inflamed mucosa of patients with inflammatory bowel disease.

<p>A) Representative microphotographs showing HIF-1α, p38-MAPK and CD36 immunostaining in the damaged and non-damaged mucosa of patients with inflammatory bowel disease. Biopsy specimens of the intestine were excised, formalin-fixed, paraffin-embedded, cut into 5 µm slices, and stained with hematoxylin; B) Graph shows a quantitative analysis of the number of HIF-1α, p38-MAPK or CD36 positive cells in a total area of 0.135 mm² of the mucosa of patients with IBD. Bars in the graph represent mean± SEM (<i>n></i>3). Significant difference from the respective non-damaged mucosa is shown by *<i>P</i><0.05. C) Graphs show a positive and significant correlation between CD36 and HIF-1α (R Spearman = 0.7170, P = 0.0087**, n = 12) and p38-MAPK and CD36 (R Spearman = 0.6525, P = 0.0215*, n = 12) immunostaining at the damaged mucosa of patients with IBD. No correlation was observed between CD36 and HIF-1α (R Spearman = −0.0513, P = 0.95, n = 5), or p38-MAPK and CD36 (R Spearman = 0.5204, P = 0.2311, n = 7) immunostaining at the non-damaged mucosa.</p

Patient clinical information.

<p>Patient clinical information.</p

Role of CD36 and TSP-1 in phagocytosis mediated by macrophages.

<p>Graphs show the effects of CD36 and TSP-1 functional antibodies or control IgG on phagocytosis of apoptotic neutrophils mediated by U937 cells or THP1 cells. In both cases, blockade of CD36 or TSP-1 significantly reduced hypoxia-induced phagocytosis. Data show the intensity of fluorescence in arbitrary units (quantified by static cytometry). Bars represent mean± SEM (<i>n></i>3). Groups were compared using ANOVA followed by a Newman Keuls test. *P<0.05 shows significant difference with respect to all groups in the same graph.</p

Recruitment of HIF-1 to the promoter of <i>TSP-1</i> gene.

<p>A) Results show a representative chromatin immunoprecipitation (ChIP) experiment performed in samples from U937-derived macrophages in normoxia or hypoxia. Chromatin was immunoprecipitated with anti-HIF-1α antibody, or a non-related antibody anti-IgG as a control. An aliquot of the input chromatin is also shown. Primers specific to the promoter region for TSP-1 gene were used to amplify the DNA isolated from the ChIP assay. B) HIF-1α expression in nuclear lysates derived from non-transfected cells and from <i>miHIF1α</i> or mock-transfected U937cells exposed to normoxia or hypoxia. Interactions between HIF-1α and HRE of the TSP-1 promoter gene were examined by EMSA using synthetic oligonucleotides and nuclear lysates derived from transfected or non-transfected cells exposed to normoxia or hypoxia. Specificity was determined with excess unlabelled probed (XS) or mutated probe (n = 3).</p

Hypoxia induces TSP-1 and CD36 expression and HIF-1α stabilization through activation of p38-MAPK.

<p>U937 cells were maintained under normoxia or hypoxia in the presence or absence of SB 202190 (a p38-MAPK inhibitor, 10 µM, 24 h) and levels of proteins were determined by Western blot. Graphs show quantification of HIF-1α, TSP-1 and CD36 by densitometry. In hypoxia, cells treated with SB 202190 exhibited significantly lower protein expression of HIF-1α, TSP-1 and CD36 than cells treated with vehicle. In all cases bars represent mean± SEM (<i>n></i>3). Comparisons between groups were performed using ANOVA followed by a Newman Keuls test. *P<0.05 and ***P<0.001 with respect to all groups in the same graph and ^###P<0.001 vs. bars in normoxia.</p