M2 Macrophages Activate WNT Signaling Pathway in Epithelial Cells: Relevance in Ulcerative Colitis

Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation

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

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

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

Patient clinical information.

<p>Patient clinical information.</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

WNT2b activates epithelial-mesenchymal transition through FZD4: relevance in penetrating Crohn´s disease

BACKGROUND AND AIMS Epithelial-mesenchymal transition (EMT) has been related to fibrosis and fistula formation, common complications associated to Crohn´s disease (CD). The WNT signaling pathway mediates EMT and specific WNT/FZD interactions have been related with the activation of this process in several diseases. We aim to analyze the relevance of EMT and Wnt ligands and receptors in the penetrating behavior of CD. METHODS Intestinal surgical resections were obtained from control and CD patients with a stenotic or penetrating behavior. Fibrosis was determined by the histological analysis of collagen deposition and EMT by confocal microscopy. The expression of WNT ligands, inhibitors and FZD receptors was analyzed by RT-PCR, WB, IH and IF studies. The effects of WNT2b and the role of FZD4 in EMT were analyzed in HT29 epithelial cells. RESULTS Fibrosis and expression of EMT markers were detected in samples from CD patients irrespective of the clinical behavior. However, an increased co-localization of E-CADHERIN and VIMENTIN, an increased number of cells expressing WNT2b and a higher expression of FZD4 and WNT2b/FZD4 interaction were detected in intestinal tissue from the penetrating compared with the stenotic CD behavior. WNT2b induced EMT in HT29 cells through FZD4 activation. CONCLUSION An increased EMT, associated with increased WNT2b/FZD4 interaction is detected in intestinal tissue from CD patients with a penetrating behavior. WNT2b, through FZD4 activation induces EMT in vitro which points to a novel pharmacological target to prevent intestinal penetrating complications of CD

Novel variants in the stem cell niche factor WNT2B define the disease phenotype as a congenital enteropathy with ocular dysgenesis

WNT2B is a member of the Wnt family, a group of signal transduction proteins involved in embryologic development and stem cell renewal and maintenance. We recently reported homozygous nonsense variants in WNT2B in three individuals with severe, neonatal-onset diarrhea, and intestinal failure. Here we present a fourth case, from a separate family, with neonatal diarrhea associated with novel compound heterozygous WNT2B variants. One of the two variants was a frameshift variant (c.423del [p.Phe141fs]), while the other was a missense change (c.722 G > A [p.G241D]) that we predict through homology modeling to be deleterious, disrupting post-translational acylation. This patient presented as a neonate with severe diet-induced (osmotic) diarrhea and growth failure resulting in dependence on parenteral nutrition. Her gastrointestinal histology revealed abnormal cellular architecture particularly in the stomach and colon, including oxyntic atrophy, abnormal distribution of enteroendocrine cells, and a paucity of colonic crypt glands. In addition to her gastrointestinal findings, she had bilateral corneal clouding and atypical genital development later identified as a testicular 46,XX difference/disorder of sexual development. Upon review of the previously reported cases, two others also had anterior segment ocular anomalies though none had atypical genital development. This growing case series suggests that variants in WNT2B are associated with an oculo-intestinal (and possibly gonadal) syndrome, due to the protein’s putative involvement in multiple developmental and stem cell maintenance pathways