Jesús Cosín-Roger

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

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 miHIF-1α-targeting vector blocked the increase in mRNA expression of CD36 and TSP-1 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.

Aspirin-induced gastrointestinal damage is associated with an inhibition of epithelial cell autophagy.

BackgroundAspirin (ASA) causes gastrotoxicity by hampering the epithelial defense against luminal contents through cyclooxygenasexa0inhibition. Since cell survival in tough conditions may depend on rescue mechanisms like autophagy, we analyzed whether epithelial cells rely on this process to defend themselves from aspirin’s damaging action.MethodsRats received a single dose of ASA (150xa0mg/kg, p.o.) with or without pretreatment with the autophagy inhibitor 3-methyladenine, and gastric injury and epithelial autophagy were evaluated 3xa0h later. The effects of ASA on cell viability and autophagy were also evaluated in gastric epithelial AGS cells.ResultsBasal autophagy in the gastric mucosa was inhibited by ASA as demonstrated by increased levels of p62 and ubiquitinated proteins and total LC3 and a reduced LC3-II/LC3-I ratio. Similarly, ASA increased p62 and decreased LC3-II accumulation and the number of EmGFP/LC3B puncta in AGS cells. ASA activated the PI3K/Akt-GSK3-mTOR pathway, which phosphorylates ULK1 to prevent autophagy initiation, changes that were inhibited by the PI3K-inhibitor wortmannin. Autophagy inhibition seems to enhance the vulnerability of gastric epithelial cells as a combination of ASA with 3-methyladenine exacerbated rat gastric damage and AGS cell apoptosis.ConclusionsOur data highlight the importance of autophagy in the gastric mucosa as a protective mechanism when the epithelium is injured. In the stomach, aspirin induces mucosal damage and reduces autophagy, thus, eliminating a protective mechanism that epithelial cells could use to escape death. We hypothesize that the combination of aspirin with drugs that activate autophagy could protect against gastric damage.

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.

Autophagy stimulation prevents intestinal mucosal inflammation and ameliorates murine colitis

Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis.

Stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis

Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis.

M1 Macrophages Activate Notch Signalling in Epithelial Cells: Relevance in Crohn's Disease.

BACKGROUNDnThe Notch signalling pathway plays an essential role in mucosal regeneration, which constitutes a key goal of Crohns disease (CD) treatment. Macrophages coordinate tissue repair and several phenotypes have been reported which differ in the expression of surface proteins, cytokines and hypoxia-inducible factors (HIFs). We analysed the role of HIFs in the expression of Notch ligands in macrophages and the relevance of this pathway in mucosal regeneration.nnnMETHODSnHuman monocytes and U937-derived macrophages were polarized towards the M1 and M2 phenotypes and the expression levels of HIF-1α, HIF-2α, Jagged 1 (Jag1) and delta-like 4 (Dll4) were evaluated. The effects of macrophages on the expression of hairy and enhancer of split-1 (HES1, the main target of Notch signalling) and intestinal alkaline phosphatase (IAP, enterocyte marker) in epithelial cells in co-culture were also analysed. Phenotype macrophage markers and Notch signalling were evaluated in the mucosa of CD patients.nnnRESULTSnM1 macrophages were associated with HIF-1-dependent induction of Jag1 and Dll4, which increased HES1 protein levels and IAP activity in co-cultured epithelial cells. In the mucosa of CD patients a high percentage of M1 macrophages expressed both HIF-1α and Jag1 while M2 macrophages mainly expressed HIF-2α and we detected a good correlation between the ratio of M1/M2 macrophages and both HES1 and IAP protein levels.nnnCONCLUSIONnM1, but not M2, macrophages are associated with HIF-1-dependent induction of Notch ligands and activation of epithelial Notch signalling pathway. In the mucosa of chronic CD patients, the prevalence of M2 macrophages is associated with diminution of Notch signalling and impaired enterocyte differentiation.

The flesh ethanolic extract of Hylocereus polyrhizus exerts anti- inflammatory effects and prevents murine colitis

BACKGROUND & AIMSnIBD is a chronic disorder of the gastrointestinal tract characterized by mucosal inflammation and epithelial damage. Biologic therapy has significantly improved the course of the disease but there are still a high percentage of patients that do not respond to current therapies. We aim to determine the effects of the flesh ethanolic extract of Hylocereus polyrhizus (EH) in a mice model of colitis induced by TNBS.nnnMETHODSnBalb/c mice received TNBS (175xa0mg/kg, 100xa0μl, i.r.) and six and thirty hours later were administered with EH (1xa0g/kg, i.p.). Mice were weighted daily and after sacrificing (2 and 4 days after TNBS) we analyzed mucosal histology, myeloperoxidase activity (MPO), the expression of pro-inflammatory molecules (qPCR) and NF-κB and Iκβ-α protein levels. The chemical characterization of the EH was determined by LC-MS/MS.nnnRESULTSnThe administration of EH to TNBS-treated mice prevented (Pxa0<xa00.05) the loss of body weight and significantly reduced in the colon: a) histological damage score, b) MPO enzymatic activity c) the expression of pro-inflammatory molecules and d) Iκβ-α degradation and nuclear NF-κβ protein levels. The LC-MS analysis detected metabolites such as polyphenols and fatty acids.nnnCONCLUSIONnSystemic administration of the ethanolic extract of H. polyrhizus exerts an anti-inflammatory effect and prevents murine colitis induced by TNBS.