Sara Calatayud

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.

Mechanisms of gastroprotection by transdermal nitroglycerin in the rat

Nitric oxide (NO) donors prevent experimentally‐induced gastric mucosal damage, but their clinical utility is limited by short duration of action or unsuitability of the pharmaceutical form employed. This study analyses the gastroprotection elicited by a clinically used mode of continuous administration of an NO donor, namely the nitroglycerin patch. Application to rats of a transdermal patch that releases doses of nitroglycerin comparable to those used in man (40, 80, 160 and 400 ng min−1 rat−1) reduced gastric damage induced by indomethacin (25 mg kg−1, p.o. or s.c.). The nitroglycerin patch (160 ng  min−1 rat−1) also diminished damage by oral administration (1 ml) of acidified bile salts (100 mg kg−1 taurocholic acid in 150 mM HCl) or 50% ethanol. Transdermal nitroglycerin (160 ng min−1 rat−1) did not influence basal gastric blood flow, as measured by lasser‐doppler flowmetry, but prevented its reduction by indomethacin. Transdermal nitroglycerin (160 ng min−1 rat−1) prevented in vivo leukocyte rolling and adherence in the rat mesentery microvessels superfused with indomethacin, as evaluated by intravital microscopy. The transdermal nitroglycerin patch protects the gastric mucosa from damage by mechanisms that involve maintenance of mucosal blood flow and reduction of leukocyte‐endothelial cell interaction.

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.

Nitric oxide donors preferentially inhibit neuronally mediated rat gastric acid secretion

Continuous i.v. infusion of the nitric oxide (NO) donors, S-nitroso-glutathione (10-50 micrograms kg-1 min-1) and S-nitroso-N-acetyl-penicillamine (10 micrograms kg-1 min-1) inhibited neuronally mediated gastric acid secretion, as induced by gastric distension (20 cm water) or i.v. bolus administration of 2-deoxy-D-glucose (150 mg kg-1) in the anaesthetized rat. By contrast, gastric acid responses to i.v. infusion of submaximal doses of pentagastrin (8 micrograms kg-1 h-1) or histamine (1 mg kg-1 h-1) were not influenced by these NO donors. These findings suggest that NO does not directly influence acid secretion in vivo but could play an inhibitory modulator role in neuronally mediated acid responses.

The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD.

The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6−/− mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also exhibited decreased mucosal expression of Wnt2b, Wnt7b, and Wnt10a, diminished protein levels of nuclear β-catenin that is mainly located in crypts adjacent to damage, and reduced mRNA expression of two Wnt/β-catenin target molecules Lgr5 and c-Myc when compared with wild-type (WT) mice. Murine peritoneal macrophages treated with interleukin-4 (IL-4) and polarized toward an M2a phenotype overexpressed Wnt2b, Wnt7b, and Wnt10a in a STAT6-dependent manner. Administration of a Wnt agonist as well as transfer of properly polarized M2a macrophages to STAT6−/− mice activated the Wnt signaling pathway in the damaged mucosa and accelerated wound healing. Our results demonstrate that a STAT6-dependent macrophage phenotype promotes mucosal repair in TNBS-treated mice through activation of the Wnt signaling pathway.

Nitric oxide mediates the inhibition by interleukin-1β of pentagastrin-stimulated rat gastric acid secretion

Bolus injection of interleukin‐1β (2 μg kg−1, i.v.) inhibited acid secretion induced by intravenous infusion of pentagastrin (8 μg kg−1 h−1) in the continuously perfused stomach of the anaesthetized rat. Administration of interleukin‐1β did not modify mean systemic arterial blood pressure. Pretreatment with NG‐nitro‐l‐arginine methyl ester (l‐NAME, 2 −10 mg kg−1, i.v.), but not dexamethasone (5 mg kg−1, s.c. twice over 16 h), restored the acid secretory responses to pentagastrin. The actions of l‐NAME were reversed by the prior administration of l‐arginine (100 mg kg−1, i.v.), but not by its enantiomer d‐arginine (100 mg kg−1, i.v.). l‐NAME (5 mg kg−1, i.v.) increased blood pressure but this was not the mechanism by which interleukin‐induced acid inhibition was prevented, since similar systemic pressor responses induced by phenylephrine (10 μg kg−1 min−1, i.v.), had no such effect. These findings suggest that interleukin‐induced inhibition of acid responses to pentagastrin involves synthesis of NO from l‐arginine.

Gastrin induces leukocyte-endothelial cell interactions in vivo and contributes to the inflammation caused by Helicobacter pylori

Gastric mucosal inflammation causes hypergastrinemia, and gastrin receptors have been detected in several leukocyte types. We have analyzed whether gastrin affects the leukocyte‐endothelial cell interactions in vivo by monitoring leukocyte rolling, adhesion, and emigration in rat mesenteric venules using intravital microscopy. Mesenteric superfusion with exogenous gastrin increased these processes in a concentration‐ and time‐dependent manner, effects prevented by the cholecystokinin (CCK)‐2 receptor antagonists (proglumide, L‐365,260) but not by the CCK‐1 receptor antagonist devazepide. A similar response was induced by exogenous CCK or endogenously released gastrin. CCK‐2 receptors were localized in mesenteric macrophages and polymorphonuclear leukocytes. This effect of gastrin is not modulated by somatostatin and is independent of the endogenous release of histamine. To analyze whether hypergastrinemia elicited by Helicobacter pylori (HP) modulates the inflammation induced by the germ, rats were chronically administered with an extract of a CagA+/VacA+ strain of HP. This protocol increased gastrinemia and induced an inflammatory response in the rat mesentery. Blockade of CCK‐2 receptors attenuated this response and induced a qualitative change in the leukocyte infiltrate suggestive of a receding inflammatory process. Our results reveal a new proinflammatory role of gastrin that seems to contribute to the maintenance of the inflammation elicited by HP components.— Álvarez, A., Ibiza, S., Hernández, C., Álvarez‐Barrientos, A., Esplugues, J. V., Sara Calatayud, S. Gastrin induces leukocyte‐endothelial cell interactions in vivo and contributes to the inflammation caused by Helicobacter pylori. FASEB J. 20, E1742–E1752 (2006)

Nitric oxide and sensory afferent neurones modulate the protective effects of low-dose endotoxin on rat gastric mucosal damage.

Pretreatment (1 h) with low doses (5-40 micrograms/kg i.p.) of Escherichia coli endotoxin dose dependently reduced the gastric mucosal damage induced by a 10 min challenge with 1 ml ethanol (50% and 100%) in conscious rats. Treatment with the nitric oxide synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 5 and 10 mg/kg i.p.), significantly inhibited the protective effects of endotoxin (40 micrograms/kg i.p.). The actions of L-NAME were reversed by the prior administration of L-arginine (100 mg/kg i.p.). The protective effects of endotoxin were not influenced by pretreatment with dexamethasone (5 mg/kg s.c. twice) or indomethacin (5 mg/kg s.c.). However, ablation of sensory afferent neurons by capsaicin pretreatment (20, 30 and 50 mg/kg s.c.) abolished the mucosa protective effects of endotoxin (40 micrograms/kg). These findings suggest that the protection elicited by low doses of endotoxin against ethanol-induced mucosal damage involves synthesis of nitric oxide and activation of sensory neurones.

Hypoxic macrophages impair autophagy in epithelial cells through Wnt1: relevance in IBD

A defective induction of epithelial autophagy may have a role in the pathogenesis of inflammatory bowel diseases. This process is regulated mainly by extracellular factors such as nutrients and growth factors and is highly induced by diverse situations of stress. We hypothesized that epithelial autophagy is regulated by the immune response that in turn is modulated by local hypoxia and inflammatory signals present in the inflamed mucosa. Our results reveal that HIF-1α and Wnt1 were co-localized with CD68 in cells of the mucosa of IBD patients. We have observed increased protein levels of β-catenin, phosphorylated mTOR, and p62 and decreased expression of LC3II in colonic epithelial crypts from damaged mucosa in which β-catenin positively correlated with phosphorylated mTOR and negatively correlated with autophagic protein markers. In cultured macrophages, HIF-1 mediated the increase in Wnt1 expression induced by hypoxia, which enhanced protein levels of β-catenin, activated mTOR, and decreased autophagy in epithelial cells in co-culture. Our results demonstrate a HIF-1-dependent induction of Wnt1 in hypoxic macrophages that undermines autophagy in epithelial cells and suggest a role for Wnt signaling and mTOR pathways in the impaired epithelial autophagy observed in the mucosa of IBD patients.

Transdermal nitroglycerin prevents nonsteroidal anti-inflammatory drug gastropathy

Abstract The application of a transdermal nitroglycerin patch (2–30 μg/3 h/rat) protected, in a dose-dependent manner, the rat gastric mucosa against damage induced by the nonsteroidal anti-inflammatory agent indomethacin (20 mg/kg s.c.).