Relevance of the macrophage phenotype in mucosal regeneration in Inflammatory Bowel Disease

La enfermedad Inflamatoria Intestinal (EII) es una enfermedad crónica, recidivante, de carácter sistémico que afecta principalmente al tracto gastrointestinal. Esta patología engloba dos entidades clínicas, la Enfermedad de Crohn (EC) y la Colitis Ulcerosa (CU), que difieren en algunos aspectos pero comparten la disrupción de la barrera epitelial y la respuesta exacerbada del sistema inmunológico a la flora bacteriana. La etiología de la enfermedad es desconocida pero se sabe que factores genéticos (tanto alteraciones a nivel genético como la presencia de miRNAs), ambientales (tales como la dieta, tabaco, actividad física o la calidad del sueño), flora intestinal y sistema inmunológico contribuyen al desarrollo de la enfermedad. En los últimos años se ha evidenciado que la adecuada recuperación de la mucosa dañada constituye un objetivo clave del tratamiento ya que permite prolongar de forma significativa los periodos de remisión clínica. En la presente tesis nos planteamos como principal objetivo analizar la relevancia de la inmunidad innata, en particular de los macrófagos, en la regeneración de la mucosa intestinal en la EII. Nuestros resultados demuestran la co-existencia de diferentes fenotipos macrofágicos, un fenotipo pro-inflamatorio M1 y un fenotipo antiinflamatorio M2, tanto en la mucosa de pacientes crónicos con EII como en un modelo murino de colitis aguda, siendo prevalente el fenotipo M2 sobre el fenotipo M1 en la mucosa dañada. El análisis de la expresión de mediadores específicamente implicados en los mecanismos de regeneración en las criptas intestinales reveló que la expresión de ligandos Wnt es selectiva de macrófagos M2 y la de ligandos Notch se asocia a macrófagos M1 con lo que estas células parecen modular de forma diferencial las rutas de señalización Wnt y Notch en la mucosa. A pesar de la similitud encontrada en la expresión de receptores de superficie en el fenotipo M2 entre la colitis crónica y la aguda, la función desempeñada por este fenotipo macrofágico parece diferir dependiendo de la fase del proceso inflamatorio, probablemente como consecuencia de una diferente producción de citocinas. En la mucosa de pacientes con EII observamos una prevalencia de macrófagos M2 sobre macrófagos M1 asociada a la activación de la ruta Wnt y la inhibición de la ruta Notch lo que determina una inhibición de la autofagia epitelial y una dañada diferenciación enterocítica. Por el contrario, en el modelo agudo de colitis los macrófagos M2 parecen desempeñar un papel fundamental en la regeneración de la mucosa a través de la síntesis de ligandos Wnt y la activación de la ruta Wnt en el epitelio. En conclusión, el presente trabajo demuestra la relevancia de los macrófagos en la regeneración de la mucosa intestinal en la EII. Estas células, de manera fenotipo-dependiente, se asocian con la inducción de ligandos Wnt y Notch que al señalizar sobre las células epiteliales de las criptas intestinales regulan los procesos de proliferación, diferenciación y autofagia, fundamentales en los mecanismos de regeneración mucosa. Una mejor caracterización funcional de los fenotipos macrofágicos en la mucosa de pacientes con EII ayudará a establecer nuevas aproximaciones celulares en la terapia de la enfermedad inflamatoria intestinal

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

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

Background: The Notch signalling pathway plays an essential role in mucosal regeneration, which constitutes a key goal of Crohn's 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. Methods: Human 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. Results: M1 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. Conclusion: M1, 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. Key Words: MacrophagesCrohn's diseasemucosal healingNotch signallin

HIF-Overexpression and Pro-Inflammatory Priming in Human Mesenchymal Stromal Cells Improves the Healing Properties of Extracellular Vesicles in Experimental Crohn’s Disease

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have therapeutic potential in the treatment of several immune disorders, including ulcerative colitis, owing to their regenerative and immunosuppressive properties. We recently showed that MSCs engineered to overexpress hypoxia-inducible factor 1-alpha and telomerase (MSC-T-HIF) and conditioned with pro-inflammatory stimuli release EVs (EVMSC-T-HIFC) with potent immunomodulatory activity. We tested the efficacy of EVMSC-T-HIFC to repolarize M1 macrophages (Mφ1) to M2-like macrophages (Mφ2-like) by analyzing surface markers and cytokines and performing functional assays in co-culture, including efferocytosis and T-cell proliferation. We also studied the capacity of EVMSC-T-HIFC to dampen the inflammatory response of activated endothelium and modulate fibrosis. Finally, we tested the therapeutic capacity of EVMSC-T-HIFC in an acute colitis model. EVMSC-T-HIFc induced the repolarization of monocytes from Mφ1 to an Mφ2-like phenotype, which was accompanied by reduced inflammatory cytokine release. EVMSC-T-HIFc-treated Mφ1 had similar effects of immunosuppression on activated peripheral blood mononuclear cells (PBMC) as Mφ2, and reduced the adhesion of PBMCs to activated endothelium. EVMSC-T-HIFc also prevented myofibroblast differentiation of TGF-β-treated fibroblasts. Finally, administration of EVMSC-T-HIFc promoted healing in a TNBS-induced mouse colitis model in terms of preserving colon length and intestinal mucosa architecture and altering the ratio of Mφ1/ Mφ2 infiltration. In conclusion, EVMSC-T-HIFC have effective anti-inflammatory properties, making them potential therapeutic agents in cell free-based therapies for the treatment of Crohn’s disease and likely other immune-mediated inflammatory diseases

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

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

Relevancia fisiopatológica del fenotipo macrofágico y del factor inducible por hipoxia (HIF) en la enfermedad inflamatoria intestinal

[ES] La enfermedad inflamatoria intestinal (EII) es una patología de elevada incidencia y prevalencia cuyo tratamiento farmacológico no está clínicamente resuelto, lo que hace que el estudio de su etiopatogenia resulte de gran interés clínico. Alteraciones en la función barrera epitelial así como un elevado infiltrado de células inflamatorias son características de esta patología. En muestras humanas se ha visto expresión del factor inducible por hipoxia (HIF), tanto en células epiteliales como inflamatorias. El papel de HIF en el epitelio parece ser el de promover la función barrera epitelial. Sin embargo, poco se sabe de la función que desempeña este factor de transcripción específicamente en los macrófagos. Se sabe que estas células presentan cierta plasticidad y como consecuencia adquieren diferente patrón de activación dependiendo del microambiente en el que se encuentren. En el presente trabajo planteamos determinar el perfil de expresión de HIF-1¿ y HIF-2¿ así como de diferentes fenotipos macrofágicos en la mucosa intestinal de pacientes con EII. A continuación, analizaremos posibles correlaciones entre estos dos parámetros y, por último, en células aisladas, evaluaremos el efecto de cada fenotipo macrofágico sobre la proliferación de células epiteliales. Nosotros hipotetizamos que el perfil macrofágico así como la expresión de HIF-1¿ y HIF-2¿ cambiará en función de la fase de la enfermedad y, como consecuencia, su influencia en la función barrera epitelial. Consideramos que conocer el papel que la expresión génica inducida por HIF en los diferentes mecanismos de daño y reparación de la mucosa ayudará a sentar las bases para el establecimiento de nuevas dianas terapéuticas en el tratamiento de la enfermedad inflamatoria intestinal.[EN] Inflammatory bowel disease (IBD) is a highly prevalent pathology in developed societies. Pharmacological treatment of this pathology continues to present problems, and so the study of its ethiopathogenesis is of great clinical interest. Changes in epithelial barrier function and an increased leukcoyte infiltration are characteristics of this pathology. Expression of hypoxia inducible factor (HIF) has been observed in both epithelial and inflammatory cells in the mucosa of patients with IBD. In the epithelium, HIF seems to be involved in promoting epithelial barrier function. However, less is known about the precise role of this transcription factor in macrophages of the lamina propria. It is known that these cells exhibit some plasticity and, consequently, acquire different activation pattern depending on the microenvironment in which they are. In this study we aim to determine the pattern of expression of HIF-1¿, HIF-2¿ and macrophage phenotypes in intestinal mucosa of IBD patients. Next, we analyze possible correlations between these two parameters and, finally, in isolated cells we evaluate the effect of each macrophage phenotype on epithelial cell proliferation. We hypothesize that the macrophage phenotype and the expression of HIF-1¿ and HIF-2¿ will change depending on the phase of the disease and, therefore, their influence on epithelial barrier function. We believe that knowing the role of HIF-induced gene expression in the mechanisms of injury and mucosal repair will help to establish new therapeutic targets in the treatment of inflammatory bowel disease.Cosín Roger, J. (2012). Relevancia fisiopatológica del fenotipo macrofágico y del factor inducible por hipoxia (HIF) en la enfermedad inflamatoria intestinal. http://hdl.handle.net/10251/17882Archivo delegad

Metabolomics as a Promising Resource Identifying Potential Biomarkers for Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a relapsing chronic disorder of the gastrointestinal tract characterized by disruption of epithelial barrier function and excessive immune response to gut microbiota. The lack of biomarkers providing early diagnosis or defining the status of the pathology difficulties an accurate assessment of the disease. Given the different metabolomic profiles observed in IBD patients, metabolomics may reveal prime candidates to be studied, which may help in understanding the pathology and identifying novel therapeutic targets. In this review, we summarize the most current advances describing the promising metabolites such as lipids or amino acids found through untargeted metabolomics from serum, faecal, urine and biopsy samples

Is the Macrophage Phenotype Determinant for Fibrosis Development?

Fibrosis is a pathophysiological process of wound repair that leads to the deposit of connective tissue in the extracellular matrix. This complication is mainly associated with different pathologies affecting several organs such as lung, liver, heart, kidney, and intestine. In this fibrotic process, macrophages play an important role since they can modulate fibrosis due to their high plasticity, being able to adopt different phenotypes depending on the microenvironment in which they are found. In this review, we will try to discuss whether the macrophage phenotype exerts a pivotal role in the fibrosis development in the most important fibrotic scenarios

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