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

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

M2 macrophages increase in the mucosa of chronic UC patients.

<p>A) Representative photographs showing CD68, CD86 and CD206 immunostaining in paraffin-embedded sections of damaged and non-damaged mucosa of patients with UC, magnification 60X. B) Graphs show quantitative analysis of CD68, CD86 and CD206 positive cells performed in a total area of 0.3 mm² of consecutive slides. Bars represent mean±SEM. *<i>P</i><0.05 and ***<i>P</i><0.001 vs the respective non-damaged mucosa and ^##<i>P</i><0.01 and ^###<i>P</i><0.001 vs the respective mucosa in newly diagnosed patients. C) Graphs show percentage of CD86+/CD68+ cells and CD206+/CD68+ cells in newly diagnosed (n=8) and chronic patients with UC (n=12). Bars represent mean±SEM. *<i>P</i><0.05 <i>vs</i> the respective non-damaged mucosa and ^&&<i>P</i><0.01 and ^&&&<i>P</i><0.001 <i>vs</i> the CD86/CD68 in the respective mucosa in the same graph. </p

Histological score in the mucosa of patients with UC.

<p>A) Representative photographs showing histological score assigned to biopsies, magnification 10X. B) Graphs show histological score in damaged and non-damaged mucosa of newly diagnosed (n=8) and chronic UC patients (n=12). Bars represent mean±SEM. **<i>P</i><0.01 <i>vs</i> respective non-damaged mucosa.</p

M2 macrophages decrease alkaline phosphatase activity through Wnt signalling pathways.

<p>A) Graphs show alkaline phosphatase activity in Caco-2 cells co-cultured with macrophages or with an empty insert (n=5). Some cells were treated with XAV939, 1µM (n=4). Bars represent mean±SEM. **<i>P</i><0.01 <i>vs</i> Caco-2 cells co-cultured with an empty insert and ^##<i>P</i><0.01 <i>vs</i> Caco-2 cells co-cultured with M2 macrophages. B) Graphs show alkaline phosphatase activity in Caco-2 cells and HT29 cells 24h after treatment with Wnt1 20ng/ml. Bars represent mean±SEM. *<i>P</i><0.05 and ** <i>P</i><0.01 <i>vs</i> cells treated with vehicle.</p

An impaired alkaline phosphatase activity and increased Wnt signalling in damaged mucosa of patients with UC.

<p>A) Graph shows alkaline phosphatase activity determined from non-damaged and damaged mucosa of chronic patients with UC. Bars represent mean±SEM. *<i>P</i><0.05 vs the non-damaged mucosa (n=3). B) Three representative photographs showing co-localization of Wnt1 and CD206 in the mucosa of chronic patients with UC, magnification 160X. C) Representative photographs showing nuclear immunostaining of β-catenin and c-Myc in epithelial cells of the same crypt in consecutive slides. Magnification 10X, 60X and 160X. D) Total and nuclear protein from frozen non-damaged and damaged mucosa were extracted and expression of β-catenin was analyzed by Western blot. A representative western blot showing total and nuclear protein levels of β-catenin in the non-damaged and damaged mucosa of the same patients. Graphs show quantification of the protein expression of total (n=5) and nuclear β-catenin (n=5). E) A representative western blot showing β-catenin expression after immunoprecipitation of Tcf-4 in the non-damaged (ND) and damaged (D) mucosa of three UC patients. F) Total mRNA from frozen non-damaged and damaged mucosa was extracted. Graphs show mRNA expression of <i>Lgr5</i> and <i>c-Myc</i>. Bars represent mean±SEM. n=7. *<i>P</i><0.05 and **<i>P</i><0.01 vs non-damaged mucosa. G) There is a positive and significant correlation between the number of CD206 macrophages and the intensity of β-catenin immunostaining.</p

M2 macrophages activate the Wnt signalling pathway in epithelial cells.

<p>Caco-2 cells were co-cultured for 24h with M1, M2 or non-polarized macrophages derived from U937 cells or an empty insert. A) A representative western blot and graph showing protein expression of total, cytoplasmatic and nuclear β-catenin (n=4) in Caco-2 cells after co-culture. B) A representative western blot of β-catenin after immunoprecipitation of Tcf-4 of three experiments. C) Graphs show the mRNA expression levels of <i>c-Myc</i> (n=7) and <i>Lgr5</i> (n=7) in Caco-2 cells after co-culture. D) Graphs show the mRNA expression level of <i>Wnt1</i> in M2 macrophages treated with <i>miWnt1</i> or <i>mock</i>. E) Caco-2 cells were co-cultured with transfected M2 macrophages. Graphs show quantification of protein expression of nuclear β-catenin by western blot (n=5) and mRNA expression of <i>Lgr5</i> (n=5) and <i>c-Myc</i> (n=5). Two representative western blots showing nuclear β-catenin in Caco-2 cells. Bars represent mean±SEM. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs</i> Caco-2 cells co-cultured with an empty insert. ^#<i>P</i><0.05 and ^##<i>P</i><0.01 <i>vs</i> Caco-2 cells co-cultured with M1 macrophages or non-polarized macrophages. ^$<i>P</i><0.05 vs M2 macrophages transfected with mock and ^&<i>P</i><0.05 and ^&&<i>P</i><0.01 vs Caco-2 cells co-cultured with M2 macrophages transfected with mock.</p

SUCNR1 Mediates the Priming Step of the Inflammasome in Intestinal Epithelial Cells: Relevance in Ulcerative Colitis

Intestinal epithelial cells (IECs) constitute a defensive physical barrier in mucosal tissues and their disruption is involved in the etiopathogenesis of several inflammatory pathologies, such as Ulcerative Colitis (UC). Recently, the succinate receptor SUCNR1 was associated with the activation of inflammatory pathways in several cell types, but little is known about its role in IECs. We aimed to analyze the role of SUCNR1 in the inflammasome priming and its relevance in UC. Inflammatory and inflammasome markers and SUCNR1 were analyzed in HT29 cells treated with succinate and/or an inflammatory cocktail and transfected with SUCNR1 siRNA in a murine DSS model, and in intestinal resections from 15 UC and non-IBD patients. Results showed that this receptor mediated the inflammasome, priming both in vitro in HT29 cells and in vivo in a murine chronic DSS-colitis model. Moreover, SUNCR1 was also found to be involved in the activation of the inflammatory pathways NF&#1082;B and ERK pathways, even in basal conditions, since the transient knock-down of this receptor significantly reduced the constitutive levels of pERK-1/2 and pNF&#1082;B and impaired LPS-induced inflammation. Finally, UC patients showed a significant increase in the expression of SUCNR1 and several inflammasome components which correlated positively and significantly. Therefore, our results demonstrated a role for SUCNR1 in basal and stimulated inflammatory pathways in intestinal epithelial cells and suggested a pivotal role for this receptor in inflammasome activation in UC