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.

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.

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.

Background: Fibrosis is a common complication of Crohn’s disease (CD) in which macrophages play a central role. Epithelial-mesenchymal transition (EMT) and the WNT pathway have been associated with fibrosis. We aim to analyse the relevance of the tissue microenvironment in macrophage phenotype and the EMT process. Methods: Intestinal surgical resections are obtained from control and CD patients with stenotic or penetrating behaviour. Cytokine’s expression, macrophage phenotype, EMT markers and WNT signalling pathway are determined by WB, RT-PCR, ELISA or Cytometry. U937 cells are treated with IFNγ, TNFα, IL1β, IL4 or IL10 and co-cultured with HT29 cells and, in some cases, are treated with XAV939 or miFZD4. The expression of macrophage, EMT and WNT pathway markers in U937 or HT29 cells is analysed by WB or RT-PCR. Results: IFNγ, WNT6, CD16 and CD86 are increased in the intestinal tissue of CD patients. IFNγ-treated U937 activated the EMT process and WNT pathway in HT29 cells, and the EMT process is mediated by FZD4. Conclusions: An IFNγ-rich microenvironment polarises macrophages, which induces EMT through the WNT pathway.

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 NFкB 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 pNFкB 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.

Background Fibrosis represent the main complications related to Crohn's disease (CD). Notch signalling mediate fibrogenic process, including epithelial-mesenchymal transition (EMT) and fibroblast senescence but its role in CD fibrosis is currently unknown. Previous studies have shown a high expression of NOTCH4, NOTCH3, DLL3 and DLL4 in CD fibrotic tissue. DLL4 mainly activates transcription factors involved in EMT, and macrophages could act as a possible source of DLL4 (Edo, et al., 2022. JCC (i200)).The general aim of the present study is to determine the possible potential of Notch pathway as a therapeutic target in intestinal fibrosis associated with CD. Specifically, we pretend: to analyze the localization of NOTCH3/4 receptors in the intestinal tissue of patients with CD complicated; to study the relevance of NOTCH3/4 receptors in the EMT; to study the relevance of Notch pathway in the senescence of intestinal fibroblast. Methods We have analyzed in intestinal samples from CD patients with complicated lesions: the localization of NOTCH3/4 receptors by IH and the protein expression of senescence markers (BCL2 and P53) by WB. We carry out in vitro studies and analyze: the protein expression of HES1 (effector Notch pathway) and EMT markers in DLL4-HT29 treated cells transfected with miNOTCH3 or miNOTCH4; and the protein expression of senescence proteins in HSIF fibroblasts treated with DLL4 or DLL3. Results NOTCH3 was located preferentially in muscular areas -muscularis mucosa, endothelium and muscularis externa- with a striking staining of infiltrated cells in the mucosa of the unaffected area. NOTCH4 is found more specifically in the crypts of the mucosa, as well as in cells of the lamina propria of the unaffected mucosa. The expression of senescence proteins in the tissue showed elevated levels of BCL2 and P53, compared to the unaffected tissue of the same patient. DLL4 increased the protein expression of EMT markers in HT29 cells, and NOTCH4 silencing significantly reverted the expression of these EMT markers. NOTCH3 silencing produced no significant changes after DLL4-HT29 treatment. DLL3, and not DLL4, produced in intestinal fibroblasts a significant increase in the protein expression levels of BCL2, and P53, compared to the vehicle (Figure). Conclusion NOTCH pathway is involved in the regulation of key cellular functions and processes essential for the pathogenesis of intestinal fibrosis in CD patients. DLL4-NOTCH4 interaction triggers transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells, while DLL3 seems to have a more relevant role in activation of senescence in fibroblasts.

Background Crohn’s Disease (CD) is a subtype of IBD characterized by a chronic transmural inflammation of the gastrointestinal tract associated with several complications being intestinal fibrosis the most frequent. CD patients present microbiota dysbiosis and altered metabolomic profiles. GPCRs constitute a family of receptors which could be involved in inflammatory and fibrotic processes associated to CD. We aim to characterize microbiota composition, tissue metabolomic profile and metabolite-sensing GPCRs expression in ileal resections from fibrotic CD patients. Methods Ileal resections from B2-CD (n=21) and non-IBD (n=13) patients were obtained. Microbiota characterization was performed by 16S rRNA gene Illumina Miseq sequencing. Bioinformatic analysis of sequencing data was performed using constrained correspondence analysis and non-parametric Wilcoxon test to compare species proportions. Bacterial load was estimated by qPCR. Metabolomic analysis was performed by NMR. Results are expressed as μg metabolite/g tissue. Murine intestinal fibrosis was induced in C67BL/6 mice by: a) the heterotopic intestinal transplant model and b) chronic administration of 4 cycles of increasing DSS percentages. Gene expression of GPCRs was analyzed by qPCR. Data were expressed as fold induction vs control (mean±SEM) and compared by a t-test. Correlations were analyzed with the Spearman coefficient. Results First, microbiota analysis revealed a reduction in bacterial diversity and load in fibrotic CD patients. Then, in B2-CD samples we found at genus level Enterococcus genera significantly decreased and at species level Ruminococcus bromii and Faecalibacterium prausnitzii also reduced compared to controls. From the metabolomic analysis, altered levels of metabolites were found in ileal resections from fibrotic CD patients as summarized in Table 1. Next, B2-CD patients exhibited differential expression of metabolite-sensing GPCRs vs non-IBD as shown in Table 2. Moreover, gene expression of fibrotic markers was analyzed in B2-CD patients and significantly increased levels of COL1A1 (13.22±4.38), COL3A1 (1.84±0.52), and COL4A1(7.75±2.19), were found vs controls. Of interest, GPR81, GPR84, GPR4 and GPR68 positively correlated with profibrotic markers, specifically with COL1A1 and COL4A1. Finally, in line with human results, we also analyzed the expression of metabolite-sensing GPCRs in two different murine colitis models and results obtained are represented in Table 3. Conclusion Fibrotic CD patients exhibit microbial dysbiosis, joined with altered levels of metabolites and gene expression of metabolite-sensing GPCRs, which are also affected in murine colitis models. Their correlation with profibrotic markers points them as protagonists of intestinal fibrosis.

Background Ulcerative colitis (UC) is characterized by a diffuse, continuous, and chronic inflammation of mucosa and submucosa layers in the colon whose etiology is still unknown. Intestinal microbiota dysbiosis and alterations in the metabolomic profile have been reported in mucosal biopsies from Inflammatory Bowel Disease (IBD) patients. We aim to characterize the microbiota composition and metabolomic profile in colonic resections of UC patients. Methods Colonic resections from UC (n=18) and non-IBD (n=20) patients were obtained. Microbiota identification, composition and classification was performed by 16S rRNA gene Illumina Miseq sequencing. The bioinformatic analysis of sequencing data was performed using constrained correspondence analysis (CCA) and non-parametric Wilcoxon test to compare species and genera proportions. Bacterial load was estimated by qPCR. Metabolomic analysis was performed using Nuclear Magnetic Resonance (NMR) to study polar metabolites and Gas-Chromatography or Liquid-Chromatography Mass-Spectrometry to study non-polar metabolites. Results Microbiota analysis revealed differences at genus and species level between UC and non-IBD patients. Cellulosimicrobium was found at 10-fold higher levels in UC samples while Escherichia genus was 3.88 times more abundant in controls. A reduction in bacterial load was seen in UC samples. Levels of short-chain fatty acids (FA), medium-chain FA, long-chain FA, carboxylic acids and amino acids were obtained from the metabolomics analysis and a positive correlation between propionic, aspartic and hydroxybutyric acids and Cellulosimicrobium and Pseudomonas was found (Fig1B). Gut commensals such as unclassified Agathobacter, Blautia faecis, Oscillospiraceae or Faecalibacterium prausnitzii positively correlated with the abundance of butyric acid, acetic acid and succinic acid; meanwhile Subdoligranulum, Lachnospireace or unclassified Blautia negatively correlated with some medium or long chain fatty acids (Fig1B). Fig1. Correlations between bacterial species and metabolites in (A) controls and (B) UC patients. Performed with mixOmics library in R. Conclusion Significant differences in microbiota composition and abundance are found in UC patients, which undergo a clear gut dysbiosis in colonic tissue. Correlation analysis suggests Cellulosimicrobium and Pseudomonas as sources of propionic, aspartic and hydroxybutyric acid. These results point at Cellulosimicrobium as a potential candidate in UC pathology.