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Perianal fistula represents one of the most disabling manifestations of Crohn's Disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation.Surgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing (RNA-seq). Genes significantly modulated were validated by RT-PCR, WB, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype and ECM organization was investigated with endogenous Lentivirus-induced over-expression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein (rhTSG-6) to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed.A markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and metalloproteinases (MMPs) and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared to area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains (HCs) from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TSG-6 expression and expression of mechanosensitive genes in fistula tissue. The over-expression of TSG-6 in Caco-2 cells promoted migration, epithelial mesenchymal transition (EMT), transcription factor SNAIL and hyaluronan synthase (Has) levels; while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with rhTSG-6 protein promoted collagen release, increase of SNAIL, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli.By mediating changes in the ECM organization, TSG-6 triggers the EMT transcription factor SNAIL through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.

Background: Pomegranate (Punica granatum) can be used to prepare a bioactive extract exerting anti-inflammatory activities. Clinical studies demonstrated an improvement in clinical response in inflammatory bowel disease (IBD) patients when pomegranate extract (PG) was taken as a complement to standard medications. However, the molecular mechanisms underlying its beneficial effects are still scarcely investigated. This study investigates the effect of PG on bacterial biofilm formation and the promotion of mucosal wound healing. Methods: The acute colitis model was induced in C57BL/6N mice by 3% dextran sodium sulfate administration in drinking water for 5 days. During the recovery phase of colitis, mice received saline or PG (200 mg/kg body weight) by oral gavage for 11 days. Colitis was scored daily by evaluating body weight loss, bleeding, and stool consistency. In vivo intestinal permeability was evaluated by fluorescein isothiocyanate-conjugated dextran assay, bacterial translocation was assessed by fluorescence in situ hybridization on tissues, whereas epithelial and mucus integrity were monitored by immunostaining for JAM-A and MUC-2 markers. Bacterial biofilm formation was assessed using microfluidic devices for 24 or 48 h. Primary fibroblasts were isolated from healthy and inflamed areas of 8 IBD patients, and Caco-2 cells were stimulated with or without PG (5 μg/mL). Inflammatory mediators were measured at the mRNA and protein level by RT-PCR, WB, or Bio-plex multiplex immunoassay, respectively. Results: In vivo, PG boosted the recovery phase of colitis, promoting a complete restoration of the intestinal barrier with the regeneration of the mucus layer, as also demonstrated by the absence of bacterial spread into the mucosa and the enrichment of crypt-associated fibroblasts. Microfluidic experiments did not highlight a specific effect of PG on Enterobacterales biofilm formation, even though Citrobacter freundii biofilm was slightly impaired in the presence of PG. In vitro, inflamed fibroblasts responded to PG by downregulating the release of metalloproteinases, IL-6, and IL-8 and upregulating the levels of HGF. Caco-2 cells cultured in a medium supplemented with PG increased the expression of SOX-9 and CD44, whereas in the presence of HGF or plated with a fibroblast-conditioned medium, they displayed a decrease in SOX-9 and CD44 expression and an increase in AXIN2, a negative regulator of Wnt signaling. Conclusions: These data provide new insight into the manifold effects of PG on promoting mucosal homeostasis in IBD by affecting pathogen biofilm formation and favoring the regeneration of the intestinal barrier through the regulation of the crosstalk between epithelial and stromal cells.

The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of β-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and β039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the β/γ+β globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the β-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

Additional file 4: Supplementary Figure 4. Gene expression analysis in colon-rectal cancer cell lines and in S1PR2-overexpressing RKO cells. (A) S1PR2 mRNA expression levels in the indicated CRC cell lines. (B) Representative images of migratory and invasive cells (magnification, 4x) are shown. Cell migration and invasiveness were reported as the number of cells in 40 fields of view. (C) Relative expression of metalloproteinases 1–2 (MMP1–2) in S1PR2-overexpressing RKO and scramble cells by qRT-PCR (2 -∆∆Ct method). Data are shown as means ± SEM and are representative of 2 independent experiments, each constituted by three replicates. (D) Ex vivo macroscopic examination of organs (liver, adipose tissue, mesenteric lymph nodes (MLN) and xenograft tumor) in nude mice injected with S1PR2-overexpressing and scramble cells by an imaging system (IVIS) using GFP labeled cells. Data are representative of 2 independent experiments; n = 4.

Additional file 5: Supplemental Figure 5. Bodyweight changes in S1PR2−/− and S1PR2+/+ mice after 7 days of X-ray irradiation at 9 Gy. Data are presented as the mean ± SEM, and significance was evaluated by the Mann-Whitney test. Each group of data is representative of 3 experiments (n = 4).

Additional file 3: Supplementary Figure 3. Analysis of survival rate of the colonic mucosa of S1PR2−/− and S1PR2+/+ mice after AOM/DSS induced colitis-associated cancer. The tissues (healthy and tumor regions) were evaluated by immunohistochemical analysis with an anti-cleaved caspase-3 antibody. The images were acquired by the DotSlide system at 20x objective. Apoptotic cells (blue arrows) were examined microscopically at 40X magnification. Data are presented as the mean ± SEM, and significance was evaluated by the Mann-Whitney test. Data is representative of 2 experiments (n = 6).

Additional file 1: Supplementary Figure 1. S1P levels in human colorectal cancer. Quantification of Sphingosine 1 phosphate (S1P) in adenocarcinomas and healthy colon tissue by spectrometry assay (n = 7). The levels of S1P are reported as pmol/mg. Data are presented as the mean ± SEM, and significance was evaluated by performing a non-parametric test.

Additional file 6. Corresponds to supplementary information related to the material and methods.

Additional file of Unveiling role of sphingosine-1-phosphate receptor 2 as a brake of epithelial stem cell proliferation and a tumor suppressor in colorectal cancer

BackgroundSphingosine-1-phosphate receptor 2 (S1PR2) mediates pleiotropic functions encompassing cell proliferation, survival, and migration, which become collectively de-regulated in cancer. Information on whetherS1PR2participates in colorectal carcinogenesis/cancer is scanty, and we set out to fill the gap.MethodsWe screened expression changes of S1PR2 in human CRC and matched normal mucosa specimens [N = 76]. We compared CRC arising in inflammation-driven and genetically engineered models in wild-type (S1PR2+/+) and S1PR2 deficient (S1PR2−/−) mice. We reconstituted S1PR2 expression in RKO cells and assessed their growth in xenografts. Functionally, we mimicked the ablation of S1PR2 in normal mucosa by treating S1PR2+/+organoids with JTE013 and characterized intestinal epithelial stem cells isolated from S1PR2−/−Lgr5-EGFP- mice.ResultsS1PR2 expression was lost in 33% of CRC; in 55%, it was significantly decreased, only 12% retaining expression comparable to normal mucosa. Both colitis-induced and genetic Apc+/minmouse models of CRC showed a higher incidence in size and number of carcinomas and/or high-grade adenomas, with increased cell proliferation in S1PR2−/−mice compared to S1PR2+/+controls. Loss of S1PR2 impaired mucosal regeneration, ultimately promoting the expansion of intestinal stem cells. Whereas its overexpression attenuated cell cycle progression, it reduced the phosphorylation of AKT and augmented the levels of PTEN.ConclusionsIn normal colonic crypts, S1PR2 gains expression along with intestinal epithelial cells differentiation, but not in intestinal stem cells, and contrasts intestinal tumorigenesis by promoting epithelial differentiation, preventing the expansion of stem cells and braking their malignant transformation. Targeting of S1PR2 may be of therapeutic benefit for CRC expressing high Lgr5.Graphical Abstract. Schematic drawing of the role of S1PR2 in normal mucosa and colorectal cancer. In the normal mucosa, S1PR2 is highly expressed by differentiated cells at the upper region of both colon and intestinal crypts (S1PR2 ON), but not by the undifferentiated stem cell at the base of the crypts (S1PR2 OFF), in which acts as a negative proliferative regulator promoting epithelial differentiation. Its loss leads to the expansion of stem cells and reduced levels of PTEN and Axin-2, two negative regulators respectively of PI3K/AKT and Wnt signaling that control β-catenin signaling. The translocation of β-catenin into the nucleus promotes the transcription of target genes involved in the proliferation and malignant transformation. Thereby, S1PR2 works in the intestine as a tumor suppressor

The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6−/− red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562 cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation. To obtain an overview on processes downstream to Sox6 expression, we performed a differential proteomic analysis on human erythroid K562 cells overexpressing Sox6. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24 A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these proteins during erythropoiesis.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6−/−-MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6−/−-MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.