Gut virome-colonising Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo.

OBJECTIVES: Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis. DESIGN: HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferation assay, chromatin immunoprecipitation assay with sequencing and RNA-sequencing were performed on in vitro models of the gut barrier. HBx-silencing experiments were performed in vitro and in vivo. RESULTS: HBx was detected in about 45% of patients with UC and found to induce colonic inflammation in mice, while its silencing reverted the colitis phenotype in vivo. HBx acted as a transcriptional regulator in epithelial cells, provoking barrier leakage and altering both innate and adaptive mucosal immunity ex vivo and in vivo. CONCLUSION: This study described HBx as a contributor to the UC pathogenesis and provides a new perspective on the virome as a target for tailored treatments.

Relationships Between Intestinal Ultrasound Parameters and Histopathologic Findings in a Prospective Cohort of Patients With Crohn's Disease Undergoing Surgery.

OBJECTIVES: Recognition of intestinal lesions with substantial fibrosis is strategic for optimal management of patients with Crohn's disease (CD). We aimed to assess the relationships between intestinal ultrasound parameters and histopathologic findings in a prospective cohort of patients with CD undergoing surgery. METHODS: Seventeen consecutive adult CD patients with involvement of the terminal ileum or the sigmoid colon who underwent bowel resective surgeries were enrolled and performed intestinal ultrasound (IUS) within 30 days prior to surgery. Uni- and multivariable analyses were used to assess the relationships between IUS parameters and histopathological elements of lesions. RESULTS: Sensitivity, specificity, accuracy, PPV and NPV (95% CI) of IUS in detecting stricturing and penetrating complications (surgical specimen as reference standard) were 93% (68-100), 86% (42-100), 91% (71-99), 93% (68-100) and 86% (42-100), and 78% (40-97), 92% (64-100), 86% (65-97), 88% (47-100) and 86% (57-98), respectively. Only the presence of hyperechogenic spiculates was statistically significantly associated with collagen content (b = 7.29, 95% CI = 1.88/12.69, P = .012), while only the presence of vascular signals at color Doppler (Limberg score 3 or 4) was significantly associated with active inflammation (OR = 10.0, 95% CI = 0.9/108.6, P = .037). There was a strong correlation between IUS and histological measurements of the wall thickness (r = 0.67, P = .01). CONCLUSIONS: The presence of hyperechogenic spiculates was associated with the presence of fibrosis, while the presence of marked vascular signals was associated with the presence of inflammation. Wall thickness measured by IUS was reliable and reproducible in comparison with histological measurement.

TNF-Stimulated Gene-6 Is a Key Regulator in Switching Stemness and Biological Properties of Mesenchymal Stem Cells.

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.

Lymphatic endothelium contributes to colorectal cancer growth via the soluble matrisome component GDF11.

Colorectal cancer (CRC) is one of the most malignant tumors worldwide. Stromal cells residing in the tumor microenvironment strongly contribute to cancer progression through their crosstalk with cancer cells and extracellular matrix. Here we provide the first evidence that CRC-associated lymphatic endothelium displays a distinct matrisome-associated transcriptomic signature, which distinguishes them from healthy intestinal lymphatics. We also demonstrate that CRC-associated human intestinal lymphatic endothelial cells regulate tumor cell growth via growth differentiation factor 11, a soluble matrisome component which in CRC patients was found to be associated with tumor progression. Our data provide new insights into lymphatic contribution to CRC growth, aside from their conventional role as conduits of metastasis.

MFSD2A Promotes Endothelial Generation of Inflammation-Resolving Lipid Mediators and Reduces Colitis in Mice.

BACKGROUND & AIMS: Alterations in signaling pathways that regulate resolution of inflammation (resolving pathways) contribute to pathogenesis of ulcerative colitis (UC). The resolution process is regulated by lipid mediators, such as those derived from the ω-3 docosahexaenoic acid (DHA), whose esterified form is transported by the major facilitator superfamily domain containing 2A (MFSD2A) through the endothelium of brain, retina, and placenta. We investigated if and how MFSD2A regulates lipid metabolism of gut endothelial cells to promote resolution of intestinal inflammation. METHODS: We performed lipidomic and functional analyses of MFSD2A in mucosal biopsies and primary human intestinal microvascular endothelial cells (HIMECs) isolated from surgical specimens from patients with active, resolving UC and healthy individuals without UC (controls). MFSD2A was knocked down in HIMECs with small hairpin RNAs or overexpressed from a lentiviral vector. Human circulating endothelial progenitor cells that overexpress MFSD2A were transferred to CD1 nude mice with dextran sodium sulfate-induced colitis, with or without oral administration of DHA. RESULTS: Colonic biopsies from patients with UC had reduced levels of inflammation-resolving DHA-derived epoxy metabolites compared to healthy colon tissues or tissues with resolution of inflammation. Production of these metabolites by HIMECs required MFSD2A, which is required for DHA retention and metabolism in the gut vasculature. In mice with colitis, transplanted endothelial progenitor cells that overexpressed MFSD2A not only localized to the inflamed mucosa but also restored the ability of the endothelium to resolve intestinal inflammation, compared with mice with colitis that did not receive MFSD2A-overexpressing endothelial progenitors. CONCLUSIONS: Levels of DHA-derived epoxides are lower in colon tissues from patients with UC than healthy and resolving mucosa. Production of these metabolites by gut endothelium requires MFSD2A; endothelial progenitor cells that overexpress MFSD2A reduce colitis in mice. This pathway might be induced to resolve intestinal inflammation in patients with colitis.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis.

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PDE4 Inhibition and Inflammatory Bowel Disease: A Novel Therapeutic Avenue.

BACKGROUND: In the last few decades, a better knowledge of the inflammatory pathways involved in the pathogenesis of Inflammatory Bowel Disease (IBD) has promoted biological therapy as an important tool to treat IBD patients. However, in spite of a wider spectrum of biological drugs, a significant proportion of patients is unaffected by or lose their response to these compounds, along with increased risks of infections and malignancies. For these reasons there is an urgent need to look for new pharmacological targets. The novel Phosphodiesterase 4 (PDE4) inhibitors have been recently introduced as new modulators of intracellular signals and gene transcription for the treatment of IBD. AIM: To discuss and describe the state of the art of this new class of compounds in the IBD field, with particular attention to apremilast. METHODS: Published articles selected from PubMed were comprehensively reviewed, with key words including apremilast, inflammatory disease, IBD, psoriasis, psoriatic arthritis, pathogenesis, therapies, and treatment. RESULTS: PDE4 inhibitors generate elevated intracellular levels of cyclic Adenosine Monophosphate (cAMP), that consequently down-regulate the release of pro-inflammatory cytokines in the mucosa of IBD patients. The newly developed apremilast is one of these drugs and has already been approved for the treatment of dermatologic/rheumatologic inflammatory conditions; studies in psoriasis and psoriatic arthritis have in fact demonstrated its clinical activity. However, no clinical trials have yet been published on the use of apremilast in IBD. CONCLUSION: In light of the similarity of pro-inflammatory signaling pathways across the gut, the skin, and joints, apremilast is likely supposed to show its efficacy also in IBD.

Vascular endothelial growth factor C disrupts the endothelial lymphatic barrier to promote colorectal cancer invasion.

BACKGROUND & AIMS: Colorectal cancer (CRC) is highly metastatic. Metastases spread directly into local tissue or invade distant organs via blood and lymphatic vessels, but the role of lymphangiogenesis in CRC progression has not been determined. Lymphangiogenesis is induced via vascular endothelial growth factor C (VEGFC) activation of its receptor, VEGFR3; high levels of VEGFC have been measured in colorectal tumors undergoing lymphangiogenesis and correlated with metastasis. We investigated VEGFC signaling and lymphatic barriers in human tumor tissues and mice with orthotopic colorectal tumors. METHODS: We performed immunohistochemical, immunoblot, and real-time polymerase chain reaction analyses of colorectal tumor specimens collected from patients; healthy intestinal tissues collected during operations of patients without CRC were used as controls. CT26 CRC cells were injected into the distal posterior rectum of BALB/c-nude mice. Mice were given injections of an antibody against VEGFR3 or an adenovirus encoding human VEGFC before orthotopic tumors and metastases formed. Lymph node, lung, and liver tissues were collected and evaluated by flow cytometry. We measured expression of vascular endothelial cadherin (CDH5) on lymphatic vessels in mice and in human intestinal lymphatic endothelial cells. RESULTS: Levels of podoplanin (a marker of lymphatic vessels), VEGFC, and VEGFR3 were increased in colorectal tumor tissues, compared with controls. Mice that expressed VEGFC from the adenoviral vector had increased lymphatic vessel density and more metastases in lymph nodes, lungs, and livers, compared with control mice. Anti-VEGFR3 antibody reduced numbers of lymphatic vessels in colons and prevented metastasis. Expression of VEGFC compromised the lymphatic endothelial barrier in mice and endothelial cells, reducing expression of CDH5, increasing permeability, and increasing trans-endothelial migration by CRC cells. Opposite effects were observed in mice and cells when VEGFR3 was blocked. CONCLUSIONS: VEGFC signaling via VEGFR3 promotes lymphangiogenesis and metastasis by orthotopic colorectal tumors in mice and reduces lymphatic endothelial barrier integrity. Levels of VEGFC and markers of lymphatic vessels are increased in CRC tissues from patients, compared with healthy intestine. Strategies to block VEGFR3 might be developed to prevent CRC metastasis in patients.

Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine.

BACKGROUND & AIMS: Mesenchymal stem cells (MSCs) are pluripotent cells that can promote expansion of immune regulatory cells and might be developed for the treatment of immune disorders, including inflammatory bowel diseases. MSCs were reported to reduce colitis in mice; we investigated whether MSC localization to the intestine and production of paracrine factors, including tumor necrosis factor-induced protein 6 (TSG6), were required for these effects. METHODS: MSCs were isolated from bone marrow (BM-MSCs) of 4- to 6-week-old C57BL/6, C57BL/6-green fluorescent protein, or Balb/c Tsg6-/- male mice. Colitis was induced by ad libitum administration of dextran sulfate sodium for 10 days; after 5 days the mice were given intraperitoneal injections of BM-MSCs or saline (controls). Blood samples and intestinal tissues were collected 24, 48, 96, and 120 hours later; histologic and flow cytometry analyses were performed. RESULTS: Injection of BM-MSCs reduced colitis in mice, increasing body weight and reducing markers of intestinal inflammation, compared with control mice. However, fewer than 1% of MSCs reached the inflamed colon. Most of the BM-MSCs formed aggregates in the peritoneal cavity. The aggregates contained macrophages and B and T cells, and produced immune-regulatory molecules including FOXP3, interleukin (IL)10, transforming growth factor-ß, arginase type II, chemokine (C-C motif) ligand 22 (CCL22), heme oxygenase-1, and TSG6. Serum from mice given BM-MSCs, compared with mice given saline, had increased levels of TSG6. Injection of TSG6 reduced the severity of colitis in mice, along with the numbers of CD45+ cells, neutrophils and metalloproteinase activity in the mucosa, while increasing the percentage of Foxp3CD45+ cells. TSG6 injection also promoted the expansion of regulatory macrophages that expressed IL10 and inducible nitric oxide synthase, and reduced serum levels of interferon-γ, IL6, and tumor necrosis factor. Tsg6-/- MSCs did not suppress the mucosal inflammatory response in mice with colitis. CONCLUSIONS: BM-MSCs injected into mice with colitis do not localize to the intestine but instead form aggregates in the peritoneum where they produce immunoregulatory molecules, including TSG6, that reduce intestinal inflammation. TSG6 is sufficient to reduce intestinal inflammation in mice with colitis.

The urokinase plasminogen activator receptor (uPAR) controls macrophage phagocytosis in intestinal inflammation.

OBJECTIVE: Inflammation plays crucial roles in the pathogenesis of several chronic inflammatory disorders, including Crohn's disease (CD) and UC, the two major forms of IBD. The urokinase plasminogen activator receptor (uPAR) exerts pleiotropic functions over the course of both physiological and pathological processes. uPAR not only has a key role in fibrinolysis but also modulates the development of protective immunity. Additionally, uPAR supports extracellular matrix degradation and regulates cell migration, adhesion and proliferation, thus influencing the development of inflammatory and immune responses. This study aimed to evaluate the role of uPAR in the pathogenesis of IBD. DESIGN: The functional role of uPAR was assessed in established experimental models of colitis. uPAR deficiency effects on cytokine release, polarisation and bacterial phagocytosis were analysed in colonic macrophages. uPAR expression was analysed in surgical specimens collected from normal subjects and patients with IBD. RESULTS: In mice, uPAR expression is positively regulated as colitis progresses. uPAR-KO mice displayed severe inflammation compared with wild-type littermates, as indicated by clinical assessment, endoscopy and colon histology. The absence of uPAR led to an increased production of inflammatory cytokines by macrophages that showed an M1 polarisation and impaired phagocytosis. In human IBD, CD68(+) macrophages derived from the inflamed mucosa expressed low levels of uPAR. CONCLUSIONS: These findings point to uPAR as an essential component of intestinal macrophage functions and unravel a new potential target to control mucosal inflammation in IBD.