Microbiota profiling in esophageal diseases: Novel insights into molecular staining and clinical outcomes.

Gut microbiota is recognized nowadays as one of the key players in the development of several gastro-intestinal diseases. The first studies focused mainly on healthy subjects with staining of main bacterial species via culture-based techniques. Subsequently, lots of studies tried to focus on principal esophageal disease enlarged the knowledge on esophageal microbial environment and its role in pathogenesis. Gastro Esophageal Reflux Disease (GERD), the most widespread esophageal condition, seems related to a certain degree of mucosal inflammation, via interleukin (IL) 8 potentially enhanced by bacterial components, lipopolysaccharide (LPS) above all. Gram- bacteria, producing LPS), such as Campylobacter genus, have been found associated with GERD. Barrett esophagus (BE) seems characterized by a Gram- and microaerophils-shaped microbiota. Esophageal cancer (EC) development leads to an overturn in the esophageal environment with the shift from an oral-like microbiome to a prevalently low-abundant and low-diverse Gram--shaped microbiome. Although underinvestigated, also changes in the esophageal microbiome are associated with rare chronic inflammatory or neuropathic disease pathogenesis. The paucity of knowledge about the microbiota-driven mechanisms in esophageal disease pathogenesis is mainly due to the scarce sensitivity of sequencing technology and culture methods applied so far to study commensals in the esophagus. However, the recent advances in molecular techniques, especially with the advent of non-culture-based genomic sequencing tools and the implementation of multi-omics approaches, have revolutionized the microbiome field, with promises of implementing the current knowledge, discovering more mechanisms underneath, and giving insights into the development of novel therapies aimed to re-establish the microbial equilibrium for ameliorating esophageal diseases..

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.

A multi-omic analysis reveals the esophageal dysbiosis as the predominant trait of eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic immune-mediated rare disease, characterized by esophageal dysfunctions. It is likely to be primarily activated by food antigens and is classified as a chronic disease for most patients. Therefore, a deeper understanding of the pathogenetic mechanisms underlying EoE is needed to implement and improve therapeutic lines of intervention and ameliorate overall patient wellness. METHODS: RNA-seq data of 18 different studies on EoE, downloaded from NCBI GEO with faster-qdump ( https://github.com/ncbi/sra-tools ), were batch-corrected and analyzed for transcriptomics and metatranscriptomics profiling as well as biological process functional enrichment. The EoE TaMMA web app was designed with plotly and dash. Tabula Sapiens raw data were downloaded from the UCSC Cell Browser. Esophageal single-cell raw data analysis was performed within the Automated Single-cell Analysis Pipeline. Single-cell data-driven bulk RNA-seq data deconvolution was performed with MuSiC and CIBERSORTx. Multi-omics integration was performed with MOFA. RESULTS: The EoE TaMMA framework pointed out disease-specific molecular signatures, confirming its reliability in reanalyzing transcriptomic data, and providing new EoE-specific molecular markers including CXCL14, distinguishing EoE from gastroesophageal reflux disorder. EoE TaMMA also revealed microbiota dysbiosis as a predominant characteristic of EoE pathogenesis. Finally, the multi-omics analysis highlighted the presence of defined classes of microbial entities in subsets of patients that may participate in inducing the antigen-mediated response typical of EoE pathogenesis. CONCLUSIONS: Our study showed that the complex EoE molecular network may be unraveled through advanced bioinformatics, integrating different components of the disease process into an omics-based network approach. This may implement EoE management and treatment in the coming years.

Tofacitinib Inhibits Leukocyte Trafficking Across the Intestinal Endothelial Barrier in a Specific Cohort of Ulcerative Colitis Patients.

The JAK/STAT inhibitor tofacitinib, recently approved for the treatment of ulcerative colitis, is found to modulate the intestinal endothelial barrier functions in directing the leukocyte adhesion and transmigration in ulcerative colitis patients displaying high levels of endothelial STAT3/STAT6 phosphorylation.

GPR120 prevents colorectal adenocarcinoma progression by sustaining the mucosal barrier integrity.

GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by ω-3 Polyunsaturated Fatty Acids (PUFAs), and expressed in many cell types. Its role in the context of colorectal cancer (CRC) is still puzzling with many controversial evidences. Here, we explored the involvement of epithelial GPR120 in the CRC development. Both in vitro and in vivo experiments were conducted to mimic the conditional deletion of the receptor from gut epithelium. Intestinal permeability and integrity of mucus layer were assessed by using Evans blue dye and immunofluorescence for MUC-2 protein, respectively. Microbiota composition, presence of lipid mediators and short chain fatty acids were analyzed in the stools of conditional GPR120 and wild type (WT) mice. Incidence and grade of tumors were evaluated in all groups of mice before and after colitis-associated cancer. Finally, GPR120 expression was analyzed in 9 human normal tissues, 9 adenomas, and 17 primary adenocarcinomas. Our work for the first time highlights the role of the receptor in the progression of colorectal cancer. We observed that the loss of epithelial GPR120 in the gut results into increased intestinal permeability, microbiota translocation and dysbiosis, which turns into hyperproliferation of epithelial cells, likely through the activation of ß -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. these results demonstrate that the epithelial GPR120 receptor is essential to maintain the mucosal barrier integrity and to prevent CRC developing. Therefore, our data pave the way to GPR120 as an useful marker for the phenotypic characterization of CRC lesions and as new potential target for CRC prevention.

mTOR-Dependent Stimulation of IL20RA Orchestrates Immune Cell Trafficking through Lymphatic Endothelium in Patients with Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory condition that can affect different portions of the gastrointestinal tract. Lymphatic drainage was demonstrated to be dysfunctional in CD pathogenesis, ultimately causing the failure of the resolution of intestinal inflammation. To investigate the molecular mechanisms underlying these dysfunctions, we isolated human intestinal lymphatic endothelial cells (HILECs) from surgical specimens of patients undergoing resection for complicated CD (CD HILEC) and from a disease-free margin of surgical specimens of patients undergoing resection for cancer (healthy HILEC). Both cell types underwent transcriptomic profiling, and their barrier functionality was tested using a transwell-based co-culture system between HILEC and lamina propria mononuclear cells (LPMCs). Results showed CD HILEC displayed a peculiar transcriptomic signature that highlighted mTOR signaling as an orchestrator of leukocyte trafficking through the lymphatic barrier of CD patients. Moreover, we demonstrated that LPMC transmigration through the lymphatic endothelium of patients with CD depends on the capability of mTOR to trigger interleukin 20 receptor subunit α (IL20RA)-mediated intracellular signaling. Conclusively, our study suggests that leukocyte trafficking through the intestinal lymphatic microvasculature can be controlled by modulating IL20RA, thus leading to the resolution of chronic inflammation in patients with CD.

Activation of the VEGFC/VEGFR3 Pathway Induces Tumor Immune Escape in Colorectal Cancer.

Colorectal cancer is a major cause of cancer-related death in Western countries and is associated with increased numbers of lymphatic vessels (LV) and tumor-associated macrophages (TAM). The VEGFC/VEGFR3 pathway is regarded as the principal inducer of lymphangiogenesis and it contributes to metastases; however, no data are available regarding its role during primary colorectal cancer development. We found that both VEGFC and VEGFR3 were upregulated in human nonmetastatic colorectal cancer, with VEGFR3 expressed on both LVs and TAMs. With the use of three different preclinical models of colorectal cancer, we also discovered that the VEGFC/VEGFR3 axis can shape both lymphatic endothelial cells and TAMs to synergistically inhibit antitumor immunity and promote primary colorectal cancer growth. Therefore, VEGFR3-directed therapy could be envisioned for the treatment of nonmetastatic colorectal cancer. SIGNIFICANCE: The prolymphangiogenic factor VEGFC is abundant in colorectal cancer and activates VEGFR3 present on cancer-associated macrophages and lymphatic vessels; activation of VEGFR3 signaling fosters cancer immune escape, resulting in enhanced tumor growth.

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.