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INTRODUCTION: The immune mechanisms underlying human autoimmune atrophic gastritis (AAG) are poorly understood. We sought to assess immune mucosal alterations in patients with AAG. METHODS: In 2017-2021, we collected gastric corpus biopsies from 24 patients with AAG (median age 62 years, interquartile range 56-67, 14 women), 26 age-matched and sex-matched healthy controls (HCs), and 14 patients with Helicobacter pylori infection (HP). We investigated the lamina propria mononuclear cell (LPMC) populations and the mucosal expression of thymic stromal lymphopoietin (TSLP) and nicotinamide phosphoribosyltransferase (NAMPT). Ex vivo cytokine production by organ culture biopsies, under different stimuli (short TSLP and zinc-l-carnosine), and the gastric vascular barrier through plasmalemma vesicle-associated protein-1 (PV1) were also assessed. RESULTS: In the subset of CD19+ LPMC, CD38+ cells (plasma cells) were significantly higher in AAG compared with HC. Ex vivo production of tumor necrosis factor (TNF)-α, interleukin (IL)-15, and transforming growth factor β1 was significantly higher in AAG compared with HC. At immunofluorescence, both IL-7R and TSLP were more expressed in AAG compared with HC and HP, and short TSLP transcripts were significantly increased in AAG compared with HC. In the supernatants of AAG corpus mucosa, short TSLP significantly reduced TNF-α, while zinc-l-carnosine significantly reduced interferon-γ, TNF-α, IL-21, IL-6, and IL-15. NAMPT transcripts were significantly increased in AAG compared with HC. PV1 was almost absent in AAG, mildly expressed in HC, and overexpressed in HP. DISCUSSION: Plasma cells, proinflammatory cytokines, and altered gastric vascular barrier may play a major role in AAG. TSLP and NAMPT may represent potential therapeutic targets, while zinc-l-carnosine may dampen mucosal inflammation.

Background and Aims The SARS-CoV-2 pandemic has overwhelmed the treatment capacity of the health care systems during the highest viral diffusion rate. Patients reaching the emergency department had to be either hospitalized (inpatients) or discharged (outpatients). Still, the decision was taken based on the individual assessment of the actual clinical condition, without specific biomarkers to predict future improvement or deterioration, and discharged patients often returned to the hospital for aggravation of their condition. Here, we have developed a new combined approach of omics to identify factors that could distinguish coronavirus disease 19 (COVID-19) inpatients from outpatients. Methods Saliva and blood samples were collected over the course of two observational cohort studies. By using machine learning approaches, we compared salivary metabolome of 50 COVID-19 patients with that of 270 healthy individuals having previously been exposed or not to SARS-CoV-2. We then correlated the salivary metabolites that allowed separating COVID-19 inpatients from outpatients with serum biomarkers and salivary microbiota taxa differentially represented in the two groups of patients. Results We identified nine salivary metabolites that allowed assessing the need of hospitalization. When combined with serum biomarkers, just two salivary metabolites (myo-inositol and 2-pyrrolidineacetic acid) and one serum protein, chitinase 3-like-1 (CHI3L1), were sufficient to separate inpatients from outpatients completely and correlated with modulated microbiota taxa. In particular, we found Corynebacterium 1 to be overrepresented in inpatients, whereas Actinomycetaceae F0332, Candidatus Saccharimonas, and Haemophilus were all underrepresented in the hospitalized population. Conclusion This is a proof of concept that a combined omic analysis can be used to stratify patients independently from COVID-19.

The translocation of bacterial components from the intestinal lumen into the portal circulation is crucial in the pathogenesis of alcoholic liver disease (ALD). Recently the important role of the gut vascular barrier (GVB) was elucidated in alcoholic liver disease. Here we report about the influence of A. muciniphila supplementation in experimental ALD on the GVB. Ethanol feeding was associated with increased Pv-1, indicating altered endothelial barrier function, whereas A. muciniphila administration tended to restore GVB. To further investigate GVB in experimental ALD, β-catenin gain-of-function mice, which display an enhanced GVB, were ethanol-fed. β-catenin gain-of-function mice were not protected from ethanol-induced liver injury, suggest an alternative mechanism of ethanol-induced GVB disruption. The description of the GVB in ALD could pave the way for new therapeutic options in the future.

Background: The gut-liver-axis presents the pathophysiological hallmark for multiple liver diseases and has been proposed to be modulated during stress and shock. Access to the gut-liver-axis needs crossing of the mucus and gut-vascular barrier. The role of β-adrenoreceptor-activation for both barriers has not been defined and is characterized here. Methods: Splanchnic β-adrenergic stimulation was achieved by chronic intraperitoneal application of isoproterenol via alzet-pump in vivo. The intestinal permeability and gut-vascular barrier function was assessed in ileal loop experiments. The extravasation of predefined sizes of fluorescence isothiocyanate (FITC)-dextran molecules in ileal microcirculation was evaluated by intravital confocal laser endomicroscopy in vivo. Mucus parameters thickness, goblet cell count and mucin-expression were assessed by stereomicroscopy, immunostaining and RNA-sequencing respectively. Ileal lamina propria (LP) as well as mesenteric lymph node mononuclear cells was assessed by FACS. Results: Healthy mice lack translocation of 4 kDa-FITC-dextran from the small intestine to the liver, whereas isoproterenol-treated mice demonstrate pathological translocation (PBT). Mucus layer is reduced in thickness with loss of goblet-cells and mucin-2-staining and -expression in isoproterenol-treated animals under standardized gnotobiotic conditions. Isoproterenol disrupts the gut vascular barrier displaying Ileal extravasation of large-sized 70- and 150 kDa-FITC-dextran. This pathological endothelial permeability and accessibility induced by isoproterenol associates with an augmented expression of plasmalemmal-vesicle-associated-protein-1 in intestinal vessel. Ileal LP after isoproterenol treatment contains more CD11c+-dendritic cells (DC) with increased appearance of CCR7+ DC in mesenteric lymph nodes. Conclusions: Isoproterenol impairs the intestinal muco-epithelial and endothelial-vascular barrier promoting PBT to the liver. This barrier dysfunction on multiple levels potentially can contribute to liver injury induced by catecholamines during states of increased β-adrenergic drive.

Immune privilege is a complex process that protects organs from immune-mediated attack and damage. It is accomplished by a series of cellular barriers that both control immune cell entry and promote the development of tolerogenic immune cells. In this Review, we describe the vascular endothelial and epithelial barriers in organs that are commonly considered to be immune privileged, such as the brain and the eye. We compare these classical barriers with barriers in the intestine, which share features with barriers of immune-privileged organs, such as the capacity to induce tolerance and to protect from external insults. We suggest that when intestinal barriers break down, disruption of other barriers at distant sites can ensue, and this may underlie the development of various neurological, metabolic and intestinal disorders.

Background and aimsPathological bacterial translocation (PBT) in liver cirrhosis (LC) is the hallmark for spontaneous bacterial infections increasing mortality several-fold. Factors known to contribute to PBT in LC are among others an increased intestinal permeability of which however, the mucus layer has not been addressed so far in detail. A clear route of translocation for luminal intestinal bacteria is yet to be defined but we hypothesize that the recently described gut vascular barrier (GVB) is impaired in experimental portal hypertension leading to increased accessibility of the vascular compartment for translocating bacteria.ResultsHealthy and pre-hepatic portal-hypertensive (PPVL) mice lack translocation of FITC-dextran and GFP-Escherichia colifrom the small intestine to the liver whereas bile-duct-ligated (BDL) and CCl4-induced cirrhotic mice demonstrate pathological translocation which is not altered by prior thoracic-duct ligation. Mucus layer is reduced in thickness with loss of goblet-cells and Muc2-staining and expression in cirrhotic but not PPVL-mice associated with bacterial overgrowth in inner mucus layer and pathological translocation of GFP-E.colithrough the ileal epithelium. GVB is profoundly altered in BDL and CCl4-mice with Ileal extravasation of large-sized 150 kDa-FITC-dextran but only minor in PPVL-mice. This pathological endothelial permeability and accessibility in cirrhotic mice associates with an augmented expression of PV1 in intestinal vessels. OCA but not fexaramine stabilizes the GVB whereas both FXR-agonists ameliorate gut-liver-translocation of GFP-E.coli.ConclusionsLiver cirrhosis but not portal hypertension per se grossly impairs the endothelial and muco-epithelial barriers promoting PBT to the portal-venous circulation. Both barriers appear FXR-modulated with –agonists reducing PBT via the portal-venous route.

Graphical abstract, Highlights • During diet-induced dysbiosis the gut vascular barrier is disrupted. • Gut vascular barrier disruption is responsible for the translocation of bacteria or bacterial products systemically. • Inhibiting gut vascular barrier disruption prevents the development of non-alcoholic steatohepatitis. • Obeticholic acid can control gut vascular barrier disruption both in a preventive and therapeutic way., Background & Aims Fatty liver disease, including non-alcoholic fatty liver (NAFLD) and steatohepatitis (NASH), has been associated with increased intestinal barrier permeability and translocation of bacteria or bacterial products into the blood circulation. In this study, we aimed to unravel the role of both intestinal barrier integrity and microbiota in NAFLD/NASH development. Methods C57BL/6J mice were fed with high-fat diet (HFD) or methionine-choline-deficient diet for 1 week or longer to recapitulate aspects of NASH (steatosis, inflammation, insulin resistance). Genetic and pharmacological strategies were then used to modulate intestinal barrier integrity. Results We show that disruption of the intestinal epithelial barrier and gut vascular barrier (GVB) are early events in NASH pathogenesis. Mice fed HFD for only 1 week undergo a diet-induced dysbiosis that drives GVB damage and bacterial translocation into the liver. Fecal microbiota transplantation from HFD-fed mice into specific pathogen-free recipients induces GVB damage and epididymal adipose tissue enlargement. GVB disruption depends on interference with the WNT/β-catenin signaling pathway, as shown by genetic intervention driving β-catenin activation only in endothelial cells, preventing GVB disruption and NASH development. The bile acid analogue and farnesoid X receptor agonist obeticholic acid (OCA) drives β-catenin activation in endothelial cells. Accordingly, pharmacologic intervention with OCA protects against GVB disruption, both as a preventive and therapeutic agent. Importantly, we found upregulation of the GVB leakage marker in the colon of patients with NASH. Conclusions We have identified a new player in NASH development, the GVB, whose damage leads to bacteria or bacterial product translocation into the blood circulation. Treatment aimed at restoring β-catenin activation in endothelial cells, such as administration of OCA, protects against GVB damage and NASH development. Lay summary The incidence of fatty liver disease is reaching epidemic levels in the USA, with more than 30% of adults having NAFLD (non-alcoholic fatty liver disease), which can progress to more severe non-alcoholic steatohepatitis (NASH). Herein, we show that disruption of the intestinal epithelial barrier and gut vascular barrier are early events in the development of NASH. We show that the drug obeticholic acid protects against barrier disruption and thereby prevents the development of NASH, providing further evidence for its use in the prevention or treatment of NASH.

Background & Aims Pathological bacterial translocation (PBT) in cirrhosis is the hallmark of spontaneous bacterial infections, increasing mortality several-fold. Increased intestinal permeability is known to contribute to PBT in cirrhosis, although the role of the mucus layer has not been addressed in detail. A clear route of translocation for luminal intestinal bacteria is yet to be defined, but we hypothesize that the recently described gut-vascular barrier (GVB) is impaired in experimental portal hypertension, leading to increased accessibility of the vascular compartment for translocating bacteria. Materials Cirrhosis was induced in mouse models using bile-duct ligation (BDL) and CCl4. Pre-hepatic portal-hypertension was induced by partial portal vein ligation (PPVL). Intestinal permeability was compared in these mice after GFP-Escherichia coli or different sized FITC-dextrans were injected into the intestine. Results Healthy and pre-hepatic portal-hypertensive (PPVL) mice lack translocation of FITC-dextran and GFP-E. coli from the small intestine to the liver, whereas BDL and CCl4-induced cirrhotic mice demonstrate pathological translocation, which is not altered by prior thoracic-duct ligation. The mucus layer is reduced in thickness, with loss of goblet cells and Muc2-staining and expression in cirrhotic but not PPVL mice. These changes are associated with bacterial overgrowth in the inner mucus layer and pathological translocation of GFP-E. coli through the ileal epithelium. GVB is profoundly altered in BDL and CCl4-mice with Ileal extravasation of large-sized 150 kDa-FITC-dextran, but only slightly altered in PPVL mice. This pathological endothelial permeability and accessibility in cirrhotic mice is associated with augmented expression of PV1 in intestinal vessels. OCA but not fexaramine stabilizes the GVB, whereas both FXR-agonists ameliorate gut to liver translocation of GFP-E. coli. Conclusions Cirrhosis, but not portal hypertension per se, grossly impairs the endothelial and muco-epithelial barriers, promoting PBT to the portal-venous circulation. Both barriers appear to be FXR-modulated, with FXR-agonists reducing PBT via the portal-venous route. Lay summary For intestinal bacteria to enter the systemic circulation, they must cross the mucus and epithelial layer, as well as the gut-vascular barrier. Cirrhosis disrupts all 3 of these barriers, giving bacteria access to the portal-venous circulation and thus, the gut-liver axis. Diminished luminal bile acid availability, cirrhosis and the associated reduction in farnesoid x receptor (FXR) signaling seem, at least partly, to mediate these changes, as FXR-agonists reduce bacterial translocation via the portal-venous route to the liver in cirrhosis.

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases. Copyright © 2021 Elsevier Inc. All rights reserved.

It has been widely demonstrated that tolerance against gut microbiota is compartmentalized to mucosal sites where microbes mostly reside. How the commensal bacteria are excluded from the entrance into the blood stream via intestinal capillaries that are located beneath the gut epithelium was not clear. We recently described the existence of a new anatomical structure, the 'gut vascular barrier' (GVB), both in murine and human intestines that plays a fundamental role in avoiding indiscriminate trafficking of bacteria from the gut into the blood circulation. The vascular barrier integrity could be altered by Salmonella typhimurium, a pathogen capable of systemic dissemination, through the modulation of the Wnt/β-catenin signaling pathway. Here we have analyzed the differences in gut endothelial gene expression profiles during Salmonella infection and have identified some interesting characteristics of endothelial to mesenchymal transition. These findings add new insights in the gut-liver axis.

A gut bacterial containment system Trillions of bacteria selectively inhabit our guts, but how do our bodies keep them contained? Spadoni et al. describe a “gut-vascular barrier” that prevents intestinal microbes from accessing the liver and the bloodstream in mice (see the Perspective by Bouziat and Jabri). Studies with human samples and in mice revealed that the cell biology of the gut-vascular barrier shares similarities with the blood-brain barrier of the central nervous system. Pathogenic bacteria such as Salmonella typhimurium could penetrate the gut-vascular barrier in mice, gaining access to the liver and bloodstream, in a manner dependent on the Salmonella pathogenicity island 2–type III secretion system. Science , this issue p. 830 ; see also p. 742

Cetuximab is a monoclonal antibody that is effective in the treatment of metastatic colorectal cancer (mCRC). Cetuximab blocks epidermal growth factor receptor (EGFR)-ligand interaction and inhibits downstream RAS-ERK activation. However, only some activating mutations in RAS affect cetuximab efficacy, and it is not clear what else mediates treatment success. Here we hypothesized that cetuximab induces immunogenic cell death (ICD) that activates a potent antitumor response. We found that cetuximab, in combination with chemotherapy, fostered ICD in CRC cells, which we measured via the endoplasmic reticulum (ER) stress response and an increase in phagocytosis by dendritic cells. ICD induction depended on the mutational status of the EGFR signaling pathway and on the inhibition of the splicing of X-box binding protein 1 (XBP1), an unfolded protein response (UPR) mediator. We confirmed the enhanced immunogenicity elicited by cetuximab in a mouse model of human EGFR-expressing CRC. Overall, we demonstrate a new, immune-related mechanism of action of cetuximab that may help to tailor personalized medicine.

Thymic stromal lymphopoietin (TSLP) is produced by epithelial cells and keratinocytes, and is involved in immune homeostasis or inflammation. The mechanism through which TSLP regulates intestinal inflammation is unclear. Here, we report that mouse dendritic cells (DCs) express TSLP both in vitro and in vivo in response to Toll-like receptor ligation in a MyD88-dependent fashion. TSLP is produced by the CD103(+) subset of tolerogenic gut DCs and is downregulated during experimental colitis. TSLP produced by DCs acts directly on T cells by reducing their capacity to produce interleukin (IL)-17 and fostering the development of Foxp3(+) T cells. Consistently, TSLP protects against colitis development through a direct action on T cells, as adoptive transfer of naïve T cells from TSLPR(-/-) to SCID mice results in a more severe colitis, with increased frequency of IL-17-producing T cells and inflammatory cytokines. Hence, we describe a new anti-inflammatory role of TSLP in the gut.

Objective: In mice, a subpopulation of gut dendritic cells (DCs) expressing CD103 drives the development of regulatory T (T reg ) cells. Further, it was recently described that the cross-talk between human intestinal epithelial cells (IECs) and DCs helps in maintaining gut immune homeostasis via the induction of non-inflammatory DCs. In this study, an analysis was carried out to determine whether IECs could promote the differentiation of CD103 + tolerogenic DCs, and the function of primary CD103 + DCs isolated from human mesenteric lymph nodes (MLNs) was evaluated. Methods: Monocyte-derived DCs (MoDCs) and circulating CD1c + DCs were conditioned or not with supernatants from Caco-2 cells or IECs isolated from healthy donors or donors with Crohn’s disease and analysed for their ability to induce T reg cell differentiation. In some cases, transforming growth factor β (TGFβ), retinoic acid (RA) or thymic stromal lymphopoietin (TSLP) were neutralised before conditioning. CD103 + and CD103 − DCs were sorted by fluorescence-activated cell sorting (FACS) from MLNs and used in T reg cell differentiation experiments. Results: It was found that human IECs promoted the differentiation of tolerogenic DCs able to drive the development of adaptive Foxp3 + T reg cells. This control was lost in patients with Crohn’s disease and paralleled a reduced expression of tolerogenic factors by primary IECs. MoDCs differentiated with RA or IEC supernatant upregulated the expression of CD103. Consistently, human primary CD103 + DCs isolated from MLNs were endowed with the ability to drive T reg cell differentiation. This subset of DCs expressed CCR7 and probably represents a lamina propria-derived migratory population. Conclusions: A population of tolerogenic CD103 + DCs was identified in the human gut that probably differentiate in response to IEC-derived factors and drive T reg cell development.