Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life.

BACKGROUND: Perinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel disease (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2'-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis. RESULTS: In pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring. CONCLUSIONS: Our findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS.

The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and ß1-7) but exists as different subtypes depending on the cell type. In immune cells, for instance, constitutive catalytic proteasome subunits can be replaced by the so-called immuno-catalytic subunits, giving rise to the immunoproteasome. Proteasome activity is also altered by post-translational modifications (PTMs) and by genetic variants. Immunochemical methods are commonly used to investigate these PTMs whereby protein-tagging is necessary to monitor their effect on 20S assembly. Here, we present a new miniaturized workflow combining top-down and bottom-up mass spectrometry of immunopurified 20S proteasomes that analyze the proteasome assembly status as well as the full proteoform footprint, revealing PTMs, mutations, single nucleotide polymorphisms (SNPs) and induction of immune-subunits in different biological samples, including organoids, biopsies and B-lymphoblastoid cell lines derived from patients with proteasome-associated autoinflammatory syndromes (PRAAS). We emphasize the benefits of using top-down mass spectrometry in preserving the endogenous conformation of protein modifications, while enabling a rapid turnaround (1 h run) and ensuring high sensitivity (1-2 pmol) and demonstrate its capacity to semi-quantify constitutive and immune proteasome subunits.

Epithelial production of elastase is increased in inflammatory bowel disease and causes mucosal inflammation.

Imbalance between proteases and their inhibitors plays a crucial role in the development of Inflammatory Bowel Diseases (IBD). Increased elastolytic activity is observed in the colon of patients suffering from IBD. Here, we aimed at identifying the players involved in elastolytic hyperactivity associated with IBD and their contribution to the disease. We revealed that epithelial cells are a major source of elastolytic activity in healthy human colonic tissues and this activity is greatly increased in IBD patients, both in diseased and distant sites of inflammation. This study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. We demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier. Epithelial ELA2A hyperactivity also modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcripts, while reducing the expression of pro-resolving and repair factor genes. ELA2A thus appears as a novel actor produced by intestinal epithelial cells, which can drive inflammation and loss of barrier function, two essentials pathophysiological hallmarks of IBD. Targeting ELA2A hyperactivity should thus be considered as a potential target for IBD treatment.

Colitis Linked to Endoplasmic Reticulum Stress Induces Trypsin Activity Affecting Epithelial Functions.

BACKGROUND AND AIMS: Intestinal epithelial cells [IECs] from inflammatory bowel disease [IBD] patients exhibit an excessive induction of endoplasmic reticulum stress [ER stress] linked to altered intestinal barrier function and inflammation. Colonic tissues and the luminal content of IBD patients are also characterized by increased serine protease activity. The possible link between ER stress and serine protease activity in colitis-associated epithelial dysfunctions is unknown. We aimed to study the association between ER stress and serine protease activity in enterocytes and its impact on intestinal functions. METHODS: The impact of ER stress induced by Thapsigargin on serine protease secretion was studied using either human intestinal cell lines or organoids. Moreover, treating human intestinal cells with protease-activated receptor antagonists allowed us to investigate ER stress-resulting molecular mechanisms that induce proteolytic activity and alter intestinal epithelial cell biology. RESULTS: Colonic biopsies from IBD patients exhibited increased epithelial trypsin-like activity associated with elevated ER stress. Induction of ER stress in human intestinal epithelial cells displayed enhanced apical trypsin-like activity. ER stress-induced increased trypsin activity destabilized intestinal barrier function by increasing permeability and by controlling inflammatory mediators such as C-X-C chemokine ligand 8 [CXCL8]. The deleterious impact of ER stress-associated trypsin activity was specifically dependent on the activation of protease-activated receptors 2 and 4. CONCLUSIONS: Excessive ER stress in IECs caused an increased release of trypsin activity that, in turn, altered intestinal barrier function, promoting the development of inflammatory process.