Colon impairments and inflammation driven by an altered gut microbiota leads to social behavior deficits rescued by hyaluronic acid and celecoxib.

BACKGROUND: The exact mechanisms linking the gut microbiota and social behavior are still under investigation. We aimed to explore the role of the gut microbiota in shaping social behavior deficits using selectively bred mice possessing dominant (Dom) or submissive (Sub) behavior features. Sub mice exhibit asocial, depressive- and anxiety-like behaviors, as well as systemic inflammation, all of which are shaped by their impaired gut microbiota composition. METHODS: An age-dependent comparative analysis of the gut microbiota composition of Dom and Sub mice was performed using 16S rRNA sequencing, from early infancy to adulthood. Dom and Sub gastrointestinal (GI) tract anatomy, function, and immune profiling analyses were performed using histology, RT-PCR, flow cytometry, cytokine array, and dextran-FITC permeability assays. Short chain fatty acids (SCFA) levels in the colons of Dom and Sub mice were quantified using targeted metabolomics. To support our findings, adult Sub mice were orally treated with hyaluronic acid (HA) (30 mg/kg) or with the non-steroidal anti-inflammatory agent celecoxib (16 mg/kg). RESULTS: We demonstrate that from early infancy the Sub mouse gut microbiota lacks essential bacteria for immune maturation, including Lactobacillus and Bifidobacterium genera. Furthermore, from birth, Sub mice possess a thicker colon mucin layer, and from early adulthood, they exhibit shorter colonic length, altered colon integrity with increased gut permeability, reduced SCFA levels and decreased regulatory T-cells, compared to Dom mice. Therapeutic intervention in adult Sub mice treated with HA, celecoxib, or both agents, rescued Sub mice phenotypes. HA treatment reduced Sub mouse gut permeability, increased colon length, and improved mouse social behavior deficits. Treatment with celecoxib increased sociability, reduced depressive- and anxiety-like behaviors, and increased colon length, and a combined treatment resulted in similar effects as celecoxib administered as a single agent. CONCLUSIONS: Overall, our data suggest that treating colon inflammation and decreasing gut permeability can restore gut physiology and prevent social deficits later in life. These findings provide critical insights into the importance of early life gut microbiota in shaping gut immunity, functionality, and social behavior, and may be beneficial for the development of future therapeutic strategies.

Ex vivo intestinal permeability assay (X-IPA) for tracking barrier function dynamics.

The intestinal epithelial barrier facilitates homeostatic host-microbiota interactions and immunological tolerance. However, mechanistic dissections of barrier dynamics following luminal stimulation pose a substantial challenge. Here, we describe an ex vivo intestinal permeability assay, X-IPA, for quantitative analysis of gut permeability dynamics at the whole-tissue level. We demonstrate that specific gut microbes and metabolites induce rapid, dose-dependent increases to gut permeability, thus providing a powerful approach for precise investigation of barrier functions.

Diet-induced modifications to human microbiome reshape colonic homeostasis in irritable bowel syndrome.

Changes in microbiome composition are associated with a wide array of human diseases, turning the human microbiota into an attractive target for therapeutic intervention. Yet, clinical translation of these findings requires the establishment of causative connections between specific microbial taxa and their functional impact on host tissues. Here, we infuse gut organ cultures with longitudinal microbiota samples collected from therapy-naive patients with irritable bowel syndrome (IBS) under a low-fermentable oligo-, di-, mono-saccharides and polyols (FODMAP) diet. We show that post-diet microbiota regulates intestinal expression of inflammatory and neuro-muscular gene sets. Specifically, we identify Bifidobacterium adolescentis as a diet-sensitive pathobiont that alters tight junction integrity and disrupts gut barrier functions. Collectively, we present a pathway discovery platform for mechanistic dissection and identification of functional diet-host-microbiota modules. Our data support the hypothesis that the gut microbiota mediates the beneficial effects of a low-FODMAP diet and reinforce the potential feasibility of microbiome-based therapies in IBS.

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

Gut-Ex-Vivo system as a model to study gluten response in celiac disease.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.