Loris Riccardo Lopetuso

Commensal Clostridia: leading players in the maintenance of gut homeostasis

The gastrointestinal tract is a complex and dynamic network where an intricate and mutualistic symbiosis modulates the relationship between the host and the microbiota in order to establish and ensure gut homeostasis. Commensal Clostridia consist of gram-positive, rod-shaped bacteria in the phylum Firmicutes and make up a substantial part of the total bacteria in the gut microbiota. They start to colonize the intestine of breastfed infants during the first month of life and populate a specific region in the intestinal mucosa in close relationship with intestinal cells. This position allows them to participate as crucial factors in modulating physiologic, metabolic and immune processes in the gut during the entire lifespan, by interacting with the other resident microbe populations, but also by providing specific and essential functions. This review focus on what is currently known regarding the role of commensal Clostridia in the maintenance of overall gut function, as well as touch on their potential contribution in the unfavorable alteration of microbiota composition (dysbiosis) that has been implicated in several gastrointestinal disorders. Commensal Clostridia are strongly involved in the maintenance of overall gut function. This leads to important translational implications in regard to the prevention and treatment of dysbiosis, to drug efficacy and toxicity, and to the development of therapies that may modulate the composition of the microflora, capitalizing on the key role of commensal Clostridia, with the end goal of promoting gut health.

Gut Microbial Flora, Prebiotics, and Probiotics in IBD: Their Current Usage and Utility

Inflammatory bowel diseases are chronic diseases affecting the gastrointestinal tract, whose major forms are represented by Crohns disease (CD) and ulcerative colitis (UC). Their etiology is still unclear, although several factors have been identified as major determinants for induction or relapses. Among these, the role of the “forgotten organ”, gut microbiota, has become more appreciated in recent years. The delicate symbiotic relationship between the gut microbiota and the host appears to be lost in IBD. In this perspective, several studies have been conducted to assess the role of prebiotics and probiotics in gut microbiota modulation. This is a minireview aimed to address in an easy format (simple questions-simple answers) some common issues about the theme. An update on the role of selected constituents of gut microbiota in the pathogenesis of IBD is presented together with the analysis of the efficacy of gut microbiota modulation by prebiotics and probiotics administration in the management of IBD.

The gut barrier: new acquisitions and therapeutic approaches.

The intestinal barrier serves 2 critical functions for the survival of the individual: first, it allows nutrient absorption and second, it defends the body from dangerous macromolecule penetration. It is a complex multilayer system, consisting of an external “anatomic” barrier and an inner “functional” immunological barrier. The interaction of these 2 barriers enables equilibrated permeability to be maintained. Many factors can alter this balance: gut microflora modifications, mucus layer alterations, and epithelial damage can increase intestinal permeability, allowing the translocation of luminal content to the inner layer of intestinal wall. Several techniques are now available that enable us to study gut permeability: “in vitro” models (Caco-2 and HT29-MTX cells) and “in vivo” not invasive tests (sugar tests and radioisotope scanning tests) are used to estimate permeability and to suggest molecular pathophysiological mechanisms of intestinal permeability in health and diseases. Many medicinal products used in the treatment of gastrointestinal diseases have also found to play an active role in modulate intestinal permeability: corticosteroids, 5-aminosalicylic acid, anti–tumor necrosis factor, probiotics, and mucosal protectors, like gelatin tannate. This review will particularly address the role of the gut barrier in maintaining intestinal permeability (microbiota, mucus, and epithelial cells), the techniques used for estimating intestinal permeability and the therapeutic approaches able to modify it.

Opposing Functions of Classic and Novel IL-1 Family Members in Gut Health and Disease

In addition to their well-established role(s) in the pathogenesis of gastrointestinal (GI)-related inflammatory disorders, including inflammatory bowel disease (IBD) and inflammation-associated colorectal cancer (CRC), emerging evidence confirms the critical involvement of the interleukin-1 (IL-1) cytokine family and their ligands in the maintenance of normal gut homeostasis. In fact, the paradigm that IBD occurs in two distinct phases is substantiated by the observation that classic IL-1 family members, such as IL-1, the IL-1 receptor antagonist (IL-1Ra), and IL-18, possess dichotomous functions depending on the phase of disease, as well as on their role in initiating vs. sustaining chronic gut inflammation. Another recently characterized IL-1 family member, IL-33, also possesses dual functions in the gut. IL-33 is upregulated in IBD and potently induces Th2 immune responses, while also amplifying Th1-mediated inflammation. Neutralization studies in acute colitis models, however, have yielded controversial results and recent reports suggest a protective role of IL-33 in epithelial regeneration and mucosal wound healing. Finally, although little is currently known regarding the potential contribution of IL-36 family members in GI inflammation/homeostasis, another IL-1 family member, IL-37, is emerging as a potent anti-inflammatory cytokine with the ability to down-regulate colitis. This new body of information has important translational implications for both the prevention and treatment of patients suffering from IBD and inflammation-associated CRC.

Emerging role of the interleukin (IL)-33/ST2 axis in gut mucosal wound healing and fibrosis.

Interleukin (IL)-33 (IL-1F11) is the newest member of the IL-1Family of cytokines and has been best characterized as a potent inducer of T helper (Th)2 immune responses. Increasing evidence, however, indicates that IL-33 also represents an important mediator of mucosal healing and epithelial restoration and repair. As such, IL-33 follows the trend of several innate-type cytokines, including members of the IL-1Family (for example, IL-1α, IL-1β, and IL-18), that possess dichotomous roles of inducing a potent proinflammatory response, while also promoting protection and the return to immune homeostasis. This dual function is best depicted in the gut mucosa and is dependent upon the immunological/genetic status of the host and/or the type and phase of the ongoing inflammatory process. IL-33 has also been described as a prototypic ‘alarmin’ that has the ability to signal local, innate immune responses of trauma or infection in an effort to mount an effective, physiologic inflammatory reaction to induce mucosal healing and restore normal gut equilibrium. Finally, several recent studies have reported the role of IL-33 during fibrogenesis as fibrosis is commonly thought to occur as the end stage of dysregulated wound healing wherein chronic tissue damage is paired with uncontrolled activation of mesenchymal cells. Taken together, aside from its established function of promoting potent Th2 immune responses, IL-33 is emerging as an important cytokine for the induction of mucosal healing and restoration of intestinal homeostasis, as well as playing a central role in fibrosis and wound repair. The present review will focus on what is currently known regarding IL-33’s role in gut mucosal wound healing and fibrosis, as well as touch on its potential contribution to tumorigenesis and GI-related cancer, an alternate outcome of dysregulated epithelial proliferation.

The Role of Antibiotics in Gut Microbiota Modulation: The Eubiotic Effects of Rifaximin

Antibiotics are mainly used in clinical practice for their activity against pathogens, but they also alter the composition of commensal gut microbial community. Rifaximin is a non-absorbable antibiotic with additional effects on the gut microbiota about which very little is known. It is still not clear to what extent rifaximin can be able to modulate gut microbiota composition and diversity in different clinical settings. Studies based on culture-dependent techniques revealed that rifaximin treatment promotes the growth of beneficial bacteria, such as Bifidobacteria and Lactobacilli. Accordingly, our metagenomic analysis carried out on patients with different gastrointestinal and liver diseases highlighted a significant increase in Lactobacilli after rifaximin treatment, persisting in the short time period. This result was independent of the disease background and was not accompanied by a significant alteration of the overall gut microbial ecology. This suggests that rifaximin can exert important eubiotic effects independently of the original disease, producing a favorable gut microbiota perturbation without changing its overall composition and diversity.

Role of Microbiota and Innate Immunity in Recurrent Clostridium difficile Infection

Recurrent Clostridium difficile infection represents a burdensome clinical issue whose epidemiology is increasing worldwide. The pathogenesis is not yet completely known. Recent observations suggest that the alteration of the intestinal microbiota and impaired innate immunity may play a leading role in the development of recurrent infection. Various factors can cause dysbiosis. The causes most involved in the process are antibiotics, NSAIDs, acid suppressing therapies, and age. Gut microbiota impairment can favor Clostridium difficile infection through several mechanisms, such as the alteration of fermentative metabolism (especially SCFAs), the alteration of bile acid metabolism, and the imbalance of antimicrobial substances production. These factors alter the intestinal homeostasis promoting the development of an ecological niche for Clostridium difficile and of the modulation of immune response. Moreover, the intestinal dysbiosis can promote a proinflammatory environment, whereas Clostridium difficile itself modulates the innate immunity through both toxin-dependent and toxin-independent mechanisms. In this narrative review, we discuss how the intestinal microbiota modifications and the modulation of innate immune response can lead to and exacerbate Clostridium difficile infection.

Locally injected Infliximab ameliorates murine DSS colitis: Differences in serum and intestinal levels of drug between healthy and colitic mice

BACKGROUND Infliximab is effective in human and murine IBD, but its pharmacodynamic is still poorly known. The aim of this study was to assess the affinity of infliximab to murine TNF-α, its role in murine colitis when administered intra-rectally and its levels in the blood, gut mucosa and stool of healthy and sick mice. METHODS An ELISA kit was built in order to assess the affinity of infliximab to human or murine-TNF-α. Human IgG were used as controls. DSS model of colitis on C57BL/6 mice was used to assess clinical efficacy of infliximab administered intravenously or by enema. Stool, serum and colon samples were collected to assess infliximab levels and histology for Rachmilewitz score. RESULTS Infliximab showed a good affinity both for human-TNF-α and murine-TNF-α. In DSS colitic mice infliximab ameliorated the severity of colitis, regardless of the administration route. In comparison with colitic mice, healthy mice displayed higher serum and mucosal infliximab levels, while detectable levels of infliximab were found in faeces, particularly in colitic mice. CONCLUSION Our data support murine models to study infliximab pharmacokinetics and dynamics. Measurable levels of infliximab can be found at different concentrations in blood, intestinal mucosa and stool from healthy and sick mice, thus infliximab pharmacokinetics could have a major impact in human IBD.

Gelatin tannate ameliorates acute colitis in mice by reinforcing mucus layer and modulating gut microbiota composition: Emerging role for 'gut barrier protectors' in IBD?

Background Gelatin tannate, a gelatin powder containing tannic acids, is commonly employed as an intestinal astringent. Neither information nor animal model exist to confirm its efficacy or unravel mechanisms of action. Objective To evaluate the action of gelatin tannate in murine dextran sodium sulphate (DSS)-induced acute colitis. Methods Mice were exposed to DSS and received gelatin tannate by gavage. At sacrifice, colon histological degree of inflammation was assessed. Stool samples were cultured for microbiological analysis. Colon samples were analysed by two-photon confocal microscopy and atomic force microscopy. Elisa was performed on murine serum to assess lipopolysaccharide and peptidoglycan levels. Results Gelatin tannate treatment reduced disease activity, bodyweight loss, and preserved colonic length. It produced a decrease in the amount of enterobacteria and enterococci. At confocal microscopy, intestinal samples from healthy and treated mice displayed similar structure in mucus layer thickness and composition; samples from placebo group had no mucus layer or a thinner stratus. Atomic force microscopy confirmed these findings. Treated mice showed lower blood LPS levels vs. control. Conclusions Gelatin tannate decreased the severity of colitis. Acting as a gut barrier enhancer, it re-establishes gut homeostasis by recovering intestinal permeability and mucus layer integrity in gut mucosa and by modulating microbiota composition.

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology.