Periodic Reporting for period 4 - INVADERS (Mucus-Penetrating Microbiota: Characterization, Mechanism and Therapeutic in Metabolic Disease)
Periodo di rendicontazione: 2024-04-01 al 2024-09-30
Humanity is facing an epidemic of inter-related metabolic disorders, including obesity, insulin resistance, hyperglycemia, hyperlipidemia, and hepatic steatosis, that altogether have major impact on the promotion of cardiovascular diseases. The increasing incidence of these complex metabolic disorders and their highly morbid, chronic and costly downstream diseases threatens to overwhelm the world’s health care systems and economies, making it a top public health priority in dire need of investigation.
The intestinal tract is inhabited by a large and diverse community of bacteria, collectively referred to as the intestinal microbiota. When stably maintained at an appropriately safe distance from the epithelial cell monolayer, the microbiota provides important benefits to its host. However, disturbance of the microbiota-host relationship, promoted by genetic or non-genetic factors, can alter intestinal homeostasis and drive chronic low-grade intestinal inflammation, ultimately leading to metabolic abnormalities. We previously reported that a ubiquitous class of food additives, emulsifiers, detrimentally impact the microbiota resulting in its encroachment into the mucus layer that associated with low-grade inflammation and development of metabolic disorders.
The central goal of this proposal is to investigate the hypothesis that bacteria that penetrate the inner part of the mucus layer, referred as invaders, promote development of metabolic alterations.
We herein propose to identify mucus-invaders, in preclinical models and clinical conditions, and investigate mechanisms by which they promote inflammatory and metabolic abnormalities. Furthermore, we propose to define original approaches to modulate the intestinal microbiota in order to counteract microbiota encroachment and protect against associated metabolic abnormalities.
The intestinal tract is inhabited by a large and diverse community of bacteria, collectively referred to as the intestinal microbiota. When stably maintained at an appropriately safe distance from the epithelial cell monolayer, the microbiota provides important benefits to its host. However, disturbance of the microbiota-host relationship, promoted by genetic or non-genetic factors, can alter intestinal homeostasis and drive chronic low-grade intestinal inflammation, ultimately leading to metabolic abnormalities. We previously reported that a ubiquitous class of food additives, emulsifiers, detrimentally impact the microbiota resulting in its encroachment into the mucus layer that associated with low-grade inflammation and development of metabolic disorders.
The central goal of this proposal is to investigate the hypothesis that bacteria that penetrate the inner part of the mucus layer, referred as invaders, promote development of metabolic alterations.
We herein propose to identify mucus-invaders, in preclinical models and clinical conditions, and investigate mechanisms by which they promote inflammatory and metabolic abnormalities. Furthermore, we propose to define original approaches to modulate the intestinal microbiota in order to counteract microbiota encroachment and protect against associated metabolic abnormalities.
Over the full course of the project, we have made significant and transformative progress on all proposed scientific objectives, yielding advances that have substantially impacted both basic understanding and translational perspectives in the field of host-microbiota interactions in metabolic and inflammatory diseases.
Aim 1.
We initially established ex vivo assays to systematically evaluate the impact of dietary emulsifiers on human microbiotas, leveraging both in vitro microbiota models (MBRA) and gnotobiotic mice. Our pioneering work revealed that exposure to emulsifiers consistently alters the compositional and functional landscape of human microbiotas. Notably, we identified both susceptible and resistant microbiota phenotypes and began to elucidate the underlying molecular determinants and bacterial players underlying this differential response. Advanced techniques, including laser capture microdissection and high-resolution spatial profiling, allowed the robust identification and isolation of mucus-invading bacteria in both preclinical models and human biopsies. These efforts led to the first comprehensive catalog of mucus-infiltrating "invaders" in the context of metabolic disorders, several of which were found to possess heightened pro-inflammatory and metabolic disease-promoting capacities when transplanted into germ-free.
Aim 2.
Building on our initial discoveries, we successfully demonstrated that both Akkermansia muciniphila supplementation and immunization strategies targeting flagellin represent viable modalities to block microbiota encroachment. Our studies—culminating in multiple high-profile publications—established that A. muciniphila can prevent emulsifier-induced metabolic derangement by preserving mucosal barrier integrity and modulating local immune responses. Similarly, our translational work revealed that vaccination against flagellin not only reduced bacterial penetration into the mucus but also conferred substantial protection against both colitis and obesity in preclinical models. Mechanistic insights indicated that these interventions restructure microbiota spatial organization and function, and our findings provide a solid foundation for future microbiome-targeted therapies.
Aim 3.
We have now completed extensive recruitment, sample collection, and spatial-microbiome profiling from well-characterized human cohorts with metabolic disease, and innovative clinical trials assessing the effect of emulsifier consumption in humans (as recently published in Gastroenterology 2022). This work confirmed our initial hypotheses drawn from animal models: patients with metabolic syndrome harbor an increased burden of mucus-infiltrating bacteria. Longitudinal and interventional analyses have provided strong evidence that these "invader" taxa are associated with disease severity and can actively drive metabolic inflammation and barrier disruption. For the first time, we isolated and characterized human-derived bacteria that, when transferred to recipients, recapitulate key aspects of metabolic pathophysiology, providing a crucial proof-of-concept for causality.
Exploitation and Dissemination
The results of this project have been widely disseminated across >60 peer-reviewed publications—including in prestigious journals (see publication list attached)—as well as through invited plenary talks, major symposia, and extensive outreach activities targeting clinicians, regulatory bodies (e.g. EFSA, WHO), and the general public. Several key data sets—such as those establishing inter-individual variability in microbiota response to emulsifiers and personalization of fiber interventions—have already been taken up in translational clinical protocols and dietary guideline discussions. Intellectual property has been protected through patents.
Aim 1.
We initially established ex vivo assays to systematically evaluate the impact of dietary emulsifiers on human microbiotas, leveraging both in vitro microbiota models (MBRA) and gnotobiotic mice. Our pioneering work revealed that exposure to emulsifiers consistently alters the compositional and functional landscape of human microbiotas. Notably, we identified both susceptible and resistant microbiota phenotypes and began to elucidate the underlying molecular determinants and bacterial players underlying this differential response. Advanced techniques, including laser capture microdissection and high-resolution spatial profiling, allowed the robust identification and isolation of mucus-invading bacteria in both preclinical models and human biopsies. These efforts led to the first comprehensive catalog of mucus-infiltrating "invaders" in the context of metabolic disorders, several of which were found to possess heightened pro-inflammatory and metabolic disease-promoting capacities when transplanted into germ-free.
Aim 2.
Building on our initial discoveries, we successfully demonstrated that both Akkermansia muciniphila supplementation and immunization strategies targeting flagellin represent viable modalities to block microbiota encroachment. Our studies—culminating in multiple high-profile publications—established that A. muciniphila can prevent emulsifier-induced metabolic derangement by preserving mucosal barrier integrity and modulating local immune responses. Similarly, our translational work revealed that vaccination against flagellin not only reduced bacterial penetration into the mucus but also conferred substantial protection against both colitis and obesity in preclinical models. Mechanistic insights indicated that these interventions restructure microbiota spatial organization and function, and our findings provide a solid foundation for future microbiome-targeted therapies.
Aim 3.
We have now completed extensive recruitment, sample collection, and spatial-microbiome profiling from well-characterized human cohorts with metabolic disease, and innovative clinical trials assessing the effect of emulsifier consumption in humans (as recently published in Gastroenterology 2022). This work confirmed our initial hypotheses drawn from animal models: patients with metabolic syndrome harbor an increased burden of mucus-infiltrating bacteria. Longitudinal and interventional analyses have provided strong evidence that these "invader" taxa are associated with disease severity and can actively drive metabolic inflammation and barrier disruption. For the first time, we isolated and characterized human-derived bacteria that, when transferred to recipients, recapitulate key aspects of metabolic pathophysiology, providing a crucial proof-of-concept for causality.
Exploitation and Dissemination
The results of this project have been widely disseminated across >60 peer-reviewed publications—including in prestigious journals (see publication list attached)—as well as through invited plenary talks, major symposia, and extensive outreach activities targeting clinicians, regulatory bodies (e.g. EFSA, WHO), and the general public. Several key data sets—such as those establishing inter-individual variability in microbiota response to emulsifiers and personalization of fiber interventions—have already been taken up in translational clinical protocols and dietary guideline discussions. Intellectual property has been protected through patents.
Aim 1.
We identified specific bacterial taxa and strains that penetrate the inner mucus layer in response to dietary and environmental factors, especially processed food additives such as emulsifiers. Mechanistic studies detailed how these microbes cross mucus barriers, pinpointing bacterial factors and host interactions, as well as their distinct pro-inflammatory/metabolic profiles. We provided the first causal demonstration that invader-enriched consortia transfer can induce obesity, metabolic syndrome, and inflammation in naive mice.
Aim 2.
Our finding that Akkermansia muciniphila supplementation and flagellin-based vaccination both protect against mucus disruption and its metabolic consequences opens new microbiome-modulating strategies. Anti-encroachment approaches were validated in animal models and translated into early-phase clinical research.
Aim 3.
We showed that spatial microbiota alterations in metabolic syndrome patients predict clinical severity and dietary response. IP-protected biomarkers are being developed for stratifying those most likely to benefit from barrier-protective strategies. Our work advances the blueprint for precision nutrition and microbiome-personalized interventions in health and disease, as recognized by recent reviews and editorials.
Summary:
The ERC project has generated major insights into the diet-microbiota-barrier axis in inflammatory and metabolic diseases. Results are widely disseminated and translated, with a legacy in both fundamental science and intervention development.
We identified specific bacterial taxa and strains that penetrate the inner mucus layer in response to dietary and environmental factors, especially processed food additives such as emulsifiers. Mechanistic studies detailed how these microbes cross mucus barriers, pinpointing bacterial factors and host interactions, as well as their distinct pro-inflammatory/metabolic profiles. We provided the first causal demonstration that invader-enriched consortia transfer can induce obesity, metabolic syndrome, and inflammation in naive mice.
Aim 2.
Our finding that Akkermansia muciniphila supplementation and flagellin-based vaccination both protect against mucus disruption and its metabolic consequences opens new microbiome-modulating strategies. Anti-encroachment approaches were validated in animal models and translated into early-phase clinical research.
Aim 3.
We showed that spatial microbiota alterations in metabolic syndrome patients predict clinical severity and dietary response. IP-protected biomarkers are being developed for stratifying those most likely to benefit from barrier-protective strategies. Our work advances the blueprint for precision nutrition and microbiome-personalized interventions in health and disease, as recognized by recent reviews and editorials.
Summary:
The ERC project has generated major insights into the diet-microbiota-barrier axis in inflammatory and metabolic diseases. Results are widely disseminated and translated, with a legacy in both fundamental science and intervention development.