Microbiota, leaky gut syndrome and gut-related diseases

1) Good morning and thank you for attending this im7th International Immunonutrition Workshop in Carovigno.I would like to thank Prof. Jirillo that invited me to lecture on the importance of gut microbiota and gut-related diseases.
2) Summary
 3) Human microbiome research has the potential to transform the practice of medicine, fundamentally shifting the ways in which we think not only about human health, illness, and disease, but also about clinical practice and public health interventions. What are the new insights and main components of the intestinal ecosystem and its functions?
 4) Let’s start from the microbiome. Analyzing new scientific studies the question arise on “how much are we only human”. Our gut contains more than 1000 different species that compose a complex eco-system.
 5) Humans can be considered meta-horganisms where the number of symbionts are 10-fold greater than human cells, metabolically and immunologically integrated, with a more than 1 kg active biomass
 6) Symbionts colonize many sites of human body, creating a complex network of micro-ecosystems each characterized by different organisms, exchanges of matter and energy; Such micro-ecosystems can be found on our skin, genitourinary tract, mouth, pharinx and all gastrointestinal tracts. They constitute potential therapeutical targets for innovative bio-therapeutical agents that will be listed later.
7) Human microbiota starts from a sterile environment guaranteed by the mother placental barrier. Earlier research suggested that term fetuses are sterile and that the initial bacterial colonization of the newborn GIT occurs only after the baby transits through the birth canal. However, recent studies have demonstrated that the colonization and/or contact of the fetus with the maternal GIT microbiota may start in utero
8) Arumugam and others studied human microbiome through faecal metagenomes of individuals coming from 4 countries. They identified three robust clusters (then called enterotypes ) that are not nation or continent-specific. Enterotypes are identifiable by the variation in the levels of one of three genera: Bacteroides, Prevotella and Ruminococcus
9) Real inoculation of the baby body goes on through the contact of the mother skin and faecal microbiota, through the mothers nipples during breast-feeding and with the contact with other relatives skin. But it takes some months before the real native core microbiota is established. This happens in a time range that goes from 4th to 36th months after birth. This core microbiota will be a unique fingerprint of the individual although it could be modified during the lifespan.
 10) There are two fundamental concepts we need to consider to fully understand gut-ecosystem: The concept of intestinal ecosystem’s functional level and the concept of physical and physiological gut barrier. The intestinal ecosystem is composed by 4 different functional levels: intestinal lumen, mucus layer (internal and esternal, endotelium made by a single layer of enterocytes and the submucosa, where most of the enteric immunitary and nervous system is located.
11) The human intestinal mucosa is composed of a simple layer of columnar epithelial cells with different characteristic for each tract, as well as the underlying lamina propria and muscular mucosa. The tight junction, a component of the apical junctional complex, seals the paracellular space between epithelial cells.
12) Tight juntions are one of the pillars of gut selective permeability, closing gaps between endothelial cells and directing nutrients and big molecules through intracellular absorption pathays. Tight junctions consist of several intracytoplasmic or intra- or extracellular proteins, such as occludine and claudine, and another third category: JAM (junctional adhesion molecules). Finally, the intracytoplasmic actin fibers ensure the integrity at the apical side of the enterocyte.
13) We can say that gut permeability is the complex result of different components and it is a highly regulated process
14) Our symbiont bacteria, viruses and fungi lives in the lumen and in the mucus layer. Sometimes traslocation in the submucosa occurs leading to pathological presence of bacteria in blood and other tissues like pericardium. Mucus layer is composed by a hard base, 50 μm thick, firmly adherent to the epithelial cells and a thinner outer layer which is the usual habitat of the commensal flora. The inner mucus layer is dense and does not allow bacteria to penetrate, thus keeping the epithelial cell surface free from bacteria. Other bacteria can be found in the lumen content and are the major components of faecal mass.
15) Let’s see what are the main effects of gut microbiota on human health. Gut entothelium and mucus layer create a physical and physiological barrier toward the outside (that is inside us). Microbiota is responsible for developement of immunocompetence and tolerance. It is responsible for synthesis of vitamins and metabolic modulators. Metabolic/Trophic function, Drug methabolism and Behavior conditioning;
16) Development of Native Core Microbiota and development of immune systems goes together during the first 2-3 years of life
17) In early life, composition of the microbiota profoundly influences the development and maturation of the gastrointestinal tract (GIT) mucosa and GI ecosystem. Therefore, strategies to manipulate the microbiota during infancy may prevent development of some diseases later in adult life. After vaginal birth, the colonization of the neonate GIT continues through contact with maternal feces and vaginal bacteria, leading to a relatively simple microbial community that is influenced by feeding type (breast vs. formula feeding). Maternal GIT microbiota, vaginal microbiota, and breast milk composition are influenced by maternal diet. Alterations of the maternal GIT microbiota composition via supplementation with probiotics and prebiotics have been shown; however, transfer of these benefits to the offspring remains to be demonstrated. 18) The intestines contain immune cells that are important for maintaining the health of the digestive tract. The GALT and the LP are where the majority of the immune cells reside. During conditions of health, the GALT supports the development of anti-inflammatory adaptive immune responses towards harmless antigens originating from food and the intestinal microbiota. A delicate homeostasis is created and allows us to live with our intestinal flora without immune reactions to the food we eat.
19) It is estimated that 60% of the immune cells in the submucosal zone lie under the epithelium. These cells largely consist of 2 important organization types: constantly active cells, such as the transepithelial M-cells, and submucosal macrophages which, as sensors, are permanently susceptible to signals from the microbiota.
 20) The gut makes a huge investment in maintaining an extensive and highly active immune system. Specialized M-cells constantly transport gut bacteria and antigens from the gut lumen into the lymphoid tissue. Dendritic Cells in the Lamina propria reach through epithelial cells and also sample gut bacteria and antigens. Potentially tissue-damaging T-cell responses may be inhibited by immunosuppressive cytokines and regulatory T cells.
21) How the host-gut microbiota balance is mantained? And what triggers unbalances?
 22) Let’s start from the causes and give a look to the most common triggers that may act over a genetic predisposition. We saw that genetic predisposition and the vertical transfer of the core microbiota from the mother can strongly influence the microbial communities of the subject. Life conditions can trigger substantial changes of the microbiota and expression of the microbial flora: diet, physical exercise, infections, stress, environmental toxines, for example. Any of this changes have as end result the breaking of the symbiontic link between the human and the symbionts.
23) Microbes-gut link alterations leads to dysbiosis. Dysbiosis can be considered the second step in etiopathogenesis of many chronical diseases, even those apparently not linked to gut health.
24) Dysbiosis is the intermediate step from mirobiota alteration toward leaky gut and its consequences.
25) Many diseases are associated to unbalanced gut microbiota and more and more are joining the list. Sometimes the link between the disease and the alteration of gut ecosystem is not direct and fully understood.
26) We should not forget that gut microbiota is composed not only by bacteria. Fungal and viral community importance has been underestimated for too long. Bacterofages in the future could be used to control and influence bacterial community. Dysbiosis of fungal microbiota is associated with inflammation and could lead to fungal overgrowth as it happens in Candidosis
 27) Candidosis is commonly considered and treated as a simple parassitosis but we should re-consider the pathogenetical role of minor fungal microbioma unbalances for the general conditions of gut microbiota and human health
28) This slide shows some complex interactions network undergoing life of symbiontic communities. Here we see how Saccharomices boulardii can interfere with the activity of pathogenics microbes with different mechanisms: anti-toxinic effect, preservation of tight juntions, modulation of inflammatory signals, interference with adherence of pathogens. Dysbiosis is a strong risk factor for GI infections and associated with after infection complications.
29) This study of Spiller (2009) shows that when gastrointestinal infections occur the unbalance can proceed toward recovery or induce a post-infectious IBS. Post infectious IBS accounts for at least 5 to 10% of total IBS and its consequences can last for years
30) Potentially, probiotics maintain or restore gut micro-ecology during or after antibiotic treatment through: receptor competition, competition for nutrients, inhibition of epithelial and mucosal adherence of pathogens. introduction of lower colonic pH favoring the growth of nonpathogenic species, stimulation of immunity, or production of antimicrobial substances. Reinforcing gut microbiota can help preventing acute GI infections like traveler diarrhea and Antibiotic Associated Diarrhea and some organisms have better evidences of preventive activity: (Lactobacilli among bacteria and Saccharomices among fungi).
31) Microbiota of IBS and healthy subjects are significantly different
 32) Some groups of strains dramatically decrease, like bacteroidetes, other slightly increase. A Firmicutes icrease of 5% it is shown in this 2011 study on IBS patients.
 33) Unbalanced microbiota together with a leaky epithelial barrier allows bacteria and/or bacterial products access to the submucosal compartment where mast cells and immune cells (lymphocytes) are activated, releasing mast cell proteases, chemokines, and cytokines, which can activate sensory neurons. This, in turn, can result in local reflexes that affect motor and secretory functions or lead to enhanced visceral sensation centrally.
34) This extraordinary picture (thanks to prof Barbara of Bologna university) shows how Mast Cell-Nerve Vicinity Correlates with Abdominal Pain in IBS. Abdominal pain is a very common symptom of intestinal functional disorders classified under Roma 3 criteria
 35) We know for sure that composition, distribution and bio-diversity of intestinal flora is completely alterated in all chronic inflammatory gut diseases, in IBS and many gastrointestinal functional disorders.
 36) We can affirm that bio-diversity of intestinal flora is always reduced in gastrointestinal tract disorders with proliferation of some pathogenic strains and reduction of Bacteroidetes prevalence.
 37) LGS and Dysbiosis can interfere with metabolic processes, in the first place disturbance of the carbohydrate-lipid metabolism. Progression from triggers to dysbioses , followed by a disturbed barrier function, which in its turn leads to a hyperpermeability, specifically for bacterial antigenes, endotoxins, composed of lipopolysaccharides (LPS), which pass through the barrier via disrupted junctions and cause endotoxemia. This bacterial endotoxemia involves a disturbance of the metabolic signal cascade, specifically GLP1 and GLP2, the carbohydrate regulation, modification of the lipogenesis and of fat storage. This leads to low grade inflammation and clearly contributes to the development of type II diabetes.
 38) The integrity of the intestinal barrier is also linked with overweight and obesity.
39) Hyper-permeability is a trigger which changes the so-called ponderostate and the increases fat storage for the same calorie intake. IHP enhances adipocyte signals into energy storage, which leads to overweight, hyperplasia and hypertrophy of the adipocytes.
 40) Finally, there is a link between HPS and immune diseases. We know the reason for this: hyperpermeability allow the antigenes to pass through, they start overactivating the submucosal immune system, which creates inflammatory signals from a distance, which activate super antigenes and many clones, applied in autoimmune pathology. This is well identified in inflammatory diseases, psoriasis and in many autoimmune diseases. Theoretically, the antigenes are filtered by the tight junctions, but in case of HPS, the antigenes and large molecules pass through the barrier.
 41) Another disorder linked to microbiota that we should not forget is chronic fatigue syndrome. Fibromyalgia is often linked with IHPS. Normalization of the LGS is clinically associated with early and improved remission scores.
 42) Finally, in neuropsychiatric disorders an increased IHP involves inflammatory factors which release cytokines in de glia of the brains and particularly activate enzymes which cause tryptophan or thyrosin to degradate. This way, amino acids are diverted from their metabolic task, that is the synthesis of neurotransmitters, to pathogenic pathways, more specifically these of kineurine. In short, LGS involves a decrease of the neurotransmitters production and neurotoxicity. In the last decade, articles have been published which show a clear connection between LGS and clinical signs of depression, major and chronic depression, which do not react well to the classical antidepressant treatment
43) Let’s give a look at the liver. Small intestinal bacterial overgrowth (SIBO), present in a variety of liver diseases, and dysbiosis lead to an enhanced release of pro-inflammatory cytokines. Increased intestinal permeability, also well described in liver disease, enhances translocation of bacteria, endotoxin, or pro-inflammatory products such as LPS from gram-negative bacteria, which reach the liver through the portal vein or, in the presence of portal-systemic shunting, access the systemic circulation directly.
44) Gut microbiota unbalances are present at all levels in the progression of non alcholic liver disease to nash to cirrosis up to the terminal stage of liver carcinoma.
45) Science many times proceeds on the shoulders of visionary individuals that can foresee a glimpse of the general picture. It was like that with the «Leaky gut syndrome», that was conceived far before the real discovery of permeability mechanisms. The “leaky gut syndrome» spent many years in the backstage of science until a italian researcher Prof. Alessio Fasano discovered by chance, the zonulines and opened the way of understanding mechanisms of gut permeability regulation.
 46) Currently we know that some bacterial toxin, alcol, non steroideal drugs, and many nutrients can modulate gut permeability ; so we have a strong confirm that diet is one of the best weapons to mantain and restore gut balance. Nutrients can influence: mucus production, endotelial integrity, junctions stability, microbial balance
47) How? Modulating ATP production, tight junctions protein production, membrane fluidity and microbic trophysm;
 48) Nutrient’s ability to fight digestive tract disorders depends on how much the balance has been corrupted and the disease chronicized. More severe unbalances can benefit of a integrative nutritional approach and specific diets
 49) In this proof-of-concept study, conducted at Rome University, a combination of pre- and probiotics (L. acidophilus NCFM, B lactis Bi-07), vitamins, minerals, antioxidants and anti-inflammatory agents (Nutrimonium®) was added to the common diet of 33 subjects with D-IBS or M-IBS and increased intestinal permeability (measured with 51CrEDTA Test) for 60 days. After the treatment, each patient repeated the intestinal permeability test with 51CrEDTA. Global health state with a slightly modified version of EQ-5D VAS (0-100 mm) was evaluated at baseline and after the treatment.
50) Restoring the impaired gut barrier may be challenging, especially since there are several therapeutic targets to hit, as gut microbiota, intestinal mucus, intestinal immune cells, tight-junction function, et cetera. A multimodal approach, with a combination of different healing agents, seems to be effective both in improving intestinal permeability and in ameliorating symptoms.
 51) I would like to mention the epigenetic role of nutrients that is usually underestimated and can infulence health of offspring. Vitamine D has a pleiotropic effect and acts on more than 2.000 genes not only on bone and muscles but on every bodly district. Its activity can be prolonged vertically to the next generation.
52) New frontiers of clinical intervention on microbiota includes faecal transplants. This new technique showed good results on antibiotic resistant infections from Clostridium difficile.
 53) Then the technique was applied to other digestive tract disorders like ulcerative colitis, Ibs and later to other systemic diseases like chronic fatigue and multiple sclerosis. At the moment more than 300 centers in the world have ongoing applied researches on Faecal transplants or microbiome transplantation.
54) FDA is now regulating the procedure in order to guarantee safety for the patients and currently in Italy the procedure is considered for the regulatory aspects a «tissue transplantation»
 55) Biotherapy is currently a functional approach to rebalance gut microbiota together with lifestile intervections (diet and physical excercise)
56) GUT Microbiota is a complex organ enrolled in crucial tropho-metabolic, barrier and immunological functions, Balance between different bacterial species and between bacteria and host strongly influences human health. An unbalanced gut microbiota (Dysbiosis) is involved in the pathogenesis of most GI disorders. Microbiota assessment and remodulation are key strategies for restablishment of a correct host-bacteria cross talk.