2.1. Nutrients, Food Additives, Bugs and Us
Many environmental factors impact the gut microbiome. Geography, lifecycle, mode of delivery, infant feeding, stress, exercise, hygiene, infections, pharmaceuticals and food are some examples [9
]. Diet has emerged as one of the most relevant factors in influencing the gut microbiome. In reality, nutritional customs have a critical impact on human health, affecting an individual’s risk for various chronic diseases. The ‘westernization’ of worldwide eating and lifestyle modifications is associated with an increasing rate of cardiovascular, cancerous, metabolic and allergic diseases. Moreover, an individual’s lifestyle selection can markedly affect the progression and manifestation of autoimmune diseases [10
]. In light of these outcomes, it is logical that the search for alternative therapies to combat such diseases would include inquiries into lifestyle modifications [14
]. Nutrition, from as early as in utero, through the neonatal period, and up to adulthood, has a profound effect on the shape and trajectory of our intestinal microbiome. New genetic technologies and bioinformatics reveal the immense influence the enteric microbiome has on our early development, intestinal homeostasis, behaviors and susceptibility to and recovery from human diseases. When nutrients enter the human intestinal bioreactor much of human physiology is changed, including major effects on the gut microbiota composition, diversity and metabolomic product secretion.
Significant changes in the gut microbiome have been primarily associated with the intake of fiber from fruits, vegetables and other plants. In this regard, De Filippo et al., compared the gut microbiome of African to Western children [15
]. Upon comparison of a vegetarian diet (low fat, low animal protein, abundant in starch, plant polysaccharides and fiber) to a Western diet (plentiful in animal protein, sugar, starch and fat, but short in fiber), relevant discrepancies were depicted in the four major phyla: Actinobacteria
increased in the African group while Firmicutes
were more plentiful in the European branch of the study. Interestingly, the African children exclusively harbored short fatty acid (SCFA)-producing bacteria that use xylen, xylose and carboxymethylcellulose, thus producing four times more SCFA. SCFA was described as an anti-inflammatory at the gut levels [16
]. De Filippo et al., suggested that the African children’s microbiome co-evolved with their diet to assist with energy harvest by producing higher levels of SCFA [15
]. When the fecal flora of adult vegetarian/vegan subjects were compared to an omnivorous diet, the first group disclosed a lower microbial count of Bifidobacterium
, Escherichia coli
and lower pH, compared to the second group [17
]. The highly enriched indigestible carbohydrate and fiber diet of the vegetarian/vegan subjects is the origin of the higher SCFA content, resulting in the lower stool pH. It is well known that dietary fibers are related to the high production of SCFAs by the gut microbiota and, in turn, with the induction of immune tolerance [18
Despite our growing knowledge, less is known about the interplay of nutrients and gut microbiota in immune-mediated diseases. Dietary milk, carbohydrates, fats, protein, fiber, fruit, vegetables, animal proteins, sodium chloride and aluminum [19
] were investigated as potential inducing factors in Crohn’s disease [20
]. Cow milk, fruit and berry juices, and n3-polyunsaturated fatty acids were explored in type one diabetes. Even the incidence of multiple sclerosis was positively associated with the consumption of milk, animal fat and meat, total energy intake and resulting obesity [21
Contrary to disease induction, multiple nutrients were suggested as acting as anti-inflammatory agents, and thus might have protective or preventive effects. These include, at least in rheumatoid arthritis, fish and primrose oils, black cumin, fenugreek, licorice, coriander, tomato, carrot, sweet potato, broccoli, green tea, rosemary, hazelnut, walnut, wheat germ and dates. In celiac disease (CD), long chain ω-3 fatty acids, plant flavonoids and carotenoids appeared to modulate oxidative stress, inflammatory mediators and gene expression. More so, phytonutrients such as lycopene, quercitine, vitamin C and tyrosol were suggested to protect against the cytotoxic effects of gliadin. Nevertheless, the majority of investigations have been equivocal or circumstantial and do not yet validate any of these nutrients as causal factors [22
]. It should be noted that those nutritional epidemiological studies have not integrated microbiome stool analysis, therefore the role played by a specific nutrient on the microbe’s composition and function is far from being elucidated.
It seems that the dietary exposome is far from clarifying the microbiome behavior and the human reactome.
In addition to food and nutrients, the industrial food processing additives also affect enteric eco-events. Glucose, salt, emulsifiers, organic solvents, gluten, microbial transglutaminase, and nanoparticles, which are increasingly used in industrial food processing, impact microbiota composition. They are also considered to breach the enteric tight junction (TJ) integrity and are potential inducers of the autoimmune cascade [10
]. More so, microbial transglutaminase (mTg) that functionally imitates the tissue transglutaminase (tTg) (the autoantigen of CD), was lately shown to be immunogenic in celiac disease patients [12
A distinctive place should be dedicated to gluten, a universally consumed nutrient. Considering the analogous increase in world-wide gluten intake and chronic, non-infectious diseases incidences, it is proposed that gluten might have biologically detrimental effects [24
]. In fact, gluten has multiple side effects, affecting human health, characterized by gluten dependent digestive and extra-digestive signs and complaints that may be arbitrated by immunological reactions and primed by gastrointestinal inadequacy. In the enteric lumen, it affects the microbiome composition and diversity and enhances intestinal permeability. Gluten is immunogenic and cytotoxic, pro-inflammatory and drives the innate and adaptive immune systems. On the cellular level it augments apoptosis, decreases viability and differentiation and influences nucleic acid and glycoprotein synthesis. It has many systemic effects as a pro-inflammatory, and affects epigenetic pathways. On therapeutic level, a gluten-free diet, in certain non-celiac autoimmune diseases patients (type one diabetes, rheumatoid arthritis, multiple sclerosis, psoriasis, autoimmune hepatitis and thyroiditis) may be helpful to reduce gluten’s disadvantageous effects [24
]. It appears that early diagnosis of CD, on gluten withdrawal, is protective for other associated autoimmune diseases [25
], an effect not seen on late CD diagnosis [26
]. Most recently, even in the veterinarian world, a gluten-free diet improved the epileptoid cramping syndrome in Border Terrier dogs [27
Finally, in a more optimistic approach, based on the influences of the Western lifestyle on adiposity, glucose metabolism, oxidative stress and inflammation, bacterial strains and their metabolic products that are beneficial under this lifestyle were selected as the most promising probiotic isolates [28
]. It seems that we are beginning to unravel the importance of the microbial key components that might hinder the evolution of human chronic diseases.
In summary, depleted microbial biodiversity of the gut microbiota in people consuming a Western diet is linked to increasing incidence of obesity, coronary vascular disease, stroke, metabolic syndrome, autoimmune diseases as well as an increased risk of malignancies. Improving dietary habits towards a long-term consumption of a “healthy” versus an “unhealthy” diet, will impact substantially the microbiota/dysbiota balance. The popular sentence, “we will be what we eat or what we were fed” [29
] should include the microbiome as a gate keeper between food and mankind’s health. The association between this agrarian-based diet with specific bacterial taxa, a surge in microbial richness, at the taxonomic and genetic levels, and improved health compared to Western diets has been consistently established. In view of the food effects, it makes sense that the quest for nutritional therapies to abate the initiation and progression of chronic diseases would include explorations into more holistic lifestyle changes.
After the description of the dietary influence on the microbiota profile, the following will expend on the microbial reactome, setting the stage for the gut-microbial–brain axis.
2.2. Microbial Metabolome as Mobilome
Although food affects the compound and diversity of the intestinal microbiome, more significant are its impacts on the metabolome. The enteric ecosystem, overloaded with microorganisms and compacted immune system cells can be viewed as an isolated compartment on its own. Under dysbiotic states, however, the microbiome/dysbiome equilibrium is changed and results in an abnormal interaction between the bugs and us. Some enteric microbiome dwellers have been associated with specific chronic human conditions including autoimmune diseases and food allergies [30
]. Changing a single microbial species and/or the entire commensal community can modify the outcome of a specific autoimmune disease due to the imbalance of detrimental/protective immune responses [32
]. A list of specific bacterial species, related to defined animal models of autoimmune diseases and their functions, in relation to disease development, was most recently reported [30
]. However, no phenotype–microbial relationship or cause-and-effect relationship, to our ample knowledge, was established for any of those chronic conditions.
The gut microbiota produce endless and constantly changing metabolites that impact host physiology and susceptibility to disease, however, the causative molecular events remain largely unknown. Nutrition-induced alterations in the composition of the enteric microbiota can modulate the recruitment of regulatory versus effector immune responses at the intestinal level and ameliorate the health outcome. Prebiotics, probiotics and dietary fiber are the main means for prophylactic and therapeutic intervention against intestinal inflammation [33
]. Most recently, the relationships between diet, the microbiota, metabolomics, and gene function was further clarified on an animal model and in CD [16
]. It was shown that bacterial colonization modulates global histone acetylation and methylation in various host tissues in a diet-dependent manner: intake of a “Western-type” diet deprives many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Supplementation of germ-free mice with SCFAs, a major metabolite of gut microbial fermentation, was sufficient to renew chromatin modification status and transcriptional reactions associated with colonization [34
]. In fact, the most studied metabolic products, that have beneficial effects, are SCFAs. SCFA-mediated signaling pathways are vital for enteric bacterial communication with the host. They regulate immune functions, intestinal hormone production, lipogenesis and many more luminal and systemic influences [16
]. Interestingly, butyrate promotes colonic health and helps to prevent cancer [35
Acetate, propionate, butyrate, and pentanoate, with two, three, four, and five carbon atoms, respectively, are SCFAs, largely made by bacterial fermentation of non-digestible polysaccharides like starches and fibers in the colonic lumen. After being absorbed by the gross intestine epithelium, where the preferred fuel source of colonocytes is butyrate, they enter the bloodstream through the portal vein of the host and/or the distal colon. Then, they are distributed to peripheral organs where they are taken up, metabolized and used in multiple cellular responses [16
]. Contrary to the variability in the loss of diversity of the microbiome repertoire in autoimmune diseases, less is known about the source of the luminal metabolites. SCFA production is greatly related to food, but the specific microbial species rate of SCFA output is yet unknown. A metabolic signature in the lumen and stools of specific and total SCFAs, in CD, for example was described [16
]. However, a long-term gluten-free diet did not completely restore the microbiome in the metabolome of CD children [36
]. One of the actions of the luminal SCFAs is the increase of mucosal immune tolerance by the activation of G-protein-coupled receptors and the subsequent activation of T regulatory cells [37
Despite the proposal that probiotics (e.g., Lactobacillus and Bifidobacterium) may alter the metabolism in the colon by enhancing the production of SCFAs, we are far from “rebiosis” or the answer to the question: how can bacterial diversity and functionality be restored in dysbiotic or in pathobiotic circumstances?
However, SCFAs are not the only metabolic products. The list of diet-dependent, microbial-originated metabolic products that improve or deteriorate human health is constantly increasing [38
]. Food rich in phosphatidylcholine is a main source of choline. Catabolism of choline by the gut microbiome induces the formation of gas and trimethylamine, which is metabolized by the liver into trimethylamine oxide, a small molecule that is firmly related to the increased risk for coronary vascular diseases [39
]. Red meat rich l
-carnitine also induces trimethylamine oxide production [40
]. The importance of the metabolome in predicting host dysbiosis was recently evaluated [41
]. Using machine learning techniques and computational predictions, the authors showed that the aggregates predicted the community enzyme function profile and that modeled metabolomes of a microbiota are more predictive of dysbiosis than either observed microbiome community composition or predicted enzyme function repertoires. Table 1
summarizes some of the gut microbiotic beneficial and harmful metabolites in physiological and pathological conditions, respectively [42
Overall, the metabolomic profile has deep implications for comprehending the complex interactions between diets, gut microbiota and host health. This brings a potential promise of nutritional manipulations of the gut microbiome and its metabolites as a way to improve health and treat diseases. Nutrigenetics, nutrigenomics, personal diets or purified metabolomic compounds are a few of the future therapeutic strategies to improve nutrient, metabolome, and gut performance for the benefits of mankind [44
2.3. Post-Translational Modification of Naïve Proteins
Post-translational modification of proteins (PTMP) dominates numerous pathways related to cellular metabolism, representing a key regulator of autoimmunity and potentially of allergy [21
Bacteria have an astounding capability for accommodation and survival strategies, comprising different utterances of the transcriptome and proteome, disparities in growth rate, and withstanding extreme conditions. PTMP contributes significantly to this adaptability and microbial life cycle modifications. Additionally, bacterial PTMP represents a substantial importance to the host. Their enzymatic capacities to transform the naïve/self or non-self-peptides to autoimmunogenic or allergenic forms, is extensive. A large list of enzymes synthesized by dysbiotic populations, capable of PTMP, was published lately [30
A known example of PTMP is the tissue transglutaminase (tTg) in CD or peptidylarginine deiminases in rheumatic arthritis, where deamidation/crosslinking of gliadin or citrullination occur, respectively [46
]. In CD, the autoantigen is tTg, capable of deamidating or cross-linking gliadin [23
]. This PTMP takes place below the epithelium, where neo-epitopes of gliadin docked on the tTg are created, provoking anti-tTg or anti neo-epitope tTg autoantibodies synthesis. Those are well established biomarkers of CD [48
]. Recently, a family member of tTg, the microbial Tg, abundantly used by the processed food industry, was described to be a potent inducer of specific antibodies in CD patients [12
]. More so, the same food ingredient has been suggested as a new environmental trigger and potential inducer of CD [12
]. Very recently, only CD patients, and not controls, were shown to raise specific antibodies against the cross-linked complex between the microbial Tg and the gliadin [12
]. Moreover, PTMP is an important intestinal luminal event that potentially contributes to the extraintestinal phenotype development in CD [21
]. In rheumatoid arthritis, citrullination of peptides, by the bacterial enzyme peptidylarginine deiminases is a prototype of PTMP. Most recently, we put forward the hypothesis that the cerebral tTg or potentially the microbial Tg might be involved in neurodegenerative diseases [50
]. Being a universal protein crosslinker and translational modifier of peptides, the tTg and/or the microbial Tg can crosslink various peptides, to be deposited in the brain, in a folded or misfolded configuration, thus imitating neurodegenerative processes [52
]. The intestinal microbiota, dysbiota, pathobiome, probiotics and processed food contribute to the luminal bacterial origin Tg daily cargo [50
]. It is hypothesized that those bacterial enzymes potentially steer neurodegenerative and neuroinflammatory diseases via intestinal luminal events. By crosslinking naïve proteins, the enzyme can potentially create neo-epitopes that are not only immunogenic but may also be pathogenic, activating some pathological pathways in the cascade of chronic CNS disease induction. The detrimental activities of the bacterial Tg may represent a new mechanism in the gut–microbiome–brain axis and might open novel therapeutic strategies to combat those degenerative diseases. In fact, tTg is a disease-modifying factor in neurodegenerative diseases, because tTg might enzymatically stabilize aberrant aggregates of proteins involved in those conditions. The enzyme contributes to the aggregation of huntingtin protein, insoluble neurofibrillary tangles and β-amyloid plaques, or α-synuclein in Huntington’s disease, in Alzheimer’s disease and in Parkinson’s disease, respectively. Tg is additionally involved in neurotransmitter release states like the botulinum and tetanus neurotoxins activities [53
Concerning allergies, tTg is involved in wheat allergy [54
], PTMP participates in mugwort pollen allergy [55
] and delay type hypersensitivity [56
]. Neo-epitope formation by PTMP is shared in autoimmunity as neo-immunogens, as well as in allergies, as neo-allergens.
Returning to the microbial Tg, it has been proposed that the whole family of microbial Tgs are proteases and that the eukaryotic Tgs have evolved from an ancestral protease [57
]. Taking into account that proteases and anti-proteases are drivers of PTMP and can potentially breach tight junction integrity [45
], the microbial Tg becomes a potential enhancer of intestinal permeability, thus setting the stage for the next section.
2.4. Increased Intestinal Permeability: The Leaky Gut
The intestinal barrier illustrates a vast surface of 400 m2
, where billions of microbes confront the vastest immune apparatus in the human body. Humans support a very complex microbial ecosystem peacefully coexisting with the microbiotic cargo, which tightly interacts with the underlying immune systems. It has been proposed that the human genome cannot support all duties and functions required to survive, since the gut microbiota is crucial to maintaining health and protecting against the pathobiome and numerous diseases [59
]. Our symbiotic microbiome endows multiple metabolic capacities that the mammalian genome-dependent metabolome lacks. One of the microbiome-dependent pivotal functions is to maintain the functional integrity of the intestinal barrier [45
]. The gut barrier is composed of the mucus layer, epithelial layer and the underlying lamina propria. The tight junction (TJ) machinery is situated between the enterocytes, connecting the gut epithelial cells and regulating the paracellular permeability. It prevents the loss of water, electrolytes and small molecular nutrients, and the entry of antigens, toxins and microorganisms inside our body. Such opposing functions are much regulated, microbiome-dependent, extremely orchestrated and evolutionarily conserved under normal conditions. Its key role in avoiding inflammatory responses to the microbiota is heavily dependent on the fine-tuned mucosal and systemic immune networks for microbial recognition and tolerance induction. Loss of this barrier may result in enhanced epithelial permeability to gut microbiota or additional luminal components, which may lead to the phenomenon of molecular mimicry, a well-known pathway of autoimmunogenesis.
Numerous and various categories of potential TJ disruptors exist. Some of them are listed in Table 2
. In fact, multiple human conditions have been associated with dysbiotic alterations or reductions of the microbiota’s diversity, including cancer, inflammatory bowel diseases, food allergies and other atopic conditions, critical illness, irritable bowel syndrome, non-celiac or celiac gluten sensitivity, and metabolic diseases such as diabetes mellitus type two and obesity, cardiovascular, non-alcoholic fatty liver, or non-alcoholic steatohepatitis diseases and neuropathologies [45
]. Additionally, TJ functional impairment is a primary defect in autoimmune diseases [63
]. Intestinal permeability is increased in many of them: ulcerative colitis, Crohn’s disease, CD, inflammatory joint disease, ankylosing spondylitis, juvenile onset arthritis, psoriatic arthritis, diabetes mellitus type one and primary biliary cirrhosis. In fact, the loss of the protective capacity of the mucosal barriers that interact with the outside world is necessary for autoimmunity, allergy, inflammatory, metabolic and some cancer diseases to develop [10
]. Since balanced homeostasis is the physiological rule and since many TJ distractors exist, counteracting environmental factors that protect or improve TJ functions should operate. Table 3
summarizes the factors that protect intestinal permeability and might present potential new therapeutic strategies.
Taking together, TJ dysfunction, frequently called ‘leaky gut’, and its pathophysiological consequences on the pathogenesis of chronic human diseases is constantly unraveled, but many aspects remain unclear. Is it a cause, consequence or co-evolutional phenomenon that the gut ecosystems drive [2
]? Accumulating information suggests that intestinal luminal eco-events, whereof the microbiome is a major one, might alter the regulatory mechanisms of the TJ. This results in a leaky gut, thus shattering the balance between tolerance and immunity to non-self-antigens. Metabolomic products, microbial constituents, transformed neo-epitope peptides, immunogenic/pro-inflammatory molecules, toxins, allergens, carcinogens, drugs, pathobionts and nutritional products can potentially be transported systemically, reaching remote organs, including the brain [2
]. Figure 1
illustrates schematically the factors that are associated with increasing (enhancers) or decreasing (protectors) of intestinal permeability at the TJ level. Breached TJ integrity might represent a crucial step in the gut–brain hinge.