Immunonutritional contribution of gut microbiota to fatty liver disease

Nutrición Hospitalaria Revisión ISSN (electrónico): 1699-5198 - ISSN (papel): 0212-1611 - CODEN NUHOEQ S.V.R. 318 Immunonutritional contribution of gut microbiota to fatty liver disease Contribución inmunonutricional de la microbiota intestinal a la hepatopatía grasa Ricardo Fernández-Musoles1, Aurora García-Tejedor1 and José Moisés Laparra2 1Food Science and Health Group, Valencian International University. Valencia, Spain. 2Instituto Madrileño de Estudios Avanzados en Alimentación (IMDEA Alimentación). Grupo de Inmunonutrición Molecular. Madrid, Spain Keywords: Biomarkers. Gastrointestinal. Global health. Microbiome. Phenotype(s). Abstract Non-alcoholic fatty liver disease (NAFLD) is indisputably the most widespread liver disease worldwide, leading to a significant increase in patient morbidity, mortality, and health care utilization. The gut microbiota and its genome (microbiome) have emerged as a novel modulator of the immunometabolic processes that NAFLD implies, but microbiota-targeted interventions have resulted both astounding and at the same time unsuccessful. The most relevant alteration appears to be the overgrowth of Gram-negative bacteria, characterized by an increased ratio of Firmicutes to Bacteroidetes, although current evidence indicates speciesand strain-specific effects influencing energy harvest, the host’s innate and adaptive immune systems, and epigenetic regulation as determinants of the immunomodulatory milieu in NAFLD. The genera Lactobacillus and Bifidobacterium deserve special attention since many of their probiotic strains are marketed for human consumption, even more so when considering that, in conjunction with prebiotics, they are potential modulators of gut microbiota composition and/or metabolic activity. Here, a better understanding of the major intestinal microbial factors with a detrimental or preventive role in NAFLD, and of the dynamic interplay between gut microbiome and host factors, appears crucial in defining the exposome for the prevention and treatment of NAFLD and associated diseases such as metabolic syndrome, type-2 diabetes, and obesity. Palabras clave: Biomarcadores. Gastrointestinal. Salud global. Microbioma. Fenotipo(s). Resumen La esteatosis hepática no alcohólica (NAFLD, por sus siglas en inglés) es indiscutiblemente la patología hepática más extendida a escala mundial y conlleva un aumento significativo de la utilización de la atención médica de los pacientes, así como de la morbilidad y la mortalidad. La microbiota intestinal y su genoma (microbioma) se han revelado como uno de los factores moduladores de los procesos inmunometabólicos subyacentes que desencadenan la NAFLD. Las intervenciones dirigidas a modificar la composición y/o la actividad de la microbiota han resultado sorprendentes y, al mismo tiempo, infructuosas. La disbiosis más relevante en la patología es un aumento de la proporción entre Firmicutes y Bacteroidetes. La evidencia actual indica que los efectos específicos de la especie y la cepa influyen en el resultado funcional de la microbiota sobre el metabolismo de los nutrientes, la rama innata y la adaptativa del sistema inmune, y la regulación epigenética del genoma humano en relación al NAFLD. Los géneros Lactobacillus y Bifidobacterium merecen especial atención ya que muchas cepas probióticas de estos géneros se comercializan para consumo humano, e incluso más si se considera que, junto con los prebióticos, son moduladores potenciales de la composición de la microbiota intestinal y/o su actividad metabólica. En este contexto, una mejor comprensión de los principales factores microbianos con papel perjudicial o preventivo en la NAFLD, y de la interacción dinámica entre el microbioma intestinal y los factores del huésped, parece crucial para definir el exposoma de la prevención y el tratamiento de la NAFLD y sus enfermedades asociadas, como el síndrome metabólico, la diabetes de tipo 2 y la obesidad. Correspondence: José Moisés Laparra Llopis. Instituto Madrileño de Estudios Avanzados en Alimentación (IMDEA Alimentación). Grupo de Inmunonutrición Molecular. Ctra. de Cantoblanco, 8. 28049 Madrid, Spain e-mail: [email protected] Fernández-Musoles R, García-Tejedor A, Laparra JM. Immunonutritional contribution of gut microbiota to fatty liver disease. Nutr Hosp 2020;37(1):193-206 DOI: http://dx.doi.org/10.20960/nh.02775 Received: 05/07/2019 • Accepted: 13/10/2019 Author´s contribution: JML thanks MICINN for his ‘Ramon y Cajal’ contract (RYC-2015-18083). This work was supported by grants RYC-2015-18083 from the Spanish Ministry of Science and Innovation (MICINN, Spain). ©Copyright 2020 SENPE y ©Arán Ediciones S.L. Este es un artículo Open Access bajo la licencia CC BY-NC-SA (http://creativecommons.org/licenses/by-nc-sa/4.0/). Conflict of interest: the authors declare no conflict of interest. 194 R. Fernández-Musoles et al. [Nutr Hosp 2020;37(1):193-206] INTRODUCTION Lifestyle and dietary habits are major contributors to the growing incidence of non-communicable diseases. As a consequence of a dramatic increase in risk factors such as obesity, type-2 diabetes and altered food supply and preferences, non-alcoholic fatty liver disease (NAFLD) is one of the most important causes of liver disease worldwide, affecting an estimated ≥ 24% of most populations (1). Genetic predisposition must be also placed in the context of environmental factors. Nowadays, accumulating evidence describes a ‘lean’ manifestation of NAFLD, which was initially described in Asian populations and affects at least 5% of the population in western societies (2). This ‘lean NAFLD phenotype’ represents, among others, a ‘metabolically obese, normal weight phenotype’ (1,2). A significant body of data suggests that dietary composition and its interaction with the gut microbiota – and eventually the immune system – may predispose individuals to NAFLD, rather than total calorie intake, with a continuous positive balance promoting obesity, fatty liver, and finally NASH and/or metabolic syndrome. Over the past ten years, different mouse models – germfree, antibiotic-treated and with or without colonization by specific microbial strains (3-5) – have been used to prove metabolic and immune effects derived from microbes for hosts. Here, imbalances in the gut microbiota have been linked to major risk factors for NAFLD (6,7). However, direct evidence for an association between functional alterations in the gut microbiota composition, as well as the microbiota-related molecular signature, and NAFLD and NASH remains largely inferential (3,5). In this scenario, surrounding environmental conditions, including diet (within 24 h), have proven to significantly impact microbial genome (microbiome) influencing host metabolic stress and dietary energy expenditure (8). Otherwise, long-term dietary habits were more clearly associated with imbalances in microbial levels categorized into enterotypes. The metabolic activity of gut microbiota has led to consider it as a ‘supraorgan’ likely comparable to the liver. Notably, gut microbiome exhibits high variability between individuals (i.e., genders) and may constitute a source of variation for the effects observed, but evidence at the population level is incompletely characterized. The liver is a central organ for the turnover and transformation of the metabolites supplied from the intestine. In addition, the intestine constitutes an important source of immune signals that stem from the intestinal mucosa and immune system. This so-called ‘gut-liver’ axis is sensitive to the gut microbiota, which modulates energy supply and homeostasis, ectopic fat deposition, intestinal and fatty tissue inflammation, and immune maturation (9-11). The regulation of the energy balance operates both in the central nervous system and at peripheral sites, including adipose tissue and the liver, playing a critical role in the development of NAFL/NASH and liver fibrosis (12,13). In this sense, contradictory findings have been reported suggesting an important role for the diversity of gut microbiota as well as its composition, worsening or improving, both qualitatively and quantitatively, liver lipid accumulation (14,15). Besides, only a few studies have investigated the role (bidirectional) of the gut microbiota in host lipid metabolism (16) to establish steady-state commensalism and tissue metabolic homeostasis (17). Preclinical data also support the relevance of preand post-natal nutrition to the developmental programming of NAFLD (18). The importance of perinatal outcomes in microbiota modulation at critical periods for development have been highlighted for their potential role in the prevention (19) and treatment of human disease. This is increasingly thought to occur through epigenetic changes as integral underlying mechanisms. Here, it can be established that an indirect gut microbiota-mediated epigenetic regulation may modify host immunometabolic responses and, ultimately, gene expression (20,21). These are novel perspectives connecting the intrauterine milieu to risk factors for NAFLD, i.e., diabetes, obesity, and hyperlipidemia, as well as for metabolic syndrome as a whole. However, how immunonutritional food components influence these aspects in NAFLD remains poorly understood. In relation to the above, several recent reviews (22,23) have focused on reporting experimental and clinical evidence of intestinal dysbiosis in NAFLD. From these studies, conclusions were drawn on the therapeutic potential of manipulating the gut microbiota to decrease the incidence and prevalence of fatty liver disease. However, relatively few protocols have been registered in relation to gut microbiota manipulation in NAFLD patients (24). There is still an open debate about how and what microbes or their genomes may modulate NAFLD severity and (consequently) energy expenditure or nutrient fate to prevent metabolic and immune complications. Currently obtained results are promising, but as yet they do not permit definitive clinical conclusions in relation to their clear efficacy in microbiome-targeted therapies (24,25). This review provides a comprehensive overview about the role of the gut microbiota as an important contributor to immunometabolic factors influencing liver disease. To this end a comprehensive update of the dynamic interplay between diet, nutrients, and microbes, as well as the microbial phylogenetic changes that occur in obese versus lean individuals is reported. Particularly, this manuscript is focused on the immune function and includes the current knowledge on the functional outcome obtained from modifications in microbial ecosystems. EXPOSOME AND LIVER FUNCTION The mammalian gut harbours highly complex microbial communities, which develop multifaceted interactions in the health and nutrition of the host and are influenced by the exposome (i.e., environmental exposures to various exogenous – diet – and endogenous factors), and have influential effects on both metabolism and immunity. Microbial products derive from either saccharolytic or proteolytic fermentation and may affect the gut-liver axis via multiple mechanisms, hence contributing to the pathogenesis of NAFLD. Different microbial-mediated processes have received major attention, including: a) efficiency of energy harvest, i.e., utilization of nutrients (fermentation of complex dietary carbohydrates) favouring short-chain fatty acid (SCFA) production (5); b) modula- 195IMMUNONUTRITIONAL CONTRIBUTION OF GUT MICROBIOTA TO FATTY LIVER DISEASE [Nutr Hosp 2020;37(1):193-206] tion of low-grade liver (and adipose tissue) inflammation, e.g., via activation of free fatty acid receptor 2 (FFA2, a G-protein-coupled receptor also known as GPR43) (26); c) impact on the innate immune response, such as endotoxin production from Gram-negative bacteria (27), and the adaptive immune response, by binding to leukocyte receptors (butyrate binding to GPR41 and GPR43) suppressing lymphocyte proliferation, to inhibit cytokine production by Th1-lymphocytes and to induce T-lymphocyte apoptosis (28). SCFAs can regulate lipogenesis through their interaction with the endocannabinoid system or stimulation of orexigenic hormone release (e.g., leptin) in adipose tissue (29). Also, they can reduce or increase insulin resistance (30) via a GPR40-mediated and calcium-dependent mechanism regulating the expression of PPARd. Otherwise, the impact or modulatory role of bacterial metabolites originating from a proteolytic metabolism on host physiology has received much less attention. Microbes gradually shift to their proteolytic metabolism as carbohydrates are depleted in the intestinal lumen. Bacterial protein degradation products are considered deleterious for the host’s physiology due to the production of phenolic compounds (i.e., indole) derived from aromatic amino acid degradation. Numerous Gram-negative, as well as Gram-positive, bacterial species able to produce indole have been identified (including Escherichia coli, Clostridium spp., and Bacteroides spp), including Lactobacilli and Bifidobacteria that are considered beneficial for the host’s health. Accumulating evidence suggests that microbial tryptophan catabolites resulting from proteolysis have an impact on mammalian gut immune homeostasis through binding to the aryl hydrocarbon receptor (AHR), stimulate hormone (i.e., GLP-1) secretion, and modulate gastrointestinal motility (31). Furthermore, adequate tryptophan metabolism can prevent redox imbalances due to decreased production of NAD equivalents, which are used to compensate the fermentation of carbohydrates. Nutritional deficiency of choline has also been associated with changes in the microbiome of the human gut and the development of fatty liver, as previously found in rodent models of NASH (32). Accordingly, changes have been reported in various physiological processes such as lipid metabolism, enterohepatic circulation of bile acids, and cholesterol metabolism (33). These observations were accompanied by increased numbers of the Gram-negative bacteria Gammaproteobacteria and Erysipelotrichi, whose presence has been associated with increases in fat content in the liver (34). The Erysipelotrichaceae family is relatively distant, from a phylogenetic point of view, from the Clostridiales order, for which oxidative enzymes contributing to the production of oxysterols have been identified. This bacterial metabolic potential opens new avenues to the microbial contribution to synergistic lipotoxic effects leading to mitochondrial function impairment (35). Collectively, existing data demonstrate that the gut microbiome can affect the liver, the adipose tissue, and metabolic diseases both in a beneficial and in detrimental way. However, owing to the complexity and multifaceted nature of the interactions between gut microbiota and its microbiome with the host’s nutritional and health status, no human in vivo data on a direct link between microbial-derived metabolites, liver function, and NAFLD progression are available. ENERGY HARVEST AND FATE OF NUTRIENTS The commensal microbiota regulates nutrient acquisition and utilization in symbiosis with the host, providing additional enzymes (36,37). Here, it is implied a regulated expression of various genes that are involved in the utilization of dietary carbohydrates and lipids. Metagenomic studies identified gut microbial enzymes specialized in the utilization of otherwise non-digestible nutritional carbohydrates and host-derived glycoconjugates (e.g., mucins, proteoglycans). These genes participate in the deconjugation and dehydroxylation of bile acids, in cholesterol reduction (the latter being mainly converted to coprostanol by intestinal fermentative bacteria, either directly by the enzyme cholesterol reductase or via an alternative indirect pathway), in the biosynthesis of vitamins and isoprenoids, and in the metabolism of amino acids and xenobiotics (36,38). Thus, novel human microbiome-targeted interventions have been proposed, while an effective approach has yet to be successfully developed (24). Human trials support potential positive roles for prebiotics (i.e., non-digestible food ingredients, mostly oligosaccharides that stimulate growth and activity in several specific, beneficial intestinal bacteria) enhancing NAFLD/NASH progression likely through their modulatory effects on glucose homeostasis and lipid metabolism (5). Also, the few existing studies revealed changes in the microbiome after very short-term dietary interventions (10 days) with high-fat/low fibre versus lowfat/high fibre (39). However, direct evidence of the influence of defined prebiotic structures on specific microbial genes and their physiological functions is lacking. Nowadays, prebiotic contribution to establishing a ‘score’ of disease and validating its use in clinical practice remains elusive. The potential contribution of microbial fermentation has also been a matter of controversy since it improves energy harvest from the diet, which may contribute as much as 10% to daily calorie intake (40). Studies in rodents clearly showed the causative role of the gut microbiota in adiposity and insulin resistance, linking changes in host phenotypes to variations in gut microbial composition (14,4143). Thus, germ-free animals did not develop diet-induced obesity and related comorbidities, like visceral adipose tissue inflammation with macrophage infiltration and insulin resistance (42). Otherwise, these mice gained excess fat mass when conventionalized with microbiota from lean non-germ-free animals kept on a normal diet (14). The persistent lean phenotype of the germ-free animals displayed increased skeletal muscle and liver levels of phosphorylated AMP-activated protein kinase (AMPK), accompanied by its downstream targets involved in fatty-acid oxidation (acetylCoA carboxylase, carnitine-palmitoyl transferase) (14). Fat mass gain was greater when microbiota from ob/ob mice (obese due to defective leptin signalling) was transferred to germ-free animals (41). The ob/ob microbiome showed a higher proportion of environmental gene tags encoding many enzymes involved in breaking down oth- 196 R. Fernández-Musoles et al. [Nutr Hosp 2020;37(1):193-206] erwise indigestible dietary polysaccharides, including pathways for starch/sucrose, galactose and butanoate metabolism, indicating an improved energy harvest (41). In these scenarios, a comparison of microbial families associated to diabetes-related clinical traits revealed a positive correlation with Lactobacillaceae, while Bacteroidaceae was negatively associated with weight gain (43). Dietary supplementation (12 weeks) with prebiotic galacto-oligosaccharides (GOS) was proven effective to increase fecal numbers of Bifidobacterium spp. without improving insulin sensitivity in overweight or obese prediabetic men and women (44). Metagenomic studies in patients with T2D revealed that changes in the microbiome rather than in microbial taxa have a greater influence on the risk factors for T2D (45). MICROBIOTA IN METABOLIC AND ENDOCRINE PROCESSES Promising reports on the effectiveness of microbiome-based targeted interventions demonstrated changes in hepatic gene expression when animals were fed prebiotics (46,47). Gene expression implied that different genes are involved in diverse biochemical processes: lipogenesis, cholesterol production and mobilisation, or induction of PPARα-mediated fatty-acid oxidation. Notably, the composition of the gut microbiota, including the activity and growth of lactobacilli and bifidobacteria, which promote the production of SCFA (e.g., acetate, butyrate and propionate), appeared as a critical factor driving the magnitude and final biological outcomes. Apart from trophic effects on intestinal epithelia (48,49), reported findings remark the high dependence of biological effects in the host’s physiology on the SCFA profile produced. While the production of luminal acetate appears to be a precursor for hepatic lipid synthesis (16), propionate oppositely impacts lipid metabolism inhibiting the effects of acetate. Generally, butyrate is regarded as a healthy metabolite as it is utilized by enterocytes, positively influencing cell growth and differentiation, and promoting anti-inflammatory effects. Collectively, it was found that increasing proportions of SCFA producers significantly alleviate hepatic steatosis, decreasing the content of triglycerides and free fatty acids in rats fed a high-fat diet (50). The latter could open new interpretations to the role of lactobacilli, major contributors to the efficiency of energy harvest and nutrient utilization, and their potential contribution to the onset and progression of NAFLD. SCFAs also affect endocrine functions due to their interaction with G-protein-coupled FA receptors (i.e., GRP43 and GRP41) (51), the stimulation of which increases the production of peptide YY and leptin (orexigenic and anorexigenic peptides, respectively) (52,53). Much of the beneficial effects of prebiotics on plasma lipid and hepatic triglyceride concentrations are attributed to regulation of the expression of the different transcription factors involved in lipid metabolism (i.e., acetyl-CoA carboxylase, fatty-acid synthase, malic enzyme, and ATP citrate lyase). These effects occur via regulation of glucose metabolism (54) and insulin resistance (55). This could point to enhanced enteroendocrine function in L-cells (mainly located in the ileum and colon) (56) as mediators of the trophic effects associated with reduced mucosal inflammation (48,57) and induction of the antidiabetic (PPAR-mediated regulation of Akt pathway influencing GLUT4 expression in liver) and anorexigenic incretin glucagon-like peptide 1 (GLP-1). Prebiotics have also been proven to influence physiological endocrine systems up-regulating the production of GLP-2 and expression of the endocannabinoid system (eCB) (58). Thus alleviating gut permeability, plasma lipopolysaccharides (endotoxaemia) from Gram-negative bacteria (LPS) and cytokines decreased the hepatic expression of inflammatory cytokines (e.g., TNF-α, MCP-I) and oxidative stress markers in obese (leptin-deficient) mice. It has been recently shown that a dysregulated expression of eCB 1 and 2 in the colon and liver is associated to metabolic imbalances derived from sirtuin 3 (SIRT3) deficiency (59). These observations were followed by increased endotoxaemia in correlation with gut microbiota dysbiosis. Yet the underlying mechanism linking SIRT3 with gut microbiota remains obscure, and SIRT3 deficiency may be attenuated by sodium butyrate. Prebiotic-treated obese (ob/ ob) mice exhibited a normalized eCB system tone (expression of eCB-1r, plasma eCB levels or adipose tissue eCB content) in both the gut and adipose tissue, as well as endotoxaemia (29). Figure 2 summarizes the interrelationship between prebiotics, gut permeability and eCB-induced adipogenesis and NAFL. Relatively rapid shifts in gut microbiota composition and their related functional outcomes in response to high-sugar and highfat diets have been reported (8,60,61). These responses modify both gene expression and the resultant metabolite generation and flow in the host. At the same time, alterations in the composition of the gut microbiota influence strain-specific susceptibility to diet-induced metabolic disease (43). This bidirectional interaction determines the microbial capacity for processing dietary sugars and for generating hydrophobic bile acids and, thereby susceptibility to metabolic disease. The impact of metabolic disease on bacterial functional outcomes is reflected, whether worsening or improving the disease, in the role of GPR43 as sensor for excessive dietary energy (15). Apparently, this receptor loses its inhibitory influence on insulin signaling, reducing fat accumulation in adipocytes. An example is vancomycin-treated mice, which display microbial changes relatively similar to obese patients, with a significantly reduced diversity of the gut microbiota and a markedly increased abundance of Lactobacillus spp (62). However, these bacterial changes were accompanied by significant increases in body weight and colonic GPR43 expression. Microbial alterations in obese people (60,63,64) favor higher glycoside hydrolase activities and a higher activity of host transport proteins importing the breakdown products of indigestible carbohydrates. MICROBIOME AND LIVER FUNCTION Grain-based food, sugars and dairy products are the nutritional staples of western diet, which produce high glycemic and fat loads that have been recognized as aggravating factors of fatty liver disease. 197IMMUNONUTRITIONAL CONTRIBUTION OF GUT MICROBIOTA TO FATTY LIVER DISEASE [Nutr Hosp 2020;37(1):193-206] Figure 1. Beneficial effects of short-chain fatty acids (SCFA) derived from bacterial fermentation of dietary fructo-oligosaccharides and galacto-oligosaccharides on non-alcoholic fatty liver (NAFL) development. Figure 2. Interrelationship between prebiotics, gut permeability, and influence of the endocannabinoid (eCB) system in adipogenesis and NAFL development. 198 R. Fernández-Musoles et al. [Nutr Hosp 2020;37(1):193-206] Western diets, with the concomitant specific changes in gut microbiota to pro-inflammatory microbes (Gammaproteobacteria genera including Klebsiella spp., Enterobacter spp., and Escherichia spp.), also reduce the availability and utilization of several amino acids originating from both alimentary and endogenous proteins. These metabolic conditions favour the development of fatty liver inflammation in patients on controlled diets with lowered levels of choline (32,65). Dietary choline deficiency is an example of one (of several) nutritional factors in modern diets that may favor the development of NAFL and NASH. Most studies documented that choline is an essential nutrient with a key role on fatty liver progression by decreasing methionine levels, favoring abnormal phospholipid synthesis and lipoprotein secretion, mitochondrial dysfunction, and endoplasmic reticulum stress (32,33). In addition, choline deficiency contributes to dysregulate methionine levels and tissue polyamines, a significant part of which have their origin in the intestinal microbiota, impairing glucose, lipid, and energy homeostasis. Studies correlating the content of polyunsaturated fatty acids (PUFA) with the development and extent of NAFL have produced divergent results (66-68). Dietary deficiency of ω-3 PUFA caused imbalances on microbial composition in mice, with decreased cecal numbers of Lactobacillus spp. and Bacteroidetes-Prevotella spp. (67). However, ω-3-depleted mice displayed a slightly increased proportion of Bifidobacterium spp., which was significantly associated with increased insulin resistance and steatosis. Here, dietary supplementation with fructo-oligosaccharides (FOS), for only 24 days, increased the proportion of lactobacilli, contributing to restore an efficient fat storage without lessening liver steatosis (67). Thus, these studies demonstrate different effects of prebiotic fibres in fatty liver disease depending on dietary microbiome adaptations. Also, the results may be interpreted to suggest a direct role for microbial fermentation in steatosis, insulin resistance, and down-regulation of hepatic inflammatory regulators (i.e., peroxisome proliferator-activated receptor (PPAR)-α, and receptor gamma coactivator α). Collectively, it can be delineated a significant bulk of evidence supporting the bidirectional impact of either nutrient availability or gut microbial imbalances on each other to trigger profound effects on mammalian lipid metabolism. Table I summarizes the effects observed in serum and liver lipids, as well as in the biomarkers associated to NAFL, in different animal models fed a high-fat diet. Indirectly, gut microbiota can modify the lipid profile in the enterohepatic circulation affecting overall hepatic lipid metabolism. Solely microbial transformation of cholesterol and bile acids constitute the origin of coprostanol and secondary bile acids – deoxycholic acid (3α,12α-dihydroxy-5β-cholan-24-oic acid) and lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid). Under physiological conditions these bile acids are absorbed but human hepatic metabolic capacities cannot remove them from the bile acid pool causing hepatic toxicity (69). 7α-dehydroxylation is the most quantitatively important bacterial bile salt biotransformation in the human colon, and 16S rDNA phylogenetic analyses have led to classify most competent intestinal bacteria into the genus Clostridium (70). The order Clostridiales, including Clostridium XIVa and IV subgroups, within the superfamily Lachnospiraceae is peripherally related to the family Erysipelotrichaceae within phylum Firmicutes (71) mostly associated to obese individuals (72). Unfortunately, there are scarce studies addressing to what extent these differences in hydroxysteroid dehydrogenases could be responsible, at least in part, for the significant detrimental effects in NAFLD as 7β-dehydroxylation activity has also been described in Bacteroides distasonis (73). Despite the direct effect of bile acids helping in the intestinal absorption of lipids and fat-soluble vitamins, they also play a role in regulating lipid-glucose homeostasis, inflammation, and thereby innate immune cells polarization via specific nuclear receptors (FXR) and G-protein-coupled receptors (TGR5). FXR is highly expressed in the liver, intestine, kidney and adrenal gland, and may play a role in reducing hepatic triglyceride levels via different gene pathways, including FGF21 (74), ChREBP (75), PPARα (76), and AKR1B7 (77). TGR5 is highly expressed in the gallbladder, intestine (primarily in the ileum and colon), and liver, where it has been shown to antagonize the activity of NFkB in macrophages and hepatic Kupffer cells (78). Moreover, in intestinal L-cells TGR5 induces GLP1 secretion, and in the liver TGR5 attenuated triglyceride accumulation through a yet-to-be-determined mechanism (79). MICROBIAL IMBALANCES ASSOCIATED WITH LIVER DISEASE A direct link between NAFL/NASH status and imbalances in the composition of the gut microbiota is now being established (3,27). The most relevant alteration is an increased ratio of Firmicutes to Bacteroidetes, associated to the overgrowth of Gram-negative bacteria and decreased proportions of Bifidobacterium spp. in the cecal contents of obese humans (63,80). However, contrasting results have also been obtained from clinical studies where the proportion of Bacteroides was found significantly increased in subjects with liver fibrosis (81). In a similar way to the procedures followed to uncover the role of gut microbiota in obese and T2D patients, ‘infection’ models served to establish the microbial contribution to the induction of adaptive and innate immune memory (82). This bacterial contribution seems to occur at the epigenetic, metabolic, or functional level. However, despite its potentially important role, to the best of our knowledge no population-based evidence is available regarding the ability of the mucosal immune system to co-operate with systemic immunity in response to certain commensals in the setting of NAFL. Scientific evidence available in the literature tried to establish a relationship between bacterial translocation through the intestinal barrier and NAFL aggravated symptoms and disease progression (83,84). The overgrowth of small intestinal bacteria (SIBO) is directly associated with an increase in intestinal permeability affecting nutrient absorption and utilization. Notwithstanding this, the impact and magnitude of intestinal functional impairment seem to be associated to the NAFLD phenotype. 199IMMUNONUTRITIONAL CONTRIBUTION OF GUT MICROBIOTA TO FATTY LIVER DISEASE [Nutr Hosp 2020;37(1):193-206] Table I. Examples of the effects of prebiotic fibres on serum and liver lipids and biomarkers associated to NAFL in animal models fed a high-fat diet Animals Diet Duration (weeks) Serum lipids Liver physiology Reference Wistar rats High-fat diet supplemented with 10% oligofructose 3 ↓ triglycerides and ↓total cholesterol triglycerides unchanged (136) High-fat diet supplemented with 10% oligofructose 3-5 ↓ postprandial triglycerides ↓ triglycerides (49) High-fat diet supplemented with 5% inulin 4-12 ↓ triglycerides, total cholesterol unchanged ↓ triglycerides and total cholesterol (137) High-fat/high-sucrose diet supplemented with 5% inulin 8↓ triglycerides, total cholesterol unchanged ↓ triglycerides; ↓ AST, ALT (138) 5% synthesized inulin, 5% resistant maltodextrin, high-fat/high-sucrose diet, high-fat/high-sucrose diet 5% synthesized inulin, high-fat/high-sucrose diet 5% resistant maltodextrin, colfibrate 0, 0.1 or 0.25% was added to each diet after 1 week 3 ↓ triglycerides with inulin degree of polymerization 16 in high-fat/highsucrose diet ↓ triglycerides and total cholesterol with inulin and resistant starch high-fat/highsucrose diet; colfibrate + inulin ↓ triglycerides in high-fat/high-sucrose diet (139) Balb/cJ mic D-galactose + 5% fructo-oligosaccharides 7 Not determined ↓ triglycerides (140) Sprague-Dawley rats High-fat diet supplemented with lactulose 8 ↓ triglycerides, but not total cholesterol, with lactulose ↓ ALT, AST and hepatic Inflammation scores (141) Sucrose, sucrose + 10% fructo-oligosaccharides 3 ↓ postprandial free fatty acids and triglycerides, but not total cholesterol or free cholesterol ↓ liver weight (54) Lean and obese JCR:La-cp rats Standard diet supplemented with 10%, 20% 50 inulin:50 fructo-oligosaccharides 10 ↓ triglycerides in obese rats ↓ liver weight and triglycerides in obese group (142) 200 R. Fernández-Musoles et al. [Nutr Hosp 2020;37(1):193-206] For example, a study population (n = 35 patients) with biopsy-proven NAFLD determined the presence of a “leaky” intestine with evidence of increased SIBO (83). Furthermore, a cross-sectional study performed in outpatients with metabolic risk factors for NAFLD development (type-2 diabetes, obesity, dyslipidemia) did not find any significant association between SIBO and NAFLD or hepatic steatosis (85). However, it was found that central obesity was significantly associated with SIBO. Metagenome-wide association studies revealed that gut microbiota in T2D patients was characterized by decreased numbers of some universal butyrate-producing bacteria (Eubacterium rectale, Faecalibacterium prausnitzii, Roseburia intestinalis and R. inulinivorans). Besides this, an increase in various opportunistic pathogens (Bacteroides caccae, Clostrium hathewayi, Clostridium ramosum, Clostridium symbiosum, Eggerthella lenta, and Escherichia coli) was quantified, as well as an enrichment of other microbial functions conferring sulphate reduction and oxidative stress resistance. Obesity was associated with reduced bacterial diversity, with lower numbers of Bacteroidetes and increased numbers of Actinobacteria (30,63,86). Some other components of the obesity-associated gut microbiome are distributed among Proteobacetria, Fusobacteria and Verrucomicrobia (72). Otherwise, contrasting data are reported in relation to the proportion of Methanobrevibacter smithii in obese subjects (63,72). Unexpectedly, M. smithii and Escherichia coli were found to negatively correlate with body mass index in overweight and obese people (87). Recent extensive studies have revealed that molecular hydrogen (H2) has a great impact on obesity development with a decreased expression of H2S-synthesizing enzymes and reduced H2S levels in adipose tissues in mice. Also, significant increases in some other bacterial groups such as Staphylococcus aureus (88,89) and Faecalibacterium prausnitzii (90) have been associated with NAFL comorbidities. However, a decreased abundance of both the Faecalibacterium and Ruminococcus group was recently demonstrated in ‘lean’ NASH patients (91). The role of Lactobacillus spp. in obesity has been a subject of controversy, with part of the risk for causing public health problems in humans being attributed to the promotion of consumption of products containing these bacteria. Analyses on the proportions of Lactobacillus spp. evidenced contrasting changes in this microbial population. While significant increases (63,92), particularly in the species L. reuteri and L. ingluviei (63,93), have been found in overweight people, significant reductions in the fecal concentrations of the Lactobacillus gasseri subgroup were observed in obese infants at baseline (80). Nowadays few interesting data are available regarding the role of dietary supplementation with probiotics in the management of obesity, as most probiotics for human consumption belong to the Lactobacillus genus. In this sense, a recent study in obese children who received daily supplementation with L. casei (Shirota strain) microbial effectiveness contributing to weight loss while improving lipid metabolism was demonstrated. Notably, these effects could be associated to a significant increase in the fecal numbers of Bifidobacterium spp. and acetic acid concentration (80). These results suggest that microbial metabolic associations are important to determine the functional outcomes of gut microbiota composition in the development of metabolic diseases. Table II summarizes the most significant bacterial groups associated with overweight in obese animals and humans. The specific role of gut microbiota components and their bioactive factors remains unclear in NAFLD. In addition, the direct role of gut microbiome in the development of obesity and its associated phenotypes has been demonstrated to be transmissible by fecal transplantation into germ-free mice (43,94). Here, bacterial colonization in obesity and associated disorders appears to be dependent on host metabolic responses to diet. In addition to the gut microbiota metabolic participation in the modulation of lipid metabolism, it has also been associated with an indirect regulatory influence in the central nervous system (CNS), the so-called ‘gut-brain’ axis. Thereby, microbial-mediated CNS control could influence food intake and expenditure, the regulation of hepatic lipogenesis, and gut integrity, as well as hepatic low-grade inflammation. Preclinical studies have shown that microbial colonization of germ-free mice can reduce the levels of circulating fasting-induced adipose factor (Fiaf), and the levels of phosphorylated AMP-activated protein kinase (both in the skeletal muscle and liver), thus contributing to fat storage (7,95) (Fig. 1). Taken together, these functional outcomes of gut microbiota beyond energy harvest from diet have led to the concept that gut microbiota serves as an important regulator of energy homeostasis and ectopic fat deposition. Nevertheless, the functional characterization of transplanted microbial communities suggest that variation in metabolic outcomes is under the control of both host genetics and gut microbiota (43). Data from scarce nutritional interventions in humans also strongly suggest the microbiota-mediated positive effects in patients with nonalcoholic steatohepatitis (NASH) (96). A study in 66 patients who received a symbiotic mixture of Bifidobacterium longum with FOS for 24 weeks concluded that, compared to lifestyle modification alone, dietary supplementation with the symbiotic formulation significantly reduced NASH activity index, serum concentrations of LDL cholesterol, inflammatory markers, and insulin resistance. Otherwise, knockout mice for phosphatidylethanolamine N-methyltransferase (PEMT) develop severe NASH (97). However, these mice are protected from obesity and whole-body insulin resistance. Here, the study of the influence of feeding prebiotics to animals with metabolic risk factors for NAFLD development has been inferential. Feeding the prebiotic kojibiose (2-O-α-D-glucopyranosyl-α-D-glucopyranose) to hyperglycaemic animals treated with arachidic acid significantly reduced the plasma levels of lyso-phosphatidylcholine (98). These changes were accompanied by body weight loss and increased numbers of Bifidobacterium spp. (by 12%), Bacteroides spp. (by 2%), and Enterobacteriaceae (by 4%) relative to control animals. POTENTIAL DUAL ROLE OF PROBIOTICS To date there is a controversial debate concerning the speciesor strain-dependent role, whether positive or negative, of Lactobacillus spp. in body weight gain (92,99). Different investi- 201IMMUNONUTRITIONAL CONTRIBUTION OF GUT MICROBIOTA TO FATTY LIVER DISEASE [Nutr Hosp 2020;37(1):193-206] gations point out specific alterations in gut microbiota composition causing markedly opposite regulatory effects on lipogenesis associated with increased numbers of Lactobacillus spp., and thereby Firmicutes in fecal contents. This is of particular importance since most probiotics for human consumption belong to this genus. While some species (L. acidophilus and L. reuteri) have been associated with obesity-associated gut microbiota (63,64), others (L. plantarum and L. gasseri) are associated with weight loss in obese humans and animals (63). Feeding L. ingluievi to mice preceded an increase in weight and increased FAS and TNF-α transcript levels in the liver (93). L. acidophilus or L. casei were likely protective against diabetes since animals fed with these probiotics did not show any evidence of metabolic syndrome such as insulin resistance (100). However, in some cases strain-specific beneficial effects have been reported, for example, in patients dietary supplemented with L. plantarum 299v or a yoghurt supplemented with L. acidophilus 145, B. longum 913 and oligofructose (101,102). Most prominent effects significantly reduced serum concentrations of LDL-cholesterol (by 9.6%) and fibrinogen (101) or increased HDL-cholesterol (by 25.8%) (102). Feeding L. rhamnosus GG and L. sakei NR28, supplemented within a normal chow, to mice resulted in a significant reduction of epididymal fat mass, as well as obesity-related biomarkers like AA, FAS, and steatoryl-CoA desaturase-1 in the liver (103). Taken together, all studies seem to highlight strain-dependent effects on lipid metabolism and liver/adipose tissue inflammation for Bifidobacterium and Bacteroides spp. that counteract NAFL/NASH. Another important aspect is that most species of these genera possess β-glucosidase activity contributing to nutrient utilization. Otherwise, metagenomic studies have also demonstrated the expression of β-glucuronidase activity in some species of Firmicutes (104). β-glucuronidase activity might contribute to the production of toxins and/or mutagens resulting from increased intestinal reuptake and hepatic retention of usually deconjugated, potentially toxic bile acids. However, the role of bacterial β-glucosidases in the development of NAFL/NASH needs further study, since opposite bacterial patterns have been found as a function of the aetiology of fatty liver disease. For example, enteric dysbiosis with relative abundance of Bacteroidetes and Verrucomicrobia compared to Firmicutes, a pattern that should correlate with amelioration of NAFL/NASH or obesity, has been associated with alcohol-induced steatosis and steatohepatitis (105). INNATE IMMUNITY NAFLD has been strongly associated with gut injury and increased intestinal permeability, and NASH frequently recurs in patients who underwent weight reduction surgery to treat obesity. These conditions favour translocation of bacterial antigens across the intestinal mucosal wall into the portal circulation in 25-70% of patients with liver disease, enhancing hepatic inflammation and disease progression to NASH (9,11). Studies in animals and humans indicate that gut-derived bacterial endotoxin (i.e., lipopolysaccharide, LPS) and other cell wall components play importTable II. Summary of the most significant bacterial groups associated with overweight and obese animals and humans Bacterial group Increased Reduced Reference Prevotellaceae  (72) Staphylococcus aureus Bifidobacterium (88) Faecalibacterium prausnitzii (90) Lactobacillus ingluviei (93) Firmicutes Bacteroidetes (143) Enterobacteriaceae Bifidobacterium Escherichia coli Bacteroidetes (86) Bacteroidetes (144) Bacteroidetes (145) Staphylococcus aureus Bacteroides fragilis (146) Lactobacillus Staphylococcus aureus Bifidobacterium (89) Enterobacteriaceae Bacteroides Escherichia coli Clostridium histolyticum Bacteroides-Prevotella (147) Eubacterium rectale Clostridium coccoides Lactobacillus acidophilus Lactobacillus plantarum (63) Lactobacillus fermentum Lactobacillus gasseri Lactobacillus ingluviei Lactobacillus reuteri Bifidobacterium animalis (64) Methanobrevibacter smithii Lactobacillus reuteri Bifidobacterium animalis (148) Methanobrevibacter smithii Escherichia coli Firmicutes Bacteroidetes (149) Firmicutes Bacteroidetes (151) Lactobacillus casei/ paracasei (148) Lactobacillus plantarum Firmicutes Bacteroidetes (151) Fusobacteria Verrucomicrobia Proteobacteria Enterobacteriaceae (152) Actinobacteria Bacteroidetes (153) Firmicutes Verrucomicrobia  Faecalibacterium prausnitzii 