Gastroenterología y Hepatología 45 (2022) 724---734 Gastroenterología y Hepatología www.elsevier.es/gastroenterologia REVIEW Metabolic-associated fatty liver disease: From simple steatosis toward liver cirrhosis and potential complications. Proceedings of the Third Translational Hepatology Meeting, organized by the Spanish Association for the Study of the Liver (AEEH) Rocío Gallego-Durán a,b,1 , Agustín Albillos a,c , Javier Ampuero a,b,d , María Arechederra a,e,f , Rafael Bañares a,g,h,i , Ana Blas-García a,j,k , Genoveva Berná l,m,n , Esther Caparrós a,o,p , Teresa C. Delgado a,q , Juan Manuel Falcón-Pérez a,r,s , Rubén Francés a,o,p , Maite G. Fernández-Barrena a,e,f , Isabel Graupera a,t , Paula Iruzubieta a,u , Yulia A. Nevzorova a,h,v,w , Rubén Nogueiras x,y,z , Rocío I.R. Macías a,aa , Franz Martín l,m,n , Guadalupe Sabio bb , Germán Soriano a,cc , Javier Vaquero a,h , Francisco Javier Cubero a,h,v,1 , Jordi Gracia-Sancho a,dd,ee,∗,1 a Biomedical Research Network on Hepatic and Digestive Diseases (CIBEREHD), Instituto de Salud Carlos III, Madrid, Spain b SeLiver Group, Instituto de Biomedicina de Sevilla/Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de Sevilla, Sevilla, Spain c Gastroenterology and Hepatology Department, Ramón y Cajal Hospital/Alcalá University/IIS Ramón y Cajal (IRYCIS), Madrid, Spain d UCM Digestive Diseases, Virgen del Rocío University Hospital, Sevilla, Spain e Program of Hepatology, Centre of Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain f IdiSNA, Navarra Institute for Health Research, Pamplona, Spain g Gregorio Marañón Hospital, Madrid, Spain h IIS Gregorio Marañón (IiSGM), Madrid, Spain i Department of Medicine, Complutense University School of Medicine, Madrid, Spain j FISABIO --- University Hospital Dr Peset, Valencia, Spain Abbreviations: AEEH, Spanish Association for the Study of the Liver; MAFLD, metabolic-associated fatty liver disease; MASH, metabolic- associated steatohepatitis; HCC, hepatocellular carcinoma; T2DM, type 2 diabetes mellitus; SM, metabolic syndrome; HVPG, hepatic venous pressure gradient; PH, portal hypertension; LSECs, liver sinusoidal endothelial cells; HSCs, hepatic stellate cells; HFD, high fat diet; ECM, extracellular matrix; FXR, farnesoid X receptor; TGR5, Takeda G protein-coupled receptor 5; BMI, body mass index; DAMPS, damage- associated molecular patterns; PAMPS, pathogen-associated molecular patterns; MCJ, methylation-controlled J protein; KCs, Kupffer cells; TCA, tricarboxylic acid; ROS, reactive oxygen species; RNS, reactive nitrosative species; O2 , superoxide radicals; OH, hydroxyl radicals; H2 O2 , hydrogen peroxide radicals; SOD, superoxide dismutase; CAT, catalase; GPX, glutathione peroxidase; GST, glutathione-S-transferase; SCFA, short chain fatty acids; TLR, Toll-like receptor (TLR); PMBCs, peripheral mononuclear blood cells; MAIT, mucosal-associated invariant T (MAIT); ihTh17, inflammatory hepatic T helper 17; EVs, extracellular vesicles; DILI, drug-induced liver injury; CCl4 , carbon tetrachloride. ∗ Corresponding author. E-mail address: jordi.gracia@idibaps.org (J. Gracia-Sancho). 1 Co-senior authors. https://doi.org/10.1016/j.gastrohep.2022.02.005 0210-5705/© 2022 The Authors. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). Gastroenterología y Hepatología 45 (2022) 724---734 k Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain l Biomedical Research Network on Diabetes and Related Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III, Madrid, Spain m Andalusian Center of Molecular Biology and Regenerative Medicine-CABIMER, Seville, Spain n University Pablo Olavide-University of Seville-CSIC, Seville, Spain o Clinical Medicine Department, Miguel Hernández University, San Juan De Alicante, Spain p IIS Isabial, Alicante General Hospital, Alicante, Spain q Liver Disease Lab, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain r Exosomes Laboratory & Metabolomics Platform, Center for Cooperative Research in Biosciences (CIC bioGUNE), Derio, Spain s Basque Foundation for Sciences, Ikerbasque, Bilbao, Spain t Liver Unit, Hospital Clínic de Barcelona, IDIBAPS Biomedical Research Institute, Barcelona, Spain u Aparato Digestivo, Marqués de Valdecilla Hospital, Santander, Spain v Department of Immunology, Opthalmology and ENT, Complutense University School of Medicine, Madrid, Spain w Department of Internal Medicine III, University Hospital RWTH, Aachen, Germany x Biomedical Research Network on Obesity and Nutrition (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain y Department of Physiology, CIMUS, University of Santiago de Compostela, Spain z IIS Santiago de Compostela, Spain aa Experimental Hepatology and Drug Targeting (HEVEPHARM) Group, University of Salamanca, IBSAL, Salamanca, Spain bb Myocardial Pathophysiology, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain cc Hospital de la Santa Creu i Sant Pau, Barcelona, Spain dd Liver Vascular Biology, IDIBAPS Biomedical Research Institute, Barcelona, Spain ee Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Switzerland Received 1 February 2022; accepted 21 February 2022 Available online 3 March 2022 KEYWORDS Abstract: This is a meeting report of the 3rd Translational Hepatology Meeting held in Alicante, MAFLD; Spain, in October 2021. The meeting, which was organized by the Spanish Association for the MASH; Study of the Liver (AEEH), provided an update on the recent advances in the field of basic and Steatohepatitis; translational hepatology, with a particular focus on the molecular and cellular mechanisms and Fibrosis; therapeutic targets involved in metabolic-associated fatty liver disease (MAFLD), metabolic- Hepatocellular associated steatohepatitis (MASH), cirrhosis and end-stage hepatocellular carcinoma (HCC). carcinoma © 2022 The Authors. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). PALABRAS CLAVE Resumen Este es el resumen de la 3a Reunión de Hepatología Traslacional celebrada en Ali- cante, España, en octubre de 2021. La reunión, organizada por la Asociación Española para el Esteatosis; Estudio del Hígado (AEEH), ofreció una actualización de los recientes avances en el campo de Esteatohepatitis; la Hepatología básica y traslacional, con un enfoque en los mecanismos moleculares y celulares Fibrosis; y las dianas terapéuticas implicadas en la enfermedad del hígado graso asociada a disfunción Carcinoma metabólica (MAFLD), la esteatohepatitis asociada a disfunción metabólica (MASH), y las etapas hepatocelular terminales como la cirrosis y el carcinoma hepatocelular (HCC). © 2022 Los Autores. Publicado por Elsevier España, S.L.U. Este es un artı́culo Open Access bajo la licencia CC BY-NC-ND (http://creativecommons.org/licenses/by-nc-nd/4.0/). Definition, comorbidities and pathophysiology of the leading causes of liver cirrhosis and hepatocellu- lar carcinoma (HCC) worldwide. This disease has surfaced Introduction as a growing public health issue, since MAFLD is becom- ing increasingly prevalent in parallel with the pandemics of obesity and diabetes. Prevalence of MAFLD in Spain is Metabolic-associated fatty liver disease (MAFLD), formerly predicted to increase up to 27.6% and metabolic-associated known as non-alcoholic fatty liver disease (NAFLD),1 is one 725 R. Gallego-Durán, A. Albillos, J. Ampuero et al. steatohepatitis (MASH), the inflammatory advanced stage of defined in other aetiologies for MAFLD patients: Previous this disease, will reach about 6% of the Spanish population data from retrospective studies showed that HVPG in MAFLD in 2030, whereas mortality and advanced liver disease will is not as accurate as in other aetiologies,15,16 therefore the double in 2030.2 thorough characterization of advanced MAFLD patients with MAFLD and MASH are strongly associated to risk fac- longitudinal assessment would help to identify the best cut- tors such as obesity, type 2 diabetes mellitus (T2DM), offs and to assess their prognostic value. (iii) Evaluation dyslipidaemias and metabolic syndrome (SM)3 and recip- of the usefulness of non-invasive tests for the follow-up rocally, patients with significant fibrosis in the context in MAFLD patients in order to assess disease progression, of MAFLD have been proven to be at risk of developing clinically significant portal hypertension (PH) or signs of both T2DM and arterial hypertension.4 Besides, mounting oesophageal varices: Recent Baveno Consensus proposed the evidence from both preclinical and human studies has high- use of non-invasive tests to avoid invasive assessment in lighted the relationship of this disease with inflammatory patients with PH signs, but data coming from MAFLD patients phenotypes, especially related to immune-mediated inflam- are still scarce.17 The prospective longitudinal assessment matory diseases, such as Crohn’s disease,5 coeliac disease6 of patients with MAFLD will generate data regarding non- or hidradenitis suppurativa.7 invasive tests and transient elastography and their role in follow-up to predict clinical outcomes. (iv) Multidisciplinary management of advanced-MAFLD patients: Management of MAFLD pathophysiology comorbidities in MAFLD patients is key in order to avoid disease progression. Among them, the incorporation of MAFLD is a complex entity involving numerous genetic, epi- combined treatments and monitorization for potential side- genetic and environmental factors. This disease begins with effects, the identification of the best time-frame and the the accumulation of triglycerides and other lipids in the main best clinical approach (surgical vs. endoscopic) and the best hepatic cell type, the hepatocyte, and this can progress from candidates for obesity in MAFLD patients are crucial for simple steatosis to steatohepatitis, cirrhosis or even liver MAFLD management.18---20 cancer.8 Initially, obesity induces insulin resistance and chronic inflammation that promotes lipolysis of adipose tissue.9 Excess blood fatty acids begin to accumulate in the hepato- cytes, causing the onset of the so-called fatty liver. These lipids come from the systemic circulation, although they are Role of sinusoidal cells in MAFLD also synthesized de novo within the hepatocyte which, in addition, decreases both their degradation and export. Stor- Non-parenchymal liver cells, mainly liver sinusoidal age of lipids in the form of triglycerides is not particularly endothelial cells (LSECs), hepatic stellate cells (HSCs) and harmful, but other lipids such as diacylglycerols, choles- resident macrophages, play essential roles maintaining terol, phosphatidylcholines and certain saturated fatty acids liver homeostasis and their de-regulation represent a key are particularly toxic. underlying mechanism of all hepatopathies. Indeed, the At the same time, insulin resistance and inflammation complexity of the hepatic sinusoidal milieu is defined in adipose tissue lead to increased secretion of adipokines by specific functions of each cell type together with and inflammatory cytokines that generate a state of chronic intense paracrine communication between them.21 In inflammation.10 This inflammation, in the liver, is coupled the context of fatty liver disease, all sinusoidal cells with lipotoxicity due to lipid accumulation promoting the become dysfunctional. Different studies have described activation of stress kinases and hepatocyte cell death- the rapid de-differentiation of LSECs upon administration related pathways.11 Then, the repair mechanisms including of high fat diet (HFD) in animal models, which become proliferation and fibrosis are activated, which will be deci- vasoconstrictor and pro-inflammatory. Subsequently, the sive for the disease to develop into liver cirrhosis or HCC. dysfunctional endothelium activates and recruits local and systemic myeloid cells, thus promoting an amplification of Main unresolved clinical issues in MAFLD the damage, and paracrinally affects HSCs which start to become activated.22 As MAFLD progresses and together with Epidemiological modeling studies have shown that MAFLD hepatocyte dysfunction and death, sinusoidal dysfunction- incidence and prevalence is rapidly increasing and ality is further aggravated resulting in disease-modifying advanced-MAFLD will be one of the main causes of consequences including the development of hepatic micro- liver-related complications, liver transplantations and liver- circulatory dysfunction and PH, exacerbation of hepatic related deaths in the near future.12 The main challenges that and systemic inflammation, and synthesis and release of clinicians will face in this context are: (i) Identification of large amounts of extracellular matrix (ECM) components patients that will progress to advanced-NAFLD and rapid- resulting in hepatic fibrosis. Interestingly, recent studies progressors: the main strategy to overcome this challenge propose a change in the paradigm demonstrating that the would be to understand deeply the intrahepatic and extra- biomechanical properties of the chronically injured liver hepatic mechanisms of disease progression and discover new (i.e. high stiffness and high vascular tone) are not only non-invasive test with pathophysiological profile13,14 ; (ii) consequences of the disease but active players in the aggra- Determination of hepatic venous pressure gradient (HVPG) vation and perpetuation of sinusoidal cells dysfunctionality as the main predictor for clinical decompensation in patients and liver disease,23 and therefore potential targets for with MAFLD, and validation of the current cut-offs of HVPG therapy. 726 Gastroenterología y Hepatología 45 (2022) 724---734 Role of fat in MAFLD Metabolic alterations of bile acids during MAFLD progression In 2020, an international expert consensus panel proposed a new definition of fatty liver, MAFLD, which is based on Bile acids are key components of bile that perform essential a set of positive diagnostic criteria for fatty liver disease functions beside facilitate the absorption of dietary lipids. associated with metabolic dysfunction.1 The guiding sign is Thus, they participate in the homeostasis of hepatic lipid the hepatic steatosis, which can be evidenced by biopsy, and glucose metabolism and energy expenditure acting as imaging or blood biomarker. To date, there are no data sup- signaling molecules through nuclear and membrane recep- porting an association between the degree of steatosis and tors such as the nuclear farnesoid X receptor (FXR) and the the risk of MAFLD progression or clinical outcomes. How- membrane Takeda G protein-coupled receptor 5 (TGR5).33 ever, experimental studies showed that PH may begin to Several studies have described alterations in develop in the absence of fibrosis.24 Indeed, preliminary serum/plasma bile acid levels and/or in the propor- data in MAFLD patients suggest that the degree of steato- tion of molecular species in MAFLD patients,34 which sis may be associated with PH, but the clinical relevance of overall indicated that bile acids may play a role in the this subclinical PH (6---9.5 mmHg) is unknown until date.25 pathophysiology and progression of MAFLD. However, the On the other hand, the liver fat content could be relevant results are not consistent, mainly because there is a for treatment monitoring. Emerging data support the use of strong association of this metabolic condition with obesity, magnetic resonance imaging derived proton density fat frac- insulin resistance, and T2DM, where alterations in bile acid tion (MRI-PDFF), a non-invasive and quantitative measure of metabolism already occur, but also because some studies liver fat content, for treatment response assessment in NASH do not have appropriate patient matching, with control trials.26,27 Finally, visceral fat constitutes a relevant type of groups presenting lower body mass index (BMI) or fasting fat in MAFLD pathogenesis, since the expansion of this fat glycemia. The comparison of fasting plasma bile acids in is associated with MAFLD progression and development of obese subjects with and without steatohepatitis matched cardiovascular disease, the main cause of mortality among for BMI and insulin resistance found no differences in total MAFLD patients. bile acid levels or in the proportion of molecular species,35 but a more complete study concluded that plasma bile acid concentrations were elevated in steatohepatitis patients with severe insulin resistance.36 Future studies may clarify the role of bile acids in the development of MAFLD and their potential therapeutic utility. Immune and systemic inflammatory disorders in MAFLD Advanced MASH --- pathophysiology of decompensated cirrhosis Evolution has selected those species best endowed to survive and reproduce in a given environment, both Decompensated cirrhosis is associated to poor prognosis from a metabolic (nutrition) and an immune/inflammatory specially when acute-on-chronic liver failure occurs. Even (defense against pathogens) point of view. This would though the mechanisms associated to this condition are par- explain the close relationship between metabolism, immu- tially understood, several interacting key factors have been nity and inflammation along the entire evolutionary chain up identified. to humans.28 Moreover, humans are adapted to the lack of (i) Portal hypertension: Portal pressure is determined nutrients after millions of years of being hunter-gatherers. by the interaction between vascular resistance and por- The excess of nutrients is a consequence of the emergence tal blood flow. Increased vascular resistance, the initial of agriculture, livestock and the industrial revolution, very factor in PH development, has two components. The first recent phenomena leading to a new situation to which we is structural, associated to the architectural disturbance have not yet adapted. This would be the evolutionary expla- characteristics of cirrhosis (fibrosis, parenchymal extinc- nation for the current epidemic of obesity and MAFLD.28 tion, sinusoidal capillarization, etc.).37 The second is the Systemic inflammation in obesity is triggered by ischemia of dynamic component caused by the dysregulation of liver vas- hypertrophic adipose tissue, whose capillaries are not suf- cular tone and by the activation of contractile cells such ficient to oxygenate it properly. However, in later stages, as myofibroblasts and hepatic stellate cells. PH is the most various organs including the liver contribute to the systemic important factor in early stages of cirrhosis and its worsen- inflammation that in turn affect the rest of the body.28,29 ing is strongly related to disease progression.38 (ii) Systemic This explains the frequent coincidence of MAFLD with extra- hemodynamics: PH promotes marked splanchnic vasodila- hepatic diseases such as psoriasis, cardiovascular events (a tion leading to effective hypovolemia and hyperdynamic major cause of mortality in patients with MAFLD) and cog- circulation, activation of homeostatic compensating sys- nitive impairment.28,30 tems (sympathetic nervous system, non-osmotic secretion Systemic inflammation in MAFLD has a variable inten- of antidiuretic hormone, and activation of renin-angiotensin sity in a given patient due to the fluctuating nature of the system), sodium and water retention, and renal failure.39 In injuries causing the disease.31 As a consequence, determin- advanced stages, cardiac systolic and diastolic function may ing the parameters of systemic inflammation is not yet useful be also affected, contributing to circulatory derangements. in the diagnosis, prognosis or treatment of patients in clini- (iii) Systemic inflammation: There is growing evidence indi- cal practice,32 although it may be in the future.29 cating the existence of marked systemic inflammation in 727 R. Gallego-Durán, A. Albillos, J. Ampuero et al. cirrhosis. Initially triggered by damage-associated molecular HSCs (the primary fibrogenic cells) get activated and lose patterns (DAMPS) and pathogen-associated molecular pat- their intracellular lipids normally observed when they are in terns (PAMPS) overproduction, the continuous stimulation of a quiescent stage.49 The mechanism mediating the reduc- innate immune system cells induces the overproduction of tion of fatty acids in activated HSCs remain largely unknown inflammatory mediators that extended damage to different but there seems to be related with a decrease in the expres- organs. Interestingly, the intensity of inflammatory response sion of adipogenic genes.50 Identifying the causal pathways is associated with acute-on-chronic liver failure (ACLF) mediating this crucial event for the activation of these fibro- development and prognosis.40 Additionally, albumin dys- genic cells might open new avenues to find potential targets function, caused not only by a decrease in its synthesis but for the treatment of fibrosis. also due to several posttranscriptional changes, increases the severity of inflammation and worsens prognosis.41 (iv) Mitochondrial (dys)function in MAFLD Metabolic alterations and mitochondrial dysfunction: The exacerbated systemic inflammation promotes a catabolic Besides, mitochondria play a plethora of functions in the state, similar to the observed in sepsis and severe trauma, liver which include regulation of cellular signaling, ener- with mitochondrial dysfunction and shifting of ATP pro- getics and redox balance. In patients with fatty liver, fatty duction from oxidative phosphorylation to a less efficient acids are preferably oxidized through fatty acid oxidation aerobic glycolysis. Additionally, the increase in energetic into acetyl-CoA and further metabolized through the tricar- needs derived from systemic inflammation lead to periph- boxylic acid (TCA) cycle51 and oxidative phosphorylation52 eral organs to hypometabolism, dysfunction and failure.42 rather than safely disposed through ketogenesis.53 Under these circumstances, excessive oxidative burst in the mito- Carcinogenesis in MAFLD chondria results in exacerbated production of reactive oxygen species and oxidative stress. Increased oxidative Among other complications, individuals with MAFLD are at stress markers with lowering of the hepatic antioxidant higher risk than healthy individuals of malignancies, pre- machinery and decreased mitochondria biogenesis cause dominantly HCC, but also other extra-hepatic cancers.43 mitochondrial damage. In fact, mitochondria structural and Indeed, although the incidence of HCC in MAFLD patients functional impairment with reduced respiratory capacity is lower than that of other liver diseases, the ongoing global and decreased activity of the respiratory complexes are hall- epidemic of MAFLD is causing a worldwide increase in HCC marks of NASH.52 In the last years, even though therapies incidence.44 Unlike other aetiologies, a high percentage of targeting mitochondria have been proposed for the treat- MAFLD patients develop HCC without cirrhosis.44 MAFLD- ment of MAFLD, these are still rather experimental. In fact, related HCC molecular features are not yet well defined, it has been recently shown that the inhibition of Glutam- however as far as it is known to date, they do not differ inase 1,54 a mitochondrial enzyme, as well as silencing an much compared to another HCC aetiologies. Notably, these endogenous inhibitor of the complex I of the electron trans- tumors display higher rates of ACVR2A mutations, and are port chain, the methylation-controlled J protein (MCJ),55 enriched in bile and fatty acid signaling, oxidative stress and ameliorates liver steatosis in mouse models of diet-induced inflammation, and present a higher fraction of Wnt/TGF- MAFLD by different underlying mechanisms. ␤ proliferation subclass tumors.45 Even more pronounced than HCCs of other aetiologies, the majority of MAFLD- Oxidative stress and cell death associated HCCs are diagnosed at advanced or very advanced stage.46 Importantly, recent evidence shows that although As introduced above, oxidative stress is the result of an immunotherapy improved survival of advanced HCCs, it was imbalance between the production of reactive oxygen (ROS) not superior in patients with non-viral HCC, particularly and nitrosative (RNS) species and the antioxidant capac- NASH---HCC, probably owing to NASH-related aberrant T cell ity. Oxidative stress includes superoxide (O2 •− ), hydroxyl activation.47 Future research is still needed to better under- (• OH), and hydrogen peroxide (H2 O2 ) radicals. Apart from stand MAFLD-related HCC and to develop specific biomarkers mitochondria, other subcellular structures or organelles, and therapeutic options. including the plasma membrane, endoplasmic reticulum, and peroxisomes contribute to the production of oxidative stress. The antioxidant system may rely on enzymatic and Update on the mechanisms of liver injury in non-enzymatic reactions. The enzymatic system comprises MAFLD superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione-S-transferase (GST).56 Adipose tissue and liver crosstalk Excessive oxidative stress can result in lipid peroxidation and cause damage to proteins and DNA. Peroxidation of mem- Currently, it is well-accepted that there is a close crosstalk brane lipids leads to both functional and structural damage, between the adipose tissue and the liver.48 The excess of fat which finally results in cell death. is commonly correlated with a generalized proinflammatory The mode of cell death is not only pivotal in directing state and the elevated production of adipokines is likely to the severity but also the outcome of liver injury. Apoptosis, play a role in the pathogenesis of MAFLD, which can progress necrosis, necroptosis, autophagy, pyroptosis and ferrop- to advanced stages such as fibrosis. However, different cells tosis 57 overlap and even crosstalk in a variety of liver in the liver display a divergent response to the excess of fat. diseases including MAFLD/MASH, where apoptotic effectors While hepatocytes store more fatty acids (namely steatosis), CASP3/6/7/8 are predominantly involved in intrinsic (via 728 Gastroenterología y Hepatología 45 (2022) 724---734 lipotoxicity and organelle stress) as well as in extrinsic metabolism, insulin resistance, mitochondrial disfunction (via cell surface receptors) apoptosis thus driving inflam- and oxidative stress thus participating in the development mation, whilst the role of necroptosis via the involvement and progression of fatty liver to MASH.65 Additionally, epi- of RIPK1/3/MLKL might lead to fibrosis and metabolic transcriptomics describe chemical RNA modifications, also changes.58 dynamic and reversible, that controls its structure and func- tion without affecting its sequence. To date, more than 100 Inflammation and fibrogenesis different chemical RNA modifications have been identified, being N6-methyladenosine (m6 A) the most characterized As described above, hepatocyte and endothelial injury trig- m6 A modification that plays an important role in glucose and gers multiple proinflammatory and profibrogenic pathways lipid homeostasis, while some m6 A regulators are involved and promotes the release of extracellular vesicles, activat- in the progression of MAFLD.66 ing other liver cell populations and contributing to MAFLD A deep knowledge and integrative analysis of the genetic, progression. Monocyte recruitment to the damaged liver and epigenetic and epitranscriptomics modifiers and events can its polarization in inflammatory macrophages is promoted help enormously for individual risk stratification and con- by chemokine release by Kupffer cells (KCs) and other acti- stitute the basis for further developing prevention and vated non-parenchymal cells including HSCs. The diversity treatment strategies. of liver macrophage subsets and their plasticity adapting to changes in the microenvironment explain their differ- Nutritional geometry in MAFLD ent functional responses in MAFLD, in which they regulate inflammation, fibrosis, and tumor progression, as well as The understanding of the effect of nutrients on MAFLD, tissue repair.59 Activated HSC are the main source of ECM in order to define nutritional interventions for treatment and drive the fibrotic response to damage and the devel- and prevention, is unclear.67 Nutritional geometry is a novel opment of cirrhosis. Besides some well-known proliferative approach that analyzes how nutrients and foods can be com- and profibrogenic cytokines, novel routes are emerging as bined in a system that allows us to know the interaction of important regulators of HSC activation, such as Hippo-Yap foods to regulate the properties of diets that affect health. and Notch pathways.60,61 It consists of the graphical representation of n-dimensions Recent studies are unveiling the enormous complexity of nutrients in a diet and their comparison by response of liver biology. In this line, two different popula- surfaces of different physiological or health/disease param- tions of liver-resident macrophages with inflammatory and eters. Using this geometric approach, it was shown that in immunoregulatory functions have been described, and sev- humans energy intake increases as protein intake decreases, eral subclasses of HSCs have been also defined in murine a phenomenon known as ‘‘protein leverage’’.68 A study with models of steatohepatitis, including a subclass of activated mice showed that macronutrient composition of diets deter- HSCs with properties similar to inflammatory fibroblasts mines the probability of having fatty liver disease, such associated with cancer. Although further research is needed, that diets low in proteins and high in fats are the strongest these transcriptomic analyzes provide new insights into cell drivers of MAFLD.69 This may indicate, that for MAFLD treat- heterogeneity and its role in liver disease, and offer hope ment, we should consider weight loss and the proportion that specific cell subtypes may be targeted by precision of macronutrients, their quality and the overall intake of drugs to reduce inflammation or fibrosis.62,63 energy. Further studies are warranted to evaluate whether MAFLD patients have an adequate protein intake, with a How to approach MAFLD higher energy intake coming from the consumption of high- fat foods. Genetics, epigenetics and other risk factors Role of the microbiome in immunological and A really critical unanswered question is why there are cer- inflammatory alterations in MAFLD tain patients that progress to severe symptomatic states, whereas another important group does not. The reasons Intestinal microbiota dysbiosis has a deep impact in for this interindividual variability are not completely under- the hepatic immune response in MAFLD. Though healthy stood but can be at least partially attributed to differences gut microbial phylum ratio is higher in Bacteroides vs. in genetic background, epigenetic modifications and also Firmicutes, MAFLD and further advanced stages show a dis- in new recently described events known as epitranscrip- turbance in this ratio toward Firmicutes and Proteobacteria tomics. Different variants of genes mainly implicated in phylum, with increasing bacterial abundance and decreas- the cellular metabolism of lipids in the liver define the ing bacterial diversity.70 The unbalance of short chain fatty genetic risk factors for MAFLD. As such, the most relevant acids (SCFA) production, bile acids pool and dysbiotic micro- loci affecting MAFLD are PNPLA3 (rs738409 C>G), TM6SF2 bial products induce a potent liver immune response via TLR (rs58542926 C>T), GCKR (rs1260326), MBOAT7 (rs641738 activation of hepatocytes, KCs and HSC.71 In a multicenter C>T) or HSD17B13 (rs6834314) among others.64 Epigenetics study, MAFLD patients with BMI above 30 kg/m2 showed a is the second branch able to explain the variability, partici- higher number of different antigens in serum and increased pating in the development and progression of fatty liver to toll-like receptor (TLR) expression in peripheral mononu- MASH. Methylation of DNA, chemical modification of histone clear blood cells (PMBCs) both at RNA and protein levels.72 tails and non-coding RNA-mediated regulation are the princi- Independently of BMI, when bacterial antigen was present pal epigenetic events, many of them involved in hepatic lipid in serum, there was a significant increase of TNF-␣ and IL-6. 729 R. Gallego-Durán, A. Albillos, J. Ampuero et al. Though antigen-specific response of resident hepatic practice in the most recent MAFLD European and American immune cells is essential, mucosal-associated invariant T guidelines.84,85 Further insights into the natural history of (MAIT) cells responding to bacterial riboflavin metabolite are MAFLD, development of specific therapies, better validation emerging potential contributors. They produce IL-17 and IL- studies, and further technological improvements may all 22 favoring the intestinal barrier in steady state and access be needed for ‘‘omics’’ to fully integrate into clinical the liver to promote regulatory macrophage activation as practice. shown in MCD murine model.73 Interestingly, new poten- tial contribution in fueling MAFLD has been proposed by Technological advances in the study of MAFLD described inflammatory hepatic Th17 (ihTh17) in obesity.74 This CXCR3+ subset shows increased glycolytic capacity and Over the past century, novel technological advances have produces IL-17, IFN-␥ and TNF-␣, driving to NAFLD worsen- driven discoveries related to both hepatocyte organiza- ing. tion and function. This includes hepatocyte separation techniques, novel immunohistochemistry and microscopy Methods and technologies for the study of approaches, cell sorting and single cell RNA sequencing, MAFLD among others.86 Despite its high prevalence, this disease is still lacking from pharmacological therapies to prevent and treat the MAFLD outbreak, therefore, preclinical research Omics and exosomes is crucial to identify and test new therapeutic agents. The absence of models able to reflect the unique cellular struc- Extracellular vesicles (EVs) constitute a novel biological ture recapitulating the liver microenvironment constitutes entity that has awaked great interest to identify biomarkers, a significant limiting factor in MAFLD. Thus, novel 3D models and as active players in the development of liver diseases. have been established from different cells sources, includ- Omics technologies have been widely applied to character- ing spheroids, derived from different hepatic cell types, ize the content and function of EVs secreted by liver cells in and hepatic organoids, produced by stem cell differenti- different pathological scenarios including drug-induced liver ation in parenchymal and non-parenchymal liver cells.87 injury (DILI), NAFLD and MS.75 Thus, transcriptomics and Organoids are 3D physiological in vitro structures that reca- proteomics of these EVs have provided several low invasive pitulate morphological and functional features of in vivo candidate biomarkers for cirrhosis in serum76 and urine.77 tissues and offer significant advantages over traditional cell The transcriptomic analysis of EVs and the cells that secrete culture methods.88 Besides, livers-on-a chip are designed to those EVs made possible the identification of a sorting RNA mimic the physiological microenvironment of the hepatic signal that can incorporate the RNAs into the EVs to be lobule, even reproducing blood circulation.89 More recently, exported out of the cells.78 Another important contribution it has been successfully developed a microfluidic NASH-on- of the omics technologies to the hepatic EVs, in this case a-chip platform that recapitulates the main NASH histologic done by metabolomics has been the demonstration that hep- endpoints in a single chip and that can emerge as a human- atic EVs carry several active enzymes that are able to modify relevant, in vitro platform to study disease pathogenesis the serum metabolic composition that could have important and develop novel anti-NASH drugs.90 Finally, precision cut implications for endothelial functioning.79 The integration of liver slices from rat or human origin retain the structure several ‘‘omics’’ technologies combined in different exper- and cellular composition of the native liver and represent imental settings including the analysis of the cells, the EVs an improved system to study liver fibrosis compared to two- secreted by those cells, and the cells exposed to those EVs dimensional mono- or co-cultures.91 allow to dissect the EVs-mediated mechanisms underlying the development and progression of liver diseases and it provides novel therapeutics targets. Animal models of MAFLD Application of a wide range of ‘‘omics’’ to MAFLD has provided a huge amount of information valuable from a clin- Given the epidemic of ‘‘Diabesity’’ (obesity and T2DM) ical perspective.82 First, ‘‘omics’’ have greatly enhanced and concomitant meteoric rise in MAFLD there is an our knowledge of the pathophysiology and mechanisms urgent need in preclinical animal models. The theoret- of the disease, revealing relevant risk factors such as ical ‘‘ideal’’ MAFLD model should: (i) fully recapitulate polymorphisms of PNPLA3 or other genes80 and potential the liver phenotype (macrovesicular steatosis, inflamma- epigenomic81 and metabolomic82 pathways amenable to tion, hepatocellular ballooning and fibrosis) plus features of diagnostic or therapeutic exploitation. Second, information the associated metabolic syndrome (dyslipidemia, adiposity, from ‘‘omics’’ may help to predict the response to specific insulin resistance), (ii) have the ability to further progress to therapeutic interventions.83 Finally, ‘‘omics’’ have a great advanced fibrosis, cirrhosis and ultimately HCC, (iii) be sta- potential for identifying non-invasive biomarkers for the ble, reliable and reproducible, (iv) have high success rate diagnosis, staging and monitoring of MAFLD. In this sense, and low mortality, (v) be simple and feasible.92 specific miRNAs, metabolomic/lipidomic factors, and the All existing models of MAFLD can be broadly classified combination of different ‘‘omics’’ have shown considerable into: ability to detect and differentiate the stages of disease.82 Despite their potential, no ‘‘omics’’-based tools have 1. Dietary models including: (a) overnutrition-diets with demonstrated to be superior to current and more simple high fat and/or rich in saturated fatty acids, fructose and tools outside of the trials in which they were described, cholesterol (HFD, Western diet, ALIOS)93 ; (b) deficient and they are not recommended for routine medical diets lacking methionine and/or choline (MCD, CDAA). 730 Gastroenterología y Hepatología 45 (2022) 724---734 2. Genetic models with different genetic alterations lead- References ing to hepatic lipid accumulation (ob/ob, db/db, PTEN knock-out, DIAMOND mice). 1. Eslam M, Newsome PN, Sarin SK, Anstee QM, Targher G, 3. ‘‘Hybrid’’ or intensified models presenting the combina- Romero-Gomez M, et al. A new definition for metabolic tion of dietary or genetic factors with other hepatotoxins dysfunction-associated fatty liver disease: An international (carbon tetrachloride --- CCl4 , alcohol, DUAL model, expert consensus statement. 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