Advertisement

Immune mechanisms linking metabolic injury to inflammation and fibrosis in fatty liver disease – novel insights into cellular communication circuits

      Summary

      Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease and is emerging as the leading cause of cirrhosis, liver transplantation and hepatocellular carcinoma (HCC). NAFLD is a metabolic disease that is considered the hepatic manifestation of the metabolic syndrome; however, during the evolution of NAFLD from steatosis to non-alcoholic steatohepatitis (NASH), to more advanced stages of NASH with liver fibrosis, the immune system plays an integral role. Triggers for inflammation are rooted in hepatic (lipid overload, lipotoxicity, oxidative stress) and extrahepatic (gut-liver axis, adipose tissue, skeletal muscle) systems, resulting in unique immune-mediated pathomechanisms in NAFLD. In recent years, the implementation of single-cell RNA-sequencing and high dimensional multi-omics (proteogenomics, lipidomics) and spatial transcriptomics have tremendously advanced our understanding of the complex heterogeneity of various liver immune cell subsets in health and disease. In NAFLD, several emerging inflammatory mechanisms have been uncovered, including profound macrophage heterogeneity, auto-aggressive T cells, the role of unconventional T cells and platelet-immune cell interactions, potentially yielding novel therapeutics. In this review, we will highlight the recent discoveries related to inflammation in NAFLD, discuss the role of immune cell subsets during the different stages of the disease (including disease regression) and integrate the multiple systems driving inflammation. We propose a refined concept by which the immune system contributes to all stages of NAFLD and discuss open scientific questions arising from this paradigm shift that need to be unravelled in the coming years. Finally, we discuss novel therapeutic approaches to target the multiple triggers of inflammation, including combination therapy via nuclear receptors (FXR agonists, PPAR agonists).

      Keywords

      Introduction

      Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, affecting roughly 25% of the global population.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      ,
      • Sanyal A.J.
      Past, present and future perspectives in nonalcoholic fatty liver disease.
      NAFLD has a strong association with obesity, type 2 diabetes, and hypertension, and is therefore considered the hepatic manifestation of the metabolic syndrome.
      • Sanyal A.J.
      Past, present and future perspectives in nonalcoholic fatty liver disease.
      In parallel with the rising global prevalence of obesity and type 2 diabetes, the burden of NAFLD is projected to increase rapidly over the coming decade.
      • Estes C.
      • Razavi H.
      • Loomba R.
      • Younossi Z.
      • Sanyal A.J.
      Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease.
      ,
      • Estes C.
      • Anstee Q.M.
      • Arias-Loste M.T.
      • Bantel H.
      • Bellentani S.
      • Caballeria J.
      • et al.
      Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016-2030.
      NAFLD is emerging as a leading cause of liver transplantation.
      • Wong R.J.
      • Singal A.K.
      Trends in liver disease etiology among adults awaiting liver transplantation in the United States, 2014-2019.
      NAFLD encompasses a spectrum of liver pathologies ranging from steatosis, to the inflammatory form non-alcoholic steatohepatitis (NASH), which is characterised by lobular inflammation and hepatocyte ballooning (with or without fibrosis), to more advanced stages including cirrhosis and hepatocellular carcinoma (HCC).
      • Diehl A.M.
      • Day C.
      Cause, pathogenesis, and treatment of nonalcoholic steatohepatitis.
      NAFLD is the fastest growing cause of HCC in Europe and the USA.
      • Huang D.Q.
      • El-Serag H.B.
      • Loomba R.
      Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention.
      HCC is the third leading cause of cancer-related death and can occur even in the absence of cirrhosis in patients with NAFLD.
      • Akinyemiju T.
      • Abera S.
      • Ahmed M.
      • Alam N.
      • Alemayohu M.A.
      • Allen C.
      • et al.
      The burden of primary liver cancer and underlying etiologies from 1990 to 2015 at the global, regional, and national level: results from the global burden of disease study 2015.
      • Yang J.D.
      • Hainaut P.
      • Gores G.J.
      • Amadou A.
      • Plymoth A.
      • Roberts L.R.
      A global view of hepatocellular carcinoma: trends, risk, prevention and management.
      • Sung H.
      • Ferlay J.
      • Siegel R.L.
      • Laversanne M.
      • Soerjomataram I.
      • Jemal A.
      • et al.
      Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      NAFLD is considered a multisystem metabolic disease
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      ; morbidity and mortality in patients are reciprocally influenced by comorbidities and risk factors. The prevalence of NAFLD among individuals with type 2 diabetes is 55% and conversely, patients with NAFLD are 2 to 5 times more likely to develop type 2 diabetes.
      • Francque S.
      • Szabo G.
      • Abdelmalek M.F.
      • Byrne C.D.
      • Cusi K.
      • Dufour J.-F.
      • et al.
      Nonalcoholic steatohepatitis: the role of peroxisome proliferator-activated receptors.
      ,
      • Younossi Z.M.
      • Golabi P.
      • de Avila L.
      • Paik J.M.
      • Srishord M.
      • Fukui N.
      • et al.
      The global epidemiology of NAFLD and NASH in patients with type 2 diabetes: a systematic review and meta-analysis.
      Cardiovascular disease is the leading cause of death in patients with NAFLD (40–45% of deaths), and vice versa, NAFLD is an independent risk factor for cardiovascular disease.
      • Anstee Q.M.
      • Mantovani A.
      • Tilg H.
      • Targher G.
      Risk of cardiomyopathy and cardiac arrhythmias in patients with nonalcoholic fatty liver disease.
      The strong impact of metabolic status on fatty liver disease has even prompted an intense debate about whether NAFLD should be “re-defined” as “metabolic dysfunction-associated fatty liver disease” (MAFLD).
      • Eslam M.
      • Newsome P.N.
      • Sarin S.K.
      • Anstee Q.M.
      • Targher G.
      • Romero-Gomez M.
      • et al.
      A new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement.
      The involvement of multiple organs in this clinical syndrome indicates common bi-directional pathomechanisms. Indeed, the liver is involved in the development of metabolic dysregulation and may exacerbate insulin resistance, adipose tissue dysfunction and gut dysbiosis, leading to a vicious circle whereby extrahepatic factors further trigger liver damage, resulting in metabolic and inflammatory insults.
      • Francque S.
      • Szabo G.
      • Abdelmalek M.F.
      • Byrne C.D.
      • Cusi K.
      • Dufour J.-F.
      • et al.
      Nonalcoholic steatohepatitis: the role of peroxisome proliferator-activated receptors.
      On the other hand, storage of fat in adipose tissue or liver may protect from systemic cardio- and neurovascular disease. This subpopulation of patients is referred to as healthy obese individuals.
      • Bluher M.
      Metabolically healthy obesity.
      Furthermore, a substantial proportion of patients with morbid obesity show normal liver histology, underlining the complex pathophysiology of these conditions.
      • Ahrens M.
      • Ammerpohl O.
      • von Schonfels W.
      • Kolarova J.
      • Bens S.
      • Itzel T.
      • et al.
      DNA methylation analysis in nonalcoholic fatty liver disease suggests distinct disease-specific and remodeling signatures after bariatric surgery.
      ,
      • von Schonfels W.
      • Beckmann J.H.
      • Ahrens M.
      • Hendricks A.
      • Rocken C.
      • Szymczak S.
      • et al.
      Histologic improvement of NAFLD in patients with obesity after bariatric surgery based on standardized NAS (NAFLD activity score).
      Specific pathways for differential disease manifestations in the context of metabolic risk relate, for instance, to the TM6SF2 and PNPLA3 risk alleles for NAFLD, which in turn can protect against vascular morbidity.
      • Xia M.
      • Ma S.
      • Huang Q.
      • Zeng H.
      • Ge J.
      • Xu W.
      • et al.
      NAFLD-related gene polymorphisms and all-cause and cause-specific mortality in an Asian population: the Shanghai Changfeng Study.
      ,
      • DiCorpo D.
      • LeClair J.
      • Cole J.B.
      • Sarnowski C.
      • Ahmadizar F.
      • Bielak L.F.
      • et al.
      Type 2 diabetes partitioned polygenic scores associate with disease outcomes in 454,193 individuals across 13 cohorts.
      NAFLD is the most prevalent chronic liver disease globally and a major cause of liver cirrhosis, liver transplantation and hepatocellular carcinoma.
      The clinical management of NAFLD is currently restricted to lifestyle interventions and difficult-to-sustain weight loss, although numerous novel therapies are being evaluated with some promising trial results.
      • Vuppalanchi R.
      • Noureddin M.
      • Alkhouri N.
      • Sanyal A.J.
      Therapeutic pipeline in nonalcoholic steatohepatitis.
      The complex and multifactorial pathogenesis of NAFLD make it a challenging disease, and an improved understanding of disease mechanisms will form the basis for the development of better therapeutics. The strongest predictors of liver-related mortality in NAFLD are the presence and stage of liver fibrosis.
      • Angulo P.
      • Kleiner D.E.
      • Dam-Larsen S.
      • Adams L.A.
      • Bjornsson E.S.
      • Charatcharoenwitthaya P.
      • et al.
      Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease.
      • Dulai P.S.
      • Singh S.
      • Patel J.
      • Soni M.
      • Prokop L.J.
      • Younossi Z.
      • et al.
      Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis.
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      • Vilar-Gomez E.
      • Calzadilla-Bertot L.
      • Wai-Sun Wong V.
      • Castellanos M.
      • Aller-de la Fuente R.
      • Metwally M.
      • et al.
      Fibrosis severity as a determinant of cause-specific mortality in patients with advanced nonalcoholic fatty liver disease: a multi-national cohort study.
      • Sanyal A.J.
      • Van Natta M.L.
      • Clark J.
      • Neuschwander-Tetri B.A.
      • Diehl A.
      • Dasarathy S.
      • et al.
      Prospective study of outcomes in adults with nonalcoholic fatty liver disease.
      In addition, inflammation is a key driver of disease progression and fibrosis development,
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      evidenced by a strong correlation between the presence of NASH and stage 4 fibrosis.
      • Ekstedt M.
      • Hagström H.
      • Nasr P.
      • Fredrikson M.
      • Stål P.
      • Kechagias S.
      • et al.
      Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up.
      Inflammatory mechanisms are involved along the entire spectrum of NAFLD but particularly at more advanced disease stages, including cirrhosis and during the transition to HCC.
      • Peiseler M.
      • Tacke F.
      Inflammatory mechanisms underlying nonalcoholic steatohepatitis and the transition to hepatocellular carcinoma.
      • Schuster S.
      • Cabrera D.
      • Arrese M.
      • Feldstein A.E.
      Triggering and resolution of inflammation in NASH.
      • Kazankov K.
      • Jørgensen S.M.D.
      • Thomsen K.L.
      • Møller H.J.
      • Vilstrup H.
      • George J.
      • et al.
      The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
      NAFLD in patients is characterised by profound clinical and histological heterogeneity (rapid vs. slow progression, lean vs. obese NASH, or NASH-associated HCC with and without cirrhosis). Underlying this heterogeneous clinical presentation, inflammation in NAFLD rarely progresses in a linear fashion but fluctuates between flares and resolution. This might explain why the more dynamic parameter ‘inflammation’ is a less strong prognostic feature than the more static parameter ‘fibrosis’ on liver histology, when captured at a single timepoint.
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      Research in recent years has identified many cellular and molecular targets, however, translation of these findings into disease-modifying treatments has proven challenging. Liver disease research has been propelled forward by the recent technological advances in single-cell multi-omics.
      • Saviano A.
      • Henderson N.C.
      • Baumert T.F.
      Single-cell genomics and spatial transcriptomics: discovery of novel cell states and cellular interactions in liver physiology and disease biology.
      With respect to immune mechanisms in NAFLD, employing these novel tools has enabled the identification of unrecognised cell subsets and has increased our awareness of immune cell heterogeneity. Herein, we summarise the current knowledge of the major liver immune cells and their role in NAFLD and review novel concepts of immune-mediated mechanisms in NASH and NASH-to-HCC transition. We propose a conceptual framework that integrates hepatic inflammatory pathways with extrahepatic triggers of inflammation and we discuss unanswered scientific questions.

      Inflammation in NAFLD

      Liver inflammation is a hallmark of progressive NAFLD in patients; numerous rodent studies have mechanistically demonstrated involvement of immune cells in advancing NASH and fibrosis (Table 1).
      • Arab J.P.
      • Arrese M.
      • Trauner M.
      Recent insights into the pathogenesis of nonalcoholic fatty liver disease.
      Recent technological advances including single-cell multi-omics, spatial transcriptomics, multiparametic flow and mass cytometry, as well as multiplex immunofluorescence and intravital microscopy, have provided new insights into how the immune cell composition is reshaped during steatohepatitis both in murine models and patients with various degrees of NAFLD.
      • Saviano A.
      • Henderson N.C.
      • Baumert T.F.
      Single-cell genomics and spatial transcriptomics: discovery of novel cell states and cellular interactions in liver physiology and disease biology.
      ,
      • Ramachandran P.
      • Matchett K.P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Henderson N.C.
      Single-cell technologies in hepatology: new insights into liver biology and disease pathogenesis.
      • Malehmir M.
      • Pfister D.
      • Gallage S.
      • Szydlowska M.
      • Inverso D.
      • Kotsiliti E.
      • et al.
      Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.
      • Davis R.P.
      • Surewaard B.G.J.
      • Turk M.
      • Carestia A.
      • Lee W.Y.
      • Petri B.
      • et al.
      Optimization of in vivo imaging provides a first look at mouse model of non-alcoholic fatty liver disease (NAFLD) using intravital microscopy.
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      We now recognise that NAFLD-associated inflammation mirrors its systemic character, as multiple organ systems fuel liver inflammation (Fig. 1).
      • Anstee Q.M.
      • Reeves H.L.
      • Kotsiliti E.
      • Govaere O.
      • Heikenwalder M.
      From NASH to HCC: current concepts and future challenges.
      ,
      • Peiseler M.
      • Tacke F.
      Inflammatory mechanisms underlying nonalcoholic steatohepatitis and the transition to hepatocellular carcinoma.
      ,
      • Huby T.
      • Gautier E.L.
      Immune cell-mediated features of non-alcoholic steatohepatitis.
      The conceptual framework of the pathogenesis of NAFLD proposes that overwhelming metabolic energy substrates, carbohydrates and free fatty acids lead to toxic lipid accumulation in the liver.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      Hepatic steatosis develops because of increased liver triglyceride storage and hepatic de novo lipogenesis resulting in lipotoxicity. NASH onset is linked to oxidative stress, reactive oxygen species, mitochondrial dysfunction and endoplasmic reticulum stress resulting in hepatocellular metabolic dysfunction and injury.
      • Hardy T.
      • Oakley F.
      • Anstee Q.M.
      • Day C.P.
      Nonalcoholic fatty liver disease: pathogenesis and disease spectrum.
      Hepatocellular stress induces different modes of cell death including apoptosis, necrosis and necroptosis, and ultimately the release of damage-associated molecular patterns (Fig. 1).
      • Arab J.P.
      • Arrese M.
      • Trauner M.
      Recent insights into the pathogenesis of nonalcoholic fatty liver disease.
      ,
      • Schwabe R.F.
      • Luedde T.
      Apoptosis and necroptosis in the liver: a matter of life and death.
      In addition, hepatocyte senescence, i.e. a specific form of cell cycle arrest, can also lead to the release of inflammatory signals, termed the senescence-associated secretory phenotype.
      • Engelmann C.
      • Tacke F.
      The potential role of cellular senescence in non-alcoholic fatty liver disease.
      Detection of tissue perturbance by immune sentinels and the ensuing immune response induce further damage of stressed hepatocytes, resulting in a vicious cycle and the full picture of necroinflammation. This framework was initially coined the ‘two-hit hypothesis’.
      • Day C.P.
      • James O.F.
      Steatohepatitis: a tale of two "hits"?.
      However, overnutrition and insulin resistance can directly influence immune cell activation and composition in different organ systems.
      Table 1Summary of the major immune cell subsets involved in the pathogenesis of NAFLD derived from patient data and murine models.
      PopulationFindings in human NAFLDMechanisms in rodent NAFLD
      Monocytes/MacrophagesAccumulation of CCR2+ pro-inflammatory macrophages

      CD9+TREM-2+ scar/lipid-associated macrophages (function?)

      Presence of Timd4+/- KC subsets
      Pro-inflammatory role in aggravating NASH, hepatocyte damage (CCR2+)

      Replenishment of emKCs with more pro-inflammatory moKCs

      Early KC activation via DAMPs and PAMPs

      Distinct KC2 subset increases in steatosis, metabolic gene profile
      NeutrophilsAccumulation of neutrophils in liver biopsies

      Markers of NETs increased in patients

      Myeloperoxidase increased in human NASH

      Neutrophil chemoattractants increased in NASH livers
      Promote NASH via the release of effector molecules (proteases, elastase, myeloperoxidase, ROS)

      Blocking NETs beneficial in mice

      Myeloperoxidase deficient mice or neutrophil depletion protected from NASH
      DCsIncreased XCR1+ cDC1s correlate with NASH severity

      cDC2s correlate with lobular inflammation and hepatocyte ballooning
      Increased XCR1+ cDC1s in mice, specific ablation alleviates NASH

      T cellsCXCR6+PD-1+CD8+ T cells increased in NASH

      Auto-aggressive killing of hepatocytes

      IFN-producing CD4+ T cells increased in patients with NASH

      Th17 cells are increased in patients

      Treg cells are decreased in patients
      CD8+ T cells promote NASH by aggravating injury

      IFN-producing CD4+ T cells, promote NASH in mice

      Th17 cells promote NASH in mice

      PD-1+CD8+ T cells promote NASH-HCC transition

      Loss of CD4+ T cells promotes HCC
      NKT cellsIncreased in human NASH and cirrhosisCXCR6+ iNKT cells promote steatohepatitis
      MAIT cellsIncreased in liver, decreased in blood in human NASH

      Accumulation in fibrotic niche of patients with cirrhosis
      Depletion of MAIT cells aggravates NASH suggesting a protective effect
      B cellsBAFF was elevated in serum of patients with NAFLD

      B cells increased in human NAFLD with activated profile

      Increased anti-OSE titres in patients with NASH
      B-cell depletion ameliorated NASH

      IgA-producing B cells drive NASH

      Blocking BAFF in mice ameliorated steatohepatitis

      TNF-α and IL-6 producing B cells promote NASH
      γδ T cellsNo alterations in γδ T-cell numbers in patients with NASHγδ T cells increased in murine NASH

      Tcrd-/- mice are protected from experimental NASH
      PlateletsPlatelet aggregates in human NASH, signs of platelet activationAnti-platelet therapy improved experimental NASH

      Platelets recruited immune cells via CD44 and GPIba
      Anti-OSE, anti-oxidative stress-derived epitopes; BAFF, B cell activating factor; CCR2, C-C chemokine receptor type 2; cDC, conventional dendritic cells; CXCR6, C-X-C chemokine receptor type 6; DAMPs, damage-associated molecular patterns; DCs, dendritic cells; emKC, embryonic KC; IFN, interferon; KC, Kupffer cells; MAIT, mucosal-associated invariant T; moKC, monocyte-derived KC; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NETs, neutrophil extracellular traps; NKT cells, natural killer T cells; OSE, oxidation-specific epitopes; PAMPs, pathogen-associated molecular patterns; PD-1, Programmed cell death protein 1; ROS, reactive oxygen species; Th, T helper; TNF, tumor necrosis factor; TREM-2, Triggering receptor expressed on myeloid cells 2.
      Figure thumbnail gr1
      Fig. 1Triggers of inflammation in NAFLD.
      Intra- and extrahepatic factors trigger inflammation in NAFLD. Hypercaloric diet, obesity, lifestyle, and genetic risk predispose individuals to NAFLD. Overload of hepatocytes with FFAs and increased de novo lipogenesis lead to lipid accumulation in hepatocytes. Fat overload in the liver induces lipotoxicity resulting in ER stress, oxidative stress, ROS production and mitochondrial damage. Stressed hepatocytes release pro-inflammatory mediators and DAMPs resulting in robust immune cell activation and infiltration, further damaging hepatocytes. Different forms of cell death occur as well as hepatocyte senescence, triggering a more pronounced immune response. Cholangiocytes might also release inflammatory mediators. Liver inflammation is also propagated by multiple extrahepatic systems including the adipose tissue, gut, skeletal muscle and bone marrow. BECs, biliary epithelial cells; DAMPs, damage-associated molecular patterns; ER, endoplasmic reticulum; EVs, extracellular vesicles; FGF, fibroblast growth factor; FFAs, free fatty acids; LPS, lipopolysaccharide; NAFLD, non-alcoholic fatty liver disease; SCFAs, short-chain fatty acids; TNF, tumour necrosis factor.
      Inflammation in NAFLD is regulated by multiple intrahepatic and extrahepatic factors (Fig. 1).
      • Schuster S.
      • Cabrera D.
      • Arrese M.
      • Feldstein A.E.
      Triggering and resolution of inflammation in NASH.
      As a multisystem disease, extrahepatic factors include organ crosstalk with inflammatory signals being derived from the gut, adipose tissue, skeletal muscle and bone marrow (Fig. 1).
      • Azzu V.
      • Vacca M.
      • Virtue S.
      • Allison M.
      • Vidal-Puig A.
      Adipose tissue-liver cross talk in the control of whole-body metabolism: implications in nonalcoholic fatty liver disease.
      • Hundertmark J.
      • Krenkel O.
      • Tacke F.
      Adapted immune responses of myeloid-derived cells in fatty liver disease.
      • Marra F.
      • Svegliati-Baroni G.
      Lipotoxicity and the gut-liver axis in NASH pathogenesis.
      • Chakravarthy M.V.
      • Siddiqui M.S.
      • Forsgren M.F.
      • Sanyal A.J.
      Harnessing muscle-liver crosstalk to treat nonalcoholic steatohepatitis.
      • Nachit M.
      • Kwanten W.J.
      • Thissen J.P.
      • Op De Beeck B.
      • Van Gaal L.
      • Vonghia L.
      • et al.
      Muscle fat content is strongly associated with NASH: a longitudinal study in patients with morbid obesity.
      Particularly the intestinal microbiome has garnered much attention in recent years, as NAFLD, NASH and the metabolic syndrome show a strong association with intestinal dysbiosis.
      • Boursier J.
      • Mueller O.
      • Barret M.
      • Machado M.
      • Fizanne L.
      • Araujo-Perez F.
      • et al.
      The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota.
      ,
      • Aron-Wisnewsky J.
      • Vigliotti C.
      • Witjes J.
      • Le P.
      • Holleboom A.G.
      • Verheij J.
      • et al.
      Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders.
      More recently, alterations of the intestinal virome and mycobiome were linked to NAFLD.
      • Demir M.
      • Lang S.
      • Hartmann P.
      • Duan Y.
      • Martin A.
      • Miyamoto Y.
      • et al.
      The fecal mycobiome in non-alcoholic fatty liver disease.
      ,
      • Lang S.
      • Demir M.
      • Martin A.
      • Jiang L.
      • Zhang X.
      • Duan Y.
      • et al.
      Intestinal virome signature associated with severity of nonalcoholic fatty liver disease.
      As an intrahepatic factor, the biliary compartment is increasingly recognised as a potential driver of inflammation in NAFLD (Fig. 1).
      • Zhou T.
      • Kundu D.
      • Robles-Linares J.
      • Meadows V.
      • Sato K.
      • Baiocchi L.
      • et al.
      Feedback signaling between cholangiopathies, ductular reaction, and non-alcoholic fatty liver disease.
      Patients with progressive NAFLD frequently exhibit biliary epithelial cell proliferation and a so-called “ductular reaction” that correlates with portal inflammation, NASH activity and fibrosis.
      • Richardson M.M.
      • Jonsson J.R.
      • Powell E.E.
      • Brunt E.M.
      • Neuschwander-Tetri B.A.
      • Bhathal P.S.
      • et al.
      Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction.
      Ductular reaction refers to histological biliary cell proliferation or hyperplasia and is rooted either in proliferation of cholangiocytes, hepatic progenitor cells or transdifferentiation of hepatocytes to cholangiocytes, which is still a topic of debate.
      • Zhou T.
      • Kundu D.
      • Robles-Linares J.
      • Meadows V.
      • Sato K.
      • Baiocchi L.
      • et al.
      Feedback signaling between cholangiopathies, ductular reaction, and non-alcoholic fatty liver disease.
      Furthermore, NASH patients with cholestasis had more advanced histological disease compared to age- and sex-matched controls without cholestasis.
      • Sorrentino P.
      • Tarantino G.
      • Perrella A.
      • Micheli P.
      • Perrella O.
      • Conca P.
      A clinical-morphological study on cholestatic presentation of nonalcoholic fatty liver disease.
      NASH cirrhosis was characterised by reactive biliary cells that expressed the chemoattractant C-C motif chemokine ligand (CCL)2, suggesting a role in immune cell recruitment.
      • Chiba M.
      • Sasaki M.
      • Kitamura S.
      • Ikeda H.
      • Sato Y.
      • Nakanuma Y.
      Participation of bile ductular cells in the pathological progression of non-alcoholic fatty liver disease.
      In acute biliary injury in mice and cholestatic liver diseases in humans, high CCL2 expression by biliary epithelial cells was confirmed and, mechanistically, cholangiocytes mediated monocyte recruitment via CCL2 and the integrin αvβ6; vice versa, monocytes induced biliary cell proliferation and regeneration.
      • Guillot A.
      • Guerri L.
      • Feng D.
      • Kim S.J.
      • Ahmed Y.A.
      • Paloczi J.
      • et al.
      Bile acid-activated macrophages promote biliary epithelial cell proliferation through integrin alphavbeta6 upregulation following liver injury.
      This suggests an intriguing cellular crosstalk between cholangiocytes and immune cells, potentially driving inflammation in NAFLD (Fig. 1).
      Given that the immune system is fundamental in maintaining liver homeostasis, its role during liver inflammation in the different stages of NAFLD is conflicting and might vary during disease progression. During early stages of NAFLD, inflammatory mechanisms clearly drive disease progression,
      • Schuster S.
      • Cabrera D.
      • Arrese M.
      • Feldstein A.E.
      Triggering and resolution of inflammation in NASH.
      at the same time the liver attempts to resolve inflammation and initiate repair. Ductular reaction is a sign of ongoing liver repair in NAFLD, and the importance of liver macrophages (for example) in this response has been demonstrated.
      • Boulter L.
      • Govaere O.
      • Bird T.G.
      • Radulescu S.
      • Ramachandran P.
      • Pellicoro A.
      • et al.
      Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease.
      Furthermore, regression of disease, e.g. clearance of cell debris or resolution of fibrosis, is actively controlled by inflammatory mechanisms.
      • Schuster S.
      • Cabrera D.
      • Arrese M.
      • Feldstein A.E.
      Triggering and resolution of inflammation in NASH.
      While inflammatory cytokines and mediators are often increased in NASH – suggesting an overall increase in inflammation –patients with NAFLD may develop HCC, pointing to a loss of sufficient anti-tumour immune surveillance and to imbalanced inflammation.
      The pathogenesis of NALFD is multifactorial and comprises metabolic dysregulation, progressive inflammation and fibrogenesis.
      For most of the major immune cell subsets, we now have data derived from human studies and rodent models that report on cell frequencies in the blood and/or liver, as well as mechanistic studies investigating their role in steatohepatitis (Table 1). However, it is noteworthy that whether an altered immune cell subset is cause or consequence in NASH is difficult to disentangle, especially considering that many different rodent models are used, and cell/organ-specific loss-of-function experiments are scarce. Nonetheless, it is worth detailing the proposed roles that different inflammatory cells play in the pathogenesis of NAFLD. Moreover, it should be noted that immune cell-driven NASH is a multistage process, which interconnects different immune cells, analogous to the metastatic cascade. Thus, to develop immune-targeted therapies at the various stages of pathology, it will be important to identify the gaps in our current knowledge and to assemble the ‘big picture’ of inflammation in NAFLD.

      Monocyte and macrophage heterogeneity in NASH

      Most organs are equipped with a population of tissue-resident macrophages with unique functions that are essential in maintaining homeostasis.
      • Wynn T.A.
      • Chawla A.
      • Pollard J.W.
      Macrophage biology in development, homeostasis and disease.
      The liver-resident macrophages, Kupffer cells (KCs), are the most abundant population of tissue-resident macrophages in the human body.
      • Dixon L.J.
      • Barnes M.
      • Tang H.
      • Pritchard M.T.
      • Nagy L.E.
      Kupffer cells in the liver.
      • Krenkel O.
      • Tacke F.
      Liver macrophages in tissue homeostasis and disease.
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      KCs reside in liver sinusoids and are critical sentinels in the liver.
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      In humans, KCs are less well-characterised than in the mouse system and no bona fide KC marker has been identified. A recently published spatial proteogenomic cell atlas of healthy livers found that human monocytes/macrophages form a single continuum without the clear separation of a distinct population identifiable as KCs.
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      In healthy human livers, using single-cell RNA-sequencing, hepatic macrophages clustered as CD68+MARCO+ and CD68+MARCO- subsets.
      • MacParland S.A.
      • Liu J.C.
      • Ma X.Z.
      • Innes B.T.
      • Bartczak A.M.
      • Gage B.K.
      • et al.
      Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.
      ,
      • Zhao J.
      • Zhang S.
      • Liu Y.
      • He X.
      • Qu M.
      • Xu G.
      • et al.
      Single-cell RNA sequencing reveals the heterogeneity of liver-resident immune cells in human.
      The CD68+MARCO+ subset was deemed KCs and expressed genes predominantly involved in immune tolerance, whereas the CD68+MARCO- macrophages resembled pro-inflammatory macrophages in mice with higher expression of pro-inflammatory genes. In addition, 2 distinct human KC populations, which differed based on expression of Timd4 (T cell immunoglobulin and mucin domain-containing 4), were identified using single-cell sequencing.
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      The ontogeny of different human liver macrophages is unknown. Analogous to the mouse system, studies using single-cell technologies have identified resident KCs (CD68+MARCO+Timd4+) and monocyte-derived macrophages (CD68+MARCO-Timd4-) with a more pro-inflammatory gene signature.
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      ,
      • MacParland S.A.
      • Liu J.C.
      • Ma X.Z.
      • Innes B.T.
      • Bartczak A.M.
      • Gage B.K.
      • et al.
      Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations.
      Supporting this view, long-lived donor KCs were identified after HLA-mismatched liver transplantation.
      • Pallett L.J.
      • Burton A.R.
      • Amin O.E.
      • Rodriguez-Tajes S.
      • Patel A.A.
      • Zakeri N.
      • et al.
      Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes.
      In the healthy murine liver, KCs are characterised based on markers such as F4/80, C-type lectin domain family 4 member F (CLEC4F) and Tim4.
      • Scott C.L.
      • Zheng F.
      • De Baetselier P.
      • Martens L.
      • Saeys Y.
      • De Prijck S.
      • et al.
      Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells.
      KCs are yolk sac-derived and maintained locally by self-renewal; in the mouse system, there is little contribution of bone marrow-derived macrophages to the pool of resident KCs.
      • Heymann F.
      • Tacke F.
      Immunology in the liver--from homeostasis to disease.
      In the context of liver inflammation and NASH, the pool of liver macrophages is expanded by the recruitment of monocytes that give rise to a phenotypically distinct population of monocyte-derived macrophages.
      • Guillot A.
      • Tacke F.
      Liver macrophages: old dogmas and new insights.
      In humans, classical (CD14highCD16neg), intermediate (CD14highCD16low) and non-classical monocytes (CD14lowCD16high) are differentiated.
      • Jakubzick C.V.
      • Randolph G.J.
      • Henson P.M.
      Monocyte differentiation and antigen-presenting functions.
      In the mouse system, 2 subsets of monocytes have been described, pro-inflammatory CCR2highLy6Chigh monocytes and patrolling CX3CR1+Ly6Clow monocytes.
      • Guilliams M.
      • Mildner A.
      • Yona S.
      Developmental and functional heterogeneity of monocytes.
      In human NAFLD, macrophages are considered key players and an increase in periportal macrophages was an early histological hallmark.
      • Gadd V.L.
      • Skoien R.
      • Powell E.E.
      • Fagan K.J.
      • Winterford C.
      • Horsfall L.
      • et al.
      The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease.
      C-C motif chemokine receptor (CCR)2+ inflammatory macrophages accumulated in the periportal area in patients with NASH and correlated with disease severity and fibrosis.
      • Krenkel O.
      • Puengel T.
      • Govaere O.
      • Abdallah A.T.
      • Mossanen J.C.
      • Kohlhepp M.
      • et al.
      Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis.
      In mouse models of NASH, monocytes are rapidly recruited to the liver and differentiate into monocyte-derived macrophages (Fig. 2). CCR2+ monocytes are important drivers of liver injury and their therapeutic inhibition reduces the severity of NASH.
      • Krenkel O.
      • Tacke F.
      Liver macrophages in tissue homeostasis and disease.
      ,
      • Krenkel O.
      • Puengel T.
      • Govaere O.
      • Abdallah A.T.
      • Mossanen J.C.
      • Kohlhepp M.
      • et al.
      Therapeutic inhibition of inflammatory monocyte recruitment reduces steatohepatitis and liver fibrosis.
      ,
      • Baeck C.
      • Wehr A.
      • Karlmark K.R.
      • Heymann F.
      • Vucur M.
      • Gassler N.
      • et al.
      Pharmacological inhibition of the chemokine CCL2 (MCP-1) diminishes liver macrophage infiltration and steatohepatitis in chronic hepatic injury.
      Figure thumbnail gr2
      Fig. 2Macrophage heterogeneity in NAFLD.
      The figure shows the different fates and cell subsets of monocytes and macrophages during different stages of NAFLD. Embryonic KCs store lipids, subsequently lose the ability to self-replicate and undergo cell death. Monocyte-derived KCs replenish the niche. At the same time, a distinct population of LAMs emerges in proximity to steatosis or as SAMs in the fibrotic niche. CCR2-expressing pro-inflammatory macrophages infiltrate the liver and propagate injury. Among resident KCs, steatosis favours the population of KC2s. Already on the level of bone marrow precursors, NAFLD is characterised by a unique inflammatory gene expression. emKCs, embryonic Kupffer cells; HSCs, hepatic stellate cells; LAMs, lipid-associated macrophages; moKCs, monocyte-derived Kupffer cells; NAFLD, non-alcoholic fatty liver disease; SAMs, scar-associated macrophages.
      KCs are thought to instigate steatohepatitis as early responders by releasing pro-inflammatory mediators such as tumour necrosis factor (TNF)-α and CCL2.
      • Tosello-Trampont A.C.
      • Landes S.G.
      • Nguyen V.
      • Novobrantseva T.I.
      • Hahn Y.S.
      Kuppfer cells trigger nonalcoholic steatohepatitis development in diet-induced mouse model through tumor necrosis factor-α production.
      Chemical depletion of KCs during onset of experimental NASH attenuated inflammation and reduced the severity of liver damage, emphasising the importance of KCs in NASH initiation.
      • Huang W.
      • Metlakunta A.
      • Dedousis N.
      • Zhang P.
      • Sipula I.
      • Dube J.J.
      • et al.
      Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance.
      In addition, free fatty acid-induced release of mitochondrial DNA triggered NLRP3 (NOD-, LLR- and pyrin domain-containing protein 3) inflammasome activation in KCs, resulting in pro-inflammatory interleukin (IL)-1β secretion and progression of experimental NASH.
      • Pan J.
      • Ou Z.
      • Cai C.
      • Li P.
      • Gong J.
      • Ruan X.Z.
      • et al.
      Fatty acid activates NLRP3 inflammasomes in mouse Kupffer cells through mitochondrial DNA release.
      Damage-associated molecular patterns released from dying hepatocytes and pathogen-associated molecular patterns such as lipopolysaccharide (LPS) have been shown to play an important role in NASH pathogenesis.
      • Friedman S.L.
      • Neuschwander-Tetri B.A.
      • Rinella M.
      • Sanyal A.J.
      Mechanisms of NAFLD development and therapeutic strategies.
      ,
      • Schuster S.
      • Cabrera D.
      • Arrese M.
      • Feldstein A.E.
      Triggering and resolution of inflammation in NASH.
      LPS activated myeloid cells and thus promoted NASH via toll-like receptor 4 (TLR-4) signalling in a methionine- and choline-deficient (MCD) diet mouse model.
      • Csak T.
      • Velayudham A.
      • Hritz I.
      • Petrasek J.
      • Levin I.
      • Lippai D.
      • et al.
      Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice.
      A mechanistic link between intestinal dysbiosis and NAFLD is gut barrier dysfunction, which leads to increased translocation of microbial products from the ‘leaky gut’. Indeed, patients with NASH have increased endotoxin levels,
      • Farhadi A.
      • Gundlapalli S.
      • Shaikh M.
      • Frantzides C.
      • Harrell L.
      • Kwasny M.M.
      • et al.
      Susceptibility to gut leakiness: a possible mechanism for endotoxaemia in non-alcoholic steatohepatitis.
      and NASH severity has been shown to correlate with dysbiosis and a shift in the metabolic function of gut microbiota.
      • Boursier J.
      • Mueller O.
      • Barret M.
      • Machado M.
      • Fizanne L.
      • Araujo-Perez F.
      • et al.
      The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota.
      Lamina propria macrophages expressing the fractalkine receptor CX3CR1 are critical in maintaining intestinal barrier integrity
      • Honda M.
      • Surewaard B.G.J.
      • Watanabe M.
      • Hedrick C.C.
      • Lee W.Y.
      • Brown K.
      • et al.
      Perivascular localization of macrophages in the intestinal mucosa is regulated by Nr4a1 and the microbiome.
      ; gut barrier loss and more severe steatohepatitis were observed in Cx3cr1-deficient mice.
      • Schneider K.M.
      • Bieghs V.
      • Heymann F.
      • Hu W.
      • Dreymueller D.
      • Liao L.
      • et al.
      CX3CR1 is a gatekeeper for intestinal barrier integrity in mice: limiting steatohepatitis by maintaining intestinal homeostasis.
      Furthermore, intestinal dysbiosis disrupts the levels of microbial metabolites, which are critical in maintaining intestinal epithelial integrity and immune homeostasis. In mice fed a high-fat diet, microbiota-derived tryptophan metabolites were depleted in the serum and liver; a lack of these metabolites resulted in increased inflammatory cytokine production by hepatic macrophages and hepatocytes.
      • Krishnan S.
      • Ding Y.
      • Saedi N.
      • Choi M.
      • Sridharan G.V.
      • Sherr D.H.
      • et al.
      Gut microbiota-derived tryptophan metabolites modulate inflammatory response in hepatocytes and macrophages.
      During the early stages of murine NASH, resident KCs partake in lipid storage, which ultimately renders them incapable of self-renewal, triggering their cell death (Fig. 2).
      • Malehmir M.
      • Pfister D.
      • Gallage S.
      • Szydlowska M.
      • Inverso D.
      • Kotsiliti E.
      • et al.
      Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.
      ,
      • Daemen S.
      • Gainullina A.
      • Kalugotla G.
      • He L.
      • Chan M.M.
      • Beals J.W.
      • et al.
      Dynamic shifts in the composition of resident and recruited macrophages influence tissue remodeling in NASH.
      • Remmerie A.
      • Martens L.
      • Thoné T.
      • Castoldi A.
      • Seurinck R.
      • Pavie B.
      • et al.
      Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver.
      • Seidman J.S.
      • Troutman T.D.
      • Sakai M.
      • Gola A.
      • Spann N.J.
      • Bennett H.
      • et al.
      Niche-specific reprogramming of epigenetic landscapes drives myeloid cell diversity in nonalcoholic steatohepatitis.
      • Tran S.
      • Baba I.
      • Poupel L.
      • Dussaud S.
      • Moreau M.
      • Gélineau A.
      • et al.
      Impaired Kupffer cell self-renewal alters the liver response to lipid overload during non-alcoholic steatohepatitis.
      A series of elegant studies recently demonstrated that in murine NASH, embryonic KCs are gradually lost and subsequently replaced by monocyte-derived KCs (Fig. 2).
      • Remmerie A.
      • Martens L.
      • Thoné T.
      • Castoldi A.
      • Seurinck R.
      • Pavie B.
      • et al.
      Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver.
      • Seidman J.S.
      • Troutman T.D.
      • Sakai M.
      • Gola A.
      • Spann N.J.
      • Bennett H.
      • et al.
      Niche-specific reprogramming of epigenetic landscapes drives myeloid cell diversity in nonalcoholic steatohepatitis.
      • Tran S.
      • Baba I.
      • Poupel L.
      • Dussaud S.
      • Moreau M.
      • Gélineau A.
      • et al.
      Impaired Kupffer cell self-renewal alters the liver response to lipid overload during non-alcoholic steatohepatitis.
      Monocyte-derived KCs lacked Timd4 expression, which is reminiscent of monocyte-derived KCs that repopulate the liver after genetic ablation of embryonic KCs in healthy mice.
      • Scott C.L.
      • Zheng F.
      • De Baetselier P.
      • Martens L.
      • Saeys Y.
      • De Prijck S.
      • et al.
      Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells.
      ,
      • Bonnardel J.
      • T'Jonck W.
      • Gaublomme D.
      • Browaeys R.
      • Scott C.L.
      • Martens L.
      • et al.
      Stellate cells, hepatocytes, and endothelial cells imprint the Kupffer cell identity on monocytes colonizing the liver macrophage niche.
      Embryonic and monocyte-derived KCs shared a similar gene expression profile; however, monocyte-derived KCs were more pro-inflammatory, had limited triglyceride storage and promoted liver injury.
      • Tran S.
      • Baba I.
      • Poupel L.
      • Dussaud S.
      • Moreau M.
      • Gélineau A.
      • et al.
      Impaired Kupffer cell self-renewal alters the liver response to lipid overload during non-alcoholic steatohepatitis.
      Similarly, in healthy and steatotic human livers, a population of Timd4- KCs was observed and deemed to be monocyte-derived KCs.
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      The precise mechanism of KC death is unknown. The altered environment during steatosis might impair embryonic KC self-replication, with cells unable to survive because of a lipotoxic gene signature.
      • Huby T.
      • Gautier E.L.
      Immune cell-mediated features of non-alcoholic steatohepatitis.
      An alternative suggestion was that the resident KCs lost their identity.
      • Seidman J.S.
      • Troutman T.D.
      • Sakai M.
      • Gola A.
      • Spann N.J.
      • Bennett H.
      • et al.
      Niche-specific reprogramming of epigenetic landscapes drives myeloid cell diversity in nonalcoholic steatohepatitis.
      Inflammation in NAFLD is regulated by intrahepatic factors (lipid accumulation, lipotoxicity, oxidative stress, cell death mechanisms, activation of cholangiocytes and hepatic stellate cells) and extrahepatic factors (gut-liver axis, adipose tissue inflammation, skeletal muscle, bone marrow precursors).
      In mice, KCs might be a more heterogeneous population than previously thought. Using single-cell transcriptomics and proteomics, 2 distinct subsets of embryonic KCs were identified based on CD206 and endothelial cell adhesion molecule (ESAM) expression. The authors termed these cells KC1 (CD206loESAM-) and KC2 (CD206+ESAM+) (Fig. 2).
      • Blériot C.
      • Barreby E.
      • Dunsmore G.
      • Ballaire R.
      • Chakarov S.
      • Ficht X.
      • et al.
      A subset of Kupffer cells regulates metabolism through the expression of CD36.
      KC2s expressed genes involved in metabolic processes, such as lipid metabolism. Mice fed a high-fat diet (HFD) showed a relative increase of KC2s in steatosis. KC2s induced oxidative stress via scavenger receptor CD36 and their depletion prevented steatosis and reversed obesity in mice.
      • Blériot C.
      • Barreby E.
      • Dunsmore G.
      • Ballaire R.
      • Chakarov S.
      • Ficht X.
      • et al.
      A subset of Kupffer cells regulates metabolism through the expression of CD36.
      Another scavenger receptor, macrophage scavenger receptor 1 (Msr1), expressed by liver macrophages was also identified as a critical molecule in the pathogenesis of NAFLD.
      • Govaere O.
      • Petersen S.K.
      • Martinez-Lopez N.
      • Wouters J.
      • Van Haele M.
      • Mancina R.M.
      • et al.
      Macrophage Scavenger Receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.
      Mice lacking Msr1 had milder steatohepatitis and Msr1-mediated uptake of saturated fatty acids induced a pro-inflammatory response in macrophages. In patients with NAFLD, Msr1 transcript levels were correlated with disease activity.
      • Govaere O.
      • Petersen S.K.
      • Martinez-Lopez N.
      • Wouters J.
      • Van Haele M.
      • Mancina R.M.
      • et al.
      Macrophage Scavenger Receptor 1 mediates lipid-induced inflammation in non-alcoholic fatty liver disease.
      These novel observations indicate that among the resident KCs in homeostasis, functionally distinct subsets exist; during obesity and lipid excess, KC2s might promote steatosis and steatohepatitis. On the spectrum of tolerance and immunogenicity, KC2s might therefore be less tolerogenic, and with respect to NAFLD, the KC2 subset was involved in metabolic disturbance. A potential caveat in these studies is the sole liver focus: blocking scavenger receptors or depleting the KC2 subset might decrease liver steatosis but could lead to lipid deposition elsewhere. While for many years it was known that KCs could alter their phenotype (tolerogenic or inflammatory) depending on the host’s need, the emergence of KC subsets shows there might be tremendous heterogeneity.
      In fibrotic human livers, a distinct macrophage subset was identified based on expression of CD9 and triggering receptor expressed on myeloid cells 2 (TREM-2).
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      Localised in the fibrotic niche, these cells were termed ‘scar-associated macrophages’ (SAMs).
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      In silico trajectory analysis suggested that SAMs were monocyte-derived cells. Their location in the fibrotic niche led to the hypothesis that SAMs might promote liver fibrosis. Indeed, TREM-2+CD9+ SAMs expressed multiple pro-fibrotic genes and were shown to activate hepatic stellate cells in vitro.
      • Ramachandran P.
      • Dobie R.
      • Wilson-Kanamori J.R.
      • Dora E.F.
      • Henderson B.E.P.
      • Luu N.T.
      • et al.
      Resolving the fibrotic niche of human liver cirrhosis at single-cell level.
      The aforementioned proteogenomic liver cell atlas also identified a distinct macrophage subset in healthy human livers, characterised by CD9 and TREM-2 expression, referred to as ‘lipid-associated macrophages’ (LAMs).
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      Furthermore, LAMs were found across different species, including human, murine and macaque livers. In healthy human livers, LAMs were found periportally and in close proximity to bile ducts, while in steatotic livers, these cells accumulated pericentrally in areas of steatosis and increased numerically.
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      In murine steatohepatitis, infiltrating monocytes have at least 2 distinct fates: as monocyte-derived KCs, replenishing lost embryonic KCs, or as LAMs (Fig. 2).
      • Remmerie A.
      • Martens L.
      • Thoné T.
      • Castoldi A.
      • Seurinck R.
      • Pavie B.
      • et al.
      Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver.
      • Seidman J.S.
      • Troutman T.D.
      • Sakai M.
      • Gola A.
      • Spann N.J.
      • Bennett H.
      • et al.
      Niche-specific reprogramming of epigenetic landscapes drives myeloid cell diversity in nonalcoholic steatohepatitis.
      • Tran S.
      • Baba I.
      • Poupel L.
      • Dussaud S.
      • Moreau M.
      • Gélineau A.
      • et al.
      Impaired Kupffer cell self-renewal alters the liver response to lipid overload during non-alcoholic steatohepatitis.
      ,
      • Zigmond E.
      • Varol C.
      Two roads diverge in the sick liver, monocytes travel both.
      The factors controlling which fate an infiltrating monocyte will undergo are unknown, but it is plausible that the liver microenvironment provides the respective signals.
      • Sakai M.
      • Troutman T.D.
      • Seidman J.S.
      • Ouyang Z.
      • Spann N.J.
      • Abe Y.
      • et al.
      Liver-derived signals sequentially reprogram myeloid enhancers to initiate and maintain Kupffer cell identity.
      LAMs in murine NASH express genes involved in antigen presentation, extracellular matrix remodelling, endocytosis, and lysosomal degradation.
      • Xiong X.
      • Kuang H.
      • Ansari S.
      • Liu T.
      • Gong J.
      • Wang S.
      • et al.
      Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis.
      LAMs express a high level of the chemokine osteopontin,
      • Remmerie A.
      • Martens L.
      • Thoné T.
      • Castoldi A.
      • Seurinck R.
      • Pavie B.
      • et al.
      Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver.
      which was previously found to be upregulated in human and murine NASH.
      • Kiefer F.W.
      • Zeyda M.
      • Gollinger K.
      • Pfau B.
      • Neuhofer A.
      • Weichhart T.
      • et al.
      Neutralization of osteopontin inhibits obesity-induced inflammation and insulin resistance.
      • Glass O.
      • Henao R.
      • Patel K.
      • Guy C.D.
      • Gruss H.J.
      • Syn W.K.
      • et al.
      Serum interleukin-8, osteopontin, and monocyte chemoattractant protein 1 are associated with hepatic fibrosis in patients with nonalcoholic fatty liver disease.
      • Honda M.
      • Kimura C.
      • Uede T.
      • Kon S.
      Neutralizing antibody against osteopontin attenuates non-alcoholic steatohepatitis in mice.
      A frequent histological feature of NASH is the formation of hepatic crown-like structures (hCLS), essentially a giant macrophage formation around large lipid droplets.
      • Itoh M.
      • Kato H.
      • Suganami T.
      • Konuma K.
      • Marumoto Y.
      • Terai S.
      • et al.
      Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.
      LAMs have been implicated in the formation of these hCLS
      • Daemen S.
      • Gainullina A.
      • Kalugotla G.
      • He L.
      • Chan M.M.
      • Beals J.W.
      • et al.
      Dynamic shifts in the composition of resident and recruited macrophages influence tissue remodeling in NASH.
      ,
      • Olona A.
      • Mukhopadhyay S.
      • Hateley C.
      • Martinez F.O.
      • Gordon S.
      • Behmoaras J.
      Adipoclast: a multinucleated fat-eating macrophage.
      ; loss of LAMs prevented the formation of hCLS and was associated with increased fibrosis in a high-fat high-sucrose model of NASH.
      • Daemen S.
      • Gainullina A.
      • Kalugotla G.
      • He L.
      • Chan M.M.
      • Beals J.W.
      • et al.
      Dynamic shifts in the composition of resident and recruited macrophages influence tissue remodeling in NASH.
      The biological function of LAMs is incompletely understood. Analogous to SAMs, their presence in the fibrotic area and expression of osteopontin suggested a disease-promoting role.
      • Remmerie A.
      • Martens L.
      • Thoné T.
      • Castoldi A.
      • Seurinck R.
      • Pavie B.
      • et al.
      Osteopontin expression identifies a subset of recruited macrophages distinct from Kupffer cells in the fatty liver.
      In contrast, a recent study identified TREM-2-expressing liver macrophages that regulated energy supply and mitochondrial function.
      • Hou J.
      • Zhang J.
      • Cui P.
      • Zhou Y.
      • Liu C.
      • Wu X.
      • et al.
      TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis.
      TREM-2-deficient mice exhibited accelerated NASH development, and increased body weight and triglyceride levels.
      • Hou J.
      • Zhang J.
      • Cui P.
      • Zhou Y.
      • Liu C.
      • Wu X.
      • et al.
      TREM2 sustains macrophage-hepatocyte metabolic coordination in nonalcoholic fatty liver disease and sepsis.
      LAMs seem to be a conserved population across tissues, as a population of TREM-2-expressing macrophages has been found in adipose tissue of humans and mice with obesity.
      • Jaitin D.A.
      • Adlung L.
      • Thaiss C.A.
      • Weiner A.
      • Li B.
      • Descamps H.
      • et al.
      Lipid-associated macrophages control metabolic homeostasis in a trem2-dependent manner.
      Depletion of TREM-2 macrophages resulted in severe metabolic dysfunction, suggesting a regulatory function of LAMs in adipose tissue. The precise role of LAMs in NAFLD is obscure and future investigations might uncover more heterogeneity with respect to recruited liver macrophages. Furthermore, based on expression of CD9 and TREM-2, it is likely that LAMs and SAMs refer to the same subset of recruited macrophages. Metabolic disturbances during obesity and steatohepatitis already affected myeloid cell precursors in bone marrow by imprinting a distinct NAFLD-associated inflammatory phenotype on the transcriptional level (Fig. 2).
      • Krenkel O.
      • Hundertmark J.
      • Abdallah A.T.
      • Kohlhepp M.
      • Puengel T.
      • Roth T.
      • et al.
      Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related steatohepatitis.
      Overall, altered metabolism induces common gene signature changes in the myeloid compartment of bone marrow, adipose and hepatic tissue, reciprocally nurturing inflammation in NAFLD.

      Metabolic disturbance attracts neutrophils to the liver in NAFLD

      Neutrophils are critical first responders of the innate immune system; however, in chronic inflammatory diseases, their ability to liberate toxic molecules including proteases, oxidants, cytokines and neutrophil extracellular traps (NETs) might contribute to tissue damage.
      • Jorch S.K.
      • Kubes P.
      An emerging role for neutrophil extracellular traps in noninfectious disease.
      • Peiseler M.
      • Kubes P.
      More friend than foe: the emerging role of neutrophils in tissue repair.
      • Soehnlein O.
      • Steffens S.
      • Hidalgo A.
      • Weber C.
      Neutrophils as protagonists and targets in chronic inflammation.
      The role of neutrophils in NASH is incompletely understood. In human NASH, neutrophilic infiltration is frequently observed in the liver.
      • Gadd V.L.
      • Skoien R.
      • Powell E.E.
      • Fagan K.J.
      • Winterford C.
      • Horsfall L.
      • et al.
      The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease.
      ,
      • Rensen S.S.
      • Slaats Y.
      • Nijhuis J.
      • Jans A.
      • Bieghs V.
      • Driessen A.
      • et al.
      Increased hepatic myeloperoxidase activity in obese subjects with nonalcoholic steatohepatitis.
      Furthermore, patients with NASH had higher hepatic expression of neutrophil chemoattractants such as C-X-C motif chemokine ligand (CXCL)1, IL-8 and E-selectin compared to patients with steatosis.
      • Bertola A.
      • Bonnafous S.
      • Anty R.
      • Patouraux S.
      • Saint-Paul M.C.
      • Iannelli A.
      • et al.
      Hepatic expression patterns of inflammatory and immune response genes associated with obesity and NASH in morbidly obese patients.
      Neutrophil elastase plasma concentrations also correlated with increased severity of NASH.
      • Mirea A.M.
      • Toonen E.J.M.
      • van den Munckhof I.
      • Munsterman I.D.
      • Tjwa E.
      • Jaeger M.
      • et al.
      Increased proteinase 3 and neutrophil elastase plasma concentrations are associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes.
      A study using the MCD diet and high-fat high-cholesterol diet models of NASH provided evidence of the early involvement of neutrophils, as their depletion alleviated NASH.
      • Zang S.
      • Wang L.
      • Ma X.
      • Zhu G.
      • Zhuang Z.
      • Xun Y.
      • et al.
      Neutrophils play a crucial role in the early stage of nonalcoholic steatohepatitis via neutrophil elastase in mice.
      Mice deficient in myeloperoxidase or neutrophil elastase had reduced liver damage in the MCD and Western diet models.
      • Pulli B.
      • Ali M.
      • Iwamoto Y.
      • Zeller M.W.
      • Schob S.
      • Linnoila J.J.
      • et al.
      Myeloperoxidase-hepatocyte-stellate cell cross talk promotes hepatocyte injury and fibrosis in experimental nonalcoholic steatohepatitis.
      ,
      • Chen J.
      • Liang B.
      • Bian D.
      • Luo Y.
      • Yang J.
      • Li Z.
      • et al.
      Knockout of neutrophil elastase protects against western diet induced nonalcoholic steatohepatitis in mice by regulating hepatic ceramides metabolism.
      NETosis is a more recently discovered killing mechanism of neutrophils,
      • Brinkmann V.
      • Reichard U.
      • Goosmann C.
      • Fauler B.
      • Uhlemann Y.
      • Weiss D.S.
      • et al.
      Neutrophil extracellular traps kill bacteria.
      ,
      • Papayannopoulos V.
      Neutrophil extracellular traps in immunity and disease.
      with increasing evidence implicating NETs in NASH (Fig. 3). Markers of NET formation were elevated in patients with NASH and correlated with NASH severity.
      • Miele L.
      • Alberelli M.A.
      • Martini M.
      • Liguori A.
      • Marrone G.
      • Cocomazzi A.
      • et al.
      Nonalcoholic fatty liver disease (NAFLD) severity is associated to a nonhemostatic contribution and proinflammatory phenotype of platelets.
      A recent study found NETs during early stages of experimental NASH in the MCD/high-fat diet model, while concurrent DNase treatment alleviated NASH severity.
      • Zhao X.
      • Yang L.
      • Chang N.
      • Hou L.
      • Zhou X.
      • Yang L.
      • et al.
      Neutrophils undergo switch of apoptosis to NETosis during murine fatty liver injury via S1P receptor 2 signaling.
      Interestingly, in a set of in vitro experiments, sphingosine 1 phosphatase receptor 2 redirected neutrophils towards production of NETs. NETs have frequently been reported in patients with obesity, type 2 diabetes and insulin resistance.
      • Jorch S.K.
      • Kubes P.
      An emerging role for neutrophil extracellular traps in noninfectious disease.
      ,
      • Papayannopoulos V.
      Neutrophil extracellular traps in immunity and disease.
      ,
      • D’Abbondanza M.
      • Martorelli E.E.
      • Ricci M.A.
      • De Vuono S.
      • Migliola E.N.
      • Godino C.
      • et al.
      Increased plasmatic NETs by-products in patients in severe obesity.
      ,
      • Delgado-Rizo V.
      • Martínez-Guzmán M.
      • Iñiguez-Gutierrez L.
      • García-Orozco A.
      • Alvarado-Navarro A.
      • Fafutis-Morris M.
      Neutrophil extracellular traps and its implications in inflammation: an overview.
      Moreover, hyperglycaemia seems to predispose neutrophils to produce NETs, which might explain poor healing of diabetic wounds.
      • Wong S.L.
      • Demers M.
      • Martinod K.
      • Gallant M.
      • Wang Y.
      • Goldfine A.B.
      • et al.
      Diabetes primes neutrophils to undergo NETosis, which impairs wound healing.
      However, NETosis in metabolic dysregulation, as it pertains to NASH, might merely be a bystander mechanism without causal effect. In mice fed a HFD, blocking NET formation did not dampen adipose tissue inflammation or liver steatosis.
      • Braster Q.
      • Silvestre Roig C.
      • Hartwig H.
      • Beckers L.
      • den Toom M.
      • Döring Y.
      • et al.
      Inhibition of NET release fails to reduce adipose tissue inflammation in mice.
      Single-cell RNA-sequencing (scRNA-seq) technologies enable the characterisation of the cellular heterogeneity of immune cells in NAFLD-associated inflammation.
      Neutrophil depletion in a murine model of toxic liver injury had no effect on fibrosis development in mice.
      • Moles A.
      • Murphy L.
      • Wilson C.L.
      • Chakraborty J.B.
      • Fox C.
      • Park E.J.
      • et al.
      A TLR2/S100A9/CXCL-2 signaling network is necessary for neutrophil recruitment in acute and chronic liver injury in the mouse.
      As neutrophils are thought to be short-lived cells, it is still unclear if the abundant population of neutrophils in NASH livers is constantly replenished by fresh bone marrow-derived neutrophils, which is more likely – or if these cells can be sustained for days or even weeks in steatohepatitis. Neutrophils are found in abundance during different stages of NAFLD and a disease-promoting role was suggested in various rodent studies, especially at the onset of NASH.
      • Hwang S.
      • Yun H.
      • Moon S.
      • Cho Y.E.
      • Gao B.
      Role of neutrophils in the pathogenesis of nonalcoholic steatohepatitis.
      However, their mere presence does not signify a causal role in human NASH. Lastly, to date, neutrophils are considered a fairly homogenous population despite their known pro- and anti-inflammatory functions, and single-cell studies of neutrophils in liver disease are lacking.
      • Hwang S.
      • Yun H.
      • Moon S.
      • Cho Y.E.
      • Gao B.
      Role of neutrophils in the pathogenesis of nonalcoholic steatohepatitis.
      Figure thumbnail gr3
      Fig. 3Recent advances in understanding cellular immune-mediated mechanisms in NAFLD.
      The figure depicts the reshaping of immune cells during NAFLD, based on recent advances mainly from mouse models. Neutrophils accumulate early during NASH and release NETs that induce Tregs to suppress tumour immunity. DCs, particularly cDC1s increase and corelate with NASH severity. DCs might induce naïve CD8+ T cells in hepatic lymph nodes. Pre-DC precursors are elevated in bone marrow and blood. CD8+ T cells are critical effector cells as CXCR6+CD8+ auto-aggressive T cells directly kill hepatocytes. Platelets form aggregates on Kupffer cells via CD44 and hyaluronan and guide immune cells. B cells, particularly B2 cells contribute to NASH by converting to IgA+ plasma cells and producing anti-OSE antibodies. IgA+ plasma cells promote HCC by suppressing CD8+ T cells. CD4+ T cells are selectively depleted in NASH resulting in hepatocarcinogenesis. Th17 cells drive NASH pathology as pro-inflammatory cells. Unconventional T cells play a role in NASH, but their precise contributions need further evaluation. Anti-OSE, anti-oxidative stress epitopes; cDC, conventional dendritic cells; DCs, dendritic cells; FFAs, free fatty acids; HA, hyaluronan; HCC, hepatocellular carcinoma; KCs, Kupffer cells; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; NETs, neutrophil extracellular traps; NKT, natural killer T; OSE, oxidation-specific epitopes; Teffs, effector T cells; Th17, T helper 17; Tregs, regulatory T cells.

      Dendritic cells are a critical link between multiple disease-driving systems

      Dendritic cells (DCs) connect innate and adaptive immunity by integrating information about tissue environment. As the key antigen-presenting cells of the immune system, DCs can migrate to lymphoid organs to present antigens to T cells.
      • Cabeza-Cabrerizo M.
      • Cardoso A.
      • Minutti C.M.
      • Costa MPd
      • Sousa CRe
      Dendritic cells revisited.
      Within the unique hepatic microenvironment, many DC–T-cell interactions occur directly in the liver; however, hepatic DCs are less efficient at stimulating T-cell activation than DCs in other tissues, favouring tolerance.
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      DCs are a heterogeneous population; 2 subsets of conventional (classical) DCs (cDC1s and cDC2s) and plasmacytoid DCs have been distinguished and are identified by a combination of markers, which differ between mice and humans.
      • Eisenbarth S.C.
      Dendritic cell subsets in T cell programming: location dictates function.
      ,
      • Lurje I.
      • Hammerich L.
      • Tacke F.
      Dendritic cell and T cell crosstalk in liver fibrogenesis and hepatocarcinogenesis: implications for prevention and therapy of liver cancer.
      Hepatic DCs preferentially localise to the periportal region
      • Guilliams M.
      • Bonnardel J.
      • Haest B.
      • Vanderborght B.
      • Wagner C.
      • Remmerie A.
      • et al.
      Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.
      and accumulate in patients with NASH.
      • Haas J.T.
      • Vonghia L.
      • Mogilenko D.A.
      • Verrijken A.
      • Molendi-Coste O.
      • Fleury S.
      • et al.
      Transcriptional network analysis implicates altered hepatic immune function in NASH development and resolution.
      ,
      • Henning J.R.
      • Graffeo C.S.
      • Rehman A.
      • Fallon N.C.
      • Zambirinis C.P.
      • Ochi A.
      • et al.
      Dendritic cells limit fibroinflammatory injury in nonalcoholic steatohepatitis in mice.
      A growing number of studies suggest a disease-promoting role for DCs in NASH based on single-cell sequencing. An abundance of cDCs and specifically chemokine X-C receptor 1 (XCR1)-expressing cDC1s were found in patients with NASH and the frequency of these cells correlated with NASH severity.
      • Deczkowska A.
      • David E.
      • Ramadori P.
      • Pfister D.
      • Safran M.
      • At The B.
      • et al.
      XCR1(+) type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.
      Hepatic cDC1s also increased in mice fed different steatogenic diets.
      • Deczkowska A.
      • David E.
      • Ramadori P.
      • Pfister D.
      • Safran M.
      • At The B.
      • et al.
      XCR1(+) type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.
      In addition, NASH induced an increase of cDC progenitors in the bone marrow and blood. Specific depletion of cDC1s attenuated experimental steatohepatitis in mice. Paired sequencing from liver-draining lymph nodes revealed strong interactions of cDC1s with naïve T cells, promoting inflammatory programming of naïve T cells.
      • Deczkowska A.
      • David E.
      • Ramadori P.
      • Pfister D.
      • Safran M.
      • At The B.
      • et al.
      XCR1(+) type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.
      In addition, cDC2s were also elevated in human and murine steatosis.
      • Deczkowska A.
      • David E.
      • Ramadori P.
      • Pfister D.
      • Safran M.
      • At The B.
      • et al.
      XCR1(+) type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.
      In contrast, a study investigating the role of cDC1s using Batf3-deficient mice, which lack cDC1s, and the MCD diet reported a regulatory role for cDC1s, as Batf3-deficient mice progressed rapidly towards steatohepatitis and had an increased influx of inflammatory cells.
      • Heier E.C.
      • Meier A.
      • Julich-Haertel H.
      • Djudjaj S.
      • Rau M.
      • Tschernig T.
      • et al.
      Murine CD103(+) dendritic cells protect against steatosis progression towards steatohepatitis.
      Fibrosis progression was unaffected, but expression of genes involved in lipid metabolism was altered, and adoptive transfer of CD103+ cDC1s reversed the phenotype.
      • Heier E.C.
      • Meier A.
      • Julich-Haertel H.
      • Djudjaj S.
      • Rau M.
      • Tschernig T.
      • et al.
      Murine CD103(+) dendritic cells protect against steatosis progression towards steatohepatitis.
      One caveat is that the global deletion of Batf3 might have other effects on liver inflammation. An emerging concept is the influence of the metabolic microenvironment on function and polarisation of DCs.
      • Brombacher E.C.
      • Everts B.
      Shaping of dendritic cell function by the metabolic micro-environment.
      DCs with higher lipid content showed a more pro-inflammatory phenotype in patients with NASH compared to DCs containing lower levels of lipids.
      • Ibrahim J.
      • Nguyen A.H.
      • Rehman A.
      • Ochi A.
      • Jamal M.
      • Graffeo C.S.
      • et al.
      Dendritic cell populations with different concentrations of lipid regulate tolerance and immunity in mouse and human liver.
      A study analysing the transcriptional network and immune profiles in patients with NASH identified changes in genes regulating inflammatory processes, antigen presentation and cytotoxic cells.
      • Haas J.T.
      • Vonghia L.
      • Mogilenko D.A.
      • Verrijken A.
      • Molendi-Coste O.
      • Fleury S.
      • et al.
      Transcriptional network analysis implicates altered hepatic immune function in NASH development and resolution.
      Particularly, changes in cDC1s, cDC2s and CD8+ T cells were identified, and cDC2s and CD8+ T cells correlated with lobular inflammation and hepatocyte ballooning. Despite these efforts, the precise role of DCs in NAFLD is still ambiguous, reflecting the different subsets studied and the lack of specific markers, which complicate the interpretation of depletion experiments.
      • Peiseler M.
      • Tacke F.
      Inflammatory mechanisms underlying nonalcoholic steatohepatitis and the transition to hepatocellular carcinoma.
      However, recent patient-derived data showing an increase of cDCs suggest a disease-promoting role in the inflammatory cascade.

      Inflammation in NASH is driven by “auto-aggressive” effector T cells, T helper cells and activated B cells

      Lymphocytic infiltration is frequently observed in liver biopsies of patients with NASH, often as focal lymphocytic aggregates consisting of T and B cells, resembling ectopic lymphoid structures.
      • Sutti S.
      • Albano E.
      Adaptive immunity: an emerging player in the progression of NAFLD.
      CD8+ T cells are critical effector cells of the adaptive immune system, which are important for killing cancerous or infected cells in an MHC I-restricted antigen-specific fashion.
      • Wong P.
      • Pamer E.G.
      CD8 T cell responses to infectious pathogens.
      Patients with NASH had a striking hepatic infiltration of CD8+ T cells.
      • Bhattacharjee J.
      • Kirby M.
      • Softic S.
      • Miles L.
      • Salazar-Gonzalez R.M.
      • Shivakumar P.
      • et al.
      Hepatic natural killer T-cell and CD8+ T-cell signatures in mice with nonalcoholic steatohepatitis.
      ,
      • Ghazarian M.
      • Revelo X.S.
      • Nøhr M.K.
      • Luck H.
      • Zeng K.
      • Lei H.
      • et al.
      Type I interferon responses drive intrahepatic T cells to promote metabolic syndrome.
      In another study, increased CD8+ T cells in liver and blood were reported and circulating CD8+ T cells showed a marked activation profile with increased expression of perforin, interferon (IFN)-γ and TNF-α.
      • Haas J.T.
      • Vonghia L.
      • Mogilenko D.A.
      • Verrijken A.
      • Molendi-Coste O.
      • Fleury S.
      • et al.
      Transcriptional network analysis implicates altered hepatic immune function in NASH development and resolution.
      In Rag1-/- mice, which lack mature B cells, T cells and natural killer (NK) T cells, dietary NASH induced by choline-deficient high-fat diet (CDHFD) feeding is milder with reduced steatosis, parenchymal injury and inflammation.
      • Wolf M.J.
      • Adili A.
      • Piotrowitz K.
      • Abdullah Z.
      • Boege Y.
      • Stemmer K.
      • et al.
      Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
      CD8+ T cells and NKT cells promoted liver damage in concert,
      • Bhattacharjee J.
      • Kirby M.
      • Softic S.
      • Miles L.
      • Salazar-Gonzalez R.M.
      • Shivakumar P.
      • et al.
      Hepatic natural killer T-cell and CD8+ T-cell signatures in mice with nonalcoholic steatohepatitis.
      ,
      • Wolf M.J.
      • Adili A.
      • Piotrowitz K.
      • Abdullah Z.
      • Boege Y.
      • Stemmer K.
      • et al.
      Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
      and depletion of CD8+ T cells or NKT cell deficiency led to milder steatohepatitis. Intrahepatic CD8+ T cells were identified as regulators of hepatic gluconeogenesis.
      • Ghazarian M.
      • Revelo X.S.
      • Nøhr M.K.
      • Luck H.
      • Zeng K.
      • Lei H.
      • et al.
      Type I interferon responses drive intrahepatic T cells to promote metabolic syndrome.
      In early stages of steatosis, CD8+ T cells did not meaningfully contribute to liver injury but mediated metabolic dysregulation and insulin resistance. CD8-deficient mice had improved metabolic parameters and adoptive transfer of CD8+ T cells isolated from HFD-fed livers induced worsening of glucose metabolism.
      • Ghazarian M.
      • Revelo X.S.
      • Nøhr M.K.
      • Luck H.
      • Zeng K.
      • Lei H.
      • et al.
      Type I interferon responses drive intrahepatic T cells to promote metabolic syndrome.
      CD8+ T cells further exhibited the ability to directly activate hepatic stellate cells in vivo and in vitro in mice with NASH.
      • Breuer D.A.
      • Pacheco M.C.
      • Washington M.K.
      • Montgomery S.A.
      • Hasty A.H.
      • Kennedy A.J.
      CD8(+) T cells regulate liver injury in obesity-related nonalcoholic fatty liver disease.
      This study noted a difference regarding the role of CD8+ T cells between models of obese and lean NASH. In mice fed a CDHFD, a lean NASH model, intrahepatic CD8+ T cells were elevated, however, long-term depletion had no effect on liver injury or hepatic stellate cell activation, contrasting with the results from obese NASH mice and indicating that metabolic conditions influence T-cell phenotype.
      • Breuer D.A.
      • Pacheco M.C.
      • Washington M.K.
      • Montgomery S.A.
      • Hasty A.H.
      • Kennedy A.J.
      CD8(+) T cells regulate liver injury in obesity-related nonalcoholic fatty liver disease.
      Using single-cell sequencing to characterise the intrahepatic T-cell repertoire in patients with NASH and in mice fed different NASH diets, a recent study identified a conserved and largely expanded population of CD8+ T cells with markers of tissue residency (CXCR6), exhaustion (programmed cell death 1 [PD-1]) and effector function (granzyme B).
      • Dudek M.
      • Pfister D.
      • Donakonda S.
      • Filpe P.
      • Schneider A.
      • Laschinger M.
      • et al.
      Auto-aggressive CXCR6(+) CD8 T cells cause liver immune pathology in NASH.
      In a set of mechanistic experiments, the authors identified increased IL-15 signalling in the steatotic liver, which induced downregulation of the transcription factor FOXO1 (forkhead box O1) and upregulation of CXCR6. Metabolic stimuli including acetate and extracellular ATP from dying hepatocytes promoted these cells to release pro-inflammatory cytokines. Furthermore, CXCR6+ CD8+ T cells induced the direct killing of hepatocytes via Fas-FasL interactions (Fig. 3).
      • Dudek M.
      • Pfister D.
      • Donakonda S.
      • Filpe P.
      • Schneider A.
      • Laschinger M.
      • et al.
      Auto-aggressive CXCR6(+) CD8 T cells cause liver immune pathology in NASH.
      This killing mechanism was referred to as “auto-aggression” as it was independent of MHC-class-I and thus fundamentally different from the antigen-specific immunity provided by protective CD8+ T cells. Auto-aggressive T cells underline the unavoidable reciprocal impact of metabolism and immunity in NASH as these T cells are rendered ‘hyperactivated’ by metabolic stimuli. It is unclear if there is an evolutionary benefit of having “auto-aggressive” T cells, which might be overturned in NASH. It is intriguing to speculate that the auto-aggressive CD8+ T cells found in NASH are primed by aberrant XCR1-expressing DCs, leading to a feed forward loop of liver inflammation. The current body of literature identifies CD8+ T cells as important effector cells in NAFLD: these cells promote metabolic dysregulation and insulin resistance early during steatosis, while “auto-aggressive” T cells directly cause hepatocyte death at later stages.
      Cell death of resident embryonic Kupffer cells and replenishment by monocyte-derived phagocytes is an emerging concept in experimental steatohepatitis.
      CD4+ T helper (Th) cells are broadly categorised into Th1, Th2, Th17 and regulatory T cells (Tregs); their balance is critical to maintaining liver immune tolerance, and dysregulation of regulatory and effector T helper cells is a hallmark of chronic liver diseases.
      • Ficht X.
      • Iannacone M.
      Immune surveillance of the liver by T cells.
      The role of CD4+ T cells in NAFLD is less clear and studies using single-cell technologies will likely provide novel insights in the coming years. Numerous studies reported that Th1 and Th17 cells are elevated in patients with NASH.
      • Hirsova P.
      • Bamidele A.O.
      • Wang H.
      • Povero D.
      • Revelo X.S.
      Emerging roles of T cells in the pathogenesis of nonalcoholic steatohepatitis and hepatocellular carcinoma.
      Particularly, IFN-γ-expressing CD4+ T cells were enriched in NASH and mice deficient in IFN-γ fed the MCD diet had milder steatohepatitis and reduced infiltration of inflammatory macrophages.
      • Luo X.Y.
      • Takahara T.
      • Kawai K.
      • Fujino M.
      • Sugiyama T.
      • Tsuneyama K.
      • et al.
      IFN-γ deficiency attenuates hepatic inflammation and fibrosis in a steatohepatitis model induced by a methionine- and choline-deficient high-fat diet.
      ,
      • Rau M.
      • Schilling A.K.
      • Meertens J.
      • Hering I.
      • Weiss J.
      • Jurowich C.
      • et al.
      Progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis is marked by a higher frequency of Th17 cells in the liver and an increased Th17/resting regulatory T cell ratio in peripheral blood and in the liver.
      In a humanised mouse model fed a high-fat high-calorie diet, human CD4+ T cells accumulated in the liver and their depletion reduced liver inflammation and fibrosis.
      • Her Z.
      • Tan J.H.L.
      • Lim Y.S.
      • Tan S.Y.
      • Chan X.Y.
      • Tan W.W.S.
      • et al.
      CD4(+) T cells mediate the development of liver fibrosis in high fat diet-induced NAFLD in humanized mice.
      OX40 is a costimulatory molecule that promotes T-cell division and survival.
      • Croft M.
      • So T.
      • Duan W.
      • Soroosh P.
      The significance of OX40 and OX40L to T-cell biology and immune disease.
      Mice fed different steatogenic diets had increased OX40 expression in CD4+ T cells and OX40-deficient mice were protected from steatohepatitis via Th1 and Th17 differentiation and suppressed monocyte recruitment.
      • Sun G.
      • Jin H.
      • Zhang C.
      • Meng H.
      • Zhao X.
      • Wei D.
      • et al.
      OX40 regulates both innate and adaptive immunity and promotes nonalcoholic steatohepatitis.
      Furthermore, patients with NASH had increased OX40 levels in the serum.
      In humans, intrahepatic Th17 cells were increased across the entire spectrum of NAFLD, and higher in NASH compared to steatosis.
      • Rau M.
      • Schilling A.K.
      • Meertens J.
      • Hering I.
      • Weiss J.
      • Jurowich C.
      • et al.
      Progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis is marked by a higher frequency of Th17 cells in the liver and an increased Th17/resting regulatory T cell ratio in peripheral blood and in the liver.
      Bariatric surgery improved steatosis, NASH, and led to a normalisation of Th17 cell frequencies.
      • Rau M.
      • Schilling A.K.
      • Meertens J.
      • Hering I.
      • Weiss J.
      • Jurowich C.
      • et al.
      Progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis is marked by a higher frequency of Th17 cells in the liver and an increased Th17/resting regulatory T cell ratio in peripheral blood and in the liver.
      In mice lacking IL-17, HFD or MCD diet feeding reduced hepatocellular damage and liver inflammation, while steatosis was unchanged.
      • Giles D.A.
      • Moreno-Fernandez M.E.
      • Stankiewicz T.E.
      • Cappelletti M.
      • Huppert S.S.
      • Iwakura Y.
      • et al.
      Regulation of inflammation by IL-17A and IL-17F modulates non-alcoholic fatty liver disease pathogenesis.
      • Harley I.T.
      • Stankiewicz T.E.
      • Giles D.A.
      • Softic S.
      • Flick L.M.
      • Cappelletti M.
      • et al.
      IL-17 signaling accelerates the progression of nonalcoholic fatty liver disease in mice.
      • Tang Y.
      • Bian Z.
      • Zhao L.
      • Liu Y.
      • Liang S.
      • Wang Q.
      • et al.
      Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease.
      Interestingly, a microbiome signature known to induce IL-17 production was sufficient to accelerate liver damage.
      • Harley I.T.
      • Stankiewicz T.E.
      • Giles D.A.
      • Softic S.
      • Flick L.M.
      • Cappelletti M.
      • et al.
      IL-17 signaling accelerates the progression of nonalcoholic fatty liver disease in mice.
      A subset of inflammatory hepatic CXCR3+Th17 (ihTh17) cells promoted obesity and steatohepatitis in murine models.
      • Moreno-Fernandez M.E.
      • Giles D.A.
      • Oates J.R.
      • Chan C.C.
      • Damen M.
      • Doll J.R.
      • et al.
      PKM2-dependent metabolic skewing of hepatic Th17 cells regulates pathogenesis of non-alcoholic fatty liver disease.
      These ihTh17 cells exhibited enhanced glycolytic activity and production of inflammatory cytokines IFN-γ, TNF-α and IL-17. Therapeutically, glycolysis inhibition, ihTh17 cell-specific ablation of IFN-γ or interference with the CXCR3 axis protected against steatohepatitis.
      • Moreno-Fernandez M.E.
      • Giles D.A.
      • Oates J.R.
      • Chan C.C.
      • Damen M.
      • Doll J.R.
      • et al.
      PKM2-dependent metabolic skewing of hepatic Th17 cells regulates pathogenesis of non-alcoholic fatty liver disease.
      Moreover, feeding mice a high-fat high-fructose diet resulted in hepatic and adipose tissue infiltration of Th17 cells and reduced adipose tissue Tregs.
      • Van Herck M.A.
      • Vonghia L.
      • Kwanten W.J.
      • Jule Y.
      • Vanwolleghem T.
      • Ebo D.G.
      • et al.
      Diet reversal and immune modulation show key role for liver and adipose tissue T cells in murine nonalcoholic steatohepatitis.
      These T-cell disruptions in adipose tissue and liver were sustained even after switching mice to normal chow.
      Tregs, important immune regulatory cells, were reduced in patients with NAFLD compared to healthy controls.
      • Rau M.
      • Schilling A.K.
      • Meertens J.
      • Hering I.
      • Weiss J.
      • Jurowich C.
      • et al.
      Progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis is marked by a higher frequency of Th17 cells in the liver and an increased Th17/resting regulatory T cell ratio in peripheral blood and in the liver.
      In murine models, data on the role of Tregs are controversial. In one study, Tregs were reduced in HFD-fed mice, while adoptive Treg transfer before LPS challenge reduced subsequent liver inflammation.
      • Ma X.
      • Hua J.
      • Mohamood A.R.
      • Hamad A.R.
      • Ravi R.
      • Li Z.
      A high-fat diet and regulatory T cells influence susceptibility to endotoxin-induced liver injury.
      Tregs were shown to be more prone to apoptosis in steatohepatitis.
      • Sutti S.
      • Albano E.
      Adaptive immunity: an emerging player in the progression of NAFLD.
      Furthermore, increased levels of leptin reduced Treg frequency in obese individuals and mice.
      • Francisco V.
      • Pino J.
      • Campos-Cabaleiro V.
      • Ruiz-Fernández C.
      • Mera A.
      • Gonzalez-Gay M.A.
      • et al.
      Obesity, fat mass and immune system: role for leptin.
      In contrast, a recent study reported increased intrahepatic Tregs in mice fed a CDHFD and given a diethylnitrosamine injection.
      • Wang H.
      • Zhang H.
      • Wang Y.
      • Brown Z.J.
      • Xia Y.
      • Huang Z.
      • et al.
      Regulatory T-cell and neutrophil extracellular trap interaction contributes to carcinogenesis in non-alcoholic steatohepatitis.
      Furthermore, high-fat high-carbohydrate feeding of BALB/c mice resulted in increased intrahepatic Tregs, however, adoptive Treg transfer exacerbated experimental NASH.
      • Dywicki J.
      • Buitrago-Molina L.E.
      • Noyan F.
      • Davalos-Misslitz A.C.
      • Hupa-Breier K.L.
      • Lieber M.
      • et al.
      The detrimental role of regulatory T cells in nonalcoholic steatohepatitis.
      Patients with NAFLD exhibit a pronounced imbalance between intrahepatic Th17 cells and Tregs
      • Rau M.
      • Schilling A.K.
      • Meertens J.
      • Hering I.
      • Weiss J.
      • Jurowich C.
      • et al.
      Progression from nonalcoholic fatty liver to nonalcoholic steatohepatitis is marked by a higher frequency of Th17 cells in the liver and an increased Th17/resting regulatory T cell ratio in peripheral blood and in the liver.
      ; however, it is mechanistically unclear if this is driven by local immune dysregulation or induced by extrahepatic factors. Interestingly, recent work identified microbial bile acid metabolism as a critical regulator of the Th17/Treg balance in the intestine.
      • Song X.
      • Sun X.
      • Oh S.F.
      • Wu M.
      • Zhang Y.
      • Zheng W.
      • et al.
      Microbial bile acid metabolites modulate gut RORgamma(+) regulatory T cell homeostasis.
      ,
      • Hang S.
      • Paik D.
      • Yao L.
      • Kim E.
      • Trinath J.
      • Lu J.
      • et al.
      Bile acid metabolites control TH17 and Treg cell differentiation.
      Furthermore, secondary bile acids were critical in inducing peripheral Tregs in the colon and thereby regulating colonic immune homeostasis.
      • Campbell C.
      • McKenney P.T.
      • Konstantinovsky D.
      • Isaeva O.I.
      • Schizas M.
      • Verter J.
      • et al.
      Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells.
      It remains to be investigated if altered bile acid metabolism in NAFLD also skews the T-cell balance locally in the liver or if these effects are regulated distally in the colon via the gut-liver axis.
      T-cell alterations were also found in the intestine of patients with NAFLD, with fewer CD4+ and CD8+ T cells in patients, and an increase in inflammatory cytokines and disruption of tight junctions, potentially linking intestinal inflammation to gut barrier dysfunction.
      • Jiang W.
      • Wu N.
      • Wang X.
      • Chi Y.
      • Zhang Y.
      • Qiu X.
      • et al.
      Dysbiosis gut microbiota associated with inflammation and impaired mucosal immune function in intestine of humans with non-alcoholic fatty liver disease.
      Accordingly, treatment with a gut local anti-inflammatory agent dampened bowel inflammation, decreased insulin resistance and improved liver steatosis in mice fed a HFD.
      • Luck H.
      • Tsai S.
      • Chung J.
      • Clemente-Casares X.
      • Ghazarian M.
      • Revelo X.S.
      • et al.
      Regulation of obesity-related insulin resistance with gut anti-inflammatory agents.
      B lymphocytes have important immunological functions, including production of antibodies, antigen presentation and cytokine secretion. The B cell compartment of the liver is incompletely understood, likely owing to the fact that B cells make up only 5% of intrahepatic lymphocytes in humans; however, their numbers in mice are much higher.
      • Freitas-Lopes M.A.
      • Mafra K.
      • David B.A.
      • Carvalho-Gontijo R.
      • Menezes G.B.
      Differential location and distribution of hepatic immune cells.
      B cells are a heterogeneous population that can be broadly divided into B1 and B2 cells. B2 cells arise from haematopoietic progenitors in bone marrow and migrate to secondary lymphoid organs, where they are aided by CD4+ T helper cells to generate high-affinity antibodies, before maturing into antibody-producing plasma cells. B1 cells are more ‘innate-like’ and produce ‘natural’ antibodies rapidly following antigen encounter, independent of T-cell help.
      • Cyster J.G.
      • Allen C.D.C.
      B cell responses: cell interaction dynamics and decisions.
      An additional population of B cells with immunosuppressive properties are regulatory B cells.
      • Catalan D.
      • Mansilla M.A.
      • Ferrier A.
      • Soto L.
      • Oleinika K.
      • Aguillon J.C.
      • et al.
      Immunosuppressive mechanisms of regulatory B cells.
      In NASH, B cells are found within the inflammatory infiltrate of patients and are activated during the onset of steatohepatitis.
      • Bruzzi S.
      • Sutti S.
      • Giudici G.
      • Burlone M.E.
      • Ramavath N.N.
      • Toscani A.
      • et al.
      B2-Lymphocyte responses to oxidative stress-derived antigens contribute to the evolution of nonalcoholic fatty liver disease (NAFLD).
      In patients with NASH, B cells accumulated in the liver, showed an activated profile and correlated with disease severity.
      • Barrow F.
      • Khan S.
      • Fredrickson G.
      • Wang H.
      • Dietsche K.
      • Parthiban P.
      • et al.
      Microbiota-driven activation of intrahepatic B cells aggravates NASH through innate and adaptive signaling.
      In mice, the transcriptional landscape of B cells reflected a broadly activated pro-inflammatory phenotype, including increased levels of pro-inflammatory cytokines, such as TNF-α and IL-6. B-cell depletion ameliorated NASH induced by a high-fat high-carbohydrate diet. Mechanistically, B cell-specific deletion of MyD88 (myeloid differentiation primary response 88) ameliorated NASH, while the B-cell response also involved direct B cell-receptor signalling, indicating both innate and adaptive mechanisms of activation in NASH.
      • Barrow F.
      • Khan S.
      • Fredrickson G.
      • Wang H.
      • Dietsche K.
      • Parthiban P.
      • et al.
      Microbiota-driven activation of intrahepatic B cells aggravates NASH through innate and adaptive signaling.
      Lastly, faecal microbiota transplantation of human NAFLD microbiota into mice led to accumulation and activation of intrahepatic B cells, indicating that the microbiome might partake in activating B cells in NASH.
      • Barrow F.
      • Khan S.
      • Fredrickson G.
      • Wang H.
      • Dietsche K.
      • Parthiban P.
      • et al.
      Microbiota-driven activation of intrahepatic B cells aggravates NASH through innate and adaptive signaling.
      Lipid-associated macrophages (LAMs) expressing TREM2 and CD9 accumulate in steatotic livers.
      Furthering the role of B2 cells in NASH, serum levels of B cell-activating factor (BAFF), which controls survival of B2 cells,
      • Mackay F.
      • Browning J.L.
      BAFF: a fundamental survival factor for B cells.
      were elevated in patients with NASH and correlated with the degree of fibrosis.
      • Miyake T.
      • Abe M.
      • Tokumoto Y.
      • Hirooka M.
      • Furukawa S.
      • Kumagi T.
      • et al.
      B cell-activating factor is associated with the histological severity of nonalcoholic fatty liver disease.
      In mice, neutralising BAFF reduced steatohepatitis.
      • Bruzzi S.
      • Sutti S.
      • Giudici G.
      • Burlone M.E.
      • Ramavath N.N.
      • Toscani A.
      • et al.
      B2-Lymphocyte responses to oxidative stress-derived antigens contribute to the evolution of nonalcoholic fatty liver disease (NAFLD).
      Another study found IgA-expressing plasma cells, which differentiate from B2 cells after activation, to be increased in livers of patients with NASH (Fig. 3).
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      Interestingly, these cells expressed high levels of IL-10 and promoted hepatocarcinogenesis by suppressing CD8+ T-cell responses. Furthermore, IgA levels were shown to predict the degree of fibrosis in NASH
      • McPherson S.
      • Henderson E.
      • Burt A.D.
      • Day C.P.
      • Anstee Q.M.
      Serum immunoglobulin levels predict fibrosis in patients with non-alcoholic fatty liver disease.
      and mice lacking IgA exhibited milder steatohepatitis.
      • Shalapour S.
      • Lin X.J.
      • Bastian I.N.
      • Brain J.
      • Burt A.D.
      • Aksenov A.A.
      • et al.
      Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity.
      B cells in murine NASH co-localised with T-cell clusters, presented epitopes to CD4+ T cells (in turn activating the myeloid compartment via pro-inflammatory mediators), and developed into plasma cells producing anti-OSE (oxidative stress-derived epitopes) IgG.
      • Bruzzi S.
      • Sutti S.
      • Giudici G.
      • Burlone M.E.
      • Ramavath N.N.
      • Toscani A.
      • et al.
      B2-Lymphocyte responses to oxidative stress-derived antigens contribute to the evolution of nonalcoholic fatty liver disease (NAFLD).
      Patients with NASH had elevated anti-OSE antibody titers, which positively correlated with liver inflammation.
      • Albano E.
      • Mottaran E.
      • Vidali M.
      • Reale E.
      • Saksena S.
      • Occhino G.
      • et al.
      Immune response towards lipid peroxidation products as a predictor of progression of non-alcoholic fatty liver disease to advanced fibrosis.
      While the presence of B cells in NASH is established, potential mechanisms of activation and effector function are less well understood.
      • Barrow F.
      • Khan S.
      • Wang H.
      • Revelo X.S.
      The emerging role of B cells in the pathogenesis of NAFLD.
      B2 cells accumulated in adipose tissue, contributing to local inflammation via the production of pathogenic IgG antibodies, activation of T cells and local macrophages and signalling via the leukotriene B4 receptor.
      • Ying W.
      • Wollam J.
      • Ofrecio J.M.
      • Bandyopadhyay G.
      • El Ouarrat D.
      • Lee Y.S.
      • et al.
      Adipose tissue B2 cells promote insulin resistance through leukotriene LTB4/LTB4R1 signaling.
      ,
      • Winer D.A.
      • Winer S.
      • Shen L.
      • Wadia P.P.
      • Yantha J.
      • Paltser G.
      • et al.
      B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies.
      Furthermore, loss of regulatory B cells during obesity resulted in adipose tissue inflammation and insulin resistance.
      • Nishimura S.
      • Manabe I.
      • Takaki S.
      • Nagasaki M.
      • Otsu M.
      • Yamashita H.
      • et al.
      Adipose natural regulatory B cells negatively control adipose tissue inflammation.
      Recent single-cell data suggests B cells could activate hepatic stellate cells, as ligand-receptor interactome analyses of NASH livers identified increased CXCL12 expression by activated stellate cells; a potential target could be CXCR4 expressed by B cells.
      • Xiong X.
      • Kuang H.
      • Ansari S.
      • Liu T.
      • Gong J.
      • Wang S.
      • et al.
      Landscape of intercellular crosstalk in healthy and NASH liver revealed by single-cell secretome gene analysis.
      ,
      • Barrow F.
      • Khan S.
      • Wang H.
      • Revelo X.S.
      The emerging role of B cells in the pathogenesis of NAFLD.
      A role for B cells in activating stellate cells and promoting liver fibrosis was previously reported.
      • Thapa M.
      • Chinnadurai R.
      • Velazquez V.M.
      • Tedesco D.
      • Elrod E.
      • Han J.H.
      • et al.
      Liver fibrosis occurs through dysregulation of MyD88-dependent innate B-cell activity.
      Hence, in addition to antibody production, B cell-derived cytokine production and stellate cell activation seem to be important in NASH and NASH-associated fibrosis.
      • Barrow F.
      • Khan S.
      • Wang H.
      • Revelo X.S.
      The emerging role of B cells in the pathogenesis of NAFLD.
      Collectively, recent studies relying on single-cell transcriptomic data have clearly established a role for CD8+ T cells, CD4+ T cells and B cells in the immune pathogenesis of NASH.

      Innate lymphoid cells as novel participants in NASH pathogenesis

      Innate lymphoid cells (ILCs) are lymphocytes that lack the antigen-specific receptors of T and B cells. As tissue-resident cells, ILCs are integrated into tissues and their ability to rapidly respond to stress signals by producing cytokines makes them an integral part of liver immunity.
      • Wang Y.
      • Zhang C.
      The roles of liver-resident lymphocytes in liver diseases.
      ILCs consists of 5 subsets – NK cells, ILC1s, ILC2s, ILC3s and lymphoid tissue inducer cells
      • Vivier E.
      • Artis D.
      • Colonna M.
      • Diefenbach A.
      • Di Santo J.P.
      • Eberl G.
      • et al.
      Innate lymphoid cells: 10 years on.
      – based on developmental and functional trajectories. The tissue residency phenotype of ILCs emphasises that these cells are critical in organ homeostasis and metabolism, besides their important immune function. Emerging evidence links ILCs to metabolic dysregulation in obesity, insulin resistance and NAFLD.
      • Chen Y.
      • Tian Z.
      Roles of hepatic innate and innate-like lymphocytes in nonalcoholic steatohepatitis.
      NK cells are critical for host defense and kill target cells by secreting perforins and granzymes.
      • Orange J.S.
      Formation and function of the lytic NK-cell immunological synapse.
      In humans, these cells are further divided into CD56dim cells, which circulate and can directly kill pathogens, or CD56bright cells, which are found in tissues and mainly act via production of cytokines.
      • Freud A.G.
      • Mundy-Bosse B.L.
      • Yu J.
      • Caligiuri M.A.
      The broad spectrum of human natural killer cell diversity.
      CD56dim NK cells were decreased in peripheral blood but increased in the liver of patients with NAFLD.
      • Diedrich T.
      • Kummer S.
      • Galante A.
      • Drolz A.
      • Schlicker V.
      • Lohse A.W.
      • et al.
      Characterization of the immune cell landscape of patients with NAFLD.
      Other studies failed to detect differences in the frequency of peripheral and hepatic NK cells between patients with NASH and healthy individuals.
      • Martínez-Chantar M.L.
      • Delgado T.C.
      • Beraza N.
      Revisiting the role of natural killer cells in non-alcoholic fatty liver disease.
      ,
      • Stiglund N.
      • Strand K.
      • Cornillet M.
      • Stål P.
      • Thorell A.
      • Zimmer C.L.
      • et al.
      Retained NK cell phenotype and functionality in non-alcoholic fatty liver disease.
      In keeping with the multisystem character of inflammation in NAFLD, NK cells accumulated in adipose tissue of mice fed a HFD, where they produced pro-inflammatory cytokines and polarised adipose tissue macrophages towards a pro-inflammatory phenotype.
      • Boulenouar S.
      • Michelet X.
      • Duquette D.
      • Alvarez D.
      • Hogan A.E.
      • Dold C.
      • et al.
      Adipose type one innate lymphoid cells regulate macrophage homeostasis through targeted cytotoxicity.
      ,
      • Lee B.C.
      • Kim M.S.
      • Pae M.
      • Yamamoto Y.
      • Eberlé D.
      • Shimada T.
      • et al.
      Adipose natural killer cells regulate adipose tissue macrophages to promote insulin resistance in obesity.
      NK cell depletion improved insulin resistance in mice, identifying NK cells as potential mediators of adipose tissue inflammation.
      • Lee B.C.
      • Kim M.S.
      • Pae M.
      • Yamamoto Y.
      • Eberlé D.
      • Shimada T.
      • et al.
      Adipose natural killer cells regulate adipose tissue macrophages to promote insulin resistance in obesity.
      While these studies provide evidence that NK cells promote obesity and insulin resistance, the role of NK cells in NASH is controversial.
      • Martínez-Chantar M.L.
      • Delgado T.C.
      • Beraza N.
      Revisiting the role of natural killer cells in non-alcoholic fatty liver disease.
      In a murine study using the MCD diet, NK cells seemed to be protective, by altering the liver macrophage compartment, and depletion of NK cells resulted in accelerated fibrogenesis.
      • Tosello-Trampont A.C.
      • Krueger P.
      • Narayanan S.
      • Landes S.G.
      • Leitinger N.
      • Hahn Y.S.
      NKp46(+) natural killer cells attenuate metabolism-induced hepatic fibrosis by regulating macrophage activation in mice.
      Data from human NK cells further support the influence of the microenvironment on NK cell activity, which is severely altered in obesity.
      • Martínez-Chantar M.L.
      • Delgado T.C.
      • Beraza N.
      Revisiting the role of natural killer cells in non-alcoholic fatty liver disease.
      In obese children and adults with NAFLD, NK cells exhibited a metabolically stressed phenotype, including increased glycolysis and reactive oxygen species production resulting in loss of cytotoxicity.
      • Michelet X.
      • Dyck L.
      • Hogan A.
      • Loftus R.M.
      • Duquette D.
      • Wei K.
      • et al.
      Metabolic reprogramming of natural killer cells in obesity limits antitumor responses.
      ,
      • Tobin L.M.
      • Mavinkurve M.
      • Carolan E.
      • Kinlen D.
      • O'Brien E.C.
      • Little M.A.
      • et al.
      NK cells in childhood obesity are activated, metabolically stressed, and functionally deficient.
      Interestingly, lipid accumulation in NK cells caused immune ‘paralysis’ by inhibiting mTOR-mediated glycolysis. In vivo, the lipotoxic environment impaired NK cell-mediated tumour suppression, potentially providing a link to hepatocarcinogenesis in NASH.
      • Michelet X.
      • Dyck L.
      • Hogan A.
      • Loftus R.M.
      • Duquette D.
      • Wei K.
      • et al.
      Metabolic reprogramming of natural killer cells in obesity limits antitumor responses.
      Recent studies found ILC2s in visceral adipose tissue of humans and mice, maintained by IL-33 and obesity was characterised by loss of ILC2s.
      • Brestoff J.R.
      • Kim B.S.
      • Saenz S.A.
      • Stine R.R.
      • Monticelli L.A.
      • Sonnenberg G.F.
      • et al.
      Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity.
      Furthermore, ILC2s contribute to metabolic homeostasis by sustaining eosinophils and polarising macrophages to regulate adiposity and insulin resistance.
      • Brestoff J.R.
      • Kim B.S.
      • Saenz S.A.
      • Stine R.R.
      • Monticelli L.A.
      • Sonnenberg G.F.
      • et al.
      Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity.
      ,
      • Molofsky A.B.
      • Nussbaum J.C.
      • Liang H.E.
      • Van Dyken S.J.
      • Cheng L.E.
      • Mohapatra A.
      • et al.
      Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages.
      In humans with liver fibrosis, ILC2s correlated with severity of fibrosis.
      • McHedlidze T.
      • Waldner M.
      • Zopf S.
      • Walker J.
      • Rankin A.L.
      • Schuchmann M.
      • et al.
      Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis.
      In mice, hepatocyte injury induced IL-33 signalling, resulting in ILC2 activation and propagation of fibrosis via IL-13-dependent stellate cell activation.
      • McHedlidze T.
      • Waldner M.
      • Zopf S.
      • Walker J.
      • Rankin A.L.
      • Schuchmann M.
      • et al.
      Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis.
      During development of obesity, IFN-γ-producing ILC1/NK cells accumulate in adipose tissue, skewing the immune milieu towards a more pro-inflammatory phenotype and thereby promoting insulin resistance.
      • Lee B.C.
      • Kim M.S.
      • Pae M.
      • Yamamoto Y.
      • Eberlé D.
      • Shimada T.
      • et al.
      Adipose natural killer cells regulate adipose tissue macrophages to promote insulin resistance in obesity.
      ,
      • O'Sullivan T.E.
      • Rapp M.
      • Fan X.
      • Weizman O.E.
      • Bhardwaj P.
      • Adams N.M.
      • et al.
      Adipose-resident group 1 innate lymphoid cells promote obesity-associated insulin resistance.
      ,
      • Wensveen F.M.
      • Jelenčić V.
      • Valentić S.
      • Šestan M.
      • Wensveen T.T.
      • Theurich S.
      • et al.
      NK cells link obesity-induced adipose stress to inflammation and insulin resistance.
      IL-22 deficiency by ILC3s resulted in aggravation of metabolic disorders in obese mice and restoration of IL-22-producing ILC3s improved insulin sensitivity, preserved the intestinal barrier and decreased inflammation in adipose tissue of obese mice.
      • Wang X.
      • Ota N.
      • Manzanillo P.
      • Kates L.
      • Zavala-Solorio J.
      • Eidenschenk C.
      • et al.
      Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes.
      It had previously been shown that IL-22 ameliorated experimental liver fibrosis by inducing stellate cell senescence via upregulation of signal transducer and activator of transcription (STAT)3.
      • Kong X.
      • Feng D.
      • Wang H.
      • Hong F.
      • Bertola A.
      • Wang F.S.
      • et al.
      Interleukin-22 induces hepatic stellate cell senescence and restricts liver fibrosis in mice.
      Furthermore, the therapeutic potential of IL-22 was recently suggested in a murine study using overexpression of the neutrophil chemoattractant CXCL1 and feeding of HFD as a model system.
      • Hwang S.
      • He Y.
      • Xiang X.
      • Seo W.
      • Kim S.J.
      • Ma J.
      • et al.
      Interleukin-22 ameliorates neutrophil-driven nonalcoholic steatohepatitis through multiple targets.
      Although not linked to specific cell types, IL-22 treatment reduced inflammation in mice.
      • Hwang S.
      • He Y.
      • Xiang X.
      • Seo W.
      • Kim S.J.
      • Ma J.
      • et al.
      Interleukin-22 ameliorates neutrophil-driven nonalcoholic steatohepatitis through multiple targets.
      Conventional dendritic cells (cDCs) and particularly cDC1s are elevated in NAFLD bridging innate and adaptive immunity.

      Diverse populations of unconventional T cells emerge in steatohepatitis

      Unconventional T cells are a heterogeneous group of lymphocytes of the liver immune system (Fig. 3). The most important subsets of unconventional T cells include mucosal-associated invariant T (MAIT) cells, γδ T cells and NKT cells.
      • Pellicci D.G.
      • Koay H.F.
      • Berzins S.P.
      Thymic development of unconventional T cells: how NKT cells, MAIT cells and γδ T cells emerge.
      Unconventional T cells account for roughly 10% of circulating T cells, however, in the liver they represent the majority of T cells.
      • Pellicci D.G.
      • Koay H.F.
      • Berzins S.P.
      Thymic development of unconventional T cells: how NKT cells, MAIT cells and γδ T cells emerge.
      There are key differences between humans and mice as MAIT cells represent between 15–45% of liver T cells in humans, but are rare in mice, and conversely invariant NKT (iNKT) cells constitute between 30–50% of intrahepatic T cells in mice but less than 1% in healthy humans.
      • Heymann F.
      • Tacke F.
      Immunology in the liver--from homeostasis to disease.
      The role of MAIT cells during NASH is still obscure and warrants further investigation. One study noted an increase of intrahepatic MAIT cells in patients and a decrease in peripheral blood.
      • Li Y.
      • Huang B.
      • Jiang X.
      • Chen W.
      • Zhang J.
      • Wei Y.
      • et al.
      Mucosal-associated invariant T cells improve nonalcoholic fatty liver disease through regulating macrophage polarization.
      In addition, MAIT cells were increased in the fibrotic septa of patients with cirrhosis due to alcohol-related steatohepatitis and NASH.
      • Hegde P.
      • Weiss E.
      • Paradis V.
      • Wan J.
      • Mabire M.
      • Sukriti S.
      • et al.
      Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver.
      As functional studies on the role of MAIT cells in NASH are limited, most of our understanding is derived from mouse studies. Depletion of MAIT cells in the MCD diet model in mice aggravated liver injury; their protective effect seemed to be mediated by polarising macrophages towards an anti-inflammatory phenotype. On the contrary, during obesity, MAIT cells promoted adipose tissue inflammation by skewing macrophages towards a pro-inflammatory phenotype, resulting in pronounced insulin resistance, dysbiosis and metabolic dysfunction.
      • Toubal A.
      • Kiaf B.
      • Beaudoin L.
      • Cagninacci L.
      • Rhimi M.
      • Fruchet B.
      • et al.
      Mucosal-associated invariant T cells promote inflammation and intestinal dysbiosis leading to metabolic dysfunction during obesity.
      Furthermore, MAIT cell inhibition served as a potential treatment in this study. These results indicate different mechanisms of immune regulation of MAIT cells depending on the tissue niche. In mouse models of liver fibrosis induced by bile duct ligation or chronic toxicity, a pro-inflammatory and pro-fibrogenic role for MAIT cells (via induction of hepatic stellate cells) was ascribed
      • Hegde P.
      • Weiss E.
      • Paradis V.
      • Wan J.
      • Mabire M.
      • Sukriti S.
      • et al.
      Mucosal-associated invariant T cells are a profibrogenic immune cell population in the liver.
      and further confirmed in humans by ex vivo coculture experiments.
      • Bottcher K.
      • Rombouts K.
      • Saffioti F.
      • Roccarina D.
      • Rosselli M.
      • Hall A.
      • et al.
      MAIT cells are chronically activated in patients with autoimmune liver disease and promote profibrogenic hepatic stellate cell activation.
      NKT cells can be divided into type I NKT (iNKT) cells and type II NKT cells.
      • Bandyopadhyay K.
      • Marrero I.
      • Kumar V.
      NKT cell subsets as key participants in liver physiology and pathology.
      The relative abundance of iNKT cells and their ability to recognise lipid-based antigens presented by the MHC class-I-like molecule CD1d make iNKT cells an interesting player in the inflammatory response during steatohepatitis. Indeed, hepatic NKT cell numbers were increased in patients with NASH.
      • Wolf M.J.
      • Adili A.
      • Piotrowitz K.
      • Abdullah Z.
      • Boege Y.
      • Stemmer K.
      • et al.
      Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
      ,
      • Syn W.K.
      • Oo Y.H.
      • Pereira T.A.
      • Karaca G.F.
      • Jung Y.
      • Omenetti A.
      • et al.
      Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
      Similarly, in murine steatohepatitis, NKT cells were enriched and mice deficient in CD1d had milder steatohepatitis.
      • Syn W.K.
      • Oo Y.H.
      • Pereira T.A.
      • Karaca G.F.
      • Jung Y.
      • Omenetti A.
      • et al.
      Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
      Mechanistically, it was shown that NKT cells mediated steatosis by secretion of the cytokine LIGHT which prompted free fatty acid uptake by hepatocytes (Fig. 3).
      • Wolf M.J.
      • Adili A.
      • Piotrowitz K.
      • Abdullah Z.
      • Boege Y.
      • Stemmer K.
      • et al.
      Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.
      NKT cells express the chemokine receptor CXCR6 and during experimental steatohepatitis, CXCR6 and its ligand CXCL16 were shown to be critical in the recruitment of NKT cells to the liver.
      • Wehr A.
      • Baeck C.
      • Heymann F.
      • Niemietz P.M.
      • Hammerich L.
      • Martin C.
      • et al.
      Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis.
      Mechanistically, NKT cells expressed pro-inflammatory cytokines, CXCR6 deficiency reduced liver fibrosis and adoptive transfer of NKT cells aggravated liver fibrosis in steatohepatitis.
      • Wehr A.
      • Baeck C.
      • Heymann F.
      • Niemietz P.M.
      • Hammerich L.
      • Martin C.
      • et al.
      Chemokine receptor CXCR6-dependent hepatic NK T Cell accumulation promotes inflammation and liver fibrosis.
      Given the opposing distribution of NKT cells and MAIT cells between humans and mice, findings about NKT cells from rodent models need to be interpreted with caution, and NKT cells have been suggested as the murine counterpart of MAIT cells in humans from a functional perspective.
      • Kurioka A.
      • Walker L.J.
      • Klenerman P.
      • Willberg C.B.
      MAIT cells: new guardians of the liver.
      γδ T cells express a T-cell receptor (TCR) γ-chain and δ-chain; many aspects of their biology are incompletely understood and the frequency of γδ T cells varies between humans and mice. Furthermore, both humans and mice have CD1d-restricted γδ T cells that recognise lipid antigens.
      • Pellicci D.G.
      • Koay H.F.
      • Berzins S.P.
      Thymic development of unconventional T cells: how NKT cells, MAIT cells and γδ T cells emerge.
      In human NAFLD, the frequency of hepatic γδ T cells was unchanged.
      • Diedrich T.
      • Kummer S.
      • Galante A.
      • Drolz A.
      • Schlicker V.
      • Lohse A.W.
      • et al.
      Characterization of the immune cell landscape of patients with NAFLD.
      In mice fed high-fat high-cholesterol diet, hepatic γδ T cells were increased, and the production of IL-17A was identified as a disease-promoting factor, as specific depletion of IL-17 in γδ T cells alleviated NASH (Fig. 3).
      • Li F.
      • Hao X.
      • Chen Y.
      • Bai L.
      • Gao X.
      • Lian Z.
      • et al.
      The microbiota maintain homeostasis of liver-resident γδT-17 cells in a lipid antigen/CD1d-dependent manner.
      Since CD1d-deficient mice also have impaired γδ T cells, the protective effect observed in these mice might be partially attributed to altered γδ T cells.
      • Syn W.K.
      • Agboola K.M.
      • Swiderska M.
      • Michelotti G.A.
      • Liaskou E.
      • Pang H.
      • et al.
      NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.
      IL-17 producing γδ T cells depend on CCR6 and its ligand CCL20 for recruitment to the liver.
      • Haas J.D.
      • González F.H.
      • Schmitz S.
      • Chennupati V.
      • Föhse L.
      • Kremmer E.
      • et al.
      CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells.
      Expression of CCR6 and CCL20 was increased in human cirrhosis, and mice deficient in CCR6 had aggravated liver fibrosis and inflammation in 2 models of chronic liver injury, including the MCD diet.
      • Hammerich L.
      • Bangen J.M.
      • Govaere O.
      • Zimmermann H.W.
      • Gassler N.
      • Huss S.
      • et al.
      Chemokine receptor CCR6-dependent accumulation of γδ T cells in injured liver restricts hepatic inflammation and fibrosis.
      In this study, adoptive transfer of γδ T cells reduced liver inflammation, likely by promoting apoptosis of activated hepatic stellate cells.

      Platelets bridge innate and adaptive immune cells in NASH

      Platelets are essential in hemostasis and wound healing; however, more recently their role in inflammation and infection has been appreciated.
      • Wong C.H.
      • Jenne C.N.
      • Petri B.
      • Chrobok N.L.
      • Kubes P.
      Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.
      Using intravital microscopy, a crucial contribution to the KC-mediated capture of pathogens was observed.
      • Wong C.H.
      • Jenne C.N.
      • Petri B.
      • Chrobok N.L.
      • Kubes P.
      Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.
      KC–platelet adhesion was mediated via platelet receptor GPIIb, and blocking this interaction led to rapid mortality in mice with bloodstream infections. Platelets play a role in the pathogenesis of obesity, metabolic syndrome and atherosclerosis.
      • Gawaz M.
      • Langer H.
      • May A.E.
      Platelets in inflammation and atherogenesis.
      ,
      • Santilli F.
      • Vazzana N.
      • Liani R.
      • Guagnano M.T.
      • Davì G.
      Platelet activation in obesity and metabolic syndrome.
      Platelets were also investigated in NASH and anti-platelet therapy alleviated experimental NASH in rats and mice.
      • Fujita K.
      • Nozaki Y.
      • Wada K.
      • Yoneda M.
      • Endo H.
      • Takahashi H.
      • et al.
      Effectiveness of antiplatelet drugs against experimental non-alcoholic fatty liver disease.
      During steatosis, platelets accumulate in the liver early on through CD44 expressed on their surface, binding to hyaluronic acid presented on the cell surface of stressed hepatocytes, liver sinusoidal endothelial cells and KCs.
      • Malehmir M.
      • Pfister D.
      • Gallage S.
      • Szydlowska M.
      • Inverso D.
      • Kotsiliti E.
      • et al.
      Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.