Recreating gut-liver axis during NAFLD onset by using a Caco-2/HepG2 co-culture system

Abstract

Nonalcoholic fatty liver disease (NAFLD) onset and its progression towards nonalcoholic steatohepatitis (NASH) features increased intestinal permeability and leaky gut, thereby favoring the escape of endotoxin [lipopolysaccharides (LPS)] from the gut to the liver. The aim of this study was to resemble the crosstalk between intestine and liver during NAFLD by using an in vitro model of co-culture system. Enterocytes (Caco-2) were seeded on Transwell filters (pore size: 0.4 μm) and cultured for 21 days to constitute a confluent monolayer, and then they were co-cultivated with hepatocytes (HepG2) for an additional 24 h. Caco-2 on the apical chamber were exposed to LPS and/or a mixture of palmitic and oleic acid (PAOA) for 24 h. FITC-4000 dextrans (FD4) permeability across Caco-2 monolayer was increased by the treatment of Caco-2 cells with PAOA and LPS, consistently with tight junction-associated proteins reduction. Caco-2 exposure to PAOA/LPS promoted ApoB, triglyceride (TG), and free fatty acid secretion in basolateral media. In turn, HepG2 co-cultured with Caco-2 exposed to LPS, PAOA, or both accumulated lipid droplets and increased intracellular TG content. Likewise, Caco-2 released pro-inflammatory cytokines in basolateral media. These events triggered endoplasmic reticulum (ER) and oxidative stress, enhancing reactive oxygen species (ROS), H₂O₂, aldehyde derivate production, and ROS-induced DNA damage in HepG2 cells. Hence, Caco-2/HepG2 co-culture system may faithfully reproduce the breach in the intestinal barrier integrity that occurs in NAFLD, thus resulting in the increased inflammatory response and ER and oxidative and stress, which promote the switch towards NASH.

Keywords

NAFLD / NASH / gut-liver axis / intestinal permeability / leaky gut / co-culture system

References

[1] Meroni M,Paolini E.MAFLD definition underestimates the risk to develop HCC in genetically predisposed patients.J Intern Med2021:374-6

[2] Younossi Z.Contribution of alcoholic and nonalcoholic fatty liver disease to the burden of liver-related morbidity and mortality.Gastroenterology2016;150:1778-85

[3] Eslam M,George J.International Consensus PanelMAFLD: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease.Gastroenterology2020;158:1999-2014.e1

[4] Younossi ZM,Abdelatif D,Henry L.Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology2016;64:73-84

[5] Wong RJ,Cheung R.Nonalcoholic steatohepatitis is the second leading etiology of liver disease among adults awaiting liver transplantation in the United States.Gastroenterology2015;148:547-55

[6] Dongiovanni P,Longo M,Fracanzani AL.Genetics, immunity and nutrition boost the switching from NASH to HCC.Biomedicines2021;9:1524 PMCID:PMC8614742

[7] Tilg H.Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.Hepatology2010;52:1836-46

[8] Meroni M,Dongiovanni P.Alcohol or gut microbiota: who is the guilty?.Int J Mol Sci2019;20:4568 PMCID:PMC6770333

[9] Poeta M,Vajro P.Gut-liver axis derangement in non-alcoholic fatty liver disease.Children (Basel)2017;4:66 PMCID:PMC5575588

[10] Meroni M,Dongiovanni P.The role of probiotics in nonalcoholic fatty liver disease: a new insight into therapeutic strategies.Nutrients2019;11:2642 PMCID:PMC6893730

[11] Giorgio V,Principessa L.Intestinal permeability is increased in children with non-alcoholic fatty liver disease, and correlates with liver disease severity.Dig Liver Dis2014;46:556-60

[12] Miele L,La Torre G.Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease.Hepatology2009;49:1877-87

[13] Lindstedt G,Gustafsson BE.Mucus in intestinal contents of germfree rats.J Exp Med1965;121:201-13 PMCID:PMC2137981

[14] Wang L,Stärkel P.Intestinal REG3 lectins protect against alcoholic steatohepatitis by reducing mucosa-associated microbiota and preventing bacterial translocation.Cell Host Microbe2016;19:227-39 PMCID:PMC4786170

[15] Cani PD,Geurts L.Involvement of gut microbiota in the development of low-grade inflammation and type 2 diabetes associated with obesity.Gut Microbes2012;3:279-88 PMCID:PMC3463487

[16] Boulangé CL,Chilloux J,Dumas ME.Impact of the gut microbiota on inflammation, obesity, and metabolic disease.Genome Med2016;8:42 PMCID:PMC4839080

[17] Del Chierico F,Vernocchi P.Gut microbiota profiling of pediatric nonalcoholic fatty liver disease and obese patients unveiled by an integrated meta-omics-based approach.Hepatology2017;65:451-64

[18] Delarue J.Nonalcoholic fatty liver disease: roles of the gut and the liver and metabolic modulation by some dietary factors and especially long-chain n-3 PUFA.Mol Nutr Food Res2016;60:147-59

[19] Bibbò S,Dore MP,Newton EE.Gut microbiota as a driver of inflammation in nonalcoholic fatty liver disease.Mediators Inflamm2018;2018:9321643 PMCID:PMC5833468

[20] Puri P.The intestinal microbiome in nonalcoholic fatty liver disease.Clin Liver Dis2018;22:121-32

[21] Borrelli A,Tuccillo FM.Role of gut microbiota and oxidative stress in the progression of non-alcoholic fatty liver disease to hepatocarcinoma: current and innovative therapeutic approaches.Redox Biol2018;15:467-79 PMCID:PMC5975181

[22] Buzzetti E,Tsochatzis EA.The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD).Metabolism2016;65:1038-48

[23] Schnabl B.Interactions between the intestinal microbiome and liver diseases.Gastroenterology2014;146:1513-24 PMCID:PMC3996054

[24] Ferreira DF,Prist IH,de Souza HP.Novel role of TLR4 in NAFLD development: modulation of metabolic enzymes expression.Biochim Biophys Acta2015;1851:1353-9

[25] Cai C,Li P.NLRP3 deletion inhibits the non-alcoholic steatohepatitis development and inflammation in Kupffer cells induced by palmitic acid.Inflammation2017;40:1875-83

[26] Ray K.NAFLD. Leaky guts: intestinal permeability and NASH.Nat Rev Gastroenterol Hepatol2015;12:123

[27] Meroni M,Rametta R.Genetic and epigenetic modifiers of alcoholic liver disease.Int J Mol Sci2018;19:3857 PMCID:PMC6320903

[28] De Gregorio V,Corrado B.Intestine-liver axis on-chip reveals the intestinal protective role on hepatic damage by emulating ethanol first-pass metabolism.Front Bioeng Biotechnol2020;8:163 PMCID:PMC7090126

[29] Breemen RB, Li Y. Caco-2 cell permeability assays to measure drug absorption.Expert Opin Drug Metab Toxicol2005;1:175-85

[30] Garcia MN,Cook JD.The Caco-2 cell culture system can be used as a model to study food iron availability.J Nutr1996;126:251-8

[31] Tayyeb JZ,Mensink RP.Butyric acid added apically to intestinal Caco-2 cells elevates hepatic ApoA-I transcription and rescues lower ApoA-I expression in inflamed HepG2 cells co-cultured in the basolateral compartment.Biomolecules2021;11:71 PMCID:PMC7825706

[32] Meroni M,Longo M.Mboat7 down-regulation by hyper-insulinemia induces fat accumulation in hepatocytes.EBioMedicine2020;52:102658 PMCID:PMC7026742

[33] Sambuy Y,Ranaldi G,Stammati A.The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.Cell Biol Toxicol2005;21:1-26

[34] Twarog C,O'Brien PJ.A head-to-head Caco-2 assay comparison of the mechanisms of action of the intestinal permeation enhancers: SNAC and sodium caprate (C₁₀).Eur J Pharm Biopharm2020;152:95-107

[35] He S,Xu L.Protective effects of ferulic acid against heat stress-induced intestinal epithelial barrier dysfunction in vitro and in vivo.PLoS One2016;11:e0145236 PMCID:PMC4760716

[36] Longo M,Paolini E.TM6SF2/PNPLA3/MBOAT7 loss-of-function genetic variants impact on NAFLD development and progression both in patients and in in vitro models.Cell Mol Gastroenterol Hepatol2021;13:759-88 PMCID:PMC8783129

[37] Longo M,Meroni M.α-lipoic acid improves hepatic metabolic dysfunctions in acute intermittent porphyria: a proof-of-concept study.Diagnostics (Basel)2021;11:1628 PMCID:PMC8468570

[38] Kessoku T,Tanaka K.The role of leaky gut in nonalcoholic fatty liver disease: a novel therapeutic target.Int J Mol Sci2021;22:8161 PMCID:PMC8347478

[39] Barcia AM.Triglyceride-rich lipoproteins as agents of innate immunity.Clin Infect Dis2005;41 Suppl 7:S498-503

[40] Harris HW,Kumwenda ZL.The lipemia of sepsis: triglyceride-rich lipoproteins as agents of innate immunity.J Endotoxin Res2000;6:421-30

[41] Meroni M,Erconi V.mir-101-3p downregulation promotes fibrogenesis by facilitating hepatic stellate cell transdifferentiation during insulin resistance.Nutrients2019;11:2597 PMCID:PMC6893471

[42] Dongiovanni P,Baselli GA.Insulin resistance promotes Lysyl Oxidase Like 2 induction and fibrosis accumulation in non-alcoholic fatty liver disease.Clin Sci (Lond)2017;131:1301-15

[43] Parlati L,Guillou H.New targets for NAFLD.JHEP Rep2021;3:100346 PMCID:PMC8507191

[44] Ceccarelli S,Mina M.LPS-induced TNF-α factor mediates pro-inflammatory and pro-fibrogenic pattern in non-alcoholic fatty liver disease.Oncotarget2015;6:41434-52 PMCID:PMC4747165

[45] Ashraf NU.Endoplasmic reticulum stress and Oxidative stress in the pathogenesis of Non-alcoholic fatty liver disease.Free Radic Res2015;49:1405-18

[46] Fogh J,Orfeo T.One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.J Natl Cancer Inst1977;59:221-6

[47] Ferruzza S,Scarino ML.A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium.Toxicol In Vitro2012;26:1252-5

[48] Javitt NB.Hep G2 cells as a resource for metabolic studies: lipoprotein, cholesterol, and bile acids.FASEB J1990;4:161-8

[49] Brahe LK,Larsen LH.Is butyrate the link between diet, intestinal microbiota and obesity-related metabolic diseases?.Obes Rev2013;14:950-9