Fructose regulates the pentose phosphate pathway and induces an inflammatory and resolution phenotype in Kupffer cells

Abstract Over-consumption of fructose in adults and children has been linked to increased risk of non-alcoholic fatty liver disease (NAFLD). Recent studies have highlighted the effect of fructose on liver inflammation, fibrosis, and immune cell activation. However, little work summarizes the direct impact of fructose on macrophage infiltration, phenotype, and function within the liver. We demonstrate that chronic fructose diet decreased Kupffer cell populations while increasing transitioning monocytes. In addition, fructose increased fibrotic gene expression of collagen 1 alpha 1 (Col1a1) and tissue metallopeptidase inhibitor 1 (Timp1) as well as inflammatory gene expression of tumor necrosis factor alpha (Tnfa) and expression of transmembrane glycoprotein NMB (Gpnmb) in liver tissue compared to glucose and control diets. Single cell RNA sequencing (scRNAseq) revealed fructose elevated expression of matrix metallopeptidase 12 (Mmp12), interleukin 1 receptor antagonist (Il1rn), and radical S-adenosyl methionine domain (Rsad2) in liver and hepatic macrophages. In vitro studies using IMKC and J774.1 cells demonstrated decreased viability when exposed to fructose. Additionally, fructose increased Gpnmb, Tnfa, Mmp12, Il1rn, and Rsad2 in unpolarized IMKC. By mass spectrometry, C13 fructose tracing detected fructose metabolites in glycolysis and the pentose phosphate pathway (PPP). Inhibition of the PPP further increased fructose induced Il6, Gpnmb, Mmp12, Il1rn, and Rsad2 in nonpolarized IMKC. Taken together, fructose decreases cell viability while upregulating resolution and anti-inflammatory associated genes in Kupffer cells

DataSheet_1_Myeloid cell MHC I expression drives CD8+ T cell activation in nonalcoholic steatohepatitis.pdf

Background & aimsActivated CD8+ T cells are elevated in Nonalcoholic steatohepatitis (NASH) and are important for driving fibrosis and inflammation. Despite this, mechanisms of CD8+ T cell activation in NASH are largely limited. Specific CD8+ T cell subsets may become activated through metabolic signals or cytokines. However, studies in NASH have not evaluated the impact of antigen presentation or the involvement of specific antigens. Therefore, we determined if activated CD8+ T cells are dependent on MHC class I expression in NASH to regulate fibrosis and inflammation.MethodsWe used H2Kb and H2Db deficient (MHC I KO), Kb transgenic mice, and myeloid cell Kb deficient mice (LysM Kb KO) to investigate how MHC class I impacts CD8+ T cell function and NASH. Flow cytometry, gene expression, and histology were used to examine hepatic inflammation and fibrosis. The hepatic class I immunopeptidome was evaluated by mass spectrometry.ResultsIn NASH, MHC class I isoform H2Kb was upregulated in myeloid cells. MHC I KO demonstrated protective effects against NASH-induced inflammation and fibrosis. Kb mice exhibited increased fibrosis in the absence of H2Db while LysM Kb KO mice showed protection against fibrosis but not inflammation. H2Kb restricted peptides identified a unique NASH peptide Ncf2 capable of CD8+ T cell activation in vitro. The Ncf2 peptide was not detected during fibrosis resolution.ConclusionThese results suggest that activated hepatic CD8+ T cells are dependent on myeloid cell MHC class I expression in diet induced NASH to promote inflammation and fibrosis. Additionally, our studies suggest a role of NADPH oxidase in the production of Ncf2 peptide generation.</p

Table_2_Myeloid cell MHC I expression drives CD8+ T cell activation in nonalcoholic steatohepatitis.pdf

Background & aimsActivated CD8+ T cells are elevated in Nonalcoholic steatohepatitis (NASH) and are important for driving fibrosis and inflammation. Despite this, mechanisms of CD8+ T cell activation in NASH are largely limited. Specific CD8+ T cell subsets may become activated through metabolic signals or cytokines. However, studies in NASH have not evaluated the impact of antigen presentation or the involvement of specific antigens. Therefore, we determined if activated CD8+ T cells are dependent on MHC class I expression in NASH to regulate fibrosis and inflammation.MethodsWe used H2Kb and H2Db deficient (MHC I KO), Kb transgenic mice, and myeloid cell Kb deficient mice (LysM Kb KO) to investigate how MHC class I impacts CD8+ T cell function and NASH. Flow cytometry, gene expression, and histology were used to examine hepatic inflammation and fibrosis. The hepatic class I immunopeptidome was evaluated by mass spectrometry.ResultsIn NASH, MHC class I isoform H2Kb was upregulated in myeloid cells. MHC I KO demonstrated protective effects against NASH-induced inflammation and fibrosis. Kb mice exhibited increased fibrosis in the absence of H2Db while LysM Kb KO mice showed protection against fibrosis but not inflammation. H2Kb restricted peptides identified a unique NASH peptide Ncf2 capable of CD8+ T cell activation in vitro. The Ncf2 peptide was not detected during fibrosis resolution.ConclusionThese results suggest that activated hepatic CD8+ T cells are dependent on myeloid cell MHC class I expression in diet induced NASH to promote inflammation and fibrosis. Additionally, our studies suggest a role of NADPH oxidase in the production of Ncf2 peptide generation.</p

Table_1_Myeloid cell MHC I expression drives CD8+ T cell activation in nonalcoholic steatohepatitis.pdf

Background & aimsActivated CD8+ T cells are elevated in Nonalcoholic steatohepatitis (NASH) and are important for driving fibrosis and inflammation. Despite this, mechanisms of CD8+ T cell activation in NASH are largely limited. Specific CD8+ T cell subsets may become activated through metabolic signals or cytokines. However, studies in NASH have not evaluated the impact of antigen presentation or the involvement of specific antigens. Therefore, we determined if activated CD8+ T cells are dependent on MHC class I expression in NASH to regulate fibrosis and inflammation.MethodsWe used H2Kb and H2Db deficient (MHC I KO), Kb transgenic mice, and myeloid cell Kb deficient mice (LysM Kb KO) to investigate how MHC class I impacts CD8+ T cell function and NASH. Flow cytometry, gene expression, and histology were used to examine hepatic inflammation and fibrosis. The hepatic class I immunopeptidome was evaluated by mass spectrometry.ResultsIn NASH, MHC class I isoform H2Kb was upregulated in myeloid cells. MHC I KO demonstrated protective effects against NASH-induced inflammation and fibrosis. Kb mice exhibited increased fibrosis in the absence of H2Db while LysM Kb KO mice showed protection against fibrosis but not inflammation. H2Kb restricted peptides identified a unique NASH peptide Ncf2 capable of CD8+ T cell activation in vitro. The Ncf2 peptide was not detected during fibrosis resolution.ConclusionThese results suggest that activated hepatic CD8+ T cells are dependent on myeloid cell MHC class I expression in diet induced NASH to promote inflammation and fibrosis. Additionally, our studies suggest a role of NADPH oxidase in the production of Ncf2 peptide generation.</p