BACKGROUND AND AIMS:Unresolved inflammation and fibrosis are defining features of metabolic dysfunction-associated steatohepatitis (MASH), a progressive form of steatotic liver disease that can advance to cirrhosis and hepatocellular carcinoma. While innate immune mechanisms in MASH have been extensively characterized, the role of CD4+ T cells remains poorly understood despite their central function in orchestrating immune responses through effector and regulatory mechanisms. APPROACH AND RESULTS:Integrated single-cell proteomic, transcriptomic, and functional analyses were used to investigate the CD4+ T-cell landscape in murine and human MASH. We delineated a profound shift in the differentiation of intrahepatic and peripheral CD4+ T cells toward Th1, regulatory, and cytotoxic phenotypes in murine and human MASH. Notably, hepatic CD4+ T cells exhibited heightened effector activity and elevated secretion of proinflammatory cytokines, thus amplifying inflammatory signaling cascades. The CD4+ T-cell reprogramming in MASH also included the induction of the co-stimulatory receptor OX40. In parallel, OX40 ligand (OX40L)-expressing monocyte-derived macrophages and dendritic cells accumulated in MASH livers, establishing a feed-forward CD4+ T cell-myeloid activation loop. Therapeutic blockade of the OX40-OX40L axis, in turn, reversed liver pathology in mice with established MASH and reduced disease markers in an ex vivo human liver model. Furthermore, genetic depletion or functional inhibition of CD4+ T cells attenuated fibrosis, accompanied by decreased infiltration of monocyte-derived macrophages. CONCLUSIONS:These studies provide a comprehensive single-cell proteogenomic atlas of CD4+ T cells in MASH and identify an OX40-dependent CD4+ T cell-macrophage axis as a promising therapeutic target for the treatment of MASH and liver fibrosis.

BACKGROUND & AIMS CD4+ T helper 1 (Th1) cells are involved in human inflammatory bowel disease (IBD) pathogenesis; however, mechanisms governing the persistent inflammatory function of these cells are unclear, leading us to examine how metabolism governs Th1 cell-induced IBD. METHODS Th1 cells supplemented with methyl pyruvate (MePyr) were analyzed to define how enforced mitochondrial pyruvate metabolism and subsequent glycogen synthase kinase 3β (GSK3β) deactivation reprogram cellular state. Re-analysis of the inflamed ileal single-cell RNA sequencing dataset from Crohn’s disease patients was performed to assess non-Treg CD4+ T cell metabolic gene signature. We assessed the capacity of a repurposed GSK3β inhibitor to restrain pathogenic CD4+ T cell-driven murine colitis. RESULTS Effector Th1 cells exhibit a distinct metabolic program exemplified by glucose-driven glycolysis but low mitochondrial respiration. MePyr deactivates GSK3β, glycolysis, and histone H3 acetylation on cytokine promoter region, resulting in reduced interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) expression in Th1 cells with concomitant gain of regulatory T cell-like program. GSK3β inhibition with LY2090314 mirrored the anti-inflammatory effect of MePyr in a manner reversible by acetate supplementation, implying that GSK3β potentially sustains glycolysis-derived acetyl-coenzyme A needed for histone acetylation and Th1 cell inflammatory response. Interleukin-21 exacerbates Th1 cell inflammatory response by maintaining a GSK3β-driven glycolytic program. The Th1 cell metabolic gene signature downregulated by MePyr or GSK3β inhibition in vitro is enriched in refractory Crohn’s disease patients. GSK3β inhibition with LY2090314 retrains T cell-induced colitis in mice. CONCLUSIONS MePyr impairs GSK3β-mediated glycolysis and Th1 cell immune response. GSK3β inhibition may mitigate Th1 cell-induced human IBD. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. * GSK3β : glycogen synthase kinase 3 beta VDAC1 : voltage-dependent anion channel 1 MePyr : methyl pyruvate 2-DG : 2-deoxy-Ɒ-glucose GAPDH : glyceraldehyde 3-phosphate dehydrogenase IBD : inflammatory bowel disease IL : interleukin i.p. : intraperitoneal mRNA : messenger RNA H3K27ac : histone H3 lysine 27 acetylation H3K9ac : histone H3 lysine 9 acetylation OXPHOS : oxidative phosphorylation FCCP : carbonyl cyanide-p-trifluoromethoxy phenylhydrazone TCA : tricarboxylic acid ECAR : extracellular acidification rate OCR : oxygen consumption rate ETC : electron transport chain PBMC : peripheral blood mononuclear cell PLA : proximity ligation assay scRNA-seq : single-cell RNA sequencing TCR : T cell receptor CD3 : cluster of differentiation 3 CD28 : cluster of differentiation 28 Th : T helper TNF-α : tumor necrosis factor alpha IFN-γ : interferon-gamma Th1 : Type 1 T helper Th17 : IL-17-producing T helper iTreg : induced regulatory T cell WT : wild-type [1]: pending:yes

BACKGROUND & AIMS:Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by toxic lipid-induced cellular stress (lipotoxicity), which culminates in lethal and sublethal hepatocyte injury and a sterile fibroinflammatory response. We previously reported that pharmacological inhibition of glycogen synthase kinase 3 (GSK3) ameliorates murine MASH. However, the hepatocyte-specific role of GSK3β in lipotoxic injury and the fibroinflammatory response in MASH remains unclear. METHODS:We generated hepatocyte-specific Gsk3β knockout (Gsk3βΔHep) mice by crossing Gsk3βfl/fl mice with albumin-Cre mice. Mice were fed either a choline-deficient high-fat diet (CDHFD) or a high-fat, fructose, and cholesterol (FFC) diet to induce MASH. RESULTS:Metabolic parameters and hepatic lipidomic were similar between FFC-fed Gsk3βΔHep and Gsk3βfl/fl mice. The NanoString Metabolic Pathways Panel on liver tissues showed upregulation of NAD, mitochondrial function, and oxidative phosphorylation signaling pathways in FFC-fed Gsk3βΔHep mice compared with Gsk3βfl/fl mice. In vitro studies in palmitate-treated hepatocytes showed that mitochondrial morphology, biogenesis, contact with lipid droplets, and respiration were improved, whereas mitochondrial DNA release and Ferroptosis Suppressor Protein 1 (FSP1) phase separation were reduced with pharmacological GSK3 inhibition or in hepatocytes isolated from Gsk3βΔHep mice. Similarly, liver injury, lipid peroxidation, ferroptosis markers, and circulating mitochondrial DNA levels were reduced in Gsk3βΔHep mice with MASH. Furthermore, Gsk3βΔHep mice with MASH had reduced hepatic expression of proinflammatory genes, myeloid cell infiltration, NETosis, and showed significant downregulation of fibrosis signaling pathways. CONCLUSIONS:Gsk3βΔHep reduced liver injury, mitochondrial DNA release, inflammation, and fibrosis in mice with MASH, secondary to improved mitochondrial bioenergetics and reduced ferroptosis. Therefore, GSK3β may be a potential therapeutic target for human MASH.

Unresolved inflammation and fibrosis are the two key features of metabolic dysfunction-associated steatohepatitis (MASH), a progressive form of steatotic liver disease that can evolve into cirrhosis and liver cancer. Although innate immunity has been well studied in MASH, the role of CD4+ T cells remains underexplored despite their potential to coordinate immune responses by providing help to other immune cells, promoting inflammation, or regulating immune activity through effector and regulatory subsets. To better understand the role of CD4+ T cells in the pathogenesis of MASH, we comprehensively characterized hepatic CD4+ T cells in murine and human MASH at a single-cell protein, transcriptional, and functional level. Mass cytometry and CITE-sequencing revealed a marked shift in intrahepatic CD4+ T-cell composition in MASH, with enrichment of Th1, regulatory, and cytotoxic CD4+ T cells. Similar phenotypic changes were mirrored in the peripheral blood and validated in human MASH samples. Functional assays demonstrated increased production of IFNγ and TNFα by hepatic CD4+ T cells, highlighting their proinflammatory effector activity. Transcriptomic profiling identified Tnfrsf4 (OX40) upregulation in hepatic CD4+ T cells during MASH. Therapeutic blockade of the OX40L-OX40 axis reversed hepatic fibrosis and improved histologic disease scores in mice with established MASH, and also decreased inflammatory markers in a human ex vivo liver model. Together, these studies provide a proteogenomic single-cell atlas for hepatic CD4+ T cells and uncover a CD4+ T cell-dependent immunopathogenic circuit as a promising immunotherapeutic target to alleviate MASH and liver fibrosis.

Background & Aims: Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by excessive circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to liver sinusoidal endothelial cells (LSECs) and transendothelial migration (TEM) are crucial in the inflammatory process. Under lipotoxic stress, LSECs develop a proinflammatory phenotype known as endotheliopathy. However, mediators of endotheliopathy remain unclear. Methods: Primary mouse LSECs isolated from C57BL/6J mice fed chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH resulting from a choline-deficient high-fat diet (CDHFD) or FFC diet were also treated with two structurally distinct GSK3 inhibitors (LY2090314 and elraglusib [9-ING-41]). Results: Integrated pathway analysis of the mouse LSEC proteome and transcriptome indicated that leukocyte TEM and focal adhesion were the major pathways altered in MASH. Kinome profiling of the LSEC phosphoproteome identified glycogen synthase kinase (GSK)-3β as the major kinase hub in MASH. GSK3β-activating phosphorylation was increased in primary human LSECs treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependent mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through the LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC- and CDHFD-fed mice, respectively. Immunophenotyping using cytometry by mass cytometry by time of flight of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib showed reduced infiltration of proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells. Conclusion: GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and could serve as a potential therapeutic strategy for treating human MASH. Impact and Implications: LSECs under lipotoxic stress in MASH develop a proinflammatory phenotype known as endotheliopathy, with obscure mediators and functional outcomes. The current study identified GSK3 as the major driver of LSEC endotheliopathy, examined its pathogenic role in myeloid cell-associated liver inflammation, and defined the therapeutic efficacy of pharmacological GSK3 inhibitors in murine MASH. This study provides preclinical data for the future investigation of GSK3 pharmacological inhibitors in human MASH. The results of this study are important to hepatologists, vascular biologists, and investigators studying the mechanisms of inflammatory liver disease and MASH, as well as those interested in drug development.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

BACKGROUND & AIMS:Incapacitated regulatory T cells (Tregs) contribute to immune-mediated diseases. Inflammatory Tregs are evident during human inflammatory bowel disease (IBD); however, mechanisms driving the development of these cells and their function are not well understood. Therefore, we investigated the role of cellular metabolism in Tregs relevant to gut homeostasis. METHODS:Using human Tregs, we performed mitochondrial ultrastructural studies via electron microscopy and confocal imaging, biochemical and protein analyses using proximity ligation assay, immunoblotting, mass cytometry and fluorescence-activated cell sorting, metabolomics, gene expression analysis, and real-time metabolic profiling utilizing Seahorse XF analyzer. We utilized Crohn's disease single-cell RNA sequencing dataset to infer therapeutic relevance of targeting metabolic pathways in inflammatory Tregs. We examined the superior functionality of genetically-modified Tregs in CD4+ T cell-induced murine colitis models. RESULTS:Mitochondria-endoplasmic reticulum (ER) appositions, known to mediate pyruvate entry into mitochondria via VDAC1, are abundant in Tregs. VDAC1 inhibition perturbed pyruvate metabolism, eliciting sensitization to other inflammatory signals reversible by membrane-permeable methyl pyruvate (MePyr) supplementation. Notably, IL-21 diminished mitochondria-ER appositions, resulting in enhanced enzymatic function of glycogen synthase kinase 3 β (GSK3β), a putative negative regulator of VDAC1, and a hypermetabolic state that amplified Treg inflammatory response. MePyr and GSK3β pharmacologic inhibitor (LY2090314) reversed IL-21-induced metabolic rewiring and inflammatory state. Moreover, IL-21-induced metabolic genes in Tregs in vitro were enriched in human Crohn's disease intestinal Tregs. Adoptively transferred Il21r-/- Tregs efficiently rescued murine colitis in contrast to wild-type Tregs. CONCLUSIONS:IL-21 triggers metabolic dysfunction associated with Treg inflammatory response. Inhibiting IL-21-induced metabolism in Tregs may mitigate CD4+ T cell-driven chronic intestinal inflammation.

BACKGROUND:NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis. METHODS AND RESULTS:Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis. CONCLUSIONS:Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

Abstract Background Inflammatory regulatory T cells (Tregs) are one of the hallmarks of therapy resistance. Yet mechanisms leading to inflammatory Treg development and its contribution to inflammatory bowel disease (IBD) are unexplored. Methods Tregs derived from healthy PBMCS were characterized by immune and mitochondrial phenotyping. Transcripts were validated in dataset from IBD intestinal lesions. Wildtype (WT) and IL21 receptor-deleted (Il21r−/−) Tregs were examined in murine models of CD4+ T cell-induced colitis. Results Through in vitro cytokine challenge of Tregs and subsequent analysis, we found that IL21 induces Tregs co-expressing TNF, IFNγ and IL17. Transcriptomic and metabolomic integration revealed upregulation of mRNA and metabolites associated with glycolysis, and amino acid, nucleotide and lipid metabolism. Moreover, IL21-induced metabolic transcripts were more enriched in Treg cluster within scRNA-seq dataset from intestinal lesions of anti-TNF non-responders vs. non-responders. Mechanistically, cell imaging and real-time bioenergetic analysis revealed disruption to mitochondria-ER ultrastructure and function, coinciding with glycogen synthase kinase 3β (GSK3β) activation and glycolytic switch rather than pyruvate-mediated oxidative phosphorylation. GSK3β inhibition or methyl pyruvate supplementation rescued IL21-induced metabolic and inflammatory phenotypes. In contrast to WT Tregs, murine Il21r−/− Treg transfer efficiently prevented and rescued CD4+CD45Rbhigh T cell-induced colitis. Conclusions IL21-induced metabolic dysfunction can incapacitate Treg immunosuppressive function. Desensitizing Tregs to IL21 through various therapeutic approaches can alleviate IBD. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) K01DK124358, the Pilot & Feasibility Award by the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery Career Development Award, and David F. and Margaret T. Grohne Cancer Immunology and Immunotherapy (to AOB).

BACKGROUND & AIMS:Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS:Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS:We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION:G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

IL-21 potently engages human Tregs in a hypermetabolic state that augments inflammatory cytokine production via induction of mitochondrial dysfunction. Therefore desensitizing CD4 T cells to detrimental cues, such as IL-21, may also augment Treg function during human IBD.