Lee, Hyun Se Kim

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.

Abstract Senescent cells secrete proinflammatory factors known as the senescence-associated secretory phenotype (SASP), contributing to tissue dysfunction and aging. Mitochondrial dysfunction is a key feature of senescence, influencing SASP via mitochondrial DNA (mtDNA) release and cGAS/STING pathway activation. Here, we demonstrate that mitochondrial RNA (mtRNA) also accumulates in the cytosol of senescent cells, activating RNA sensors RIG-I and MDA5, leading to MAVS aggregation and SASP induction. Inhibition of these RNA sensors significantly reduces SASP factors. Furthermore, BAX and BAK plays a key role in mtRNA leakage during senescence, and their deletion diminishes SASP expression in vitro and in a mouse model of Metabolic Dysfunction Associated Steatohepatitis (MASH). These findings highlight mtRNA's role in SASP regulation and its potential as a therapeutic target for mitigating age-related inflammation.

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol–induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE + recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8 + T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in 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).

Abstract Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). Recently, mutations in p300 and CBP were identified that abrogate their function as an acetyltransferase but not the ability to mediate crotonylation. These mutants can activate transcription independent of their ability to acetylate histones, demonstrating that crotonylation can also lead to transcriptional activation. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have both decrotonylase and deacetylase activity, but the relative importance of each function in T cells is not known. Genetic knockout of HDAC3 using CD2 icre mice results in a severe block in T cell development and spontaneous development of severe colitis. Examination of HDAC3 cKO mice has shown an aberrant ratio of Treg:Th17 cells within the gut, driving inflammation and loss of mucosal barrier integrity. Our lab has also determined that HDAC3 is required for at least three aspects of T cell development: CD4+ CD8+ (DP) cell survival, positive selection, and CD4+ lineage development. Recently, a mutation was identified in either HDAC1 or HDAC3 that eradicated deacetylase activity but retained decrotonylase activity (“VRPP”). Through this mutation we were able to generate a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP. Strikingly, this mouse does not develop colitis, although T cell development is still impaired. Thus, HDAC3 decrotonylase activity alone is sufficient to impede the IBD phenotype. Support by grants from National Institute of Allergy and Infectious Diseases (R01 AI150100)

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 Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Interestingly, ABCB7 appeared to be dispensable for splenic B cell homeostasis, as CD23-cre ABCB7 cKO mice had normal splenic B cell populations. However, stimulated ABCB7-deficient splenic B cells from CD23-cre ABCB7 cKO mice had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination. Supported by 1R21 AI157328-01 to V.S.S., T32AI007425 to M.J.L.

Abstract Inflammatory bowel disorders (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) affect between 1–3 million people in the United States with prevalence on the rise. Patients present with symptoms including diarrhea, abdominal cramps, and weight loss. Many factors are implicated in pathogenesis, including host immune response. Identification of molecules that regulate IBD is critical in understanding pathogenesis. ST8Sia6 is a sialic acid transferase that adds terminal a2,8 disialic acids to cell surface glycoproteins, and we have found that ST8Sia6 knockout (KO) mice exhibit increased spontaneous small bowel lamina propria CD4 TH1 polarization, increased inflammatory cytokine production, and decreased IgA class switching of Peyer’s patch B cells, indicative of inflammation. Additionally, adult ST8Sia6 KO mice have shortened small bowels, likely due to baseline inflammation. These mice do not exhibit obvious baseline symptoms, but ST8Sia6 KO mice exhibit increased weight loss and decreased survival when challenged with dextran sodium sulfate (DSS)-induced colitis, which was also enhanced when co-housed with wildtype (WT) mice and after antibiotic treatment. These DSS-challenged ST8Sia6 KO mice exhibit increased histological damage and immune infiltration of the gut as compared to WT controls. The accelerated DSS-colitis is also observed when ST8Sia6-deficient bone marrow is transplanted into WT recipients, indicating that the effect is mediated specifically by immune cells. This data demonstrates a role for ST8Sia6 in regulating the host immune response to inflammatory bowel disorders, and further elucidating this role will aid in developing a mechanistic understanding of IBD pathogenesis. Supported by grants from NIH R01 CA243545

Abstract The lifetime risk for colorectal cancer in the United States is approximately 4%. Individuals with Inflammatory Bowel Disease, including Ulcerative Colitis and Crohn’s Disease, have a substantially increased risk of developing colorectal cancer. The mechanisms for accelerated tumorigenesis due to enhanced inflammation are not fully characterized. Siglecs (sialic acid immunoglobulin lectin-like proteins) are a family of inhibitory receptors that are negative regulators of the immune response. Siglec-E is an inhibitory receptor that is expressed by innate immune cells, including monocytes, macrophages, neutrophils and dendritic cells. Interestingly, mice deficient in Siglec-E have accelerated development of tumors and reduced survival in a spontaneous mouse model of colorectal cancer (TS4-cre LSL-KRasG12D APClox468/wt). While tumors develop at approximately 6 months in mice with Siglec-E, tumors develop at approximately two months in Siglec-E knockout mice. Initial results indicate that Siglec-E knockout mice also have accelerated gut inflammation using the DSS colitis model as compared to WT mice. Current studies are examining inflammation that develops during tumorigenesis in Siglec-E knockout mice.