Background and Aims The tumor microenvironment drives many malignant features of pancreatic ductal adenocarcinoma (PDAC). The fibroblasts within pancreatic tumors promote tissue remodeling, immune suppression, and resistance to therapy. However, the interactions between stromal populations and pancreatic cancer cells are less understood in the liver, the most frequent site of PDAC metastasis. Methods To address this, we employ single cell transcriptomics to compare primary pancreatic vs. liver PDAC lesions. Using a ligand-receptor interaction tool, we assess upregulated pathways in liver PDAC. Results Here, we identify the expression of hepatocyte growth factor (HGF) in fibroblasts and its receptor MET in cancer cells are both markedly increased in the PDAC liver niche. Using functional assays, we validate that mitogenic MET signaling is activated in PDAC cells by liver-derived fibroblasts. Importantly, the inhibition of MET signaling leads to reduced tumor growth in immune competent mouse models. Conclusions Collectively, our data demonstrates that liver stromal-epithelial crosstalk networks engage in signaling pathways distinct from primary pancreatic tumors, highlighting opportunities to develop new treatments for metastatic disease.
BACKGROUND & AIMS:Obesity and consumption of a high-fat diet (HFD) are established risk factors for colorectal cancer (CRC). However, the cellular mechanisms linking dietary lipid exposure to intestinal stem cell (ISC) dysregulation remain incompletely defined. We therefore investigated whether HFD perturbs plasma membrane homeostasis to potentiate colonocyte stemness in mice. METHODS:Mice were placed on a chronic 12-week HFD or control (low-fat diet; LFD) feeding regimen, or a short-term 1-week HFD exposure. In addition, genetically obese leptin receptor-deficient (db/db) versus (db/+) wildtype mice were examined. Membrane free cholesterol and rigidity were quantified in colonic crypts and Lgr5+ ISCs utilizing Filipin III and Di-4-ANEPPDHQ dyes, respectively. Membrane receptor nanoclustering of epidermal growth factor receptor (EGFR) and low-density lipoprotein receptor-related protein 6 (LRP6) was assessed by super-resolution microscopy. β-catenin activation, stem cell frequency, proliferation, and organoid-forming efficiency were also evaluated by immunostaining, flow cytometry, and ex vivo organoid assays. RESULTS:Chronic HFD feeding in mice increased membrane free cholesterol content and rigidity in colonic stem cells, and enhanced EGFR and LRP6 receptor nanoclustering. This alteration in plasma membrane homeostasis resulted in the overactivation of β-catenin and expansion of the ISC niche, e.g., stem cell proliferation and organoid-forming efficiency. Notably, genetically induced obesity in db/db mice did not recapitulate the HFD membrane phenotype, highlighting a diet-specific effect. CONCLUSIONS:Collectively, these findings identify plasma membrane remodeling as a previously unrecognized mechanism linking dietary lipids to aberrant ISC homeostasis and CRC risk.
Background /Aims Severe obesity is a major global health challenge and a key driver of hepatic steatosis through systemic insulin resistance and enhanced de novo lipogenesis. Excessive hepatic triglyceride accumulation represents a central metabolic consequence of this condition. This study aimed to identify immune-derived regulators of liver lipid metabolism and to define the mechanistic role of interleukin-13 (IL-13) in obesity-associated liver disease progression. Methods IL-13 levels and related signaling pathways were analyzed in plasma samples from patients with ≥class II obesity and liver biopsies from patients with hepatic steatosis and steatohepatitis. The effects of IL-13 depletion on lipid metabolism were examined in mice, while its mechanistic role in fat metabolism was studied in primary hepatocytes. Results In a cohort of severely obese patients, plasma IL-13 levels rose after weight loss and positively associated with FGF19, FGF21, and IGF-1. IL-13-deficient mice showed worsened liver triglyceride accumulation, lower polyunsaturated fatty acids and altered expression of PPAR-dependent lipid genes. Primary murine hepatocytes revealed that IL-13 decreases triglyceride content through IL-13Rα1 and a JNK-dependent c-JUN/FOSL2 axis, which enhances β-oxidation and ketogenesis. FOSL2 was required for maximal PPAR activity and was upregulated in livers of patients with hepatic steatosis and steatohepatitis. Conclusions These data demonstrate that IL-13 is a critical immunometabolic regulator of hepatic lipid homeostasis. Targeting the IL-13-FOSL2-PPAR signaling pathway may offer a novel therapeutic approach to reduce hepatic steatosis.
Background and aims The unfolded protein response (UPR) is an essential cellular program maintaining intestinal homeostasis by acting on several cell types. However, whether the UPR sensor IRE1 acts on dendritic cells (DCs) and other antigen presenting cells (APCs) for shaping intestinal immunity remains poorly defined. Methods Here, we dissect the role of the RNase domain of the UPR sensor IRE1 in intestinal CD11c+ APCs, using IRE1 RNase reporter mice and conditional loss-of-function models for IRE1 and its downstream transcription factor XBP1s. Results We identify robust IRE1 RNase activity in intestinal conventional DC subsets, DCs associated to crypto patches and isolated lymphoid follicles (CIA-DCs) and macrophages, independently of microbial signals. Targeted ablation of IRE1 RNase in the intestinal CD11c+ compartment provoked spontaneous features of type 3 immunity in the small intestine, characterized by increased antimicrobial peptides and neutrophil recruitment, as well as elevated production of IL-17 and IL-22 by Th17 cells and group 3 innate lymphoid cells (ILC3s). This dysregulated activation required IRE1 RNase but occurred independently of XBP1s, implicating Regulated IRE1 Dependent Decay (RIDD) rather than canonical XBP1s signaling. Mechanistically, distinct IRE1 RNase-deficient intestinal APC subtypes such as cDCs exhibited increased Il6, Il1b and Ccl22 transcript levels, whereas cDCs and CIA-DCs showed marked reductions in IL-22BP expression, a profile known to increase IL-22 bioavailability and enhance Th17/ILC3 responses. Conclusions Collectively, our findings uncover IRE1 RNase in intestinal CD11c+ APC subtypes as a critical checkpoint restraining type 3 immunity and maintaining intestinal immunity in steady state, highlighting its potential as therapeutic target for intestinal inflammation.
Background and Aims EGFR signaling maintains intestinal homeostasis by modulating proliferation and differentiation within the stem cell compartment with excess EGFR signaling predisposing to neoplasia. EGFR protein levels are central to EGFR function. These levels are negatively regulated by LRIG1, which accelerates receptor internalization and degradation, supporting a tumor suppressor role for LRIG1. LRIG3 is a less studied family member that has been reported, in a context-dependent manner, to both cooperate with and oppose the effects of LRIG1 on EGFR. Methods To examine the function of LRIG3 relative to LRIG1 on EGFR levels and downstream signaling in vivo, we generated an Lrig3 floxed allele and intercrossed these mice with Lrig1CreERT2 mice that are Lrig1 null in the homozygous state. This allowed us to assess the effects of Lrig3 loss in Lrig1-null intestinal cells. We also tested how LRIG3 depletion in Lrig1-expressing cells affects colorectal tumors in Lrig1CreERT2/+; Apcfl/+ mice. Results Unlike the heightened EGFR and Wnt activity caused by Lrig1 loss, loss of Lrig3 in Lrig1Null cells markedly reduced activity in both pathways. In this setting, a widening of the intestinal crypt base was observed with the appearance of intermediate-lineage secretory cells at the expense of stem cells. In marked contrast to multiple distal colonic tumors in Lrig1CreERT2/+; Apcfl/+ mice, deletion of a single Lrig3 allele in Lrig1 haploinsufficient mice decreased EGFR signaling and markedly reduced colonic tumor formation. Conclusions These findings indicate that LRIG3 and LRIG1 oppose one another to balance EGFR signaling, maintain intestinal homeostasis and minimize the likelihood colonic neoplasia.
BACKGROUND:Acute pancreatitis (AP) is a potentially life-threatening inflammatory disorder characterized by trypsin overactivation within acinar cells. While SIRT2 is linked to inflammation, its precise role in AP remains poorly understood. METHODS:Rats were pretreated with AGK2 (SIRT2 inhibitor), Ferrostatin-1 (Fer-1, ferroptosis inhibitor), 1α,25(OH)2D3 (IER3 inhibitor), AAV-SIRT2, or AAV-sh-SIRT2 before L-arginine-induced AP. RNA-seq was performed to identify potential downstream targets of SIRT2. In vitro, SIRT2 and IER3 expression was manipulated by siRNA or overexpression plasmids. The effects on ferroptosis and inflammation were assessed using Western blot, qPCR, immunofluorescence, and assays measuring ROS, Fe2+, mitochondrial membrane potential (MMP), and lipid peroxidation. Regulatory mechanisms among SIRT2, IER3, and ferroptosis were further investigated using co-immunoprecipitation (Co-IP) and dual-luciferase assays. RESULTS:AGK2 or Fer-1 alone reduced pancreatic damage, inflammation, and ferroptosis, with combination therapy showing synergy. SIRT2 or IER3 inhibition was protective, upregulating SLC7A11/GPX4, reducing ROS/Fe2+/lipid peroxides, and restoring mitochondrial function. Conversely, overexpression of SIRT2 or IER3 produced opposite effects. AGK2 and 1α,25(OH)2D3 reversed damage from SIRT2 overexpression. Co-IP confirmed SIRT2 deacetylates and upregulates IER3. Luciferase assays showed IER3 transcriptionally represses SLC7A11. Clinically, serum SIRT2 and IER3 were elevated in AP patients and correlated with disease severity and inflammation. CONCLUSION:SIRT2 enhances IER3 expression via deacetylation, inhibiting the SLC7A11/GPX4 axis and promoting ferroptosis, thereby exacerbating AP. Serum SIRT2 and IER3 are potential biomarkers for AP severity. This study reveals a novel SIRT2/IER3-mediated mechanism driving ferroptosis in AP.
The advent of biologics and targeted oral small molecules has transformed the management of patients with inflammatory bowel disease, leading to increased rates of mucosal healing, reduced hospital admissions, and a diminished need for surgery. In contrast to biologics that specifically target a receptor or its substrate, Janus Kinase (JAK) inhibitors (JAKis) interfere with several shared signalling pathways and therefore exert broader effects. JAK-STAT signalling occupies a central position in multiple pro-inflammatory pathways and plays a pivotal role in the pathophysiology of autoimmune and inflammatory diseases. However, the pleiotropic effects of JAKis have greatly complicated the identification of cellular mediators of beneficial, adverse, or off-target effects, and this remains a challenge to date. This review summarises current knowledge on the mechanisms of action of JAKis for the treatment of IBD, outlined per major cell type implicated in the pathophysiology of the disease. Deeper understanding of their effects on the diverse mucosal cell types is crucial to elucidate therapeutic mechanisms and explain treatment non-response.
BACKGROUND & AIMS Metabolic dysfunction-associated steatohepatitis (MASH), a chronic liver disease, is characterized by persistent low-grade inflammation, partially driven by gut-derived lipopolysaccharide (LPS). Although repeated LPS exposure can induce endotoxin tolerance in innate immune cells, its role in chronic liver diseases remains unclear. Acyloxyacyl hydrolase (AOAH) is an endogenous enzyme that inactivates LPS, potentially modulating this process. We aimed to investigate how AOAH regulates endotoxin tolerance in Kupffer cells (KCs) and how this affects hepatic inflammation and fibrosis during MASH progression. METHODS AOAH-deficient (AOAH-/-) mice and wild-type controls were subjected to multiple dietary MASH models. Inflammatory responses, fibrosis, and transcriptomic changes in liver tissues and isolated KCs were analyzed. Endotoxin tolerance was modulated through β-glucan administration or LPS preconditioning. LPS bioactivity was assessed using TLR4-reporter cell assays. RESULTS LPS-preconditioned KCs exhibited reduced pro-inflammatory cytokine production and transcriptional suppression of inflammatory pathways, indicating tolerance. Despite slight elevation of plasma LPS levels in MASH, upregulation of hepatic AOAH positively correlated with disease severity, suggesting enhanced LPS inactivation but impaired establishment of tolerance. In contrast, AOAH-deficient KCs displayed reinforced endotoxin tolerance, leading to diminished hepatic inflammation and fibrosis. Reversal of tolerance using β-glucan reactivated inflammatory and fibrogenic responses in AOAH-deficient mice, whereas tolerance induction by low-dose LPS preconditioning mitigated MASH pathology, supporting the protective role of macrophage tolerance in chronic liver injury. CONCLUSIONS Endotoxin tolerance in KCs represents a protective mechanism against chronic liver inflammation and fibrosis. AOAH regulates this state by limiting bioactive LPS, thereby modulating the establishment of endotoxin tolerance and downstream inflammatory and fibrotic responses. Enhancing macrophage tolerance by utilizing LPS may offer a novel therapeutic avenue to control the progression of MASH.
Background & Aims Trimethylamine N-oxide (TMAO) is a host-microbial co-metabolite that significantly increases following Roux-en-Y gastric bypass (RYGB). While TMAO is associated with cardiovascular diseases, its role in colorectal cancer (CRC) remains unclear. Methods FabplCre;Apc15lox/+ mice, a genetically altered CRC model, together with murine macrophages and human colonic cancer cells (HCT-116) were used to investigate TMAO impact on colonic tumorigenesis. Results TMAO supplementation significantly reduced the colonic tumor load in male, but not female mice. Consistently, dietary TMAO resulted in higher retention of circulating TMAO in males, which was significantly and inversely correlated with tumor loads. Furthermore, TNF-α-expressing cell frequencies in the colonic intraepithelial lymphocytes (IEL) were significantly lower in TMAO-supplemented male mice compared to controls, suggesting that circulating TMAO could play a protective effect against colonic tumor growth via down-regulation of TNF-α-expressing cells. This was supported by in vitro observations that TMAO reduced lipopolysaccharides (LPS)-stimulated TNF-α production from macrophages. TMAO exerted no effects on cell proliferation (Ki67) and DNA damage (γH2AX) of HCT-116 cells. Conclusion Our study leads us to conclude that higher retention of circulating TMAO has a protective effect against CRC in a sex-dependent manner, highlighting the importance of understanding the complex relationship amongst the concentrations of host-microbial cometabolite TMAO, its systemic circulation, and its biological function in modulating CRC risk.
BACKGROUND & AIMS:Although the majority of human pancreatic cancers are classified as pancreatic ductal adenocarcinomas, tumors are comprised of diverse dedifferentiated tumor cell states that dynamically interact with an equally complex microenvironment, making it imperative to understand, in vivo, how normal epithelial identity is (dys)regulated during tumorigenesis. METHODS:Here, we integrate data from mouse and human pancreatic ductal adenocarcinoma with a unique zebrafish model of pancreatic cancer to elucidate evolutionarily conserved mechanisms regulating dedifferentiation during pancreatic tumorigenesis. RESULTS:Histologic and single-cell transcriptional profiling of heterogeneous tumors developing in dual-transgenic ptf1a:Gal4-VP16; UAS:mKO2- KRASG12D adult zebrafish revealed mixed populations of progenitor-like cells with acinar features, progenitor-like cells with ductal features and undifferentiated progenitor-like cells, providing the opportunity to interrogate specific transcriptional and epigenetic modifiers controlling neoplastic pancreatic cellular differentiation. Screening a panel of chromatin modifiers and chromatin readers for differential expression in these distinct cell clusters revealed hdac9b to be upregulated in the progenitor-like cells enriched for ductal features. RNA velocity trajectories supported an acinar cell-of-origin for these hdac9b-expressing progenitor-like cells. The treatment of fish with the class IIA histone deacetylase inhibitor TMP195 confirmed a functional role of histone deacetylase activity in regulating neoplastic cell differentiation through repression of the ductal-like and progenitor cell states and the associated reactivation of acinar gene expression. Cross-species analyses in autochthonous murine tumors confirmed an early, evolutionarily conserved role for Hdac9 in pancreatic tumorigenesis, marking tumor cell subpopulations characterized by a loss of epithelial differentiation and a basal subtype identity. In human pancreatic cancer, higher HDAC9 expression is significantly correlated with poorly differentiated tumor histology and predicts shorter overall survival in patients with pancreatic ductal adenocarcinoma with classical subtype-enriched tumors. CONCLUSIONS:These findings suggest a highly conserved role for HDAC9 and class IIA histone deacetylases in vertebrate pancreatic tumorigenesis and may lead to new strategies for reactivating (normal acinar-epithelial) differentiation programs to intercept and treat pancreatic ductal adenocarcinoma.
BACKGROUND & AIMS:Inflammatory bowel disease involves intractable intestinal inflammation often refractory to current pharmacotherapies. We investigated the therapeutic potential of a novel, orally available inhibitor targeting α-1,6-fucosyltransferase-the sole glycosyltransferase catalyzing core fucosylation of N-glycans in mammals-as an anti-inflammatory agent. METHODS:α-1,6-Fucosyltransferase inhibitor efficacy was evaluated in 2 murine models: a trinitrobenzene sulfonic acid-induced colitis model and a naive CD4+ T-cell adoptive transfer model using recombination activating gene 2 (Rag2)-deficient mice. Amelioration of trinitrobenzene sulfonic acid-induced colitis by the α-1,6-fucosyltransferase inhibitor was also evaluated in LckcreFut8fl/fl mice in which α-1,6-fucosyltransferase is conditionally abrogated in T cells. Splenic CD4+ T cells from wild-type mice were treated with the α-1,6-fucosyltransferase inhibitor to evaluate its effects on cell signaling, cytokine production, and T helper 1, 2, and 17 differentiation. In vivo safety was assessed in wild-type mice. RESULTS:Oral administration of the α-1,6-fucosyltransferase inhibitor effectively decreased T-cell core fucosylation and ameliorated trinitrobenzene sulfonic acid-induced colitis and colitis induced by adoptive T-cell transfer. These clinical effects were accompanied by decreased T helper 1 and 2 cytokine production from CD4+ T cells. Trinitrobenzene sulfonic acid-induced colitis was attenuated in LckcreFut8fl/fl mice, demonstrating that the ameliorative effect of α-1,6-fucosyltransferase inhibition is directly mediated by T cells. In vitro, the inhibitor suppressed T-cell signal transduction and T helper 1, 2, and 17 cell differentiation. No apparent hepatorenal toxicity or intestinal mucus layer disruption was observed. CONCLUSIONS:α-1,6-Fucosyltransferase inhibition modulated T-cell core fucosylation and reduced inflammation in murine colitis models without apparent toxicity, indicating that targeting α-1,6-fucosyltransferase may offer a novel therapeutic approach for inflammatory bowel disease by disrupting proinflammatory T-cell responses.
BACKGROUND & AIMS:The role of diet in the pathogenesis of inflammatory bowel diseases remains unclear. Most dietary interventions for inflammatory bowel disease improve symptoms without consistent mucosal healing; exceptions include exclusive enteral nutrition and exclusion diets in patients with Crohn's disease. Oxalate, a naturally occurring compound found in all plant foods, is absorbed through the gut and has been shown to activate systemic and renal proinflammatory immune responses. However, the impact of intestinal oxalate on innate immune response in the context of inflammatory bowel disease is unknown. METHODS:We measured gene expression, stool oxalate content, and dietary intake in people with inflammatory bowel diseases and controls. Complementary studies were conducted in mouse models of chemically induced colitis, spontaneous colitis, and ex vivo cell culture systems to evaluate the relationships observed between oxalate transporter expression, stool oxalate content, and mucosal inflammation. RESULTS:Intestinal oxalate transporters, SLC26A2 and SLC26A3 are consistently downregulated across inflammatory bowel disease subtypes (Crohn's disease, ulcerative colitis), inflammatory bowel disease tissues (colon, ileum), and in mouse models of experimental colitis irrespective to experimental diet assignment. In patients with Crohn's disease, we observed higher stool oxalate content despite variable dietary oxalate intake. Altered expression of SLC26A6 corresponded to stricturing behavior of disease in patients with Crohn's disease. Altered expression corresponded more directly to higher stool oxalate content and increased disease activity in mice fed an oxalate supplemented diet. CONCLUSIONS:Dietary oxalate may exacerbate innate immune responses in susceptible individuals. Expression of oxalate transporters may be a disease-associated biological signal of both sensitivity to dietary oxalate and possibly, pathogenesis and clinical trajectory of patients with inflammatory bowel disease. This warrants future exploration of the role of dietary oxalate in mucosal inflammation in inflammatory bowel diseases.
BACKGROUND & AIMS:Perianal fistulizing Crohn's disease is a severe morbidity with rectal involvement and a higher prevalence in African Americans compared with European Americans. In this study, we sought to define the distinguishing cellular and molecular features between inflamed rectal Crohn's disease with and without perianal fistula across populations that give new insight into disease severity or progression. METHODS:Rectal mucosal biopsies were obtained for single-cell sequencing from 50 individuals (262k cells); 18 with Crohn's disease of the rectum with fistula, 25 with Crohn's disease of the rectum without fistula and any other perianal complications, and 7 from individuals with no past or present mucosal disease (controls). Spatial transcriptomics were conducted and analyzed by Sopa and Squidpy, whereas immunofluorescence was processed with QuPath. Patient-derived rectal organoids were used to test inflammatory signaling. RESULTS:A major distinction between inflamed Crohn's disease of the rectum with and without fistula was the decreased phagocytic signature, antigen processing profile, and unique morphologic structures of proinflammatory CD4+CD68+ macrophages. In fistulizing Crohn's disease, these macrophages formed large aggregates in the lamina propria near epithelial crypts and appeared to disrupt cellular networks by engaging in signaling with other cell subtypes. At the population level, the most significant differences in cellular pathways were detected between African Americans and European Americans with inflamed Crohn's disease of the rectum and fistula, and between patients with and without fistulizing disease among African Americans. CONCLUSIONS:During inflamed Crohn's disease of the rectum with fistula, CD4+ macrophages alter cellular networks in the lamina propria while forming large cell aggregates, likely mediating complex interactions with T cells and possibly microbial antigens that are involved in the more severe pathologic conditions associated with fistula tract formation and progression.
BACKGROUND & AIMS:Current treatments of chronic hepatitis B are rarely curative. Capsid assembly modulators target capsid formation by the hepatitis B virus core protein. Class A capsid assembly modulators induce abnormal core protein assembly, but how they affect core protein dynamics and inhibit hepatitis B virus replication is only partially understood. METHODS:Human liver chimeric mice and hepatitis B virus-susceptible cells were infected to monitor dynamic changes and the fate of core protein and assembled capsids. We analyzed hepatitis B virus and protein dynamics over 4 weeks under treatment of the class A capsid assembly modulator molecule HAP_R01. RESULTS:We found an altered nuclear-cytoplasmic distribution of hepatitis B virus core protein upon treatment with HAP_R01. This effect was confirmed in primary human hepatocytes and in hepatitis B virus-infected liver-humanized mice. Mechanism-of-action studies in hepatitis B virus-permissive cells demonstrated that HAP_R01 primarily targets newly synthesized core protein and affects capsid assembly, core protein localization, and solubility in a dose- and time-dependent manner. At 50 nM, HAP_R01 promoted assembly of hepatitis B virus genome-free capsids and nuclear accumulation of core protein. At ≥500 nM, HAP_R01 treatment reduced soluble core protein and capsid levels, but markedly increased insoluble core protein levels, resulting in an overall intracellular accumulation of core protein. Insoluble nuclear agglomerations of core protein were deposited in promyelocytic leukemia nuclear bodies. Treating infected cells for 31 days significantly reduced an established covalently closed circular DNA pool and secreted hepatitis B surface antigen and hepatitis B e antigen levels, by inhibiting covalently closed circular DNA replenishment. CONCLUSIONS:As an exemplary class A capsid assembly modulator, HAP_R01 inhibits hepatitis B virus genome replication by perturbing capsid assembly and by inducing insoluble core protein accumulation in the nucleus that affects replenishment of the covalently closed circular DNA pool.
BACKGROUND & AIMS:Lactylation has been implicated in the repair and regeneration of multiple organs, including the skin and the heart. The liver serves as a vital organ for lactate metabolism, whereas the roles of lactate and lactylation during liver regeneration remain unclear. METHODS:Mice were subjected to 70% partial hepatectomy, and the liver-to-body weight ratio and proliferative markers were measured. Protein-protein interactions were detected by co-immunoprecipitation. The pivotal site of GCN5 for catalysis was predicted by theoretical modeling. CUT&Tag and bulk RNA sequencing assays were used to identify downstream genes regulated by H3K9 lactylation. Murine hepatocyte organoids and human liver tissues were used to validate the effects of H3K9 lactylation. RESULTS:Lactate supplementation notably improved the liver-to-body weight ratio and upregulated hepatic levels of Ki-67, proliferating cell nuclear antigen, and 5-ethynyl-2'-deoxyuridine incorporation at 48 hours after partial hepatectomy, whereas hepatocyte-specific Ldha knockout mice showed impaired liver regenerative capacity. In vitro experiments indicated that increased intracellular lactate upregulated H3 lactylation and promoted hepatocyte proliferation. Mechanistically, GCN5, rather than P300 or MOF, was identified as the pivotal enzyme catalyzing H3K9 lactylation. Furthermore, lactate treatment promoted the interaction between GCN5 and H3K9 during liver regeneration. Glu639 was identified as the critical amino acid residue in GCN5 for catalyzing H3K9 lactylation. CUT&Tag and bulk RNA sequencing results further revealed that H3K9 lactylation upregulated the expression of Lipin1 and its mediated transcription of fatty acid oxidation-related genes. The proregenerative effects of lactate and H3K9 lactylation were validated using murine hepatocyte organoids and human liver tissues. CONCLUSIONS:Lactate facilitates liver regeneration through promoting GCN5-mediated H3K9 lactylation, which upregulates Lipin1 transcription and subsequent Lipin1-mediated fatty acid oxidation in hepatocytes.
BACKGROUND & AIMS:Constitutive androstane receptor transcriptionally regulates xenobiotic detoxification, metabolism, and hepatocyte proliferation. Although constitutive androstane receptor activation promotes regeneration and its loss impairs recovery after extreme hepatectomy (86%-90% liver resection), its temporal role during standard liver regeneration following two-thirds partial hepatectomy remains unclear. METHODS:We quantified DNA synthesis, gene expression, and bile acid concentrations and composition in Car knockout and wild-type mice across regenerative time points after partial hepatectomy. RESULTS:Car transcript expression is dynamically regulated during regeneration, peaking at 12 hours and plummeting below basal levels by 72 hours post-partial hepatectomy. Despite this, Car knockout livers regenerate comparably to wild-type livers. Notably, Car knockout mice display an exaggerated increase in hepatic and systemic bile acid levels after partial hepatectomy, revealing an unexpected metabolic role for constitutive androstane receptor. Pharmacologic reduction of bile acid levels with cholestyramine in Car knockout mice attenuated 5-bromo-2'-deoxyuridine incorporation and proliferation-associated gene expression. Analysis of single-cell RNA sequencing data further showed that Car and its target genes are enriched in hypermetabolic, rather than proliferating, hepatocyte population during regeneration. CONCLUSIONS:Constitutive androstane receptor exerts a temporally regulated metabolic role during liver regeneration. Car deletion amplifies the post-hepatectomy bile acid surge, which acts as a compensatory proproliferative signal to sustain regeneration.
BACKGROUND & AIMS:Immune checkpoint inhibitor therapy is frequently limited by severe gastrointestinal toxicity. Although the immune mechanisms driving late-stage, overt mucosal inflammation are increasingly recognized, the early microbiota-metabolic events that prime epithelial barrier dysfunction remain poorly defined. METHODS:We employed an integrated multiomics approach in a murine model of early immune checkpoint inhibitor-induced injury, with validation in a clinical cohort of patients with immune checkpoint inhibitor colitis. Mechanisms were investigated using in vitro receptor screening, genetically engineered mice with altered myosin light chain kinase 1 activity, and pharmacologic intervention with repurposed low-dose tacrolimus. Subcutaneous melanoma models were utilized to evaluate the relationship between barrier modulation and antitumor outcomes. RESULTS:Early immune checkpoint inhibitor treatment induced a distinct dysbiotic state characterized by Pseudomonadota expansion, which was associated with the accumulation of bioactive lysophosphatidic acid-a metabolic signature also observed in human patients. Mechanistically, we identified lysophosphatidic acid receptor 2 as the key epithelial sensor that transduces this metabolic signal into pathological myosin light chain kinase 1 activation, driving the priming phase of barrier dysfunction. Genetic ablation of myosin light chain kinase 1 prevented this early barrier loss and was associated with improved tumor control and favorable regional immune alterations. Conversely, constitutive activation of myosin light chain kinase 1 exacerbated barrier disruption, which correlated with accelerated tumor progression and a systemic immunosuppressive shift. Furthermore, repurposing low-dose tacrolimus as a selective modulator to disrupt FKBP8-dependent myosin light chain kinase 1 recruitment effectively mitigated barrier injury without dampening antitumor responses. CONCLUSIONS:The dysbiosis-associated lysophosphatidic acid-lysophosphatidic acid receptor 2-myosin light chain kinase axis acts as an early metabolic trigger for the priming phase of lysophosphatidic acid-induced barrier dysfunction. Modulating this upstream initiation step provides a temporal and mechanism-based strategy to uncouple intestinal toxicity from anti-tumor efficacy.
