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The inflammatory response is a significant factor in acetaminophen (APAP)-induced acute liver injury. And it can be mediated by macrophages of different origins. However, whether Kupffer cells and mononuclear-derived macrophages play an injury or protective role in APAP hepatotoxicity is still unclear. In this study, C57/BL6N mice were performed to establish the APAP acute liver injury model. Intervention experiments were also carried out using clodronate liposomes or TREM2 knockout. We found that APAP overdose triggered the activation of inflammatory factors and enhanced the expression of the RIPK1-MLKL pathway in mice's livers. Moreover, our study showed that inflammation-related protein expression was increased after clodronate liposome administration or TREM2 knockout. The RIPK1-MLKL-mediated necroptosis was also significantly activated after the elimination of Kupffer cells or the inhibition of mononuclear-derived macrophages. More importantly, clodronate liposomes treatment and TREM2 deficiency all worsen APAP-induced liver damage in mice. In conclusion, the results indicate that Kupffer cells and mononuclear macrophages play a protective role in APAP-induced liver injury by regulating necroptosis. Therefore, macrophages hold as a potential therapeutic target for APAP-induced liver damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment., Design: We identified TLR8 -expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients., Results: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12 ) and downregulated genes associated with the KC identity (eg, SPIC ). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition., Conclusion: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection., Competing Interests: Competing interests: FZ and BT received grants from Assembly, Beam Therapeutics, Blue Jay and JnJ; FZ had consulting activities with Assembly, Blue Jay and GSK. YCH receives grants from Gilead Sciences and Sysmex. FZ is an associate editor of the journal. SD, LL, NK, JJW, AHL, and SPF are or were employees of Gilead Sc., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Macrophages make up a heterogeneous population of immune cells that exhibit diverse phenotypes and functions in health and disease. Although macrophage epigenomic and transcriptomic profiles have been reported, the proteomes of distinct macrophage populations under various pathological conditions remain largely elusive. Here, we employed a label-free proteomic approach to characterize the diversity of the hepatic macrophage pool in an experimental model of CCl 4 -induced liver fibrosis. We found a decrease in the proportion of liver resident embryo-derived KCs (EmKCs), and a drastic increase in the proportion of monocyte-derived KCs (MoKCs) and CLEC2 - Macs. Proteomic profiling revealed that MoKCs largely resembled EmKCs, whereas CLEC2 - Macs exhibited greater proteomic alternations compared with EmKCs, suggesting two distinct destinations for monocyte differentiation during liver fibrosis. Furthermore, CLEC2 - Macs were characterized by increased expression of proteins associated with inflammatory response, antigen processing and presentation processes, which may be involved in the pathogenesis of liver fibrosis. Collectively, our study provides insights into the considerable heterogeneity within the hepatic macrophage pool during liver fibrosis.

Background: Didymin is a dietary flavonoid originally discovered by our group as a potent anti-ulcerative colitis (UC) agent. However, whether didymin plays a protective role in UC-associated inflammatory liver injury is still unclear., Purpose: This study aimed to evaluate the therapeutic potential of didymin on UC-associated inflammatory liver injury and explore the underlying mechanism., Study Design and Methods: Colitis model was established in C57BL/6 mice by exposure to DSS, and didymin was administrated intragastrically for consecutive 10 days. The inflammatory liver injury was assessed by levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) in serum and histopathological damage in the liver. In vitro Kupffer cells and RAW264.7 cells challenged with lipopolysaccharides (LPS) were used to explore the modulatory activity of didymin on pro-inflammatory cytokines secretion and Notch1 signaling pathway activation., Results: Didymin significantly mitigated liver coefficiency, ALT and AST levels in serum, and the hepatic histopathological damage caused by DSS-induced acute and chronic colitis. The mRNA expressions of pro-inflammatory factors including Tnf, Il1, and Il6 in liver tissues, Kupffer cells, and RAW264.7 cells stimulated by the influx of LPS was significantly deprived after didymin treatment. Mechanistically, didymin obstructed the protein expression, nuclear translocation of notch intracellular domain 1 (Notch1-ICD) and mRNA expression of hairy and enhancer of split 1 (Hes1). Further, the inhibitory mechanism of the Notch1-Hes1 pathway was dependent on c-Cbl-mediated Notch1-ICD lysosomal degradation., Conclusion: Our study verified for the first time that didymin could prevent UC-associated diseases, such as inflammatory liver injury, and the mechanism was related to facilitating Notch1 lysosomal degradation rather than proteasome degradation via promoting protein expression of c-Cbl in macrophages. Our findings that the inhibition of Notch1 signaling transduction helps to alleviate UC-associated liver injury provides possible therapeutics for the treatment of colitis and also furnishes a research paradigm for the study of flavonoids with similar structures., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier GmbH.)

Inflammatory signals lead to recruitment of circulating monocytes and induce their differentiation into pro-inflammatory macrophages. Therefore, whether blocking inflammatory monocytes can mitigate disease progression is being actively evaluated. Here, we employ multiple lineage-tracing models and show that monocyte-derived macrophages (mo-mac) are the major population of immunosuppressive, liver metastasis-associated macrophages (LMAM), while the proportion of Kupffer cells (KC) as liver-resident macrophages is diminished in metastatic nodules. Paradoxically, genetic ablation of mo-macs results in only a marginal decrease in LMAMs. Using a proliferation-recording system and a KC-tracing model in a monocyte-deficient background, we find that LMAMs can be replenished either via increased local macrophage proliferation or by promoting KC infiltration. In the latter regard, KCs undergo transient proliferation and exhibit substantial phenotypic and functional alterations through epigenetic reprogramming following the vacating of macrophage niches by monocyte depletion. Our data thus suggest that a simultaneous blockade of monocyte recruitment and macrophage proliferation may effectively target immunosuppressive myelopoiesis and reprogram the microenvironment towards an immunostimulatory state., (© 2024. The Author(s).)

Purpose: Tumor recurrence after radiofrequency ablation (RFA) affects the survival rate of patients and limits its clinical application. Tumor recurrence around the ablation area may be related to the thermal injury of hepatocytes (HCs) around the tumor, but the specific mechanism is still unclear., Methods: A liver cancer thermal injury mouse model was established via RFA in the C57BL/6 mice. Primary HCs and Kupffer cells (KCs) were isolated and cultured to assess their sensitivity to thermal injury via the MTT assay. Flow cytometry was used to assess macrophage polarization. Furthermore, Western blotting and co-immunoprecipitation (co-IP) were utilized to evaluate the protein expression of intracellular signaling pathway. Finally, Transwell and wound healing assays was conducted to evaluate the invasion potential of liver cancer cells., Results: Our findings revealed that RFA-induced liver thermal injury promoted the upregulation and secretion of HMGB1 in HCs. HMGB1 had a protective effect on HCs thermal injury, potentially mediated through autophagy regulation. Heat-injured HCs release HMGB1, which activates the TREM1/JAK2/STAT3 signaling pathway in KCs, thus fostering an immunosuppressive tumor microenvironment (TME). Moreover, HMGB1 secretion by heat-injured HCs exacerbates the migration and invasion of HCC cells by influencing macrophage polarization., Conclusion: RFA-induced thermal injury triggers HMGB1 release from HCs, driving macrophage M2 polarization and increasing the invasion ability of liver cancer cells. These findings reveal a potential therapeutic target for combating liver cancer recurrence following thermal ablation., (© 2024. The Author(s).)

Septic patients, coupling severe disseminated intravascular coagulation (DIC) and thrombocytopenia, have poor prognoses and higher mortality. The platelet glycoprotein Ibα (GPIbα) is involved in thrombosis, hemostasis, and inflammation response. However, it remains unclear whether the GPIbα cytoplasmic tail regulates sepsis-mediated platelet activation and inflammation, especially in Staphylococcus aureus ( S. aureus ) and Escherichia coli ( E. coli ) infections. Using a mouse model of S. aureus -induced bacteremia, we found that both 10 amino acids of GPIbα C-terminal sequence deficiency and pharmacologic inhibition of protein kinase C (PKC) alleviated pathogenesis by diminishing platelet activation and aggregate formation. Furthermore, the GPIbα cytoplasmic tail promoted the phagocytosis of platelets by Kupffer cells in vivo. The genetically deficient GPIbα cytoplasmic tail also downregulated inflammatory cytokines and reduced liver damage, ultimately improving the survival rate of the septic mice. Our results illustrate that the platelet GPIbα cytoplasmic domain exacerbates excessive platelet activation and inflammation associated with sepsis through a PKC-dependent pathway. Thus, our findings provide insights for the development of effective therapeutic strategies using PKC inhibitor treatment against bacterial infection.

The liver is the largest solid organ in the body and is primarily composed of hepatocytes (HCs), endothelial cells (ECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs), which spatially interact and cooperate with each other to maintain liver homeostasis. However, the complexity and molecular mechanisms underlying the crosstalk between these different cell types remain to be revealed. Here, we generated mice with conditional deletion of Gdf2 (also known as Bmp9 ) and Bmp10 in different liver cell types and demonstrated that HSCs were the major source of GDF2 and BMP10 in the liver. Using transgenic ALK1 (receptor for GDF2 and BMP10) reporter mice, we found that ALK1 is expressed on KCs and ECs other than HCs and HSCs, and GDF2 and BMP10 secreted by HSCs promote the differentiation of KCs and ECs and maintain their identity. Pdgfb expression was significantly upregulated in KCs and ECs after Gdf2 and Bmp10 deletion, ultimately leading to HSCs activation and liver fibrosis. ECs express several angiocrine factors, such as BMP2, BMP6, Wnt2, and Rspo3, to regulate HC iron metabolism and metabolic zonation. We found that these angiocrine factors were significantly decreased in ECs from Gdf2/Bmp10 HSC-KO mice, which further resulted in liver iron overload and disruption of HC zonation. In summary, we demonstrated that HSCs play a central role in mediating liver cell-cell crosstalk via the production of GDF2 and BMP10, highlighting the important role of intercellular interaction in organ development and homeostasis., Competing Interests: DZ, ZH, XL, HW, QH, YW, FY, WY, DL, SY, CH, YH, LT No competing interests declared, (© 2024, Zhao, Huang, Li et al.)

Metabolic dysfunction-associated steatohepatitis is well accepted as a potential precursor of hepatocellular carcinoma. Previously, we reported that fibroblast growth factor 21 (FGF21) revealed a novel anti-inflammatory activity via inhibiting the TLR4-IL-17A signaling, which could be a potential anticarcinogenetic mechanism to prevent to MASH-HCC transition. Here, we set out to determine whether FGF21 has a major impact on Kupffer cells' (KCs) ability during MASH-HCC transition. We found aberrant hepatic FGF21 and KC pool in human MASH-HCC. Lack of FGF21 up-regulated ALOX15, which converted the oxidized fatty acids to induce excessive KC death and mobilization of monocyte-derived macrophages (MoMFs) for KC replacement. Lack of FGF21 oversupplied free fatty acids for sphingosine-1-phosphate (S1P) cascade synthesis to mediate MASH-HCC transition via S1P-YAP signaling and cross-talk between tumor cells and macrophages. In conclusion, lack of FGF21 accelerated MASH-HCC transition via the S1P-AP signaling. Compromised MoMFs could present as tumor-associated macrophage phenotype rendering tumor immune microenvironment for MASH-HCC transition.

A STING (stimulator of interferon genes) agonist GSK3996915 under investigation in early discovery for hepatitis B was orally dosed to a mouse model for understanding the parent drug distribution in liver, the target organ. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to quantify the distribution of GSK3996915 in liver collected from mice administered a single oral dose at 90 mg/kg. GSK3996915 was detected with a zonal distribution localized in the portal triad and highly concentrated in the main bile ducts, indicating clearance through biliary excretion. High spatial resolution imaging showed the distribution of the parent drug localized to the cellular populations in the sinusoids, including the Kupffer cells. Additionally, a series of drug-related metabolites were observed to be localized in the central zones of the liver. These results exemplify the potential of utilizing MALDI IMS for measuring not only quantitative drug distribution and target exposure but also drug metabolism and elimination in a single suite of experiments. SIGNIFICANCE STATEMENT: An integrated imaging approach utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) complemented with immunohistochemistry (IHC) and histology was used to address the question of target exposure at the cellular level. Localized quantification of the parent drug in the target organ and identification of potential metabolites in the context of tissue histology were also achieved in one experimental suite to support characterization of pharmacokinetic properties of the drug in the early discovery stage., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Trichloroethylene (TCE) is a common environmental contaminant that can induce occupational dermatitis medicamentosa-like TCE (ODMLT), where the liver damage is the most common complication. The study aims to uncover the underlying mechanism of TCE-sensitization-induced liver damage by targeting specific exosomal microRNAs (miRNAs). Among the enriched serum exosomal miRNAs of ODMLT patients, miR-205-5p had a significant correlation coefficient with the liver function damage indicators. Moreover, retinoic acid receptor-related orphan receptor α (RORα) was identified as a direct target of miR-205-5p via specific binding. Further experiments showed that kupffer cells (KCs) underwent M1 phenotypic and functional changes in liver injury induced by TCE which were alleviated by reducing the expression of miR-205-5p. However, this alleviation was reversed by the RORα antagonist SR1001. In vitro experiments showed that miR-205-5p promoted M1 polarization of macrophages and enhanced the secretion of inflammatory factors by regulating RORα. An increase in RORα reversed the polarization direction of M1-type macrophages and reduced the secretion of proinflammatory factors. In addition, pretreatment of mice with SR1078, a specific RORα agonist, effectively blocked M1 polarization of KCs and reduced the severity of TCE-induced liver injury. Our study uncovers that miR-205-5p regulates KC M1 polarization by targeting RORα in immune liver injury induced by TCE sensitization, providing new insight into the molecular mechanisms and new therapeutic targets for ODMLT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The global prevalence of type 2 diabetes mellitus (T2DM) continues to rise. Therefore, it has become a major concern health issue worldwide. T2DM leads to various complications, including metabolic-associated fatty liver disease (MAFLD). However, comprehensive studies on MAFLD as a diabetic complication at different stages are still lacking. Using advanced single-cell RNA-seq technology, we explored changes of livers in two T2DM murine models. Our findings revealed that increase activation of hepatic stellate cells (HSCs) exacerbated the development of MAFLD to steatohepatitis by upregulating transforming growth factor β1 induced transcript 1 ( Tgfb1i1 ). Upregulated thioredoxin-interacting protein ( Txnip ) contributed to hepatocyte damage by impairing reactive oxygen species clearance. Additionally, the capillarization of liver sinusoidal endothelial cells correlated with Fabp4 overexpression in endothelial cells. A novel subset of Kupffer cells (KCs) that expressed Cd36 exhibited an activated phenotype, potentially participating in inflammation in the liver of diabetic mice. Furthermore, ligand-receptor pair analysis indicated that activated HSCs interacted with hepatocytes or KCs through Thbs2 and Lamb2 in late-stage diseases. The reduction in cell-cell interactions within hepatocytes in diabetic mice, reflects that the mechanisms regulating liver homeostasis is disrupted. This research underscores the importance of dynamics in diabetic MAFLD, and provides new insights for targeted therapies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The liver macrophage population comprises resident Kupffer cells (KCs) and monocyte-derived macrophages with distinct pro- or anti-inflammatory properties that affect the severity and course of liver diseases. The mechanisms underlying macrophage differentiation and functions in metabolic dysfunction-associated steatotic liver disease and/or steatohepatitis (MASLD/MASH) remain mostly unknown. Using single-cell RNA sequencing (scRNA-seq) and fate mapping of hepatic macrophage subpopulations, we unraveled the temporal and spatial dynamics of distinct monocyte and monocyte-derived macrophage subsets in MASH. We revealed a crucial role for the Notch-Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) signaling pathway in controlling the monocyte-to-macrophage transition, with Rbpj deficiency blunting inflammatory macrophages and monocyte-derived KC differentiation and conversely promoting the emergence of protective Ly6C lo monocytes. Mechanistically, Rbpj deficiency promoted lipid uptake driven by elevated CD36 expression in Ly6C lo monocytes, enhancing their protective interactions with endothelial cells. Our findings uncover the crucial role of Notch-RBPJ signaling in monocyte-to-macrophage transition and will aid in the design of therapeutic strategies for MASH treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease characterized by multilineage immune dysregulation, which subsequently causes inflammation, fibrosis, and even cirrhosis of liver. Due to the limitation of traditional assays, the local hepatic immunopathogenesis of PBC has not been fully characterized. Here, we utilize single-cell RNA sequencing technology to depict the immune cell landscape and decipher the molecular mechanisms of PBC patients. We reveal that cholangiocytes and hepatic stellate cells are involved in liver inflammation and fibrosis. Moreover, Kupffer cells show increased levels of inflammatory factors and decreased scavenger function related genes, while T cells exhibit enhanced levels of inflammatory factors and reduced cytotoxicity related genes. Interestingly, we identify a liver-resident Th1-like population with JAK-STAT activation in the livers of both PBC patients and murine PBC model. Finally, blocking the JAK-STAT pathway alleviates the liver inflammation and eliminates the liver-resident Th1-like cells in the murine PBC model. In conclusion, our comprehensive single-cell transcriptome profiling expands the understanding of pathological mechanisms of PBC and provides potential targets for the treatment of PBC in patients., (© 2024. The Author(s).)

Infection by bacteria leads to tissue damage and inflammation, which need to be tightly controlled by host mechanisms to avoid deleterious consequences. It is previously reported that TMEM16F, a calcium-activated lipid scramblase expressed in various immune cell types including T cells and neutrophils, is critical for the control of infection by bacterium Listeria monocytogenes (Lm) in vivo. This function correlated with the capacity of TMEM16F to repair the plasma membrane (PM) damage induced in T cells in vitro, by the Lm toxin listeriolysin O (LLO). However, whether the protective effect of TMEM16F on Lm infection in vivo is mediated by an impact in T cells, or in other cell types, is not determined. Herein, the immune cell types and mechanisms implicated in the protective effect of TMEM16F against Lm in vivo are elucidated. Cellular protective effects of TMEM16F correlated with its capacity of lipid scrambling and augment PM fluidity. Using cell type-specific TMEM16F-deficient mice, the indication is obtained that TMEM16F expressed in liver Kupffer cells (KCs), but not in T cells or B cells, is key for protection against Listeria in vivo. In the absence of TMEM16F, Listeria induced PM rupture and fragmentation of KCs in vivo. KC death associated with greater liver damage, inflammatory changes, and dysregulated liver metabolism. Overall, the results uncovered that TMEM16F expressed in Kupffer cells is crucial to protect the host against Listeria infection. This influence is associated with the capacity of Kupffer cell-expressed TMEM16F to prevent excessive inflammation and abnormal liver metabolism., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive form of nonalcoholic fatty liver disease characterised by fat accumulation, inflammation, oxidative stress, fibrosis, and impaired liver regeneration. In this study, we found that heme oxygenase-1 (HO-1) is induced in both MASH patients and in a MASH mouse model. Further, hepatic carbon monoxide (CO) levels in MASH model mice were >2-fold higher than in healthy mice, suggesting that liver HO-1 is activated as MASH progresses. Based on these findings, we used CO-loaded red blood cells (CO-RBCs) as a CO donor in the liver, and evaluated their therapeutic effect in methionine-choline deficient diet (MCDD)-induced and high-fat-diet (HFD)-induced MASH model mice. Intravenously administered CO-RBCs effectively delivered CO to the MASH liver, where they prevented fat accumulation by promoting fatty acid oxidation via AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor induction. They also markedly suppressed Kupffer cell activation and their corresponding anti-inflammatory and antioxidative stress activities in MASH mice. CO-RBCs also helped to restore liver regeneration in mice with HFD-induced MASH by activating AMPK. We confirmed the underlying mechanisms by performing in vitro experiments in RAW264.7 cells and palmitate-stimulated HepG2 cells. Taken together, CO-RBCs show potential as a promising cellular treatment for MASH., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Toll-like receptor 9 (Tlr9) is a pathogen recognition receptor detecting unmethylated DNA derivatives of pathogens and damaged host cells. It is therefore an important modulator of innate immunity. Here we investigated the role of Tlr9 in fibrogenesis and progression of hepatocellular carcinoma in chronic liver disease., Materials and Methods: We treated mice with a constitutive deletion of Tlr9 (Tlr9 -/- ) with DEN/CCl 4 for 24 weeks. As a second model, we used hepatocyte-specific Nemo knockout (Nemo Δhepa ) mice and generated double knockout (Nemo Δhepa Tlr9 -/- ) animals., Results: We show that Tlr9 is in the liver primarily expressed in Kupffer cells, suggesting a key role of Tlr9 in intercellular communication during hepatic injury. Tlr9 deletion resulted in reduced liver fibrosis as well as tumor burden. We observed down-regulation of hepatic stellate cell activation and consequently decreased collagen production in both models. Tlr9 deletion was associated with decreased apoptosis and compensatory proliferation of hepatocytes, modulating the initiation and progression of hepatocarcinogenesis. These findings were accompanied by a decrease in interferon-β and an increase in chemokines having an anti-tumoral effect., Conclusions: Our data define Tlr9 as an important receptor involved in fibrogenesis, but also in the initiation and progression of hepatocellular carcinoma during chronic liver diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hypercholesterolemia is a major risk factor for atherosclerosis and associated cardiovascular diseases. The liver plays a key role in the regulation of plasma cholesterol levels and hosts a large population of tissue-resident macrophages known as Kupffer cells (KCs). KCs are located in the hepatic sinusoids where they ensure key functions including blood immune surveillance. However, how KCs homeostasis is affected by the build-up of cholesterol-rich lipoproteins that occurs in the circulation during hypercholesterolemia remains unknown. Here, we show that embryo-derived KCs (EmKCs) accumulate large amounts of lipoprotein-derived cholesterol, in part through the scavenger receptor CD36, and massively expand early after the induction of hypercholesterolemia. After this rapid adaptive response, EmKCs exhibit mitochondrial oxidative stress and their numbers gradually diminish while monocyte-derived KCs (MoKCs) with reduced cholesterol-loading capacities seed the KC pool. Decreased proportion of EmKCs in the KC pool enhances liver cholesterol content and exacerbates hypercholesterolemia, leading to accelerated atherosclerotic plaque development. Together, our data reveal that KC homeostasis is perturbed during hypercholesterolemia, which in turn alters the control of plasma cholesterol levels and increases atherosclerosis., (© 2024. The Author(s).)

Methionine adenosyltransferase 2 A (MAT2A) and MAT2B are essential for hepatic stellate cells (HSCs) activation. Forkhead box M1 (FOXM1) transgenic mice develop liver inflammation and fibrosis. Here we examine if they crosstalk in male mice. We found FOXM1/MAT2A/2B are upregulated after bile duct ligation (BDL) and carbon tetrachloride (CCl 4 ) treatment in hepatocytes, HSCs and Kupffer cells (KCs). FDI-6, a FOXM1 inhibitor, attenuates the development and reverses the progression of CCl 4 -induced fibrosis while lowering the expression of FOXM1/MAT2A/2B, which exert reciprocal positive regulation on each other transcriptionally. Knocking down any of them lowers HSCs and KCs activation. Deletion of FOXM1 in hepatocytes, HSCs, and KCs protects from BDL-mediated inflammation and fibrosis comparably. Interestingly, HSCs from Foxm1 Hep-/- , hepatocytes from Foxm1 HSC-/- , and HSCs and hepatocytes from Foxm1 KC-/- have lower FOXM1/MAT2A/2B after BDL. This may be partly due to transfer of extracellular vesicles between different cell types. Altogether, FOXM1/MAT2A/MAT2B axis drives liver inflammation and fibrosis., (© 2024. The Author(s).)

Macrophages have been recognized as pivotal players in the progression of MASLD/MASH. However, the molecular mechanisms underlying their multifaceted functions in the disease remain to be further clarified. In the current study, we developed a new mouse model with YAP activation in macrophages to delineate the effect and mechanism of YAP signaling in the pathogenesis of MASLD/MASH. Genetically modified mice, featuring specific depletion of both Mst1 and Mst2 in macrophages/monocytes, were generated and exposed to a high-fat diet for 12 weeks to induce MASLD. Following this period, livers were collected for histopathological examination, and liver non-parenchymal cells were isolated and subjected to various analyses, including single-cell RNA-sequencing, immunofluorescence and immunoblotting and qRT-PCR to investigate the impact of YAP signaling on the progression of MASLD. Our data revealed that Mst1/2 depletion in liver macrophages enhanced liver inflammation and fibrosis in MASLD. Using single-cell RNA-sequencing, we showed that YAP activation via Mst1/2 depletion upregulated the expressions of both pro-inflammatory genes and genes associated with resolution/tissue repair. We observed that YAP activation increases Kupffer cell populations (i.e., Kupffer-2 and Kupffer-3) which are importantly implicated in the pathogenesis of MASLD/MASH. Our data indicate that YAP activation via Mst1/2 deletion enhances both the pro-inflammatory and tissue repairing functions of Kupffer-1 and -2 cells at least in part through C1q. These YAP-regulatory mechanisms control the plasticity of liver macrophages in the context of MASLD/MASH. Our findings provide important evidence supporting the critical regulatory role of YAP signaling in liver macrophage plasticity and the progression of MASLD. Therefore, targeting the Hippo-YAP pathway may present a promising therapeutic strategy for the treatment of MASH., (© 2024 Federation of American Societies for Experimental Biology.)

Both DNA 5-methylcytosine (5mC) and RNA N6-methyladenosine (m 6 A) modifications are reported to participate in cellular stress responses including inflammation. Phosphoenolpyruvate carboxykinase 2 (PCK2) is upregulated in Kupffer cells (KCs) to facilitate the proinflammatory phosphorylation signaling cascades upon LPS stimulation, yet the role of 5mC and m 6 A in PCK2 upregulation remain elusive. Here, we report that the significantly augmented PCK2 mRNA and protein levels are associated with global 5mC demethylation coupled with m 6 A hypermethylation in LPS-activated KCs. The suppression of 5mC demethylation or m 6 A hypermethylation significantly alleviates the upregulation of PCK2 and proinflammatory cytokines in LPS-challenged KCs. Further reciprocal tests indicate 5mC demethylation is upstream of m 6 A hypermethylation. Specifically, CpG islands in the promoters of PCK2 and RNA methyltransferase (METTL3 and METTL14) genes are demethylated, while the 3'UTR of PCK2 mRNA is m6A hypermethylated, in LPS-stimulated KCs. These modifications contribute to the transactivation of the PCK2 gene as well as increased PCK2 mRNA stability and protein production via a m 6 A-mediated mechanism with IGF2BP1 as the reader protein. These results indicate that DNA 5mC and RNA m6A collaborate to upregulate PCK2 expression, respectively, at the transcriptional and post-transcriptional levels during KC activation.

Proinflammatory hepatic macrophage activation plays a key role in the development of nonalcoholic steatohepatitis (NASH). This involves increased embryonic hepatic Kupffer cell (KC) death, facilitating the replacement of KCs with bone marrow-derived recruited hepatic macrophages (RHMs) that highly express proinflammatory genes. Moreover, phago/efferocytic activity of KCs is diminished in NASH, enhancing liver inflammation. However, the molecular mechanisms underlying these changes in KCs are not known. Here, we show that hypoxia-inducible factor 2α (HIF-2α) mediates NASH-associated decreased KC growth and efferocytosis by enhancing lysosomal stress. At the molecular level, HIF-2α stimulated mammalian target of rapamycin (mTOR)- and extracellular signal-regulated kinase-dependent inhibitory transcription factor EB (TFEB) phosphorylation, leading to decreased lysosomal and phagocytic gene expression. With increased metabolic stress and phago/efferocytic burden in NASH, these changes were sufficient to increase lysosomal stress, causing decreased efferocytosis and lysosomal cell death. Of interest, HIF-2α-dependent TFEB regulation only occurred in KCs but not RHMs. Instead, in RHMs, HIF-2α promoted mitochondrial reactive oxygen species production and proinflammatory activation by increasing ANT2 expression and mitochondrial permeability transition. Consequently, myeloid lineage-specific or KC-specific HIF-2α depletion or the inhibition of mTOR-dependent TFEB inhibition using antisense oligonucleotide treatment protected against the development of NASH in mice. Moreover, treatment with an HIF-2α-specific inhibitor reduced inflammatory and fibrogenic gene expression in human liver spheroids cultured under a NASH-like condition. Together, our results suggest that macrophage subtype-specific effects of HIF-2α collectively contribute to the proinflammatory activation of liver macrophages, leading to the development of NASH.

The hepatic sinusoids are composed of liver sinusoidal endothelial cells (LSECs), which are surrounded by hepatic stellate cells (HSCs) and contain liver-resident macrophages called Kupffer cells, and other patrolling immune cells. All these cells communicate with each other and with hepatocytes to maintain sinusoidal homeostasis and a spectrum of hepatic functions under healthy conditions. Sinusoidal homeostasis is disrupted by metabolites, toxins, viruses, and other pathological factors, leading to liver injury, chronic liver diseases, and cirrhosis. Alterations in hepatic sinusoids are linked to fibrosis progression and portal hypertension. LSECs are crucial regulators of cellular crosstalk within their microenvironment via angiocrine signaling. This review discusses the mechanisms by which angiocrine signaling orchestrates sinusoidal homeostasis, as well as the development of liver diseases. Here, we summarise the crosstalk between LSECs, HSCs, hepatocytes, cholangiocytes, and immune cells in health and disease and comment on potential novel therapeutic methods for treating liver diseases., (Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: The present study aimed to explore the regulatory effects of artesunate on macrophage polarization in sepsis., Methods: Cell models and mice models were established using lipopolysaccharide (LPS), followed by treatment with various concentrations of artesunate. The phenotype of the macrophages was determined by flow cytometry. RNA immunoprecipitation was used to confirm the binding between MALAT1 and polypyrimidine tract-binding protein 1 (PTBP1), as well as between PTBP1 and interferon-induced helicase C domain-containing protein 1 (IFIH1)., Results: Treatment with artesunate inhibited M1 macrophage polarization in Kupffer cells subjected to LPS stimulation by downregulating MALAT1. Furthermore, MALAT1 abolished the inhibitory effect of artesunate on M1 macrophage polarization by recruiting PTBP1 to promote IFIH. In vivo experiments confirmed that artesunate alleviated septic liver injury by affecting macrophage polarization via MALAT1., Conclusion: The present study showed that artesunate alleviates LPS-induced sepsis in Kupffer cells by regulating macrophage polarization via the lncRNA MALAT1/PTBP1/IFIH1 axis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver pathology worldwide, closely associated with obesity and metabolic disorders. Increasing evidence suggests that macrophages play a crucial role in the development of MASLD. Several human studies have shown an inverse correlation between circulating lysosomal acid lipase (LAL) activity and MASLD. LAL is the sole enzyme known to degrade cholesteryl esters (CE) and triacylglycerols in lysosomes. Consequently, these substrates accumulate when their enzymatic degradation is impaired due to LAL deficiency (LALD). This study aimed to investigate the role of hepatic LAL activity and liver-resident macrophages (i.e., Kupffer cells (KC)) in MASLD. To this end, we analyzed lipid metabolism in hepatocyte-specific (hep)Lal-/- mice and depleted KC with clodronate treatment. When fed a high-fat/high-cholesterol diet (HF/HCD), hepLal-/- mice exhibited CE accumulation and an increased number of macrophages in the liver and significant hepatic inflammation. KC were depleted upon clodronate administration, whereas infiltrating/proliferating CD68-expressing macrophages were less affected. This led to exacerbated hepatic CE accumulation and dyslipidemia, as evidenced by increased LDL-cholesterol concentrations. Along with proteomic analysis of liver tissue, these findings indicate that hepatic LAL-D in HF/HCD-fed mice leads to macrophage infiltration into the liver and that KC depletion further exacerbates hepatic CE concentrations and dyslipidemia., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Coix seed, the dry mature seed kernel of the gramineous plant coix (Coix lacryma-jobi L. var. ma-yuen Stapf), is widely consumed as a traditional Chinese medicine and functional food in China and South Korea. We have previously demonstrated the protective effect of coixol, a polyphenolic compound extracted from coix, against Toxoplasma gondii (T. gondii) infection-induced lung injury. However, the protective effect of coixol on hepatic injury induced by T. gondii infection have not yet been elucidated., Aim of the Study: This study explores the impact of coixol on T. gondii infection-induced liver injury and elucidates the underlying molecular mechanisms., Materials and Methods: Female BALB/c mice and Kupffer cells (KCs) were employed to establish an acute T. gondii infection model in vivo and an inflammation model in vitro. The study examined coixol's influence on the T. gondii-derived heat shock protein 70 (T.g.HSP70)/toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway in T. gondii-infected liver macrophages. Furthermore, a co-culture system of KCs and NCTC-1469 hepatocytes was developed to observe the impact of liver macrophages infected with T. gondii on hepatocyte injury., Results: Coixol notably inhibited the proliferation of tachyzoites and the expression of T.g.HSP70 in mouse liver and KCs, and attenuated pathological liver injury. Moreover, coixol decreased the production of high mobility group box 1, tumor necrosis factor-α, and inducible nitric oxide synthase by suppressing the TLR4/NF-κB signaling pathway in vitro and in vivo. Coixol also mitigated KCs-mediated hepatocyte injury., Conclusions: Coixol protects against liver injury caused by T. gondii infection, potentially by diminishing hepatocyte injury through the suppression of the inflammatory cascade mediated by the T.g.HSP70/TLR4/NF-κB signaling pathway in KCs. These findings offer new perspectives for developing coixol as a lead compound for anti-T. gondii drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background & Aims: Liver macrophages fulfill various homeostatic functions and represent an essential line of defense against pathogenic insults. However, it remains unclear whether a history of infectious disease in the liver leads to long-term alterations to the liver macrophage compartment., Methods: We utilized a curable model of parasitic infection invoked by the protozoan parasite Trypanosoma brucei brucei to investigate whether infection history can durably reshape hepatic macrophage identity and function. Employing a combination of fate mapping, single-cell CITE-sequencing, single-nuclei multiome analysis, epigenomic analysis, and functional assays, we studied the alterations to the liver macrophage compartment during and after the resolution of infection., Results: We show that T. brucei brucei infection alters the composition of liver-resident macrophages, leading to the infiltration of monocytes that differentiate into various infection-associated macrophage populations with divergent transcriptomic profiles. Whereas infection-associated macrophages disappear post-resolution of infection, monocyte-derived macrophages engraft in the liver, assume a Kupffer cell (KC)-like profile and co-exist with embryonic KCs in the long-term. Remarkably, the prior exposure to infection imprinted an altered transcriptional program on post-resolution KCs that was underpinned by an epigenetic remodeling of KC chromatin landscapes and a shift in KC ontogeny, along with transcriptional and epigenetic alterations in their niche cells. This reprogramming altered KC functions and was associated with increased resilience to a subsequent bacterial infection., Conclusion: Our study demonstrates that a prior exposure to a parasitic infection induces trained immunity in KCs, reshaping their identity and function in the long-term., Impact and Implications: Although the liver is frequently affected during infections, and despite housing a major population of resident macrophages known as Kupffer cells (KCs), it is currently unclear whether infections can durably alter KCs and their niche cells. Our study provides a comprehensive investigation into the long-term impact of a prior, cured parasitic infection, unveiling long-lasting ontogenic, epigenetic, transcriptomic and functional changes to KCs as well as KC niche cells, which may contribute to KC remodeling. Our data suggest that infection history may continuously reprogram KCs throughout life with potential implications for subsequent disease susceptibility in the liver, influencing preventive and therapeutic approaches., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The role of IBA in regulating the recovery of liver cancer was investigated using a rat model of liver cancer and an intraoperative blood return model (IBA). SD rats were used to construct the IBA model. Kupffer cells were isolated from liver cancer tissues, and their biological characteristics were analyzed by flow cytometry. Comet assay was used to detect DNA damage in tumor cells; clone formation assay and transwell assay were used to detect tumor cell proliferation and migration ability. Western blot analysis was used to determine the changes in related signaling pathways. After the IBA treatment, the production of KCs was significantly promoted in rat liver cancer tissues, and the expression levels of cell cycle arrest proteins P53, AEN and CDKN1A were also significantly increased. In tumor cells, IBA induced cell cycle arrest and cellular DNA damage in a p53-mediated manner. In addition, the proliferation and migration of cancer cells were also significantly inhibited. Similar to the in vivo data, the expression of TP53, AEN and CDKN1A was also up-regulated. Our study showed that IBA can inhibit the malignant transformation of hepatocellular carcinoma by modulating the function-dependent p53-mediated pathway of tumor cells and KCs.

Purinergic signaling has emerged as an important paracrine-autocrine intercellular system that regulates physiological and pathological processes in practically all organs of the body. Although this system has been thoroughly defined since the nineties, recent research has made substantial advances regarding its role in aspects of liver physiology. However, most studies have mainly targeted the entire organ, 70% of which is made up of parenchymal cells or hepatocytes. Because of its physiological role, the liver is exposed to toxic metabolites, such as xenobiotics, drugs, and fatty acids, as well as to pathogens such as viruses and bacteria. Under injury conditions, all cell types within the liver undergo adaptive changes. In this context, the concentration of extracellular ATP has the potential to increase dramatically. Indeed, this purinergic response has not been studied in sufficient detail in non-parenchymal liver cells. In the present review, we systematize the physiopathological adaptations related to the purinergic system in chronic liver diseases of non-parenchymal liver cells, such as hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, and cholangiocytes. The role played by non-parenchymal liver cells in these circumstances will undoubtedly be strategic in understanding the regenerative activities that support the viability of this organ under stressful conditions.

While macrophage heterogeneity during metabolic dysfunction-associated steatohepatitis (MASH) has been described, the fate of these macrophages during MASH regression is poorly understood. Comparing macrophage heterogeneity during MASH progression vs regression, we identified specific macrophage subpopulations that are critical for MASH/fibrosis resolution. We elucidated the restorative pathways and gene signatures that define regression-associated macrophages and establish the importance of TREM2 + macrophages during MASH regression. Liver-resident Kupffer cells are lost during MASH and are replaced by four distinct monocyte-derived macrophage subpopulations. Trem2 is expressed in two macrophage subpopulations: i) monocyte-derived macrophages occupying the Kupffer cell niche (MoKC) and ii) lipid-associated macrophages (LAM). In regression livers, no new transcriptionally distinct macrophage subpopulation emerged. However, the relative macrophage composition changed during regression compared to MASH. While MoKC was the major macrophage subpopulation during MASH, they decreased during regression. LAM was the dominant macrophage subtype during MASH regression and maintained Trem2 expression. Both MoKC and LAM were enriched in disease-resolving pathways. Absence of TREM2 restricted the emergence of LAMs and formation of hepatic crown-like structures. TREM2 + macrophages are functionally important not only for restricting MASH-fibrosis progression but also for effective regression of inflammation and fibrosis. TREM2 + macrophages are superior collagen degraders. Lack of TREM2 + macrophages also prevented elimination of hepatic steatosis and inactivation of HSC during regression, indicating their significance in metabolic coordination with other cell types in the liver. TREM2 imparts this protective effect through multifactorial mechanisms, including improved phagocytosis, lipid handling, and collagen degradation., Competing Interests: Competing interests statement:R.L. serves as a consultant to Aardvark Therapeutics, Altimmune, Arrowhead Pharmaceuticals, AstraZeneca, Cascade Pharmaceuticals, Eli Lilly, Gilead, Glympse bio, Inipharma, Intercept, Inventiva, Ionis, Janssen Inc., Lipidio, Madrigal, Neurobo, Novo Nordisk, Merck, Pfizer, Sagimet, 89 bio, Takeda, Terns Pharmaceuticals and Viking Therapeutics. C.K.G. is a founder and member of the SAB of Asteroid Pharmaceuticals. A.E.F. is an employee and stockholder of Novo Nordisk. C.K.G. is a stockholder of Asteroid Therapeutics. R.L. is a co-founder and equity holder of LipoNexus Inc. R.L. received research grants from Arrowhead Pharmaceuticals, Astrazeneca, Boehringer-Ingelheim, Bristol-Myers Squibb, Eli Lilly, Galectin Therapeutics, Gilead, Intercept, Hanmi, Intercept, Inventiva, Ionis, Janssen, Madrigal Pharmaceuticals, Merck, Novo Nordisk, Pfizer, Sonic Incytes and Terns Pharmaceuticals.

In mice, the first liver-resident macrophages, known as Kupffer cells (KCs), are thought to derive from yolk sac (YS) hematopoietic progenitors that are specified prior to the emergence of the hematopoietic stem cell (HSC). To investigate human KC development, we recapitulated YS-like hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks. Single-cell RNA sequencing (scRNA-seq) of engrafted YS-macrophages revealed a homogeneous MARCO-expressing KC gene signature and low expression of monocyte-like macrophage genes. In contrast, human cord blood (CB)-derived macrophage progenitors generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis in vivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSC-derived, YS-like progenitors., Competing Interests: Declaration of interests G.M. Keller is a founding investigator and a paid consultant for BlueRock Therapeutics LP and a paid consultant for VistaGen Therapeutics and Apiary Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Dysregulated fatty acid metabolism is closely linked to the development of alcohol-associated liver disease (ALD). KCs, which are resident macrophages in the liver, play a critical role in ALD pathogenesis. However, the effect of alcohol on fatty acid metabolism in KCs remains poorly understood. The current study aims to investigate fatty acid metabolism in KCs and its potential effect on ALD development., Methods: Wild-type C57BL/6 mice were fed a Lieber-DeCarli ethanol liquid diet for 3 days. Then, the liver injury and levels of intrahepatic bacteria were assessed. Next, we investigated the effects and underlying mechanisms of ethanol exposure on fatty acid metabolism and the phagocytosis of KCs, both in vivo and in vitro. Finally, we generated KCs-specific Fasn knockout and overexpression mice to evaluate the impact of FASN on the phagocytosis of KCs and ethanol-induced liver injury., Results: Using Bodipy493/503 to stain intracellular neutral lipids, we found significantly reduced lipid levels in KCs from mice fed an alcohol-containing diet for 3 days and in RAW264.7 macrophages exposed to ethanol. Mechanistically, alcohol exposure suppressed sterol regulatory element-binding protein 1 transcriptional activity, thereby inhibiting fatty acid synthase (FASN)-mediated de novo lipogenesis in macrophages both in vitro and in vivo. We show that genetic ablation and pharmacologic inhibition of FASN significantly impaired KC's ability to take up and eliminate bacteria. Conversely, KCs-specific Fasn overexpression reverses the impairment of macrophage phagocytosis caused by alcohol exposure. We also revealed that KCs-specific Fasn knockout augmented KCs apoptosis and exacerbated liver injury in mice fed an alcohol-containing diet for 3 days., Conclusions: Our findings indicate the crucial role of de novo lipogenesis in maintaining effective KCs phagocytosis and suggest a therapeutic target for ALD based on fatty acid synthesis in KCs., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

The simultaneous abuse of alcohol-cocaine is known to cause stronger and more unpredictable cellular damage in the liver, heart, and brain. However, the mechanistic crosstalk between cocaine and alcohol in liver injury remains unclear. The findings revealed cocaine-induced liver injury and inflammation in both marmosets and mice. Of note, co-administration of cocaine and ethanol in mice causes more severe liver damage than individual treatment. The metabolomic analysis confirmed that hippuric acid (HA) is the most abundant metabolite in marmoset serum after cocaine consumption and that is formed in primary marmoset hepatocytes. HA, a metabolite of cocaine, increases mitochondrial DNA leakage and subsequently increases the production of proinflammatory factors via STING signaling in Kupffer cells (KCs). In addition, conditioned media of cocaine-treated KC induced hepatocellular necrosis via alcohol-induced TNFR1. Finally, disruption of STING signaling in vivo ameliorated co-administration of alcohol- and cocaine-induced liver damage and inflammation. These findings postulate intervention of HA-STING-TNFR1 axis as a novel strategy for treatment of alcohol- and cocaine-induced excessive liver damage., (© 2024. The Author(s).)

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathologic syndrome characterized by excessive fat deposition in hepatocytes and a major cause of end-stage liver disease. Autophagy is a metabolic pathway responsible for degrading cytoplasmic products and damaged organelles, playing a pivotal role in maintaining the homeostasis and functionality of hepatocytes. Recent studies have shown that pharmacological intervention to activate or restore autophagy provides benefits for liver function recovery by promoting the clearance of lipid droplets (LDs) in hepatocytes, decreasing the production of pro-inflammatory factors, and inhibiting activated hepatic stellate cells (HSCs), thus improving liver fibrosis and slowing down the progression of NAFLD. This article summarizes the physiological process of autophagy, elucidates the close relationship between NAFLD and autophagy, and discusses the effects of drugs on autophagy and signaling pathways from the perspectives of hepatocytes, kupffer cells (KCs), and HSCs to provide assistance in the clinical management of NAFLD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shen, Yang, Wang, Chen, Chen, Zhang, Xiong and Leng.)

Impaired self-renewal of Kupffer cells (KCs) leads to inflammation in metabolic dysfunction-associated steatohepatitis (MASH). Here, we identify neutrophil cytosolic factor 1 (NCF1) as a critical regulator of iron homeostasis in KCs. NCF1 is upregulated in liver macrophages and dendritic cells in humans with metabolic dysfunction-associated steatotic liver disease and in MASH mice. Macrophage NCF1, but not dendritic cell NCF1, triggers KC iron overload, ferroptosis, and monocyte-derived macrophage infiltration, thus aggravating MASH progression. Mechanistically, elevated oxidized phospholipids induced by macrophage NCF1 promote Toll-like receptor (TLR4)-dependent hepatocyte hepcidin production, leading to increased KC iron deposition and subsequent KC ferroptosis. Importantly, the human low-functional polymorphic variant NCF1 90H alleviates KC ferroptosis and MASH in mice. In conclusion, macrophage NCF1 impairs iron homeostasis in KCs by oxidizing phospholipids, triggering hepatocyte hepcidin release and KC ferroptosis in MASH, highlighting NCF1 as a therapeutic target for improving KC fate and limiting MASH progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Kupffer cells (KC),an important subset of immune cells in the liver,are essential for maintaining tissue homeostasis and responding quickly to liver damage.The complement receptor of the immunoglobulin superfamily (CRIg) is a receptor protein on the KC membrane.CRIg can not only capture pathogens in the blood flowing through the liver by complement binding but also mediate immune responses by regulating immune cells in the liver.Recent studies have confirmed the role of CRIg in regulating liver immunity.This article reviews the main modes of action of CRIg and the research progress of CRIg in regulating liver immunity.

Innate immune activation is critical for initiating hepatic inflammation during nonalcoholic steatohepatitis (NASH) progression. However, the mechanisms by which immunoregulatory molecules recognize lipogenic, fibrotic, and inflammatory signals remain unclear. Here, we show that high-fat diet (HFD)-induced oxidative stress activates Foxo1, YAP, and Notch1 signaling in hepatic macrophages. Macrophage Foxo1 deficiency (Foxo1 M-KO ) ameliorated hepatic inflammation, steatosis, and fibrosis, with reduced STING, TBK1, and NF-κB activation in HFD-challenged livers. However, Foxo1 and YAP double knockout (Foxo1/YAP M-DKO ) or Foxo1 and Notch1 double knockout (Foxo1/Notch1 M-DKO ) promoted STING function and exacerbated HFD-induced liver injury. Interestingly, Foxo1 M-KO strongly reduced TGF-β1 release from palmitic acid (PA)- and oleic acid (OA)-stimulated Kupffer cells and decreased Col1α1, CCL2, and Timp1 expression but increased MMP1 expression in primary hepatic stellate cells (HSCs) after coculture with Kupffer cells. Notably, PA and OA challenge in Kupffer cells augmented LIMD1 and LATS1 colocalization and interaction, which induced YAP nuclear translocation. Foxo1 M-KO activated PGC-1α and increased nuclear YAP activity, modulating mitochondrial biogenesis. Using chromatin immunoprecipitation (ChIP) coupled with massively parallel sequencing (ChIP-Seq) and in situ RNA hybridization, we found that NICD colocalizes with YAP and targets Mb21d1 (cGAS), while YAP functions as a novel coactivator of the NICD, which is crucial for reprogramming STING function in NASH progression. These findings highlight the importance of the macrophage Foxo1-YAP-Notch1 axis as a key molecular regulator that controls lipid metabolism, inflammation, and innate immunity in NASH., (© 2024. The Author(s).)

Background and Aims: In obesity, depletion of KCs expressing CRIg (complement receptor of the Ig superfamily) leads to microbial DNA accumulation, which subsequently triggers tissue inflammation and insulin resistance. However, the mechanism underlying obesity-mediated changes in KC complement immune functions is largely unknown., Approach and Results: Using KC-specific deactivated Cas9 transgenic mice treated with guide RNA, we assessed the effects of restoring CRIg or the serine/arginine-rich splicing factor 3 (SRSF3) abundance on KC functions and metabolic phenotypes in obese mice. The impacts of weight loss on KC responses were evaluated in a diet switch mouse model. The role of SRSF3 in regulating KC functions was also evaluated using KC-specific SRSF3 knockout mice. Here, we report that overexpression of CRIg in KCs of obese mice protects against bacterial DNA accumulation in metabolic tissues. Mechanistically, SRSF3 regulates CRIg expression, which is essential for maintaining the CRIg+ KC population. During obesity, SRSF3 expression decreases, but it is restored with weight loss through a diet switch, normalizing CRIg+ KCs. KC SRSF3 is also repressed in obese human livers. Lack of SRSF3 in KCs in lean and obese mice decreases their CRIg+ population, impairing metabolic parameters. During the diet switch, the benefits of weight loss are compromised due to SRSF3 deficiency. Conversely, SRSF3 overexpression in obese mice preserves CRIg+ KCs and improves metabolic responses., Conclusions: Restoring SRSF3 abundance in KCs offers a strategy against obesity-associated tissue inflammation and insulin resistance by preventing bacterial DNA accumulation., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Ligand-receptor recognition serves as the fundamental driving force for active targeting, yet it is still constrained by off-target effects. Herein, we demonstrate that circumventing or blocking the mononuclear phagocyte system (MPS) are both viable strategies to address off-target effects. Naturally derived lignin nanoparticles (LNPs) show great potential to block MPS due to its good stability, low toxicity, and degradability. We further demonstrate the impact of LNPs dosage on in vivo tumor targeting and antitumor efficacy. Our results show that a high dose of LNPs (300 mg/kg) leads to significant accumulation at the tumor site for a duration of 14 days after intravenous administration. In contrast, the low-dose counterparts (e.g., 50, 150 mg/kg) result in almost all LNPs accumulating in the liver. This discovery indicates that the liver is the primary site of LNP capture, leaving only the surplus LNPs the chance to reach the tumor. In addition, although cell membrane-engineered LNPs can rapidly penetrate tumors, they are still prone to capture by the liver during subsequent circulation in the bloodstream. Excitingly, comparable therapeutic efficacy is obtained for the above two strategies. Our findings may offer valuable insights into the targeted delivery of drugs for disease treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Ischemia-reperfusion injury (IRI) poses a significant challenge to liver transplantation (LT). The underlying mechanism primarily involves overactivation of the immune system. Heat shock protein 110 (HSP110) functions as a molecular chaperone that helps stabilize protein structures., Methods: An IRI model was established by performing LT on Sprague-Dawley rats, and HSP110 was silenced using siRNA. Hematoxylin-eosin staining, TUNEL, immunohistochemistry, ELISA and liver enzyme analysis were performed to assess IRI following LT. Western blotting and quantitative reverse transcription-polymerase chain reaction were conducted to investigate the pertinent molecular changes., Results: Our findings revealed a significant increase in the expression of HSP110 at both the mRNA and protein levels in the rat liver following LT (P < 0.05). However, when rats were injected with siRNA-HSP110, IRI subsequent to LT was notably reduced (P < 0.05). Additionally, the levels of liver enzymes and inflammatory chemokines in rat serum were significantly reduced (P < 0.05). Silencing HSP110 with siRNA resulted in a marked decrease in M1-type polarization of Kupffer cells in the liver and downregulated the NF-κB pathway in the liver (P < 0.05)., Conclusions: HSP110 in the liver promotes IRI after LT in rats by activating the NF-κB pathway and inducing M1-type polarization of Kupffer cells. Targeting HSP110 to prevent IRI after LT may represent a promising new approach for the treatment of LT-associated IRI., Competing Interests: Competing interest No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article., (Copyright © 2023 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease globally. Non-alcoholic steatohepatitis (NASH) represents an extremely progressive form of NAFLD, which, without timely intervention, may progress to cirrhosis or hepatocellular carcinoma. Presently, a definitive comprehension of the pathogenesis of NAFLD/NASH eludes us, and pharmacological interventions targeting NASH specifically remain constrained. The aetiology of NAFLD encompasses a myriad of external factors including environmental influences, dietary habits and gender disparities. More significantly, inter-organ and cellular interactions within the human body play a role in the development or regression of the disease. In this review, we categorize the influences affecting NAFLD both intra- and extrahepatically, elaborating meticulously on the mechanisms governing the onset and progression of NAFLD/NASH. This exploration delves into progress in aetiology and promising therapeutic targets. As a metabolic disorder, the development of NAFLD involves complexities related to nutrient metabolism, liver-gut axis interactions and insulin resistance, among other regulatory functions of extraneous organs. It further encompasses intra-hepatic interactions among hepatic cells, Kupffer cells (KCs) and hepatic stellate cells (HSCs). A comprehensive understanding of the pathogenesis of NAFLD/NASH from a macroscopic standpoint is instrumental in the formulation of future therapies for NASH., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Acute cholestatic liver injury (ACLI) is a disease associated with bile duct obstruction that causes liver inflammation and apoptosis. Although G protein-coupled bile acid receptor1 (Gpbar-1) has diverse metabolic roles, its involvement in ACLI-associated immune activation remains unclear. Liver tissues and blood samples from 20 patients with ACLI and 20 healthy individuals were analyzed using biochemical tests, H&E staining, western blotting, and immunohistochemistry to verify liver damage and expression of Gpbar-1. The expression of Gpbar-1, cAMP/PKA signaling, and the NLRP3 inflammasome was tested in wild-type (WT) and Gpbar-1 knockdown (si-Gpbar-1) mice with ACLI induced by bile duct ligation (BDL) and in primary Kupffer cells (KCs) with or without Gpbar-1-siRNA. The results showed that total bile acids and Gpbar-1 expressions were elevated in patients with ACLI. Gpbar-1 knockdown significantly worsened BDL-induced acute hepatic damage, inflammation, and liver apoptosis in vivo. Knockdown of Gpbar-1 heightened KC sensitivity to lipopolysaccharide (LPS) stimulation. Gpbar-1 activation inhibited LPS-induced pro-inflammatory responses in normal KCs but not in Gpbar-1-knockdown KCs. Notably, NLRP3-ASC inflammasome expression was effectively enhanced by Gpbar-1 deficiency. Additionally, Gpbar-1 directly increased intracellular cAMP levels and PKA phosphorylation, thus disrupting the NLRP3-ASC inflammasome. The pro-inflammatory characteristic of Gpbar-1 deficiency was almost neutralized by the NLRP3 inhibitor CY-09. In vitro, M1 polarization was accelerated in LPS-stimulated Gpbar-1-knockdown KCs. Therapeutically, Gpbar-1 deficiency exacerbated BDL-induced ACLI, which could be rescued by inhibition of the NLRP3-ASC inflammasome. Our study reveal that Gpbar-1 may act as a novel immune-mediated regulator of ACLI by inhibiting the NLRP3-ASC inflammasome., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests. All authors agree to the author list., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hepatic ischemia-reperfusion injury (HIRI) represents a major risk factor in liver transplantation and resection surgeries. Kupffer cells (KCs) produce proinflammatory cytokines and lead to hepatic neutrophil infiltration in the liver, which is one of the leading causes of HIRI. Mid1 is involved in immune infiltration, but the role of Mid1 remains poorly understood. Herin, our study aimed to investigate the effect of Mid1 on HIRI progression. Male C57BL/6 mice aged 6 weeks were used for the HIRI model established. The function of Mid1 on liver injury and hepatic inflammation was evaluated. In vitro, KCs were used to investigate the function and mechanism of Mid1 in modulating KC inflammation upon lipopolysaccharide (LPS) stimulation. We found that Mid1 expression was up-regulated upon HIRI. Mid1 inhibition alleviated liver damage, as evidenced by neutrophil infiltration, intrahepatic inflammation, and hepatocyte apoptosis. In vitro experiments further revealed that Mid1 knockdown reduced the secretion of proinflammatory cytokines and chemokines in KCs. Moreover, silenced-Mid1 suppressed proinflammatory responses by the inhibition of NF-κB, JNK, and p38 signaling pathways. Taken together, Mid1 contributes to HIRI via regulating the proinflammatory response of KCs and inducing neutrophil infiltration. Targeting Mid1 may be a promising strategy to protect against HIRI., (© 2024 Federation of American Societies for Experimental Biology.)

Intrahepatic accumulation dominates organ distribution for most nanomedicines. However, obscure intrahepatic fate largely hampers regulation on their in vivo performance. Herein, PEGylated liposomal doxorubicin is exploited to clarify the intrahepatic fate of both liposomes and the payload in male mice. Kupffer cells initiate and dominate intrahepatic capture of liposomal doxorubicin, following to deliver released doxorubicin to hepatocytes with zonated distribution along the lobule porto-central axis. Increasing Kupffer cells capture promotes doxorubicin accumulation in hepatocytes, revealing the Kupffer cells capture-payload release-hepatocytes accumulation scheme. In contrast, free doxorubicin is overlooked by Kupffer cells, instead quickly distributing into hepatocytes by directly crossing fenestrated liver sinusoid endothelium. Compared to free doxorubicin, liposomal doxorubicin exhibits sustained metabolism/excretion due to the extra capture-release process. This work unveils the pivotal role of Kupffer cells in intrahepatic traffic of PEGylated liposomal therapeutics, and quantitively describes the intrahepatic transport/distribution/elimination process, providing crucial information for guiding further development of nanomedicines., (© 2024. The Author(s).)

Sepsis is a lethal systemic inflammatory disease against infection that lacks effective therapeutic approaches. Liver resident macrophage Kupffer cell (KC)-initiated bacterial clearance is crucial for the host to defend against infection. However, it remains unclear whether this process also governs the antibacterial therapy of sepsis that would be used to improve therapeutic outcomes. Here, we found that copper-doped carbon dots (Cu-CDs) exhibited superior antibacterial capabilities in vitro but displayed limited therapeutic effects in septic mice due to their limited ability to target the liver and restore KC antimicrobial capacity. Thus, we developed a composite nanodrug of copper-doped carbon dot-loaded apoVs (CC-apoVs) that combined the antibacterial ability of Cu-CDs and liver KC targeting features of apoV. Moreover, intravenous injection of CC-apoVs markedly alleviated the systemic infection and decreased the mortality of septic mice compared to Cu-CD and apoV infusion alone. Mechanistically, CC-apoV injection rescued impaired liver KCs during sepsis and enhanced their ability to capture and kill bloodborne bacteria. In addition, apoV-promoted macrophage killing of bacteria could be blocked by the inhibition of small GTPase Rab5. This study reveals a liver KC-targeted therapeutic strategy for sepsis and provides a nanodrug CC-apoV to improve the host antibacterial defense and amplify the therapeutic effect of the nanodrug.

Partial hepatectomy is an essential surgical technique used to treat advanced liver diseases such as liver tumors, as well as for performing liver transplants from living donors. However, postoperative complications such as bleeding, abdominal adhesions, wound infections, and inadequate liver regeneration pose significant challenges and increase morbidity and mortality rates. A self-repairing mixed hydrogel (O 5 H 2 /Cu 2+ /SCCK), containing stem cell derived cytokine (SCCK) derived from human umbilical cord mesenchymal stem cells (HUMSCs) treated with the traditional Chinese remedy Tanshinone IIA (TSA), is developed. This SCCK, in conjunction with O 5 H 2 , demonstrates remarkable effects on Kupffer cell activation and extracellular matrix (ECM) remodeling. This leads to the secretion of critical growth factors promoting enhanced proliferation of hepatocytes and endothelial cells, thereby facilitating liver regeneration and repair after partial hepatectomy. Furthermore, the hydrogel, featuring macrophage-regulating properties, effectively mitigates inflammation and oxidative stress damage in the incision area, creating an optimal environment for postoperative liver regeneration. The injectability and strong adhesion of the hydrogel enables rapid hemostasis at the incision site, while its physical barrier function prevents postoperative abdominal adhesions. Furthermore, the hydrogel's incorporation of Cu 2+ provides comprehensive antibacterial effects, protecting against a wide range of bacteria types and reducing the chances of infections after surgery., (© 2024 Wiley‐VCH GmbH.)

Obesity-induced chronic low-grade inflammation, also known as metaflammation, results from alterations of the immune response in metabolic organs and contributes to the development of fatty liver diseases and type 2 diabetes. The diversity of tissue-resident leukocytes involved in these metabolic dysfunctions warrants an in-depth immunophenotyping in order to elucidate disease etiology. Here, we present a 30-color, full spectrum flow cytometry panel, designed to (i) identify the major innate and adaptive immune cell subsets in murine liver and white adipose tissues and (ii) discriminate various tissue-specific myeloid subsets known to contribute to the development of metabolic dysfunctions. This panel notably allows for distinguishing embryonically-derived liver-resident Kupffer cells from newly recruited monocyte-derived macrophages and KCs. Furthermore, several adipose tissue macrophage (ATM) subsets, including perivascular macrophages, lipid-associated macrophages, and pro-inflammatory CD11c + ATMs, can also be identified. Finally, the panel includes cell-surface markers that have been associated with metabolic activation of different macrophage and dendritic cell subsets. Altogether, our spectral flow cytometry panel allows for an extensive immunophenotyping of murine metabolic tissues, with a particular focus on metabolically-relevant myeloid cell subsets, and can easily be adjusted to include various new markers if needed., (© 2024 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

Abstract: Proinflammatory hyperactivation of Kupffer cells (KCs) is foremost involved in the pathogenesis of sepsis-induced liver injury. Our previous study found that stimulator of interferon genes (STING) signaling was activated in KCs in response of lipopolysaccharide (LPS) and knocking down dynamin-related protein 1 (DRP1) in KCs effectively inhibited the activation of STING signaling and the subsequent production of proinflammatory cytokines. In this study, we demonstrated that in vivo treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of DRP1, alleviated cecal ligation and puncture (CLP)-induced liver injury with the improvement of liver pathology and function. Moreover, we found that STING in liver was mainly concentrated in KCs and STING signaling was significantly activated in KCs after CLP. The STING deficiency effectively ameliorated liver injury and decreased the mortality of septic mice, which were reversely worsened by the enhanced activation of STING with DMXAA. The further study showed that Mdivi-1 markedly attenuated STING signaling activation in KCs and inhibited systemic inflammatory response. Importantly, DMXAA application in CLP mice blunted Mdivi-1's liver protection effect. Taken together, our study confirmed Mdivi-1 effectively alleviated CLP-induced liver injury partially through inhibiting STING signaling activation in KCs, which provides new insights and a novel potential pharmacological therapeutic target for treating septic liver injury., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

Patients with metastatic colorectal cancer often have poor outcomes, primarily due to hepatic metastasis. Colorectal cancer (CRC) cells have the ability to secrete cytokines and other molecules that can remodel the tumor microenvironment, facilitating the spread of cancer to the liver. Kupffer cells (KCs), which are macrophages in the liver, can be polarized to M2 type, thereby promoting the expression of adhesion molecules that aid in tumor metastasis. Our research has shown that huachanshu (with bufalin as the main active monomer) can effectively inhibit CRC metastasis. However, the underlying mechanism still needs to be thoroughly investigated. We have observed that highly metastatic CRC cells have a greater ability to induce M2-type polarization of Kupffer cells, leading to enhanced metastasis. Interestingly, we have found that inhibiting the expression of IL-6, which is highly expressed in the serum, can reverse this phenomenon. Notably, bufalin has been shown to attenuate the M2-type polarization of Kupffer cells induced by highly metastatic Colorectal cancer (mCRC) cells and down-regulate IL-6 expression, ultimately inhibiting tumor metastasis. In this project, our aim is to study how high mCRC cells induce M2-type polarization and how bufalin, via the SRC-3/IL-6 pathway, can inhibit CRC metastasis. This research will provide a theoretical foundation for understanding the anti-CRC effect of bufalin., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As a highly promising nanomaterial, exploring the impact of the liver, a vital organ, stands out as a crucial focus in the examination of its biological effects. Kupffer cells (KCs) are one of the first immune cells to contact with exotic-substances in liver. Therefore, this study investigates the immunomodulatory effects and mechanisms of polyethylene glycol-modified graphene oxide (GO-PEG) on KCs. Initial RNA-seq and KEGG pathway analyses reveal the inhibition of the TOLL-like receptor, TNF-α and NOD-like receptor pathways in continually stimulated KCs exposed to GO-PEG. Subsequent biological experiments validate that a 48-hour exposure to GO-PEG alleviates LPS-induced KCs immune activation, characterized by a shift in polarization from M1 to M2. The underlying mechanism involves the absorption of double-stranded RNA/single-stranded RNA, inhibiting the activation of TLR3 and TLR7 in KCs. Employing a Kupffer/AML12 cell co-culture model and animal studies, it is observed that GO-PEG indirectly inhibit oxidative stress, mitochondrial dysfunction, and apoptosis in AML12 cells, partially mitigating systemic inflammation and preserving liver tissue/function. This effect is attributed to the paracrine interaction between KCs and hepatocytes. These findings suggest a meaningful and effective strategy for treating liver inflammation, particularly when combined with anti-inflammatory drugs., (© 2024 Wiley‐VCH GmbH.)