The role of Kupffer cells in hepatic diseases

Kupffer cells (KCs) constitute 80–90% of the tissue macrophages present in the body. Essential to innate and adaptive immunity, KCs are responsible for the swift containment and clearance of exogenous particulates and immunoreactive materials which are perceived as foreign and harmful to the body. Similar to other macrophages, KCs also sense endogenous molecular signals that may result from perturbed homeostasis of the host. KCs have been implicated in host defense and the pathogenesis of various hepatic diseases, including endotoxin tolerance, liver transplantation, nonalcoholic fatty liver disease, and alcoholic liver disease. In this review, we summarized some novel findings associated with the role of KCs in hepatic diseases, such as the origin and mechanisms KCs polarization, molecular basis for caspase-1 activation called “non-canonical inflammasome pathway” involving the cleavage of Gsdmd by caspase-11, the important role of microRNA in liver transplantation, and so on. A better understanding of KCs biological characteristics and immunologic function in liver homeostasis and pathology may pave the way to investigate new diagnostic and therapeutic approaches for hepatic diseases.     

Innate lymphocytes: pathogenesis and therapeutic targets of liver diseases and cancer

The liver is a lymphoid organ with unique immunological properties, particularly, its predominant innate immune system. The balance between immune tolerance and immune activity is critical to liver physiological functions and is responsible for the sensitivity of this organ to numerous diseases, including hepatotropic virus infection, alcoholic liver disease, nonalcoholic fatty liver disease, autoimmune liver disease, and liver cancer, which are major health problems globally. In the past decade, with the discovery of liver-resident natural killer cells, the importance of innate lymphocytes with tissue residency has gradually become the focus of research. In this review, we address the current knowledge regarding hepatic innate lymphocytes with unique characteristics, including NK cells, ILC1/2/3s, NKT cells, γδ T cells, and MAIT cells, and their potential roles in liver homeostasis maintenance and the progression of liver diseases and cancer. A better understanding of the immunopathogenesis of hepatic innate lymphocytes will be helpful for proposing effective treatments for liver diseases and cancer.     

Increased serum enzyme levels associated with kupffer cell reduction with no signs of hepatic or skeletal muscle injury

Macrophage colony-stimulating factor (M-CSF) is a hematopoietic growth factor that is responsible for the survival and proliferation of monocytes and the differentiation of monocytes into macrophages, including Kupffer cells (KCs) in the liver. KCs play an important role in the clearance of several serum enzymes, including aspartate aminotransferase and creatine kinase, that are typically elevated as a result of liver or skeletal muscle injury. We used three distinct animal models to investigate the hypothesis that increases in the levels of serum enzymes can be the result of decreases in KCs in the apparent absence of hepatic or skeletal muscle injury. Specifically, neutralizing M-CSF activity via a novel human monoclonal antibody reduced the CD14(+)CD16(+) monocyte population, depleted KCs, and increased aspartate aminotransferase and creatine kinase serum enzyme levels in cynomolgus macaques. In addition, the treatment of rats with clodronate liposomes depleted KCs and led to increased serum enzyme levels, again without evidence of tissue injury. Finally, in the osteopetrotic (Csf1(op)/Csf1(op)) mice lacking functional M-CSF and having reduced levels of KCs, the levels of serum enzymes are higher than in wild-type littermates. Together, these findings support a mechanism for increases in serum enzyme levels through M-CSF regulation of tissue macrophage homeostasis without concomitant histopathological changes in either the hepatic or skeletal system.     

Activation of α2 adrenoceptor attenuates lipopolysaccharide-induced hepatic injury

Sepsis induces hepatic injury but whether alpha-2 adrenoceptor (α2-AR) modulates the severity of sepsis-induced liver damage remains unclear. The present study used lipopolysaccharide (LPS) to induce hepatic injury and applied α2-AR agonist dexmedetomidine (DEX) and/or antagonist yohimbine to investigate the contribution of α2-AR in LPS-induced liver injury. Our results showed that LPS resulted in histological and functional abnormality of liver tissue (ALT and AST transaminases, lactate), higher mortality, an increase in proinflammatory cytokines (IL-1β, IL-6 & TNF-α), as well as a change in oxidative stress (MDA, SOD). Activation of α2-AR by dexmedetomidine (DEX) attenuated LPS-induced deleterious effects on the liver and block of α2-AR by yohimbine aggravated LPS-induced liver damage. Our data suggest that α2-AR plays an important role in sepsis-induced liver damage and activation of α2-AR with DEX could be a novel therapeutic avenue to protect the liver against sepsis-induced injury.     

Altered Endothelin-1 Signaling in Production of Thromboxane A2 in Kupffer Cells from Bile Duct Ligated Rats

Kupffer cells (KCs), the liver resident macrophages accounting for 80–90% of the total population of fixed tissue macrophages in the body, not only play a key role in host defense via removing particulate materials from the portal circulation, but may also contribute to the pathogenesis of various liver diseases. We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A₂ (TXA₂) in early fibrosis induced by one-week bile duct ligation (BDL). Production of TXA₂ is controlled by cytosolic phospholipase A₂ (cPLA₂) that is activated by the interaction of entothelin-1 (ET-1) with its G-protein coupled ET receptor B (ETBR). However, the signaling pathways that contribute to the ET-1-induced activation of cPLA₂ and production of TXA₂ in KCs in the normal healthy or injured livers are not yet clear, which are investigated in the present study using isolated KCs from one-week BDL or sham rats. The pharmacological inhibition of cPLA₂ or chelation of intracellular calcium abrogated the ET-1 induction of TXA₂ from KCs. Compared to those from sham rats, KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1, increased ETBR and Gαi subunit but decreased Gαq and Gα11 expression. Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA₂ in sham KCs but only slightly in BDL KCs. In contrast, inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA₂ in BDL KCs but had no effect in sham KCs. Lastly, inhibition of PLC or PKC abrogated ET-1 induction of TXA₂ in KCs from both sham and BDL groups. The hepatic stress (such as BDL) induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms, particularly a shift from Gq induced signaling to Gi induced signaling, in the activation of cPLA₂ and production of TXA₂ in response to ET-1.     

Invariant natural killer T cells contribute to chronic-plus-binge ethanol-mediated liver injury by promoting hepatic neutrophil infiltration

Neutrophil infiltration is a hallmark of alcoholic steatohepatitis; however, the underlying mechanisms remain unclear. We previously reported that chronic-plus-binge ethanol feeding synergistically induces hepatic recruitment of neutrophils, which contributes to liver injury. In this paper, we investigated the roles of invariant natural killer T (iNKT) cells in chronic-plus-binge ethanol feeding-induced hepatic neutrophil infiltration and liver injury. Wild-type and two strains of iNKT cell-deficient mice (CD1d- and Jα18-deficient mice) were subjected to chronic-plus-binge ethanol feeding. Liver injury and inflammation were examined. Chronic-plus-binge ethanol feeding synergistically increased the number of hepatic iNKT cells and induced their activation, compared with chronic feeding or binge alone. iNKT cell-deficient mice were protected from chronic-plus-binge ethanol-induced hepatic neutrophil infiltration and liver injury. Moreover, chronic-plus-binge ethanol feeding markedly upregulated the hepatic expression of several genes associated with inflammation and neutrophil recruitment in wild-type mice, but induction of these genes was abrogated in iNKT cell-deficient mice. Importantly, several cytokines and chemokines (e.g., MIP-2, MIP-1, IL-4, IL-6 and osteopontin) involved in neutrophil infiltration were upregulated in hepatic NKT cells isolated from chronic-plus-binge ethanol-fed mice compared to pair-fed mice. Finally, treatment with CD1d blocking antibody, which blocks iNKT cell activation, partially prevented chronic-plus-binge ethanol-induced liver injury and inflammation. Chronic-plus-binge ethanol feeding activates hepatic iNKT cells, which play a critical role in the development of early alcoholic liver injury, in part by releasing mediators that recruit neutrophils to the liver, and thus, iNKT cells represent a potential therapeutic target for the treatment of alcoholic liver disease.     

From nonalcoholic fatty liver disease to metabolic-associated fatty liver disease: Big wave or ripple?

There is some dissatisfaction with the term “nonalcoholic fatty liver disease (NAFLD),” which overemphasizes alcohol and underemphasizes the importance of metabolic risk factors in this disease. Recently, a consensus recommended “metabolic (dysfunction)-associated fatty liver disease (MAFLD)” as a more appropriate term to describe fatty liver diseases (FLD) associated with metabolic dysfunction. During the definition change from NAFLD to MAFLD, subjects with FLD and metabolic abnormalities, together with other etiologies of liver diseases such as alcohol, virus, or medication who have been excluded from the NAFLD criteria, were added to the MAFLD criteria, while subjects with FLD but without metabolic abnormality, who have been included in the NAFLD criteria, were excluded from the MAFLD criteria. This means that there is an emphasis on the metabolic dysfunction in MAFLD which may underestimate the prognostic value of hepatic steatosis itself, whereas the MAFLD criteria might better identify subjects who are at a higher risk of hepatic or cardiovascular outcomes. However, non-metabolic risk NAFLD subjects who are excluded from the MAFLD criteria are missed from the diagnosis, and their potential risk can be the cause of future diseases. Although huge controversies remain, this review focused on summarizing recent studies that compared the clinical and prognostic characteristics between subjects with NAFLD and MAFLD.     

Inflammatory processes in the liver: divergent roles in homeostasis and pathology

The hepatic immune system is designed to tolerate diverse harmless foreign moieties to maintain homeostasis in the healthy liver. Constant priming and regulation ensure that appropriate immune activation occurs when challenged by pathogens and tissue damage. Failure to accurately discriminate, regulate, or effectively resolve inflammation offsets this balance, jeopardizing overall tissue health resulting from an either  overly tolerant or an overactive inflammatory response. Compelling scientific and clinical evidence links dysregulated hepatic immune and inflammatory responses upon sterile injury to several pathological conditions in the liver, particularly nonalcoholic steatohepatitis and ischemia-reperfusion injury. Murine and human studies have described interactions between diverse immune repertoires and nonhematopoietic cell populations in both physiological and pathological activities in the liver, although the molecular mechanisms driving these associations are not clearly understood. Here, we review the dynamic roles of inflammatory mediators in responses to sterile injury in the context of homeostasis and disease, the clinical implications of dysregulated hepatic immune activity and therapeutic developments to regulate liver-specific immunity.     

Macrophage Phenotype in Liver Injury and Repair

Macrophages hold a critical position in the pathogenesis of liver injury and repair, in which their infiltrations is regarded as a main feature for both acute and chronic liver diseases. It is noted that, based on the distinct phenotypes and origins, hepatic macrophages are capable of clearing pathogens, promoting/or inhibiting liver inflammation, while regulating liver fibrosis and fibrolysis through interplaying with hepatocytes and hepatic stellate cells (HSC) via releasing different types of pro‐ or anti‐inflammatory cytokines and growth factors. Macrophages are typically categorized into M1 or M2 phenotypes by adapting to local microenvironment during the progression of liver injury. In most occasions, M1 macrophages play a pro‐inflammatory role in liver injury, while M2 macrophages exert an anti‐inflammatory or pro‐fibrotic role during liver repair and fibrosis. In this review, we focused on the up‐to‐date information about the phenotypic and functional plasticity of the macrophages and discussed the detailed mechanisms through which the phenotypes and functions of macrophages are regulated in different stages of liver injury and repair. Moreover, their roles in determining the fate of liver diseases were also summarized. Finally, the macrophage‐targeted therapies against liver diseases were also be evaluated.     

Neutrophil Depletion Blocks Early Collagen Degradation in Repairing Cholestatic Rat Livers

Biliary obstruction results in a well-characterized cholestatic inflammatory and fibrogenic process; however, the mechanisms and potential for liver repair remain unclear. We previously demonstrated that Kupffer cell depletion reduces polymorphonuclear cell (neutrophil) (PMN) and matrix metalloproteinase (MMP)8 levels in repairing liver. We therefore hypothesized that PMN-dependent MMP activity is essential for successful repair. Male Sprague-Dawley rats received reversible biliary obstruction for 7 days, and the rat PMN-specific antibody RP3 was administered 2 days before biliary decompression (repair) and continued daily until necropsy, when liver underwent morphometric analysis, immunohistochemistry, quantitative RT-PCR, and in situ zymography. We found that RP3 treatment did not reduce Kupffer cell or monocyte number but significantly reduced PMN number at the time of decompression and 2 days after repair. RP3 treatment also blocked resorption of type I collagen. In addition, biliary obstruction resulted in increased expression of MMP3, MMP8, and tissue inhibitor of metalloproteinase 1. Two days after biliary decompression, both MMP3 and tissue inhibitor of metalloproteinase 1 expression declined toward sham levels, whereas MMP8 expression remained elevated and was identified in bile duct epithelial cells by immunohistochemistry. PMN depletion did not alter the hepatic expression of these genes. Conversely, collagen-based in situ zymography demonstrated markedly diminished collagenase activity following PMN depletion. We conclude that PMNs are essential for collagenase activity and collagen resorption during liver repair, and speculate that PMN-derived MMP8 or PMN-mediated activation of intrinsic hepatic MMPs are responsible for successful liver repair.Extra- and/or intrahepatic bile duct obstruction induces a pattern of liver injury composed of bile duct epithelial cell hyperplasia, periportal fibrosis and an inflammatory cell infiltrate^1,2 that includes monocytes³ and polymorphonuclear cells (neutrophils) (PMNs).^4,5 A number of congenital and acquired illnesses result in obstruction of bile flow, which results in cholestatic liver injury and predisposes adults and children to the development of hepatic fibrosis and cirrhosis. Many studies have improved our understanding of the molecular regulation of hepatic fibrosis and until recently, hepatic fibrosis was considered to be the immobile extracellular matrix scar preceding cirrhosis. Clinical and experimental reports have since suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. Consequently, therapeutic interventions to promote resolution of hepatic injury remain elusive.Despite significant efforts to determine specific cellular and molecular mechanisms of cholestatic injury and intrinsic repair, our understanding is still far from complete.^6,7 Current hypotheses have focused on the central role of resident tissue macrophages or Kupffer cells (KCs) as the key mediators of cholestatic liver injury.^8–10 Activated KCs orchestrate the up-regulation of complex network of proinflammatory cytokines such as tumor necrosis factor α, transforming growth factor β, interleukin-1, and interleukin-6 and recruit systemic macrophages and PMNs to the site of injury. Subsequent to KC activation is the activation of hepatic stellate cells to α-smooth muscle actin (α-SMA)-positive myofibroblasts, which preferentially deposit collagen type I, leading to a marked increase in collagen type I relative to other matrix components in the fibrotic scar.¹¹Successful intrinsic repair of liver fibrosis involves remodeling and breakdown of multiple extracellular matrix components, with degradation of the predominant component, collagen type-I, being particularly important for recovery of normal liver architecture and function. Matrix metalloproteinases (MMPs) are zinc-dependent endoproteinases known to digest components of the extracellular matrix in a substrate-specific fashion. In the rat, MMP8 and MMP13 are the primary collagenases capable of digesting collagen type I. Hepatic stellate cells and KCs, located in the sinusoids, are the primary producers of MMP13. MMP8, also known as PMN collagenase, is known to be localized in PMNs, chondrocytes, endothelial cells, and rheumatoid synovial fibroblasts and can be induced in circulating mononuclear phagocytes.¹²Using a rat model of biliary obstruction and decompression to simulate chronic cholestatic fibrotic injury and subsequent fibrinolytic repair we have used a strategy of KC and PMN depletion to study the cellular and molecular mechanisms involved in hepatic matrix metabolism. We have shown that KC depletion alone does not inhibit intrinsic repair mechanisms of fibrotic degradation. KC depletion in conjunction with a reduction in the number of PMNs in the liver, however, was sufficient to inhibit fibrotic degradation. Moreover, we have also shown that fibrosis resolution, following biliary decompression, is concurrent with a increase in matrix metalloproteinase (MMP)8 expression and activity^13,14 without a contribution from the other main collagenase, MMP13 identified in rat liver. These findings may suggest a more meaningful effector role for PMNs and MMP8 in the resorption of matrix compared with KCs and MMP 13 during repair.Cholestatic liver injury is further characterized by an inflammatory infiltrate that includes PMNs, which localize to portal regions.^5,13,15 The peri-portal region is a prominent location of matrix deposition during injury and collagen resorption during repair. Our results reveal that unlike KC and recruited macrophage clearance from the liver after biliary decompression, PMNs persist after decompression and remain elevated during repair. The temporal association of PMNs colocalized in the portal region of collagen fibrosis and concurrent demonstration of elevated PMN collagenase (MMP8) gene expression and matrix degradation activity further supports an important biological role for PMN during resolution of hepatic fibrosis. We hypothesize that PMNs are crucial for successful hepatic repair following cholestatic injury. To test this hypothesis we used a unique rat model of reversible extrahepatic cholestatic injury consisting of bile duct obstruction that rapidly develops fibrosis during injury, followed by biliary decompression resulting in intrinsic resolution of fibrosis.^14,16,17 Here we report that PMNs are necessary for liver repair in this model. We also show that PMN depletion results in decreased levels of direct collagenase activity, universally considered an important component involved in resolution of fibrosis. We conclude that PMNs are needed for successful repair either by directly degrading collagen fibrotic matrix via MMP8 release and activity or by activating and regulating intrinsic hepatic collagenase degradation.     

Macrophage and perisinusoidal cell kinetics in acute liver injury.

Perisinusoidal cells (PSCs) are currently regarded as the major source of extracellular matrix proteins during hepatic fibrogenesis in response to liver injury. However, the cellular mechanisms underlying their response to injury are not fully understood. One hypothesis is that the PSCs are stimulated by peptide growth factors produced by hepatic macrophages (Kupffer cells) in response to parenchymal cell damage. In this study we have investigated the kinetics of the PSC and macrophage populations in acute carbon tetrachloride-induced hepatic injury in rats. PSCs were identified immunohistochemically by detection of cytoplasmic desmin; monocytes and macrophages were detected using the monoclonal antibodies ED1 and ED2; cells in S phase were identified by immunohistochemical detection of nuclear-incorporated bromodeoxyuridine. The results showed an expansion of the desmin-positive PSC population, predominantly within the damaged perivenular zones, which reached a peak on days 3 and 4 following administration of carbon tetrachloride; this was contributed to by local PSC proliferation. The PSC response was preceded by an expansion of the macrophage population resulting from both local macrophage proliferation and influx of blood monocytes. These results are in keeping with the hypothesis that the PSC response to acute liver injury is mediated, at least in part, by hepatic macrophages.     

Serum from CCl4-induced acute rat injury model induces differentiation of ADSCs towards hepatic cells and reduces liver fibrosis

Cellular therapies hold promise to alleviate liver diseases. This study explored the potential of allogenic serum isolated from rat with acute CCl₄ injury to differentiate adipose derived stem cells (ADSCs) towards hepatic lineage. Acute liver injury was induced by CCl₄ which caused significant increase in serum levels of VEGF, SDF1α and EGF. ADSCs were preconditioned with 3% serum isolated from normal and acute liver injury models. ADSCs showed enhanced expression of hepatic markers (AFP, albumin, CK8 and CK19). These differentiated ADSCs were transplanted intra-hepatically in CCl₄-induced liver fibrosis model. After one month of transplantation, fibrosis and liver functions (alkaline phosphatase, ALAT and bilirubin) showed marked improvement in acute injury group. Elevated expression of hepatic (AFP, albumin, CK 18 and HNF4a) and pro survival markers (PCNA and VEGF) and improvement in liver architecture as deduced from results of alpha smooth muscle actin, Sirius red and Masson’s trichome staining was observed.     

Impact of renal ischemia/reperfusion injury on the rat Kupffer cell as a remote cell: A biochemical,histological, immunohistochemical,and electron microscopic study

Almost all transplanted solid organs are exposed to some degree of ischemia-reperfusion (IR) damage. It is interesting to know that this IR damage affects various remote tissues including the liver and resulted in serious adverse effects. Liver injury triggers different responses of liver tissue especially Kupffer cells (KCs). The goal of this current study is to assess the biochemical and morphological changes of hepatic KCs after the induction of renal ischemia-reperfusion (RIR) and point out their role in remote liver injury after RIR.Sixteen male Sprague-Dawley rats were randomly divided into two equal groups: Group I; sham group. Group II; renal ischemia reperfusion (IR) group in which rats were exposed to renal ischemia for 45 min followed by renal reperfusion for 48 h. Three rats from each group were subjected to charcoal injection to evaluate KCs activity. Specimens of rat liver from each group were obtained and processed for biochemical, light microscopic and ultramicroscopic examination. The current results showed elevated serum levels of AST and ALT. The liver HGF-α protein expression increased in IR group compared to the sham group. In IR group, numerous charcoal labeled KCs were observed mainly localized around the central vein. Scanning electron micrographs showed complex primary and secondary foot process of the KCs. Ultrastructural study showed KCs with multiple cytoplasmic vacuoles, lysosomes and mitochondria, rough endoplasmic reticulum and ribosomes. Immuno-histochemical study showed more tumor necrosis factor-α (TNF-α) expression in KCs than the sham group. These results collectively demonstrated that renal IR produced biochemical and morphological changes in the liver KCs and theses cells might have a role in the remote liver injury after renal IR. This might be one of the mechanisms through which RIR affects the liver.     

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.     

Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats

Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMN- and CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not α-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN- and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) and KC co-localized in the liver in the neighbor of HSCs. The double α-SMA- and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for α-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver fibrosis models.     

Gut Bacteria Drive Kupffer Cell Expansion via MAMP-Mediated ICAM-1 Induction on Sinusoidal Endothelium and Influence Preservation-Reperfusion Injury after Orthotopic Liver Transplantation

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.More than 100 trillion, largely colon-restricted, autochthonous bacteria comprise the gut microbiome.¹ They not only help shape gut morphologic features and mucosal immunity² but also contribute to the development of the extraintestinal immune system. For example, germ-free (GF) rodents exhibit smaller, less cellular spleens and lower systemic antibody levels,³ and the gut-derived microbe-associated molecular pattern (MAMP) peptidoglycan (PDG) can prime the systemic innate immunity.⁴ Extraintestinal effects of the gut microbiome are thought to be mediated by MAMPs, which are recognized by germline encoded pattern recognition receptors (PRRs) expressed on cells throughout the body, including Kupffer cells (KCs), hepatocytes, and liver sinusoidal endothelial cells (LSECs).⁵Gut-derived MAMPs reach the liver via blood from the portal vein and first encounter PRR-bearing KCs, the most abundant of all tissue macrophage populations, and sinusoidal endothelium. Despite a valid assumption that MAMPs reach the liver via the portal venous blood, the relative physiologic composition of various portal venous MAMPs under homeostatic conditions and their effects, if any, on LSEC and KC populations, including their physiologic activation/maturation state, are poorly understood. Interactions among gut-derived MAMPs, LSECs, and KCs, however, have the potential to control KC activation status and signaling pathways and trigger protein secretion that can profoundly influence all nearby cells, including hepatocytes, stellate cells, LSECs, and intrahepatic leukocytes. Consequently, gut-derived MAMP interactions with LSECs and KCs can significantly influence susceptibility to injury and hepatic physiologic and pathophysiologic responses to environmental challenges. Included are host defense, regeneration, fibrogenesis, carcinogenesis, toxin exposure, ischemia/reperfusion injury, and immunologic tolerance.⁶Particular bacterial species (ie, Clostridium species, segmented filamentous bacteria) and specific MAMPs (ie, polysaccharide A, PDG) can induce the development of lymphoid tissue and immune cell subsets within the gut,² and MAMPs translocate to the liver via portal circulation under homeostatic conditions. Therefore, we tested the hypothesis that gut bacteria orchestrate the development of the normal KC population of the liver and, in turn, help determine the susceptibility to injury and response to environmental challenges. Results indicate that the number and activation/maturational status of KCs directly correlate with density of gut bacteria and susceptibility to injury, as indicated by the extent of cold preservation/reperfusion injury after orthotopic liver transplantation. This is mediated via gut-derived MAMPs that increase LSEC intercellular adhesion molecule 1 (ICAM-1) and lymphatic vessel endothelial receptor 1 (LYVE-1) expression, which in turn recruit KCs from circulating KC progenitors. Most importantly, broad-spectrum antibiotic therapy can decrease both the gut microbiome and liver KC population, which in turn influences a wide variety of hepatic immunology, drug reactions, physiologic processes, and susceptibility to injury.     

Cross-regulation by TLR4 and T cell Ig mucin-3 determines severity of liver injury in a CCl4-induced mouse model

Acute liver injury is a common pathological basis for a variety of acute liver diseases in the clinic, which can eventually lead to liver fibrosis and even liver failure. In this study, we found that T cell Ig and mucin domain protein 3 (Tim-3) and TLR4 receptors play important roles in CCl4-induced acute liver injury. Tim-3 is a negative regulator that is expressed by T cells and macrophages. Using antibodies against Tim-3 (anti-Tim-3 Ab), we studied the Tim-3 signal in an animal model of acute liver injury and found that a large number of inflammatory factors were upregulated. In vitro experimental data shown that anti-Tim-3 Ab treatment increased interferon-ɣ production by concanavalin A (ConA)-stimulated spleen T cells, and we found that the expression level of interleukin (IL)-6 was increased in a macrophage/spleen T cell coculture system, while administration of galectin-9 (Gal-9, a Tim-3 ligand) reduced the IL-6 production. This indicates the importance of the Tim-3/Gal-9 signalling pathway in maintaining hepatic homeostasis. The Tim-3 signalling pathway inhibits TLR4-mediated NF-κB activity, and an anti-Tim-3 Ab does not affect the liver injury in TLR4-deficient mice. Regulation between Tim-3 and TLR4 determines the severity of liver damage. The negative regulation of Tim-3 reflects the protective mechanisms of patients with impaired liver function, and these results provide important information about innate and adaptive responses in the regulation of liver damage. This finding is potentially important for the study of early liver injury.     

Taurine Attenuates Liver Injury by Downregulating Phosphorylated p38 MAPK of Kupffer Cells in Rats with Severe Acute Pancreatitis

This study was undertaken to clarify the effects of taurine on liver injury in rats with severe acute pancreatitis (SAP). Rats were randomly assigned to three groups: a sham operation (SO), a SAP (established by infusion of 5% taurocholate), and a SAP given taurine (Taur). At 12 and 24 h post-operation, taurine pretreatment significantly attenuated hepatic tissue injury induced by SAP, and concurrently, serum alanine aminotransferase, aspartate transaminase, and amylase levels were significantly reduced by taurine pretreatment. Compared with the SO group, the total and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) expression and nuclear factor-κB (NF-κB) activity of Kupffer cells (KCs) were significantly higher in the SAP group, but taurine pretreatment inhibited the total and phosphorylated p38 MAPK expression and NF-κB activity of KCs in the SAP group. The increase of tumor necrosis factor-α and interleukin-lβ in cultured supernate of the SAP rat-derived KCs was also significantly inhibited by taurine pretreatment. These results suggest that taurine pretreatment ameliorated liver injury in rats with SAP mainly by inhibiting phosphorylated p38 MAPK and NF-κB activity in KCs, which may play an important role in liver injury.     

Hydroxysafflor yellow A attenuates ischemia/reperfusion-induced liver injury by suppressing macrophage activation

Hydroxysafflor yellow A (HSYA), a major constituent in the hydrophilic fraction of the safflower plant, can retard the progress of hepatic fibrosis. However, the anti-inflammatory properties and the underlying mechanisms of HSYA on I/R-induced acute liver injury are unknown. Inhibiting macrophage activation is a potential strategy to treat liver ischemia/reperfusion (I/R) injury. In this study, we investigated the therapeutic effect of HSYA on liver I/R injury and the direct effect of HSYA on macrophage activation following inflammatory conditions. The therapeutic effects of HSYA on I/R injury were tested in vivo using a mouse model of segmental (70%) hepatic ischemia. The mechanisms of HSYA were examined in vitro by evaluating migration and the cytokine expression profile of the macrophage cell line RAW264.7 exposed to acute hypoxia and reoxygenation (H/R). Results showed that mice pretreated with HSYA had reduced serum transaminase levels, attenuated inflammation and necrosis, reduced expression of inflammatory cytokines, and less macrophage recruitment following segmental hepatic ischemia. In vitro HSYA pretreated RAW264.7 macrophages displayed reduced migratory response and produced less inflammatory cytokines. In addition, HSYA pretreatment down-regulated the expression of matrix matalloproteinase-9 and reactive oxygen species, and inhibited NF-κB activation and P38 phosphorylation in RAW264.7 cells. Thus, these data suggest that HSYA can reduce I/R-induced acute liver injury by directly attenuating macrophage activation under inflammatory conditions.