Jinzheng Li

An efficient method to isolate and culture mouse Kupffer cells.

Kupffer cells (KCs) play an essential role in the physiological and pathological functions of the liver. Although the isolation methods of KCs have been well-described, most of them are sophisticated and time-consuming. In addition, these methods are mainly used for isolating the KCs of the human and rat. In this study, a three-step procedure was applied to isolate KCs in sufficient number and purity from mouse liver, including the techniques of enzymatic tissue treatment, gradient centrifugation, and selective adherence. F4/80 immunofluorescence and flow cytometry were used for cell identification. The combination method resulted in a satisfactorily high yield of 5-6×10(6) KCs per liver, over 92.0% positive for F4/80 and 98.5% viable cells. After 24h of culturing, the KCs showed typical macrophage morphologic features such as irregular shape, transparent cytoplasm and kidney-like nucleus. The phagocytic assay showed that the isolated cells exhibited strong phagocytosis activity. The KCs we isolated were functionally intact and exhibited a concentration dependent TNF-α production induced by LPS. The method we described is an effective method to isolate mouse KCs in high purity and yield, which consuming fewer collagenase and time without altering the functional capacity of the KCs.

The role of Kupffer cells in hepatic diseases.

&NA; 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. HighlightsCytoplasmic LPS can activate macrophage via TLR4‐independent pathway that results in activation of the enzyme caspase‐11.The negative regulators associated with endotoxin tolerance in KCs include IRAK‐M, SHIP1, and SCOS1 and Twist‐2.KCs are responsible for the initiation of the pro‐inflammatory response in IRI.KCs are involved in liver immune tolerance through inducing apoptosis of T cells.KCs release IL‐1&bgr; that promotes development of NAFLD by activating TLR4, TLR9 and NLRP3 inflammasome pathways.

Therapeutic potential of stem cell in liver regeneration

Liver transplantation is the only life-saving procedure for patients with end-stage liver disease. However, its potential benefits are hampered by many disadvantages, such as the relative shortage of donors, operative risks, and high costs. These issues have prompted the search for new alternative therapies for irreversible liver disease. Stem cell therapy, with the ability for self-renewal and potential for multilineage differentiation, is a promising alternative approach. Several studies have demonstrated that transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells leads to donor cell-mediated repopulation of the liver and improved survival rates in experimental models of liver disease. However, a registered clinical application based on stem cell technology will take at least an additional 5 to 10 years because of some limitations; e.g. the lack of suitable cell sources and risk of teratoma formation. This review summarizes the general understanding of the therapeutic potentials of stem cells in liver disease, including the sources, mechanisms, and delivery methods of hepatic stem cells in liver regeneration, and discusses some challenges for their therapeutic application.

NLRP3 Deletion Inhibits the Non-alcoholic Steatohepatitis Development and Inflammation in Kupffer Cells Induced by Palmitic Acid

The cleavage and secretion of pro-inflammatory cytokines IL-1β and IL-18 is regulated by NLRP3 (NACHT, LRR, and PYD domain-containing protein 3) inflammasome activation. Kupffer cells (KCs) are implicated in the pathogenesis of various liver diseases, such as non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease, and liver fibrosis. However, the role of NLRP3 played in the non-alcoholic steatohepatitis (NASH) has yet to be evaluated. In the present study, methionine–choline-deficient (MCD) diet was used to establish the mice NASH model. The expression levels of F4/80 and NLRP3 in liver tissues were evaluated, and the IL-1β and IL-18 in serum were also evaluated. KCs were isolated from wild-type (WT) mice and NLRP3 knockout (NLRP3−/−) mice and then randomly divided into two groups: the control and palmitic acid (PA) groups. The expression levels of NLRP3, ASC, and caspase-1 in KCs were determined by RT-PCR, western blotting, and immunofluorescence. The levels of IL-1β and IL-18 in the supernatant (SN) of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). We found that KCs and NLRP3 play pro-inflammatory roles in the progression of NASH, probably through secretions of IL-1β and IL-18 by KCs induced by PA. PA could act as a kind of damage-associated molecular patterns to elevate the messenger RNA and protein expression levels of NLRP3, ASC, and caspase-1 in KCs from WT mice. In the contrast, NLRP3 deletion could inhibit the NLRP3 inflammasome upregulation and activation in KCs induced by PA. Furthermore, the levels of pro-inflammatory cytokines IL-1β and IL-18 in the SN of KCs from WT mice were all elevated with the stimulation of PA, and the increase of these cytokines in the SN was blocked by NLRP3 deletion. In conclusion, our novel findings demonstrate that NLRP3 plays a pivotal role in NASH development and pro-inflammatory cytokines IL-1β and IL-18 secretion induced by PA stimulation, and NLRP3 might be an effective potential target for the treatment of liver inflammatory diseases associated with NLRP3 inflammasome activation.

Up-regulation of Galectin-9 in vivo results in immunosuppressive effects and prolongs survival of liver allograft in rats

BACKGROUND AND OBJECTIVEnAcute rejection is still a major cause of graft dysfunction and would jeopardize recipients post-transplantation survival. Current studies demonstrate that Galectin-9 (Gal-9) is associated with down-regulation of pro-inflammatory cytokines, thus, possesses a negative immune regulatory role. However, the specific role of Gal-9 in liver transplant remains unknown.nnnMETHODSnTo establish acute rejection models of rat liver transplantation (Lewis-BN, n = 45), recipients were randomly divided into following three groups: the transfected group (n = 15); the blank plasmid group (n = 15); and the control group (n = 15). The transfected group was perfused with Ad-galectin-9 through the portal vein during the cold ischemia period. The blank plasmid group was perfused with non-target vector, and the control group was perfused with saline. The acute rejection was assayed by pathological examination; Gal-9, T-bet, RORγt, GATA3 and Foxp3 mRNA expression was detected by real-time PCR; Western blots and enzyme-linked immunosorbent assays were performed to measure IFN-γ, IL-10 and IL-17 expression.nnnRESULTSnThe pathological change of the transfected group was ameliorated than that of the other two groups. The Gal-9 mRNA level in the transfected group was much higher than that in the other two groups (*P < 0.05); T-bet and RORγt mRNA levels were significantly lower in the transfected group than in the other two groups while GATA3 and Foxp3 were not shown statistics significances (*P < 0.05). The IFN-γ and IL-17 levels in the transfected group were significantly lower than in the other two groups (*P < 0.05 for both protein and serum levels).nnnCONCLUSIONnUp-regulation of Gal-9 in vivo turns immune system toward immnuosuppression and prolongs rats liver allograft survival by the diminishment of Th1/Th17.

Inhibition of NLRP3 inflammasome by thioredoxin-interacting protein in mouse Kupffer cells as a regulatory mechanism for non-alcoholic fatty liver disease development

NOD-like receptor (NLR) NLRP3 inflammasome activation has been implicated in the progression of non-alcoholic fatty liver disease (NAFLD) from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). It has been also shown that palmitic acid (PA) activates NLRP3 inflammasome and promotes interleukin-1β (IL-1β) secretion in Kupffer cells (KCs). However, the specific mechanism of the NLRP3 inflammasome activation is unclear. We studies the molecular mechanisms by investigating the roles of Thioredoxin-interacting protein (TXNIP) and NLRP3 on NAFLD development in patients, high-fat diet (HFD)-induced NAFL and methionine choline deficient (MCD) diet-induced NASH in wild type (WT), TXNIP−/− (thioredoxin-interacting protein) and NLRP3−/− mice, and isolated KCs. We found that the expressions of NLRP3 and TXNIP in human liver tissues were higher in NASH group than in NAFL group. Furthermore, co-immunoprecipitation analyses show that activation of the TXNIP-NLRP3 inflammasome protein complex occurred in KCs of NASH WT mice rather than NAFL WT mice, thus suggesting that the formation and activation of this protein complex is mainly involved in the development of NASH. NLRP3−/− mice exhibited less severe NASH than WT mice in MCD diet model, whereas TXNIP deficiency enhanced NLRP3 inflammasome activation and exacerbated liver injury. PA triggered the activation and co-localization of the NLRP3 inflammasome protein complex in KCs isolated from WT and TXNIP−/− but not NLRP3−/− mice, and most of the complex co-localized with mitochondria of KCs following PA stimulation. Taken together, our novel findings indicate that TXNIP plays a protective and anti-inflammatory role in the development of NAFLD through binding and suppressing NLRP3.

Exogenous vascular endothelial growth factor delivery prior to endothelial precursor cell transplantation in orthotopic liver transplantation–induced hepatic ischemia/reperfusion injury

Vascular endothelial growth factor (VEGF) promotes angiogenesis in vivo. We hypothesized that exogenous delivery of VEGF prior to bone marrow–derived endothelial precursor cell (EPC) transplantation may improve orthotopic liver transplantation (OLT)–induced hepatic ischemia/reperfusion injury (HIRI). OLT between Sprague Dawley donor rats and inbred LEW Wistar recipient rats was performed in 6 experimental groups to comparatively assess the effects of the VEGF gene: an untreated normal control group, a surgical control group, a liposomal control group, a VEGF group receiving only the liposome‐encapsulated VEGF plasmid, an EPC group receiving only EPCs, and an EPC+VEGF group receiving the liposome‐encapsulated VEGF plasmid followed by EPCs. VEGF plasmid delivery to liver tissue, endogenous VEGF, and vascular endothelial growth factor receptor (VEGFR) expression, liver transaminase levels, hepatocellular injury levels, apoptosis, apoptotic biomarkers, hepatotrophic mitogens, angiogenesis, and nitric oxide synthase (NOS) activity were assayed after OLT. Exogenous VEGF gene delivery prior to EPC transplantation significantly increased endogenous VEGF and VEGFR expression, significantly reduced liver transaminase levels, significantly reduced hepatocellular injury levels, significantly reduced hepatic apoptosis levels, and significantly reduced several apoptotic biomarkers (ie, B cell lymphoma 2–associated X protein/B cell lymphoma 2 ratio, caspase 3 activity, and heat shock protein 70 expression) in post‐OLT–induced HIRI. Moreover, VEGF gene delivery prior to EPC transplantation significantly increased hepatotrophic mitogen expression (ie, epidermal growth factor, heparin‐binding epidermal growth factor–like growth factor, hepatocyte growth factor, and transforming growth factor α), angiogenesis, and NOS activity in post‐OLT–induced HIRI. In conclusion, exogenous liposomal delivery of the VEGF gene prior to bone marrow–derived EPC transplantation may be an effective strategy in decreasing OLT‐induced HIRI. Liver Transplantation 23 804–812 2017 AASLD.

Augmenter of liver regeneration attenuates acute rejection after rat liver transplantation

BACKGROUNDnThe role of augmenter of liver regeneration (ALR) on liver transplantation immune regulation remains unknown.nnnMETHODSnMale Lewis and Brown-Norway (BN) rats were assigned to allograft group (Lewis-to-BN liver transplantation), isograft group (BN-to-BN), and ALR group (Lewis-to-BN, ALR, 100xa0μg/kg/d, intramuscular injection postoperatively). Rats were sacrificed at indicated times for assessment of cytokines production, T-cell (TC) activation and apoptosis. Kupffer cells (KCs) and TCs were isolated from grafts to assess cytokine expression. Effect of ALR and KCs on TCs was monitored by co-culture ofxa0(3)H-thymidine TCs.nnnRESULTSn(1) Treatment with ALR significantly decreased interleukin-2 and interferon-γ expression, promoted TC apoptosis, and prolonged the survival of allografts; (2) KCs in ALR group and isograft group that had significantly increased interleukin-10 and decreased tumor necrosis factor-α expression were able to inhibit TC proliferation and induce their apoptosis relative to KCs in the allograft group; (3) ALR and KCs directly inhibited TC proliferation and activation and induced TC apoptosis.nnnCONCLUSIONSnALR could inhibit TC proliferation and function both inxa0vivo and inxa0vitro and attenuate acute rejection after liver transplantation.

Differential metabonomic profiles of primary hepatocellular carcinoma tumors from alcoholic liver disease, HBV-infected, and HCV-infected cirrhotic patients

Our objective was to comparatively profile the metabolite composition of primary hepatocellular carcinoma (HCC) tumors from alcoholic liver disease (ALD), hepatitis B virus (HBV)-infected, and hepatitis C virus (HCV)-infected cirrhotic patients. Primary HCC tumors were collected from ALD, HBV-infected, and HCV-infected cirrhotic patients (n=20 each). High-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy and metabonomic data analysis were performed to compare HCC tumors from the three groups. Sensitivity analyses were performed to determine the effects of diabetes, high body mass index, and smoking status. Metabonomic pathway analyses were conducted to identify dysregulated pathways. Three metabolites were significantly differentiated between ALD and HBV-infected patients, which were distinguishable by changes in ketone body, glycerolipid, and phenylalanine metabolism. Five metabolites were significantly differentiated between ALD and HCV-infected patients, which were distinguishable by changes in ketone body, alanine/aspartate/glutamate, and phenylalanine metabolism. Six metabolites were significantly differentiated between HBV-infected and HCV-infected patients, which were distinguishable by changes in ketone body, tyrosine, and alanine/aspartate/glutamate metabolism. In conclusion, this is the first study to demonstrate that the metabolic phenotypes of primary HCC tumors vary significantly across ALD, HBV-infected, and HCV-infected cirrhotic patients.Our objective was to comparatively profile the metabolite composition of primary hepatocellular carcinoma (HCC) tumors from alcoholic liver disease (ALD), hepatitis B virus (HBV)-infected, and hepatitis C virus (HCV)-infected cirrhotic patients. Primary HCC tumors were collected from ALD, HBV-infected, and HCV-infected cirrhotic patients (n=20 each). High-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy and metabonomic data analysis were performed to compare HCC tumors from the three groups. Sensitivity analyses were performed to determine the effects of diabetes, high body mass index, and smoking status. Metabonomic pathway analyses were conducted to identify dysregulated pathways. Three metabolites were significantly differentiated between ALD and HBV-infected patients, which were distinguishable by changes in ketone body, glycerolipid, and phenylalanine metabolism. Five metabolites were significantly differentiated between ALD and HCV-infected patients, which were distinguishable by changes in ketone body, alanine/aspartate/glutamate, and phenylalanine metabolism. Six metabolites were significantly differentiated between HBV-infected and HCV-infected patients, which were distinguishable by changes in ketone body, tyrosine, and alanine/aspartate/glutamate metabolism. In conclusion, this is the first study to demonstrate that the metabolic phenotypes of primary HCC tumors vary significantly across ALD, HBV-infected, and HCV-infected cirrhotic patients.

TIM‑4 blockade of KCs combined with exogenous TGF‑β injection helps to reverse acute rejection and prolong the survival rate of mice receiving liver allografts

An acute reaction response (AR) following liver transplantation (LT) is caused by immune responses that are primarily mediated by T lymphocytes. Kupffer cells (KCs) are the largest antigen presenting cell (APC) group in vivo and are the primary modulators of the inflammatory or tolerogenic immune response in liver tissues. T cell immunoglobulin-domain and mucin-domain-4 (TIM-4), the only TIM protein not expressed on T cells, is expressed on APCs; suggesting that it mediates the various immune responses. However, to the best of our knowledge, the role of TIM-4 expressed by KCs in LT injury remains unknown. The present study aimed to explore whether and how TIM-4 expressed by KCs is involved in the AR of liver allografts. Orthotopic liver transplantation (OLT) was performed in mice to establish a model of AR and results demonstrated that LT may lead to the augmented expression of TIM-4 in activated KCs. It was also revealed that TIM-4 blockade markedly attenuated AR injury in vivo via the nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways. In addition, levels of transforming growth factor-β (TGF-β) were increased following TIM-4 blockade. Furthermore, in a KC/cluster of differentiation (CD)4+ T cell co-culture system, blocking TIM-4 inhibited T helper 2 (Th2) differentiation, stimulated the conversion of naive (CD)4+ T cells into CD4+CD25+Forkhead box protein p3+ T regulatory cells and suppressed interleukin-4/signal transducer and activator of transcription 6/transcription factor gata3 signaling. These effects were enhanced following the addition of TGF-β. It was also demonstrated that LT mouse models treated with TIM-4 blockade in combination with exogenous TGF-β injections, increased the survival times of mice and enhanced the amelioration of AR in LT. These results indicate that blocking the expression of TIM-4 by KCs via exogenous TGF-β injection may be an effective therapeutic strategy to inhibit the AR of liver allografts.