Isolated Kupffer Cells Research Articles

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism

ObjectiveAchieving 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...

Kupffer cell pyroptosis mediated by METTL3 contributes to the progression of alcoholic steatohepatitis.

Chronic alcohol consumption is a major risk factor for alcoholic steatohepatitis (ASH). Previous studies have shown that direct injury of hepatocytes is the key factor in its occurrence and development. However, our study shows that the role of Kupffer cells in ASH cannot be ignored. We isolated Kupffer cells from the livers of ASH mice and found that alcohol consumption induced Kupffer cell pyroptosis and increased the release of interleukin-1β (IL-1β). Furthermore, we screened the related m6A enzyme methyltransferase-like 3 (METTL3) from liver Kupffer cells, and found that silencing METTL3 alleviated inflammatory cytokine eruption by Kupffer cell pyroptosis in ASH mice. In vitro, we silenced METTL3 with lentivirus in BMDMs and RAW264.7 cells and confirmed that METTL3 could reduce pyroptosis by influencing the splicing of pri-miR-34A. Together, our results revealed a critical role of KC pyroptosis in ASH and highlighted the mechanism by which METLL3 relieves cell pyroptosis, which could be a promising therapeutic strategy for ASH.

The imbalance of liver resident macrophages polarization promotes chronic autoimmune hepatitis development in mice.

Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. At present, it is largely unknown how the innate immune cells influence AIH development. To inquiry about mechanism of liver resident macrophages in AIH development, thus offering a new direction for AIH targeted treatment. The liver resident macrophages were eliminated by clodronate liposomes in AIH liver tissues, followed by HE and Picrosirius assay to detect liver fibrosis and lymphocyte infiltration. The liver resident macrophages polarization was detected by Immunohistochemistry and qPCR. The collagenase digestion was used to isolate Kupffer cells from AIH mice liver tissues and pro-/anti-inflammatory cytokines were determined by qPCR. M2 macrophages were the dominant phenotype at early immune response stage and hepatic inflammation was progressively aggravated after depletion of liver resident macrophages. M2 macrophages could effectively delay the development of AIH and could be polarized to M1 macrophages at the disease progresses. TLR2 ligands could promote M2 macrophages producing anti-inflammatory cytokines, whereas TLR4 ligands could promote M1 macrophages producing proinflammatory cytokines. The change of TLR2 and TLR4 ligands could lead to continuous high expression of TLR4 and decreased expression of TLR2 in macrophages to further affect liver resident macrophages polarization state. TLR2 and TLR4 ligands mediated liver resident macrophages polarization to favor chronic autoimmune hepatitis development.

Tax1BP1 limits hepatic inflammation and reduces experimental hepatocarcinogenesis

The nuclear factor kappa beta (NFκB) signaling pathway plays an important role in liver homeostasis and cancer development. Tax1-binding protein 1 (Tax1BP1) is a regulator of the NFκB signaling pathway, but its role in the liver and hepatocellular carcinoma (HCC) is presently unknown. Here we investigated the role of Tax1BP1 in liver cells and murine models of HCC and liver fibrosis. We applied the diethylnitrosamine (DEN) model of experimental hepatocarcinogenesis in Tax1BP1+/+ and Tax1BP1−/− mice. The amount and subsets of non-parenchymal liver cells in in Tax1BP1+/+ and Tax1BP1−/− mice were determined and activation of NFκB and stress induced signaling pathways were assessed. Differential expression of mRNA and miRNA was determined. Tax1BP1−/− mice showed increased numbers of inflammatory cells in the liver. Furthermore, a sustained activation of the NFκB signaling pathway was found in hepatocytes as well as increased transcription of proinflammatory cytokines in isolated Kupffer cells from Tax1BP1−/− mice. Several differentially expressed mRNAs and miRNAs in livers of Tax1BP1−/− mice were found, which are regulators of inflammation or are involved in cancer development or progression. Furthermore, Tax1BP1−/− mice developed more HCCs than their Tax1BP1+/+ littermates. We conclude that Tax1BP1 protects from liver cancer development by limiting proinflammatory signaling.

Resveratrol Modulates the Inflammatory Profile of Immune Responses and Circulating Endothelial Cells' (CECs') Population During Acute Whole Body Gamma Irradiation.

Wistar rats were whole body irradiated with a single dose of 2 Gy post administration with 10 or 100 mg/kg of resveratrol (RSV) intraperitoneally for 30 days. Rats’ livers were dissected and processed to analyze immune response profiles of Th1, Th2, Th9, Th17, and Th22 by flow cytometry. In addition, peripheral blood samples were collected and circulating endothelial cells (CECs) were counted as an indicator for endothelial damage. Results demonstrated that resveratrol at 100 mg/kg enhanced liver immunological response influenced by irradiation by inducing Th2 immune response that was revealed by an increase in IL-10 secretion to more than 5,000 pmol/ml post irradiation. Results also indicated that RSV, at a dose of 100 mg/kg, decreased levels of the main pro-inflammatory cytokines such as INF-γ, IL-22, IL-17A, and GM-CSF post irradiation. In addition, the same RSV was bound to upregulate the expression of IL-10 mRNA in isolated Kupffer cells (KCs) and their secretion of IL-10 post irradiation. The result demonstrated that KCs were the central source of this anti-inflammatory response mediated mainly by IL10. These results, proposed for the first time, clearly states that RSV promotes IL-10 mediated immune resolution by Kupffer cells and not by hepatocytes. This implies that KCs have a crucial role in radiotherapy. Additionally, this study showed that RSV had an anti-apoptotic effect through re-increasing the number of CECs, which is implicated in irradiation damage. Result of the current work discloses novel findings about the potential of RSV as a radio-protector agent of a natural origin and suggests novel roles of KCs as a pharmacological target during radiation exposure.

Fructose Metabolism and Regulation of Extracellular Matrix Protein Gene Expression in Activated Macrophages

Abstract Objectives Recently fructose has been linked to the development of Nonalcoholic Fatty Liver Disease (NAFLD), but the mechanisms behind the progression remain to be elucidated. Kupffer cells have been identified as regulators of hepatic inflammation and extracellular matrix (ECM) proteins. Activated macrophages are known to express tissue inhibitor of metalloproteinase (TIMP1), which inhibits matrix metalloproteinase 9 (MMP9) activity of reconstructing ECM which leads to fibrosis. In humans and mouse models, TIMP1 expression is positively correlated with the progression of NAFLD. Based on these findings, we hypothesize that fructose regulates TIMP1 gene expression in activated proinflammatory macrophages. Methods Using an in vitro model of J774 macrophages and primary Kupffer cells, cells were treated to induce an M1 phenotype in the presence of glucose or fructose. Cells were harvested for RNA and RT-PCR was conducted to measure ECM gene expression. Isolated Kupffer cells were collected from C57BL/6J mice fed a high fat diet (HFD) supplemented with 30% fructose or 30% glucose and analyzed for ECM expression. To examine fructose uptake and intra- and extracellular metabolites, mass spectrophotometry and nuclear magnetic resonance was conducted. Results Timp1 gene expression was significantly increased in J77.4 cells treated with fructose compared to glucose. Surprisingly, fructose treatment decreased Mmp9 gene expression. Likewise, fructose treatment increased TIMP1 protein expression in isolated Kupffer cells. In vivo, isolated hepatic macrophages from mice fed HF and high fructose diet had elevated Timp1 gene expression compared to mice fed high glucose diet. Extracellular levels of lactate decreased by 1.5-fold in fructose treated J77.4 cells compared to glucose. Metabolites involved in the TCA cycle and mitochondrial function were decreased when treated with fructose compared to glucose in non-activated macrophages. However, fructose treatment increased intracellular methanol and acetate levels in M1 macrophages compared to glucose. Conclusions Our data suggest that fructose upregulates Timp1 expression and possibly decreases mitochondrial function while increasing acetate and methanol production, identifying new mechanisms by which fructose drives the progression of NAFLD. Funding Sources Kenan Institute North Carolina University.

M-CSF Receptor Antagonists Inhibit the Initiation and Progression of Hepatocellular Carcinoma in Mice.

The aim of this study was to investigate the effects of the macrophage colony-stimulating factor (M-CSF) receptor antagonist on hepatic carcinogenesis in mice. Mice were injected with diethylnitrosamine (DEN) and treated with M-CSF receptor antagonist GW2580 (GW) or a saline vehicle just after (early treated group) or 2 weeks after (late treated group) DEN injection. Animals were sacrificed after 28 weeks and incidence of tumor was assessed. Isolated Kupffer cells were co-cultured with M-CSF in the presence or absence of GW, and the concentration of VEGF was measured. The incidence of tumors was significantly blunted both in the early- and the late-treated groups. In addition, angiogenesis within the tumor was also suppressed in both groups. The concentration of VEGF increased in Kupffer cells treated with M-CSF compared to those cultured without M-CSF. This increase was blunted by GW. M-CSF and its receptor could be novel molecular targets for hepatocellular carcinoma.

P-292 - S-allyl-glutathione suppresses liver fibrosis by inhibition of excess skewing polarization of macrophages in rats

Purpose Sustained hepatic inflammation derived from parenchymal liver injury is a major driving force of fibrogenesis. We synthesized S-allyl-glutathione (SAG) which regulates macrophage (MO) polarization, and investigated whether SAG can improve liver fibrosis. Method Liver fibrosis was induced by CCl4 injection twice a week for 9 weeks in rats. SAG was administered as a mixed diet from the starting CCl4. To study fibrosis regression, rats were orally administered SAG for 5 weeks after terminating CCl4. In vitro studies were performed effects of SAG (10 -14M~) on M1 or M2 polarization of isolated Kupffer cells (KCs) or bone marrow derived MO (BMDM), and its interactions with isolated hepatic stellate cells (HSCs). Result and Discussion SAG markedly inhibited liver fibrogenesis in a dose-dependent manner and various fibrosis markers. In the fibrolysis stage, SAG accelerated fibrolysis after development of liver chirrosis and decreased the amount of M2 MO. Both Col1A1 and HSP47 mRNA in day 6 HSCs were inhibited by the conditioned medium of SAG-treated MO with M2 inducers onto day 3 HSCs. SAG did not directly inhibit HSC activation. In the M1 MO, SAG also inhibited. These findings suggested that SAG could improve liver cirrhosis via the attenuation of excess polarization in KCs.

An efficient method to isolate Kupffer cells eliminating endothelial cell contamination and selective bias.

Multicolor flow cytometry and cell sorting are powerful immunologic tools for the study of hepatic mϕ, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of mϕ and endothelial cell reporter mice, flow cytometry, and confocal imaging, we have shown that conventional flow‐cytometric strategies for identification of Kupffer cells (KCs) leads to inclusion of a significant proportion of CD31hi endothelial cells. These cells were present regardless of the method used to prepare cells for flow cytometry and represented endothelium tightly adhered to remnants of KC membrane. Antibodies to endothelial markers, such as CD31, were vital for their exclusion. This result brings into focus recently published microarray datasets that identify high expression of endothelial cell‐associated genes by KCs compared with other tissue‐resident mϕ. Our studies also revealed significant and specific loss of KCs among leukocytes with commonly used isolation methods that led to enrichment of proliferating and monocyte‐derived mϕ. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.

Spred2 Deficiency Exacerbates D-Galactosamine/Lipopolysaccharide -induced Acute Liver Injury in Mice via Increased Production of TNF\u03b1

Acute liver injury (ALI) is characterized by hepatocyte damage and inflammation. In the present study, we examined whether the absence of Sprouty-related EVH1-domain-containing protein 2 (Spred2), a negative regulator of the Ras/Raf/ERK/MAPK pathway, influences ALI induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). Compared to wild-type mice, Spred2−/− mice developed exacerbated liver injury represented by enhanced hepatocyte damage and inflammation. Enhanced ERK activation was observed in Spred2−/−-livers, and the MEK/ERK inhibitor U0126 ameliorated ALI. Hepatic tumour necrosis factor α (TNFα) and interleukin (IL)-1β levels were increased in Spred-2−/−-livers, and the neutralization of TNFα dramatically ameliorated ALI, which was associated with decreased levels of endogenous TNFα and IL-1β. When mice were challenged with D-GalN and TNFα, much severer ALI was observed in Spred2−/− mice with significant increases in endogenous TNFα and IL-1β in the livers. Immunohistochemically, Kupffer cells were found to produce TNFα, and isolated Kupffer cells from Spred2−/− mice produced significantly higher levels of TNFα than those from wild-type mice after LPS stimulation, which was significantly decreased by U0126. These results suggest that Spred2 negatively regulates D-GalN/LPS-induced ALI under the control of TNFα in Kupffer cells. Spred2 may present a therapeutic target for the treatment of ALI.

Development of Kupffer cell targeting type-I interferon for the treatment of hepatitis via inducing anti-inflammatory and immunomodulatory actions

Because of its multifaceted anti-inflammatory and immunomodulatory effects, delivering type-I interferon to Kupffer cells has the potential to function as a novel type of therapy for the treatment of various types of hepatitis. We report herein on the preparation of a Kupffer cell targeting type-I interferon, an albumin-IFNα2b fusion protein that contains highly mannosylated N-linked oligosaccharide chains, Man-HSA(D494N)-IFNα2b, attached by combining albumin fusion technology and site-directed mutagenesis. The presence of this unique oligosaccharide permits the protein to be efficiently, rapidly and preferentially distributed to Kupffer cells. Likewise IFNα2b, Man-HSA(D494N)-IFNα2b caused a significant induction in the mRNA levels of IL-10, IL-1Ra, PD-L1 in RAW264.7 cells and mouse isolated Kupffer cells, and these inductions were largely inhibited by blocking the interferon receptor. These data indicate that Man-HSA(D494N)-IFNα2b retained the biological activities of type-I interferon. Man-HSA(D494N)-IFNα2b significantly inhibited liver injury in Concanavalin A (Con-A)-induced hepatitis model mice, and consequently improved their survival rate. Moreover, the post-administration of Man-HSA(D494N)-IFNα2b at 2 h after the Con-A challenge also exerted hepato-protective effects. In conclusion, this proof-of-concept study demonstrates the therapeutic effectiveness and utility of Kupffer cell targeting type-I interferon against hepatitis via its anti-inflammatory and immunomodulatory actions.

Astaxanthin prevents ischemia-reperfusion injury of the steatotic liver in mice.

Steatosis has a low tolerance against ischemia-reperfusion injury (IRI). To prevent IRI in the steatotic liver, we attempted to elucidate the protective effect of astaxanthin (ASTX) in the steatotic liver model by giving mice a methionine and choline-deficient high fat (MCDHF) diet. Levels of lipid peroxidation and apoptosis, the expression of inflammatory cytokines and heme oxygenase (HO)-1, in the liver were assessed. Reactive oxygen species (ROS), inflammatory cytokines, apoptosis-related proteins and members of the signaling pathway were also examined in isolated Kupffer cells and/or hepatocytes from the steatotic liver. ASTX decreased serum ALT and AST levels, the amount of TUNEL, F4/80, or 4HNE-positive cells and the mRNA levels of inflammatory cytokines in MCDHF mice by IRI. Moreover, HO-1 and HIF-1α, phosphorylation of Akt and mTOR expressions were increased by ASTX. The inflammatory cytokines produced by Kupffer, which were subjected to hypoxia and reoxygenation (HR), were inhibited by ASTX. Expressions of Bcl-2, HO-1 and Nrf2 in hepatocytes by HR were increased, whereas Caspases activation, Bax and phosphorylation of ERK, MAPK, and JNK were suppressed by ASTX. Pretreatment with ASTX has a protective effect and is a safe therapeutic treatment for IRI, including for liver transplantation of the steatotic liver.

Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma

ABSTRACTKupffer cells represent the first line of defense against tumor cells in the liver. Myeloid-derived suppressor cells (MDSC) have recently been observed in the liver parenchyma of tumor-bearing animals. The present study investigates the function of the MDSC subsets, and their impact on Kupffer cell phenotype and function. RIL-175 mouse hepatocellular carcinoma (HCC) cells were injected into the median liver lobe of C57BL/6 mice. Three weeks later, the median lobe hosting the tumor nodule was removed, and Kupffer cells and MDSCs were sorted from the remaining liver. Mouse livers devoid of HCC served as control. Kupffer cells expressed less co-stimulatory CD86 and MHCII and more co-inhibitory CD274 molecules in HCC-bearing livers than in control livers. Corresponding to this phenotype, Kupffer cells from HCC-bearing mice were less efficient in their function as antigen-presenting cells. Three CD11b+ cell populations were identified and sorted from HCC-bearing mice. These cells had various phenotypes with different levels of MDSC-specific surface markers (Ly6Ghigh cells, Gr1high cells, and Ly6Clow cells), and may be considered as bonafide MDSCs given their suppression of antigen-specific T cell proliferation. Primary isolated Kupffer cells in co-culture with the three MDSC subsets showed a decrease in CCL2 and IL-18 secretion, and an increase in IL-10 and IL-1β secretion, and an increased expression of CD86, CD274, and MHCII. In conclusion, these data demonstrated the existence of three MDSC subsets in HCC-bearing animals. These cells altered Kupffer cell function and may decrease the migration and activation of anticancer effector cells in the liver.

PICK1 confers anti-inflammatory effects in acute liver injury via suppressing M1 macrophage polarization.

Protein interacting with C kinase 1 (PICK1) is a scaffolding protein mainly implicated in neurological diseases, however, the function of PICK1 in acute liver injury (ALI) remains unknown. Our study found a dramatical decrease in mRNA and protein levels of PICK1 in liver tissues and isolated Kupffer cells (KCs) from the liver in mice with ALI. Furthermore, pretreatment the mice with ALI with FSC-231, a pharmacological inhibitor of PICK1, could significantly augment inflammatory response. Furthermore, invitro studies showed that both lipopolysaccharide (LPS) and interferon gamma (IFN-γ) significantly reduced the expression of PICK1, while IL-4 elevated its expression in RAW 264.7cells. Additionally, over-expression of PICK1 inhibited the expression of M1 biomarkers by suppressing NF-κB activity, and enhanced the expression of M2 biomarkers by promoting STAT6 activity. In contrast, knockdown of PICK1 or FSC-231 pretreatment promoted M1 polarization and suppressed M2 polarization. Besides, caveolin-1 was identified as a potential target gene controlled by PICK1 in RAW 264.7cells. Mechanistic investigation revealed a dual role of PICK1 in regulating macrophage polarization and implied PICK1 as a potential therapeutic target in ALI.

Identification of potential biomarkers for post-traumatic complications released after trauma–hemorrhage from murine Kupffer cells and its investigation in lung and liver

Context: Early diagnosis of complications after severe trauma by specific biomarkers remains difficult.Objective: Identify potential new biomarkers for early diagnosis of post-traumatic complications.Material and methods: Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR.Results: Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5.Conclusion: Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.

Dietary advanced glycation end-products aggravate non-alcoholic fatty liver disease.

AIMTo determine if manipulation of dietary advanced glycation end product (AGE), intake affects non-alcoholic fatty liver disease (NAFLD) progression and whether these effects are mediated via RAGE.METHODSMale C57Bl6 mice were fed a high fat, high fructose, high cholesterol (HFHC) diet for 33 wk and compared with animals on normal chow. A third group were given a HFHC diet that was high in AGEs. Another group was given a HFHC diet that was marinated in vinegar to prevent the formation of AGEs. In a second experiment, RAGE KO animals were fed a HFHC diet or a high AGE HFHC diet and compared with wildtype controls. Hepatic biochemistry, histology, picrosirius red morphometry and hepatic mRNA were determined.RESULTSLong-term consumption of the HFHC diet generated significant steatohepatitis and fibrosis after 33 wk. In this model, hepatic 4-hydroxynonenal content (a marker of chronic oxidative stress), hepatocyte ballooning, picrosirius red staining, α-smooth muscle actin and collagen type 1A gene expression were all significantly increased. Increasing the AGE content of the HFHC diet by baking further increased these markers of liver damage, but this was abrogated by pre-marination in acetic acid. In response to the HFHC diet, RAGE-/- animals developed NASH of similar severity to RAGE+/+ animals but were protected from the additional harmful effects of the high AGE containing diet. Studies in isolated Kupffer cells showed that AGEs increase cell proliferation and oxidative stress, providing a likely mechanism through which these compounds contribute to liver injury.CONCLUSIONIn the HFHC model of NAFLD, manipulation of dietary AGEs modulates liver injury, inflammation, and liver fibrosis via a RAGE dependent pathway. This suggests that pharmacological and dietary strategies targeting the AGE/RAGE pathway could slow the progression of NAFLD.

Ankaflavin ameliorates steatotic liver ischemia-reperfusion injury in mice

It is well-known that steatotic liver is more susceptible to ischemia-reperfusion (I/R) injury during liver transplantation, liver resection and other liver surgeries. The increasing incidence of non-alcoholic fatty liver disease (NAFLD) decreases the availability of liver donors. Although steatotic liver is now accepted as a source of liver for transplantation, NAFLD exacerbates the liver injury after liver surgery. The present study was to investigate the protective role of ankaflavin in steatotic liver I/R injury. The model of fatty liver mice was induced with high fat diet in four weeks, ankaflavin or vehicle (saline) was administrated by gavage once a day for one week. The animals were subjected to partial hepatic I/R. Blood samples were collected to measure serum aminotransferases. The liver tissues were used to examine liver steatosis, apoptosis of hepatocytes, hepatic oxidative stress, Kupffer cells and inflammatory cytokines. The effects of ankaflavin on inflammatory cytokines were evaluated in isolated Kupffer cells from the steatotic liver. Ankaflavin reduced liver steatosis in high fat diet mice. Compared with normal mice, I/R induced more damage to the mice with steatosis, such as hepatocyte apoptosis, inflammatory cytokines (TNF-alpha, IL-6 and IL-1 beta), serum aminotransferases and thiobarbituric acid reactive substances. Importantly, ankaflavin administration significantly attenuated these changes. In addition, ankaflavin significantly decreased the proliferation of Kupffer cells and the expression of TNF-alpha, IL-6 and IL-1 beta protein in isolated Kupffer cells stimulated by TNF-alpha. Ankaflavin has protective effects against I/R injury through anti-inflammatory, anti-oxidant and anti-apoptotic mechanisms in fatty livers, these effects are at least partially mediated by inhibiting Kupffer cell functions.

Kupffer Cell Isolation for Nanoparticle Toxicity Testing.

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle toxicity testing in immortalized cell lines or primary cells have shown discrepancies, highlighting the need to extend the use of primary cells for in vitro assays. This protocol describes the isolation of mouse liver macrophages, named Kupffer cells, and their use to study nanoparticle toxicity. Kupffer cells are the most abundant macrophage population in the body and constitute part of the reticulo-endothelial system (RES), responsible for the capture of circulating nanoparticles. The Kupffer cell isolation method reported here is based on a 2-step perfusion method followed by purification on density gradient. The method, based on collagenase digestion and density centrifugation, is adapted from the original protocol developed by Smedsrød et al. designed for rat liver cell isolation and provides high yield (up to 14 x 10(6) cells per mouse) and high purity (>95%) of Kupffer cells. This isolation method does not require sophisticated or expensive equipment and therefore represents an ideal compromise between complexity and cell yield. The use of heavier mice (35-45 g) improves the yield of the isolation method but also facilitates remarkably the procedure of portal vein cannulation. The toxicity of functionalized carbon nanotubes f-CNTs was measured in this model by the modified LDH assay. This method assesses cell viability by measuring the lack of structural integrity of Kupffer cell membrane after incubation with f-CNTs. Toxicity induced by f-CNTs can be measured consistently using this assay, highlighting that isolated Kupffer cells are useful for nanoparticle toxicity testing. The overall understanding of nanotoxicology could benefit from such models, making the nanoparticle selection for clinical translation more efficient.

β-Caryophyllene alleviates d-galactosamine and lipopolysaccharide-induced hepatic injury through suppression of the TLR4 and RAGE signaling pathways

Agastache rugosa (A. rugosa, Labiatae), a perennial herb spread throughout Korean fields, is widely consumed as a wild edible vegetable and is used in folk medicine. This study examined the hepatoprotective mechanisms of β-caryophyllene (BCP), a major bicyclic sesquiterpene of A. rugosa, against d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic failure. Mice were given an intraperitoneal injection of BCP (50, 100 and 200mg/kg) 1h before GalN (800mg/kg)/LPS (40μg/kg) injection and were killed 1h or 6h after GalN/LPS injection. GalN/LPS markedly increased mortality and serum aminotransferase activity, both of which were attenuated by BCP. BCP also attenuated increases in serum tumor necrosis factor-α, interleukin 6, and high-mobility group protein B1 levels by GalN/LPS. GalN/LPS significantly increased toll-like receptor (TLR) 4 and receptor for advanced glycation end products (RAGE) protein expression, extracellular signal-related kinase, p38 and c-Jun N-terminal kinase phosphorylation, nuclear factor κB (NF-κB), early growth response protein-1, and macrophage inflammatory protein-2 protein expression. These increases were attenuated by BCP. Furthermore, BCP suppressed increased TLR4 and RAGE protein expression and proinflammatory cytokines production in LPS-treated isolated Kupffer cells. Our findings suggest that BCP protects against GalN/LPS-induced liver injury through down-regulation of the TLR4 and RAGE signaling.

Acquiring Kupffer Cells in Mice Using a MACS-Based Method

ObjectiveThis study sought to establish a new method to isolate Kupffer cells (KCs) by magnetic activated cell sorting (MACS). MethodsNonparenchymal cells were acquired from C57BL/6 mice livers by a perfusion system in vivo and then stained with F4/80+ fluorescein isothiocyanate and CD11c− phycoerythrin antibodies. After incubating with immunomagnetic beads, F4/80+CD11c− KCs were obtained by MACS selection. The purity was evaluated by flow cytometry, and the morphological features and vitality were analyzed in in vitro cultures. ResultsCompared with traditional methods, acquiring KCs by MACS was characterized by economy, efficiency, and high purity. The F4/80+CD11c− KCs cultured in vitro also showed the typical adherent shape and excellent phagocytic ability. ConclusionsWith the 2-step method using immunomagnetic beads, we provide a new method by which KCs can be obtained from mouse liver with high purity and distinct phenotype of F4/80+ CD.