Human Kupffer Cells Research Articles

The variances in cytokine production profiles from non- or activated THP-1, Kupffer cell and human blood derived primary macrophages following exposure to either alcohol or a panel of engineered nanomaterials.

The portfolio of cytokines is key to the function of macrophages as sentries of the innate immune system as well as being critical for the transition from innate to adaptive immunity. Cytokine bias is critical in the fate of macrophages into a continuum of inflammatory to anti-inflammatory macrophages. Due to advances in the field of toxicology, increasingly advanced multi-cellular in vitro safety assessment models are being developed in order to allow for a better predication of potential adverse effects in humans with many of these models include a macrophage population. The selection of the correct macrophage cells in these advanced in vitro models is critical for a physiologically relevant and realistic immune response. In this study we investigated cytokine response profile (IL1-β, IL6, IL10 and TNF-α) of activated and non-activated THP-1 (immortalized monocyte-like cell line), primary human Kupffer cells (liver resident macrophages) and human primary peripheral blood mononuclear cells following exposure of a panel of nanomaterials or ethanol. The data demonstrated that the THP-1 cell line are not great cytokine producers. The PBMC appear to be a good in vitro surrogate for circulating/pro-inflammatory macrophages but are not a suitable replacement for Kupffer cells. The findings from this study highlight the necessity for the selection of appropriate macrophages populations to meet the specific physiological requirements of in vitro experiment.

Human 3D multicellular microtissues: An upgraded model for the in vitro mechanistic investigation of inflammation-associated drug toxicity

Inflammation is one of the factors that may increase the sensitivity of hepatic cells to acetaminophen (APAP) induced toxicity. To investigate the mechanisms, we exposed 3-dimensional (3D) Human Liver Microtissues, a co-culture of primary human hepatocytes (PHH) and Kupffer cells (KCs), to 0, 0.5 (low), 5 (median) and 10 mM (high dose) APAP for 24 h, with/without lipopolysaccharide (LPS). Microarray-technology was used to evaluate the transcriptome changes. In the presence of LPS, the median-dose of APAP is sufficient to inhibit the expression of respiratory chain- and antioxidant-related genes, suggesting the involvement of reactive oxygen species (ROS) and oxidative stress. Furthermore, the median- and high-dose of APAP inhibited the expression of Fc fragment receptor (FcγR)-coding genes, regardless of the presence of LPS. The toll-like receptor 4 (TLR4) expression, however, was continuously elevated after the LPS/APAP co-exposures, which may result in reduced KC-phagocytosis and unbalanced cytokine patterns. Compared to the treatment with LPS only, LPS/APAP co-exposures induced the production of interleukin (IL)-8, a pro-inflammatory cytokine, but suppressed the secretion of IL-6, a cytokine regulating hepatic regeneration, along with the increase in APAP dosages. In addition to the disrupted mitochondrial functions, the presence of LPS exacerbated APAP toxicity. These findings suggest that 3D Microtissues are a suitable model for the mechanistic exploration of inflammation-associated drug toxicity.

Akt1 and Akt2 Isoforms Play Distinct Roles in the Development of Inflammation and Fibrosis during Alcoholic Liver Disease

Akt isoforms (Akt1, Akt2, and Akt3), the key phosphorylating kinases, play vital and unique roles in all aspects of alcoholic liver disease (ALD). In vitro, cell specific silencing of Akt isoforms in acetaldehyde+LPS and/or ethanol+LPS treated human hepatic stellate cells (HSC), Kupffer cells and/or VL17A hepatocyte cultures; and in vivo, Akt1 and Akt2 silencing in a chronic ethanol/binge/lipopolysaccharide (EBL) induced mouse liver injury model using pharmacological inhibitors, were used to gain a better understanding of how each Aktisoform differentially regulates cellular functions during ALD. This study involves hepatocyte and Kupffer cells inflammation, and hepatic stellate cells proliferation, migration and fibrogenesis, which cumulatively lead to the pathogenesis of ALD. Herein, our in vitro results showed that siRNA‐directed silencing of Akt2, but not Akt1, significantly suppressed cell inflammatory markers in HSC and Kupffer cells. While Akt1 and Akt2 inhibited cell proliferation in HSC; Akt2 alone inhibited cell migration in HSC; and Akt1 and Akt2, but not Akt3 inhibited the fibrogenic markers in hepatocytes and HSC. Moreover, although Akt isoforms show extensive sequence similarity, they are localized in different subcellular compartments in HSC. On the other hand, our in vivo results showed that EBL treatment in wild‐type C57BL/6 mice stimulated all three isoforms of Akt with concomitant increases in phosphorylated species of phosphoinositide dependent kinase‐1, mammalian target‐o‐rapamycin complex 2, and phosphatidylinositol‐3‐kinase, resulting in increases in inflammatory, proliferative, and fibrogenic genes. In addition, pharmacological blocking of Akt2, but not Akt1, inhibited EBL‐induced inflammation; and of Akt1 and Akt2 inhibited the pro‐fibrogenic markers as well as fibrosis. Our finding suggest that Akt isoforms play unique roles against inflammation, cell proliferation, migration, and fibrogenesis during EBL‐induced liver injury. These in vitro and in vivo results also suggest that close attention must be paid when targeting all Akt isoforms as a therapeutic intervention.Support or Funding InformationNIAAA/NIH K01 award ‐ number: K01AA025140‐02This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Generation of mature kupffer cells from human induced pluripotent stem cells

Liver macrophages, Kupffer cells (KCs), play a critical role in drug-induced liver injury (DILI) and liver diseases including cholestasis, liver fibrosis and viral hepatitis. Application of KCs in in vitro models of DILI and liver diseases is hindered due to limited source of human KCs. In vivo, KCs originate from MYB-independent macrophage progenitors, which differentiate into liver-specific macrophages in response to hepatic cues in the liver. Here, we recapitulated KCs ontogeny by differentiation of MYB-independent iPSCs to macrophage-precursors and exposing them to hepatic cues to generate iPSC-derived KCs (iKCs). iKCs expressed macrophage markers (CD11/CD14/CD68/CD163/CD32) at 0.3–5 folds of primary adult human KCs (pKCs) and KC-specific CLEC-4F, ID1 and ID3. iKCs phagocytosed and secreted IL-6 and TNFα upon stimulation at levels similar to pKCs but different from non-liver macrophages. Hepatocyte-iKCs co-culture model was more sensitive in detecting hepatotoxicity induced by inflammation-associated drugs, Acetaminophen and Trovafloxacin, and Chlorpromazine-induced cholestasis when compared to hepatocytes alone. Overall, iKCs were mature, liver-specific and functional. Furthermore, donor-matched iKCs and iPSC-hepatocyte co-culture exhibited minimal non-specific background response compared to donor-mismatched counterpart. iKCs offer a mature renewable human cell source for liver-specific macrophages, useful in developing in vitro model to study DILI and liver diseases such as cholestasis.

The small molecule drug diminazene aceturate inhibits liver injury and biliary fibrosis in mice

There is no established medical therapy to treat biliary fibrosis resulting from chronic inflammation in the biliary tree. We have recently shown that liver-specific over-expression of angiotensin converting enzyme 2 (ACE2) of the renin angiotensin system (RAS) ameliorated liver fibrosis in mice. Diminazene aceturate (DIZE), a small molecule drug approved by the US Food and Drug Administration, which is used to treat human trypanosomiasis, has been shown to have antifibrotic properties by enhancing ACE2 activity. In this study we sought to determine the therapeutic potential of DIZE in biliary fibrosis using bile duct ligated and multiple drug resistant gene-2 knockout mice. Additionally, human hepatic stellate (LX-2) and mouse Kupffer (KUP5) cell lines were used to delineate intracellular pathways. DIZE treatment, both in vivo and in vitro, markedly inhibited the activation of fibroblastic stellate cells which was associated with a reduced activation of Kupffer cells. Moreover, DIZE-inhibited NOX enzyme assembly and ROS generation, activation of profibrotic transcription factors including p38, Erk1/2 and Smad2/3 proteins and proinflammatory and profibrotic cytokine release. These changes led to a major reduction in biliary fibrosis in both models without affecting liver ACE2 activity. We conclude that DIZE has a potential to treat biliary fibrosis.

Akt Isoforms in the Pathogenesis of Alcoholic Liver Disease

Alcoholic liver disease (ALD) represents a major public health problem with substantial morbidity and mortality. Chronic Ethanol/Binge/LPS (EBL) liver mouse model resembles a relevant “2‐hit” model of alcoholic liver disease. In this model, LPS is selected as the second hit agent because in the initial step EtOH increases gut permeability leading to generation of endotoxins that activate NFκB in Kupffer cells (KC), resulting in the production of proinflammatory cytokines; TNFα and IL1β. Moreover, in the hepatocytes, EtOH is oxidized to its toxic metabolite, acetaldehyde (ACE) that leads to the generation of reactive oxygen species and increased oxidative stress, resulting in the activation of hepatic stellate cells (HSC), which causes HSC proliferation, migration, and up regulation of the fibrogenic genes; PDGF β‐receptor, α‐SMA, extracellular matrix proteins and epigenetic repressor gene, methyl‐CpG binding protein. In contrast, adipogenic gene, PPARγ is suppressed resulting in the loss of vitamin A, and their trans‐differentiation from quiescent lipid storing cells to active myofibroblastic phenotype. Akt isoforms, the key phosphorylating kinases, seem to play vital roles in all aspects of this pathogenic process. Although Akt has been the subject of extensive investigations, very few studies have addressed the specific role/s of each isoform and it is generally assumed that the Akt isoforms play redundant and overlapping roles. This study proposes to identify what specific roles each of these isoforms of Akt plays in alcoholic liver injury utilizing both in in vitro human HSC and in vivo mouse models. Our results were as follows: In vitro: In the EtOH/LPS and/or ACE/LPS model of the independent cultures of human HSC, KC and VL17A hepatocytes, siRNA‐directed silencing of (A) Akt2, but not Akt1, significantly suppressed cell inflammatory markers in HSC and KC; (B) Akt1 and Akt2 inhibited cell proliferation in HSC; (C) Akt2 alone inhibited cell migration in HSC; (D) Akt1 and Akt2, but not Akt3 inhibited the fibrogenic markers in hepatocytes and HSC. Moreover, although Akt isoforms show extensive sequence similarity, they are localized in different subcellular compartments in normal HSC with their distinct functions. In vivo: EBL treatment in wild‐type C57BL/6 mice (a) stimulated all three isoforms of Akt (b) with concomitant increases in phosphorylated species of phosphoinositide‐dependent kinase‐1 and mammalian target of rapamycin complex 2, and phosphatidylinositol 3‐kinase, resulting in increases in inflammatory, proliferative, and fibrogenic genes. In addition, pharmacological blocking (c) of Akt2, but not Akt1, inhibited EBL‐induced inflammation; and (d) of Akt1 and Akt2 inhibited the fibrogenic markers. In conclusion, each isoform of Akt has a specific regulatory function in EBL‐mediated liver injury. Akt1 and Akt2 play crucial roles in EBL‐induced HSC fibrogenesis and proliferation; (ii) only Akt2 appears to be involved in inflammation and migration; and (iii) Akt3 may not play a significant role in EBL‐induced liver injury.Support or Funding InformationThanks to NIH/NIAAA‐funded researchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serum IgA Immune Complexes Promote Proinflammatory Cytokine Production by Human Macrophages, Monocytes, and Kupffer Cells through FcαRI–TLR Cross-Talk

IgA is predominantly recognized to play an important role in host defense at mucosal sites, where it prevents invasion of pathogens by neutralization. Although it has recently become clear that IgA also mediates other immunological processes, little remains known about the potential of IgA to actively contribute to induction of inflammation, particularly in nonmucosal organs and tissues. In this article, we provide evidence that immune complex formation of serum IgA plays an important role in orchestration of inflammation in response to pathogens at various nonmucosal sites by eliciting proinflammatory cytokines by human macrophages, monocytes, and Kupffer cells. We show that opsonization of bacteria with serum IgA induced cross-talk between FcαRI and different TLRs, leading to cell type-specific amplification of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-23. Furthermore, we demonstrate that the increased protein production of cytokines was regulated at the level of gene transcription, which was dependent on activation of kinases Syk and PI3K. Taken together, these data demonstrate that the immunological function of IgA is substantially more extensive than previously considered and suggest that serum IgA-induced inflammation plays an important role in orchestrating host defense by different cell types in nonmucosal tissues, including the liver, skin, and peripheral blood.

Immune microenvironment modulation via the interaction of human invariant natural killer T cell (iNKT cells) and Kupffer cells (KC), dendritic cells (DC) in colorectal liver metastasis

Abstract Aims To investigate the immune microenvironment modulation via the interaction between human iNKT cells and KC, DC in colorectal liver metastasis. Methods Different subtypes of iNKT cells, KC and DC from the core of tumor (CT), tumor invasive margin (IM), and adjacent normal liver tissue (NL) were analyzed by BD LSRFortessa™, TMA, and immunofluorescence(n=138). Ex vivo 3D co-culture, Quansys Multiplex array, and Elisa array were performed. Results In metastasis, CXCR6+iNKT positively correlated with better prognosis. In addition, iNKT cells were enriched at IM, especially iNKT2. Intriguingly, although there were few iNKT cells in CT, the iNKT/Lymphocytes rate increased to ~5 folds and expressed elevated IL9, IFNγ, and FasL compared to NL and IM. This implied the critical role of iNKT cells in tumor control. Both DC and KC could drive iNKT activation, the proportion of BDCA-3+DCs significantly increased from NL to CT; though they expressed higher CXCL16 compared to other subtypes, the capability of activating iNKT cells via CD1d was less strong than plasmacytoid DC(pDC). pDCs showed significantly elevated α-GalCer: CD1d complex formation in an ex vivo iNKT cells activation assay, which implied its critical role of activating capability of pDC. However, pDCs, as well as BDCA-1+DCs, decreased from NL to CT. In this study, KC was found also activating iNKT cells. Interestingly, iNKT expressed Th2 cytokines when activated by KC, however, Th1 biased response could be found when activated with DC. In addition, KC subsets also differed with location. Furthermore, transmembrane CD1d density and intra/tmCXCL16 expression, Th1/2/17 biased immune responses of KC and DC were associated with the lipid metabolism preferences of these cells.

Nucleic acid-based polymers effective against hepatitis B Virus infection in patients don\u2019t harbor immunostimulatory properties in primary isolated liver cells

Nucleic acid polymers (NAPs) block the release of subviral particles from hepatocytes, a mechanism consistent with their antiviral activity against hepatitis B virus (HBV) in patients. Analysis of immunostimulatory properties of NAPs were conducted with several NAP species: REP 2006, the prototypic degenerate NAP [dN]40, containing TLR9-stimulatory CpG; REP 2055 a clinically active NAP with a sequence [dAdC]20 devoid of CpG content; REP 2139 (also clinically active) and REP 2165 (REP 2055 analogues further rendered immunologically inactive by replacing cytidine with 5-methylcytidine and incorporating 2′-O methylation of riboses). These analyses revealed pro-inflammatory responses in human peripheral blood mononuclear cells with REP 2006 and with REP 2139 and REP 2165 only at high dose but displayed no significant antiviral activity. In primary isolated human hepatocytes and liver sinusoidal endothelial cells no significant inflammatory or antiviral responses were detected for any NAPs. In human Kupffer cells pro-inflammatory activity was observed with REP 2006 and REP 2055, whereas a weak but significant induction of interferon genes was only observed with REP 2006 at the highest concentration. We therefore hypothesize that the antiviral activity of NAPs optimized to treat HBV infection in patients cannot be explained by direct induction of innate antiviral responses.

Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-β/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

Modeling Therapeutic Antibody-Small Molecule Drug-Drug Interactions Using a Three-Dimensional Perfusable Human Liver Coculture Platform.

Traditional in vitro human liver cell culture models lose key hepatic functions such as metabolic activity during short-term culture. Advanced three-dimensional (3D) liver coculture platforms offer the potential for extended hepatocyte functionality and allow for the study of more complex biologic interactions, which can improve and refine human drug safety evaluations. Here, we use a perfusion flow 3D microreactor platform for the coculture of cryopreserved primary human hepatocytes and Kupffer cells to study the regulation of cytochrome P450 3A4 isoform (CYP3A4) activity by chronic interleukin 6 (IL-6)-mediated inflammation over 2 weeks. Hepatocyte cultures remained stable over 2 weeks, with consistent albumin production and basal IL-6 levels. Direct IL-6 stimulation that mimics an inflammatory state induced a dose-dependent suppression of CYP3A4 activity, an increase in C-reactive protein (CRP) secretion, and a decrease in shed soluble interleukin-6 receptor (IL-6R) levels, indicating expected hepatic IL-6 bioactivity. Tocilizumab, an anti-IL-6R monoclonal antibody used to treat rheumatoid arthritis, has been demonstrated clinically to impact small molecule drug pharmacokinetics by modulating cytochrome P450 enzyme activities, an effect not observed in traditional hepatic cultures. We have now recapitulated the clinical observation in a 3D bioreactor system. Tocilizumab was shown to desuppress CYP3A4 activity while reducing the CRP concentration after 72 hours in the continued presence of IL-6. This change in CYP3A4 activity decreased the half-life and area under the curve up to the last measurable concentration (AUClast) of the small molecule CYP3A4 substrate simvastatin hydroxy acid, measured before and after tocilizumab treatment. We conclude that next-generation in vitro liver culture platforms are well suited for these types of long-term treatment studies and show promise for improved drug safety assessment.

Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens.

The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1β and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1β and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1β and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.

P102: Antiviral activity of human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells against Hepatitis C virus is restricted to toll‐like receptor 3 stimulation and involves type I interferons

Journal of Viral HepatitisVolume 22, Issue S2 p. 71-72 Poster PresentationFree Access P102: Antiviral activity of human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells against Hepatitis C virus is restricted to toll-like receptor 3 stimulation and involves type I interferons First published: 25 June 2015 https://doi.org/10.1111/jvh.96_12425AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume22, IssueS2Special Issue: 15th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 26th to 28th, 2015, in Berlin, GermanyJune 2015Pages 71-72 RelatedInformation

Hepatitis C Virus Induces Interleukin-1β (IL-1β)/IL-18 in Circulatory and Resident Liver Macrophages

Hepatitis C virus (HCV)-mediated chronic liver disease is a global health problem, and inflammation is believed to be an important player in disease pathogenesis. HCV infection often leads to severe fibrosis/cirrhosis and hepatocellular carcinoma, although the mechanisms for advancement of disease are not fully understood. The proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 have critical roles in establishment of inflammation. In this study, we examined induction of IL-1β/IL-18 secretion following HCV infection. Our results demonstrated that monocyte-derived human macrophages (THP-1) incubated with cell culture-grown HCV enhance the secretion of IL-1β/IL-18 into culture supernatants. A similar cytokine release was also observed for peripheral blood mononuclear cell (PBMC)-derived primary human macrophages and Kupffer cells (liver-resident macrophages) upon incubation with HCV. THP-1 cells incubated with HCV led to caspase-1 activation and release of proinflammatory cytokines. Subsequent studies demonstrated that HCV induces pro-IL-1β and pro-IL-18 synthesis via the NF-κB signaling pathway in macrophages. Furthermore, introduction of HCV viroporin p7 RNA into THP-1 cells was sufficient to cause IL-1β secretion. Together, our results suggested that human macrophages exposed to HCV induce IL-1β and IL-18 secretion, which may play a role in hepatic inflammation.

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism.

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.

Adiponectin upregulates hepatocyte CMKLR1 which is reduced in human fatty liver

Objective: Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. Methods: Primary human hepatocytes and liver of patients with and without liver steatosis were used. Human liver tissue was obtained according to the guidelines of the charitable state-controlled foundation Human Tissue & Cell Research (HTCR) with the patient's informed consent. To induce NASH mice were fed a methionine-choline deficient diet for 6 weeks. Results: CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human fatty liver and in fibrotic liver of mice fed a methionine-choline deficient diet CMKLR1 protein was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analysed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL–6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFbeta tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein, and CMKLR1 was suppressed in the liver of adiponectin deficient mice. Conclusion: These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.

Adiponectin upregulates hepatocyte CMKLR1 which is reduced in human fatty liver

Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human and rodent fatty liver and in fibrotic liver of mice fed a methionine–choline deficient diet CMKLR1 was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analyzed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL-6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFβ tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein in PHH and hepatic CMKLR1 when injected into wild type mice. Further, CMKLR1 was suppressed in the liver of adiponectin deficient mice. These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.

Subcellular localization and regulation of StarD4 protein in macrophages and fibroblasts

StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.

Differential cytokine responses from primary human Kupffer cells following infection with wild-type or vaccine strain yellow fever virus

Wild-type yellow fever virus (YFV) infections result in a hepatotropic disease which is often fatal, while vaccination with the live-attenuated 17-D strain results in productive infection yet is well-tolerated with few adverse events. Kupffer cells (KCs) are resident liver macrophages that have a significant role in pathogen detection, clearance and immune signaling. Although KCs appear to be an important component of YF disease, their role has been under-studied. This study examined cytokine responses in KCs following infection with either wild-type or vaccine strains of YFV. Results indicate that KCs support replication of both wild-type and vaccine strains, yet wild-type YFV induced a prominent and prolonged pro-inflammatory cytokine response (IL-8, TNF-α and RANTES/CCL5) with little control by a major anti-inflammatory cytokine (IL-10). This response was significantly reduced in vaccine strain infections. These data suggest that a differentially regulated infection in KCs may play a critical role in development of disease.

The influence of a 6 mT static magnetic field on apoptotic cell phagocytosis depends on monocyte/macrophage differentiation

In a previous work we showed that a 6 mT static magnetic field (SMF) interferes with monocyte/macrophage 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of promonocytes (U937 cells) and monocytes (THP-1 cells). In this study we investigated whether in the same cells and under the same conditions, phagocytosis of apoptotic cells is influenced by 6 mT SMF exposure. Fluid phase endocytosis and phagocytosis of latex particles were also analyzed for comparison. The results indicate that SMF exposure has effects on phagocytosis but not on fluid phase endocytosis, and that these effects are greater at the late stages of macrophage differentiation (THP-1 > U937 cells). The phagocytosis index and rate of phagocytosis decreased under SMF exposure while the number of latex particles bound to the plasma membrane of TPA-differentiated U937 and THP-1 cells increased. Conversely, the rate of phagocytosis of apoptotic cells increased under SMF exposure, while the number of apoptotic cells bound to the plasma membrane of isolated human Kupffer cells, Raw 264.7 macrophages and TPA-differentiated THP-1 and U937 cells decreased. In non-differentiated U937 and THP-1 cells, the SMF exposure enhanced the number of cell-surface bound apoptotic cells and latex beads.