Liver sinusoidal cells eliminate blood-borne phage K1F.

Phage treatment has regained attention due to an increase in multiresistant bacteria. For phage therapy to be successful, phages must reach their target bacteria in sufficiently high numbers. Blood-borne phages are believed to be captured by macrophages in the liver and spleen. Since liver sinusoids also consist of specialized scavenger liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), this study investigated the contribution of both cell types in the elimination of Escherichia coli phage K1Fg10b::gfp (K1Fgfp) in mice. Circulatory half-life, organ, and hepatocellular distribution of K1Fgfp were determined following intravenous administration. Internalization of K1Fgfp and effects of phage opsonization on uptake were explored using primary mouse and human LSEC and KC cultures. When inoculated with 107 virions, >95% of the total K1Fgfp load was eliminated from the blood within 20 min, and 94% of the total retrieved K1Fgfp was localized to the liver. Higher doses resulted in slower elimination, possibly reflecting temporary saturation of liver scavenging capacity. Phage DNA was detected in both cell types, with a KC:LSEC ratio of 12:1 per population following cell isolation. Opsonization with plasma proteins increased time-dependent cellular uptake in both LSECs and KCs in vitro. Internalized phages were rapidly transported along the endocytic pathway to lysosomal compartments. Reduced viability of intracellular K1Fgfp corroborated inactivation following endocytosis. This study is the first to identify phage distribution in the liver at the hepatocellular level, confirming clearance of K1Fgfp performed mostly by KCs with a significant uptake also in LSECs.IMPORTANCEFaced with the increasing amounts of bacteria with multidrug antimicrobial resistance, phage therapy has regained attention as a possible treatment option. The phage field has recently experienced an emergence in commercial interest as research has identified new and more efficient ways of identifying and matching phages against resistant superbugs. Currently, phages are unapproved drugs in most parts of the world. For phages to reach broad clinical use, they must be shown to be clinically safe and useful. The results presented herein contribute to increased knowledge about the pharmacokinetics of the T7-like phage K1F in the mammalian system. The cell types of the liver that are responsible for rapid phage blood clearance are identified. Our results highlight the need for more research about appropriate dose regimens when phage therapy is delivered intravenously and advise essential knowledge about cell systems that should be investigated further for detailed phage pharmacodynamics.

Infection of liver sinusoidal endothelial cells with Muromegalovirus muridbeta1 involves binding to neuropilin-1 and is dynamin-dependent.

Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles, including some viruses. Endocytosis in LSEC is mainly via the clathrin-coated pit mediated route, which is dynamin-dependent. LSEC can also be a site of infection and latency of betaherpesvirus, but mode of virus entry into these cells has not yet been described. In this study we have investigated the role of dynamin in the early stage of muromegalovirus muridbeta1 (MuHV-1, murid betaherpesvirus 1, murine cytomegalovirus) infection in mouse LSECs. LSEC cultures were freshly prepared from C57Bl/6JRj mouse liver. We first examined dose- and time-dependent effects of two dynamin-inhibitors, dynasore and MitMAB, on cell viability, morphology, and endocytosis of model ligands via different LSEC scavenger receptors to establish a protocol for dynamin-inhibition studies in these primary cells. LSECs were challenged with MuHV-1 (MOI 0.2) ± dynamin inhibitors for 1h, then without inhibitors and virus for 11h, and nuclear expression of MuHV-1 immediate early antigen (IE1) measured by immune fluorescence. MuHV-1 efficiently infected LSECs in vitro. Infection was significantly and independently inhibited by dynasore and MitMAB, which block dynamin function via different mechanisms, suggesting that initial steps of MuHV-1 infection is dynamin-dependent in LSECs. Infection was also reduced in the presence of monensin which inhibits acidification of endosomes. Furthermore, competitive binding studies with a neuropilin-1 antibody blocked LSEC infection. This suggests that MuHV-1 infection in mouse LSECs involves virus binding to neuropilin-1 and occurs via endocytosis.

Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis.

Liver sinusoidal endothelial cells (LSECs) are fenestrated endothelial cells with a unique, high endocytic clearance capacity for blood-borne waste macromolecules and colloids. This LSEC scavenger function has been insufficiently characterized in liver disease. The Glmpgt/gt mouse lacks expression of a subunit of the MFSD1/GLMP lysosomal membrane protein transporter complex, is born normal, but soon develops chronic, mild hepatocyte injury, leading to slowly progressing periportal liver fibrosis, and splenomegaly. This study examined how LSEC scavenger function and morphology are affected in the Glmpgt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmpgt/gt mice, aged 4 months (peak of liver inflammation), 9-10 month, and age-matched Glmpwt/wt mice. Organs were harvested for light and electron microscopy, quantitative image analysis of ligand uptake, collagen accumulation, LSEC ultrastructure, and endocytosis receptor expression (also examined by qPCR and western blot). In both age groups, the Glmpgt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmpgt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmpgt/gt mice and age-matched controls. At the same time, immunostaining revealed differences in the stabilin-1 expression pattern in sinusoids and accumulation of stabilin-1-positive macrophages in Glmpgt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmpgt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmpgt/gt mice showed well-preserved fenestrae organized in sieve plates but the frequency of holes >400 nm in diameter was increased, especially in areas with hepatocyte damage. In both genotypes, FITC-FSA also distributed to endothelial cells of spleen and bone marrow sinusoids, suggesting that these locations may function as possible compensatory sites of clearance of blood-borne scavenger receptor ligands in liver fibrosis.

Highly oxidized albumin is cleared by liver sinusoidal endothelial cells via the receptors stabilin-1 and -2.

Oxidized albumin (oxHSA) is elevated in several pathological conditions, such as decompensated cirrhosis, acute on chronic liver failure and liver mediated renal failure. Patient derived oxidized albumin was previously shown to be an inflammatory mediator, and in normal serum levels of oxHSA are low. The removal from circulation of oxidized albumins is therefore likely required for maintenance of homeostasis. Liver sinusoidal endothelial cells (LSEC) are prominent scavenger cells specialized in removal of macromolecular waste. Given that oxidized albumin is mainly cleared by the liver, we hypothesized the LSEC are the site of uptake in the liver. In vivo oxHSA was cleared rapidly by the liver and distributed to mainly the LSEC. In in vitro studies LSEC endocytosed oxHSA much more than other cell populations isolated from the liver. Furthermore, it was shown that the uptake was mediated by the stabilins, by affinity chromatography-mass spectrometry, inhibiting uptake in LSEC with other stabilin ligands and showing uptake in HEK cells overexpressing stabilin-1 or -2. oxHSA also inhibited the uptake of other stabilin ligands, and a 2-h challenge with 100 µg/mL oxHSA reduced LSEC endocytosis by 60% up to 12 h after. Thus the LSEC and their stabilins mediate clearance of highly oxidized albumin, and oxidized albumin can downregulate their endocytic capacity in turn.

Changes in the proteome and secretome of rat liver sinusoidal endothelial cells during early primary culture and effects of dexamethasone.

INTRODUCTION: Liver sinusoidal endothelial cells (LSECs) are specialized fenestrated scavenger endothelial cells involved in the elimination of modified plasma proteins and tissue turnover waste macromolecules from blood. LSECs also participate in liver immune responses. A challenge when studying LSEC biology is the rapid loss of the in vivo phenotype in culture. In this study, we have examined biological processes and pathways affected during early-stage primary culture of rat LSECs and checked for cell responses to the pro-inflammatory cytokine interleukin (IL)-1ß and the anti-inflammatory drug dexamethasone. METHODS: LSECs from male Sprague Dawley rats were cultured on type I collagen in 5% oxygen atmosphere in DMEM with serum-free supplements for 2 and 24 h. Quantitative proteomics using tandem mass tag technology was used to examine proteins in cells and supernatants. Validation was done with qPCR, ELISA, multiplex immunoassay, and caspase 3/7 assay. Cell ultrastructure was examined by scanning electron microscopy, and scavenger function by quantitative endocytosis assays. RESULTS: LSECs cultured for 24 h showed a characteristic pro-inflammatory phenotype both in the presence and absence of IL-1ß, with upregulation of cellular responses to cytokines and interferon-γ, cell-cell adhesion, and glycolysis, increased expression of fatty acid binding proteins (FABP4, FABP5), and downregulation of several membrane receptors (STAB1, STAB2, LYVE1, CLEC4G) and proteins in pyruvate metabolism, citric acid cycle, fatty acid elongation, amino acid metabolism, and oxidation-reduction processes. Dexamethasone inhibited apoptosis and improved LSEC viability in culture, repressed inflammatory and immune regulatory pathways and secretion of IL-1ß and IL-6, and further upregulated FABP4 and FABP5 compared to time-matched controls. The LSEC porosity and endocytic activity were reduced at 24 h both with and without dexamethasone but the dexamethasone-treated cells showed a less stressed phenotype. CONCLUSION: Rat LSECs become activated towards a pro-inflammatory phenotype during early culture. Dexamethasone represses LSEC activation, inhibits apoptosis, and improves cell viability.

Autofluorescence in freshly isolated adult human liver sinusoidal cells.

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of fluorophores, especially when labelling of live cells where the quencher is not compatible.

Transcriptome and proteome profiling reveal complementary scavenger and immune features of rat liver sinusoidal endothelial cells and liver macrophages.

BACKGROUND: Liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body's most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics. RESULTS: We observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation. CONCLUSIONS: Our findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.

Dose- and time-dependent effects of triethylene glycol dimethacrylate on the proteome of human THP-1 monocytes.

Triethylene glycol dimethacrylate (TEGDMA) is commonly used in polymer resin-based dental materials. This study investigated the molecular mechanisms of TEGDMA toxicity by identifying its time- and dose-dependent effects on the proteome of human THP-1 monocytes. The effects of different concentrations (0.07-5 mM) and exposure times (0-72 h) of TEGDMA on cell viability, proliferation, and morphology were determined using a real-time viability assay, automated cell counting, and electron microscopy, and laid the fundament for choice of exposure scenarios in the proteomic experiments. Solvents were not used, as TEGDMA is soluble in cell culture medium (determined by photon correlation spectroscopy). Cells were metabolically labeled [using the stable isotope labeled amino acids in cell culture (SILAC) strategy], and exposed to 0, 0.3 or 2.5 mM TEGDMA for 6 or 16 h before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Regulated proteins were analyzed in the STRING database. Cells exposed to 0.3 mM TEGDMA showed increased viability and time-dependent upregulation of proteins associated with stress/oxidative stress, autophagy, and cytoprotective functions. Cells exposed to 2.5 mM TEGDMA showed diminished viability and a protein expression profile associated with oxidative stress, DNA damage, mitochondrial dysfunction, and cell cycle inhibition. Altered expression of immune genes was observed in both groups. The study provides novel knowledge about TEGDMA toxicity at the proteomic level. Of note, even low doses of TEGDMA induced a substantial cellular response.

Iterative Design and in Vivo Evaluation of an Oncolytic Antilymphoma Peptide.

Oncolytic peptides represent a promising new strategy within the field of cancer immunotherapy. Here we describe the systematic design and evaluation of short antilymphoma peptides within this paradigm. The peptides were tested in vitro and in vivo to identify a lead compound for further evaluation as novel oncolytic immunotherapeutic. In vitro tests revealed peptides with high activity against several lymphoma types and low cytotoxicity toward normal cells. Treated lymphoma cells exhibited a reduced mitochondrial membrane potential that resulted in an irreversible disintegration of their plasma membranes. No caspase activation or ultrastructural features of apoptotic cell death were observed. One of these peptides, 11, was shown to induce complete tumor regression and protective immunity following intralesional treatment of murine A20 B-lymphomas. Due to its selectivity for lymphoma cells and its ability to induce tumor-specific immune responses, 11 has the potential to be used in intralesional treatment of accessible lymphoma tumors.

Methods and terminology used in cell-culture studies of low-dose effects of matrix constituents of polymer resin-based dental materials.

General comprehension of terms and confounding factors associated with in vitro experiments can maximize the potential of in vitro testing of substances. In this systematic review, we present an overview of the terms and methods used to determine low-dose effects of matrix constituents in polymer resin-based dental materials in cell-culture studies and discuss the findings in light of how they may influence the comprehension and interpretation of results. Articles published between 1996 and 2015 were identified by searches in the Scopus, Web of Science, MEDLINE, PubMed, and Embase databases using keywords associated with low-dose effects, polymer resin-based materials, in vitro parameters, and dental materials. Twenty-nine articles were included. Subtoxic (n = 11), sublethal (n = 10), and nontoxic (n = 6) were the terms most commonly used to describe the low-dose effects of methacrylates. However, definition of terms varied. Most (82%) studies employed only one method to define the exposure scenario, and no agreement was seen between studies on the use of solvents. Prophylactic use of antibiotics was widespread, and mycoplasma screening was not reported. In conclusion, cell-culture conditions and tests used to define exposure scenarios have changed little in the last decades, despite development in recommendations. Nomenclature alignment is needed for a better understanding of possible biohazards of methacrylates.

Liver Sinusoidal Endothelial Cells.

The liver sinusoidal endothelial cell (LSEC) forms the fenestrated wall of the hepatic sinusoid and functions as a control post regulating and surveying the trafficking of molecules and cells between the liver parenchyma and the blood. The cell acts as a scavenger cell responsible for removal of potential dangerous macromolecules from blood, and is increasingly acknowledged as an important player in liver immunity. This review provides an update of the major functions of the LSEC, including its role in plasma ultrafiltration and regulation of the hepatic microcirculation, scavenger functions, immune functions, and role in liver aging, as well as issues that are either undercommunicated or confusingly dealt with in the literature. These include metabolic functions, including energy metabolic interplay between the LSEC and the hepatocyte, and adequate ways of identifying and distinguishing the cells.

Efficient uptake of blood-borne BK and JC polyomavirus-like particles in endothelial cells of liver sinusoids and renal vasa recta.

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells that mediate high-capacity clearance of soluble waste macromolecules and colloid material, including blood-borne adenovirus. To explore if LSECs function as a sink for other viruses in blood, we studied the fate of virus-like particles (VLPs) of two ubiquitous human DNA viruses, BK and JC polyomavirus, in mice. Like complete virions, VLPs specifically bind to receptors and enter cells, but unlike complete virions, they cannot replicate. 125I-labeled VLPs were used to assess blood decay, organ-, and hepatocellular distribution of ligand, and non-labeled VLPs to examine cellular uptake by immunohisto- and -cytochemistry. BK- and JC-VLPs rapidly distributed to liver, with lesser uptake in kidney and spleen. Liver uptake was predominantly in LSECs. Blood half-life (∼1 min), and tissue distribution of JC-VLPs and two JC-VLP-mutants (L55F and S269F) that lack sialic acid binding affinity, were similar, indicating involvement of non-sialic acid receptors in cellular uptake. Liver uptake was not mediated by scavenger receptors. In spleen, the VLPs localized to the red pulp marginal zone reticuloendothelium, and in kidney to the endothelial lining of vasa recta segments, and the transitional epithelium of renal pelvis. Most VLP-positive vessels in renal medulla did not express PV-1/Meca 32, suggesting location to the non-fenestrated part of vasa recta. The endothelial cells of these vessels also efficiently endocytosed a scavenger receptor ligand, formaldehyde-denatured albumin, suggesting high endocytic activity compared to other renal endothelia. We conclude that LSECs very effectively cleared a large fraction of blood-borne BK- and JC-VLPs, indicating a central role of these cells in early removal of polyomavirus from the circulation. In addition, we report the novel finding that a subpopulation of endothelial cells in kidney, the main organ of polyomavirus persistence, showed selective and rapid uptake of VLPs, suggesting a role in viremic organ tropism.

Autocrine prostaglandin E2 signaling promotes tumor cell survival and proliferation in childhood neuroblastoma.

BACKGROUND: Prostaglandin E(2) (PGE(2)) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE(2) production, the expression pattern and localization of PGE(2) receptors and intracellular signal transduction pathways activated by PGE(2). PRINCIPAL FINDINGS: A high expression of the PGE(2) receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE(2) and stimulation of serum-starved neuroblastoma cells with PGE(2) increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE(2) (dmPGE(2)) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE(2). Similarly, PGE(2) receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner. CONCLUSIONS: These findings demonstrate that PGE(2) acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE(2) signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.

Age-related changes in scavenger receptor-mediated endocytosis in rat liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) play an essential role in systemic waste clearance by effective endocytosis of blood-borne waste macromolecules. We aimed to study LSECs' scavenger function during aging, and whether age-related morphological changes (eg, defenestration) affect this function, in F344/BN F1 rats. Endocytosis of the scavenger receptor ligand formaldehyde-treated serum albumin was significantly reduced in LSECs from old rats. Ligand degradation, LSEC protein expression of the major scavenger receptors for formaldehyde-treated serum albumin endocytosis, stabilin-1 and stabilin-2, and their staining patterns along liver sinusoids, was similar at young and old age, suggesting that other parts of the endocytic machinery are affected by aging. Formaldehyde-treated serum albumin uptake per cell, and cell porosity evaluated by electron microscopy, was not correlated, indicating that LSEC defenestration is not linked to impaired endocytosis. We report a significantly reduced LSEC endocytic capacity at old age, which may be especially important in situations with increased circulatory waste loads.

Liver sinusoidal endothelial cells depend on mannose receptor-mediated recruitment of lysosomal enzymes for normal degradation capacity.

UNLABELLED: Liver sinusoidal endothelial cells (LSECs) are largely responsible for the removal of circulating lysosomal enzymes (LE) via mannose receptor (MR)-mediated endocytosis. We hypothesized that LSECs rely on this uptake to maintain their extraordinarily high degradation capacity for other endocytosed material. Circulatory half-life studies of (125)I-cathepsin-D in MR knockout (MR(-/-)) and wild-type mice, and endocytosis studies in LSEC cultures, showed a total dependence on the MR for effective clearance of cathepsin-D. Radioiodinated formaldehyde-treated serum albumin, a ligand for the LSEC scavenger receptors, was used to study catabolism of endocytosed material in MR(-/-) and wild-type mice. The plasma clearance, liver uptake, and the starting point for release of degradation products to blood, were similar in both experimental groups, indicating normal endocytosis and intracellular transport of scavenger receptor ligands in MR(-/-) mice. However, the rate of formaldehyde-treated serum albumin catabolism in the liver of the MR deficient animals was reduced to approximately 50% of wild-type values. A similar reduction in intracellular degradation was recorded in LSEC cultures from MR(-/-) mice compared to wild-type controls. In accordance with this, MR(-/-) LSECs had markedly and significantly reduced enzyme activities for four out of five LE tested, i.e., cathepsin-D, alpha-mannosidase, beta-hexosaminidase and arylsulfatase, but not acid phosphatase, compared to wild-type controls. Immunoblot analysis showed that the content of pro-cathepsin-D relative to total cathepsin-D in wild-type LSECs was less than one-fifth of that in hepatocytes, indicating lower endogenous LE production in the LSECs. CONCLUSION: We show for the first time that LSEC depend on MR-mediated recruitment of LE from their surroundings for effective catabolism of endocytosed macromolecules.

Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides.

BACKGROUND/AIMS: Bacterial DNA and synthetic oligonucleotides containing unmethylated motifs have become the focus of many studies due to their ability to activate cells of the innate immune system through interaction with Toll-like receptor 9 (TLR9). This study was undertaken to investigate if and how CpG-oligonucleotides (CpGs) activate liver sinusoidal endothelial cells (LSECs), known to be the main site of clearance of DNA-oligonucleotides from the circulation. METHODS: Expression of TLR9 was analyzed by RT-PCR and immunohistochemistry. Production of IL-1beta and IL-6 was measured by ELISA. RESULTS: Here we show for the first time that mouse LSECs express TLR9 mRNA and protein. Moreover, our findings suggest that CpGs are first taken up by LSECs by scavenger receptor(s)-mediated endocytosis, and then join TLR9 in the lysosomal compartments. Furthermore, we found that uptake of CpGs in LSECs results in the activation of transcription factor NF-kappaB and secretion of IL-1beta and IL-6. CONCLUSIONS: The presence of functional TLR9 in LSECs emphasizes the importance of these cells in the innate defense mechanisms of the liver.