Karen Kristine Sørensen

Liver sinusoidal endothelial cells (LSECs) which make up the fenestrated wall of the hepatic sinusoids, are active scavenger cells involved in blood waste clearance and liver immune functions. Dexamethasone is a synthetic glucocorticoid commonly used in the clinic and as cell culture supplement. However, the response is dependent on tissue, cell type, and cell state. The aim of this study was to investigate the effect of dexamethasone on primary mouse LSECs (C57BL/6J); their viability (live-dead, LDH release, caspase 3/7 assays), morphology (scanning electron microscopy), release of inflammatory markers (ELISA), and scavenging functions (endocytosis assays), and associated biological processes and pathways. We have characterized and catalogued the proteome of LSECs cultured for 1, 10, or 48 h to elucidate time-dependent and dexamethasone-specific cell responses. More than 6,000 protein IDs were quantified using tandem mass tag technology and advanced mass spectrometry (synchronous precursor selection multi-notch MS3). Enrichment analysis showed a culture-induced upregulation of stress and inflammatory markers, and a significant shift in cell metabolism already at 10 h, with enhancement of glycolysis and concomitant repression of oxidative phosphorylation. At 48 h, changes in metabolic pathways were more pronounced with dexamethasone compared to time-matched controls. Dexamethasone repressed the activation of inflammatory pathways (IFN-gamma response, TNF-alpha signaling via NF-kB, Cell adhesion molecules), and culture-induced release of interleukin-6, VCAM-1, and ICAM-1, and improved cell viability partly through inhibition of apoptosis. The mouse LSECs did not proliferate in culture. Dexamethasone treated cells showed upregulation of xanthine dehydrogenase/oxidase (Xdh), and the transcription regulator Foxo1. The drug further delayed but did not block the culture-induced loss of LSEC fenestration. The LSEC capacity for endocytosis was significantly reduced at 48 h, independent of dexamethasone, which correlated with diminished expression of several scavenger receptors and C-type lectins and altered expression of proteins in the endocytic machinery. The glucocorticoid receptor (NR3C1) was suppressed by dexamethasone at 48 h, suggesting limited effect of the drug in prolonged LSEC culture. Conclusion: The study presents a detailed overview of biological processes and pathways affected by dexamethasone in mouse LSECs in vitro.

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 (K1F ^gfp ) in mice. Circulatory half-life, organ, and hepatocellular distribution of K1F ^gfp were determined following intravenous administration. Internalization of K1F ^gfp and effects of phage opsonization on uptake were explored using primary mouse and human LSEC and KC cultures. When inoculated with 10 ⁷ virions, >95% of the total K1F ^gfp load was eliminated from the blood within 20 min, and 94% of the total retrieved K1F ^gfp 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 K1F ^gfp corroborated inactivation following endocytosis. This study is the first to identify phage distribution in the liver at the hepatocellular level, confirming clearance of K1F ^gfp performed mostly by KCs with a significant uptake also in LSECs. IMPORTANCE: Faced 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.

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 Glmp^gt/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 Glmp^gt/gt model. FITC-labelled formaldehyde-treated serum albumin (FITC-FSA), a model ligand for LSEC scavenger receptors was administered intravenously into Glmp^gt/gt mice, aged 4 months (peak of liver inflammation), 9–10 month, and age-matched Glmp^wt/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 Glmp^gt/gt mice showed multifocal liver injury and fibrosis. The uptake of FITC-FSA in LSECs was significantly reduced in Glmp^gt/gt compared to wild-type mice. Expression of LSEC receptors stabilin-1 (Stab1), and mannose receptor (Mcr1) was almost similar in liver of Glmp^gt/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 Glmp^gt/gt liver. FcγRIIb (Fcgr2b), which mediates LSEC endocytosis of soluble immune complexes was widely and significantly downregulated in Glmp^gt/gt liver. Despite increased collagen in space of Disse, LSECs of Glmp^gt/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.

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)-1β 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-1β, 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-1β 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.

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.

Infectious keratoconjunctivitis (IKC) is a common transmissible ocular disease in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus). In large outbreaks, IKC may affect tens of animals in a herd, with the most severe cases often requiring euthanasia due to the destruction of the affected eyes and permanent blindness. An experimental inoculation with cervid herpesvirus 2 (CvHV2), alone or in combination with Moraxella bovoculi, demonstrated that CvHV2 has the ability to cause clinical signs of IKC in previously unexposed reindeer. Tissues collected from upper and lower eyelids, lacrimal gland and cornea, were processed for light and transmission electron microscopy. Histopathological analysis of the eyes inoculated with CvHV2 showed widespread and severe pathological findings. Mucosal tissues from these eyes showed fibrinous and purulent exudates, hyperemia, hemorrhages, necrosis, vascular thrombosis, vascular necrosis, infiltration of mononuclear cells and neutrophils, and lymphoid follicle reaction, which matches the described histopathology of IKC in reindeer. Characteristic alpha-herpesvirus particles matching the size and morphology of CvHV2 were identified by transmission electron microscopy in the conjunctival tissue. The quantification of viral particles by qPCR revealed high copy numbers of viral DNA in all CvHV2 inoculated eyes, but also in the non-inoculated eyes of the same animals. The histopathology of eye tissues obtained from the CvHV2 inoculated reindeer and the lack of inflammation from bacterial infection, together with the detection of CvHV2 DNA in swabs from the inoculated and non-inoculated eyes of the same animals, verified that CvHV2 was the primary cause of the observed histopathological changes.

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.

Infectious keratoconjunctivitis (IKC) is a transmissible disease in semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus). It is regarded as multifactorial and a single causative pathogen has not yet been identified. From clinical outbreaks we have previously identified Cervid herpesvirus 2 (CvHV2) and Moraxella bovoculi as candidates for experimental investigations. Eighteen reindeer were inoculated in the right eye with CvHV2 (n = 5), M. bovoculi (n = 5), CvHV2 and M. bovoculi (n = 5) or sterile saline water (n = 3; controls). All animals inoculated with CvHv2, alone or in combination with M. bovoculi, showed raised body temperature, increased lacrimation, conjunctivitis, excretion of pus and periorbital oedema; clinical signs that increased in severity from day 2 post inoculation (p.i.) and throughout the experiment, until euthanasia 5–7 days p.i. Examination after euthanasia revealed corneal oedema, and three animals displayed a corneal ulcer. CvHV2 could be identified in swab samples from both the inoculated eye and the control eye from most animals and time points, indicating a viral spread from the inoculation site. This study showed that CvHV2 alone and in combination with M. bovoculi was able to cause the characteristic clinical signs of IKC in reindeer, whereas inoculation of M. bovoculi alone, originally isolated from a reindeer with IKC, did not produce clinical signs. Previous studies have suggested that herding procedures, animal stress and subsequent reactivation of latent CvHV2 infection in older animals is a plausible mechanism for IKC outbreaks among reindeer calves and young animals in reindeer herds. However, further studies are needed to fully understand the infection biology and epidemiology associated with IKC in reindeer.

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.