Histidine-rich glycoprotein promotes macrophage activation and inflammation in chronic liver disease.

UNLABELLED: Pathogen- and injury-related danger signals as well as cytokines released by immune cells influence the functional differentiation of macrophages in chronic inflammation. Recently, the liver-derived plasma protein, histidine-rich glycoprotein (HRG), was demonstrated, in mouse tumor models, to mediate the transition of alternatively activated (M2) to proinflammatory (M1) macrophages, which limit tumor growth and metastasis. We hypothesized that liver-derived HRG is a critical endogenous modulator of hepatic macrophage functionality and investigated its implications for liver inflammation and fibrosis by comparing C57BL/6N wild-type (WT) and Hrg(-/-) mice. In homeostatic conditions, hepatic macrophages were overall reduced and preferentially polarized toward the anti-inflammatory M2 subtype in Hrg(-/-) mice. Upon chronic liver damage induced by CCl4 or methionine-choline-deficient (MCD) diet, liver injury and fibrosis were attenuated in Hrg(-/-) , compared to WT, mice. Macrophage populations were reduced and skewed toward M2 polarization in injured livers of Hrg(-/-) mice. Moreover, HRG-deficient mice showed significantly enhanced hepatic vascularization by micro-computed tomography and histology, corroborating proangiogenic activities of M2-polarized liver macrophages. Purified HRG protein induced, but HRG-deficient serum prevented, M1 macrophage differentiation in vitro. Accordingly, Hrg(-/-) mice transplanted with Hrg(+/+) bone marrow, but not Hrg(-/-) -transplanted Hrg(+/+) mice, remained protected from experimental steatohepatitis. Consistent with these findings, patients with chronic hepatitis C and nonalcoholic steatohepatitis significantly up-regulated hepatocytic HRG expression, which was associated with M1 polarization of adjacent macrophages. CONCLUSIONS: Liver-derived HRG, similar to alarmins, appears to be an endogenous molecular factor promoting polarization of hepatic macrophages toward the M1 phenotype, thereby promoting chronic liver injury and fibrosis progression, but limiting angiogenesis. Therefore, controlling tissue levels of HRG or PGF might be a promising strategy in chronic inflammatory liver diseases.

Cyclic adenosine monophosphate-responsive element modulator alpha overexpression impairs function of hepatic myeloid-derived suppressor cells and aggravates immune-mediated hepatitis in mice.

UNLABELLED: Molecular factors driving immune-mediated inflammation in the liver are incompletely understood. The transcription factor, cyclic adenosine monophosphate-responsive element modulator alpha (CREMα) can endorse differentiation of T lymphocytes toward T-helper (Th)17 cells, thereby promoting autoimmunity in systemic lupus erythematosus or lung inflammation. To investigate the role of CREMα in liver disease, we subjected transgenic (Tg) mice overexpressing CREMα under control of the CD2 promoter (cremtg mice), which restrains expression mainly to lymphocytes (T, natural killer [NK], and NKT cells), to acute and chronic liver injury models. Already in steady state, Tg CREMα overexpression broadly reduced hepatic immune cell numbers by decreasing their viability, but did not affect immune cell migration or the fibrogenic response to chronic liver injury. Strikingly, cremtg mice developed more severe immune-mediated hepatitis with a higher mortality rate, compared to wild-type (wt) mice, upon concanavalin A (ConA) administration. Unlike in T cells from spleen, CREMα overexpression did not induce a predominant Th17 response in intrahepatic T cells, given that hepatic cremtg CD4+ T cells expressed less interleukin (IL)-17 than wt T cells. Reconstitution of Rag1-/- mice with Crem-/- T cells did not ameliorate ConA hepatitis. Overexpression of CREMα did not influence NK and NKT-cell effector functions either. Interestingly, a subset of monocytic myeloid-derived suppressor cells (MDSCs) also expressed CD2 and CREMα. Cremtg MDSCs isolated from liver expressed reduced inducible nitric oxide synthase and arginase 1 and displayed a reduced T-cell suppressive activity. The adoptive transfer of wt MDSCs was capable of reducing the fulminant immune-mediated liver damage in cremtg mice to wt level. CONCLUSION: These results suggest compartmental differences of T cell activation pathways between liver and other organs in autoimmunity and define a functional role of CREMα in hepatic monocytic MDSCs for the pathogenesis of immune-mediated liver disease.

Liposomal encapsulation of dexamethasone modulates cytotoxicity, inflammatory cytokine response, and migratory properties of primary human macrophages.

The encapsulation of drugs into liposomes aims to enhance their efficacy and reduce their toxicity. Corticosteroid-loaded liposomes are currently being evaluated in patients suffering from rheumatoid arthritis, atherosclerosis, colitis, and cancer. Here, using several different fluorophore-labeled formulations, we comprehensively studied the impact of liposome encapsulation of the prototypic corticosteroid dexamethasone on various primary human cells in vitro. Liposomal dexamethasone targeted several primary cell types in a dose and time-dependent manner, but specifically reduced cytotoxicity against human fibroblasts and macrophages in comparison to the solute drug. Furthermore, macrophage maturation and polarization markers were altered. Interestingly, liposomal dexamethasone induced proinflammatory cytokine secretion (specifically TNF, IL1ß, IL6) in unstimulated cells, but reduced this response under inflammatory conditions. Monocyte and macrophage migration was significantly inhibited by dexamethasone-loaded liposomes. The findings indicate that the encapsulation of dexamethasone into liposomes modulates their cellular mechanism of action, and provides important indications for follow-up in vivo investigations. FROM THE CLINICAL EDITOR: This study investigates mechanism of action of liposomal dexamethason in the treatment of inflammatory conditions. It is concluded that liposomal dexamethasone actually induces proinflammatory cytokine secretion in unstimulated cells, but reduces the same response under inflammatory conditions. Monocyte and macrophage migration was also inhibited. The findings indicate that liposomal dexamethasone may have different mechanisms of action than its native counterpart.

Correction to: Neutrophils are a main source of circulating suPAR predicting outcome in critical illness.

[This corrects the article DOI: 10.1186/s40560-019-0381-5.].

Neutrophils are a main source of circulating suPAR predicting outcome in critical illness.

BACKGROUND: Circulating levels of soluble urokinase plasminogen activation receptor (suPAR) have been proposed as a prognostic biomarker in patients with critical illness and sepsis. However, the origin of suPAR in sepsis has remained obscure. We investigated the potential cellular sources of suPAR by analyzing membrane-bound urokinase plasminogen activator receptor (uPAR, CD87) and evaluated its clinical relevance in critically ill patients. METHODS: We studied 87 critically ill patients (44 with sepsis, 43 without sepsis) from the medical intensive care unit (ICU) in comparison to 48 standard care patients with infections and 27 healthy controls in a prospective single-center non-interventional cohort study. Cellular uPAR expression of different immune cell subsets (by flow cytometry from peripheral blood) and corresponding serum suPAR concentrations were determined upon ICU admission and at day 3. Furthermore, we analyzed the effects of serum from sepsis patients on the activation and uPAR cleavage of primary human neutrophils and macrophages in vitro. RESULTS: In healthy controls, uPAR (CD87) expression was detected on nearly all blood neutrophils and monocytes, but only scarcely on lymphocytes. While uPAR expression on monocytes was maintained in ICU patients, only 58% of neutrophils from critically ill patients expressed uPAR, which was significantly lower than in healthy controls or standard care patients. Concomitantly, serum suPAR levels were significantly increased in ICU patients. We noted a clear inverse correlation between low neutrophilic uPAR and high serum suPAR in standard care and ICU patients, indicating that shedding of uPAR from activated neutrophils represents a main source of suPAR in systemic inflammation. Both low uPAR and high suPAR were closely associated with mortality in critically ill patients. Furthermore, serum from sepsis patients induced uPAR protein expression and subsequent receptor shedding on isolated primary neutrophils, but not on macrophages, in vitro. CONCLUSIONS: The inverse correlation between low uPAR surface expression on neutrophils and high serum suPAR in critically ill patients supports that neutrophils are a main source of shed suPAR proteins in systemic inflammation. Furthermore, high suPAR levels and low neutrophilic uPAR expression predict mortality in ICU patients.

Erratum: Hohlstein, P. et al. Prognostic Relevance of Altered Lymphocyte Subpopulations in Critical Illness and Sepsis. Journal of Clinical Medicine 2019, 8, 353.

The authors wish to make the following corrections to this paper [...].

Prognostic Relevance of Altered Lymphocyte Subpopulations in Critical Illness and Sepsis.

Lymphopenia and functional defects in lymphocytes may impact the prognosis in patients with critical illness or sepsis. Therefore, we prospectively analyzed peripheral blood leukocytes from 63 healthy volunteers, 50 non-critically ill standard care (SC) patients with infections, and 105 intensive care unit (ICU) patients (52 with sepsis, 53 without sepsis) using flow cytometry. Compared to healthy volunteers, SC and ICU patients showed significant leukocytosis, especially in sepsis, while lymphocyte numbers were significantly decreased. All major lymphocyte populations (B, T, and natural killer (NK) cells) decreased in ICU patients. However, we observed a relative reduction of T cells, alongside decreased CD8+ T cells, in critically ill patients, independent of sepsis. High absolute T cell counts (>0.36/nL) at ICU admission were associated with a significantly reduced mortality, independent of patient's age. Moreover, patients that survived ICU treatment showed dynamic changes within 48 h towards restoration of lymphopenia and T cell depletion, while non-surviving patients failed to restore lymphocyte counts. In conclusion, the flow-cytometric analysis of peripheral blood revealed striking changes in circulating lymphocyte subsets in critically ill patients, independent of sepsis. Lymphopenia and T cell depletion at ICU admission were associated with increased mortality, supporting their relevance as predictive biomarkers and potential therapeutic targets in intensive care medicine.

Molecular response of liver sinusoidal endothelial cells on hydrogels.

There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~97%) compared to magnetic-assisted cell sorting (~80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization.

Isolation and time lapse microscopy of highly pure hepatic stellate cells.

Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60-95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.

Therapeutic targeting of liver inflammation and fibrosis by nanomedicine.

Nanomedicine constitutes the emerging field of medical applications for nanotechnology such as nanomaterial-based drug delivery systems. This technology may hold exceptional potential for novel therapeutic approaches to liver diseases. The specific and unspecific targeting of macrophages, hepatic stellate cells (HSC), hepatocytes, and liver sinusoidal endothelial cells (LSEC) using nanomedicine has been developed and tested in preclinical settings. These four major cell types in the liver are crucially involved in the complex sequence of events that occurs during the initiation and maintenance of liver inflammation and fibrosis. Targeting different cell types can be based on their capacity to ingest surrounding material, endocytosis, and specificity for a single cell type can be achieved by targeting characteristic structures such as receptors, sugar moieties or peptide sequences. Macrophages and especially the liver-resident Kupffer cells are in the focus of nanomedicine due to their highly efficient and unspecific uptake of most nanomaterials as well as due to their critical pathogenic functions during inflammation and fibrogenesis. The mannose receptor enables targeting macrophages in liver disease, but macrophages can also become activated by certain nanomaterials, such as peptide-modified gold nanorods (AuNRs) that render them proinflammatory. HSC, the main collagen-producing cells during fibrosis, are currently targeted using nanoconstructs that recognize the mannose 6-phosphate and insulin-like growth factor II, peroxisome proliferator activated receptor 1, platelet-derived growth factor (PDGF) receptor ß, or integrins. Targeting of the major liver parenchymal cell, the hepatocyte, has only recently been achieved with high specificity by mimicking apolipoproteins, naturally occurring nanoparticles of the body. LSEC were found to be targeted most efficiently using carboxy-modified micelles and their integrin receptors. This review will summarize important functions of these cell types in healthy and diseased livers and discuss current strategies of cell-specific targeting for liver diseases by nanomedicine.