Heparan Sulfate Induces Necroptosis in Murine Cardiomyocytes: A Medical-In silico Approach Combining In vitro Experiments and Machine Learning.

Life-threatening cardiomyopathy is a severe, but common, complication associated with severe trauma or sepsis. Several signaling pathways involved in apoptosis and necroptosis are linked to trauma- or sepsis-associated cardiomyopathy. However, the underling causative factors are still debatable. Heparan sulfate (HS) fragments belong to the class of danger/damage-associated molecular patterns liberated from endothelial-bound proteoglycans by heparanase during tissue injury associated with trauma or sepsis. We hypothesized that HS induces apoptosis or necroptosis in murine cardiomyocytes. By using a novel Medical-In silico approach that combines conventional cell culture experiments with machine learning algorithms, we aimed to reduce a significant part of the expensive and time-consuming cell culture experiments and data generation by using computational intelligence (refinement and replacement). Cardiomyocytes exposed to HS showed an activation of the intrinsic apoptosis signal pathway via cytochrome C and the activation of caspase 3 (both p < 0.001). Notably, the exposure of HS resulted in the induction of necroptosis by tumor necrosis factor α and receptor interaction protein 3 (p < 0.05; p < 0.01) and, hence, an increased level of necrotic cardiomyocytes. In conclusion, using this novel Medical-In silico approach, our data suggest (i) that HS induces necroptosis in cardiomyocytes by phosphorylation (activation) of receptor-interacting protein 3, (ii) that HS is a therapeutic target in trauma- or sepsis-associated cardiomyopathy, and (iii) indicate that this proof-of-concept is a first step toward simulating the extent of activated components in the pro-apoptotic pathway induced by HS with only a small data set gained from the in vitro experiments by using machine learning algorithms.This work was supported by an intramural grant to LM (START 46/16) and EZ (START 113/17). LM has received a grant by the Deutsche Forschungsgemeinschaft (DFG, MA 7082/1–1). We thank Dr Claycomb and his coworkers for providing the HL-1 cells and a detailed documentation. The Immunohistochemistry and Confocal Microscopy Unit, a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen, within the Faculty of Medicine at the RWTH Aachen University, supported this work

Die phänotypische Diversität von Monozyten und Makrophagen in der nicht-alkoholischen Fettlebererkrankung

Non-alcoholic fatty liver disease (NAFLD) is one major clinical burden in modern western societies. Monocytes and monocyte-derived macrophages (MoMF) have been shown to drive inflammation and thereby aggravate fatty liver disease. In this study, the inflammatory phenotype of both bone marrow monocytes and their tissue resident successors was investigated during NAFLD progression. Therefore, wildtype (WT) and chemokine (C-C-motif) receptor 2 deficient (Ccr2-/-) mice were fed a high-fat, high-sugar and high-cholesterol Western diet for up to 16 weeks. NAFLD phenotype was assessed in the first step by measuring serum trans-aminase levels, liver triglycerides and influx of leukocytes into the liver tissue. Next, the inflammatory polarization of myeloid leukocytes from bone marrow and liver was investigated by multicolor flow cytometry, single cell RNA sequencing and in vitro polarization assays. Furthermore, the inflammatory polarization of monocytes and MoMF was also investigated during acute liver injury in healthy controls and Western diet fed obese mice. Acute liver injury was induced by a non-lethal overdose of acet-aminophen.It could be shown that during NAFLD progression, bone marrow monocytes infiltrate into the liver. These liver infiltrating monocytes contribute to NAFLD progression, as Ccr2-/- mice, lacking infiltrating inflammatory monocytes, showed significantly reduced liver steatosis compared to equally treated WT mice. Single cell RNA sequencing and flow cytometry analysis of myeloid bone marrow and liver leukocytes showed that monocytes, MoMF as well as dendritic cells showed a reduced gene expression of the inflammatory marker calprotectin (S100A8/A9 heterodimer). On the other hand, the anti-inflammatory marker CD301 was shown to be highly expressed by MoMF during NAFLD progression. In vitro experiments using bone marrow derived macro-phages could confirm that treatment with free fatty acids induced a similar inflammatory polarization as observed in monocytes and MoMF following Western diet feeding. Furthermore, the anti-inflammatory phenotype in bone marrow monocytes which was induced during NAFLD progression was shown to be persistent during acute liver injury following an overdose of acetaminophen. Taken together, these data suggest that bone marrow monocytes respond to changes in dietary composition during NAFLD progression, which induces an adaptive reduction of their inflammatory capacity

Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis

Activation of hepatic stellate cells (HSCs) and their trans-differentiation towards collagen-secreting myofibroblasts (MFB) promote liver fibrosis progression. During chronic liver disease, resting HSCs become activated by inflammatory and injury signals. However, HSCs/MFB not only produce collagen, but also secrete cytokines, participate in metabolism, and have biomechanical properties. We herein aimed to characterize the heterogeneity of these liver mesenchymal cells by single cell RNA sequencing. In vivo resting HSCs or activated MFB were isolated from C57BL6/J mice challenged by carbon tetrachloride (CCl4) intraperitoneally for 3 weeks to induce liver fibrosis and compared to in vitro cultivated MFB. While resting HSCs formed a homogenous population characterized by high platelet derived growth factor receptor &#946; (PDGFR&#946;) expression, in vivo and in vitro activated MFB split into heterogeneous populations, characterized by &#945;-smooth muscle actin (&#945;-SMA), collagens, or immunological markers. S100 calcium binding protein A6 (S100A6) was a universal marker of activated MFB on both the gene and protein expression level. Compared to the heterogeneity of in vivo MFB, MFB in vitro sequentially and only transiently expressed marker genes, such as chemokines, during culture activation. Taken together, our data demonstrate the heterogeneity of HSCs and MFB, indicating the existence of functionally relevant subsets in hepatic fibrosis

Data from: Differential impact of the dual CCR2/CCR5 inhibitor cenicriviroc on migration of monocyte and lymphocyte subsets in acute liver injury

A hallmark of acute hepatic injury is the recruitment of neutrophils, monocytes and lymphocytes, including natural killer (NK) or T cells, towards areas of inflammation. The recruitment of leukocytes from their reservoirs bone marrow or spleen into the liver is directed by chemokines such as CCL2 (for monocytes) and CCL5 (for lymphocytes). We herein elucidated the impact of chemokine receptor inhibition by the dual CCR2 and CCR5 inhibitor cenicriviroc (CVC) on the composition of myeloid and lymphoid immune cell populations in acute liver injury. CVC treatment effectively inhibited the migration of bone marrow monocytes and splenic lymphocytes (NK, CD4 T-cells) towards CCL2 or CCL5 in vitro. When liver injury was induced by an intraperitoneal injection of carbon tetrachloride (CCl4) in mice, followed by repetitive oral application of CVC, flow cytometric and unbiased t-SNE analysis of intrahepatic leukocytes demonstrated that dual CCR2/CCR5 inhibition in vivo significantly decreased numbers of monocyte derived macrophages in acutely injured livers. CVC also reduced numbers of Kupffer cells (KC) or monocyte derived macrophages with a KC-like phenotype, respectively, after injury. In contrast to the inhibitory effects in vitro, CVC had no impact on the composition of hepatic lymphoid cell populations in vivo. Effective inhibition of monocyte recruitment was associated with reduced inflammatory macrophage markers and moderately ameliorated hepatic necroses at 36h after CCl4. In conclusion, dual CCR2/CCR5 inhibition primarily translates into reduced monocyte recruitment in acute liver injury in vivo, suggesting that this strategy will be effective in reducing inflammatory macrophages in conditions of liver disease