Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform.

Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.

Mesenteric lymph node stromal cell-derived extracellular vesicles contribute to peripheral de novo induction of Foxp3+ regulatory T cells.

Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-ß when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-ß-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.

Proliferation status defines functional properties of endothelial cells.

Homeostasis of solid tissue is characterized by a low proliferative activity of differentiated cells while special conditions like tissue damage induce regeneration and proliferation. For some cell types it has been shown that various tissue-specific functions are missing in the proliferating state, raising the possibility that their proliferation is not compatible with a fully differentiated state. While endothelial cells are important players in regenerating tissue as well as in the vascularization of tumors, the impact of proliferation on their features remains elusive. To examine cell features in dependence of proliferation, we established human endothelial cell lines in which proliferation is tightly controlled by a doxycycline-dependent, synthetic regulatory unit. We observed that uptake of macromolecules and establishment of cell-cell contacts was more pronounced in the growth-arrested state. Tube-like structures were formed in vitro in both proliferating and non-proliferating conditions. However, functional vessel formation upon transplantation into immune-compromised mice was restricted to the proliferative state. Kaposi's sarcoma-associated herpes virus (KSHV) infection resulted in reduced expression of endothelial markers. Upon transplantation of infected cells, drastic differences were observed: proliferation arrested cells acquired a high migratory activity while the proliferating counterparts established a tumor-like phenotype, similar to Kaposi Sarcoma lesions. The study gives evidence that proliferation governs endothelial functions. This suggests that several endothelial functions are differentially expressed during angiogenesis. Moreover, since proliferation defines the functional properties of cells upon infection with KSHV, this process crucially affects the fate of virus-infected cells.

N-terminal fragment of cardiac myosin binding protein-C triggers pro-inflammatory responses in vitro.

Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage/monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage/monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1ß (IL-1ß)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NFκB signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor.

BACKGROUND & AIMS: Hepatitis B virus (HBV) is a major human pathogen restricted to hepatocytes. Expression of the specific receptor human sodium taurocholate cotransporting polypeptide (hNTCP) in mouse hepatocytes renders them susceptible to hepatitis delta virus (HDV), a satellite of HBV; however, HBV remains restricted at an early stage of replication. This study aims at clarifying whether this restriction is caused by the lack of a dependency factor or the activity of a restriction factor. METHODS: Six hNTCP-expressing mouse and human cell lines were generated and functionally characterized. By fusion with replication-supporting but non-infectable HepG2 cells, we analysed the ability of these heterokaryonic cells to fully support HBV replication by HBcAg expression and HBsAg/HBeAg secretion. RESULTS: While hNTCP expression in three mouse cell lines and the non-hepatic human HeLa cells conferred susceptibility to HDV, HBV replication was still restricted. Upon fusion of refractive cells to HepG2 cells, all heterokaryonic cells supported receptor-mediated infection with HBV. hNTCP was provided by the mouse cells and replication competence came from the HepG2 cell line. Transfection of a covalently closed circular DNA (cccDNA)-like molecule into non-susceptible cells promoted gene expression, indicating that the limiting step is upstream of cccDNA formation. CONCLUSIONS: In addition to the expression of hNTCP, establishment of HBV infection in mouse and non-hepatocytic human cell lines requires supplementation with a dependency factor and is not limited by a restriction factor. This result opens new avenues for the development of a fully permissive immunocompetent HBV mouse model.

Impaired antiviral response of adenovirus-transformed cell lines supports virus replication.

Activation of the innate immune response represents one of the most important cellular mechanisms to limit virus replication and spread in cell culture. Here, we examined the effect of adenoviral gene expression on the antiviral response in adenovirus-transformed cell lines; HEK293, HEK293SF and AGE1.HN. We demonstrate that the expression of the early region protein 1A in these cell lines impairs their ability to activate antiviral genes by the IFN pathway. This property may help in the isolation of newly emerging viruses and the propagation of interferon-sensitive virus strains.

Hepatitis C virus replication in mouse cells is restricted by IFN-dependent and -independent mechanisms.

BACKGROUND & AIMS: Current treatment strategies for hepatitis C virus (HCV) infection include pegylated interferon (IFN)-alfa and ribavirin. Approximately 50% of patients control HCV infection after treatment, but the broad range of patients' outcomes and responses to treatment, among all genotypes, indicates a role for host factors. Although the IFN system is important in limiting HCV replication, the virus has evolved mechanisms to circumvent the IFN response. However, direct, IFN-independent antiviral processes also might help control HCV replication. We examined the role of IFN-independent responses against HCV replication. METHODS: We analyzed replication of the subgenomic JFH1 replicon in embryonic fibroblasts and primary hepatocytes from mice with disruptions in genes encoding factors in the IFN-dependent and alternative antiviral pathways (signal transducers and activators of transcription 1 [STAT1], protein kinase R, interferon regulatory factors (IRF) IRF-1, IRF-3, IRF-5, IRF-7, mitochondrial antiviral signaling molecule [MAVS], and IFN receptor [IFNAR]). We also assessed the effects of expression of these factors by mouse primary hepatocytes on HCV replication. RESULTS: In addition to IRF-3- and IFN-mediated antiviral responses, IFN-independent, but IRF-1- and IRF-5-dependent mechanisms, restrict HCV replication in mouse embryonic fibroblasts. In primary hepatocytes these IFN-independent require MAVS and IRF-1. CONCLUSIONS: HCV replication is limited by interferon-mediated pathways as well pathways that are independent of type I IFNs. IRF1 and IRF5 control IFN-independent signaling events that lead to antiviral responses. We observed antiviral roles of IRF1 and IRF5 that were IFN-independent and cell-type specific. These mechanisms are important in controlling viruses that interfere with the IFN signaling because cells retain the ability to induce functional but local antiviral states through expression of interferon-stimulated genes.

Eternity and functionality - rational access to physiologically relevant cell lines.

In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines.

The C0-C1f Region of Cardiac Myosin Binding Protein-C Induces Pro-Inflammatory Responses in Fibroblasts via TLR4 Signaling.

Myocardial injury is associated with inflammation and fibrosis. Cardiac myosin-binding protein-C (cMyBP-C) is cleaved by µ-calpain upon myocardial injury, releasing C0-C1f, an N-terminal peptide of cMyBP-C. Previously, we reported that the presence of C0-C1f is pathogenic within cardiac tissue and is able to activate macrophages. Fibroblasts also play a crucial role in cardiac remodeling arising from ischemic events, as they contribute to both inflammation and scar formation. To understand whether C0-C1f directly modulates fibroblast phenotype, we analyzed the impact of C0-C1f on a human fibroblast cell line in vitro by performing mRNA microarray screening, immunofluorescence staining, and quantitative real-time PCR. The underlying signaling pathways were investigated by KEGG analysis and determined more precisely by targeted inhibition of the potential signaling cascades in vitro. C0-C1f induced pro-inflammatory responses that might delay TGFß-mediated myofibroblast conversion. TGFß also counteracted C0-C1f-mediated fibroblast activation. Inhibition of TLR4 or NFκB as well as the delivery of miR-146 significantly reduced C0-C1f-mediated effects. In conclusion, C0-C1f induces inflammatory responses in human fibroblasts that are mediated via TRL4 signaling, which is decreased in the presence of TGFß. Specific targeting of TLR4 signaling could be an innovative strategy to modulate C0-C1f-mediated inflammation.

Circulating Monocyte Subsets Are Associated With Extent of Myocardial Injury but Not With Type of Myocardial Infarction.

Inflammation is a hallmark of the period after a myocardial infarction (MI) that is either promoted or resolved by distinct subtypes of circulating inflammatory cells. The three main monocyte subpopulations play different roles inflammation. This study examined whether the type of MI (type 1 or type 2) or the extent of myocardial injury is associated with differences in monocyte subpopulations. For this purpose, peripheral whole blood from patients with a suspected MI was used for flow cytometric measurements of the monocyte subpopulations, and myocardial injury was classified by cardiac troponin levels in serum. In patients with acute coronary syndrome (n = 82, 62.2% male) similar proportions of the monocyte subsets were associated with the two types of MI, whereas total monocyte counts were increased in patients with substantial myocardial injury vs. those with minor injury (p = 0.045). This was accompanied by a higher proportion of intermediate (p = 0.045) and classical monocytes (p = 0.059); no difference was found for non-classical monocytes (p = 0.772). In patients with chronic coronary syndrome (n = 144, 66.5% male), an independent association with myocardial injury was also observed for classical monocytes (p = 0.01) and intermediate monocytes (p = 0.08). In conclusion, changes in monocyte subpopulation counts, particularly for classical and intermediate monocytes, were related to the extent of myocardial injury in acute and stable coronary artery disease but not to the type of MI.

Blood consumption in total arterial coronary artery bypass grafting.

BACKGROUND: Accumulating evidence consistently demonstrates that blood transfusion in cardiac surgery is related to decreased short- and long-term survival. We aimed to evaluate periprocedural blood loss and transfusion rates in elective, isolated total arterial coronary artery bypass grafting (CABG) using exclusively skeletonized bilateral internal mammary arteries (IMAs). METHODS: We identified 1011 consecutive patients with coronary artery disease who underwent CABG between 1/2007 and 12/2014. Of them, 595 patients who presented preoperative hemoglobin levels >9md/dl and underwent elective, isolated CABG for multi-vessel coronary artery disease were included in the study population. 419 patients (70.4%) received total arterial CABG using skeletonized bilateral IMAs, in 176 patients (29.6%) mixed CABG (single IMA & saphenous vein) was performed. Propensity score adjustment using 16 variables was applied to control for treatment effect. RESULTS: In patients undergoing total arterial CABG, heterologous blood transfusion could be avoided in 87.8% of all cases. Propensity score adjusted results showed a significantly lower incidence of erythrocyte concentrate transfusion in patients undergoing total arterial CABG compared to mixed CABG (odds ratio 2.74, 95% confidence interval 1.38-5.43, P = 0.004). There were no statistically significant differences in the rates of thrombocyte concentrate (P = 0.39) and fresh frozen plasma transfusions (P = 0.07). CONCLUSIONS: In this study, patients who underwent elective, isolated total arterial CABG using exclusively skeletonized bilateral IMAs showed reduced transfusion rates of erythrocyte concentrates compared to mixed CABG using a combination of single IMA and saphenous vein grafts. No evidence for a higher incidence of complications was found with a total arterial approach.

High-Content Immunophenotyping and Hierarchical Clustering Reveal Sources of Heterogeneity and New Surface Markers of Human Blood Monocyte Subsets.

OBJECTIVE: Blood monocyte subsets are emerging as biomarkers of cardiovascular inflammation. However, our understanding of human monocyte heterogeneity and their immunophenotypic features under healthy and inflammatory conditions is still evolving. RATIONALE: In this study, we sought to investigate the immunophenome of circulating human monocyte subsets. METHODS: Multiplexed, high-throughput flow cytometry screening arrays and computational data analysis were used to analyze the expression and hierarchical relationships of 242 specific surface markers on circulating classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes in healthy adults. RESULTS: Using generalized linear models and hierarchical cluster analysis, we selected and clustered epitopes that most reliably differentiate between monocyte subsets. We validated existing transcriptional profiling data and revealed potential new surface markers that uniquely define the classical (e.g., BLTR1, CD35, CD38, CD49e, CD89, CD96), intermediate (e.g., CD39, CD275, CD305, CDw328), and nonclassical (e.g., CD29, CD132) subsets. In addition, our analysis revealed phenotypic cell clusters, identified by dendritic markers CMRF-44 and CMRF-56, independent of the traditional monocyte classification. CONCLUSION: These results reveal an advancement of the clinically applicable multiplexed screening arrays that may facilitate monocyte subset characterization and cytometry-based biomarker selection in various inflammatory disorders.

Non-Invasive Approach for Evaluation of Pulmonary Hypertension Using Extracellular Vesicle-Associated Small Non-Coding RNA.

Extracellular vesicles are released by numerous cell types of the human body under physiological but also under pathophysiological conditions. They are important for cell-cell communication and carry specific signatures of peptides and RNAs. In this study, we aimed to determine whether extracellular vesicles isolated from patients with pulmonary hypertension show a disease specific signature of small non-coding RNAs and thus have the potential to serve as diagnostic and prognostic biomarkers. Extracellular vesicles were isolated from the serum of 23 patients with chronic thromboembolic pulmonary hypertension (CTEPH) and 23 controls using two individual methods: a column-based method or by precipitation. Extracellular vesicle- associated RNAs were analyzed by next-generation sequencing applying molecular barcoding, and differentially expressed small non-coding RNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). We identified 18 microRNAs and 21 P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) or piRNA clusters that were differentially expressed in CTEPH patients compared with controls. Bioinformatic analysis predicted a contribution of these piRNAs to the progression of cardiac and vascular remodeling. Expression levels of DQ593039 correlated with clinically meaningful parameters such as mean pulmonary arterial pressure, pulmonary vascular resistance, right ventricular systolic pressure, and levels of N-terminal pro-brain natriuretic peptide. Thus, we identified the extracellular vesicle- derived piRNA, DQ593039, as a potential biomarker for pulmonary hypertension and right heart disease.

MicroRNA expression profile of human advanced coronary atherosclerotic plaques.

MicroRNA (miR) is reported to be involved in vascular inflammation and may represent a novel class of diagnostic biomarkers in cardiovascular disease. We aimed to identify the miR expression profile in human advanced coronary atherosclerotic plaques (CAP) and to connect this expression to the processes in atherosclerosis. Microarray techniques and TaqMan polymerase chain reaction were used to analyse the global expression of 352 miRs in CAP obtained during ACS MULTI-LINK study. 11 miRs were selected on the basis of their implication in atherosclerosis, endothelial activation, and inflammation. 6 miRs were found to be differently expressed in CAP when compared to non-atherosclerotic internal mammary arteries (IMA, p < 0.05). The expression of miR-21, -92a, and -99a was verified and found to be significantly up-regulated in CAP versus IMA (p < 0.001). We also performed bioinformatic analysis and found several potential target genes of miR-92a and -99a as well as several pathways with impact on atherosclerosis which could be differently expressed due to this miRNA profile. The most up-regulated miRs are involved in processes known to be connected to atherosclerosis. Interfering with the miR expression in the artery wall is a potential way to affect atherosclerotic plaque and cardiovascular disease development.

Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.

Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-ß. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.

Expansion of functional personalized cells with specific transgene combinations.

Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.

Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes.

Liver infections with hepatotropic viruses, such as hepatitis B virus and hepatitis C virus are accompanied by viral persistence and immune failure. CD8+ T cells are crucial mediators of the intrahepatic antiviral immune response. Chronic infections of the liver and other organs correlate with T-cell exhaustion. It was previously suggested that high antigen load could result in T-cell exhaustion. We aimed at elucidating the impact of different intrahepatic antigen loads on the quality of CD8+ T-cell-mediated immunity by employing an infection-free transgenic mouse model expressing ovalbumin (Ova) as the target antigen. Adoptive transfer of OT-I cells induced a transient intrahepatic immune response toward both high and low Ova levels. However, antigen clearance was achieved only in mice expressing low antigen levels. In contrast, T cells exposed to high antigen levels underwent exhaustion and became depleted, causing antigen persistence. Moreover, when functional T cells were exposed to high intrahepatic antigen levels, a complete transition toward exhaustion was observed. Thus, this study shows that the antigen expression level in the liver correlates inversely with T-cell immunity in vivo and governs the efficiency of immune responses upon antigen presentation.