Lena Hell

Tissue factor is induced by interleukin-33 in human endothelial cells: a new link between coagulation and inflammation.

Tissue factor (TF) is the primary trigger of coagulation. Elevated levels of TF are found in atherosclerotic plaques, and TF leads to thrombus formation when released upon plaque rupture. Interleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial cells (ECs). Here, we investigated the impact of IL-33 on TF in human ECs, as a possible new link between inflammation and coagulation. IL-33 induced TF mRNA and protein in human umbilical vein ECs and coronary artery ECs. IL-33-induced TF expression was ST2- and NF-κB-dependent, but IL-1-independent. IL-33 also increased cell surface TF activity in ECs and TF activity in ECs-derived microparticles. IL-33-treated ECs reduced coagulation time of whole blood and plasma but not of factor VII-deficient plasma. In human carotid atherosclerotic plaques (n = 57), TF mRNA positively correlated with IL-33 mRNA expression (r = 0.691, p < 0.001). In this tissue, IL-33 and TF protein was detected in ECs and smooth muscle cells by immunofluorescence. Furthermore, IL-33 and TF protein co-localized at the site of clot formation within microvessels in plaques of patients with symptomatic carotid stenosis. Through induction of TF in ECs, IL-33 could enhance their thrombotic capacity and thereby might impact on thrombus formation in the setting of atherosclerosis.

Staphylococcus aureus-derived factors induce IL-10, IFN-γ and IL-17A-expressing FOXP3+CD161+ T-helper cells in a partly monocyte-dependent manner

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4+FOXP3+ T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10, IFN-γ and IL-17A in FOXP3+ cells. Further, CD161 and HELIOS separated the FOXP3+ cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3+ cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3+ cells. Together, these data show that S. aureus potently induces FOXP3+ cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3+ cells to inflammation and repression of immune-suppression upon encounter with S. aureus.

Association of platelet activation markers with cancer-associated venous thromboembolism

Abstract Venous thromboembolism (VTE) is a frequent complication in cancer patients. Platelet activation is thought to be involved in cancer-associated VTE. Here, we determined the association between evolving markers of platelet activation (soluble P-selectin [sP-selectin], soluble CD40 ligand [sCD40L], thrombospondin-1 [TSP-1] and platelet factor-4 [PF-4]) and the development of cancer-associated VTE. A nested matched case–control study was applied within a cohort of 1779 patients with different types of cancer that had been included in the Vienna Cancer and Thrombosis Study (CATS), a prospective, observational study on patients with newly diagnosed or progressive cancer after remission. Primary endpoint is symptomatic VTE during a maximum follow-up of 2 years. Cases (patients who developed VTE during follow-up) were matched in a 1:2 ratio to controls without VTE during follow-up with respect to tumor type, stage and time of observation in the study. In total, 131 VTE cases were compared to 262 controls. In logistic regression analysis, only sP-selectin was associated with risk of VTE. The odds ratios (OR) per double increase of sP-selectin, sCD40L, TSP-1 and PF-4 were 1.66 (95% confidence interval: 1.17–2.35, p = 0.005), 1.04 (0.89–1.21, p = 0.635), 1.09 (0.90–1.32, p = 0.360) and 1.03 (0.87–1.21, p = 0.737), respectively. In conclusion, sP-selectin, but not sCD40L, TSP-1 or PF-4 were associated with risk of VTE in cancer patients in this nested case–control study.

Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants.

Microvesicles (MVs) are small membrane bound vesicles released from various cell types after activation or apoptosis. In the last decades, MVs received an increased interest as biomarkers in inflammation, coagulation and cancer. However, standardized pre‐analytical steps are crucial for the minimization of artifacts in the MV analysis. Thus, this study evaluated the MV release in whole blood samples under the influence of different anticoagulants, storage time and various temperature conditions. Samples were collected from healthy probands and processed immediately, after 4, 8, 24 and 48 hours at room temperature (RT) or 4°C. To identify MV subpopulations, platelet free plasma (PFP) was stained with Annexin V, calcein AM, CD15, CD41 and CD235a. Analysis was performend on a CytoFLEX flow cytometer. Procoagulatory function of MVs was measured using a phospholipid dependent activity and a tissue factor MVactivity assay. Without prior storage, sodium citrate showed the lowest MV count compared to heparin and EDTA. Interestingly, EDTA showed a significant release of myeloid‐derived MVs (MMVs) compared to sodium citrate. Sodium citrate showed a stable MV count at RT in the first 8 hours after blood collection. Total MV counts increased after 24 hours in sodium citrated or heparinzed blood which was related to all subpopulations. Interestingly, EDTA showed stable platelet‐derived MV (PMV) and erythrocyte‐derived MV (EryMV) count at RT over a 48 h period. In addition, the procoagulatory potential increased significantly after 8‐hour storage. Based on both, this work and literature data, the used anticoagulant, storage time and storage temperature differently influence the analysis of MVs within 8 hours. To date, sodium citrated tubes are recommended for MV enumeration and functional analysis. EDTA tubes might be an option for the clinical routine due to stable PMV and EryMV counts. These new approaches need to be validated in a clinical laboratory setting before being applied to patient studies.

Citrullinated histone H3, a biomarker of neutrophil extracellular trap formation, predicts the risk of venous thromboembolism in cancer patients

Essentials Neutrophil extracellular traps (NETs) might play a role in cancer‐related coagulopathy. We determined NET biomarkers and followed cancer patients for venous thromboembolism (VTE). We found a constant association with VTE for citrullinated histone H3. Biomarkers of NET formation could reflect a novel pathomechanism of cancer‐related VTE.

IL-33 stimulates the release of procoagulant microvesicles from human monocytes and differentially increases tissue factor in human monocyte subsets

Monocytes and monocyte-derived microvesicles (MVs) are the main source of circulating tissue factor (TF). Increased monocyte TF expression and increased circulating levels of procoagulant MVs contribute to the formation of a prothrombotic state in patients with cardiovascular disease. Interleukin (IL)-33 is a pro-inflammatory cytokine involved in atherosclerosis and other inflammatory diseases, but its role in regulating thrombosis is still unclear. The aim of the present study was to investigate the effects of IL-33 on the procoagulant properties of human monocytes and monocyte-derived MVs. IL-33 induced a time- and concentration-dependent increase of monocyte TF mRNA and protein levels via binding to the ST2-receptor and activation of the NF-κB-pathway. The IL-33 treated monocytes also released CD14+TF+ MVs and IL-33 was found to increase the TF activity of both the isolated monocytes and monocyte-derived MVs. The monocytes were classified into subsets according to their CD14 and CD16 expression. Intermediate monocytes (IM) showed the highest ST2 receptor expression, followed by non-classical monocytes (NCM), and classical monocytes (CM). IL-33 induced a significant increase of TF only in the IM (p<0.01), with a tendency in NCM (p=0.06), but no increase was observed in CM. Finally, plasma levels of IL-33 were positively correlated with CD14+TF+ MVs in patients undergoing carotid endarterectomy (r=0.480; p=0.032; n=20). We hereby provide novel evidence that the proinflammatory cytokine IL-33 induces differential TF expression and activity in monocyte subsets, as well as the release of procoagulant MVs. In this manner, IL-33 may contribute to the formation of a prothrombotic state characteristic for cardiovascular disease.

Procoagulant extracellular vesicles in amniotic fluid

&NA; Embolization of amniotic fluid (AF) into the blood circulation leads to disseminated intravascular coagulation (DIC). Procoagulant phosphatidylserine (PS)‐ and tissue factor (TF)–exposing extracellular vesicles (EVs) might play an important role in AF embolism–induced DIC. It was the aim of the present study to perform analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry. We applied a prothrombinase assay (that quantifies PS exposure on EVs), an EV‐associated TF activity assay, a fibrin generation assay, a thrombin generation assay, a whole blood clotting model, and flow cytometry in AF and control plasma. We found that PS exposure on EVs was 21‐fold increased in AF compared with plasma. Also, EV‐associated TF activity was highly increased in AF compared with plasma. AF‐derived EVs activated the blood coagulation cascade via PS and TF in the fibrin and thrombin generation assays. In a whole blood clotting model, AF‐derived EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence to 232 ± 139 seconds in the absence of an anti‐TF antibody. The contact activation pathway via factor XII (FXII) was not affected. Applying flow cytometry, a subpopulation of PS+ and TF+ EVs was identified in AF but not in control plasma. In conclusion, we investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine their procoagulant potential. Taken together, our data further delineate the pathomechanisms underlying AF‐induced coagulopathy.

Myeloid but not epithelial tissue factor exerts protective anti-inflammatory effects in acid aspiration-induced acute lung injury

Essentials Tissue factor (TF) represents a central link between hemostasis and inflammation. We studied the roles of myeloid and airway epithelial TF in acid‐caused acute lung injury (ALI). TF on myeloid cells displays a non‐coagulatory role regulating the inflammatory response in ALI. Airway epithelial TF contributes to hemostatic functions, but is dispensable in ALI pathogenesis.

Androgen receptor dampens tissue factor expression via nuclear factor-κB and early growth response protein 1

Background Prostate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which aims at diminishing circulating testosterone to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. most important mediators of coagulation and VTE, but so far there is limited data on androgen receptor (AR) mediated TF gene expression.Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients. We characterized androgen receptor‐mediated tissue factor regulation in prostate epithelial cells. Androgen receptor is dampening tissue factor expression in prostate epithelial cells. Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates.

FOXP3+ CD4 T-cell maturity and responses to microbial stimulation alter with age and associate with early-life gut colonization

FOXP3+ CD4 T-cell maturity and responses to microbial stimulation alter with age and associate with early-life gut colonization