Clin Res Cardiol 108, Suppl 1, April 2019

Soluble MD-2 is associated with early death in patients with dilated cardiomyopathy and increases M1 macrophage polarization and recruitment

R. Feldtmann1, A. Kümmel1, A. Riad2, B. Chamling1, A. Strohbach1, D. Westermann3, D. Lindner3, S. B. Felix1

1Klinik und Poliklinik für Innere Medizin B, Universitätsmedizin Greifswald, Greifswald; 2Innere Medizin, DRK Krankenhaus Teterow gGmbH, Teterow; 3Klinik für Allgemeine und Interventionelle Kardiologie, Universitäres Herzzentrum Hamburg GmbH, Hamburg;

Objective: Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction and dilatation of ventricles. Among other causes, myocardial inflammation and leukocyte activation and recruitment plays a major role in development and progression of disease. Myeloid differentiation factor-2 (MD-2) is the TLR4 co-receptor and has been shown to be an important risk predictor in outcome of patients with DCM. Furthermore, it is highly expressed in various cell types and mediates TLR4 dependent inflammatory responses and activation processes. Purpose: We examined the impact of soluble MD-2 on the polarization and recruitment of monocytes and its impact on mortality of DCM patients. Methods: We included 77 DCM patients with reduced LV ejection fraction (EF<40%) and elevated LV end-diastolic diameter (Henry>117%). and quantified MD-2 levels in the sera by ELISA upon first hospital admission. Mortality was monitored up to 12 years. Of the included patients 35 died during follow-up. These were divided into “early death” and “late death” group using the median time point of death (3.5y). A third group (n=42) was still alive after the end of the follow-up. In THP-1 cells, cytokine secretion was quantified by ELISA after 72h treatment with MD-2 (5µg/mL). Bone marrow derived macrophages (BMDM) were generated from MD-2 knock out and Bl6N-wild type mice. In these cells, the NFkB p65 phosphorylation at Ser536 and changes in gene expression of different adhesion molecules was quantified after 10min (phosphorylation) and 4h (gene expression) of treatment with 1 or 10ng/mL LPS. After 15min of treatment with 10ng/mL LPS or 5µg/mL MD-2, protein kinase B (PKB) phosphorylation at Ser473 was quantified in human umbilical vein endothelial cells (HUVEC) by means of western blot. In addition, adhesion of monocytes on treated HUVEC was determined by FACS (fig. 2). Initial HUVEC treatment with MD-2 (5µg/mL) or LPS (10 or 100ng/mL) took place for 48h. Results: We found a significant increase in MD-2 levels in “early death” (591.3ng/mL ± 71.5 N=18) vs “late death” (p=0.015) (369.2ng/mL ± 46.5 N=17) and “alive” (p=<0.0001) (303.2ng/mL ± 18.1 N=42) patients. Interestingly, treatment of THP-1 cells (N=5) with MD-2 lead to a significantly increased secretion of inflammatory cytokines IL-8 (p=0.012), IP-10 (p=0.029), and MCP-1 (p=0.032). In contrast, we detected no changes in anti-inflammatory IL-4, IL-10 and IL-13 secretion. Treatment of BMDM obtained from MD-2 KO and WT mice with 10ng/mL LPS lead to a significantly increased phosphorylation of NFkB (N=4) in WT mice (p=0.022) but not in KO mice (Fig. 1) and to a significant increase in VLA-4 (p=0.006) and ICAM-1 (p= 0.049) gene expression in WT mice compared to KO mice (N=6). In HUVEC, MD-2 as well as LPS induced a significantly increased phosphorylation of PKB as compared to untreated controls (N=5) (MD-2 p=0.008; LPS p=0.008). Furthermore, treatment of HUVEC with both MD-2 and LPS lead to a significant increase in monocyte adhesion (MD-2 p=0.015; LPS p=0.0001) (N=10) (Fig. 2a,b). Conclusion: The impact of MD-2 on cardiac inflammation and macrophage recruitment has not been described yet. In this study, we showed that elevated levels of sMD-2 are associated with early death. We could demonstrate that MD-2 enhance the process of endothelial cell based monocyte recruitment. Furthermore, we could show that MD-2 induces inflammatory monocyte activity and triggers polarization of macrophages towards an inflammatory phenotype.

https://www.abstractserver.com/dgk2019/jt/abstracts//V1044.htm