Summary Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We show active chromatin remodelling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses, and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Our study therefore represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.

Aortic valve stenosis (AS) development is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. Toll-like-receptor-3 (TLR3) is a lysosomal pattern-recognition receptor that binds double-stranded RNA and promotes pro-inflammatory cellular responses. In recent years, TLR3 has emerged as a major regulator of vascular inflammation. The exact role of TLR3 in the development of AS has not been investigated. Isolated human valvular interstitial cells (VICs) were stimulated with the TLR3-agonist polyIC and the resulting pro-inflammatory and pro-osteogenic response measured. Severe AS was induced in wildtype- and TLR3−/− mice via mechanical injury of the aortic valve with a coronary springwire. TLR3 activation was achieved by polyIC injection every 24 h after wire injury, while TLR3 inhibition was realized using Compound 4a (C4a) every 48 h after surgery. Endothelial mesenchymal transition (EndoMT) of human valvular endothelial cells (VECs) was assessed after polyIC stimulation. Stimulation of human VICs with polyIC promoted a strong inflammatory and pro-osteogenic reaction. Similarly, injection of polyIC marginally increased AS development in mice after wire injury. AS induction was significantly decreased in TLR3−/− mice, confirming the role of endogenous TLR3 ligands in AS pathology. Pharmacological inhibition of TLR3 with C4a not only prevented the upregulation of inflammatory cytokines and osteogenic markers in VICs, and EndoMT in VECs, but also significantly abolished the development of AS in vivo. Endogenous TLR3 activation significantly contributes to AS development in mice. Pharmacological inhibition of TLR3 with C4a prevented AS formation. Therefore, targeting TLR3 may be a viable treatment option.

Background Aortic valve stenosis (AS) is the most common valve disease worldwide and is associated with a very high morbidity and mortality. Until today, aortic valve replacement is the only therapeutic option available. Analysis of explanted human aortic valves has shown that atherosclerosis-like lesions, that contain cholesterol crystals (CC), are present in stenotic aortic valve cusps. It has been demonstrated that CCs can activate the NLRP3 inflammasome and hereby trigger a complex, IL-1b driven, inflammatory response. 2-hydroxypropyl-β-cyclodextrin (CD) is a cyclic oligosaccharide that can increase the solubility of CCs, which results in a reduction of CC-load and therefore could inhibit the pro-inflammatory immune response. Methods Severe AS was induced in 10 weeks old C57BL/6-J (WT) and Apolipoprotein-E-deficient (ApoE) mice. Acoronary springwire was used to induce an endothelial injury under echocardiographic guidance. AS development was confirmed via ultrasound examinations. ApoE mice were fed a cholesterol-rich western diet and concomitantly received daily injections of 2g/kg/d CD via subcutaneous injection. CCs were visualized with laser reflection confocal microscopy. Serum cholesterol analysis were performed via mass GC-MS-SIM. Results In order to evaluate whether hyperlipidemia aggravates AS development, WT and ApoE mice were fed a cholesterol-rich diet and subjected to our model of wire-induced AS. Trans-aortic valve peak velocity levels of ApoE mice were significantly increased six weeks after injury compared to WT mice. Histological analysis of these mice showed large CC-deposits in the aortic valves of ApoE mice. Next, CC solubility was increased in a group of ApoE mice, control mice only received PBS injections. Interestingly, mice treated with CD displayed a significantly reduced peak blood velocity over the aortic valve compared to PBS mice. Left ventricular ejection fraction remained unchanged. Serum cholesterol analysis was performed to analyze the effect of CD on cholesterol metabolism. 27-hydroxycholesterol, an endogenous oxysterol of cholesterol metabolism, which reduces the potential for the conversion of free cholesterol into crystals was significantly increased in CD treated mice. The levels of cholesterol precursors were unchanged, indicating that CD doesn't influence de-novo synthesis of cholesterol. Intestinal absorption of cholesterol was also not affected by CD, as assessed by quantification of phytosterols in the serum of CD and PBS treated mice. Conclusion These results underline the importance of hyperlipidemia in the pathogenesis of AS. Particularly CCs seem to act as an important inflammatory trigger in the development of AS. Increasing the solubility of cholesterol through CD reduces AS development in mice and could, as it is already considered safe in human, act as a possible therapeutic option. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): DFG, German Research Foundation

Background: Our current concept of aortic valve stenosis (AS) development suggests that local chronic inflammation drives fibrosis and calcification of the valve cusps. This remodeling is driven by endothelial to mesenchymal transition (EndMT) of valvular endothelial cells (VECs) and by calcification of valvular interstitial cells (VICs). In a preliminary screening, we found Toll-like receptor 3 (TLR3) expression increased in human AS samples. Therefore, aim of this study was to investigate the role of TLR3 in the pathogenesis of AS. Methods/Results: We confirmed TLR3 expression in cultured human VICs and VECs. Upon specific TLR3 stimulation with PolyI:C, both VICs and VECs displayed a positive feedback with increased TLR3 expression. Concomitant treatment of PolyI:C-stimulated VICs and VECs with the TLR3/RNA complex inhibitor (C4a) significantly blunted this response. Importantly, the TLR3-mediated pro-inflammatory and calcifying response of VICs and EndMT by VECs was significantly reduced by TLR3-inhibtion with C4a.To examine the role or TLR3 in AS development, a wire injury induced AS mouse model was used. Valves were explanted and stained with hematoxylin/eosin, Sirius red, von Kossa, or anti CD68. PolyI:C treatment promoted AS, as demonstrated by increased valve cusp thickness and pronounced valvular inflammation. Interestingly, AS development was nearly absent in TLR3 deficient mice suggesting a critical role of endogenous TLR3 ligands in this model. Treatment of mice with C4a after wire-injury of the aortic valve significantly reduced both morphological and functional parameters of AS development. Conclusion: These findings not only support the role of endogenous TLR3 activation in the development of AS but suggest that specific TLR3 inhibition might be beneficial for the treatment or prevention of AS. Further studies are required to decipher the exact mechanisms how TLR3 contributes to AS.

Background Aortic stenosis (AS) is the most common valvular heart disease (VHD) in developed countries. The pathophysiology of calcific AS has several clinical and pathobiological findings in common with atherosclerosis including chronic inflammation and lipoprotein deposition. Histopathological examination has revealed atherosclerosis-like lesions, that mainly contain cholesterol crystals (CC) in resected calcific aortic valve cusps. Previous studies have demonstrated that CCs can activate the NLRP3 inflammasome, resulting in an IL-1 driven systemic inflammation, that leads to the development of atherosclerotic plaques. Purpose In this study, we sought to validate a novel assay for measuring the serum capacity to dissolve cholesterol crystals (CCDC) in patients with AS and to examine whether this biomarker may be associated with clinical outcomes. Methods Our study cohort included 348 patients with AS undergoing transcatheter aortic valve replacement. The CCDC was measured using flow cytometry to enumerate CC, that were added to a 50% serum solution, at baseline and after two hours of incubation. The dissolution capacity was indicated as percentage change in CC count at baseline and after incubation. The study cohort was stratified according to the median CCDC into high and low CC dissolvers. Results The study population was 47.7% female and had a mean age of 80.9±6.2 years. The primary end point, a composite of one-year all-cause mortality and major vascular complication occurred less frequently in the high CCDC group (7.0%) as compared with the low CCDC group (15.3%, p=0.01). This was mainly driven by lower rates of one-year mortality in patients with a high CCDC (7.0% vs 13.6%, p=0.05), as presented in Figure 1. Furthermore, unplanned endovascular interventions were significantly less frequent in high CC dissolvers in contrast to low CC dissolvers (12.2% vs 20.5%, p=0.04). Although LDL cholesterol (101.8±37.3 mg/dL vs 97.9±37.6 mg/dL, p=0.35) and total cholesterol levels (158.1±43.8 mg/dL vs 154.1±40.2, p=0.41) were comparable in the high and low CCDC group, only patients with a low CCDC showed a benefit from statin treatment (Figure 2). In multivariate analysis, only low CCDC (OR: 2.51 [95% CI: 1.02–6.15], p=0.05) and Albumin (OR: 0.88 [95% CI: 0.79–0.98], p=0.03) were independently associated with one-year all-cause mortality Conclusion The CCDC is a novel biomarker associated with clinical outcome in patients with AS undergoing TAVR. It may provide new insights into patient's preventative anti-inflammatory capacity and additional prognostic information to identify vulnerable patients beyond classic risk assessment. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).