SFRR-E OP 1.1:
Cardio-Pulmonary Consequences of Synergistic Exposure to Particulate Matter and Noise pollution
Prof. Dr. Andreas Daiber | University Medical Center Mainz | Germany
An excess of 8.79 million deaths worldwide have recently been attributed to air pollution, and particulate matter (PM), a constituent of air pollution, has lately been shown to have detrimental effects on human health. Noise is a problem of a modern urbanised world, but unlike air pollution, it has not been recognized widely as a serious risk factor. In order to probe the synergistic effect of PM and noise, we used a custom exposure system from TSE Systems (Germany). For the combined noise/PM exposure, C57BL/6 mice were acutely exposed to ambient PM (fully characterised particles obtained from the NIST, USA) and noise (aircraft landing and take-off sounds at random intervals), separately, as well as to both stressors simultaneously. Uptake and adverse health effects of PM were documented by endothelial dysfunction, increased blood pressure and successful uptake and body distribution was documented by IVIS imaging of fluorescent particles and MRI of iron oxide particles for nano-/micro-dimension particles. We observed that blood pressure was significantly increased in all exposed groups. An additive impairment of the endothelial function was observed in isolated aortic rings. Upregulation of the NOX2 protein was observed in the immunohistochemistry staining of aortic sections, accompanied by the increase in 3-nitrotyrosine positive protein. Increase in oxidative stress markers, such as dihydroethidine (DHE) staining and 3-nitrotyrosine, was observed in both aortic and lung sections. Activation of the antioxidant system in the combined exposure was demonstrated through an increase in protein expression of heme oxygenase-1 in the lung and in the vasculature. In addition to oxidative stress, markers of inflammation, such as COX2 and MCP1 were also found to be upregulated. Oxidative stress and inflammation seem to be aggravated by exposure to both factors, but more data are needed to fully elucidate the mechanism of the combined PM and Noise exposure.
SFRR-E OP 1.2 [YIA]:
Amplification of ROS/NET formation induces resolution of inflammation
Maximilien Euler | University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany | Germany
Although neutrophil granulocytes are usually regarded as archetypical pro-inflammatory cells, they also can exert anti-inflammatory and immune-regulatory functions. In previous studies, we showed that reactive oxygen species (ROS)-dependent formation of aggregated neutrophil extracellular traps (aggNETs) is crucial for resolution of inflammation of experimental arthritis and lupus. The underlying mechanism involves neutrophil serine proteases that degrade locally released cytokines and chemokines and thereby interrupt ongoing inflammatory processes.
In this project we aimed to employ restoration and amplification of NET formation for inducing resolution of innate- and autoimmune-driven inflammation. To this end we made use of NADPH oxidase (NOX) 2-activating sulfonamides and aminoferrocene-based NOX2-independent ROS amplifiers. Preliminary data suggest that the NOX2-activator RE-02 induces (agg)NET formation and alleviates experimental lupus and that the aminoferrocene-based prodrug MIS43 triggers (agg)NET formation and prevents chronification of gouty arthritis in mice with non-functional NOX2. Treatment with MIS43 was associated with lower local levels of inflammatory cytokines/chemokines. Furthermore, serological analysis of WT and NOX2 defective mice and transcriptomic analysis on paws from MIS43-treated mice support our findings and show induction of inflammatory resolution.
Taken together, the studies suggest a therapeutic efficiency of ROS-induction in chronic inflammatory syndromes that occur in the context of unsufficient ROS production from NOX2.
SFRR-E OP 1.3:
Mapping the modification of histones by the myeloperoxidase-derived oxidant hypochlorous acid (HOCl)
Prof. Clare L. Hawkins | University of Copenhagen | Denmark
Histones are highly basic nuclear proteins that are important to package and stabilise DNA. Histones are rich in Lys and Arg residues, which facilitate the tight wrapping and packaging of DNA in chromatin. These residues play a key role in the regulatory function of histones and are the site of a range of different post-translational modifications. Histones are also present in the extracellular environment and are a key component of neutrophil extracellular traps (NETs). In addition to histones, NETs contain myeloperoxidase, which retains its enzymatic activity and produces hypochlorous acid (HOCl). This localised production of HOCl could result in oxidative modification of the NET components, particularly the abundant histones. Therefore, in this study, we examine the reactivity of HOCl with a mixture of linker (H1) and core (H2A, H2B, H3 and H4) histones. HOCl induced histone modification in a dose- and time-dependent manner, resulting in fragmentation, aggregation, and the formation of both unstable and stable oxidation products. Under the conditions used in this study, histone H1 was the most susceptible to modification. Exposure of the histones to HOCl resulted in the formation of unstable N-chloramines together with the formation of Lys products, including nitriles and carbonyls (aminoadipic semialdehydes), at multiple on each of the histones. Chlorination and dichlorination of Tyr residues, but not Trp, were also observed, together with Met sulfoxide and Met sulfone, though relatively high yields of Met sulfoxide were also present in the non-treated histones. The formation of these oxidative products was observed on exposure of the histones to < 20-fold molar excess HOCl, which is readily achievable under patho-physiological conditions. Given that histones comprise ca. 70% of NET-associated proteins, these results could have implications for the development of diseases where aberrant NET release is dominant.
SFRR-E OP 1.4:
Gain of function effects of oxidized phospholipids make them pharmacological targets and leads
Prof. Valery Bochkov | University of Graz | Austria
Upon oxidation phospholipids acquire a number of biological activities that were not characteristic of their non-oxidized precursors. Recent in vivo studies have convincingly demonstrated causative role of endogenously generated oxidized phospholipids (OxPLs) in a number of pathologies, including those associated with acute and chronic inflammation. Our data suggest that in addition to direct induction of inflammatory mediators, an important mechanism of proinflammatory action of OxPLs is their ability to amplify effects of low concentrations of inflammatory cytokines. In particular, OxPLs act synergistically with low concentrations of TNFα thus shifting its dose-response to the left. We have identified several known non-antioxidant drug-like molecules, as well as a novel molecular scaffold, that were capable of inhibiting proinflammatory action of OxPLs. The data suggest that in addition to direct or indirect antioxidant therapy, inflammation induced by oxidative stress and lipid peroxidation can be inhibited by targeting intracellular signaling mechanisms. Paradoxically, OxPLs also demonstrated protective effects in certain types of pathology. For example, OxPLs inhibited inflammation induced by Toll-like receptors and protected lung endothelial barrier. We have synthesized non-electrophilic phospholipase-resistant alkyl-amide-OxPLs demonstrating anti-LPS and anti-edemagenic protective activity in vitro and in animal models. In summary, OxPLs represent potential targets for treatment of inflammation associated with oxidative stress. On the other hand, molecular leads mimicking OxPLs structure can be applied for pharmacological inhibition of Toll-like receptors and prevention of lung edema.
SFRR-E OP 1.5 [YIA]:
Myeloid cell derived Interleukin-6 causes vascular dysfunction, inflammation and endothelin-1 expression in mice
Dr. Johannes Wild | University Medical Center of the Johannes Gutenberg-University Mainz | Germany
Objective: The exact contribution of the pro-inflammatory cytokine Interleukin-6 (IL-6) to cardiovascular disease is poorly understood. As myeloid cells are an important source of IL-6, we aimed to analyze the influence of myeloid cell-derived IL-6 on vascular function.
Methods: We generated a mouse strain overexpressing IL-6 in lysozyme M+ myeloid cells (LysM-IL-6OE mice) which are one main source of this cytokine. For bone marrow transfer studies, BM cells from LysM-IL-6OE or control mice were transplanted into recipient mice in different concentrations. We assessed systolic blood pressure and vascular function. Furthermore, we measured ROS/RNS in whole blood after incubation with L-012 and phorbol 12,13-dibutyrate (PDBu). For visualization of reactive oxygen species in the aortas, aortic sections were stained with dihydroethidium (DHE). Vascular infiltration of inflammatory cells was investigated by flow cytometry. For in vitro studies, human pulmonary arterial endothelial cells (HPAECs) were cultivated and stimulated with IL-6/soluble IL-6 receptor with subsequent protein and RNA analyses.
Results: LysM-IL-6OE mice had normal blood pressure, but significantly impaired endothelium dependent aortic relaxation and increased aortic reactive oxygen species (ROS). Bone-marrow-transplantation studies indicated that vascular dysfunction and ROS formation correlated with IL-6 levels. Vascular dysfunction was accompanied by accumulation of neutrophils and Ly6C+ monocytes and macrophages in the aortic wall and by vascular fibrosis. Furthermore, we detected elevated endothelin 1 (ET-1) expression in LysM-IL-6OE aortas, a known mediator of vascular dysfunction. We could reproduce IL-6 driven vascular ET-1 expression in in vitro studies.
Conclusion: Myeloid cell-derived IL-6 induces vascular dysfunction mediated by vascular inflammation, increased oxidative stress, vascular fibrotic remodeling, dysregulated nitric oxide, and elevated vascular ET-1 expression. Notably, these findings were not linked to arterial hypertension.
Funding: DFG Grant KA4035-1.