C7-Subproject
Extracellular nucleic acids in severe pneumo-coccal pneumonia: Mechanisms and therapeutic approaches in combination with adrenomedullin (Fischer / Müller-Redetzky)
Pneumonia remains a significant health burden worldwide. Dysregulation of the innate immune system may lead to hyper-inflammation mediating pulmonary vascular leakage and micro-circulatory failure, which likely contributes to the unfavorable outcome for patients with severe pneumonia. Our preliminary data indicate that the endogenous peptide adrenomedullin (AM) protects mice with pneumonia (and mechanical ventilation) by stabilizing the pulmonary endo-epithelial barrier, preventing extra-pulmonary organ dysfunction, micro-vascular thrombosis and finally tissue damage. We further show that AM reduced the liberation of extracellular RNA (eRNA) and neutrophil extracellular traps (NETs) in vitro. Both eRNA and NETs may act as nucleic acid “danger associated molecular patterns” DAMPs in pneumonia contributing to pulmonary and extrapulmonary organ failure. Interrelation of AM with nucleic acid DAMPs may account in part for its protective function. The role of nucleic acids/DAMP-dependent mechanisms in the exaggerated innate immune response during pneumonia and their role in pulmonary and extrapulmonary organ failure and finally their interrelation with AM are not defined yet.
The severe inflammatory response during pulmonary infections depends - among other mechanisms - on the liberation of tumor necrosis factor (TNF)-α. Our preliminary studies demonstrated that host extracellular RNA (eRNA) promotes the liberation of TNF-α from monocytes, by activating the sheddase TNF-α-converting enzyme (TACE/ADAM17). Furthermore, eRNA is a strong permeability- and inflammation-inducing cofactor. Thus, eRNA, which we found to be released from lung epithelium by pneumolysin, may act as a nucleic acid DAMP, contributing to the pro-inflammatory host response during pneumonia. We identified sialoadhesin-1 (siglec-1) as first eRNA-binding protein on monocytes to be involved in eRNA-induced TNF-α liberation. Moreover, NETs may be considered as endogenous nucleic acids/DAMP as well, since these extracellular DNA-histone complexes not only kill invading microorganisms, but also express procoagulant and cytotoxic functions, likely to be relevant in micro-vessel thrombosis and endo-epithelial barrier breakdown during lung infection. In particular, NET-bound histones express strong cytotoxic activity, which was neutralized by the highly negatively charged polysaccharide polysialic acid (polySia). Interestingly, AM was found to suppress the eRNA-induced TNF--release from monocytes/macrophages as well as NETosis in vitro via a yet unknown process.
To characterize the underlying pathomechanisms by which nucleic acids/DAMPs are involved in pneumonia and their interrelation with protective functions of AM, the following issues will be addressed: (1) The formation of eRNA and NET, the localization of both DAMPs and their natural antagonists RNase1, DNase1 and polySia in tissue and blood samples and their correlation with disease progression will be characterized in murine Streptococcus pneumoniae pneumonia and in complementary ex vivo human lung tissue and cell culture models. (2) The functional role of the outlined nucleic acid DAMPs and their putative endogenous antagonists in pneumonia will be investigated. The contribution of eRNA will be studied by performing RNase1 or TAPI (inhibitor of ADAMs) treatment and applying siglec-1 deficient mice in murine pneumococcal pneumonia. With regard to NET/histones, elastase deficient mice (lacking NETosis), and DNase- and polySia treated mice will be characterized for the impact of NETs and histones in pneumonia-related lung- and extrapulmonary organ injury (murine pneumonia models using S. pneumoniae and P. aeruginosa). Complementary cell culture and ex vivo human lung tissue experiments will address mechanistic insights and prove the translation of findings from animal models to the human lung. (3) Finally, the impact of AM on eRNA and NET/histone formation as well as on RNase1 and polySia expression in pneumonia will be investigated. Together, these approaches will shed light on the pathogenetic role of endogenous nucleic acids/DAMPs in pneumonia and offer new causative protective functions of AM and may further implicate new adjuvant therapeutic strategies.