The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics.

Severe COVID-19 can result in pneumonia and acute respiratory failure. Accumulation of mucus in the airways is a hall mark of the disease and can result in hypoxemia. Here, we show that quantitative proteome analysis of the sputum from severe COVID-19 patients reveal high levels of neutrophil extracellular trap(s) (NETs) components, which was confirmed by microscopy. Extracellular DNA from excessive NET formation can increase sputum viscosity and can lead to acute respiratory distress syndrome (ARDS). Recombinant human DNase (rhDNase/Pulmozyme) has been shown to be beneficial in reducing sputum viscosity and improve lung function. We treated 5 COVID-19 patients presenting acute symptoms with clinically approved aerosolized Pulmozyme. No adverse reactions to the drug were seen, and improved oxygen saturation and recovery in all severely ill COVID-19 patients was observed after therapy. Immunofluorescence and proteome analysis of sputum and blood plasma samples after treatment revealed a marked reduction of NETs and a set of statistically significant proteome changes that indicate reduction of haemorrhage, plasma leakage and inflammation in the airways, and reduced systemic inflammatory state in the blood plasma of patients. Taken together, the results indicate that NETs contribute to acute respiratory failure in COVID-19 and that degrading NETs may reduce dependency on external high flow oxygen therapy in patients. Targeting NETs using rhDNase may have significant therapeutic implications in COVID-19 disease and warrants further studies.

It is well established that fibroblasts play a crucial role in pathophysiological extracellular matrix remodelling. The aim of this project is to elucidate their role in normal physiological remodelling. Specifically, the remodelling of the human cervix during pregnancy, resulting in an enabled passage of the child, is used as the model system. Fibroblast cultures were established from cervices of non-pregnant women, women after 36 weeks of pregnancy and women directly after partus. The cells were immunostained and quantified by western blots for differentiation markers. The cultures were screened for cytokine and metalloproteinase production and characterized by global proteome analysis. The cell cultures established from partal donors differ significantly from those from non-pregnant donors, which is in accordance with in vivo findings. A decrease in alpha-smooth actin and prolyl-4-hydroxylase and an increase in interleukin (IL)-6, IL-8 and matrix metalloproteinases (MMP)-1 and MMP-3 were observed in cultures from partal donors. 2D-gel electrophoresis followed by mass spectrometry showed that the expression of 59 proteins was changed significantly in cultures of partal donors. The regulated proteins are involved in protein kinase C signalling, Ca2+ binding, cytoskeletal organization, angiogenesis and degradation. Our data suggest that remodelling of the human cervix is orchestrated by fibroblasts, which are activated or recruited by the inflammatory processes occurring during the ripening cascade.

Collateral damage caused by extracellular histones has an immediate impact on morbidity and mortality in many disease models. A significant increase in the levels of extracellular histones is seen in critically ill patients with trauma and sepsis. We showed that histones are released from necrotic cells in patients with invasive skin infections. Under in vitro conditions, endogenous p33, an endothelial surface protein also known as the gC1q receptor, interacts with histones released from damaged endothelial cells. Functional analyses have revealed that recombinantly expressed p33 completely neutralizes the harmful features of histones, i.e. hemolysis of erythrocytes, lysis of endothelial cells and platelet aggregation. We also noted that mice treated with a sublethal dose of histones developed severe signs of hemolysis, thrombocytopenia and lung tissue damage already 10 min after inoculation. These complications were fully counteracted when p33 was administered together with the histones. Moreover, application of p33 significantly improved survival in mice receiving an otherwise lethal dose of histones. Together, our data suggest that treatment with p33 is a promising therapeutic approach in severe infectious diseases.

Invasive infections caused by the important pathogen Streptococcus pyogenes are often associated with disturbed blood coagulation in the human host, and may in severe cases develop into the life-threatening condition disseminated intravascular coagulation. In this study, the addition of M1 protein to human blood or purified peripheral blood mononuclear cells led to a dose-dependent increase of pro-coagulant activity, which was mediated by an upregulation of tissue factor on monocytes. Analysis of the resulting clots by transmission electron microscopy revealed that the cells were covered with a fibrin network that seemed to originate from the cell surface. Taken together, the results imply an important role for M proteins in the induction of haemostatic disorders in invasive streptococcal infectious diseases.