ABSTRACT
Background: The liver is the main producer of coagulation factors, which are activated in response to a variety of viruses, presumably to contain and limit virus spreading. Neutrophils and their extracellular traps (NETs) often build the molecular and structural basis for such thrombi that can cause adverse complications and organ damage in patients. Using the murine coronavirus m-CoV, we analysed the consequences of a coronavirus infection in the liver. Aim(s): Analysis of haemostatic alterations and immunothrombosis in a virus infected liver. Method(s): C57Bl/6 mice were nasally infected with m-CoV (MHV-A59). Livers were collected 2, 4 and 10 days after infection, and virus burden, tissue damage, thrombus formation, expression of coagulation factors and neutrophil activation were analysed via histology and qPCR. Result(s): NET formation occurred rapidly and U-shaped over the course of the infection: 1.55 thrombi/mm2 liver section were present 2 days after m-CoV infection, which decreased to 0.50 thrombi/ mm2 at day 4 and increased again to 1.04 thrombi/mm2 at day 10. Neutrophil counts significantly increased until day 4 and showed highest MPO activity on day 2. M-CoV virus is mainly present 4 days after infection and coincided with highest percentage of damaged tissue (4.58%). Although mRNAs of factors II, V, VII, VIII, IX, and X were not significantly altered, we could detect a significant peak in plasminogen mRNA on day 2 and PAI 1 mRNA on day 4. Plasma concentrations of MCP-1, IFN-gamma, IL-6 and TNF alpha were also highest at day 4. Conclusion(s): We describe a U-shaped liver thrombosis development during a coronavirus infection. Liver NETs are rapidly formed after a viral infection and are resolved due to increased plasminogen expression. Peak virus burden of the liver and increased systemic inflammation markers induce expression of liver PAI-1 and activation of recruited neutrophils, which favours NET formation anew.
ABSTRACT
Background: Thrombosis and activation of neutrophils are bystanders of a SARS-CoV-2 infection in humans. However, these events seem to appear late during the disease and might represent a skewed activation of the immune system. To study thrombosis during a SARS-CoV infection, we used the murine coronavirus mCoV-A59, which belongs to the beta coronavirus subgroup, like SARS-CoV-2. Aims: Analysis of thrombus formation during a SARS-CoV infection, and identification of cellular origin. Methods: C57Bl/6 mice were infected with the murine mCoV-A59. Lungs were collected 2, 4 and 10 days after infection, and thrombus formation and virus burden were analysed via histology and qPCR. The C57Bl/6 mouse strain represents a mild to moderate coronavirus infection model. Results: Thrombus formation occurred rapidly, with 1.73 thrombi/mm 2 lung section 2 days after SARS-CoV infection, which further increased to 3.92 thrombi/mm 2 at day 4 and stayed stable with 4.09 thrombi/ mm 2 at day 10. Compared to day 2, virus burden was already reduced 2-fold at day 4 and 100-fold at day 10. Day 4 thrombi were platelet-rich, whereas thrombi at day 10 were positive for citrullinated histone H4, a marker for neutrophil extracellular traps (NETs). This coincided with a decrease in neutrophils by 40% and increase in lung cell apoptosis of 1.8 % to 10.5 % measured by double-strand DNA breaks via TUNEL. This indicated that neutrophil activation after day 4 led to NET formation and thrombus formation generating NET rich thrombi in an environment with already massively diminished viral burden. Conclusions: We describe a biphasic process of lung thrombosis development during a coronavirus infection. Whereas the early phase thrombi are platelet rich, the late phase thrombi are associated with neutrophil activation and NET formation. We suggest that targeting specific thrombotic events during the infection will ameliorate lung tissue damage.