Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible

PAR1 contributes to influenza A virus pathogenicity in mice

Influenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PAR1 antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses

Plasminogen controls inflammation and pathogenesis of influenza virus infections via fibrinolysis

Detrimental inflammation of the lungs is a hallmark of severe influenza virus infections. Endothelial cells are the source of cytokine amplification, although mechanisms underlying this process are unknown. Here, using combined pharmacological and gene-deletion approaches, we show that plasminogen controls lung inflammation and pathogenesis of infections with influenza A/PR/8/34, highly pathogenic H5N1 and 2009 pandemic H1N1 viruses. Reduction of virus replication was not responsible for the observed effect. However, pharmacological depletion of fibrinogen, the main target of plasminogen reversed disease resistance of plasminogen-deficient mice or mice treated with an inhibitor of plasminogen-mediated fibrinolysis. Therefore, plasminogen contributes to the deleterious inflammation of the lungs and local fibrin clot formation may be implicated in host defense against influenza virus infections. Our studies suggest that the hemostatic system might be explored for novel treatments against influenza

PAR1 contributes to influenza A virus pathogenicity in mice

International audienceInfluenza causes substantial morbidity and mortality, and highly pathogenic and drug-resistant strains are likely to emerge in the future. Protease-activated receptor 1 (PAR1) is a thrombin-activated receptor that contributes to inflammatory responses at mucosal surfaces. The role of PAR1 in pathogenesis of virus infections is unknown. Here, we demonstrate that PAR1 contributed to the deleterious inflammatory response after influenza virus infection in mice. Activating PAR1 by administering the agonist TFLLR-NH2 decreased survival and increased lung inflammation after influenza infection. Importantly, both administration of a PAR1 antagonist and PAR1 deficiency protected mice from infection with influenza A viruses (IAVs). Treatment with the PAR1 agonist did not alter survival of mice deficient in plasminogen (PLG), which suggests that PLG permits and/or interacts with a PAR1 function in this model. PARE antagonists are in human trials for other indications. Our findings suggest that PAR1 antagonism might be explored as a treatment for influenza, including that caused by highly pathogenic H5N1 and oseltamivir-resistant H1N1 viruses

The deleterious role of plasminogen is independent on virus replication.

<p>(A) Virus replication of IAV A/PR/8/34 and A/Netherlands/602/09 after inoculation of A549 cells in presence or absence (triangle) of plasminogen (square) or trypsin (circle). Data represent mean ± s.e.m of three independent experiments. (B) Western blot analysis of A/PR/8/34 and A/Netherlands/602/09 HA cleavage after infection of A549 cells in presence or absence of plasminogen (PLG) or trypsin (Try). Membranes were probed with anti-HA and anti-tubulin antibodies. kDa (apparent molecular weight). NI stands for uninfected. (C) Infectious A/PR/8/34 (n = 3–5) and A/Netherlands/602/09 (n = 3) lung virus titers at the indicated time points post-inoculation of WT (black bars) or PLG-KO mice (white bars). Data represent mean ± s.e.m of 3–5 individual mice per group. n = mice per group and per time-point.</p

Effect of Ancrod treatment on inflammation and IAV pathogenesis.

<p>(A) Survival and weight loss of mice treated with Ancrod (open symbols, n = 11) or not (closed symbols, n = 11) after infection with IAV A/PR/8/34 (squares) or uninfected mice (diamonds, n = 5). Weight loss data represent weight average ± s.e.m of the above indicated number of mice. (B) Cytokines levels in the BAL were measured by ELISA after A/PR/8/34 infection of WT and PLG-KO (KO) mice treated with Ancrod (white bars) or not (black bars). Data represent mean ± s.e.m. of n = 4 mice per group. (C) Survival rate (left panels) and weight loss (right panels) of WT (squares) and PLG-KO (triangles) mice treated with Ancrod (open symbols) or not (closed symbols) after intranasal inoculation with IAV A/PR/8/34 (n = 8–10 mice per group). Weight loss data represent weight average ± s.e.m of the above indicated number of mice.</p

Schematic overview of the proposed model for Plasminogen-mediated influenza virus pathogenesis.

<p>During IAV infection, plasminogen is converted into plasmin. On the one hand, plasmin cleaves and activates the viral hemagglutinin, promoting IAV replication for some influenza strains. On the other hand, plasmin promotes inflammation via fibrinolysis and increases permeability.</p

Plasminogen-deficiency prevents severe inflammation and virus dissemination.

<p>(A) Cytokine levels in BAL were assessed by 23-multiplex Luminex kit (uninfected, white bars; infected, black bars) on the indicated days post inoculation of WT (top panel) and PLG-KO mice (bottom panel) with IAV A/PR/8/34. The levels of IL-2, IL-3, IL-4, IL-5, IL-9, IL-12(p70), IL-13, IL-17 and eotaxin were below the detection limit (not shown). Data represent mean ± s.e.m. of 2 individual mice per group from one experiment and is representative of 2 individual experiments (total n = 3–6 mice per group). (B) A/PR/8/34 virus titers in the indicated organs of WT (closed symbols) and PLG-KO mice (open symbols) was assessed 2 and 5 days post-inoculation.</p

Fibrinolysis is induced following severe influenza infections.

<p>(A) Levels of Plasminogen, Active Plasmin, FDP, D-dimer and Fibrinogen, were determined by ELISA in the BAL of A/PR/8/34 infected or uninfected (−) C57BL/6 mice after the indicated days post-inoculation. Markers were also evaluated in the BAL of WT or PLG-KO mice infected with A/Netherlands/602/09 virus. Data represent mean ± s.e.m of n = 3–6 mice per group. (B) Western blot analysis for the detection of fibrinogen and FDP in the lungs of IAV-infected mice on the indicated days post inoculation (representative of n = 3). kDa: apparent molecular weight. n = mice per group. (C) Presence of fibrinogen was assessed in the blood of mice treated or not with Ancrod by ELISA (left panel) or Western blot analysis (right panel). The results represent the mean values ± s.e.m from 3 individual animals per group for the ELISA. The western blot analysis is representative for results of 3 mice per group.</p