Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation
Juliana P. Vago, Isabella Zaidan, Luiza O. Perucci, Larissa Froede Brito, Lívia C.R. Teixeira, Camila Meirelles Souza Silva, Thaís C. Miranda, Eliza M. Melo, Alexandre S. Bruno, Celso Martins Queiroz-Junior, Michelle A. Sugimoto, Luciana P. Tavares, Laís C. Grossi, Isabela N. Borges, Ayda Henriques Schneider, Nagyung Baik, Ayda H. Schneider, André Talvani, Raphael G. Ferreira, José C. Alves-Filho, Vandack Nobre, Mauro M. Teixeira, Robert J. Parmer, Lindsey A. Miles, Lirlândia P. Sousa
Juliana P. Vago, Isabella Zaidan, Luiza O. Perucci, Larissa Froede Brito, Lívia C.R. Teixeira, Camila Meirelles Souza Silva, Thaís C. Miranda, Eliza M. Melo, Alexandre S. Bruno, Celso Martins Queiroz-Junior, Michelle A. Sugimoto, Luciana P. Tavares, Laís C. Grossi, Isabela N. Borges, Ayda Henriques Schneider, Nagyung Baik, Ayda H. Schneider, André Talvani, Raphael G. Ferreira, José C. Alves-Filho, Vandack Nobre, Mauro M. Teixeira, Robert J. Parmer, Lindsey A. Miles, Lirlândia P. Sousa
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Research Article Infectious disease

Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation

Abstract

Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla–afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.

Authors

Juliana P. Vago, Isabella Zaidan, Luiza O. Perucci, Larissa Froede Brito, Lívia C.R. Teixeira, Camila Meirelles Souza Silva, Thaís C. Miranda, Eliza M. Melo, Alexandre S. Bruno, Celso Martins Queiroz-Junior, Michelle A. Sugimoto, Luciana P. Tavares, Laís C. Grossi, Isabela N. Borges, Ayda Henriques Schneider, Nagyung Baik, Ayda H. Schneider, André Talvani, Raphael G. Ferreira, José C. Alves-Filho, Vandack Nobre, Mauro M. Teixeira, Robert J. Parmer, Lindsey A. Miles, Lirlândia P. Sousa

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Figure 3

Assessment of survival rates and inflammatory parameters in Plg–/– mice and their WT littermates during nonsevere sepsis.

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Assessment of survival rates and inflammatory parameters in Plg–/– mice ...
(A) Plg+/+ and Plg–/– mice were subjected to nonsevere (30G needle) CLP. The survival rates (n = 6 mice) were monitored for 6 days. In another set of experiments (n = 4–7), cells present in the peritoneal cavity were harvested 12 hours after CLP. (B–D) The number of total cells (B), mononuclear cells (C), and neutrophils (D) were evaluated by counting cytospin slides stained with May–Grünwald–Giemsa. (E) The number of M1 (F4/80low GR1+ CD11bmed) macrophages was determined by flow cytometry. (F) The percentage of efferocytosis was determined by morphological counting of cytospin slides treated with May–Grünwald–Giemsa stain. (G) Expression of fibrinogen in the liver was determined by Western blotting with anti–β-actin used as the loading control. (H) Densitometric analysis from Western blotting gels is also represented. (I) The levels of TNF, IL-10, and IL-6 were quantified by ELISA in cell-free peritoneal lavages. (J and K) The peritoneal fluid (J) and blood (K) samples were plated in brain–heart infusion medium for the analysis of bacterial load. (L) ALT activity was measured from plasma samples. (M) Representative slides of H&E-stained lungs of Plg+/+ and Plg–/– mice are shown. Bottom row: Higher-magnification images (scale bar: 10 μm) of the micrographs in the Upper row (scale bar: 50 μm). (N) Histopathological score (maximum score = 5) evaluated focal hemorrhage (arrow), edema (arrowhead), vascular hyperemia (#), and inflammatory infiltrate (*). Results are shown as the mean ± SEM or median of 4–7 mice per group. The experiments were performed 2 times with similar results. #P < 0.05 and ##P < 0.01 when comparing Plg+/+ and Plg–/– mice by log-rank test (survival curves), unpaired 2-tailed Student’s t test or Mann-Whitney U test. Outliers were removed from the graphs when detected.