Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis

7 figures, 1 table.[Aims]: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis.[Methods]: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times.[Results]: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα.[Conclusions]: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.This work was supported by grant SAF2017-83120-C2-1-R and SAF2014-54763-C2-2-R from the Ministry of Science, Innovation and Universities and FEDER (Group B29_17R, Aragon Government). MG and LS were supported by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM was supported by a PhD fellowship from Aragon Government, MA was supported by a post-doctoral fellowship “Juan de la Cierva-formación” from the Ministry of Science, Innovation and Universities. JP was supported by ARAID Foundation.Peer reviewe

Serum active Granzyme A: a new biomarker that contributes to the pathogenesis of peritoneal sepsis

Abstract of the work presented at the 30th ECCMID 2020, European Congress of Clinical Microbiology and Infectious Diseases, organized by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).-- www.eccmid.org .-- www.escmid.org.-- Accepted abstract 1508.[Background] Peritonitis is one of the most common leading cause of sepsis. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed in NK cells and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role of serum GzmA as a biomarker and therapeutic target in peritoneal sepsis.[Materials/methods] Concentration and enzyme activity of soluble GzmA were sequentially analyzed in serum from healthy donors and patients with peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (Wt) and GzmA-KO mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics and with serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days and the levels of serum proinflammatory cytokines and bacterial load in blood and spleen were analyzed at 6 and 24h from CLP.[Results] We have found high levels of GzmA in serum of patient with peritonitis. Most importantly, we observed that GzmA activity in serum correlates with SOFA score, suggesting that active GzmA could play an important role in sepsis development in peritonitis patients and could be a new biomarker of sepsis severity. In order to analyze the therapeutical potential of soluble GzmA in peritoneal sepsis, we used the CLP mouse model. After peritonitis induction, GzmA-KO mice exhibit increased survival compared with Wt mice, which correlated with reduced levels of proinflammatory cytokines in serum. The analysis of bacterial load in blood and spleen showed no differences between Wt and GzmA-KO mice suggesting that GzmA does not play an important role in bacterial control. Treatment with serpinb6b reduced mortality, which correlated with reduced cytokine serum levels in serum, confirming the therapeutical potential of gzmA to treat peritoneal sepsis.[Conclusions] Our findings confirm that soluble GzmA plays an important role in the pathogenesis of sepsis and could be a new therapeutic target and a biomarker for the treatment of peritoneal sepsis.Peer reviewe