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6 figures.-- Supplemenatry material available.--This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions., [Aims]: Recent in vitro findings suggest that the serine protease Granzyme K (GzmK) may act as a proinflammatory mediator. However, its role in sepsis is unknown. Here we aim to understand the role of GzmK in a mouse model of bacterial sepsis and compare it to the biological relevance of Granzyme A (GzmA)., [Methods]: Sepsis was induced in WT, GzmA-/- and GzmK-/- mice by an intraperitoneal injection of 2x108 CFU from E. coli. Mouse survival was monitored during 5 days. Levels of IL-1α, IL-1β, TNFα and IL-6 in plasma were measured and bacterial load in blood, liver and spleen was analyzed. Finally, profile of cellular expression of GzmA and GzmK was analyzed by FACS., [Results]: GzmA and GzmK are not involved in the control of bacterial infection. However, GzmA and GzmK deficient mice showed a lower sepsis score in comparison with WT mice, although only GzmA deficient mice exhibited increased survival. GzmA deficient mice also showed reduced expression of some proinflammatory cytokines like IL1-α, IL-β and IL-6. A similar result was found when extracellular GzmA was therapeutically inhibited in WT mice using serpinb6b, which improved survival and reduced IL-6 expression. Mechanistically, active extracellular GzmA induces the production of IL-6 in macrophages by a mechanism dependent on TLR4 and MyD88., [Conclusions]: These results suggest that although both proteases contribute to the clinical signs of E. coli-induced sepsis, inhibition of GzmA is sufficient to reduce inflammation and improve survival irrespectively of the presence of other inflammatory granzymes, like GzmK., This work was supported by grant SAF2017-83120-C2-1-R and PID2020-113963RBI00 from the Ministry of Science, Innovation and Universities and FEDER (Group B29_17R, Aragon Government). IU-M , MG-T and CP were supported by a PhD fellowships from Aragon Government, Ministry of Science, Innovation and Universities (FPI) and Asociacion Española contra el Cancer (AECC). MA and LS were supported by a post-doctoral fellowship “Juan de la Cierva-formación” (MA, LS) and "Juan de la Cierva-incorporacion" (MA) from the Ministry of Science, Innovation and Universities. JP was supported by ARAID Foundation.

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.

6 figures, 6 tables.-- Supplementary material available., Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis., [Methods]: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity., [Results]: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFNα and TNFα, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNFα, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity., [Conclusions]: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death., The authors would like to thank the Biobank of the Aragon Health System integrated in the Spanish National Biobanks Network and the Servicios Científico Técnicos de Citometria de Flujo del CIBA for their collaboration. Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_17R), Health National Institute Carlos III (COV20-00308), Aragón Government (Fondo COVID-19), Fundación Santander-Universidad de Zaragoza (Programa COVID-19), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente, ASPANOA and Carrera de la Mujer de Monzón. EMG is funded by Agencia Estatal de Investigación (SAF2017-83120-C2-1-R and PID2020-113963RB-I00). IUM and SH are supported by a PhD fellowship from Aragon Government, CP by a PhD fellowship from AECC, LS by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. DDM is supported by a postdoctoral fellowship 'Sara Borrell', and MA is supported by a postdoctoral fellowship 'Juan de la Cierva-incorporacion' from the Ministry of Science, Innovation and Universities. EM and BGT are supported by Rio Hortega contract. JP is supported by the ARAID Foundation.