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3 figuras.-- Póster presentado en el 42º Congreso SEI, Congreso de la Sociedad Española de Inmunología, formato virtual, 24-26 marzo de 2021, Madrid., Peritonitis is one of the most common causes of sepsis. Evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as proinflammatory mediator and could play an important role in the pathogenesis of sepsis. Here, we aim to analyze the role and therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis.

2 figures. Cytotoxic lymphocytes (CLs), and more specifically Tc and NK cells, are the main executors of cell death in the immune system, playing a key role during both immunosurveillance and immunotherapy. These cells induce regulated cell death (RCD) by different mechanisms, being granular exocytosis and expression of death ligands the most prominent and best characterized ones. Apoptosis, a traditionally considered low-inflammatory type of cell death, has been accepted for years as the paradigm of RCD induced by CLs. However, several recent studies have demonstrated that NK cells and Tc cells can also induce more inflammatory forms of cell death, namely, necroptosis, pyroptosis, and ferroptosis. Activation of these highly inflammatory types of cell death appears to critically contribute to the activation of a successful antitumour immune response. Additionally, the role of specific cell death pathways in immunogenic cell death is still under intense debate, especially considering the interconnections with other inflammatory forms of cell death. These evidences, together with the advent of new cancer immunotherapies, highlight the necessity to deepen our understanding of the link between the cell death triggered by CLs and inflammation. This knowledge will be instrumental to maximize the antitumour potential of immunotherapies, minimizing deleterious effects associated with these treatments. In this review, we will briefly summarize the main features of apoptosis, necroptosis, pyroptosis and ferroptosis, to subsequently discuss the most recent evidences about the role of these RCD pathways during the elimination of cancer cells mediated by CLs and its modulation to increase the efficacy of cancer immunotherapy. Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_17R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Health National Institute Carlos III, Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RB-I00), Fundación Inocente 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). DDM is supported by a postdoctoral fellowship ‘Sara Borrell’, LS by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM and SH are supported by a PhD fellowship from Aragon Government. MA is supported by a postdoctoral fellowship ‘Juan de la Cierva-formación’ from the Ministry of Science, Innovation and Universities, and JP is supported by the ARAID Foundation.

Trabajo presentado en el III Workshop de NanoOncología, 14-15 de Enero de 2020, en la Facultad de Medicina de la Universidad de Zaragoza (España).

Oral presentation made at the Serpins2019 Conference, held in Sevilla (Spain), 19th – 22nd September 2019.

of the poster presented at the III SYMPOSIUM IMMUNOTHERAPY CANCER, held in Zaragoza, November 7th, 2019., [Introduction]: The number of patients benefiting from Immune Checkpoint Inhibitors (ICIs) is still relatively low, restricted to specific tumour types; those presenting specific inflammatory immune cell populations at tumour microenvironment: the so-called hot tumours. Development of protocols to selectively modulate inflammation and increase tumour sensitivity to ICIs is required. Some nanoparticles (NPs) are able to modulate the immune response. Yet, their effect on tumour microenvironment and inflammation has not been explored. [Aim]: test the effect of NPs of different composition on the inflammatory response in myeloid cells in vitro and in tumours in vivo., [Methods]: M1/M2 macrophages and dendritic cells (DCs) were differentiated from mouse bone marrow. Gold and chitosan NPs (GNPs or CNCs) were synthetized and characterised. Production of inflammatory cytokines in myeloid cells and the mechanism involved were analysed in vitro. Immunomodulation in vivo was analysed in the B16 melanoma model., [Results]: GNPs are not cytotoxic in vitro at any concentration tested, in contrast to CNCs, that present cytotoxicity at high concentration. Both GNPs and CNCs were able to induce the release of TNFα and IL6 in macrophages in vitro, and, in combination with LPS, they also induced IL1β. The latter depended on the canonical caspase-1-inflammasome. NPs also induced DC maturation in vitro. GNPs increased in vivo LPS immunomodulation in tumours, modifying the tumour growth and the microenvironment, including cell populations and the profile of inflammatory cytokines., [Conclusions]: GNPs and CNCs are able to modulate the inflammatory response in tumour microenvironment in vitro and in vivo. Further studies will required to analyse if they modulate the sensitivity of tumour to ICIs.

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.

8 figures, 1 table.-- Electronic supplementary information available, If not properly regulated, the inflammatory immune response can promote carcinogenesis, as evident in colorectal cancer (CRC). Aiming to gain mechanistic insight into the link between inflammation and CRC, we perform transcriptomics analysis of human CRC, identifying a strong correlation between expression of the serine protease granzyme A (GzmA) and inflammation. In a dextran sodium sulfate and azoxymethane (DSS/AOM) mouse model, deficiency and pharmacological inhibition of extracellular GzmA both attenuate gut inflammation and prevent CRC development, including the initial steps of cell transformation and epithelial-to-mesenchymal transition. Mechanistically, extracellular GzmA induces NF-κB-dependent IL-6 production in macrophages, which in turn promotes STAT3 activation in cultured CRC cells. Accordingly, colon tissues from DSS/AOM-treated, GzmA-deficient animals present reduced levels of pSTAT3. By identifying GzmA as a proinflammatory protease that promotes CRC development, these findings provide information on mechanisms that link immune cell infiltration to cancer progression and present GzmA as a therapeutic target for CRC., This work was supported in part by FEDER/Gobierno de Aragón (group B29), Ministerio de Economia y Competitividad (SAF2014-54763-C2-1 and SAF2017-83120-C2-1-R to J.P. and SAF2014-54763-C2-2-R to E.M.G.), and Instituto de Salud Carlos III (PI13/00864 to L.M.-L.). Predoctoral grants/contracts from Fundacion Santander/Universidad de Zaragoza (to L.S. and M.A.A.), Gobierno de Aragon (to I.U.-M., L.C., L.S., and P.J.-S.), and FPU/Ministerio de Educación, Cultura y Deportes (to P.M.L.). I.U.-M. was supported by Fondo Garantía Empleo Juvenil/INAEM. M.A.A. has a Juan de la Cierva contract (Ministerio de Ciencia, Innovación y Universidades). J.P. was supported by Fundación Aragon I+D (ARAID).

2 figures., Cytotoxic lymphocytes (CLs), and more specifically Tc and NK cells, are the main executors of cell death in the immune system, playing a key role during both immunosurveillance and immunotherapy. These cells induce regulated cell death (RCD) by different mechanisms, being granular exocytosis and expression of death ligands the most prominent and best characterized ones. Apoptosis, a traditionally considered low-inflammatory type of cell death, has been accepted for years as the paradigm of RCD induced by CLs. However, several recent studies have demonstrated that NK cells and Tc cells can also induce more inflammatory forms of cell death, namely, necroptosis, pyroptosis, and ferroptosis. Activation of these highly inflammatory types of cell death appears to critically contribute to the activation of a successful antitumour immune response. Additionally, the role of specific cell death pathways in immunogenic cell death is still under intense debate, especially considering the interconnections with other inflammatory forms of cell death. These evidences, together with the advent of new cancer immunotherapies, highlight the necessity to deepen our understanding of the link between the cell death triggered by CLs and inflammation. This knowledge will be instrumental to maximize the antitumour potential of immunotherapies, minimizing deleterious effects associated with these treatments. In this review, we will briefly summarize the main features of apoptosis, necroptosis, pyroptosis and ferroptosis, to subsequently discuss the most recent evidences about the role of these RCD pathways during the elimination of cancer cells mediated by CLs and its modulation to increase the efficacy of cancer immunotherapy., Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_17R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Health National Institute Carlos III, Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RB-I00), Fundación Inocente 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). DDM is supported by a postdoctoral fellowship ‘Sara Borrell’, LS by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM and SH are supported by a PhD fellowship from Aragon Government. MA is supported by a postdoctoral fellowship ‘Juan de la Cierva-formación’ from the Ministry of Science, Innovation and Universities, and JP is supported by the ARAID Foundation.

2 Figuras, 1 Tabla, Microbiota have emerged as key modulators of both the carcinogenic process and the immune response against cancer cells, and, thus, it seems to influence the efficacy of immunotherapy. While most studies have focused on analyzing the influence of gut microbiota, its composition substantially differs from that in the lung. Here, we describe how microbial life in the lungs is associated with host immune status in the lungs and, thus, how the identification of the microbial populations in the lower respiratory tract rather than in the gut might be key to understanding the lung carcinogenic process and to predict the efficacy of different treatments. Understanding the influence of lung microbiota on host immunity may identify new therapeutic targets and help to design new immunotherapy approaches to treat lung cancer.