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5 figures.-- Supplementary information available., The contribution of the T cell-related inhibitory checkpoint PD-1 to the regulation of NK cell activity is still not clear with contradictory results concerning its expression and role in the modulation of NK cell cytotoxicity. We provide novel key findings on the mechanism involved in the regulation of PD-1 expression on NK cell membrane and its functional consequences for the elimination of cancer cells. In contrast to freshly isolated NK cells from cancer patients, those from healthy donors did not express PD-1 on the cell membrane. However, when healthy NK cells were incubated with tumor target cells, membrane PD-1 expression increased, concurrent with the CD107a surface mobilization. This finding suggested that PD-1 was translocated to the cell membrane during NK cell degranulation after contact with target cells. Indeed, cytosolic PD-1 was expressed in freshly-isolated-NK cells and partly co-localized with CD107a and GzmB, confirming that membrane PD-1 corresponded to a pool of preformed PD-1. Moreover, NK cells that had mobilized PD-1 to the cell membrane presented a significantly reduced antitumor activity on PD-L1-expressing-tumor cells in vitro and in vivo, which was partly reversed by using anti-PD-1 blocking antibodies. Our results indicate that NK cells from healthy individuals express cytotoxic granule-associated PD-1, which is rapidly mobilized to the cell membrane after interaction with tumor target cells. This novel finding helps to understand how PD-1 expression is regulated on NK cell membrane and the functional consequences of this expression during the elimination of tumor cells, which will help to design more efficient NK cell-based cancer immunotherapies., Work in the JP laboratory is funded by FEDER (Fondo Europeo de Desarrollo Regional), Gobierno de Aragón (Group B29_20R), Ministerio de Ciencia, Innovación y Universidades (MCUN), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente Inocente, ASPANOA, and Carrera de la Mujer de Monzón. Postdoctoral Juan de la Cierva Contract (MA and LS) and Predoctoral Grant from AECC (CP). JP is supported by ARAID Foundation; Fundación Agencia Aragonesa para la investigación y el Desarrollo; Fundación Científica Asociación Española Contra el Cáncer.

3 figures., NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences., This research was supported by CIBER -Consorcio Centro de Investigación Biomédica en Red- (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU, FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_20R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RB-I00), ASPANOA, Donación Javier Saura Carceller, and Carrera de la Mujer de Monzón. JP was supported by Fundacion Aragon I+D (ARAID). MA was granted by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (IJC2019-039192-I). LS was granted by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJC2020-046181-I). IU-M and SH are supported by a PhD fellowship from Aragon Government, CP by a PhD fellowship from Fundación Científica de la Asociación Española Contra el Cáncer (FC AECC), MG-T by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. CO was granted by AEI -Agencia Estatal de Investigación- (PTA2020-018510-I / AEI / 10.13039/501100011033).

5 figures, 3 tables.-- Supplementary information available., Gliotoxin is a fungal secondary metabolite with impact on health and agriculture since it might act as virulence factor and contaminate human and animal food. Homologous gliotoxin (GT) gene clusters are spread across a number of fungal species although if they produce GT or other related epipolythiodioxopiperazines (ETPs) remains obscure. Using bioinformatic tools, we have identified homologous gli gene clusters similar to the A. fumigatus GT gene cluster in several fungal species. In silico study led to in vitro confirmation of GT and Bisdethiobis(methylthio)gliotoxin (bmGT) production in fungal strain cultures by HPLC detection. Despite we selected most similar homologous gli gene cluster in 20 different species, GT and bmGT were only detected in section Fumigati species and in a Trichoderma virens Q strain. Our results suggest that in silico gli homology analyses in different fungal strains to predict GT production might be only informative when accompanied by analysis about mycotoxin production in cell cultures., This research was funded by CIBER -Consorcio Centro de Investigación Biomédica en Red-(CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea –NextGenerationEU, FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_20R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RB-I00), ASPANOA, GILEAD (GLD18/00100), Donación Javier Saura Carceller, and Carrera de la Mujer de Monzón. J.P. was supported by Fundacion Aragon I+D (ARAID). P.E.was supported by Fondo Garantıa Empleo Juvenil/INAEM. S.R. was granted funding by a DGA predoctoral contract. M.A. was granted funding by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (IJC2019-039192-I). M.P.D. was supported by PTA2019-016739-I/MCIN/AEI/10.13039/501100011033.

6 figures.-- Supplementary material available., Background: Colorectal cancer (CRC) is a heterogeneous disease with variable mutational profile and tumour microenvironment composition that influence tumour progression and response to treatment. While chemoresistant and poorly immunogenic CRC remains a challenge, the development of new strategies guided by biomarkers could help stratify and treat patients. Allogeneic NK cell transfer emerges as an alternative against chemoresistant and poorly immunogenic CRC., Methods: NK cell-related immunological markers were analysed by transcriptomics and immunohistochemistry in human CRC samples and correlated with tumour progression and overall survival. The anti-tumour ability of expanded allogeneic NK cells using a protocol combining cytokines and feeder cells was analysed in vitro and in vivo and correlated with CRC mutational status and the expression of ligands for immune checkpoint (IC) receptors regulating NK cell activity., Results: HLA-I downmodulation and NK cell infiltration correlated with better overall survival in patients with a low-stage (II) microsatellite instability-high (MSI-H) CRC, suggesting a role of HLA-I as a prognosis biomarker and a potential benefit of NK cell immunotherapy. Activated allogeneic NK cells were able to eliminate CRC cultures without PD-1 and TIM-3 restriction but were affected by HLA-I expression. In vivo experiments confirmed the efficacy of the therapy against both HLA+ and HLA− CRC cell lines. Concomitant administration of pembrolizumab failed to improve tumour control., Conclusions: Our results reveal an immunological profile of CRC tumours in which immunogenicity (MSI-H) and immune evasion mechanisms (HLA downmodulation) favour NK cell immunosurveillance at early disease stages. Accordingly, we have shown that allogeneic NK cell therapy can target tumours expressing mutations conferring poor prognosis regardless of the expression of T cell-related inhibitory IC ligands. Overall, this study provides a rationale for a new potential basis for CRC stratification and NK cell-based therapy., Work in the JP laboratory is funded by ASPANOA, CIBER (CB 2021; Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación and Union Europea.NextGenerationEU), Fundacion Inocente, Carrera de la Mujer Monzón, FEDER/Gobierno de Aragón (Group B29_17R), and Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017‐83120‐C2‐1‐R and PID2020-113963RB-I00). Predoctoral grants/contracts from Gobierno de Aragon (IU-M and JP) are supported by ARAID Foundation. EG is funded by Ministerio de Ciencia, Innovación y Universidades (MCNU), and Agencia Estatal de Investigación (PID2020-113963RB-I00). MA and LS are funded by Postdoctoral Juan de la Cierva Contract. SR, LC, SH, and IU-M are funded by predoctoral contracts from Aragon Government. PL is funded by FPU predoctoral grants from Ministerio de Ciencia, Innovación e Universidades. Work at the Catalan Institute of Oncology is funded by the entity, the Instituto de Salud Carlos III and Ministerio de Economia y Competitividad, and co-funded by FEDER funds—a way to build Europe (PI20/00767), CIBERESP (grant CB07/02/2005), H2020 grant MoTriColor, and the Agency for Management of University and Research Grants (AGAUR) of the Catalan Government grant 2017SGR723. This work is supported by COST Action CA17118.

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.

Trabajo presentado en el XXIV Congreso Nacional de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), celebrado de manera virtual del 5-11 junio de 2021., [Introducción/Objetivos]: la gliotoxina es una micotoxina producida por Aspergillus fumigatus y otros hongos del género Aspergillus. Es un metabolito secundario perteneciente a la familia de las epipolitiodioxopiperazinas, caracterizada por la presencia de un puente disulfuro interno en un anillo de piperazina, que parece ser necesario para la mayoría de las propiedades biológicas de este compuesto. Se ha demostrado que la gliotoxina es un factor de virulencia durante la aspergilosis invasiva y presenta una toxicidad notable contra las células de mamíferos. Sin embargo, las evidencias científicas sobre su potencial actividad antimicrobiana son escasas. Por lo tanto, este trabajo tiene como objetivo evaluar su eficacia antibiótica contra microorganismos de interés clínico como Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa; y antifúngico contra Candida albicans y Candida glabrata. [Material y métodos]: la sensibilidad a la gliotoxina de diferentes cepas (incluidas las farmacorresistentes) de los microorganismos indicados se analizó por diferentes métodos: métodos de difusión en disco (antibiogramas/antifungigramas), estudio de curvas de tiempo-mortalidad, métodos de microdilución y ensayo MTT. Para los aislados de Candida, los resultados fueron confirmados mediante el análisis de la muerte celular por citometría de flujo. [Resultados]: Los resultados sugieren una potente actividad antimicrobiana de gliotoxina contra Candida albicans y Staphylococcus aureus, incluso en las cepas resistentes a azoles y a meticilina, respectivamente. En el caso de Escherichia coli y Candida glabrata, la gliotoxina ejerce una acción antimicrobiana moderada. Sin embargo, Pseudomonas aeruginosa parece ser resistente a la gliotoxina. [Conclusiones]: Nuestros resultados indican que la gliotoxina es efectiva contra Candida albicans y Staphylococcus aureus por lo que podría usarse como una alternativa para el tratamiento de infecciones causadas por cepas resistentes a antibióticos/antifúngicos. Sin embargo, será necesario aclarar aún más los mecanismos por los cuales la gliotoxina ejerce este efecto y probar en modelos apropiados in vivo la viabilidad de su uso teniendo en cuenta los posibles efectos secundarios debido a su toxicidad. En este sentido, planeamos analizar su eficacia y seguridad en un modelo de infección cutánea causada por Staphylococcus aureus.

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.

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.

7 figures, 2 tables., Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of hospital-acquired and community infections and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin (GT) is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that GT shows antimicrobial activity against S. aureus in vitro, albeit its efficacy against multidrug-resistant strains such as MRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimed to evaluate the antibiotic efficacy of GT as monotherapy or in combination with other therapeutics against MRSA in vitro and in vivo using a Caenorhabditis elegans infection model., This research was funded by FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_17R), Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R), ASPANOA, GILEAD (GLD18/00100), Donación Javier Saura Carceller, and Carrera de la Mujer de Monzón. J.P. was supported by Fundacion Aragon I+D (ARAID). P.E. was supported by Fondo Garantıa Empleo Juvenil/INAEM. L.C. and S.R. were granted funding by a DGA predoctoral contract. M.A. was granted funding by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJCI-2017-31629).

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the aetiology of coronavirus disease 2019 (COVID19) pandemic. ICEP4 purified compound (ICEP4) is a recently discovered furocoumarin-related purified compound coming from roots and seeds of Angelica archangelica (herbal drug). ICEP4-related herbal preparations have been extensively used as active herbal ingredient in traditional medicine treatments in several European countries. Extraction method of patent pending ICEP4 (patent application no. GB2017123.7) has previously shown strong manufacturing robustness, long-lasting stability, and repeated chemical consistency. Here we show that ICEP4 presents a significant in vitro cytoprotective effect in highly virulent-SARS-CoV-2 challenged Vero E6 cellular cultures, using doses of 34.5 and 69 μM. No dose-related ICEP4 toxicity was observed in Vero E6 cells, M0 macrophages, B, CD4+ T and CD8+ T lymphocytes, Natural Killer (NK) or Natural Killer T (NKT) cells. No dose-related ICEP4 inflammatory response was observed in M0 macrophages quantified by IL6 and TNFα release in cell supernatant. No decrease in survival rate was observed after either 24 hr acute or 21-day chronic exposure in in vivo toxicity studies performed in C. elegans. Therefore, ICEP4 toxicological profile has demonstrated marked differences compared to others vegetal furocoumarins. Successful ICEP4 doses against SARS-CoV-2-challenged cells are within the maximum threshold of toxicity concern (TTC) of furocoumarins as herbal preparation, stated by European Medicines Agency (EMA). The characteristic chemical compounding of ICEP4, along with its safe TTC, allow us to assume that the first-observation of a natural antiviral compound has occurred. The potential druggability of a new synthetic ICEP4-related compound remains to be elucidated. However, well-established historical use of ICEP4-related compounds as herbal preparations may point towards an already-safe, widely extended remedy, which may be ready-to-go for large-scale clinical trials under the EMA emergency regulatory pathway. To the best of the authors' knowledge, ICEP4-related herbal drug can be postulated as a promising therapeutic treatment for COVID19., Iratxe Uranga-Murillo is supported by a predoctoral contract from Aragón Government. ii Dr. Maykel Arias is supported by a Juan de la Cierva postdoctoral contract. iii Dr. Julian Pardo´s laboratory is funded by FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón -Group B29_17R-, Ministerio de Ciencia, Innovación e Universidades - MCNU-, Agencia Estatal de Investigación -SAF2017‐83120‐C2‐1‐R-), Instituto de Salud Carlos III, Fundación Inocente Inocente, ASPANOA and Carrera de la Mujer de Monzón.

Comunicación oral presentada en el Congreso Virtual de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), 4-6 junio de 2020., [Introducción/Objetivos]: las bacterias resistentes a múltiples fármacos como el Staphylococcus aureus resistente a meticilina (MRSA) se encuentran entre las principales causas de infecciones adquiridas en el hospital o en la comunidad, y representan un desafío para el sistema de salud. Por lo tanto, es importante encontrar nuevos medicamentos que muestren actividad contra estas bacterias, tanto en monoterapia como en combinación con otros medicamentos antimicrobianos. La gliotoxina es una micotoxina producida por Aspergillus fumigatus y otros hongos del género Aspergillus. Alguna evidencia sugiere que la gliotoxina muestra actividad antimicrobiana, especialmente contra S. aureus. Este trabajo tiene como objetivo evaluar la eficacia antibiótica de la gliotoxina como monoterapia o en combinación con otros agentes terapéuticos contra MRSA in vitro e in vivo utilizando un modelo de infección por C. elegans., [Material y métodos]: La sensibilidad de MRSA ATCC700699 contra la gliotoxina se analizó mediante diferentes métodos: difusión en disco, curvas de mortalidad-tiempo y ensayos de microdilución. Los efectos sinérgicos de la gliotoxina con otros medicamentos antiestafilocócicos (vancomicina, ácido fusídico y linezolid) se probaron mediante un ensayo de tablero de ajedrez. Los gusanos sincronizados de C. elegans L4 se infectaron con MRSA. El cultivo se complementó por separado con gliotoxina, vancomicina o su combinación a diferentes concentraciones. La tasa de supervivencia de C. elegans se evaluó manualmente cada 12 h durante 4 días., [Resultados]: El análisis de sensibilidad de MRSA contra la gliotoxina mostró que la gliotoxina tiene una potente actividad antimicrobiana (MIC = 2 µg / ml). Además, se observó actividad sinérgica, cuando la gliotoxina se combinó con vancomicina y ácido fusídico, usando ensayos de tablero de ajedrez y curvas de muerte. Este efecto sinérgico fue confirmado por el software CalcuSyn (IC = 0.15 para 1 µg / ml de gliotoxina combinada con 2 µg / ml de vancomicina). El estudio in vivo de la infección por S. aureus en un modelo de C. elegans mostró que el tratamiento combinado de vancomicina con gliotoxina aumentó un 70% en la tasa de supervivencia de los gusanos infectados con MRSA., [Conclusiones]: nuestros resultados sugieren que la gliotoxina es efectiva contra las cepas de MRSA, lo que indica su posible uso como un nuevo fármaco contra las infecciones por MRSA.

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.

6 Figures, [Background] Elimination of cancer cells by some stimuli like chemotherapy and radiotherapy activates anticancer immunity after the generation of damage‐associated molecular patterns, a process recently named immunogenic cell death (ICD). Despite the recent advances in cancer immunotherapy, very little is known about the immunological consequences of cell death activated by cytotoxic CD8+ T (Tc) cells on cancer cells, that is, if Tc cells induce ICD on cancer cells and the molecular mechanisms involved., [Methods] ICD induced by Tc cells on EL4 cells was analyzed in tumor by vaccinating mice with EL4 cells killed in vitro or in vivo by Ag-specific Tc cells. EL4 cells and mutants thereof overexpressing Bcl-XL or a dominant negative mutant of caspase-3 and wild-type mice, as well as mice depleted of Tc cells and mice deficient in perforin, TLR4 and BATF3 were used. Ex vivo cytotoxicity of spleen cells from immunized mice was analyzed by flow cytometry. Expression of ICD signals (calreticulin, HMGB1 and interleukin (IL)-1β) was analyzed by flow cytometry and ELISA., [Results] Mice immunized with EL4.gp33 cells killed in vitro or in vivo by gp33-specific Tc cells were protected from parental EL4 tumor development. This result was confirmed in vivo by using ovalbumin (OVA) as another surrogate antigen. Perforin and TLR4 and BATF3-dependent type 1 conventional dendritic cells (cDC1s) were required for protection against tumor development, indicating cross-priming of Tc cells against endogenous EL4 tumor antigens. Tc cells induced ICD signals in EL4 cells. Notably, ICD of EL4 cells was dependent on caspase-3 activity, with reduced antitumor immunity generated by caspase-3–deficient EL4 cells. In contrast, overexpression of Bcl-XL in EL4 cells had no effect on induction of Tc cell antitumor response and protection., [Conclusions] Elimination of tumor cells by Ag-specific Tc cells is immunogenic and protects against tumor development by generating new Tc cells against EL4 endogenous antigens. This finding helps to explain the enhanced efficacy of T cell-dependent immunotherapy and provide a molecular basis to explain the epitope spread phenomenon observed during vaccination and chimeric antigen receptor (CAR)-T cell therapy. In addition, they suggest that caspase-3 activity in the tumor may be used as a biomarker to predict cancer recurrence during T cell-dependent immunotherapies., The authors would like to acknowledge the use of Servicios Científico Técnicos del CIBA (IACS-Universidad de Zaragoza) and Servicios Apoyo Investigación de la Universidad de Zaragoza.

1 Figura, 2 tablas Immune checkpoint receptors (IC) positively or negatively regulate the activation of the host immune response, preventing unwanted reactions against self-healthy tissues. In recent years the term IC has been mainly used for the inhibitory ICs, which are critical to control Natural Killer (NK) and Cytotoxic CD8+ T cells due to its high cytotoxic potential. Due to the different nature of the signals that regulate T and NK cell activation, specific ICs have been described that mainly regulate either NK cell or T cell activity. Thus, strategies to modulate NK cell activity are raising as promising tools to treat tumors that do not respond to T cell-based immunotherapies. NK cell activation is mainly regulated by ICs and receptors from the KIR, NKG2 and NCRs families and the contribution of T cell-related ICs is less clear. Recently, NK cells have emerged as contributors to the effect of inhibitors of T cell-related ICs like CTLA4, LAG3 or the PD1/PD-L1 axes in cancer patients, suggesting that these ICs also regulate the activity of NK cells under pathological conditions. Strikingly, in contrast to NK cells from cancer patients, the level of expression of these ICs is low on most subsets of freshly isolated and in vitro activated NK cells from healthy patients, suggesting that they do not control NK cell tolerance and thus, do not act as conventional ICs under non-pathological conditions. The low level of expression of T cell-related ICs in “healthy” NK cells suggest that they should not be restricted to the detrimental effects of these inhibitory mechanisms in the cancer microenvironment. After a brief introduction of the regulatory mechanisms that control NK cell anti-tumoral activity and the conventional ICs controlling NK cell tolerance, we will critically discuss the potential role of T cell-related ICs in the control of NK cell activity under both physiological and pathological (cancer) conditions. This discussion will allow to comprehensively describe the chances and potential limitations of using allogeneic NK cells isolated from a healthy environment to overcome immune subversion by T cell-related ICs and to improve the efficacy of IC inhibitors (ICIs) in a safer way. Work in the JP laboratory was funded by FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_17R and RIS3 grant LMP215-18), Fundación ASPANOA, Fundación Inocente Inocente, Ministerio de Ciencia, Innovación y Universidades (MCNU)/Agencia Estatal de Investigación (SAF2017-83120-C2-1-R). Grant from Asociacion Española frente al Cancer (AECC; CP), predoctoral grants/contracts from Fundación Santander/Universidad de Zaragoza (MA) and Ministerio de Ciencia, Innovación y Universidades (FPU; PL) and a postdoctoral Juan de la Cierva Contract (MA). JP was supported by ARAID Foundation.

1 figure, 1 table Sepsis is a serious global health problem. In addition to a high incidence, this syndrome has a high mortality and is responsible for huge health expenditure. The pathophysiology of sepsis is very complex and it is not well-understood yet. However, it is widely accepted that the initial phase of sepsis is characterized by a hyperinflammatory response while the late phase is characterized by immunosuppression and immune anergy, increasing the risk of secondary infections. Granzymes (Gzms) are a family of serine proteases classified according to their cleavage specificity. Traditionally, it was assumed that all Gzms acted as cytotoxic proteases. However, recent evidence suggests that GzmB is the one with the greatest cytotoxic capacity, while the cytotoxicity of others such as GzmA and GzmK is not clear. Recent studies have found that GzmA, GzmB, GzmK, and GzmM act as pro-inflammatory mediators. Specially, solid evidences show that GzmA and GzmK function as extracellular proteases that regulate the inflammatory response irrespectively of its ability to induce cell death. Indeed, studies in animal models indicate that GzmA is involved in the cytokine release syndrome characteristic of sepsis. Moreover, the GZM family also could regulate other biological processes involved in sepsis pathophysiology like the coagulation cascade, platelet function, endothelial barrier permeability, and, in addition, could be involved in the immunosuppressive stage of sepsis. In this review, we provide a comprehensive overview on the contribution of these novel functions of Gzms to sepsis and the new therapeutic opportunities emerging from targeting these proteases for the treatment of this serious health problem. 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 (JP-P), SAF2014-54763-C2-2-R (EG)] and Instituto de Salud Carlos III (PI16-00526, LM-L; PI18/00527, JP-P). Predoctoral grants/contracts from Fundacion Santander/Universidad de Zaragoza (LS and MA), Ministerio de Ciencia, Innovación y Universidades (MG-T). MA has a Juan de la Cierva Contract (Ministerio de Ciencia, Innovación y Universidades) and JS-M a Rio Hortega Contract (Instituto de Salud Carlos III). JP was supported by Fundación Aragon I+D (ARAID).

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.-- Abstract 6144. [Background] Multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) is among the major causes of hospital-acquired and community infections, and pose a challenge to the human health care system. Therefore, it is important to find new drugs that show activity against these bacteria, both in monotherapy and in combination with other antimicrobial drugs. Gliotoxin is a mycotoxin produced by Aspergillus fumigatus and other fungi of the Aspergillus genus. Some evidence suggests that gliotoxin shows antimicrobial activity, especially against S. aureus. This work aims to evaluate the antibiotic efficacy of gliotoxin as monotherapy or in combination with other therapeutic against MRSA in vitro and in vivo using a C. elegans infection model. [Materials/methods] MRSA ATCC700699 sensitivity against gliotoxin was analysed by different methods: disc diffusion, time-mortality curves, microdilution assays. The synergistic effects of gliotoxin with others anti-staphylococcal drugs (vancomycin, fusidic acid and linezolid) were tested by a checkerboard assay. Transformed synchronized C. elegans L4 worms were infected with MRSA. The culture was supplemented separately with gliotoxin, vancomycin or its combination to different concentrations. The survival rate of C. elegans was scored manually every 12h for 4 days. [Results] Sensitivity analysis of MRSA against gliotoxin showed that gliotoxin has a potent antimicrobial activity (MIC = 2 μg/ml). In addition, synergistic activity was observed, when the gliotoxin was combined with vancomycin and fusidic acid, using checkerboard and time-kill assays. This synergistic effect was confirmed by CalcuSyn software (CI = 0.15 for 1 μg/ml of gliotoxin combined with 2 μg/ml of vancomycin). The in vivo study of S. aureus infection in a C. elegans model showed that combined treatment of vancomycin with gliotoxin increased 70% in survival rate of the MRSA-infected worms. [Conclusions] Our results suggest that gliotoxin is effective against MRSA strains indicating their potential use as a new drug against MRSA infections.

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).

Gliotoxin (GT), a secondary metabolite produced by Aspergillus molds, has been proposed as a potential anti-tumor agent. Here we have developed a nanoparticle approach to enhance delivery of GT in tumor cells and establish a basis for its potential use as therapeutical drug. GT bound to magnetic nanoparticles (MNPs) retained a high anti-tumor activity, correlating with efficient intracellular delivery, which was increased in the presence of glucose. Our results show that the attachment of GT to MNPs by covalent bonding enhances intracellular GT delivery without affecting its biological activity. This finding represents the first step to use this potent anti-tumor agent in the treatment of cancer., This work was supported by Fondo Social Europeo (FSE; Gobierno de Aragón), ASPANOA and by grants SAF2017-83120-C2-1-R, SAF2014-54763-C2-1-R, SAF2014-54763-C2-2-R from Spanish Ministry of Economy and Competitivenes. LC was granted by a DGA predoctoral contract, EP by a Juan de la Cierva Incorporacion fellowship program (IJCI-2016-30706) and MA by a Juan de la Cierva Formación fellowship program (FJCI-2017-31629), We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

1 Figura.- 1 Tabla Pulmonary aspergillosis is a severe infectious disease caused by some members of the Aspergillus genus, that affects immunocompetent as well as immunocompromised patients. Among the different disease forms, Invasive Aspergillosis is the one causing the highest mortality, mainly, although not exclusively, affecting neutropenic patients. This genus is very well known by humans, since different sectors like pharmaceutical or food industry have taken advantage of the biological activity of some molecules synthetized by the fungus, known as secondary metabolites, including statins, antibiotics, fermentative compounds or colorants among others. However, during infection, in response to a hostile host environment, the fungal secondary metabolism is activated, producing different virulence factors to increase its survival chances. Some of these factors also contribute to fungal dissemination and invasion of adjacent and distant organs. Among the different secondary metabolites produced by Aspergillus spp. Gliotoxin (GT) is the best known and better characterized virulence factor. It is able to generate reactive oxygen species (ROS) due to the disulfide bridge present in its structure. It also presents immunosuppressive activity related with its ability to killmammalian cells and/or inactivate critical immune signaling pathways like NFkB. In this comprehensive review, we will briefly give an overview of the lung immune response against Aspergillus as a preface to analyse the effect of different secondary metabolites on the host immune response, with a special attention to GT. We will discuss the results reported in the literature on the context of the animal models employed to analyse the role of GT as virulence factor, which is expected to greatly depend on the immune status of the host: why should you hide when nobody is seeking for you? Finally, GT immunosuppressive activity will be related with different human diseases predisposing to invasive aspergillosis in order to have a global view on the potential of GT to be used as a target to treat IA. This work was supported by Fondo Social Europeo (FSE; Gobierno de Aragón), ASPANOA and by grants SAF2017-83120-C2-1-R, SAF2014- 54763-C2-1-R, SAF2014-54763-C2-2-R from Spanish Ministry of Economy and Competitivenes. MV-G was granted by a Río Hortega contract of National Institute of Health Carlos III (CM16/00236) and MA by a Juan de la Cierva contract of Spanish Ministry of Economy and Competitiveness (FJCI-2017-31629).