All about (nk cell-mediated) death in two acts and an unexpected encore: initiation, execution and activation of adaptive immunity

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

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis

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 TNFa. 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

Selective Detection of Active Extracellular Granzyme A by Using a Novel Fluorescent Immunoprobe with Application to Inflammatory Diseases

Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity in different biological fluids, confirming the presence of active forms of the soluble protease in vivo during inflammatory and infectious diseases