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.

Multiparametric in vitro and in vivo analysis of the safety profile of self-assembling peptides.

Self-assembling peptides (SAPs) have gained significant attention in biomedicine because of their unique properties and ability to undergo molecular self-assembly driven by non-covalent interactions. By manipulating their composition and structure, SAPs can form well-ordered nanostructures with enhanced selectivity, stability and biocompatibility. SAPs offer advantages such as high chemical and biological diversity and the potential for functionalization. However, studies concerning its potentially toxic effects are very scarce, a limitation that compromises its potential translation to humans. This study investigates the potentially toxic effects of six different SAP formulations composed of natural amino acids designed for nervous tissue engineering and amenable to ready cross-linking boosting their biomechanical properties. All methods were performed in accordance with the relevant guidelines and regulations. A wound-healing assay was performed to evaluate how SAPs modify cell migration. The results in vitro demonstrated that SAPs did not induce genotoxicity neither skin sensitization. In vivo, SAPs were well-tolerated without any signs of acute systemic toxicity. Interestingly, SAPs were found to promote the migration of endothelial, macrophage, fibroblast, and neuronal-like cells in vitro, supporting a high potential for tissue regeneration. These findings contribute to the development and translation of SAP-based biomaterials for biomedical applications.

CAR Immunotherapy for the treatment of infectious diseases: a systematic review.

Immunotherapy treatments aim to modulate the host's immune response to either mitigate it in inflammatory/autoimmune disease or enhance it against infection or cancer. Among different immunotherapies reaching clinical application during the last years, chimeric antigen receptor (CAR) immunotherapy has emerged as an effective treatment for cancer where different CAR T cells have already been approved. Yet their use against infectious diseases is an area still relatively poorly explored, albeit with tremendous potential for research and clinical application. Infectious diseases represent a global health challenge, with the escalating threat of antimicrobial resistance underscoring the need for alternative therapeutic approaches. This review aims to systematically evaluate the current applications of CAR immunotherapy in infectious diseases and discuss its potential for future applications. Notably, CAR cell therapies, initially developed for cancer treatment, are gaining recognition as potential remedies for infectious diseases. The review sheds light on significant progress in CAR T cell therapy directed at viral and opportunistic fungal infections.

A Subset of PD-1-Expressing CD56bright NK Cells Identifies Patients with Good Response to Immune Checkpoint Inhibitors in Lung Cancer.

(1) Despite the effectiveness of immune checkpoint inhibitors (ICIs) in lung cancer, there is a lack of knowledge about predictive biomarkers. The objective of our study is to analyze different subsets of T-lymphocytes and natural killer (NK) cells as predictive biomarkers in a cohort of patients with nonsmall cell lung cancer (NSCLC) treated with ICI. (2) This is an observational, prospective study with 55 NSCLC patients treated with ICI. A total of 43 T and NK cell subsets are analyzed in peripheral blood, including the main markers of exhaustion, differentiation, memory, activation, and inhibition. (3) Regarding the descriptive data, Granzyme B+CD4+ Treg lymphocytes stand out (median 17.4%), and within the NK populations, most patients presented cytotoxic NK cells (CD56+CD3-CD16+GranzymeB+; median 94.8%), and about half of them have highly differentiated adaptive-like NK cells (CD56+CD3-CD16+CD57+ (mean 59.8%). A statistically significant difference was observed between the expression of PD1 within the CD56bright NK cell subpopulation (CD56+CD3-CD16-PD-1+) (p = 0.047) and a better OS. (4) Circulating immune cell subpopulations are promising prognostic biomarkers for ICI. Pending on validation with a larger sample, here we provide an analysis of the major circulating T and NK cell subsets involved in cancer immunity, with promising results despite a small sample size.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

PD-1 is expressed in cytotoxic granules of NK cells and rapidly mobilized to the cell membrane following recognition of tumor cells.

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 anti-tumor 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.

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.

Adoptive NK Cell Transfer as a Treatment in Colorectal Cancer Patients: Analyses of Tumour Cell Determinants Correlating With Efficacy In Vitro and In Vivo.

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.

Integration of In Silico and In Vitro Analysis of Gliotoxin Production Reveals a Narrow Range of Producing Fungal Species.

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.

Integrated analysis of circulating immune cellular and soluble mediators reveals specific COVID19 signatures at hospital admission with utility for prediction of clinical outcomes.

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.

Biological relevance of Granzymes A and K during E. coli sepsis.

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-1ß, 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-ß 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.

Determination of the Concentration of IgG against the Spike Receptor-Binding Domain That Predicts the Viral Neutralizing Activity of Convalescent Plasma and Serum against SARS-CoV-2.

Several hundred millions of people have been diagnosed of coronavirus disease 2019 (COVID-19), causing millions of deaths and a high socioeconomic burden. SARS-CoV-2, the causative agent of COVID-19, induces both specific T- and B-cell responses, being antibodies against the virus detected a few days after infection. Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Using an in-house quantitative ELISA test, we found that plasma from 177 convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, although at very different concentrations which correlated with previous disease severity and gender. Anti-RBD IgG plasma concentrations significantly correlated with the plasma viral neutralizing activity (VN) against SARS-CoV-2 in vitro. Similar results were found using an independent cohort of serum from 168 convalescent health workers. These results validate an in-house RBD IgG ELISA test in a large cohort of COVID-19 convalescent patients and indicate that plasma from all convalescent donors does not contain a high enough amount of anti-SARS-CoV-2-RBD neutralizing IgG to prevent SARS-CoV-2 infection in vitro. The use of quantitative anti-RBD IgG detection systems might help to predict the efficacy of the passive immunization using plasma from patients recovered from SARS-CoV-2.

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 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.

How Could Antibiotics, Probiotics, and Corticoids Modify Microbiota and Its Influence in Cancer Immune Checkpoint Inhibitors: A Review.

Immunotherapy has become a new paradigm in oncology, improving outcomes for several types of cancer. However, there are some aspects about its management that remain uncertain. One of the key points that needs better understanding is the interaction between immunotherapy and gut microbiome and how modulation of the microbiome might modify the efficacy of immunotherapy. Consequently, the negative impact of systemic antibiotics and corticosteroids on the efficacy of immunotherapy needs to be clarified.

In Vitro and In Vivo Antibacterial Activity of Gliotoxin Alone and in Combination with Antibiotics against Staphylococcus aureus.

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 asMRSA or vancomycin-intermediate S. aureus (VISA) strainsis not known. This work aimedto 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.

Microbiota and Lung Cancer. Opportunities and Challenges for Improving Immunotherapy Efficacy.

The advances in molecular biology and the emergence of Next Generation Sequencing (NGS) have revealed that microbiome composition is closely related with health and disease, including cancer. This relationship affects different levels of cancer such as development, progression, and response to treatment including immunotherapy. The efficacy of immune checkpoint inhibitors (ICIs) may be influenced by the concomitant use of antibiotics before, during or shortly after treatment with ICIs. Nevertheless, the linking mechanism between microbiote, host immunity and cancer is not clear and the role of microbiota manipulation and analyses in cancer management has not been clinically validated yet. Regarding the use of microbiome as biomarker to predict ICI efficacy it has been recently shown that the use of biochemical serum markers to monitor intestinal permeability and loss of barrier integrity, like citrulline, could be useful to monitor microbiota changes and predict ICI efficacy. There are still many unknowns about the role of these components, their relationship with the microbiota, with the use of antibiotics and the response to immunotherapy. The next challenge in microbiome research will be to identify individual microbial species that causally affect lung cancer phenotypes and response to ICI and disentangle the underlying mechanisms. Thus, further analyses in patients with lung cancer receiving treatment with ICIs and its correlation with the composition of the microbiota in different organs including the respiratory tract, peripheral blood and intestinal tract could be useful to predict the efficacy of ICIs and its modulation with antibiotic use.

From Tumor Mutational Burden to Blood T Cell Receptor: Looking for the Best Predictive Biomarker in Lung Cancer Treated with Immunotherapy.

Despite therapeutic advances, lung cancer (LC) is one of the leading causes of cancer morbidity and mortality worldwide. Recently, the treatment of advanced LC has experienced important changes in survival benefit due to immune checkpoint inhibitors (ICIs). However, overall response rates (ORR) remain low in unselected patients and a large proportion of patients undergo disease progression in the first weeks of treatment. Therefore, there is a need of biomarkers to identify patients who will benefit from ICIs. The programmed cell death ligand 1 (PD-L1) expression has been the first biomarker developed. However, its use as a robust predictive biomarker has been limited due to the variability of techniques used, with different antibodies and thresholds. In this context, tumor mutational burden (TMB) has emerged as an additional powerful biomarker based on the observation of successful response to ICIs in solid tumors with high TMB. TMB can be defined as the total number of nonsynonymous mutations per DNA megabases being a mechanism generating neoantigens conditioning the tumor immunogenicity and response to ICIs. However, the latest data provide conflicting results regarding its role as a biomarker. Moreover, considering the results of the recent data, the use of peripheral blood T cell receptor (TCR) repertoire could be a new predictive biomarker. This review summarises recent findings describing the clinical utility of TMB and TCRß (TCRB) and concludes that immune, neontigen, and checkpoint targeted variables are required in combination for accurately identifying patients who most likely will benefit of ICIs.

The Multifaceted Function of Granzymes in Sepsis: Some Facts and a Lot to Discover.

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.

Extracellular Granzyme A Promotes Colorectal Cancer Development by Enhancing Gut Inflammation.

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.