Danger signals released during cold ischemia storage activate NLRP3 inflammasome in myeloid cells and influence early allograft function in liver transplantation.

BACKGROUND: Innate immunity plays a fundamental role in solid organ transplantation. Myeloid cells can sense danger signals or DAMPs released after tissue or cell damage, such as during ischemia processes. This study aimed to identify DAMPs released during cold ischemia storage of human liver and analyze their ability to activate the inflammasome in myeloid cells and the possible implications in terms of short-term outcomes of liver transplantation. METHODS: 79 samples of organ preservation solution (OPS) from 79 deceased donors were collected after cold static storage. We used different analytical methods to measure DAMPs in these end-ischemic OPS (eiOPS) samples. We also used eiOPS in the human macrophage THP-1 cell line and primary monocyte cultures to study inflammasome activation. FINDINGS: Different DAMPs were identified in eiOPS, several of which induced both priming and activation of the NLRP3 inflammasome in human myeloid cells. Cold ischemia time and donation after circulatory death negatively influenced the DAMP signature. Moreover, the presence of oligomeric inflammasomes and interleukin-18 in eiOPS correlated with early allograft dysfunction in liver transplant patients. INTERPRETATION: DAMPs released during cold ischemia storage prime and activate the NLRP3 inflammasome in liver macrophages after transplantation, inducing a pro-inflammatory environment that will complicate the outcome of the graft. The use of pharmacological blockers targeting DAMPs or the NLRP3 inflammasome in liver ischemia during static cold storage or through extracorporeal organ support could be a suitable strategy to increase the success of liver transplantation. FUNDING: Fundación Mutua Madrileña and Instituto de Salud Carlos III, Madrid, Spain.

P2X7 receptor activation impairs antitumour activity of natural killer cells.

BACKGROUND AND PURPOSE: A high number of intratumoural infiltrating natural killer (NK) cells is associated with better survival in several types of cancer, constituting an important first line of defence against tumours. Hypoxia in the core of solid tumours induces cellular stress and ATP release into the extracellular space where it triggers purinergic receptor activation on tumour-associated immune cells. The aim of this study was to assess whether activation of the purinergic receptor P2X7 by extracellular ATP plays a role in the NK cells antitumour activity. EXPERIMENTAL APPROACH: We carried out in vitro experiments using purified human NK cells triggered through P2X7 by extracellular ATP. NK cell killing activity against the tumour target cells K562 was studied by means of NK cytotoxicity assays. Likewise, we designed a subcutaneous solid tumour in vivo mouse model. KEY RESULTS: In this study we found that human NK cells, expressing a functional plasma membrane P2X7, acquired an anergic state after ATP treatment, which impaired their antitumour activity and decreased IFN-γ secretion. This effect was reversed by specific P2X7 antagonists and pretreatment with either IL-2 or IL-15. Furthermore, genetic P2rx7 knockdown resulted in improved control of tumour size by NK cells. In addition, IL-2 therapy restored the ability of NK cells to diminish the size of tumours. CONCLUSIONS AND IMPLICATIONS: Our results show that P2X7 activation represents a new mechanism whereby NK cells may lose antitumour effectiveness, opening the possibility of generating modified NK cells lacking P2X7 but with improved antitumour capacity.

Proteomics in Liver Transplantation: A Systematic Review.

Background: Although proteomics has been employed in the study of several models of liver injury, proteomic methods have only recently been applied not only to biomarker discovery and validation but also to improve understanding of the molecular mechanisms involved in transplantation. Methods: The study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology and the guidelines for performing systematic literature reviews in bioinformatics (BiSLR). The PubMed, ScienceDirect, and Scopus databases were searched for publications through April 2020. Proteomics studies designed to understand liver transplant outcomes, including ischemia-reperfusion injury (IRI), rejection, or operational tolerance in human or rat samples that applied methodologies for differential expression analysis were considered. Results: The analysis included 22 studies after application of the inclusion and exclusion criteria. Among the 497 proteins annotated, 68 were shared between species and 10 were shared between sample sources. Among the types of studies analyzed, IRI and rejection shared a higher number of proteins. The most enriched pathway for liver biopsy samples, IRI, and rejection was metabolism, compared to cytokine-cytokine receptor interactions for tolerance. Conclusions: Proteomics is a promising technique to detect large numbers of proteins. However, our study shows that several technical issues such as the identification of proteoforms or the dynamic range of protein concentration in clinical samples hinder the successful identification of biomarkers in liver transplantation. In addition, there is a need to minimize the experimental variability between studies, increase the sample size and remove high-abundance plasma proteins.

Gasdermins mediate cellular release of mitochondrial DNA during pyroptosis and apoptosis.

Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.

Addendum: MCC950 closes the active conformation of NLRP3 to an inactive state.

An Addendum to this paper has been published: https://doi.org/10.1038/s41589-021-00741-6.

CD14 release induced by P2X7 receptor restricts inflammation and increases survival during sepsis.

P2X7 receptor activation induces the release of different cellular proteins, such as CD14, a glycosylphosphatidylinositol (GPI)-anchored protein to the plasma membrane important for LPS signaling via TLR4. Circulating CD14 has been found at elevated levels in sepsis, but the exact mechanism of CD14 release in sepsis has not been established. Here, we show for first time that P2X7 receptor induces the release of CD14 in extracellular vesicles, resulting in a net reduction in macrophage plasma membrane CD14 that functionally affects LPS, but not monophosphoryl lipid A, pro-inflammatory cytokine production. Also, we found that during a murine model of sepsis, P2X7 receptor activity is important for maintaining elevated levels of CD14 in biological fluids and a decrease in its activity results in higher bacterial load and exacerbated organ damage, ultimately leading to premature deaths. Our data reveal that P2X7 is a key receptor for helping to clear sepsis because it maintains elevated concentrations of circulating CD14 during infection.

When the immune system detects an infection, it often launches an inflammatory response to fight off the disease. This defense mechanism is activated by a cascade of signaling molecules that can aggravate inflammation, causing it to damage the body's own tissues and organs. This life-threatening reaction is referred to as sepsis, and kills around 11 million people each year. New approaches are therefore needed to help alleviate the damage caused by this condition. The inflammatory response is often triggered by proteins called receptors, which sit on the surface of immune cells. When these receptors are activated, they induce cells to secrete proteins that travel around the body and activate immune cells that can eliminate the infection. In 2016, a group of researchers showed that a receptor called P2X7 stimulates the release of a signaling molecule called CD14. Patients with sepsis often have elevated amounts of CD14 in their bloodstream. Yet, it remained unclear what causes this rise in CD14 and what role this molecule plays in the development of sepsis. Now, Alarcón-Vila et al. ­ including some of the researchers involved in the 2016 study ­ have investigated the role of P2X7 in mice undergoing sepsis. This was done by puncturing the mice's intestines, causing bacteria to leak out and initiate an over-active immune response. Alarcón-Vila et al. found that mice lacking the P2X7 receptor had less CD14 and struggled to eliminate the bacterial infection from their system. This increase in bacteria caused excessive damage to the mice's organs, ultimately leading to premature death. These findings suggest that P2X7 plays an important role in preventing the onset of sepsis by helping maintain high levels of CD14 following infection. This result could help to identify new therapies that reduce the mortality rates of septic infections.

P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis.

Sepsis is characterized by a systemic inflammatory response followed by immunosuppression of the host. Metabolic defects and mitochondrial failure are common in immunocompromised patients with sepsis. The NLRP3 inflammasome is important for establishing an inflammatory response after activation by the purinergic P2X7 receptor. Here, we study a cohort of individuals with intra-abdominal origin sepsis and show that patient monocytes have impaired NLRP3 activation by the P2X7 receptor. Furthermore, most sepsis-related deaths are among patients whose NLRP3 activation is profoundly altered. In monocytes from sepsis patients, the P2X7 receptor is associated with mitochondrial dysfunction. Furthermore, activation of the P2X7 receptor results in mitochondrial damage, which in turn inhibits NLRP3 activation by HIF-1α. We show that mortality increases in a mouse model of sepsis when the P2X7 receptor is activated in vivo. These data reveal a molecular mechanism initiated by the P2X7 receptor that contributes to NLRP3 impairment during infection.

MCC950 closes the active conformation of NLRP3 to an inactive state.

NLRP3 (NOD-like receptor pyrin domain-containing protein 3) is an innate immune sensor that contributes to the development of different diseases, including monogenic autoinflammatory syndromes, gout, atherosclerosis, and Alzheimer's disease. The molecule sulfonylurea MCC950 is a NLRP3 inflammasome inhibitor with potential clinical utility. However, the mechanism of action of MCC950 remains unknown. Here, we characterize the mechanism of action of MCC950 in both wild-type and autoinflammatory-related NLRP3 mutants, and demonstrate that MCC950 closes the 'open' conformation of active NLRP3.

3D chitosan scaffolds impair NLRP3 inflammasome response in macrophages.

Chitosan (Ch) is used in different biomedical applications to promote tissue repair. However, tissue injury caused by biomaterial implantation lead to the release of danger signals that engage different inflammatory pathways on the host. Different implanted materials activate the inflammasome leading to the modulation of the immune response. Here we have studied how macroscopic biomaterials, Ch scaffolds with different chemical composition: 4% or 15% degree of acetylation (DA) modulate the activation of the NLRP3 inflammasome in vitro. For that, we assessed the NLRP3 inflammasome in bone marrow derived mouse macrophages (BMDM) and human macrophages cultured within 3D Ch scaffolds. We found that both Ch scaffolds did not trigger the NLRP3 inflammasome activation in macrophages. Furthermore, BMDMs and human macrophages cultured in both Ch scaffolds presented a reduction in the number of apoptosis-associated speck-like protein containing a caspase activating recruitment domain (ASC) specks and in IL-1ß release upon classical NLRP3 inflammasome stimulation. We also found a decrease in proIL-1ß in BMDMs after priming with LPS when cultured in Ch scaffolds with DA 4% DA after priming with LPS when compared to Ch scaffolds with 15% DA or to macrophages cultured in cell-culture plates. Our results shows that 3D Ch scaffolds with different DA impair NLRP3 inflammasome priming and activation. STATEMENT OF SIGNIFICANCE: In this research work we have assessed the role of the NLRP3 inflammasome in the macrophage response to 3D chitosan scaffolds with different degrees of acetylation (DA). To our knowledge this is the first work that demonstrates the modulatory capacity of 3D porous chitosan scaffolds in the NLRP3 inflammasome activation, because our results show that Ch scaffolds impair NLRP3 inflammasome assembly in macrophages. Interestingly, our results are in contrast with studies reported in the literature that indicate that chitosan is a powerful activator of the NLRP3 inflammasome in nanoscale chitosan products. Our studies that were performed in large scale chitosan scaffolds, stress out that the process of phagocytosis is pivotal in inflammasome assembly and activation, are rather important since they clearly illustrate the different role of the inflammasome in the biological response to large scale and nanoscale biomaterials.

P2X7 Receptor Induces Tumor Necrosis Factor-α Converting Enzyme Activation and Release to Boost TNF-α Production.

Tumor necrosis factor (TNF)-α is a major pro-inflammatory cytokine produced in response to toll-like receptor stimulation. TNF-α release is controlled by the activity of TNF-α converting enzyme (TACE) that cut membrane-bound TNF-α to shed its ectodomain as a soluble cytokine. The purinergic receptor P2X ligand-gated ion channel 7 (P2X7) is activated in response to elevated concentrations of extracellular ATP and induces different pro-inflammatory pathways in macrophages to establish an inflammatory response. P2X7 receptor promotes the activation of the inflammasome and the release of interleukin-1ß, the production of inflammatory lipids, and the generation of reactive oxygen species. In this study, we analyzed the mechanism of P2X7 receptor responsible of TNF-α release after priming macrophages with LPS doses ≤100 ng/ml. We found that P2X7 receptor increases the extracellular activity of TACE through the release of the mature form of TACE in exosomes. This effect was blocked using P2X7 receptor inhibitors or in macrophages obtained from P2X7 receptor-deficient mice. Elevation of intracellular Ca2+ and p38 mitogen-activated protein kinase after P2X7 receptor activation were involved in the release of TACE, which was able to process TNF-α on nearby expressing cells. Finally, we observed an increase of TNF-α in the peritoneal lavage of mice treated with LPS and ATP. In conclusion, P2X7 receptor induces the release of TACE in exosomes to the extracellular compartment that could amplify the pro-inflammatory signal associated to this receptor. These results are important for the development of therapeutics targeting P2X7 receptor.

The NLRP3 and Pyrin Inflammasomes: Implications in the Pathophysiology of Autoinflammatory Diseases.

Inflammasomes are multiprotein complexes that critically control different aspects of innate and adaptive immunity. Among them we could highlight the release of pro-inflammatory cytokines that induce and maintain the inflammatory response. Usually, inflammasomes result from oligomerization of a nucleotide-binding domain-like receptor (NLR) after sensing different pathogenic or endogenous sterile dangerous signals; however, other proteins such as absent in melanoma 2, retinoic acid-inducible gene I, or pyrin could also form inflammasome platforms. Inflammasome oligomerization leads to caspase-1 activation and the processing and release of the pro-inflammatory cytokines, such as interleukin (IL)-1ß and IL-18. Mutations in different inflammasomes are causative for multiple periodic hereditary syndromes or autoinflammatory diseases, characterized by acute systemic inflammatory flares not associated with infections, tumors, or autoimmunity. This review focuses on germline mutations that have been described in cryopyrin-associated periodic syndrome (CAPS) for NLRP3 or in familial Mediterranean fever (FMF) and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND) for MEFV. Besides the implication of inflammasomes in autoinflammatory syndromes, these molecular platforms are involved in the pathophysiology of different illnesses, including chronic inflammatory diseases, degenerative processes, fibrosis, or metabolic diseases. Therefore, drug development targeting inflammasome activation is a promising field in expansion.

Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process.

The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released after P2X7R stimulation goes beyond caspase-1 secretome. The characterization of P2X7R-secretome reveals a new function of this receptor through a fine-tuning of protein release. We found that P2X7R stimulation in macrophages is able to release potent anti-inflammatory proteins, such as Annexin A1, independently of their polarization state suggesting for first time a potential role for P2X7R during resolution of the inflammation and not linked to the release of pro-inflammatory cytokines. These results are of prime importance for the development of therapeutics targeting P2X7R.

Association of a characteristic membrane pattern of annexin A2 with high invasiveness and nodal status in colon adenocarcinoma.

The identification of tumor cells in lymph nodes is essential for the correct classification of patients with colorectal cancer who may benefit from adjuvant treatments. Proper classification of tumor stage becomes entangled by variables such as an insufficient number of lymph nodes examined, which can result in erroneous or missed diagnosis. The determination of pathologic factors in the primary tumor associated with positive lymph nodes is an area of research that has attempted to provide variables to solve this problem. In the present study, we observed that the localization of annexin A2 (AnxA2) in a cell membrane is the characteristic that distinguishes tumor cells with high invasiveness. Localization of AnxA2 expression was also studied in tissue specimens from 58 patients with invasive colorectal carcinoma (T3-T4), who had undergone colectomy with radical lymph node dissection. Interestingly, the membrane pattern observed in tumor cell lines was also identified in patient's tissue samples and allowed us to distinguish among different cell populations with the tumor. Univariate analysis showed that tumor deposits in pericolic fat, extramural vascular invasion, and amount of cells with AnxA2 membrane pattern in the tumor invasive edge had a significant influence on lymph node metastasis. On the contrary, multivariate analysis revealed that the number of cells with AnxA2 membrane pattern (P < 0.05) and tumor deposits (P < 0.05) was significantly associated with lymph node metastasis. Furthermore, AnxA2 cellular localization was observed in cell clusters that define tumor budding, and a significant association between both variables was detected.

Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.

The RAS to extracellular signal-regulated kinase (ERK) signal transduction cascade is crucial to cell proliferation, differentiation, and survival. Although numerous growth factors activate the RAS-ERK pathway, they can have different effects on the amplitude and duration of the ERK signal and, therefore, on the biological consequences. For instance, nerve growth factor, which elicits a larger and more sustained increase in ERK phosphorylation in PC12 cells than does epidermal growth factor (EGF), stimulates PC12 cell differentiation, whereas EGF stimulates PC12 cell proliferation. Here, we show that protein arginine methylation limits the ERK1/2 signal elicited by particular growth factors in different cell types from various species. We found that this restriction in ERK1/2 phosphorylation depended on methylation of RAF proteins by protein arginine methyltransferase 5 (PRMT5). PRMT5-dependent methylation enhanced the degradation of activated CRAF and BRAF, thereby reducing their catalytic activity. Inhibition of PRMT5 activity or expression of RAF mutants that could not be methylated not only affected the amplitude and duration of ERK phosphorylation in response to growth factors but also redirected the response of PC12 cells to EGF from proliferation to differentiation. This additional level of regulation within the RAS pathway may lead to the identification of new targets for therapeutic intervention.