Proinflammatory Cell Death Research Articles

Inhibition of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and atherosclerotic lesion development in ApoE-/- mice.

Pyroptosis is a form of pro-inflammatory cell death that can be mediated by gasdermin D (GSDMD) activation induced by inflammatory caspases such as caspase-1. Emerging evidence suggests that targeting GSDMD activation or pyroptosis may facilitate the reduction of vascular inflammation and atherosclerotic lesion development. The current study investigated the therapeutic effects of inhibition of GSDMD activation by the novel GSDMD inhibitor N-Benzyloxycarbonyl-Leu-Leu-Ser-Asp(OMe)-fluoromethylketone (Z-LLSD-FMK), the specific caspase-1 inhibitor N-Benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (Z-YVAD-FMK), and a combination of both on atherosclerosis in ApoE-/- mice fed a western diet at 5weeks of age, and further determined the efficacy of these polypeptide inhibitors in bone marrow-derived macrophages (BMDMs). In vivo studies there was plaque formation, GSDMD activation, and caspase-1 activation in aortas, which increased gradually from 6 to 18weeks of age, and increased markedly at 14 and 18weeks of age. ApoE-/- mice were administered Z-LLSD-FMK (200µg/day), Z-YVAD-FMK (200µg/day), a combination of both, or vehicle control intraperitoneally from 14 to 18weeks of age. Treatment significantly reduced lesion formation, macrophage infiltration in lesions, protein levels of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and pyroptosis-related proteins such as activated caspase-1, activated GSDMD, cleaved interleukin(IL)-1β, and high mobility group box 1 in aortas. No overt differences in plasma lipid contents were detected. In vitro treatment with these polypeptide inhibitors dramatically decreased the percentage of propidium iodide-positive BMDMs, the release of lactate dehydrogenase and IL-1β, and protein levels of pyroptosis-related proteins both in supernatants and cell lysates elevated by lipopolysaccharide + nigericin. Notably however, there were no significant differences in the above-mentioned results between the Z-LLSD-FMK group and the Z-YVAD-FMK group, and the combination of both did not yield enhanced effects. These findings indicate that suppression of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and lesion development in ApoE-/- mice.

Coexistence of apoptosis, pyroptosis, and necroptosis pathways in celiac disease.

Usually, the massive elimination of cells under steady-state conditions occurs by apoptosis, which is also acknowledged to explain the loss of enterocytes in the small intestine of celiac disease (CD) patients. However, little is known about the role of proinflammatory cell death pathways in CD. Here, we have used confocal microscopy, western blot, and RT-qPCR analysis to assess the presence of regulated cell death pathways in the duodenum of CD patients. We found an increased number of dead (TUNEL+) cells in the lamina propria of small intestine of CD patients, most of them are plasma cells (CD138+). Many dying cells expressed FAS and were in close contact with CD3+ T cells. Caspase-8 and caspase-3 expression was increased in CD, confirming the activation of apoptosis. In parallel, caspase-1, IL-1β, and GSDMD were increased in CD samples indicating the presence of inflammasome-dependent pyroptosis. Necroptosis was also present, as shown by the increase of RIPK3 and phosphorylate MLKL. Analysis of published databases confirmed that CD has an increased expression of regulated cell death -related genes. Together, these results reveal that CD is characterized by cell death of different kinds. In particular, the presence of proinflammatory cell death pathways may contribute to mucosal damage.

Record-High Ultrasound-Sensitive NO Nanogenerators for Cascade Tumor Pyroptosis and Immunotherapy.

Pyroptosis is a pro-inflammatory cell death that is associated with innate immunity promotion against tumors. Excess nitric oxide (NO)-triggered nitric stress has potential to induce pyroptosis, but the precise delivery of NO is challenging. Ultrasound (US)-responsive NO production has dominant priority due to its deep penetration, low side effects, noninvasion, and local activation manner. In this work, US-sensitive NO donor N-methyl-N-nitrosoaniline (NMA) with thermodynamically favorable structure is selected and loaded into hyaluronic acid (HA)-modified hollow manganese dioxide nanoparticles (hMnO2 NPs) to fabricate hMnO2 @HA@NMA (MHN) nanogenerators (NGs). The obtained NGs have a record-high NO generation efficiency under US irradiation and can release Mn2+ after targeting the tumor sites. Later on, cascade tumor pyroptosis and cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING)-based immunotherapy is achieved and tumor growth is effectively inhibited.

#4359 LOSS OF GASDERMIN D LEADS TO AN EXACERBATION OF KIDNEY DAMAGE IN NEPHROCALCINOSIS-RELATED CHRONIC KIDNEY DISEASE

Abstract Background and Aims Gasdermin D (GSDMD) is a membrane-targeting and pore-forming protein that is primarily expressed in immune cells. In the proinflammatory cell death process known as pyroptosis, cleaved GSDMD plays a crucial role through cell membrane pore formation, leading to the release of proinflammatory cytokines and membrane rapture. Despite this, the majority of GSDMD's other physiological functions remain to be elucidated. Previous reports have implicated GSDMD in the pathogenesis of kidney disease, utilizing some acute kidney injury (AKI) animal models and a few chronic kidney disease (CKD) animal models. However, it remains uncertain if GSDMD contributes to the pathogenesis of nephrocalcinosis-related CKD. Therefore, this study aimed to investigate the role of GSDMD in in this context. Method In our study, 8-week-old GSDMD-knockout (KO) mice and wildtype (WT) mice received an oxalate-rich diet (50 μmol/g sodium oxalate) for three weeks. All mice were sacrificed on day 21 and kidneys were harvested after the sacrifice. Plasma and urine samples were collected at different time points before the sacrifice. The glomerular filtration rate (GFR) was measured on day 0 and day 20 to assess kidney excretion function. Histological analysis was performed using Periodic Acid-Schiff (PAS) staining to assess renal injury, Sirius Red staining to check renal fibrosis, Pizzolato staining to evaluate calcium crystal deposition, F4/80 staining to evaluate the infiltration of macrophages, TUNEL staining to check for cell death, RIPK3 and phosphorylated MLKL (pMLKL) staining to detecting necroptosis. And GSDMD staining was also performed to examine the expression of GSDMD in the kidney. Results Pizzolato staining showed no differences in calcium crystal deposition between WT mice and KO mice. GFR was decreased in both WT mice and KO mice after the oxalate-rich diet (day 0 WT: 199.5±40.1 μl/min vs KO 175.0±44.8 μl/min, day 20 WT: 156.5±37.9 μl/min vs KO: 73.0±48.1 μl/min), and ΔGFR was much greater in KO mice (WT: −22.4±26.5% vs KO: −59.4±19.3%). The level of blood urea nitrogen was significantly higher in KO mice (WT: 41.9±3.90 mg/dl vs KO: 58.7±22.5 mg/dl) in accordance with the result of the GFR. PAS staining revealed more severe kidney injury in KO mice, and Sirius Red staining indicated a higher degree of renal fibrosis in KO mice. Furthermore, F4/80 staining showed greater infiltration of macrophages in KO mice. Although TUNEL staining showed no significant differences in positive cells between WT and KO, KO mice showed greater positive areas implicating more necroptosis in RIPK3 and pMLKL staining. GSDMD staining revealed that injured tubular cells during the chronic phase express GSDMD (Fig. 1). In in vitro experiments, we evaluated the functionality of macrophages such as phagocytic ability and migratory ability using bone marrow-derived macrophages (BMDMs), but we didn't find any significant disparities between BMDMs from WT mice and those from KO mice. Conclusion Genetic deletion of GSDMD resulted in more severe kidney injury, macrophage infiltration and fibrosis in mice with nephrocalcinosis-related CKD. Our results suggest a potential role for pyroptosis, especially GSDMD, in contributing to renal tubular cell necroptosis, a process downstream of calcium-oxalate crystal formation and deposition, in the progression of nephrocalcinosis-related CKD, similar to previous reports in mouse models of AKI.

Cyclic helix B peptide alleviates proinflammatory cell death and improves functional recovery after traumatic spinal cord injury

BackgroundNecroptosis and pyroptosis, two types of proinflammatory programmed cell death, were recently found to play important roles in spinal cord injury (SCI). Moreover, cyclic helix B peptide (CHBP) was designed to maintain erythropoietin (EPO) activity and protect tissue against the adverse effects of EPO. However, the protective mechanism of CHBP following SCI is still unknown. This research explored the necroptosis- and pyroptosis-related mechanism underlying the neuroprotective effect of CHBP after SCI. MethodsGene Expression Omnibus (GEO) datasets and RNA sequencing were used to identify the molecular mechanisms of CHBP for SCI. A mouse model of contusion SCI was constructed, and HE staining, Nissl staining, Masson staining, footprint analysis and the Basso Mouse Scale (BMS) were applied for histological and behavioural analyses. qPCR, Western blot analysis, immunoprecipitation and immunofluorescence were utilized to analyse the levels of necroptosis, pyroptosis, autophagy and molecules associated with the AMPK signalling pathway. ResultsThe results revealed that CHBP significantly improved functional restoration, elevated autophagy, suppressed pyroptosis, and mitigated necroptosis after SCI. 3-Methyladenine (3-MA), an autophagy inhibitor, attenuated these beneficial effects of CHBP. Furthermore, CHBP-triggered elevation of autophagy was mediated by the dephosphorylation and nuclear translocation of TFEB, and this effect was due to stimulation of the AMPK-FOXO3a-SPK2-CARM1 and AMPK-mTOR signalling pathways. ConclusionCHBP acts as a powerful regulator of autophagy that improves functional recovery by alleviating proinflammatory cell death after SCI and thus might be a prospective therapeutic agent for clinical application.

Inflammasomes: a rising star on the horizon of COVID-19 pathophysiology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a contagious respiratory virus that is the cause of the coronavirus disease 2019 (COVID-19) pandemic which has posed a serious threat to public health. COVID-19 is characterized by a wide spectrum of clinical manifestations, ranging from asymptomatic infection to mild cold-like symptoms, severe pneumonia or even death. Inflammasomes are supramolecular signaling platforms that assemble in response to danger or microbial signals. Upon activation, inflammasomes mediate innate immune defense by favoring the release of proinflammatory cytokines and triggering pyroptotic cell death. Nevertheless, abnormalities in inflammasome functioning can result in a variety of human diseases such as autoimmune disorders and cancer. A growing body of evidence has showed that SARS-CoV-2 infection can induce inflammasome assembly. Dysregulated inflammasome activation and consequent cytokine burst have been associated with COVID-19 severity, alluding to the implication of inflammasomes in COVID-19 pathophysiology. Accordingly, an improved understanding of inflammasome-mediated inflammatory cascades in COVID-19 is essential to uncover the immunological mechanisms of COVID-19 pathology and identify effective therapeutic approaches for this devastating disease. In this review, we summarize the most recent findings on the interplay between SARS-CoV-2 and inflammasomes and the contribution of activated inflammasomes to COVID-19 progression. We dissect the mechanisms involving the inflammasome machinery in COVID-19 immunopathogenesis. In addition, we provide an overview of inflammasome-targeted therapies or antagonists that have potential clinical utility in COVID-19 treatment.

Oxidative Stress Initiates Receptor-Interacting Protein Kinase-3/Mixed Lineage Kinase Domain-Like–Mediated Corneal Epithelial Necroptosis and Nucleotide-Binding Oligomerization Domain–Like Receptor Protein 3 Inflammasome Signaling during Fungal Keratitis

Fungal keratitis remains a major cause of severe visual loss in developing countries because of limited choices of therapy. The progression of fungal keratitis is a race between the innate immune system and the outgrowth of fungal conidia. Programmed necrosis (necroptosis), a type of proinflammatory cell death, has been recognized as a critical pathologic change in several diseases. However, the role and potential regulatory mechanisms of necroptosis have not been investigated in corneal diseases. The current study showed, for the first time, that fungal infection triggered significant corneal epithelial necroptosis in human/mouse/invitro models. Moreover, a reduction in excessive reactive oxygen species release effectively prevented necroptosis. NLRP3 knockout did not affect necroptosis invivo. In contrast, ablation of necroptosis via RIPK3 knockout significantly delayed migration and inhibited the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in macrophages, which enhanced the progression of fungal keratitis. Taking these findings together, the study indicated that overproduction of reactive oxygen species in fungal keratitis leads to significant necroptosis in the corneal epithelium. Furthermore, the necroptotic stimuli-mediated NLRP3 inflammasome serves as a driving force in host defense against fungal infection.

NETosis as an oncologic therapeutic target: a mini review.

Neutrophil Extracellular Traps (NETs) are a key form of pro-inflammatory cell death of neutrophils characterized by the extrusion of extracellular webs of DNA containing bactericidal killing enzymes. NETosis is heavily implicated as a key driver of host damage in autoimmune diseases where injurious release of proinflammatory enzymes damage surrounding tissue and releases 70 known autoantigens. Recent evidence shows that both neutrophils and NETosis have a role to play in carcinogenesis, both indirectly through triggering DNA damage through inflammation, and directly contributing to a pro-tumorigenic tumor microenvironment. In this mini-review, we summarize the current knowledge of the various mechanisms of interaction and influence between neutrophils, with particular attention to NETosis, and cancer cells. We will also highlight the potential avenues thus far explored where we can intercept these processes, with the aim of identifying promising prospective targets in cancer treatment to be explored in further studies.

Quercetin’in HepG2 hücrelerinde, TNF-Alfa ve LPS ile indüklenen enflamasyonda, eritropoietin, HIF-1α, HIF-2α ve piroptoz ile ilgili genlerin ekspresyon seviyeleri üzerine etkileri

Purpose: TNF-α, which increases in the circulation as a result of systemic inflammation, suppresses the production of the erythropoiesis regulating hormone, erythropoietin (EPO) and causes inflammation anemia. The anti-inflammatory activity of quercetin has been reported in different studies. However, no study has been found showing the effect of this situation on EPO gene expression. For this purpose, we evaluated the effects of quercetin on inflammation and, more importantly, its effects on EPO, suppressed in the inflammatory environment and Hypoxia-inducible factors (HIF), which are stimulators of EPO synthesis. In addition, we aimed to evaluate the effect of quercetin on pyroptosis, which is defined as pro-inflammatory programmed cell death.
 Materials and Methods: We created inflammation models with TNF-α or LPS using HepG2 cells in vitro. In these inflammation models, we evaluated the effects of quercetin on proinflammatory mediators TNF-α, IL-1α, NF-κB gene expressions, EPO, HIF-1α, HIF-2α gene expressions, as well as pyroptosis-related caspase 1 and IL-18 gene expressions. 
 Results: Quercetin showed inflammatory effects by increasing TNF-α, IL-1α, and NF-κB mRNA levels. Consistent with this inflammatory effect, EPO mRNA expression was suppressed. HIF-1α and HIF-2α mRNA levels were increased. 
 Conclusion: These results suggested that the increase in HIFs could not prevent the suppressive effect of inflammatory cytokines and NF-κB on EPO production. However, it has been observed that it tends to suppress proinflammatory cell death by decreasing caspase 1 and IL-18 mRNA levels. These results show that quercetin may show conflicting effects, and further studies are needed to test its safety.

The NLRP1 inflammasome in skin diseases.

Healthy human skin is constantly exposed to sterile and microbial agents. The skin immune system plays an important role in immune surveillance between tolerance and immune activation. This is mainly mediated by neutrophils, macrophages and most importantly lymphocytes. Keratinocytes, which form the outer skin barrier (epidermis) are also critical for cutaneous homeostasis. Being a non-professional immune cell, recognition of danger signals in keratinocytes is mediated by innate immune receptors (pattern recognition receptors, PRR). While Toll-like receptors are located on the cell membrane or the endosomes, nucleotide-binding domain and leucine-rich repeat containing gene family receptors (NLR) are intracellular PRRs. Some of these, once activated, trigger the formation of inflammasomes. Inflammasomes are multiprotein complexes and serve as platforms that mediate the release of innate cytokines after successful recognition, thereby attracting immune cells. Moreover, they mediate the pro-inflammatory cell death pyroptosis. Best characterized is the NLRP3 inflammasome. The function of inflammasomes differs significantly between different cell types (keratinocytes versus immune cells) and between different species (human versus mouse). In recent years, great progress has been made in deciphering the activation mechanisms. Dysregulation of inflammasomes can lead to diseases with varying degrees of severity. Here we focus on the structure, function, and associated pathologies of the NLRP1 inflammasome, which is the most relevant inflammasome in keratinocytes.

Membrane damage and repair: a thin line between life and death.

Bilayered membranes separate cells from their surroundings and form boundaries between intracellular organelles and the cytosol. Gated transport of solutes across membranes enables cells to establish vital ion gradients and a sophisticated metabolic network. However, an advanced compartmentalization of biochemical reactions makes cellsalso particularly vulnerable to membrane damage inflicted by pathogens, chemicals, inflammatory responses or mechanical stress. To avoid potentially lethal consequences of membrane injuries, cells continuously monitor the structural integrity of their membranes and readily activate appropriate pathways to plug, patch, engulf or shed the damaged membrane area. Here, we review recent insights into the cellular mechanisms that underly an effective maintenance of membrane integrity. We discuss how cells respond to membrane lesions caused by bacterial toxins and endogenous pore-forming proteins, with a primary focus on the intimate crosstalk between membrane proteins and lipids during wound formation, detection and elimination. We also discuss how a delicate balance between membrane damage and repair determines cell fate upon bacterial infection or activation of pro-inflammatory cell death pathways.

Regulatory T cells alleviate myelin loss and cognitive dysfunction by regulating neuroinflammation and microglial pyroptosis via TLR4/MyD88/NF-κB pathway in LPC-induced demyelination

Demyelination occurs in multiple central nervous system (CNS) disorders and is tightly associated with neuroinflammation. Pyroptosis is a form of pro-inflammatory and lytic cell death which has been observed in CNS diseases recently. Regulatory T cells (Tregs) have exhibited immunoregulatory and protective effects in CNS diseases. However, the roles of Tregs in pyroptosis and their involvement in LPC-induced demyelination have not been explicated. In our study, Foxp3-diphtheria toxin receptor (DTR) mice treated with diphtheria toxin (DT) or PBS were subjected to two-site lysophosphatidylcholine (LPC) injection. Immunofluorescence, western blot, Luxol fast blue (LFB) staining, quantitative real-time PCR (qRT-PCR) and neurobehavior assessments were performed to evaluate the severity of demyelination, neuroinflammation and pyroptosis. Pyroptosis inhibitor was further used to investigate the role of pyroptosis in LPC-induced demyelination. RNA-sequencing was applied to explore the potential regulatory mechanism underlying the involvement of Tregs in LPC-induced demyelination and pyroptosis. Our results showed that depletion of Tregs aggravated microgliosis, inflammatory responses, immune cells infiltration and led to exacerbated myelin injury as well as cognitive defects in LPC-induced demyelination. Microglial pyroptosis was observed after LPC-induced demyelination, which was aggravated by Tregs depletion. Inhibition of pyroptosis by VX765 reversed myelin injury and cognitive function exacerbated by Tregs depletion. RNA-sequencing showed TLR4/myeloid differentiation marker 88 (MyD88) as the central molecules in Tregs-pyroptosis pathway, and refraining TLR4/MyD88/NF-κB pathway alleviated the aggravated pyroptosis induced by Tregs depletion. In conclusion, our findings for the first time indicate that Tregs alleviate myelin loss and improve cognitive function by inhibiting pyroptosis in microglia via TLR4/MyD88/NF-κB pathway in LPC-induced demyelination.

IRAK-M suppresses the activation of microglial NLRP3 inflammasome and GSDMD-mediated pyroptosis through inhibiting IRAK1 phosphorylation during experimental autoimmune encephalomyelitis

The activation of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome triggers pyroptosis proinflammatory cell death in experimental autoimmune encephalomyelitis (EAE). However, the underlying mechanisms of the inflammatory processes of microglia in EAE remain unclear. Our previous studies suggested that interleukin-1 receptor-associated kinase (IRAK)-M down-regulates the toll-like receptor 4/interleukin-1 receptor signaling pathway. Here, we used IRAK-M knockout (IRAK-M−/−) mice and their microglia to dissect the role of IRAK-M in EAE. We found that deletion of IRAK-M increased the incidence rate and exacerbated the clinical symptoms in EAE mice. We then found that IRAK-M deficiency promoted the activation of microglia, activated NLRP3 inflammasomes, and enhanced GSDMD-mediated pyroptosis in the microglia of EAE. In contrast, over-expression of IRAK-M exerted inhibitory effects on neuroinflammation, NLRP3 activation, and pyroptosis. Moreover, IRAK-M deficiency enhanced the phosphorylation of IRAK1, while IRAK-M over-expression downregulated the level of phosphorylated IRAK1. Finally, we found upregulated binding of IRAK1 and TNF receptor-associated factor 6 (TRAF6) in IRAK-M−/− EAE mice compared to WT mice, which was blocked in AAVIRAK-M EAE mice. Our study reveals a complex signaling network of IRAK-M, which negatively regulates microglial NLRP3 inflammasomes and pyroptosis by inhibiting IRAK1 phosphorylation during EAE. These findings suggest a potential target for the novel therapeutic approaches of multiple sclerosis (MS)/EAE and NLRP3-related inflammatory diseases.

Microglial pyroptosis in hippocampus mediates sevolfurane-induced cognitive impairment in aged mice via ROS-NLRP3 inflammasome pathway

BackgroundPostoperative cognitive dysfunction (POCD) is a common complication with its pathophysiological mechanisms not been fully elucidated. Pyroptosis is a novel type of pro-inflammatory cell death and considered to be associated with cognitive dysfunction. Therefore, our study aimed to examine the effect of pyroptosis on sevoflurane-induced cognitive impairment in aged mice as well as its underlying mechanism. MethodsA mice model of cognitive impairment was established by sevoflurane exposure and the levels of reactive oxygen species (ROS), N-GSDMD, cleaved caspase-1, ASC, IL-1β and IL-18, and NLRP3 in hippocampus was determined. To explore the underlying mechanism, a pyroptosis inhibitor, necrosulfonamide (NSA), and a ROS scavenger, N-acetylcysteine (NAC), were administrated before sevoflurane exposure both in vitro and in vivo. Neurobehavioral tests, western blot, transmission electron microscope (TEM) observation, and immunofluorescence staining were performed. ResultsSevoflurane induced hippocampal pyroptosis in the cognitive impairment model. NSA effectively inhibited the pyroptosis and improved cognitive function. Co-labeled immunofluorescence staining suggested sevoflurane induces microglial pyroptosis. Sevoflurane induced pyroptosis accompanied with ROS accumulation in a dose-independent manner in BV2 cells, and NAC effectively reduce the levels of ROS and pyroptosis through NLRP3 inflammasome pathway in both vitro and vivo. Furthermore, NAC could also alleviate sevoflurane-induced cognitive dysfunction. ConclusionsMicroglial pyroptosis in hippocampus mediates sevolfurane-induced cognitive impairment in aged mice via ROS-NLRP3 inflammasome pathway. Both pyroptosis inhibition and ROS scavenging might be potential approaches to ameliorate sevoflurane-induced neurocognitive dysfunction.

Regulation of Inflammasome by microRNAs in Triple-Negative Breast Cancer: New Opportunities for Therapy

During the past decade, researchers have investigated the molecular mechanisms of breast cancer initiation and progression, especially triple-negative breast cancer (TNBC), in order to identify specific biomarkers that could serve as feasible targets for innovative therapeutic strategies development. TNBC is characterized by a dynamic and aggressive nature, due to the absence of estrogen, progesterone and human epidermal growth factor 2 receptors. TNBC progression is associated with the dysregulation of nucleotide-binding oligomerization domain-like receptor and pyrin domain-containing protein 3 (NLRP3) inflammasome, followed by the release of pro-inflammatory cytokines and caspase-1 dependent cell death, termed pyroptosis. The heterogeneity of the breast tumor microenvironment triggers the interest of non-coding RNAs' involvement in NLRP3 inflammasome assembly, TNBC progression and metastasis. Non-coding RNAs are paramount regulators of carcinogenesis and inflammasome pathways, which could help in the development of efficient treatments. This review aims to highlight the contribution of non-coding RNAs that support inflammasome activation and TNBC progression, pointing up their potential for clinical applications as biomarkers for diagnosis and therapy.

An Ionic Liquid Ablation Agent for Local Ablation and Immune Activation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma rapidly acquires resistance to chemotherapy, remaining a fatal disease. Immunotherapy is one of the breakthroughs in cancer treatment, which includes immune checkpoint inhibitors, chimeric antigen receptor T-cell immunotherapy, and neoantigen vaccines. However, immunotherapy has not achieved satisfactory results in the treatment of pancreatic cancer. Immunogenic death comprises proinflammatory cell death, which provides a way to enhance tumor immunogenicity and promote an immune response in solid tumors. Herein, an ionic liquid ablation agent (LAA), synthesized from choline and geranic acid, which triggers necrosis-induced immunotherapy by remodeling an immunosuppressive "cold" tumor to an immune activated "hot" tumor is described. The results indicate that LAA-treated tumor cells can enhance immunogenicity, inducing dendritic cell maturation, macrophage M1 polarization, and cytotoxic T lymphocyte infiltration. The results of the present study provide a novel strategy for solid tumor immunotherapy.

Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14.

This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

SARS-CoV-2 Infection Induces HMGB1 Secretion Through Post-Translational Modification and PANoptosis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces excessive pro-inflammatory cytokine release and cell death, leading to organ damage and mortality. High-mobility group box 1 (HMGB1) is one of the damage-associated molecular patterns that can be secreted by pro-inflammatory stimuli, including viral infections, and its excessive secretion levels are related to a variety of inflammatory diseases. Here, the aim of the study was to show that SARS-CoV-2 infection induced HMGB1 secretion via active and passive release. Active HMGB1 secretion was mediated by post-translational modifications, such as acetylation, phosphorylation, and oxidation in HEK293E/ACE2-C-GFP and Calu-3 cells during SARS-CoV-2 infection. Passive release of HMGB1 has been linked to various types of cell death; however, we demonstrated for the first time that PANoptosis, which integrates other cell death pathways, including pyroptosis, apoptosis, and necroptosis, is related to passive HMGB1 release during SARS-CoV-2 infection. In addition, cytoplasmic translocation and extracellular secretion or release of HMGB1 were confirmed via immunohistochemistry and immunofluorescence in the lung tissues of humans and angiotensin-converting enzyme 2-overexpressing mice infected with SARS-CoV-2.

OASL phase condensation induces amyloid-like fibrillation of RIPK3 to promote virus-induced necroptosis

RIPK3–ZBP1–MLKL-mediated necroptosis is a proinflammatory cell death process that is crucial for antiviral host defence. RIPK3 self-oligomerization and autophosphorylation are prerequisites for executing necroptosis, yet the underlying mechanism of virus-induced RIPK3 activation remains elusive. Interferon-inducible 2′-5′ oligoadenylate synthetase-like (OASL) protein is devoid of enzymatic function but displays potent antiviral activity. Here we describe a role of OASL as a virus-induced necroptosis promoter that scaffolds the RIPK3–ZBP1 non-canonical necrosome via liquid-like phase condensation. This liquid-like platform of OASL recruits RIPK3 and ZBP1 via protein–protein interactions to provide spatial segregation for RIPK3 nucleation. This process facilitates the amyloid-like fibril formation and activation of RIPK3 and thereby MLKL phosphorylation for necroptosis. Mice deficient in Oasl1 exhibit severely impaired necroptosis and attenuated inflammation after viral infection, resulting in uncontrolled viral dissemination and lethality. Our study demonstrates an interferon-induced innate response whereby OASL scaffolds RIPK3–ZBP1 assembly via its phase-separated liquid droplets to facilitate necroptosis-mediated antiviral immunity.

Quantifying Cell Death Induced by the NLRC4 Inflammasome.

The Nod-like Receptor (NLR) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that sense cytosolic bacterial proteins. NAIP ligation induces its association with NLRC4, leading to the assembly of the NAIP/NLRC4 inflammasome, which induces the activation of the caspase-1 protease. Caspase-1 then cleaves pro-interleukin (IL)-1β, pro-IL-18, and gasdermin D and induces a form of pro-inflammatory cell death, pyroptosis. These processes culminate in host defense against bacterial infection. Here we describe methods for activating NAIP/NLRC4 inflammasome signalling in human and murine macrophages and quantifying inflammasome-induced cell death.