Danger Signals Research Articles

A comprehensive review of sensors of radiation-induced damage, radiation-induced proximal events, and cell death.

Radiation, a universal component of Earth's environment, is categorized into non-ionizing and ionizing forms. While non-ionizing radiation is relatively harmless, ionizing radiation possesses sufficient energy to ionize atoms and disrupt DNA, leading to cell damage, mutation, cancer, and cell death. The extensive use of radionuclides and ionizing radiation in nuclear technology and medical applications has sparked global concern for their capacity to cause acute and chronic illnesses. Ionizing radiation induces DNA damage either directly through strand breaks and base change or indirectly by generating reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radiolysis of water. This damage triggers a complex cellular response involving recognition of DNA damage, cell cycle arrest, DNA repair mechanisms, release of pro-inflammatory cytokines, and cell death. This review focuses on the mechanisms of radiation-induced cellular damage, recognition of DNA damage and subsequent activation of repair processes, and the critical role of the innate immune response in resolution of the injury. Emphasis is placed on pattern recognition receptors (PRRs) and related receptors that detect damage-associated molecular patterns (DAMPs) and initiate downstream signaling pathways. Radiation-induced cell death pathways are discussed in detail. Understanding these processes is crucial for developing strategies to mitigate the harmful effects of radiation and improve therapeutic outcomes.

Ferroptosis-Mediated Inflammation Promotes Pulmonary Hypertension.

Mitochondrial dysfunction, characterized by impaired lipid metabolism and heightened reactive oxygen species generation, results in lipid peroxidation and ferroptosis. Ferroptosis is an inflammatory mode of cell death that promotes complement activation and macrophage recruitment. In pulmonary arterial hypertension (PAH), pulmonary arterial endothelial cells exhibit cellular phenotypes that promote ferroptosis. Moreover, there is ectopic complement deposition and inflammatory macrophage accumulation in the pulmonary vasculature. However, the effects of ferroptosis inhibition on these pathogenic mechanisms and the cellular landscape of the pulmonary vasculature are incompletely defined. Multiomics and physiological analyses evaluated how ferroptosis inhibition-modulated preclinical PAH. The impact of adeno-associated virus 1-mediated expression of the proferroptotic protein ACSL (acyl-CoA synthetase long-chain family member) 4 on PAH was determined, and a genetic association study in humans further probed the relationship between ferroptosis and pulmonary hypertension. Ferrostatin-1, a small-molecule ferroptosis inhibitor, mitigated PAH severity in monocrotaline rats. RNA-sequencing and proteomics analyses demonstrated ferroptosis was associated with PAH severity. RNA-sequencing, proteomics, and confocal microscopy revealed complement activation and proinflammatory cytokines/chemokines were suppressed by ferrostatin-1. In addition, ferrostatin-1 combatted changes in endothelial, smooth muscle, and interstitial macrophage abundance and gene activation patterns as revealed by deconvolution RNA-sequencing. Ferroptotic pulmonary arterial endothelial cell damage-associated molecular patterns restructured the transcriptomic signature and mitochondrial morphology, promoted the proliferation of pulmonary artery smooth muscle cells, and created a proinflammatory phenotype in monocytes in vitro. Adeno-associated virus 1-Acsl4 induced an inflammatory PAH phenotype in rats. Finally, single-nucleotide polymorphisms in 6 ferroptosis genes identified a potential link between ferroptosis and pulmonary hypertension severity in the Vanderbilt BioVU repository. Ferroptosis promotes PAH through metabolic and inflammatory mechanisms in the pulmonary vasculature.

An increase in reactive oxygen species underlies neonatal cerebellum repair.

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirm a transient increased one day after injury at postanal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed leading to reduced cerebellar growth. We also provide evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptative reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

Extracellular RIPK3 Acts as a Danger-Associated Molecular Pattern to Exaggerate Cardiac Ischemia/Reperfusion Injury.

Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease. Currently, there is no effective therapy for reducing cardiac I/R injury. Damage-associated molecular patterns are endogenous molecules released after cellular damage to exaggerate tissue inflammation and injury. RIPK3 (receptor-interacting protein kinase 3), a well-established intracellular mediator of cell necroptosis and inflammation, serves as a circulating biomarker of multiple diseases. However, whether extracellular RIPK3 also exerts biological functions in cardiac I/R injury remains totally unknown. Patients with acute myocardial infarction receiving percutaneous coronary intervention (PCI) were recruited independently in the discovery cohort (103 patients) and validation cohort (334 patients), and major adverse cardiovascular events were recorded. Plasma samples were collected before and after PCI (6 and 24 h) for RIPK3 concentration measurement. Cultured neonatal rat ventricular myocytes, macrophages and endothelial cells, and in vivo mouse models with myocardial injury induced by I/R (or hypoxia/reoxygenation) were used to investigate the role and mechanisms of extracellular RIPK3. Another cohort including patients with acute myocardial infarction receiving PCI and healthy volunteers was recruited to further explore the mechanisms of extracellular RIPK3. In the discovery cohort, elevated plasma RIPK3 levels after PCI are associated with poorer short- and long-term outcomes in patients with acute myocardial infarction, as confirmed in the validation cohort. In both cultured cells and in vivo mouse models, recombinant RIPK3 protein exaggerated myocardial I/R (or hypoxia/reoxygenation) injury, which was alleviated by the RIPK3 antibody. Mechanistically, RIPK3 acted as a damage-associated molecular pattern and bound with RAGE (receptor of advanced glycation end-products), subsequently activating CaMKII (Ca2+/calmodulin-dependent kinase II) to elicit the detrimental effects. The positive correlation between plasma RIPK3 concentrations and CaMKII phosphorylation in human peripheral blood mononuclear cells was confirmed. We identified the positive relationship between plasma RIPK3 concentrations and the risk of major adverse cardiovascular events in patients with acute myocardial infarction receiving PCI. As a damage-associated molecular pattern, extracellular RIPK3 plays a causal role in multiple pathological conditions during cardiac I/R injury through RAGE/CaMKII signaling. These findings expand our understanding of the physiological and pathological roles of RIPK3, and also provide a promising therapeutic target for myocardial I/R injury and the associated complications.

Immunological Strategies in Gastric Cancer: How Toll-like Receptors 2, -3, -4, and -9 on Monocytes and Dendritic Cells Depend on Patient Factors?

(1) Introduction: Toll-like receptors (TLRs) are key in immune response by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In gastric cancer (GC), TLR2, TLR3, TLR4, and TLR9 are crucial for modulating immune response and tumor progression. (2) Objective: This study aimed to assess the percentage of dendritic cells and monocytes expressing TLR2, TLR3, TLR4, and TLR9, along with the concentration of their soluble forms in the serum of GC patients compared to healthy volunteers. Factors such as disease stage, tumor type, age, and gender were also analyzed. (3) Materials and Methods: Blood samples from newly diagnosed GC patients and healthy controls were immunophenotyped using flow cytometry to assess TLR expression on dendritic cell subpopulations and monocytes. Serum-soluble TLRs were measured by ELISA. Statistical analysis considered clinical variables such as tumor type, stage, age, and gender. (4) Results: TLR expression was significantly higher in GC patients, except for TLR3 on classical monocytes. Soluble forms of all TLRs were elevated in GC patients, with significant differences based on disease stage but not tumor type, except for serum TLR2, TLR4, and TLR9. (5) Conclusions: Elevated TLR expression and soluble TLR levels in GC patients suggest a role in tumor pathogenesis and progression, offering potential biomarkers and therapeutic targets.

Robust and Sustained STING Pathway Activation via Hydrogel-Based In Situ Vaccination for Cancer Immunotherapy.

The stimulator of interferon genes (STING) pathway is crucial for tumor immunity, leading to the exploration of STING agonists as potential immunotherapy adjuvants. However, their clinical application faces obstacles including poor pharmacokinetics, transient activation, and an immunosuppressive tumor microenvironment (TME). Addressing these limitations, our study aims to develop an injectable silk fibroin hydrogel-based in situ vaccine. It incorporates a nanoscale STING agonist, an immunogenic cell death (ICD) inducer, and an immunomodulator to ensure their controlled and sustained release. cGAMP nanoparticles (cGAMPnps) with a core-shell structure ensure optimal delivery of cGAMP to dendritic cells (DCs), thereby activating the STING pathway and fostering DC maturation. ICD-associated damage-associated molecular patterns amplify and prolong STING activation via enhanced type I IFN and other inflammatory pathways, along with delayed degradation of cGAMP and STING. Furthermore, the STING-driven vascular normalization by cGAMPnps and ICD, in conjunction with immunomodulators like antiprogrammed cell death protein 1 antibody (anti-PD-1 Ab) or OX40 ligand (OX40L), effectively remodels the immunosuppressive TME. This in situ gel vaccine, when used independently or with surgery as neoadjuvant/adjuvant immunotherapy, enhances DC and CD8+ T-cell activation, suppressing tumor progression and recurrence across various immunologically cold tumor models. It revolutionizes the application of STING agonists in cancer immunotherapy, offering substantial promise for improving outcomes across a broad spectrum of malignancies.

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection weremeasured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus hasgreat potential to modulate the TIME in PDAC.

Hemolymph protease-17b activates proHP6 to stimulate melanization and Toll signaling in Manduca sexta

Manduca sexta hemolymph protease-6 (HP6) plays a central role in coordinating antimicrobial responses, such as prophenoloxidase (PPO) activation and Toll signaling. Our previous studies indicated that HP5 and GP6 activate proHP6 in larval hemolymph and extraembryonic tissues, respectively. Here, we report the characterization of HP17b as another HP6 activating enzyme and its regulation by multiple serpins in hemolymph. The precursor of HP17b expressed in baculovirus infected Sf9 cells became spontaneously cleaved at two sites, and these products were purified together in one preparation named HP17b′, a mixture of proHP17b, a 35 kDa intermediate, and HP17b. HP17b′ converted proHP6 to HP6. As reported before, HP6 converted precursors of PPO activating protease-1 (PAP1) and HP8 to their active forms. HP8 activates proSpӓtzle-1 to turn on Toll signaling. We found HP17b′ directly activated proSPHI and II to form a cofactor for PPO activation by PAP1. Supplementation of larval hemolymph with HP17b′, HP17b, or proHP17b significantly increased PPO activation. Adding Micrococcus luteus to the reactions did not enhance PPO activation in the reactions containing HP17b′, HP17b, or proHP17b. Using HP17b antibodies, we isolated from induced plasma HP17b fragments and associated proteins (e.g., serpin-4). Serpin-1A, 1J, 1J′, 4, 5, or 6 reduced the activation of proHP6 by HP17b’ through formation of covalent complexes with active HP17b. We detected an activity for proHP17b cleavage in hemolymph from bar-stage pharate pupae but failed to purify the protease due to its high instability. Other known HPs did not activate proHP17b in vitro. Together, these results suggest that HP17b is a clip-domain protease activated by an unknown endopeptidase in response to a danger signal and regulated by multiple serpins.

Cancer cell-selective induction of mitochondrial stress and immunogenic cell death by PT-112 in human prostate cell lines

PT-112 is a novel immunogenic cell death (ICD)-inducing small molecule currently under Phase 2 clinical development, including in metastatic castration-resistant prostate cancer (mCRPC), an immunologically cold and heterogeneous disease state in need of novel therapeutic approaches. PT-112 has been shown to cause ribosome biogenesis inhibition and organelle stress followed by ICD in cancer cells, culminating in anticancer immunity. In addition, clinical evidence of PT-112-driven immune effects has been observed in patient immunoprofiling. Given the unmet need for immune-based therapies in prostate cancer, along with a Phase I study (NCT#02266745) showing PT-112 activity in mCRPC patients, we investigated PT-112 effects in a panel of human prostate cancer cell lines. PT-112 demonstrated cancer cell selectivity, inhibiting cell growth and leading to cell death in prostate cancer cells without affecting the non-tumorigenic epithelial prostate cell line RWPE-1 at the concentrations tested. PT-112 also caused caspase-3 activation, as well as stress features in mitochondria including ROS generation, compromised membrane integrity, altered respiration, and morphological changes. Moreover, PT-112 induced damage-associated molecular pattern (DAMP) release, the first demonstration of ICD in human cancer cell lines, in addition to autophagy initiation across the panel. Taken together, PT-112 caused selective stress, growth inhibition and death in human prostate cancer cell lines. Our data provide additional insight into mitochondrial stress and ICD in response to PT-112. PT-112 anticancer immunogenicity could have clinical applications and is currently under investigation in a Phase 2 mCRPC study.

Persistent Activation of the P2X7 Receptor Underlies Chronic Inflammation and Carcinogenic Changes in the Intestine.

Aberrant signaling through damage-associated molecular patterns (DAMPs) has been linked to several health disorders, attracting considerable research interest over the last decade. Adenosine triphosphate (ATP), a key extracellular DAMP, activates the purinergic receptor P2X7, which acts as a danger sensor in immune cells and is implicated in distinct biological functions, including cell death, production of pro-inflammatory cytokines, and defense against microorganisms. In addition to driving inflammation mediated by immune and non-immune cells, the persistent release of endogenous DAMPs, including ATP, has been shown to result in epigenetic modifications. In intestinal diseases such as inflammatory bowel disease (IBD) and colorectal cancer (CRC), consequent amplification of the inflammatory response and the resulting epigenetic reprogramming may impact the development of pathological changes associated with specific disease phenotypes. P2X7 is overexpressed in the gut mucosa of patients with IBD, whereas the P2X7 blockade prevents the development of chemically induced experimental colitis. Recent data suggest a role for P2X7 in determining gut microbiota composition. Regulatory mechanisms downstream of the P2X7 receptor, combined with signals from dysbiotic microbiota, trigger intracellular signaling pathways and inflammasomes, intensify inflammation, and foster colitis-associated CRC development. Preliminary studies targeting the ATP-P2X7 pathway have shown favorable therapeutic effects in human IBD and experimental colitis.

PAMAM-Based Polymeric Immunogenic Cell Death Inducer To Potentiate Cancer Immunotherapy.

Immunogenic cell death (ICD) has been widely employed to potentiate cancer immunotherapy due to its capability to activate the anticancer immune response. Although various ICD inducers have been described, the development of synthetic materials with intrinsic ICD-inducing competency has rarely been reported. Herein, we identify a derivative of the fourth generation polyamidoamine (PAMAM) modified with multiple seven-membered heterocyclic rings, G4P-C7A, as a robust ICD inducer. G4P-C7A evokes characteristic release of damage-associated molecular patterns in tumor cells and induces efficient dendritic cell maturation. Mechanistic studies suggest that G4P-C7A can selectively accumulate in the endoplasmic reticulum and mitochondria to generate reactive oxygen species. G4P-C7A-treated tumor cells can work as potent vaccines to protect against secondary tumor implantation. Either local or systemic injection of G4P-C7A alone can effectively inhibit tumor growth by eliciting robust antitumor immune response. The combination of G4P-C7A with immunotherapeutic antibodies such as anti-PD1 (aPD-1) and anti-CD47 (aCD47) further potentiates the antitumor effect in either CT26 or 4T1 tumor model. This study offers a simple but effective strategy to induce ICD to boost cancer immunotherapy.

DAMPs Drive Fibroinflammatory Changes in the Glaucomatous ONH.

The optic nerve head (ONH) is well known to be the initial site of glaucomatous damage; however, the molecular mechanisms initiating this pathology are not fully understood. To further understand the initiating factors in glaucomatous damage we utilized a novel mouse model of glaucoma, B6.EDA+/+ mice, which constitutively express fibronectin containing the extra domain A (FN+EDA). FN+EDA is a known damage-associated molecular pattern (DAMP) that activates Toll-like receptor 4 and elicits a fibro-inflammatory response. Eyes from B6.EDA+/+ and C57BL/6J mice were evaluated for retinal ganglion cell (RGC) death, retinal nerve fiber layer (RNFL) thickness, and optic nerve (ON) damage at 12 months and 22 months of age. ONH sections were isolated using laser capture microdissection for subsequent RNA-sequencing and Gene Set Enrichment Analysis (GSEA). GSEA results were confirmed using immunohistochemical (IHC) staining. B6.EDA+/+ mice exhibit significantly higher intraocular pressure, loss of RGCs, thinning of the RNFL, and progressive levels of ON damage at 12 months and 22 months of age compared to C57BL/6J controls. Protein expression of DAMPs FN+EDA and biglycan was significantly increased in B6.EDA+/+ mice compared to C57BL/6J controls. GSEA analysis identified significantly up- and downregulated gene groupings at both 12 months and 22 months of age, and IHC staining at 12 and 18 months of age demonstrated significant increases of IFNα, IFNβ, and pSTAT1 expression in B6.EDA+/+ mice compared to C57BL/6J controls. Our study characterizes glaucomatous changes to the retina, ON, and ONH over the course of 2 years and identifies novel molecular pathways associated with these pathophysiological changes. These data illustrate the effects of FN+EDA on the fibro-inflammatory response in the aging ONH in a novel mouse model of glaucoma.

Revealing the mechanism of natural product-induced immunogenic cell death: opening a new chapter in tumor immunotherapy.

This review underscores the role of natural products in inducing immunogenic cell death (ICD) as a key strategy in tumor immunotherapy. It reveals that natural products can activate ICD through multiple pathways-apoptosis, autophagy, pyroptosis, and necroptosis-leading to the release of danger-associated molecular patterns (DAMPs), dendritic cell activation, and improved antigen presentation, which together stimulate a potent anti-tumor immune response. The study also demonstrates the enhanced therapeutic potential of combining natural products with immune checkpoint inhibitors. With a focus on translating preclinical findings into clinical practice, this review consolidates recent discoveries and suggests future research paths, offering both theoretical insights and practical guidance for advancing cancer immunotherapy.

Damage-associated molecular patterns in bacteraemic infection, including a comparative analysis with bacterial DNA, a pathogen-associated molecular pattern

Damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) are key triggers of inflammation in sepsis. However, they have rarely been studied simultaneously. Thus, in the present study of patients with bacteraemic infection, we aimed to study how DAMP dynamics are linked to disease severity and outcome and to compare diagnostic and prognostic properties of a DAMP and a previously analysed PAMP (16S rDNA). In a prospective study of adult patients hospitalized with culture-proven community-onset bacteraemic infection, caused by Streptococcus pneumonia (n = 30), Staphylococcus aureus (n = 27), or Escherichia coli (n = 26), dynamics of a PAMP, i.e. 16S rDNA, have previously been presented. For the present study, blood samples obtained on hospital days 1–2 (when blood culture was positive), 3–4, 7 ± 1, 14 ± 2, and 28 ± 4 were analysed for four different DAMPs, i.e., nuclear DNA (nDNA), mitochondrial DNA (mtDNA), heat shock protein 90 alpha (HSP90α), and extracellular high mobility group box 1 (HMGB1). Sepsis was defined according to the Sepsis-3 criteria. The study outcomes were sepsis at admission and negative outcome, defined as intensive care unit (ICU) admission and/or death within 60 days. Of 83 study patients, sepsis was noted in 41 patients (49%) and a negative outcome was noted in 17 patients (20%). nDNA had areas under the receiver operating characteristic (ROC) curves of 0.78 for sepsis and 0.76 for negative outcome, which were higher than those of the other DAMPs and additional biomarkers (CRP, IL-6, IL-8, and IL-10). The nDNA and positive 16S rDNA results on day 1–2 were correlated with each other (r = 0.68, p < 0.001). Multivariate analyses showed that high day 1–2 concentrations of both nDNA and 16S rDNA were independently associated with sepsis. In addition, high day 1–2 concentration of nDNA was independently associated with negative outcomes. While 16S rDNA dissipated from the circulation within days, nDNA concentrations remained elevated throughout the follow-up period in patients with negative outcome. In conclusion, nDNA outperformed the other DAMPs regarding sepsis detection and outcome prediction. Both nDNA (a DAMP) and 16S rDNA (a PAMP) were independently linked to sepsis; nDNA was also associated with negative outcomes and persisted elevated in such cases. This highlights nDNA as an interesting marker within sepsis pathogenesis and as a promising clinical biomarker, warranting further studies.

NINJ1-mediated Macrophage Plasma Membrane Rupture and Neutrophil Extracellular Trap Formation Contribute to Oxalate Nephropathy.

Oxalate nephropathy is characterized by calcium oxalate crystals deposition, which triggers necrosis of renal tubular epithelial cells, initiates an inflammatory cascade characterized by neutrophil and macrophage activation within the renal microenvironment. Despite the close association of immune cells with acute oxalate nephropathy, the underlying mechanisms still remain unclear. Nerve injury-induced protein 1 (NINJ1) plays an essential role in the induction of plasma membrane rupture (PMR), leading to damage-associated molecular patterns (DAMPs) release and triggering inflammation. We hypothesize that NINJ1-mediated high mobility group box 1 (HMGB1) release from macrophage PMR and neutrophil extracellular traps (NETs) formation synergistically contribute to the progression of acute oxalate nephropathy. Using a murine model of acute oxalate nephropathy, myeloid cell-specific deletion of Ninj1 mice (Ninj1fl/flvavcre) and their wild-type littermate control mice (Ninj1wt/wtvavcre) were administered intraperitoneal injection of 100 mg/kg sodium oxalate followed by drinking water with 3% sodium oxalate. Evaluation was conducted on tubular injury and inflammatory cell infiltration. In vitro studies involved isolation and culture of renal proximal tubular epithelial cells (RTECs), bone marrow-derived macrophages, and neutrophils to investigate NETs formation and HMGB1 release. Targeted deletion of Ninj1 in myeloid cells significantly mitigated oxalate-induced acute kidney injury by suppressing both HMGB1 release and NETs formation in vivo. In vitro investigations demonstrated that HMGB1 release from macrophage PMR and NETs formation in neutrophils mediated by NINJ1 oligomerization, which consequently coordinated to enhance renal tubular epithelial cell death. Our findings elucidate the pivotal role of NINJ1-dependent macrophage PMR and NETs formation in the progression of acute oxalate nephropathy, providing novel insights for its prevention and therapeutic targets.

Elevated serum mtDNA in COVID-19 patients is linked to SARS-CoV-2 envelope protein targeting mitochondrial VDAC1, inducing apoptosis and mtDNA release

Mitochondria dysfunction is implicated in cell death, inflammation, and autoimmunity. During viral infections, some viruses employ different strategies to disrupt mitochondria-dependent apoptosis, while others, including SARS-CoV-2, induce host cell apoptosis to facilitate replication and immune system modulation. Given mitochondrial DNAs (mtDNA) role as a pro-inflammatory damage-associated molecular pattern in inflammatory diseases, we examined its levels in the serum of COVID-19 patients and found it to be high relative to levels in healthy donors. Furthermore, comparison of serum protein profiles between healthy individuals and SARS-CoV-2-infected patients revealed unique bands in the COVID-19 patients. Using mass spectroscopy, we identified over 15 proteins, whose levels in the serum of COVID-19 patients were 4- to 780-fold higher. As mtDNA release from the mitochondria is mediated by the oligomeric form of the mitochondrial-gatekeeper—the voltage-dependent anion-selective channel 1 (VDAC1)—we investigated whether SARS-CoV-2 protein alters VDAC1 expression. Among the three selected SARS-CoV-2 proteins, small envelope (E), nucleocapsid (N), and accessory 3b proteins, the E-protein induced VDAC1 overexpression, VDAC1 oligomerization, cell death, and mtDNA release. Additionally, this protein led to mitochondrial dysfunction, as evidenced by increased mitochondrial ROS production and cytosolic Ca2+ levels. These findings suggest that SARS-CoV-2 E-protein induces mitochondrial dysfunction, apoptosis, and mtDNA release via VDAC1 modulation. mtDNA that accumulates in the blood activates the cGAS-STING pathway, triggering inflammatory cytokine and chemokine expression that contribute to the cytokine storm and tissue damage seen in cases of severe COVID-19.

Branched oncolytic peptides target HSPGs, inhibit metastasis, and trigger the release of molecular determinants of immunogenic cell death in pancreatic cancer.

Immunogenic cell death (ICD) can be exploited to treat non-immunoreactive tumors that do not respond to current standard and innovative therapies. Not all chemotherapeutics trigger ICD, among those that do exert this effect, there are anthracyclines, irinotecan, some platinum derivatives and oncolytic peptides. We studied two new branched oncolytic peptides, BOP7 and BOP9 that proved to elicit the release of damage-associated molecular patterns DAMPS, mediators of ICD, in pancreatic cancer cells. The two BOPs selectively bound and killed tumor cells, particularly PANC-1 and Mia PaCa-2, but not cells of non-tumor origin such as RAW 264.7, CHO-K1 and pgsA-745. The cancer selectivity of the two BOPs may be attributed to their repeated cationic sequences, which enable multivalent binding to heparan sulfate glycosaminoglycans (HSPGs), bearing multiple anionic sulfation patterns on cancer cells. This interaction of BOPs with HSPGs not only fosters an anti-metastatic effect in vitro, as demonstrated by reduced adhesion and migration of PANC-1 cancer cells, but also shows promising tumor-specific cytotoxicity and low hemolytic activity. Remarkably, the cytotoxicity induced by BOPs triggers the release of DAMPs, particularly HMGB1, IFN-β and ATP, by dying cells, persisting longer than the cytotoxicity of conventional chemotherapeutic agents such as irinotecan and daunorubicin. An in vivo assay in nude mice showed an encouraging 20% inhibition of tumor grafting and growth in a pancreatic cancer model by BOP9.

Molecular mechanisms of emerging inflammasome complexes and their activation and signaling in inflammation and pyroptosis.

Inflammasomes are multi-protein complexes that assemble within the cytoplasm of mammalian cells in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), driving the secretion of the pro-inflammatory cytokines IL-1β and IL-18, and pyroptosis. The best-characterized inflammasome complexes are the NLRP3, NAIP-NLRC4, NLRP1, AIM2, and Pyrin canonical caspase-1-containing inflammasomes, and the caspase-11 non-canonical inflammasome. Newer inflammasome sensor proteins have been identified, including NLRP6, NLRP7, NLRP9, NLRP10, NLRP11, NLRP12, CARD8, and MxA. These inflammasome sensors can sense PAMPs from bacteria, viruses and protozoa, or DAMPs in the form of mitochondrial damage, ROS, stress and heme. The mechanisms of action, physiological relevance, consequences in human diseases, and avenues for therapeutic intervention for these novel inflammasomes are beginning to be realized. Here, we discuss these emerging inflammasome complexes and their putative activation mechanisms, molecular and signaling pathways, and physiological roles in health and disease.

Human iPSC-based disease modeling studies identify a common mechanistic defect and potential therapies for AMD and related macular dystrophies.

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) primarily affect the retinal pigment epithelium (RPE) in the eye. A hallmark of AMD/MDs that drives later-stage pathologies is drusen. Drusen are sub-RPE lipid-protein-rich extracellular deposits, but how drusen forms and accumulates is not known. We utilized human induced pluripotent stem cell (iPSC)-derived RPE from patients with AMD and three distinct MDs to demonstrate that reduced activity of RPE-secreted matrix metalloproteinase 2 (MMP2) contributes to drusen in multiple maculopathies in a genotype-agnostic manner by instigating sterile inflammation and impaired lipid homeostasis via damage-associated molecular pattern molecule (DAMP)-mediated activation of receptor for advanced glycation end-products (RAGE) and increased secretory phospholipase 2-IIA (sPLA2-IIA) levels. Therapeutically, RPE-specific MMP2 supplementation, RAGE-antagonistic peptide, and a small molecule inhibitor of sPLA2-IIA ameliorated drusen accumulation in AMD/MD iPSC-RPE. Ultimately, this study defines a causal role of the MMP2-DAMP-RAGE-sPLA2-IIA axis in AMD/MDs.

Hepatocyte activation and liver injury following cerebral ischemia promote HMGB1-mediated hepcidin upregulation in hepatocytes and regulation of systemic iron levels

We previously reported that high mobility group box 1 (HMGB1), a danger-associated molecular pattern (DAMP), increases intracellular iron levels in the postischemic brain by upregulating hepcidin, a key regulator of iron homeostasis, triggering ferroptosis. Since hepatocytes are the primary cells that produce hepcidin and control systemic iron levels, we investigated whether cerebral ischemia induces hepcidin upregulation in hepatocytes. Following middle cerebral artery occlusion (MCAO) in a rodent model, significant liver injury was observed. This injury was evidenced by significantly elevated Eckhoff’s scores and increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Additionally, total iron levels were significantly elevated in the liver, with intracellular iron accumulation detected in hepatocytes. Hepcidin expression in the liver, which is primarily localized in hepatocytes, increased significantly starting at 3 h after MCAO and continued to increase rapidly, reaching a peak at 24 h. Interestingly, HMGB1 levels in the liver were also significantly elevated after MCAO, with the disulfide form of HMGB1 being the major subtype. In vitro experiments using AML12 hepatocytes showed that recombinant disulfide HMGB1 significantly upregulated hepcidin expression in a Toll-like receptor 4 (TLR4)- and RAGE-dependent manner. Furthermore, treatment with a ROS scavenger and a peptide HMGB1 antagonist revealed that both ROS generation and HMGB1 induction contributed to hepatocyte activation and liver damage following MCAO–reperfusion. In conclusion, this study revealed that cerebral ischemia triggers hepatocyte activation and liver injury. HMGB1 potently induces hepcidin not only in the brain but also in the liver, thereby influencing systemic iron homeostasis following ischemic stroke.