Potassium Efflux Research Articles

Upregulated JAK-STAT Signaling and Augmented Potassium Efflux Characterize Induced Pluripotent Stem Cell-Derived Podocytes of Black Patients with APOL1-Associated FSGS

Upregulated JAK-STAT Signaling and Augmented Potassium Efflux Characterize Induced Pluripotent Stem Cell-Derived Podocytes of Black Patients with APOL1-Associated FSGS

K+ regulates relocation of Pellino-2 to the site of NLRP3 inflammasome activation in macrophages.

Pellino proteins are E3 ubiquitin ligases involved in the innate immune system. Recently, Pellino-2 was reported to modulate the activation of the mouse Nlrp3 inflammasome. We examined the intracellular localization of human Pellino-2 in THP1-derived macrophages during activation with LPS and ATP. We observed that Pellino-2 changed intracellular localization and colocalized with the inflammasome proteins NLRP3 and ASC late in the assembly of the inflammasome. Colocalization with NLRP3 and ASC was also seen in cells maintained in potassium-free medium. The colocalization and inflammasome activation were abrogated by several potassium channel inhibitors, supporting a role for potassium efflux in modulating intracellular localization of Pellino-2. The data suggest that Pellino-2 is essential for mediating the effect of potassium efflux on inflammasome activation.

Design, synthesis, molecular docking and DFT computational insight on the structure of Piperazine sulfynol derivatives as a new antibacterial contender against superbugs MRSA

In this investigation, a series of piperazine sulfynol derivatives 5(a-l) were synthesized and amply characterized by various spectral techniques viz, LC-MS, 1H NMR, 13C NMR, and FT-IR. The energy and other quantum chemical computations of all the piperazine sulfynol derivatives were achieved using Density functional theory (DFT), Absorption, Distribution, Metabolism and Excretion (ADME), Blood Brain Barrier (BBB) and their Prediction of Activity Spectra of Computational Screening (PASS) for their possible approaches for biological applications were evaluated. The synthesized compounds were checked for the physicochemical properties such as clogP, clogS, drug-likeness, total surface area, polar surface area, H-acceptor, and H-donor parameters. The considerable change in the design with sulfonyl moiety on the piperazine core motivates to find out new antibacterial contender of the resultant molecules against superbugs-MRSA. All 5(a-l) moieties were tested for antibacterial potency against MRSA. The 5e moiety shows a MIC value of 35±0.41 µg/mL and 9.90±0.03 ZOI in mm compared to drug bacitracin 10 µg/disk (10.21±0.04) and streptomycin 10 µg/disk (14.10±0.06) ZOI in mm. The action of biocidal properties against MRSA was confirmed by an in silico docking study on the protein 1FZP and 3SRW of MRSA. The revealed data strongly recommended 5e having a good docking score, binding energy, and glide energy with high binding affinity. The antibacterial activity was validated by SEM, cellular leakage, potassium efflux and inhibitory effect on electron transport chain. The behavior of the 5e analog in haemostatic condition was well established and cytotoxicity was evaluated against L6 cell lines. The revealed data showed that 5e analog is a potential antibacterial contender against MRSA and it can be used as a futuristic drug in the eradication of MRSA infections.

Glibenclamide alleviates β adrenergic receptor activation-induced cardiac inflammation.

β-Adrenergic receptor (β-AR) overactivation is a major pathological factor associated with cardiac diseases and mediates cardiac inflammatory injury. Glibenclamide has shown anti-inflammatory effects in previous research. However, it is unclear whether and how glibenclamide can alleviate cardiac inflammatory injury induced by β-AR overactivation. In the present study, male C57BL/6J mice were treated with or without the β-AR agonist isoprenaline (ISO) with or without glibenclamide pretreatment. The results indicated that glibenclamide alleviated ISO-induced macrophage infiltration in the heart, as determined by Mac-3 staining. Consistent with this finding, glibenclamide also inhibited ISO-induced chemokines and proinflammatory cytokines expression in the heart. Moreover, glibenclamide inhibited ISO-induced cardiac fibrosis and dysfunction in mice. To reveal the protective mechanism of glibenclamide, the NLRP3 inflammasome was further analysed. ISO activated the NLRP3 inflammasome in both cardiomyocytes and mouse hearts, but this effect was alleviated by glibenclamide pretreatment. Furthermore, in cardiomyocytes, ISO increased the efflux of potassium and the generation of ROS, which are recognized as activators of the NLRP3 inflammasome. The ISO-induced increases in these processes were inhibited by glibenclamide pretreatment. Moreover, glibenclamide inhibited the cAMP/PKA signalling pathway, which is downstream of β-AR, by increasing phosphodiesterase activity in mouse hearts and cardiomyocytes. In conclusion, glibenclamide alleviates β-AR overactivation-induced cardiac inflammation by inhibiting the NLRP3 inflammasome. The underlying mechanism involves glibenclamide-mediated suppression of potassium efflux and ROS generation by inhibiting the cAMP/PKA pathway.

ML365 inhibits TWIK2 channel to block ATP-induced NLRP3 inflammasome.

Dysregulation of NLRP3 inflammasome results in uncontrolled inflammation, which participates in various chronic diseases. TWIK2 potassium channel mediates potassium efflux that has been reported to be an essential upstream mechanism for ATP-induced NLRP3 inflammasome activation. Thus, TWIK2 potassium channel could be a potential drug target for NLRP3-related inflammatory diseases. In the present study we investigated the effects of known K2P channel modulators on TWIK2 channel expressed in a heterologous system. In order to increase plasma membrane expression and thus TWIK2 currents, a mutant channel with three mutations (TWIK2I289A/L290A/Y308A) in the C-terminus was expressed in COS-7 cells. TWIK2 currents were assessed using whole-cell voltage-clamp recording. Among 6 known K2P channel modulators tested (DCPIB, quinine, fluoxetine, ML365, ML335, and TKDC), ML365 was the most potent TWIK2 channel blocker with an IC50 value of 4.07 ± 1.5 μM. Furthermore, ML365 selectively inhibited TWIK2 without affecting TWIK1 or THIK1 channels. We showed that ML365 (1, 5 μM) concentration-dependently inhibited ATP-induced NLRP3 inflammasome activation in LPS-primed murine BMDMs, whereas it did not affect nigericin-induced NLRP3, or non-canonical, AIM2 and NLRC4 inflammasomes activation. Knockdown of TWIK2 significantly impaired the inhibitory effect of ML365 on ATP-induced NLRP3 inflammasome activation. Moreover, we demonstrated that pre-administration of ML365 (1, 10, 25 mg/kg, ip) dose-dependently ameliorated LPS-induced endotoxic shock in mice. In a preliminary pharmacokinetic study conducted in rats, ML365 showed good absolute oral bioavailability with F value of 22.49%. In conclusion, ML365 provides a structural reference for future design of selective TWIK2 channel inhibitors in treating related inflammatory diseases.

Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF.

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1β (IL-1β) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1β in bone marrow-derived macrophages (BMDMs). The increased production of IL-1β was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.

Add necroptosis to your asthma action plan.

Add necroptosis to your asthma action plan.

The Pro-Tumor and Anti-Tumor Effects of NLRP3 Inflammasome as a New Therapeutic Option for Colon Cancer: A Meta-Analysis of Pre-Clinical Studies

Summary: IL-1b secretion is dependent on the assembly and activation of the NLRP3 inflammasome complex. Activation of the NLRP3 inflammasome has been suggested to require two signals. The first (S1) is provided by toll-like-receptor (TLR)-mediated NF-kappa activation and triggers the synthesis of the IL-1B precursor and assembly of NLRP3. The second (S2) can be mediated by danger signals such as stimulation of the purinergic P2X7 receptor by ATP or other stimuli leading to potassium efflux. High dose MBZ (10 lM) may activate both signals whereas low concentrations (1 lM) only can activate S2, potentially through purinergic receptors (P2R). High dose MBZ can induce S1 by activating TLR 8. Objectives: Therefore, in this review, we aimed to elaborate on these findings and explore how NLRP3 inflammasome affect CRC and which mechanism could be a potential therapeutic target. Methods: We systematically searched major indexing databases, including Pubmed/Medline, ISI web of science (WOS), Scopus, EMBASE, and Cochrane central, using standard terms without any language, study region or type restrictions. We collected information regarding all outcome measures, including assessment of IL-1β, TNF-α, IL-18, histological scoring, clinical scoring, and survival rate from animal tissue. Results: Of the 438 studies found through initial searches, 12 met the inclusion criteria. After applying the exclusion criteria, the main properties of 12 articles on 384 animals included in this meta-analysis. Of 12, eight were about anti-tumoral effect, and four were on pro-tumoral effect of inflammasome. The analysis revealed that NLPR3 inflammasome reduced TNFα (SMD:-3.75, 95% CI: -4.49, -3.02, P<0.00001, I2=10%), IL-1β (SMD:-4.65, 95% CI: -6.15, -3.16, P <0.00001, I2=62%), and IL-18 (SMD:-4.20, 95% CI: -6.42, -1.97, P =0.0002, I2=88%) significantly compared with the wild type mice. Conclusion: To translate anti-NLRP3-based anticancer agents from the bench to the bedside, it will be important not only to identify molecules that selectively target NLRP3 or its downstream pathways in malignant cells but also to consider the metabolic heterogeneity of these malignant cells and the mechanisms through which such heterogeneity is connected to cancer. Additional studies to disentangle the molecular and functional complexity of this network are urgently awaited. Funding Information: None. Declaration of Interests: None.

Identification of Tubocapsanolide A as a novel NLRP3 inhibitor for potential treatment of colitis

Increasing evidence have reported that NLRP3 inflammasome has a crucial role in various kinds of immunological diseases including colitis. However, there have only a few drug candidates directly targeting inflammasomes for the therapy of colitis. Here, we first reported that Tubocapsanolide A (TA), a natural small molecule, as a novel inhibitor of NLRP3 inflammasome for the treatment of colitis. TA inhibited the activation of NLRP3 inflammasome and suppressed the secretion of IL-1β and IL-18 in macrophages. Moreover, the ASC oligomerization was inhibited by TA. The assembly of the NLRP3 inflammasome was also restrained by TA, while had little effects on potassium and chloride efflux. Biolayer interferometry analysis showed that TA could directly bind to NLRP3. Importantly, LC-MS/MS analysis further demonstrated that TA covalently bound to the cysteine 514 residue (Cys514) of NLRP3. In vivo experiments showed that TA remarkably ameliorated DSS-induced experimental colitis in mice. However, the protection of TA against DSS-induced experimental colitis was abrogated in NLRP3-deficient (Nlrp3-/-) mice. Taken together, this study indicates TA as a novel inhibitor of NLRP3, which identifies Cys514 as a novel regulatory site of NLRP3 and suggests TA as a promising candidate compound for the treatment of colitis.

Biotransformation of soluble-insoluble lanthanum species and its induced NLRP3 inflammasome activation and chronic fibrosis

Soluble lanthanum (La)(Ⅲ) species that have been extensively used as fertilizers in agriculture can potentially get into the human body through foods and environment. Most soluble La(Ⅲ) species can rapidly transform into insoluble La(Ⅲ) species under physiological conditions, however, their potential biological behavior and chronic toxicity are rarely investigated. In the present study, insoluble La(Ⅲ) species formed under physiological condition were identified as nanoscale or microscale particles, and their major components were found to experience biotransformation process upon contact with cells. Insoluble La(Ⅲ) species could adhere to extracellular membrane or be internalized into cells, capable of activating a nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome. The underlying mechanism could be ascribed to K+ efflux and lysosomal rupture because these insoluble La(Ⅲ) species locating at extracellular membrane could reduce the unsaturated fatty acids of cell membrane, leading to potassium (K+) efflux, and those internalized into cells could consume the phospholipids of lysosomal membrane, leading to lysosomal rupture. Mice daily drinking soluble La(Ⅲ) species to mimic drinking tea process for 90 days were found to present NLRP3 inflammasome activation in liver and kidney, as a result of chronic fibrosis, which is potentially correlated to insoluble La(Ⅲ) species formation.

ATP Facilitates Staphylococcal Enterotoxin O Induced Neutrophil IL-1β Secretion via NLRP3 Inflammasome Dependent Pathways.

Staphylococcus aureus (S. aureus) is an important zoonotic food-borne pathogen causing severe invasive infections, such as sepsis, pneumonia, food poisoning, toxic shock syndrome and autoimmune diseases. Staphylococcal enterotoxin O (SEO) is a new type of enterotoxins of S. aureus with superantigenic and emetic activity. However, it is still unclear about SEO-induced host inflammatory response. Therefore, the mechanism of SEO-induced interleukin-1β (IL-1β) secretion in mouse neutrophils was investigated in this study. Our results showed that recombinant SEO had superantigenic activity with high level of gamma interferon (IFN-γ) production in mouse spleen cells and induced inflammatory cytokines expression including IL-1α, IL-1β, IL-6 and TNF-α in neutrophils under the action of ATP. In addition, SEO-induced IL-1β secretion was dependent on activation of Toll like receptor 4 (TLR4), nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways. However, SEO-induced IL-1β secretion was abolished in the neutrophils of NLRP3-/- mice compared with those of wild type mice, indicating that activation of NLRP3 inflammasome mediated IL-1β secretion during neutrophils stimulation with SEO under the action of ATP. Moreover, this process of SEO+ATP-induced IL-1β secretion was dependent on potassium (K+) efflux. Taken together, our study suggests that activation of TLR4/JNK/NLRP3 inflammasome signaling pathway mediate maturation and secretion of IL-1β and provides a new insight on S. aureus virulence factor-induced host immune response.

Recent advances in the pathophysiology of PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases; PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation, as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets.

Thyrotoxic Periodic Paralysis

Thyrotoxic Periodic Paralysis (TPP) is an emergency associated with flaccid paralysis in which the paralysis is reversible with prompt potassium replacement and the attacks are terminated when hyperthyroidism is cured. Timely diagnosis and treatment are therefore prudent. While managing patients with flaccid paralysis, physicians should be aware of TPP as potential etiology and investigate history to identify the triggering factors and provide timely and cautious treatment with replacement of potassium, further addressing permanent approaches to treating thyrotoxicosis to prevent future recurrences of TPP. We report a clinical scenario of a patient who experienced TPP.22-year-old male, laying down at home at around 1:45pm developed sudden onset bilateral lower extremity weakness and was unable to stand up. His weakness was associated with thigh pain with exertion. He was eventually able to walk a few steps, but then fell onto his knees. He reported having a carbohydrate rich lunch at noon. Notably, patient underwent surgery under general anesthesia for a deviated nasal septum the day before. His neurological examination was remarkable for giveaway proximal lower extremity weakness involving only select muscle groups. Examination and CT/CTA of head and neck findings was not consistent with acute stroke. Myopathy secondary to electrolyte imbalance, drug/toxin, infection, or inflammatory disorders, myelopathy and myasthenia gravis were considered in the differential. Labs revealed suppressed TSH <0.01uIU/mL, elevated FT4 of 6ng/dL and low serum potassium of 2.6mmol/L. CT scan of the neck revealed enlarged thyroid gland and thymic enlargement. Patient was given intravenous bolus of potassium chloride 20 mEq and serum potassium normalized in 4 hours to 3.8mmol/L. His lower extremity weakness resolved within 3hours of potassium replacement. The next day his serum potassium was 5.2mmol/L. His thyroid stimulating immunoglobulin index was elevated at 1.4 (normal <1.3). He was started on propranolol, methimazole and advised to return to the endocrinology clinic to discuss permanent treatment options for hyperthyroidism to prevent TPP. Reviewing his prior medical history, he was diagnosed with Graves’ disease 5 months prior to the emergency room evaluation and was started on methimazole. He took methimazole for 2 months and did not return to follow up until the occurrence of TPP. He did not experience symptoms of hyperthyroidism in the interim. Discussion: TPP is a rare disorder with a prevalence of 0.1-0.2% in North America. TPP commonly affects Asian and Latin Americans males. The episodes of TPP are influenced by genetic, environmental, and ethnic factors. Common environmental triggers include carbohydrate rich meals, rest after intense physical exertion, fever, infection, trauma, emotional stress, and smoking. Thymic hyperplasia has also been reported to be associated with hypokalemic periodic paralysis. Clinical Presentation: The motor weakness tends to affect proximal lower extremities as noted in our patient’s presentation and is usually associated with hyporeflexia and is painless. Cardiac arrhythmias due to hypokalemia are rare, though ventricular fibrillation have been reported, and respiratory failure requiring mechanical ventilation is a possibility. Pathophysiology: TPP has been thought to be a channelopathyassociated with increased Na+–K+ ATPase activity and loss of function mutation of the Kir 2.6 potassium efflux channel resulting in intracellular pooling of potassium and transient hypokalemia. The attacks are stimulated by thyroid hormone excess and/or hyperadrenergic activity and hyperinsulinemia, most commonly due to carbohydrate load or intravenous fluids containing dextrose. Treatment should include:1.Potassium supplementation to reverse paralysis and prevent life threatening cardiac arrhythmias. Care must be taken to not over replace potassium, as it could result in hyperkalemia when potassium channels revert to functioning normally.2.Propranolol, a non-selective betablocker works by blocking the thyroid hormone mediated adrenergic overstimulation of the Na-K ATPase channel, there by limiting the intracellular pooling of potassium.3.Definitive treatment of thyrotoxicosis should be offered. Studies comparing various modalities of treatment of thyrotoxicosis in the setting of TPP indicate permanent treatment with either radioactive iodine or thyroidectomy are often successful in preventing recurrence of TPP, as relapse events are higher in treatment with antithyroid drugs. Caution should be taken to prevent surge of thyroid hormone release following radioactive iodine treatment, as this can trigger TPP. Higher doses of radioactive iodine might be required to render hypothyroidism and prevent TPP recurrences. There is one case report of hypokalemic periodic paralysis associated with thymic hyperplasia that was treated with thymectomy. Our patient had multiple triggers leading to periodic paralysis, including a prior untreated Graves’ disease due to non-adherence to treatment during COVID-19 pandemic, general anesthesia for nasal septal surgery the day before presenting with TPP, having a carbohydrate rich meal one hour before the episode, and thymic enlargement on neck CT scan. He responded well to potassium replacement, and propranolol. He was started on methimazole and offered permanent treatment options to address hyperthyroidism.

NLRP3 and AIM2 inflammasome-triggered Th17 cell response promotes severe immunopathology in schistosomiasis

Abstract Infection with the helminth Schistosoma mansoni can cause exacerbated morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens, with T helper (Th) 17 cells playing a major role in the development of increased granulomatous hepatic immunopathology. A role of inflammasomes in intensifying disease has been reported, however, neither the types of inflammasomes involved, nor their impact on the Th17 response are known. Here we show that the observed enhanced egg-induced IL-1β secretion and pyroptotic cell death required both NLRP3 and AIM2 inflammasome activation. Schistosome genomic DNA activated AIM2, whereas reactive oxygen species, as well as potassium efflux, were the major activators of NLRP3. In addition, dual activation of caspase-1 and caspase-8 was required for egg-induced IL17 production. NLRP3 and AIM2 deficiency led to a significant reduction in Th17 responses, suggesting that both play a crucial role in triggering inflammation. We also determined that inflammasome-induced IL-1β promotes the anti-inflammatory Th2 cytokine IL-4. Our findings establish NLRP3 and AIM2 inflammasomes as central inducers of pathogenic Th17 responses in schistosomiasis.

The voltage-gated potassium channel KV1.3 regulates neutrophil recruitment during inflammation.

AimsNeutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling.Methods and resultsUsing in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3.ConclusionWe show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.

Understanding Why Post-Stroke Depression May Be the Norm Rather Than the Exception: The Anatomical and Neuroinflammatory Correlates of Post-Stroke Depression.

Ischemic Stroke precedes depression. Post-stroke depression (PSD) is a major driver for poor recovery, negative quality of life, poor rehabilitation outcomes and poor functional ability. In this systematic review, we analysed the inflammatory basis of post-stroke depression, which involves bioenergetic failure, deranged iron homeostasis (calcium influx, Na influx, potassium efflux etc), excitotoxicity, acidotoxicity, disruption of the blood brain barrier, cytokine-mediated cytotoxicity, reactive oxygen mediated toxicity, activation of cyclooxygenase pathway and generation of toxic products. This process subsequently results in cell death, maladapted, persistent neuro-inflammation and deranged neuronal networks in mood-related brain regions. Furthermore, an in-depth review likewise reveals that anatomic structures related to post-stroke depression may be localized to complex circuitries involving the cortical and subcortical regions.

Lactic Acid Fermentation Is Required for NLRP3 Inflammasome Activation.

Activation of the Nod-like receptor 3 (NLRP3) inflammasome is important for activation of innate immune responses, but improper and excessive activation can cause inflammatory disease. We previously showed that glycolysis, a metabolic pathway that converts glucose into pyruvate, is essential for NLRP3 inflammasome activation in macrophages. Here, we investigated the role of metabolic pathways downstream glycolysis – lactic acid fermentation and pyruvate oxidation—in activation of the NLRP3 inflammasome. Using pharmacological or genetic approaches, we show that decreasing lactic acid fermentation by inhibiting lactate dehydrogenase reduced caspase-1 activation and IL-1β maturation in response to various NLRP3 inflammasome agonists such as nigericin, ATP, monosodium urate (MSU) crystals, or alum, indicating that lactic acid fermentation is required for NLRP3 inflammasome activation. Inhibition of lactate dehydrogenase with GSK2837808A reduced lactate production and activity of the NLRP3 inflammasome regulator, phosphorylated protein kinase R (PKR), but did not reduce the common trigger of NLRP3 inflammasome, potassium efflux, or reactive oxygen species (ROS) production. By contrast, decreasing the activity of pyruvate oxidation by depletion of either mitochondrial pyruvate carrier 2 (MPC2) or pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) enhanced NLRP3 inflammasome activation, suggesting that inhibition of mitochondrial pyruvate transport enhanced lactic acid fermentation. Moreover, treatment with GSK2837808A reduced MSU-mediated peritonitis in mice, a disease model used for studying the consequences of NLRP3 inflammasome activation. Our results suggest that lactic acid fermentation is important for NLRP3 inflammasome activation, while pyruvate oxidation is not. Thus, reprograming pyruvate metabolism in mitochondria and in the cytoplasm should be considered as a novel strategy for the treatment of NLRP3 inflammasome-associated diseases.

Cortical spreading depolarizations in the context of subarachnoid hemorrhage and the role of ketamine

Delayed cerebral ischemia (DCI) is one of the main complications of spontaneous subarachnoid haemorrhage and one of its causes is the cortical spreading depolarizations (CSDs). Cortical spreading depolarizations are waves of neuronal and glial depolarizations in which there is loss of neuronal ionic homeostasis with potassium efflux and sodium and calcium influx. In damaged brain areas and brain areas at risk, such as those adjacent to subarachnoid haemorrhage (SAH), CSDs induce microvascular vasoconstriction and, therefore, hypoperfusion and spread of ischemia. Several studies have been devoted to minimize secondary injuries that occur hours to days after an acute insult. Ketamine, a drug until recently contraindicated in the neurosurgical population for potentially causing intracranial hypertension, has re-emerged as a potential neuroprotective agent due to its pharmacodynamic effects at the cellular level. These effects include anti-inflammatory mechanisms, and those of microthrombosis and cell apoptosis controls, and of modulation of brain excitotoxicity and CSDs. A literature review was performed at PubMed covering the period from 2002 to 2019. Retrospective studies confirmed the effects of ketamine on the control of CSDs and, consequently, of DCI in patients with SAH, but did not show improvement in clinical outcome. The influence of ketamine on the occurrence/development of DCI needs to be further confirmed in prospective randomized studies.

Tetrahedral DNA nanostructure improves transport efficiency and anti-fungal effect of histatin 5 against Candida albicans.

ObjectivesAnti‐microbial peptides (AMPs) have been comprehensively investigated as a novel alternative to traditional antibiotics against microorganisms. Meanwhile, Tetrahedral DNA nanostructures (TDNs) have gained attention in the field of biomedicine for their premium biological effects and transportation efficiency as delivery vehicles. Hence, in this study, TDN/Histatin 5 (His‐5) was synthesized and the transport efficiency and anti‐fungal effect were measured to evaluate the promotion of His‐5 modified by TDNs.Materials and MethodsTetrahedral DNA nanostructures/His‐5 complex was prepared via electrostatic attraction and characterized by transmission electron microscopy (TEM), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The anti‐fungal effect of the TDN/His‐5 complex was evaluated by determining the growth curve and colony‐forming units of C. albicans. The morphological transformation of C. albicans was observed by light microscope and scanning electron microscope (SEM). Immunofluorescence was performed, and potassium efflux was detected to mechanistically demonstrate the efficacy of TDN/His‐5.ResultsThe results showed that Histatin 5 modified by TDNs had preferable stability in serum and was effectively transported into C. albicans, leading to the increased formation of intracellular reactive oxygen species, higher potassium efflux and enhanced anti‐fungal effect against C. albicans.ConclusionsOur study showed that TDN/His‐5 was synthesized successfully. And by the modification of TDNs, His‐5 showed increased transport efficiency and improved anti‐fungal effect.

SARS-CoV-2, SARS-CoV-1, and HIV-1 derived ssRNA sequences activate the NLRP3 inflammasome in human macrophages through a non-classical pathway

SummaryMacrophages promote an early host response to infection by releasing pro-inflammatory cytokines such as interleukin-1β (IL-1β), TNF, and IL-6. The bioactivity of IL-1β is classically dependent on NLRP3 inflammasome activation, which culminates in caspase-1 activation and pyroptosis. Recent studies suggest a role for NLRP3 inflammasome activation in lung inflammation and fibrosis in both COVID-19 and SARS, and there is evidence of NLRP3 involvement in HIV-1 disease. Here, we show that GU-rich single-stranded RNA (GU-rich RNA) derived from SARS-CoV-2, SARS-CoV-1, and HIV-1 trigger a TLR8-dependent pro-inflammatory cytokine response from human macrophages in the absence of pyroptosis, with GU-rich RNA from the SARS-CoV-2 spike protein triggering the greatest inflammatory response. Using genetic and pharmacological inhibition, we show that the induction of mature IL-1β is through a non-classical pathway dependent on caspase-1, caspase-8, the NLRP3 inflammasome, potassium efflux, and autophagy while being independent of TRIF (TICAM1), vitamin D3, and pyroptosis.