Potassium Efflux Research Articles

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Potassium Efflux System Kef: Bacterial Protection against Toxic Electrophilic Compounds.

Kef couples the potassium efflux with proton influx in gram-negative bacteria. The resulting acidification of the cytosol efficiently prevents the killing of the bacteria by reactive electrophilic compounds. While other degradation pathways for electrophiles exist, Kef is a short-term response that is crucial for survival. It requires tight regulation since its activation comes with the burden of disturbed homeostasis. Electrophiles, entering the cell, react spontaneously or catalytically with glutathione, which is present at high concentrations in the cytosol. The resulting glutathione conjugates bind to the cytosolic regulatory domain of Kef and trigger activation while the binding of glutathione keeps the system closed. Furthermore, nucleotides can bind to this domain for stabilization or inhibition. The binding of an additional ancillary subunit, called KefF or KefG, to the cytosolic domain is required for full activation. The regulatory domain is termed K+ transport-nucleotide binding (KTN) or regulator of potassium conductance (RCK) domain, and it is also found in potassium uptake systems or channels in other oligomeric arrangements. Bacterial RosB-like transporters and K+ efflux antiporters (KEA) of plants are homologs of Kef but fulfill different functions. In summary, Kef provides an interesting and well-studied example of a highly regulated bacterial transport system.

Gasdermin-D activation promotes NLRP3 activation and host resistance to Leishmania infection

Intracellular parasites from the Leishmania genus cause Leishmaniasis, a disease affecting millions of people worldwide. NLRP3 inflammasome is key for disease outcome, but the molecular mechanisms upstream of the inflammasome activation are still unclear. Here, we demonstrate that despite the absence of pyroptosis, Gasdermin-D (GSDMD) is active at the early stages of Leishmania infection in macrophages, allowing transient cell permeabilization, potassium efflux, and NLRP3 inflammasome activation. Further, GSDMD is processed into a non-canonical 25 kDa fragment. Gsdmd–/– macrophages and mice exhibit less NLRP3 inflammasome activation and are highly susceptible to infection by several Leishmania species, confirming the role of GSDMD for inflammasome-mediated host resistance. Active NLRP3 inflammasome and GSDMD are present in skin biopsies of patients, demonstrating activation of this pathway in human leishmaniasis. Altogether, our findings reveal that Leishmania subverts the normal functions of GSDMD, an important molecule to promote inflammasome activation and immunity in Leishmaniasis.

Comparative Transcriptome Profiling Reveals Key MicroRNAs and Regulatory Mechanisms for Aluminum Tolerance in Olive.

Aluminum toxicity (Al) is one of the major constraints to crop production in acidic soils. MicroRNAs (miRNAs) have emerged as key regulatory molecules at post-transcriptional levels, playing crucial roles in modulating various stress responses in plants. However, miRNAs and their target genes conferring Al tolerance are poorly studied in olive (Olea europaea L.). Here, genome-wide expression changes in miRNAs of the roots from two contrasting olive genotypes Zhonglan (ZL, Al-tolerant) and Frantoio selezione (FS, Al-sensitive) were investigated by high-throughput sequencing approaches. A total of 352 miRNAs were discovered in our dataset, consisting of 196 conserved miRNAs and 156 novel miRNAs. Comparative analyses showed 11 miRNAs have significantly different expression patterns in response to Al stress between ZL and FS. In silico prediction identified 10 putative target gene of these miRNAs, including MYB transcription factors, homeobox-leucine zipper (HD-Zip) proteins, auxin response factors (ARF), ATP-binding cassette (ABC) transporters and potassium efflux antiporter. Further functional classification and enrichment analysis revealed these Al-tolerance associated miRNA-mRNA pairs are mainly involved in transcriptional regulation, hormone signaling, transportation and metabolism. These findings provide new information and perspectives into the regulatory roles of miRNAs and their target for enhancing Al tolerance in olives.

Inhibition of sphingosine-1-phosphate receptor 3 suppresses ATP-induced NLRP3 inflammasome activation in macrophages via TWIK2-mediated potassium efflux.

Inhibition of sphingosine kinase 1 (SphK1), which catalyzes bioactive lipid sphingosine-1-phosphate (S1P), attenuates NLRP3 inflammasome activation. S1P exerts most of its function by binding to S1P receptors (S1PR1-5). The roles of S1P receptors in NLRP3 inflammasome activation remain unclear. The mRNA expressions of S1PRs in bone marrow-derived macrophages (BMDMs) were measured by real-time quantitative polymerase chain reaction (qPCR) assays. BMDMs were primed with LPS and stimulated with NLRP3 activators, including ATP, nigericin, and imiquimod. Interleukin-1β (IL-1β) in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Intracellular potassium was labeled with a potassium indicator and was measured by confocal microscopy. Protein expression in whole-cell or plasma membrane fraction was measured by Western blot. Cecal ligation and puncture (CLP) was induced in C57BL/6J mice. Mortality, lung wet/dry ratio, NLRP3 activation, and bacterial loads were measured. Macrophages expressed all five S1PRs in the resting state. The mRNA expression of S1PR3 was upregulated after lipopolysaccharide (LPS) stimulation. Inhibition of S1PR3 suppressed NLRP3 and pro-IL-1β in macrophages primed with LPS. Inhibition of S1PR3 attenuated ATP-induced NLRP3 inflammasome activation, enhanced nigericin-induced NLRP3 activation, and did not affect imiquimod-induced NLRP3 inflammasome activation. In addition, inhibition of S1PR3 suppressed ATP-induced intracellular potassium efflux. Inhibition of S1PR3 did not affect the mRNA or protein expression of TWIK2 in LPS-primed BMDMs. ATP stimulation induced TWIK2 expression in the plasma membrane of LPS-primed BMDMs, and inhibition of S1PR3 impeded the membrane expression of TWIK2 induced by ATP. Compared with CLP mice treated with vehicle, CLP mice treated with the S1PR3 antagonist, TY52156, had aggravated pulmonary edema, increased bacterial loads in the lung, liver, spleen, and blood, and a higher seven-day mortality rate. Inhibition of S1PR3 suppresses the expression of NLRP3 and pro-IL-1β during LPS priming, and attenuates ATP-induced NLRP3 inflammasome activation by impeding membrane trafficking of TWIK2 and potassium efflux. Although inhibition of S1PR3 decreases IL-1β maturation in the lungs, it leads to higher bacterial loads and mortality in CLP mice.

Species- and diet-specific aquaculture wastewater nutrient profile: Implications for aquaponics and development of sustainable aquaponics diet

Aquaponic (AQP) systems use aquaculture wastewater to supply the nutrients for plant growth. Recent fish meal (FM) production and price trends call for adopting of financially and environmentally sustainable protein sources for fish feed. However, different protein sources can alter the macro-and micro-nutrient effluxes from fish tanks and their nutritional value for plant growth. Early attempts to formulate the AQP diets have focused on enhancing nutrient efflux via dietary additives such as potassium diformate or potassium chloride. Here we present an alternative approach. We tested the effect of four aquafeeds differing in protein sources (complete substitution of FM with black soldier fly meal, BSFM; poultry blood meal, PBM; and hydrolyzed feather meal, HFM) on nutrient efflux in the water from Colossoma macropomum and Clarias gariepinus. The water from PBM and HFM-fed fish showed the highest efflux of TN (0.106 and 0.097 mg.L−1.g of feed, respectively), while the efflux of soluble reactive phosphorus and calcium was highest in FM-fed fish (0.012 and 0.028 mg.L−1.g of feed, respectively). BSFM-fed fish had the highest efflux of magnesium and potassium (0.008 and 0.023 mg.L−1.g of feed, respectively). Regarding macronutrients, the species-specific effects were observed only in sulphur, with higher efflux from C. macropomum. Iron efflux was not affected by diet, while higher efflux was observed in the trial with Clarias gariepinus. Effluxes of other micronutrients showed diet- and species-specific effects, except for zinc and manganese, with similar effluxes between species. In general, we found that it is possible to manipulate the nutrient efflux from RAS using alternative sustainable aquafeed components.

Tyrosine kinase inhibitors can activate the NLRP3 inflammasome in myeloid cells through lysosomal damage and cell lysis.

Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic-induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like receptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)-including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)-activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosomal swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which activated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow-derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects.

Pannexin1 Channel-Mediated Inflammation in Acute Ischemic Stroke.

Emerging evidence suggests that inflammation mediated by the pannexin1 channel contributes significantly to acute ischemic stroke. It is believed that the pannexin1 channel is key in initiating central system inflammation during the early stages of acute ischemic stroke. Moreover, the pannexin1 channel is involved in the inflammatory cascade to maintain the inflammation levels. Specifically, the interaction of pannexin1 channels with ATP-sensitive P2X7 purinoceptors or promotion of potassium efflux mediates the activation of the NLRP3 inflammasome, triggering the release of pro-inflammatory factors such as IL-1 and IL-18, exacerbating and sustaining inflammation of brain. Also, increased release of ATP induced by cerebrovascular injury activates pannexin1 in vascular endothelial cells. This signal directs peripheral leukocytes to migrate into ischemic brain tissue, leading to an expansion of the inflammatory zone. Intervention strategies targeting pannexin1 channels may greatly alleviate inflammation after acute ischemic stroke to improve this patient population's clinical outcomes. In this review, we sought to summarize relevant studies on inflammation mediated by the pannexin1 channel in acute ischemic stroke and discussed the possibility of using brain organoid-on-a-chip technology to screen miRNAs that exclusively target the pannexin1 channel to provide new therapeutic measures for targeted regulation of pannexin1 channel to reduce inflammation in acute ischemic stroke.

Association of the P441L KCNQ1 variant with severity of long QT syndrome and risk of cardiac events.

Dysfunction of potassium voltage-gated channel subfamily Q member 1 (KCNQ1) is a primary cause of long QT syndrome type 1 (LQT1). Here, we report a missense mutation P441L in KCNQ1 C-terminus of a 37-year-old woman with severe LQT1 phenotype. Variant P441L transporting to the plasma membrane and interacting with KCNE1 were both markedly decreased, leading to potassium efflux disorder and eventually LQT1. Mutations between the C-terminal helix A and helix B of KCNQ1 have linked with low cardiac event risk, however, we firstly find variant P441L causing a severe LQT1 phenotype with a high risk of cardiac events.

Sex differences in electrophysiological properties and voltage-gated ion channel expression in the paraventricular thalamic nucleus following repeated stress.

BackgroundHabituation to repeated stress refers to a progressive reduction in the stress response following multiple exposures to the same, predictable stressor. We previously demonstrated that the posterior division of the paraventricular thalamic nucleus (pPVT) nucleus regulates habituation to 5 days of repeated restraint stress in male rats. Compared to males, female rats display impaired habituation to 5 days of restraint. To better understand how activity of pPVT neurons is differentially impacted in stressed males and females, we examined the electrophysiological properties of pPVT neurons under baseline conditions or following restraint.MethodsAdult male and female rats were exposed to no stress (handling only), a single period of 30 min restraint or 5 daily exposures to 30 min restraint. 24 h later, pPVT tissue was prepared for recordings.ResultsWe report here that spontaneous excitatory post-synaptic current (sEPSC) amplitude was increased in males, but not females, following restraint. Furthermore, resting membrane potential of pPVT neurons was more depolarized in males. This may be partially due to reduced potassium leakage in restrained males as input resistance was increased in male, but not female, rats 24 h following 1 or 5 days of 30-min restraint. Reduced potassium efflux during action potential firing also occurred in males following a single restraint as action potential half-width was increased following a single restraint. Restraint had limited effects on electrophysiological properties in females, although the mRNA for 10 voltage-gated ion channel subunits was altered in the pPVT of female rats.ConclusionsThe results suggest that restraint-induced changes in pPVT activation promote habituation in males. These findings are the first to describe a sexual dimorphism in stress-induced electrophysiological properties and voltage-gated ion channel expression in the pPVT. These results may explain, at least in part, why habituation to 5 days of restraint is disrupted in female rats.

A. caviae infection triggers IL-1β secretion through activating NLRP3 inflammasome mediated by NF-κB signaling pathway partly in a TLR2 dependent manner

ABSTRACT Aeromonas caviae, an important food-borne pathogen, induces serious invasive infections and inflammation. The pro-inflammatory IL-1β functions against pathogenic infections and is elevated in various Aeromonas infection cases. However, the molecular mechanism of A. caviae-mediated IL-1β secretion remains unknown. In this study, mouse macrophages (PMs) were used to establish A. caviae infection model and multiple strategies were utilized to explore the mechanism of IL-1β secretion. IL-1β was elevated in A. caviae infected murine serum, PMs lysates or supernatants. This process triggered NLRP3 levels upregulation, ASC oligomerization, as well as dot gathering of NLRP3 and speck-like signals of ASC in the cytoplasm. MCC950 blocked A. caviae mediated IL-1β release. Meanwhile, NLRP3 inflammasome mediated the release of IL-1β in dose- and time-dependent manners, and the release of IL-1β was dependent on active caspase-1, as well as NLRP3 inflammasome was activated by potassium efflux and cathepsin B release ways. A. caviae also enhanced TLR2 levels, and deletion of TLR2 obviously decreased IL-1β secretion. What’s more, A. caviae resulted in NF-κB p65 nuclear translocation partly in a TLR2-dependent manner. Blocking NF-κB using BAY 11-7082 almost completely inhibited NLRP3 inflammasome first signal pro-IL-1β expression. Blocking TLR2, NF-κB, NLRP3 inflammasome significantly downregulated IL-1β release and TNF-α and IL-6 levels. These data illustrate that A. caviae caused IL-1β secretion in PMs is controlled by NLRP3 inflammasome, of which is mediated by NF-κB pathway and is partially dependent on TLR2, providing basis for drugs against A. caviae.

NLRP3 Inflammasome as a Therapeutic Target for Atherosclerosis: A Focus on Potassium Outflow.

Atherosclerosis is a risk factor for various cardiovascular diseases, and is linked to high rates of morbidity and mortality across the globe. Although numerous complex processes are involved in the development and progression of atherosclerosis, the exact mechanisms behind its pathogenesis remain unclear. Inflammation and endothelial cell damage exert a lasting effect on atherosclerosis, causing lipid and fibrous tissue accumulation in the intima of the artery to form plaques, and subsequently promoting atherosclerosis. Nod-like receptor protein 3 (NLRP3) inflammatory corpuscle is thought to be the link between lipid metabolism and inflammation. Long Potassium outflow is a vital activator of NLRP3, with a simultaneous effect as a start-up and adjustment. The majority of existing drugs for atherosclerosis targeting the NLRP3 signaling pathway target IL-1, whereas drugs targeting the critical link of potassium efflux are relatively new. This review discusses the NLRP3 inflammatory corpuscle as a critical regulator of the immunological inflammatory pathway in atherosclerosis. Moreover, current knowledge on NLRP3 inflammatory corpuscle start and activation pathways were integrated, emphasizing potassium-involved outflow-related proteins. We highlight potential treatment approaches for NLRP3 inflammatory corpuscle pathways, specifically targeting potassium outflow channels of targeted drugs. Collectively, these insights indicate that targeting the NLRP3 inflammatory corpuscle is a vital anti-inflammatory therapy for treating atherosclerosis.

Stress-induced electrolyte leakage from root cells of higher plants: background, mechanism and physiological role

Electrolyte leakage from tissues is one of the central reactions of the plant organism to stress. It is observed under almost any type of stresses, both abiotic and biotic. The loss of key electrolytes can lead to significant changes in metabolism and, in some cases, to the death of cells or the whole organism. For a long time, it was believed, that electrolyte leakage is associated with disruption of cell integrity and plasma membranes degradation, and that it is an unregulated process. However, in recent years, a lot of evidence has been received that, in most cases, electrolyte leakage is inhibited by ion channel blockers and reversible. It means that it is associated with the transfer of ions through the membrane by transport proteins, such as ion channels. Recently, the experimental evidence has been obtained, that under salinity, drought, pathogen attack, excessive levels of heavy metals, hypo- and hyperthermia, as well as oxidative stress, the electrolyte leakage in plant cells is mediated by several types of cation and anion channels, including K+-selective channels (SKOR and GORK), anion channels (such as ALMT1) and a number of non-selective cation channels. It has been demonstrated that the primary reactions that induce electrolyte leakage are plasma membrane depolarisation and generation of reactive oxygen species, leading to the activation of redox-regulated outwardly rectifying K+ channels, such as SKOR and GORK. Potassium efflux is up-stream and stimulates the counterion flow (transport of anions) through the anion channels. The regulation of electrolyte leakage at the ion channel level and the corresponding selection for ion channel properties can become an important link in the directed control of stress resistance in higher plants. This can be applied in agriculture via breeding of stress-tolerant plant varieties, as well as developing modern amelioration techniques.

Epoxomicin, a Selective Proteasome Inhibitor, Activates AIM2 Inflammasome in Human Retinal Pigment Epithelium Cells.

Emerging evidence suggests that the intracellular clearance system plays a vital role in maintaining homeostasis and in regulating oxidative stress and inflammation in retinal pigment epithelium (RPE) cells. Dysfunctional proteasomes and autophagy in RPE cells have been associated with the pathogenesis of age-related macular degeneration. We have previously shown that the inhibition of proteasomes using MG-132 activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome in human RPE cells. However, MG-132 is a non-selective proteasome inhibitor. In this study, we used the selective proteasome inhibitor epoxomicin to study the effect of non-functional intracellular clearance systems on inflammasome activation. Our data show that epoxomicin-induced proteasome inhibition promoted both nicotinamide adenine dinucleotide phosphate oxidase and mitochondria-mediated oxidative stress and release of mitochondrial DNA to the cytosol, which resulted in potassium efflux-dependent absence in melanoma 2 (AIM2) inflammasome activation and subsequent interleukin-1β secretion in ARPE-19 cells. The non-specific proteasome inhibitor MG-132 activated both NLRP3 and AIM2 inflammasomes and oxidative stress predominated as the activation mechanism, but modest potassium efflux was also detected. Collectively, our data suggest that a selective proteasome inhibitor is a potent inflammasome activator in human RPE cells and emphasize the role of the AIM2 inflammasome in addition to the more commonly known NLRP3 inflammasome.

Ginsenoside Rd protects cerebral endothelial cells from oxygen-glucose deprivation/reoxygenation induced pyroptosis via inhibiting SLC5A1 mediated sodium influx

BackgroundGinsenoside Rd is a natural compound with promising neuroprotective effects. However, the underlying mechanisms are still not well-understood. In this study, we explored whether ginsenoside Rd exerts protective effects on cerebral endothelial cells after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and its potential docking proteins related to the underlying regulations. MethodCommercially available primary human brain microvessel endothelial cells (HBMECs) were used for in vitro OGD/R studies. Cell viability, pyroptosis-associated protein expression and tight junction protein degradation were evaluated. Molecular docking proteins were predicted. Subsequent surface plasmon resonance (SPR) technology was utilized for validation. Flow cytometry was performed to quantify caspase-1 positive and PI positive (caspase-1+/PI+) pyroptotic cells. ResultsGinsenoside Rd treatment attenuated OGD/R-induced damage of blood-brain barrier (BBB) integrity in vitro. It suppressed NLRP3 inflammasome activation (increased expression of NLRP3, cleaved caspase-1, IL-1β and GSDMD-N terminal (NT)) and subsequent cellular pyroptosis (caspase-1+/PI + cells). Ginsenoside Rd interacted with SLC5A1 with a high affinity and reduced OGD/R-induced sodium influx and potassium efflux in HBMECs. Inhibiting SLC5A1 using phlorizin suppressed OGD/R-activated NLRP3 inflammasome and pyroptosis in HBMECs. ConclusionGinsenoside Rd protects HBMECs from OGD/R-induced injury partially via binding to SLC5A1, reducing OGD/R-induced sodium influx and potassium efflux, thereby alleviating NLRP3 inflammasome activation and pyroptosis.

OXSR1 inhibits inflammasome activation by limiting potassium efflux during mycobacterial infection.

Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1β, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.

Why is the SARS-CoV-2 Omicron variant milder?

Why is the SARS-CoV-2 Omicron variant milder?

Pyridine coupled pyrazole analogues as lethal weapon against MRSA: An in-vitro and in-silico approach

The treatment of Methicillin-resistant staphylococcus aureus (MRSA) infections has become challenging due to the growth of multidrug resistance in the bacteria. Here we report the synthesis of pyridine-coupled pyrazoles as an antimicrobial agent against MRSA. A series of pyridine coupled pyrazoles were synthesized and synthesized compounds were characterized using FT-IR, 1H NMR, and Mass spectroscopy. The ADMET results of all the 14 active compounds are interpreted. To identify the potent compound the synthesized compounds screened for minimum inhibitory concentrations against MRSA and compared with standard drug vancomycin. Among the synthesized compounds 6d exhibited good antibacterial activity with MIC value 21 μg/mL, bacterial cell membrane damage study was studied potassium efflux, and cellular content leakage assay. Anticoagulant study for the potent compound also studied and validated by molecular docking and molecular dynamics simulation studies. The docking study of the synthesized compound was carried out and the study depicted that the pyridine ring of all the analogues binds with the various amino acids in the binding pocket of the active site of the Staphylocoagulase and PBP2a protein of MRSA.

Pyolysin of Trueperella pyogenes Induces Pyroptosis and IL-1β Release in Murine Macrophages Through Potassium/NLRP3/Caspase-1/Gasdermin D Pathway.

Trueperella pyogenes (T. pyogenes) is a commensal and an opportunistic pathogen of animals. This organism can cause inflammatory diseases, such as pneumonia, mastitis and endometritis in hosts. However, the molecular basis for the pro-inflammatory properties of this organism is still largely unknown. In the current study, using murine macrophages as model, the ability of T. pyogenes to induce pyroptosis was first determined. Then, pyolysin (PLO), a cholesterol-dependent cytolysin secreted by T. pyogenes, was found to be closely related to T. pyogenes-induced pyroptosis. Next, our work showed that PLO can form pores in the cell membrane, leading to the efflux of potassium (K+), NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated caspase-1 activation, and gasdermin D (GSDMD) cleavage. Inhibition of the K+/NLRP3/caspase-1/GSDMD pathway abolished T. pyogenes and PLO-induced IL-1β release. Taken together, these results indicate T. pyogenes-induced inflammation is related to PLO-induced pyroptosis and IL-1β release. Our work shed light on the pathogenesis of T. pyogenes and the interaction between T. pyogenes and hosts’ immune system.