Activation Of NLRP3 Inflammasome Signaling Research Articles

Interference with GPR4 inactivates NLRP3 inflammasome signaling by inhibiting LPAR1 expression to ameliorate oxygen-glucose deprivation/reoxygenation-induced inflammation and apoptosis of cardiomyocytes

Myocardial ischemia/reperfusion (MI/R) injury is a detrimental disease with high mortality worldwide. We aimed to explore the role of G protein-coupled receptor 4 (GPR4) and lysophosphatidic acid receptor 1 (LPAR1) in MI/R injury in vitro. H9c2 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) conditions to simulate the MI/R injury and GPR4 expression was detected. Then, GPR4 was knocked down and cell viability was examined with a CCK-8 assay. The activities of LDH, CK and CK-MB were detected to evaluate the damage of OGD/R-induced H9c2 cells. ELISA kits and TUNEL staining were used to examine the inflammation and apoptosis of H9c2 cells exposed to OGD/R conditions. Western blot was employed to detect the expression of proteins related to apoptosis and NLRP3 inflammasome signaling. Additionally, Co-IP analyzed the binding between GPR4 and LPAR1. Finally, LPAR1 was overexpressed to conduct the rescue experiments. Results revealed that GPR4 was upregulated in OGD/R-treated H9c2 cells and GPR4 knockdown attenuated the damage of H9c2 cells. OGD/R induced inflammation and apoptosis were markedly inhibited by GPR4 silencing, as evidenced by the decreased TNF-α, IL-6 and IL-8 levels as well as the elevated Bcl-2 expression and reduced Bax and cleaved caspase3 expression. Moreover, GPR4 bound to LPAR1 and upregulated LPAR1 expression. Interference with GPR4 inactivated the NLRP3 inflammasome signaling. Besides, LPAR1 overexpression abrogated the effects of GPR4 silencing on the damage, inflammation and apoptosis of H9c2 cells induced by OGD/R. Particularly, LPAR1 upregulation promoted the activation of NLRP3 inflammasome signaling in GPR4-silenced H9c2 cells induced by OGD/R. To be concluded, GPR4 deficiency inactivates NLRP3 inflammasome signaling by inhibiting LPAR1 expression to ameliorate OGD/R -induced inflammation and apoptosis of cardiomyocytes.

Anti-inflammatory and uric acid lowering effects of Euodiae fructus on hyperuricemia and gout mice.

The metabolic disease hyperuricemia (HUA) is caused by presence of excessive serum uric acid (UA), which leads to an increased risk of chronic kidney disease and gout. As a widely used traditional Chinese medicine, Euodiae fructus (ER) has strong anti-inflammatory and analgesic effects, however, its therapeutic effects on HUA and gout have not been investigated. To investigate the potential effects and underlying mechanisms, the effect of ER on proinflammatory cytokines and NLRP3 inflammasome activation was studied in mouse bone marrow macrophages. Moreover, a mouse model of HUA and gouty arthritis was established by coadministration of potassium oxonate (PO) and monosodium urate crystals to mice fed a high-fat diet (HFD) for 37 consecutive days. Oral administration of ER aqueous extract was given 1hour later after the injection of PO for 10 days. Our study showed that ER is a powerful NLRP3 inhibitor in mouse macrophages. Most importantly, ER (0.75g/kg) treatment substantially decreased the ankle joint thickness ratio, serum UA, creatinine and blood urea nitrogen levels (p < 0.05). Additionally, ER (0.75g/kg) dramatically reversed the increases in renal urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) as well as the decreases in organic anion transporter 1 (OAT1) and ATP binding cassette subfamily G member 2 (ABCG2) levels (p < 0.05). Moreover, ER (0.75g/kg) markedly ameliorated the production of the serum inflammatory cytokines IL-1β and TNF-α (p < 0.01), and improved the activation of NLRP3 inflammasome signaling in the kidneys. Taken together, these data indicate that ER, a powerful and specific NLRP3 inhibitor, has multiple anti-HUA, anti-gout and anti-inflammatory effects. Our investigation is designed to experimentally support the conventional use of ER-containing classical herbal formulas in the treatment of HUA-related disorders and may add a new dimension to the clinical application of ER.

Aurantio-obtusin induces hepatotoxicity through activation of NLRP3 inflammasome signaling

Aurantio-obtusin (AO) is a major anthraquinone (AQ) compound derived from Cassiae semen (CS). Although pharmacological studies have shown that the CS extracts can serve as effective agents in preclinical and clinical practice, AQ-induced hepatotoxicity in humans has attracted widespread attention. To explore whether AO induces hepatotoxicity and its underlying mechanisms, we exposed larval zebrafish and mice to AO. We found that AO delayed yolk sac absorption, and increased liver area and inflammation in the larval zebrafish. This inflammation was manifested as an increase in liver neutrophils and the up-regulated mRNA expression of interleukin-6 (Il-6) and tumor necrosis factor-α (Tnf-α) in the larval zebrafish. Furthermore, a pharmacokinetics study showed that AO was quickly absorbed into the blood and rapidly metabolized in the mice. Of note, AO induced hepatotoxicity in a gender-dependent manner, characterized by liver dysfunction, increased hepatocyte necrosis with inflammatory infiltration, and up-regulated mRNAs of Il-6, Tnf-α and monocyte chemotactic protein 1(Mcp1) in the female mice after 28-day oral administration. It also highlighted that AO triggered NOD-like receptor protein (NLRP) signaling in the female mice, as evidenced by the increased NLRP3, Caspase-1, pro-IL-1β, IL-1β and IL-18. Finally, we found that AO led to a significant increase in potassium calcium-activated channel, subfamily N, member 4 (KCNN4) and reactive oxygen species (ROS) levels, along with decreased nuclear factor kappa B p65 (NF-κB p65), in the female mouse livers. In conclusion, AO induced hepatotoxicity by activating NLRP3 inflammasome signaling, at least in part, through increased KCNN4 and ROS production, and NF-κB inhibition.

PCB118 Induces Inflammation of Islet Beta Cells via Activating ROS-NLRP3 Inflammasome Signaling.

Background Diabetes mellitus is a clinical syndrome caused by genetic and environmental factors. Growing evidence suggests that exposure to environmental endocrine disruptors and activation of NLRP3 inflammasome signaling play a vital role in diabetes. However, it is unclear how PCB118, a common environmental endocrine disruptor, contributes to the incidence of diabetes, and its specific mechanism of action is unknown. In this study, we explored whether ROS-induced NLRP3 inflammasome priming and activation were related to PCB118 exposure in mouse islet β-TC-6 cells and the mechanisms of diabetes. Methods Mouse islet β-TC-6 cells were cultured with PCB118 as a stimulating factor and ROS inhibitor N-acetyl cysteine (NAC) as an intervention. Cellular toxicity due to PCB118 was detected using the Cell Counting Kit-8; ROS was measured using DCFH-DA; the expressions of NLRP3, procaspase-1, caspase-1, pro-IL-1β, and IL-1β protein were detected by western blot; and IL-6, IL-18, and C-C chemokine ligand 2 (CCL-2) were measured by ELISA. Results PCB118 caused significant toxicity to the cells when the stimulation concentration was equal to or greater than 80 nmol/L at 72 hours (p < 0.05) and increased the levels of ROS, NLRP3, caspase-1, IL-1β, IL-6, IL-18, and CCL-2 (p < 0.05); the expressions of procaspase-1 and pro-IL-1β were downregulated in a dose-dependent manner after PCB118 exposure (p < 0.05), which was prevented by pretreatment with NAC (p < 0.05). Conclusions PCB118 can activate NLRP3 inflammasome signaling in islet beta cells via the oxidative stress pathway and cause inflammation in islet beta cells. It suggests that environmental endocrine disruptors play an important role in the inflammation of islet beta cells and may contribute to the development of diabetes through NLRP3 inflammatory signaling.

Pharmacological Effects of Fructus Ligustri Lucidi on Neuroinflammation and Exploration of Active Compound Based on the Interaction with CaSR

Background The onset of depression and the lack of effectiveness in antidepressant medication may be related to the overall activation of the neuroinflammatory response. The calcium-sensing receptor (CaSR), widely expressed in various types of cells in the nervous system, participates in inflammatory responses. Our studies had revealed that the extract of Fructus Ligustri Lucidi (FLL) could exert beneficial effects on improving bone metabolism of diabetic rats and mice via acting on renal CaSR. The effects of FLL on neuroinflammation and the role of FLL in protecting from neuropsychiatric diseases remain elusive. The present work aimed to study the pharmacological mechanisms by which FLL and its active compound inhibit neuroinflammation from the view of modulating CaSR. Methods ：Mice were treated with the phenol glycosides extract of FLL for 14 consecutive days before intraperitoneally injected with lipopolysaccharide (LPS) on the 15th day to establish acute model of depression. The allosteric sites of CaSR were predicted and its interaction with phenol glycosides was analyzed by Schördinger Maestro package. After treatment of streptozotocin-induced diabetic mice with ligustroflavone for 8 weeks, calcium balance and bone properties as well as renal CaSR signaling were measured. BV2 microglia cells were incubated with different doses of ligustroflavone before CaSR agonist cinacalcet was added to the medium to stimulate CaSR. The production of inflammatory response, like NLRP3 inflammasome, and the release of IL-1β, MCP-1, and Rantes as well as morphological changes of the cells were detected. Results Pretreatment of mice with the phenol glycosides of FLL inhibited LPS-triggered depression-like behaviors by reducing the release of inflammatory factors and attenuating the neuroinflammation via affecting the CaSR pathway. Phytochemical work clarified the main compounds like ligustroflavone contained in FLL phenol glycosides extract. Docking analysis found that ligustroflavone interacted well with the allosteric site of CaSR and had the potency as CaSR antagonist. Treatment of diabetic mice with ligustroflavone maintained calcium homeostasis by antagonizing CaSR in kidney, consequently protecting from diabetes-induced osteoporosis. Our ongoing study demonstrated that pre-incubation of microglia cells with ligustroflavone significantly inhibited the activation of microglials and repressed the cinacalcet-induced activation of NLRP3 inflammasome signaling and the increase in levels of inflammatory mediators, including cytokines and chemokines, by antagonizing CaSR. Conclusion: FLL exerted beneficial effects on protecting from depressive-like behaviors by suppressing neuroinflammation via acting on CaSR in brain tissue. Ligustroflavone might be a potential active compound as a candidate of CaSR antagonist.

The cross-talk of NLRP3 inflammasome activation and necroptotic hepatocyte death in acetaminophen-induced mice acute liver injury.

Overdose acetaminophen (APAP) can result in severe liver injury, which is responsible for nearly half of drug-induced liver injury in western countries. Previous studies have found that there existed massive hepatocellular necrosis and severe inflammatory response in APAP-induced liver injury. However, the mechanistic linkage between necroptosis and NLRP3 inflammasome pathway in APAP-induced hepatotoxicity remains poorly understood. In order to investigate the relationship between inflammation and hepatocytes death in APAP hepatotoxicity, a time-course model for APAP hepatotoxicity in C57/BL6 mice was established by intraperitoneal (i.p) injection of 300 mg/kg APAP in this study. The activity of serum enzymes and pathological changes of APAP-treated mice were evaluated, and the critical molecules in necroptosis and NF-κB-NLRP3 inflammasome signaling pathway were determined by immunoblot and immunofluorescence analysis. The results demonstrated that APAP overdose resulted in a severe liver injury. Furthermore, the expression of critical molecules in NLRP3 inflammasome and necroptosis pathways peaked at 12-24 h, and then was decreased gradually, which is consistent with the pattern of pathological injury induced by APAP. Our further investigation found that the level of IL-1β in mouse liver was closely correlated with the level of phosphorylated MLKL following exposure to APAP. Furthermore, inhibition of necroptosis with necrostatin-1 significantly suppressed the activation of NLRP3 inflammasome signaling. Taken together, our results highlighted that the cross-talk between necroptosis and NLRP3 inflammasome played a critical role for promoting APAP-induced liver injury. Inhibition of the interaction of inflammation and necroptosis by pharmaceutical methods may represent a promising therapeutic strategy for APAP-induced liver injury.

S100A12 promotes inflammation and cell apoptosis in sepsis-induced ARDS via activation of NLRP3 inflammasome signaling

Sepsis is a multiple organ dysfunction elicited by the dysregulated host immune response to microbial infection. Acute respiratory distress syndrome (ARDS) is a serious and acute inflammatory lung injury resulting from sepsis and other severe diseases. The present study aims to investigate the role of S100A12, a pro-inflammatory factor, in the pathophysiologic mechanism underlying the process in sepsis-induced ARDS. In present study, Hematoxylin and Eosin (H&E) Staining was performed to observe pathological changes. Enzyme-Linked Immunosorbent Assay (ELISA) was employed to analyze the levels of inflammatory cytokines. Western blot, immunohistochemistry (IHC) staining and reverse-transcriptase quantitative real-time PCR (RT-qPCR) were performed to determine target gene and protein expression. TUNEL assay and flow cytometry were performed to assay cell apoptosis. We found that the levels of S100A12 and soluble receptor for advanced glycation end-products (sRAGE) are upregulated in the serum of patients with Sepsis-induced ARDS and sepsis mice. Furthermore, higher cell apoptosis rate was observed in lung tissue of sepsis mice. In addition, S100A12 resulted in excessive mucins and the secretion of inflammatory cytokines secretion, and promoted the expression of chemokines and cell adhesion molecules via activating nucleotide-binding oligomerization domain (Nod) -like receptor (NLR) P3 inflammasome pathway in NHBE cells. Finally, S100A12 increased oxidative stress status and cell apoptosis in NHBE cells. Generally, the present study provides evidence that S100A12 is closely related to pathogenesis of sepsis-induced ARDS. Hence, S100A12 may be a useful biomarker of pulmonary injuries for clinical diagnosis of sepsis-induced ARDS.

Curcumin attenuates potassium oxonate-induced hyperuricemia and kidney inflammation in mice

Current evidences suggest that hyperuricemia is closely related to the overproduction or underexcretion of uric acid (UA). Curcumin (CUR), a natural polyphenol component extracted from the rhizome of Curcuma longa, has been reported to treat various symptoms such inflammation disease, seems to be efficacious in hyperuricemia. In this study, we aimed to investigate the effect of CUR on hyperuricemia and kidney inflammation in hyperuricemic mice. Administration with CUR (20 or 40 mg/kg) or allopurinol (ALL, 5 mg/kg) was given to mice orally one hour later after the injection of potassium oxonate (PO) (300 mg/kg, i.p.) for 14 days. CUR administration decreased the levels of uric acid (UA), creatinine (CRE) and blood urea nitrogen (BUN) in serum. Meanwhile, treatment with CUR effectively inhibited serum and liver xanthine oxidase (XOD) levels, and further renewed normal antioxidant enzymes activities (SOD, GSH-Px), reduced MDA accumulation in serum. Further studies showed that CUR decreased inflammatory cytokines productions (IL-1β, IL-18) in serum, as well as inhibited PO-induced the activation of NLRP3 inflammasome signaling in the kidney. In conclusion, the study revealed that CUR exhibited anti-hyperuricemic and anti-inflammatory effects through suppressing NLRP3 inflammasome activation in kidney and provided the evidence for treating hyperuricemia and associated renal inflammation.

NLRP3 Inflammasome Modulation by Melatonin Supplementation in Chronic Pristane-Induced Lupus Nephritis.

Lupus nephritis (LN) is a kidney inflammatory disease caused by systemic lupus erythematosus (SLE). NLRP3 inflammasome activation is implicated in LN pathogenesis, suggesting its potential targets for LN treatment. Melatonin, an endogenous indoleamine, is considered an important multitasking molecule that has been reported to have anti-inflammatory effects by inhibiting nuclear factor-kappa B (NF-κB)-mediated inflammatory responses in vivo. This molecule has also protective effects against the activation of the inflammasomes and, in particular, the NLRP3 inflammasome. Thus, this work evaluated the effect of melatonin on morphological alteration and NLRP3 inflammasome activation in LN pristane mouse models. To evaluate the melatonin effects in these mice, we studied the renal cytoarchitecture by means of morphological analyses and immunohistochemical expression of specific markers related to oxidative stress, inflammation and inflammasome activation. Our results showed that melatonin attenuates pristane-induced LN through restoring of morphology and attenuation of oxidative stress and inflammation through a pathway that inhibited activation of NLRP3 inflammasome signaling. Our data clearly demonstrate that melatonin has protective activity on lupus nephritis in these mice that is highly associated with its effect on enhancing the Nrf2 antioxidant signaling pathway and decreasing renal NLRP3 inflammasome activation.

Neferine inhibits LPS-ATP-induced endothelial cell pyroptosis via regulation of ROS/NLRP3/Caspase-1 signaling pathway

Oxidative stress-induced endothelial dysfunction and pyroptosis play an important role during chronic kidney disease (CKD) progression. Neferine, which is an alkaloid ingredient from the lotus seed embryo, has many biological actions such as anti-inflammatory, anticancer and antioxidant. However, the role of neferine in endothelial cell pyroptosis and the involved mechanism remain obscure. The aim is to probe the protective effects of neferine on cell pyroptosis and the involved underlying mechanism. After the HUVECs were primed with neferine treatment for 2h prior to LPS and ATP exposure for 24h, the cell proliferation was determined by BrdU; the cell LDH release was detected by LDH kits; the levels of intracellular ROS, MDA and SOD were tested by detection kits; Caspase-1 activity kit was used to determine caspase-1 activity; the contents of NLRP3, ASC, caspase-1, IL-1β, IL-18 and GSDMD were tested by RT-PCR and western blot. We found that neferine could inhibit LPS-ATP-induced oxidative stress and the activation of NLRP3 inflammasome signaling, and increased the endothelial cell viability and SOD production. siRNA which mediated the knockdown of NLRP3 promoted the neferine-induced inhibition effects of cell pyroptosis. Furthermore, these neferine-induced effects were reversed by the over-expression of NLRP3. Our findings indicated neferine may reduce ROS by anti-oxidation and inhibit LPS-ATP-induced endothelial cell pyroptosis via blocking ROS/NLRP3/Caspase-1 signaling pathway, which provides the evidence for therapeutic effect in CKD.

Chemotherapeutic Agent Paclitaxel Mediates Priming of NLRP3 Inflammasome Activation.

Paclitaxel is a chemotherapeutic drug commonly used to treat different types of cancer. In addition to its antitumor effect, paclitaxel is also known to promote Toll-like receptor (TLR) 4-dependent inflammatory responses, which may lower its chemotherapeutic efficacy. However, it remains unclear whether paclitaxel is able to affect inflammasome signaling in myeloid or cancer cells. Therefore, we examined the potential effect of paclitaxel on the activation of an inflammasome complex by examining caspase-1 activation and interleukin (IL)-1β secretion in bone marrow-derived macrophages (BMDMs). The results showed that treatment with paclitaxel alone or following LPS priming failed to trigger the secretion of active caspase-1 and IL-1β from BMDMs. However, paclitaxel could induce robust activation of caspase-1 in BMDMs in the presence of NLRP3 inflammasome-activating signal 2, such as ATP or nigericin. This paclitaxel/ATP-mediated inflammasome activation was completely abrogated in Nlrp3-deficient macrophages. Mechanistically, paclitaxel treatment induced robust activation of the TLR4 signaling cascade, including phosphorylation of IκB and JNK and upregulation of proinflammatory cytokine mRNA levels in a TLR4-dependent manner. In contrast, paclitaxel treatment alone did not induce mitochondrial damages such as the loss of the mitochondrial membrane potential and production of mitochondrial ROS. These findings suggest that paclitaxel can drive the priming of signal-mediated events for NLRP3 activation but not a second signal-triggered phenomenon such as mitochondrial damage. This suggestion was supported by the observations that paclitaxel treatment caused robust IL-1β production in macrophages in the presence of cell-free medium derived from growth of injured cells and also in the spleen of mice. Collectively, our data strongly indicate that paclitaxel is able to facilitate the activation of NLRP3 inflammasome signaling in a certain physiological environment.

Role of sweet taste receptors in the activation of NLRP3 inflammasome signaling in diabetic kidney disease

Objective To investigate the role of sweet taste receptors(STRs)in the activation of reactive oxygen species (ROS)-NLRP3 inflammasome signaling in diabetic kidney disease(DKD). Methods DKD mouse were established by high-fat diet and streptozotocin. After mouse glomerular mesangial cells(GMCs)were exposed to high glucose, STRs(T1R2/T1R3)and associated signaling components and NLRP3 inflammasome signaling components expressions were detected. After GMCs were treated with STRs inhibitor lactisole, the production of ROS was detected and the role of STRs in the activation of NLRP3 signaling was investigated. Results Under high glucose condition, the expressions of T1R2, T1R3, Gα-gustducin, phospholipase C-β2, and TRPM5 were significantly decreased in vivo and in vitro(all P<0.05)and ROS-NLRP3 signaling was activated(all P<0.05). Lactisole significantly mitigated the production of ROS and the activation of NLRP3 inflammasome signaling stimulated by high glucose in GMCs(all P<0.05). Conclusion The STRs(T1R2/T1R3)-mediated signaling pathway may be involved in the regulation of ROS-NLRP3 inflammatory signaling, suggesting that STRs may act as new therapeutic targets of DKD. (Chin J Endocrinol Metab, 2018, 34: 587-593) Key words: Sweet taste receptors; Reactive oxygen species; NLRP3 inflammasome; Diabetic kidney disease

Sweet-Taste Receptors Mediated ROS-NLRP3 Inflammasome Signaling Activation—Implications for Diabetic Nephropathy

Previous studies demonstrated that ROS-NLRP3 inflammasome signaling activation were involved in the pathogenesis of diabetic nephropathy (DN). But the mechanism by which high glucose prime ROS-NLRP3 inflammasome signaling remains debatable. Recent research has shown that taste receptor type 1, member 2 (T1R2) and member 3 (T1R3), form heterodimers to act as sweet taste receptors (STRs), are widely expressed in the extraoral tissues, and emerging evidence suggests that STRs are important sentinels of innate immunity. As a natural ligand for STRs, whether high glucose prime ROS-NLRP3 inflammasome signaling via STRs is unclear. Therefore, diabetes mellitus (DM) mouse model was induced by streptozotocin (STZ) in vivo, mouse glomerular mesangial cells (GMCs) and human proximal tubular cells were stimulated by elevated concentrations of high glucose (10, 20, and 30 mmol/L) or mannitol as the osmotic pressure (OP) control in vitro, STRs inhibitor lactisole were used as an intervention reagent to evaluated the role and mechanism of the STRs in the pathogenesis of DN. Our results showed that the expression of STRs (T1R2 and T1R3) and associated signalling components (Gα-gustducin, PLCβ2, TRPM5) were obviously down-regulated under the condition of diabetes in vivo and in vitro. Further more, lactisole signifcantly mitigated the production of intracellular ROS and reversed the decrease of Ca2+ and the activation of NLRP3 inflammasome signaling in GMCs stimulated by high glucose (p&amp;lt;0.05). These combined results support the hypothesis that high glucose induced ROS-NLRP3 inflammasome signaling activation in part via STRs, suggesting that STRs may act as new therapeutic targets of DN. Disclosure L. Zhou: None. W. Huang: None. Y. Xu: None. C. Gao: None.

Sweet Taste Receptors Mediated ROS-NLRP3 Inflammasome Signaling Activation: Implications for Diabetic Nephropathy.

Previous studies demonstrated that ROS-NLRP3 inflammasome signaling activation was involved in the pathogenesis of diabetic nephropathy (DN). Recent research has shown that sweet taste receptors (STRs) are important sentinels of innate immunity. Whether high glucose primes ROS-NLRP3 inflammasome signaling via STRs is unclear. In this study, diabetic mouse model was induced by streptozotocin (STZ) in vivo; mouse glomerular mesangial cells (GMCs) and human proximal tubular cells were stimulated by high glucose (10, 20, and 30 mmol/L) in vitro; STR inhibitor lactisole was used as an intervention reagent to evaluate the role and mechanism of the STRs in the pathogenesis of DN. Our results showed that the expression of STRs and associated signaling components (Gα-gustducin, PLCβ2, and TRPM5) was obviously downregulated under the condition of diabetes in vivo and in vitro. Furthermore, lactisole significantly mitigated the production of intracellular ROS and reversed the high glucose-induced decrease of Ca2+ and the activation of NLRP3 inflammasome signaling in vitro (p < 0.05). These combined results support the hypothesis that STRs could be involved in the activation of ROS-NLRP3 inflammasome signaling in the pathogenesis of DN, suggesting that STRs may act as new therapeutic targets of DN.

Protective Effects of Flagellin A N/C Against Radiation-Induced NLR Pyrin Domain Containing 3 Inflammasome-Dependent Pyroptosis in Intestinal Cells

Toinvestigate changes induced in mouse intestines after irradiation and to explore the potential radioprotective effects of flagellin AN/C (FlaAN/C). A mouse model of radiation-induced enteropathy was used in this study. A 10-Gy abdominal irradiation was performed on FlaAN/C- and vehicle-injected mice to explore the role of FlaAN/C in intestinal radiation injury and to study the molecular mechanism in this process. In the intestinal tissue, pathologic changes were investigated by hematoxylin and eosin staining, TUNEL staining, and immunohistochemistry; Western blotting and quantitative reverse transcription-polymerase chain reaction were used to determine the changes in protein and messenger RNA levels, respectively; and an enzyme-linked immunosorbent assay was performed to determine protein concentration in serum. The involvement of the reactive oxygen species pathway was investigated by determining superoxide dismutase, glutathione (GSH) peroxidase, GSH reductase, and GSH disulfide (GSSG) plus GSH activities. Flagellin A N/C inhibited radiation-induced reactive oxygen species production, decreased NLRP3 activity, and reduced the occurrence of caspase-1-dependent pyroptosis. The results revealed that oxidative stress, bioenergetic impairment, and subsequent NLRP3 inflammasome activation were involved in radiation-induced intestinal injury. Flagellin A N/C exerted a protective effect by blunting the activation of NLRP3 inflammasome signaling, thereby reducing the inflammatory response and occurrence of caspase-1-dependent pyroptosis in the intestine. Therefore, treatment of mice with FlaAN/C reduced radiation-induced intestinal injury. Reactive oxygen species-induced NLRP3 inflammasomes mediated radiation-induced pyroptosis of the intestinal cells, and FlaAN/C suppressed pyroptosis to protect the intestinal tissue. These results provide novel insights into the mechanisms underlying radiation-induced cytotoxicity, and FlaAN/C might be a potential preventive therapy for radiation-induced intestinal injury in patients with cancer.

Hemin-Induced Mast Cell-Extracellular Traps Impart Resistance to Therapy in a Sickle Microenvironment

Mast cell activation in the skin contributes to inflammation, neurogenic inflammation and hyperalgesia in sickle mice (Vincent et al., Blood 2013). However, the underlying cause of mast cell activation and challenges in inhibiting mast cell activation remain unclear. Hemolysis in sickle cell disease leads to excess free heme, which contributes to endothelial activation and neutrophil extracellular trap (NET) formation. We therefore hypothesized that free heme activates mast cells and extracellular trap formation in a sickle microenvironment. We examined hemin-induced activation in mast cells from the skin of HbAA-BERK control and HbSS-BERK sickle mice. Mast cells in culture were incubated with vehicle, or I ng/ml TNF-α for 4 hours, or 40 μM hemin for 2 hours, or with hemin for 2 hours after 2 hours of priming with TNF-α to create an inflammatory sickle microenvironment. Cells were stained with cell impermeable DNA dye SYTOX orange and cell permeable dye SYTO13 to visualize the extracellular DNA fibers. Incubation with TNF-α or hemin did not show significant eruption of DNA from the cell body. However, sickle mast cells incubated with both TNF-α and hemin showed distinct DNA containing fibers exploding from the cell body with the appearance of spider web like formation similar to NETs. The length of majority of individual fibers was more than 50 μm stretching to more than 500 μM, indicative of TRAP formation. This response was blunted in mast cells from control mice. Thus, mast cells in a sickle microenvironment are primed and activated, and further priming with cytokines such as TNF-α leads to hemin-induced mast cell extracellular trap (MET) formation. Sickle mast cells express significantly higher TLR4 and FcεRI as compared to control mice (Vincent et al., Blood 2013). Therefore, we examined if silencing of FcεRI and/or inhibition of TLR4 attenuated TNF-α primed/hemin-induced MET formation. TAK242 at a dose of 1 μM but not at 0.5 μM inhibited TNF-α primed/hemin-induced MET formation, but silencing of FcεRI had no effect. Since cannabinoids, imatinib, palmitoylethanolamide (PEA) and cromolyn are known mast cell inhibitors, we examined their effect on TNF-α/hemin-induced METs. Imatinib (30 - 100 μM), a known mast cell inhibitor, showed no significant effect, while cromolyn (100 μM) led to a modest decrease in MET formation. Cannabinoid CP 55,940 and PEA at a relatively low dose of 30 μM completely blocked MET formation. Functionally, imatinib, PEA and CP 55,940 reduced the release of cytokines TNF-α and RANTES from mast cells incubated with TNF-α/hemin for 24 hours. Hemin in the presence of TNF-α stimulated sustained activation of NLRP3 inflammasome signaling. Thus, mast cells in a sickle microenvironment are activated due to a rich inflammatory cytokine milieu, via activation of the inflammasome signaling upon stimulation with hemin. The unique phenomenon of MET formation is orchestrated by a combined action of inflammatory milieu and hemin, imparting resistance to common mast cell inhibitors such as cromolyn and imatinib. This new phenomenon of MET formation identified by us may in part contribute to challenges of treating sickle cell disease and other mast cell associated pathological conditions. DisclosuresNo relevant conflicts of interest to declare.

K+ efflux agonists induce NLRP3 inflammasome activation independently of Ca2+ signaling.

Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1-mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic K(+) concentration versus increased cytosolic Ca(2+) concentration ([Ca(2+)]) remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca(2+)] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K(+) efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester. The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca(2+)] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent Leu-Leu-O-methyl ester. Second, agonists for three Ca(2+)-mobilizing G protein-coupled receptors (formyl peptide receptor, P2Y2 purinergic receptor, and calcium-sensing receptor) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared with the K(+) efflux agonists. Third, the intracellular Ca(2+) buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate, widely used reagents for disruption of Ca(2+)-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca(2+) homeostasis. The results indicate that the ability of K(+) efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca(2+)] as a necessary or sufficient signal.