NLRP3 Inflammasome In Response Research Articles

Aberrant NLRP3 inflammasome response in Rett syndrome: from ex vivo to in vivo models for investigating pathogenetic mechanisms and new potential therapeutic strategies

Aberrant NLRP3 inflammasome response in Rett syndrome: from ex vivo to in vivo models for investigating pathogenetic mechanisms and new potential therapeutic strategies

Metformin inhibits activation of NLRP3 inflammasome and inflammatory response in preeclamptic rats

Backgroud: It is widely acknowledged that Metformin (MET), an established medication for managing type 2 diabetes, possesses diverse pharmacological effects. This study aims to investigate the protective effects of MET against Nω-Nitro-L-arginine methyl ester (L-NAME)-induced preeclampsia (PE). MethodsSprague Dawley (SD) rats were exposed to 200 mg/kg L-NAME with or without prior MET treatment. Histopathological analysis was performed using Hematoxylin and Eosin staining. Serum levels of inflammatory, antiangiogenic, and angiogenic factors were quantified using ELISA kits. Immunohistochemistry (IHC) staining was employed to observe NLRP3 and IL-1β expressions in placental tissues. Western blot and Quantitative Real-Time PCR (q-PCR) analyses were conducted to assess protein and mRNA expressions of NLRP3, caspase-1, ASC, and IL-1β. ResultsWe found that MET could mitigate placental histopathological deterioration and improve pregnancy outcomes in L-NAME-induced PE rat models. MET not only suppressed L-NAME-induced elevation of antiangiogenic factors but also stimulated the production of pro-angiogenic factors. Additionally, MET treatment reversed the excessive inflammatory response induced by L-NAME. Furthermore, MET inhibited the activation of the NLRP3 inflammasome triggered by L-NAME, evidenced by the downregulation of NLRP3 expression, caspase-1, and IL-1β. ConclusionsMET demonstrates a protective effect against L-NAME-induced PE rats, potentially mediated through inhibition of the inflammatory response, downregulation of NLRP3 inflammasome expression in the placenta, and regulation of the balance between anti-angiogenic and pro-angiogenic factors.

Inhibition of Immunoproteasome Attenuates NLRP3 Inflammasome Response by Regulating E3 Ubiquitin Ligase TRIM31

Excessive secretion of pro-inflammatory cytokines leads to the disruption of intestinal barrier in inflammatory bowel disease (IBD). The inflammatory cytokine tumor necrosis factor alpha (TNFα) induces the assembly of the NLRP3 inflammasome, resulting in the augmented secretion of inflammatory cytokines implicated in the pathogenesis of inflammatory bowel disease (IBD). TNFα has also been known to induce the formation of immunoproteasome (IP), which incorporates immunosubunits LMP2, LMP7, and MECL-1. Inhibition of IP activity using the IP subunit LMP2-specific inhibitor YU102, a peptide epoxyketone, decreased the protein levels of NLRP3 and increased the K48-linked polyubiquitination levels of NLRP3 in TNFα-stimulated intestinal epithelial cells. We observed that inhibition of IP activity caused an increase in the protein level of the ubiquitin E3 ligase, tripartite motif-containing protein 31 (TRIM31). TRIM31 facilitated K48-linked polyubiquitination and proteasomal degradation of NLRP3 with an enhanced interaction between NLRP3 and TRIM31 in intestinal epithelial cells. In addition, IP inhibition using YU102 ameliorated the symptoms of colitis in the model mice inflicted with dextran sodium sulfate (DSS). Administration of YU102 in the DSS-treated colitis model mice caused suppression of the NLRP3 protein levels and accompanied inflammatory cytokine release in the intestinal epithelium. Taken together, we demonstrated that inhibiting IP under inflammatory conditions induces E3 ligase TRIM31-mediated NLRP3 degradation, leading to attenuation of the NLRP3 inflammatory response that triggers disruption of intestinal barrier.

Adipose tissue IL-18 production is independent of caspase-1 and caspase-11.

Inflammation in adipose tissue, resulting from imbalanced caloric intake and energy expenditure, contributes to the metabolic dysregulation observed in obesity. The production of inflammatory cytokines, such as IL-1β and IL-18, plays a key role in this process. While IL-1β promotes insulin resistance and diabetes, IL-18 regulates energy expenditure and food intake. Previous studies have suggested that caspase-1, activated by the Nlrp3 inflammasome in response to lipid excess, mediates IL-1β production, whereas activated by the Nlrp1b inflammasome in response to energy excess, mediates IL-18 production. However, this has not been formally tested. Wild-type and caspase-1-deficient Balb/c mice, carrying the Nlrp1b1 allele, were fed with regular chow or a high-fat diet for twelve weeks. Food intake and mass gain were recorded weekly. At the end of the twelve weeks, glucose tolerance and insulin resistance were evaluated. Mature IL-18 protein levels and the inflammatory process in the adipose tissue were determined. Fasting lipid and cytokine levels were quantified in the sera of the different experimental groups. We found that IL-18 production in adipose tissue is independent of caspase-1 activity, regardless of the metabolic state, while Nlrp3-mediated IL-1β production remains caspase-1 dependent. Additionally, caspase-1 null Balb/c mice did not develop metabolic abnormalities in response to energy excess from the high-fat diet. Our findings suggest that IL-18 production in the adipose tissue is independent of Nlrp3 inflammasome and caspase-1 activation, regardless of caloric food intake. In contrast, Nlrp3-mediated IL-1β production is caspase-1 dependent. These results provide new insights into the mechanisms underlying cytokine production in the adipose tissue during both homeostatic conditions and metabolic stress, highlighting the distinct roles of caspase-1 and the Nlrp inflammasomes in regulating inflammatory responses.

Intestinal metabolite UroB alleviates cerebral ischemia/reperfusion injury by promoting competition between TRIM65 and TXNIP for binding to NLRP3 inflammasome in response to neuroinflammation

Our previous research suggests that targeting NLRP3 inflammasomes holds promise for mitigating cerebral ischemia/reperfusion injury. The gut metabolite Urolithin B (UroB) has been shown to inhibit the neuroinflammation. However, the specific role of UroB in cerebral ischemia/reperfusion injury and its potential impact on NLRP3 inflammasome remain unclear.In this study, acute stroke was simulated using the MCAO model in male Sprague-Dawley rats. UroB was intraperitoneally administered after 1 h of reperfusion. The effects of UroB on brain tissue were evaluated, including infarct volume, brain edema, and neurobehavioral changes. Western blotting and immunofluorescence were performed to investigate the effect of UroB on inflammation-related proteins. Furthermore, TRIM65 knockdown and TXNIP overexpression experiments elucidated the role of UroB in NLRP3 inflammasome activation. The ( demonstrate the neuroprotective effect of UroB in acute stroke, reducing brain tissue damage and improving motor function. Mechanistically, UroB modulated neuroinflammation by influencing TXNIP and TRIM65 protein expression, as well as competitive binding to the NLRP3 inflammasome, attenuating cerebral ischemia/reperfusion injury.In conclusion, the potential of UroB as a protective agent against cerebral ischemia/reperfusion injury in acute stroke stands out as it regulates TRIM65 and TXNIP competitive binding to the NLRP3 inflammasome. These findings suggest that UroB is a promising drug candidate for the treatment of acute stroke.

Glibenclamide-Loaded Engineered Nanovectors (GNVs) Modulate Autophagy and NLRP3-Inflammasome Activation.

Activation of the NLRP3 inflammasome in response to either exogenous (PAMPs) or endogenous (DAMPs) stimuli results in the production of IL-18, caspase-1 and IL-1β. These cytokines have a beneficial role in promoting inflammation, but an excessive activation of the inflammasome and the consequent constitutive inflammatory status plays a role in human pathologies, including Alzheimer's disease (AD). Autophagic removal of NLRP3 inflammasome activators can reduce inflammasome activation and inflammation. Likewise, inflammasome signaling pathways regulate autophagy, allowing the development of inflammatory responses but preventing excessive and detrimental inflammation. Nanotechnology led to the development of liposome engineered nanovectors (NVs) that can load and carry drugs. We verified in an in vitro model of AD-associated inflammation the ability of Glibenclamide-loaded NVs (GNVs) to modulate the balance between inflammasome activation and autophagy. Human THP1dM cells were LPS-primed and oligomeric Aß-stimulated in the presence/absence of GNVs. IL-1β, IL-18 and activated caspase-1 production was evaluated by the Automated Immunoassay System (ELLA); ASC speck formation (a marker of NLRP3 activation) was analyzed by FlowSight Imaging flow-cytometer (AMNIS); the expression of autophagy targets was investigated by RT-PCR and Western blot (WB); and the modulation of autophagy-related up-stream signaling pathways and Tau phosphorylation were WB-quantified. Results showed that GNVs reduce activation of the NLRP3 inflammasome and prevent the Aß-induced phosphorylation of ERK, AKT, and p70S6 kinases, potentiating autophagic flux and counteracting Tau phosphorylation. These preliminary results support the investigation of GNVs as a possible novel strategy in disease and rehabilitation to reduce inflammasome-associated inflammation.

VSIG4 ameliorates intestinal inflammation through inhibiting macrophages NLRP3 inflammasome and pyroptosis

Inflammatory bowel disease (IBD) is one of the most common diseases in the digestive system related to aberrant inflammation. V-set and immunoglobulin domain-containing 4 (VSIG4), a type I transmembrane receptor exclusively expressed in a subset of tissue-resident macrophages, has been reported to exert anti-inflammatory activity in immune-related diseases, which has been not explored in IBD yet. This study aims to explore the role and the potential mechanism of VSIG4 in IBD. Clinical samples were obtained from IBD patients and were examined by immunohistochemical staining. THP-1 cells were differentiated into macrophages, and then stimulated with IL-4 plus IL-13 or LPS to induce pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. Cell transfection was conducted to overexpress VSIG4. Western blot and immunofluorescence assays were performed to assess NLRP3 inflammasome- and pyroptosis-related proteins. Cytokines were measured using ELISA. A cell co-culture model of Caco-2 cells and VSIG4-mediated macrophages were established. Cell viability and apoptosis was examined by CCK-8 and flow cytometry assays, respectively. VSIG4 was downregulated in IBD and was negatively correlated with NLRP3 inflammasome. M1 macrophages exhibited higher levels of NLRP3 inflammasome, pyroptosis and inflammatory response than M2 macrophages, while VSIG4 overexpression efficiently reversed these changes in M1 macrophages. In addition, VSIG4 overexpression partly abolished M1 macrophages-induced cell viability loss, inflammatory response, apoptosis and pyroptosis in Caco-2 cells. Collectively, VSIG4 might alleviate intestinal inflammation through regulating M1/M2 macrophages, providing novel insights for the treatment of human IBD.

The Mobility of Eurasian Avian-like M2 Is Determined by Residue E79 Which Is Essential for Pathogenicity of 2009 Pandemic H1N1 Influenza Virus in Mice.

In 2009, a novel H1N1 influenza virus caused the first influenza pandemic of the 21st century. Studies have shown that the influenza M gene played important roles in the pathogenicity and transmissibility of the 2009 H1N1 pandemic ((H1N1)pdm09), whilst the underlying mechanism remains unclear. The influenza M gene encodes two proteins, matrix protein 1 and matrix protein 2, which play important roles in viral replication and assembly. In this study, it is found that the M2 protein of the (H1N1)pdm09 virus showed a lower mobility rate than the North America triple-reassortant influenza M2 protein in Polyacrylamide Gel Electrophoresis (PAGE). The site-directed mutations of the amino acids of (H1N1)pdm09 M2 revealed that E79 is responsible for the mobility rate change. Further animal studies showed that the (H1N1)pdm09 containing a single M2-E79K was significantly attenuated compared with the wild-type virus in mice and induced lower proinflammatory cytokines and IFNs in mouse lungs. Further in vitro studies indicated that this mutation also affected NLRP3 inflammasome activation. To reveal the reason why they have different mobility rates, a circular dichroism spectra assay was employed and showed that the two M2 proteins displayed different secondary structures. Overall, our findings suggest that M2 E79 is important for the virus replication and pathogenicity of (H1N1)pdm09 through NLRP3 inflammasome and proinflammatory response.

Cyanobacterial toxins and components induce expression of pro-inflammatory cytokines/chemokines and secretion of soluble cell receptors in a 3D skin cell model

Abstract Exposure to cyanobacterial toxins has been linked to several diseases and disorders including skin rashes and damage to the liver and kidneys. Previous research has shown that Microcystin-LR, the most common and toxic microcystin in fresh water, induces the NLRP3 inflammasome in response to administration to mice by oral gavage. In this study, we used a Reconstructed Human Epidermis to investigate immune system responses to cyanobacterial exposure. This 3D tissue model consisting of normal, human-derived epidermal keratinocytes was exposed to Spirulina, anatoxin-contaminated lake water, and phycocyanin. The cell supernatant was probed with a multiplex of 38 cytokines/chemokines and 14 soluble receptors to elucidate the immunological response. Our results revealed that: while most proteins were down-regulated, a number were up-regulated; the expression of many proteins were highly correlated and clustered together; and across the case study datasets, multiple cytokines/chemokines (G-CSF, GRO, IL-6, IL-8, and MCP-1) and soluble cell receptors (sCD30, sEGFR, sTNFRII and sVEGFR1, sVEGFR2 and sVEGFR3) were highly expressed when exposed to the toxins. Our findings correspond with preliminarily histological results showing tissue damage when the skin cells were exposed to the cyanobacterial components. This work shows great promise in the ability to examine the immune response to toxic exposures and aids in the development of techniques useful for studying the impact of harmful algal blooms on public health. Future studies will focus on mapping the immune pathway in response to harmful algal bloom exposure and studying the roles of each of the expressed cytokines and soluble cell receptors in the inflammatory response. This work is supported by the United States Environmental Protection Agency.

Impact of Maternal Ketogenic Diet on NLRP3 Inflammasome Response in the Offspring Brain.

The effects of maternal diet on the neuroimmune responses of the offspring remain to be elucidated. We investigated the impact of maternal ketogenic diet (KD) on the NLRP3 inflammasome response in the offspring's brain. C57BL/6 female mice were randomly allocated into standard diet (SD) and ketogenic diet (KD) groups for 30 days. After mating, the presence of sperm in the vaginal smear was considered day 0 of pregnancy, and female mice continued their respective diets during pregnancy and the lactation period. Following birth, pups were further allocated into two groups and given either LPS or intraperitoneal saline on postnatal (PN) days 4, 5 and 6; they were sacrificed on PN11 or PN21. Neuronal densities were significantly lower globally in the KD group when compared to the SD group at PN11. Neuronal density in the prefrontal cortex (PFC) and dentate gyrus (DG) regions were also significantly lower in the KD group when compared to the SD group at PN21. Following administration of LPS, the decrease in the neuronal count was more prominent in the SD group when compared to the KD group in the PFC and DG regions at PN11 and PN21. NLRP3 and IL-1β were higher in the KD group than in the SD group at PN21 in the PFC, CA1 and DG regions, and were significantly lower in the DG region of the KD group especially when compared to the SD group following LPS. Results of our study reveal that maternal KD negatively affects the offspring's brain in the mouse model. The effects of KD exhibited regional variations. On the other hand, in the presence of KD exposure, NLRP3 expression after LPS injection was lower in the DG and CA1 areas but not in the PFC when compared to SD group. Further experimental and clinical studies are warranted to elucidate the molecular mechanisms underlying the impact of antenatal KD exposure and regional discrepancies on the developing brain.

Deregulated NLRP3 inflammasome response due to a constitutive activation of NF-κB p65 pro-inflammatory pathway in lymphomonocytes of Rett syndrome patients

Deregulated NLRP3 inflammasome response due to a constitutive activation of NF-κB p65 pro-inflammatory pathway in lymphomonocytes of Rett syndrome patients

Accelerated NLRP3 inflammasome-inhibitory peptide design using a recurrent neural network model and molecular dynamics simulations

Anomalous NLRP3 inflammasome responses have been linked to multiple health issues, including but not limited to atherosclerosis, diabetes, metabolic syndrome, cardiovascular disease, and neurodegenerative disease. Thus, targeting NLRP3 and modulating its associated immune response might be a promising strategy for developing new anti-inflammatory drugs. Herein, we report a computational method for de novo peptide design for targeting NLRP3 inflammasomes. The described method leverages a long-short-term memory (LSTM) network based on a recurrent neural network (RNN) to model a valuable latent space of molecules. The resulting classifiers are utilized to guide the selection of molecules generated by the model based on circular dichroism spectra and physicochemical features derived from high-throughput molecular dynamics simulations. Of the experimentally tested sequences, 60% of the peptides showed NLRP3-mediated inhibition of IL-1β and IL-18. One peptide displayed high potency against NLRP3-mediated IL-1β inhibition. However, NLRC4 and AIM2 inflammasome-mediated IL-1β secretion was uninterrupted by this peptide, demonstrating its selectivity toward the NLRP3 inflammasome. Overall, these results indicate that deep learning and molecular dynamics can accelerate the discovery of NLRP3 inhibitors with potent and selective activity.

The thioredoxin system: Balancing redox responses in immune cells and tumors.

The thioredoxin (TRX) system is an important contributor to cellular redox balance and regulates cell growth, apoptosis, gene expression, and antioxidant defense in nearly all living cells. Oxidative stress, the imbalance between reactive oxygen species (ROS) and antioxidants, can lead to cell death and tissue damage, thereby contributing to aging and to the development of several diseases, including cardiovascular and allergic diseases, diabetes, and neurological disorders. Targeting its activity is also considered as a promising strategy in the treatment of cancer. Over the past years, immunologists have established an essential function of TRX for activation, proliferation, and responses in T cells, B cells, and macrophages. Upon activation, immune cells rearrange their redox system and activate the TRX pathway to promote proliferation through sustainment of nucleotide biosynthesis, and to support inflammatory responses in myeloid cells by allowing NF-κB and NLRP3 inflammasome responses. Consequently, targeting the TRX system may therapeutically be exploited to inhibit immune responses in inflammatory conditions. In this review, we summarize recent insights revealing key roles of the TRX pathway in immune cells in health and disease, and lessons learnt for cancer therapy.

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout.

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c+ dendritic cells

Inflammasomes are multiprotein platforms responsible for the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Mouse studies have identified inflammasome activation within dendritic cells (DC) as pivotal for driving tubulointerstitial fibrosis and inflammation, the hallmarks of chronic kidney disease (CKD). However, translation of this work to human CKD remains limited. Here, we examined the complex tubular cell death pathways mediating inflammasome activation in human kidney DC and, thus, CKD progression. Ex vivo patient-derived proximal tubular epithelial cells (PTEC) cultured under hypoxic (1% O2) conditions modelling the CKD microenvironment showed characteristics of ferroptotic cell death, including mitochondrial dysfunction, reductions in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and increases in lipid peroxidation by-product 4-hydroxynonenal (4-HNE) compared with normoxic PTEC. The addition of ferroptosis inhibitor, ferrostatin-1, significantly reduced hypoxic PTEC death. Human CD1c+ DC activated in the presence of hypoxic PTEC displayed significantly increased production of inflammasome-dependent cytokines IL-1β and IL-18. Treatment of co-cultures with VX-765 (caspase-1/4 inhibitor) and MCC950 (NLRP3 inflammasome inhibitor) significantly attenuated IL-1β/IL-18 levels, supporting an NLRP3 inflammasome-dependent DC response. In line with these in vitro findings, in situ immunolabelling of human fibrotic kidney tissue revealed a significant accumulation of tubulointerstitial CD1c+ DC containing active inflammasome (ASC) specks adjacent to ferroptotic PTEC. These data establish ferroptosis as the primary pattern of PTEC necrosis under the hypoxic conditions of CKD. Moreover, this study identifies NLRP3 inflammasome signalling driven by complex tubulointerstitial PTEC-DC interactions as a key checkpoint for therapeutic targeting in human CKD.

Mechanical Stretch-Induced NLRP3 Inflammasome Expression on Human Annulus Fibrosus Cells Modulated by Endoplasmic Reticulum Stress.

Excessive mechanical loading is a major cause of spinal degeneration, typically originating from a tear in the annulus fibrosus (AF). Endoplasmic reticulum (ER) stress and NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome have been implicated in the pathogenesis of intervertebral disc (IVD) degeneration. However, the causal relationship between the mechanical stretching of AF cells and the NLRP3 inflammasome response associated with ER stress remains scarce. To elucidate the pathogenesis and regulatory mechanisms of mechanical stretch-induced IVD degeneration, human AF cell lines were subjected to different degrees of cyclic stretching to simulate daily spinal movements. Our results indicated that 15% high cyclic stretch (HCS) induced the expression of NLRP3 and interleukin-1 beta (IL-1β) and was also responsible for the increased expression of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2) and reactive oxygen species (ROS) in human AF cells. In addition, HCS increased the expression of glucose-regulated protein 78 (GRP78), an ER stress chaperone, which was neutralized with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. In addition, HCS was found to induce thioredoxin-interacting protein (TXNIP) expression and NLRP3 inflammasome activation, which can be suppressed by si-NOX2 or the NOX2 inhibitor GSK2795039. Consequently, HCS upregulated ER stress and ROS production, leading to increased NLRP3 and IL-1β expression in human AF cells, and may further accelerate IVD degeneration.

Lysosomal dysfunction is associated with NLRP3 inflammasome activation in chronic unpredictable mild stress-induced depressive mice

NLRP3 inflammasome pathway-mediated inflammatory response is closely associated with depression. Increasing attention has been recently paid to the links between autophagy and depression, however, the relationship between autophagy and NLRP3 inflammasome in depressive behavior remain poorly understood. In the present study, the potential roles of autophagy-lysosome pathway in NLRP3 inflammasome regulation were investigated both in vivo (chronic unpredictable mild stress (CUMS)-induced depressive mouse model) and in vitro (LPS-induced cellular model) model. It demonstrated that CUMS induces depressive-like behaviors in mice, accompanied by increased expression of NLRP3 inflammasome and inflammatory responses. Meanwhile, it promoted the autophagosome marker LC3 and autophagic adapter protein p62 accumulation, accompanied by the decrease of lysosomal cathepsins B and D expression in the prefrontal cortex of mice. Notably, a significant colocalization of NLRP3 and LC3 in CUMS mice by immunofluorescence co-staining were observed. For the in vitro study, disrupting the lysosomal function with Baf A1 significantly increased the LPS-induced NLRP3 inflammasome accumulation and pro-inflammatory factors (IL-1β and IL-18) production in BV2 cells. Collectively, our results suggested that the autophagic process is related to NLRP3 inflammasome activation, and dysfunctional lysosome in autophagy-lysosomal pathway may retard NLRP3 inflammasome degradation, facilitating the production of pro-inflammatory factors, thereby contributing to depressive behavior in CUMS mice.

Linoleic Acid‐Derived Diol 12,13‐DiHOME Modulates THP‐1 Macrophage NLRP3 Inflammasome Activation

While inflammatory dysregulation is a major contributor to cardiac injury and worsened functional recovery, current anti‐inflammatory therapies have shown limited success, necessitating studies which identify gaps in our mechanistic understanding of inflammation in the heart. Essential dietary N‐3 and N‐6 polyunsaturated fatty acids (PUFAs) are metabolized into a family of PUFA epoxides and downstream diols with contrasting effects in cardiac inflammatory disease, suggesting metabolite‐ and cell‐specific roles for these molecules. Previous studies have demonstrated 12,13‐dihydroxyoctadecenoic acid (12,13‐DiHOME), a linoleic acid‐derived diol, mediates mitochondrial damage and inflammation in cardiomyocytes, though its roles in other cardiac cell types are unknown. We hypothesized that 12,13‐DiHOME enhances macrophage inflammation by promoting M1 polarization and NLRP3 inflammasome activation. To study macrophage polarization, PMA‐differentiated THP1 “M0” macrophages were incubated with vehicle, 12,13‐DiHOME (0.5 µM), M1‐polarization stimuli (10 ng/mL LPS and 20 ng/mL IFN‐gamma) or M1‐polarization stimuli + 12,13‐DiHOME for 24 hours. Using quantitative real‐time PCR we observed that 12,13‐DiHOME alone did not induce M1 polarization but M1‐associated cytokine TNF gene expression was enhanced in macrophages treated with 12,13‐DiHOME alone or M1‐polarization stimuli + 12,13‐DiHOME. The NLRP3 inflammasome response was activated in THP1 M0 macrophages by first priming with LPS (10 ng/mL) for 4.5h followed by treatment with nigericin (10 µM) for 30min. NLRP3 inflammasome activation was assessed by measuring key markers (NLRP3, caspase‐1, interleukin‐1β). We observed a concentration‐dependent increase in NLRP3 inflammasome activation when 12,13‐DiHOME was co‐treated with LPS‐priming. Next, we used epifluorescence microscopy to assess mitochondrial dysfunction, which is known to modulate macrophage NLRP3 inflammasome activation, by measuring alterations in membrane potential with TMRE (50nM). Time course analyses demonstrated no differences in mitochondrial membrane potential between LPS‐primed and LPS+12,13‐DiHOME‐primed macrophages prior to nigericin‐activation. However, LPS+12,13‐DiHOME‐primed macrophages activated with nigericin had a more rapid decline in membrane potential compared to nigericin‐activated macrophages primed with LPS alone, suggesting exaggerated mitochondrial damage. Overall, our data demonstrates 12,13‐DiHOME does not trigger macrophage polarization alone but enhances macrophage NLRP3 inflammasome activation potentially by causing mitochondrial injury.

Emodin Alleviates Arthritis Pain through Reducing Spinal Inflammation and Oxidative Stress.

Chronic pain is the predominant problem for rheumatoid arthritis patients, and negatively affects quality of life. Arthritis pain management remains largely inadequate, and developing new treatment strategies are urgently needed. Spinal inflammation and oxidative stress contribute to arthritis pain and represent ideal targets for the treatment of arthritis pain. In the present study, collagen-induced arthritis (CIA) mouse model was established by intradermally injection of type II collagen (CII) in complete Freund's adjuvant (CFA) solution, and exhibited as paw and ankle swelling, pain hypersensitivity and motor disability. In spinal cord, CIA inducement triggered spinal inflammatory reaction presenting with inflammatory cells infiltration, increased IL-1β expression, and up-regulated NLRP3 and cleaved caspase-1 levels, elevated spinal oxidative level presenting as decreased Nrf2 expression and SOD activity. To explore potential therapeutic options for arthritis pain, emodin was intraperitoneally injected for three days on CIA mice. Emodin treatment statistically elevated mechanical pain sensitivity, suppressed spontaneous pain, recovered motor coordination, decreased spinal inflammation scores and expression levels of IL-1β, increased spinal Nrf2 expression and SOD activity. Further, AutoDock data showed that emodin bind to AMPK through two electrovalent bonds. And emodin treatment increased the phosphorylated AMPK at threonine 172. In summary, emodin treatment activates AMPK, suppresses NLRP3 inflammasome response, elevates antioxidant response, inhibits spinal inflammatory reaction and alleviates arthritis pain.

BuFeiXiaoJiYin ameliorates the NLRP3 inflammation response and gut microbiota in mice with lung cancer companied with Qi-yin deficiency

IntroductionNLRP3 inflammasome responses and gut microbiota have been shown an important role in lung cancer, however, the relationship between gut microbiota and NLRP3 inflammasome responses in lung cancer with Qi-yin deficiency remains elusive.MethodsTo investigate the effect of the traditional Chinese medicine BuFeiXiaoJiYin (BFXJY) on NLRP3 inflammasome responses and dysbiosis in lung cancer with Qi-yin deficiency, the female BALB/cA-nu mice were treated with LPS and ATP to induce inflammation, and were intragastrically treated with warm Chinese medicine and smoked with shavings to induce Qi-yin deficiency, as well as were injected with 1 × 107/ml A549 cells to simulate lung cancer. Then the three different doses of BuFeiXiaoJiYin (BFXJY) and positive control (CRID3) were used for intervention in mice for 27 consecutive days. Then, we estimated the protection effect of BFXJY on lung cancer mice with Qi-yin deficiency, through deterring tumor growth, NLRP3 inflammasome, PKC signaling, and homeostasis of gut microbiota.ResultsIn this study, we found that BFXJY could inhibit the tumor growth in lung cancer with Qi-yin deficiency by reducing the production of IL-1β and IL-18 and inhibiting NLRP3 inflammasome activation, which might be associated with the inhibition of PKC signaling. Furthermore, BFXJY could promote microbial diversity and balance the microbial composition changes induced by inflammation and Qi-yin deficiency in lung cancer.ConclusionBuFeiXiaoJiYin ameliorates the NLRP3 inflammation response and gut microbiota in mice with lung cancer companied with Qi-yin deficiency. Our study provides a theoretical basis for the clinical development of therapeutic drugs targeting to treat lung cancer.