Inflammasome Activation Research Articles

The Different Responsiveness of C3- and C5-deficient Murine BM Cells to Oxidative Stress Explains Why C3 Deficiency, in Contrast to C5 Deficiency, Correlates with Better Pharmacological Mobilization and Engraftment of Hematopoietic Cells.

The liver-derived circulating in peripheral blood and intrinsic cell-expressed complement known as complosome orchestrate the trafficking of hematopoietic stem/progenitor cells (HSPCs) both during pharmacological mobilization and homing/engraftment after transplantation. Our previous research demonstrated that C3 deficient mice are easy mobilizers, and their HSPCs engraft properly in normal mice. In contrast, C5 deficiency correlates with poor mobilization and defects in HSPCs' homing and engraftment. The trafficking of HSPCs during mobilization and homing/engraftment follows the sterile inflammation cues in the BM microenvironment caused by stress induced by pro-mobilizing drugs or myeloablative conditioning for transplantation. Therefore, to explain deficiencies in HSPC trafficking between C3-KO and C5-KO mice, we evaluated the responsiveness of C3 and C5 deficient cells to low oxidative stress. As reported, oxidative stress in BM is mediated by the activation of purinergic signaling, which is triggered by the elevated level of extracellular adenosine triphosphate (eATP) and by the activation of the complement cascade (ComC). In the current work, we noticed that BM lineage negative cells (lin-) isolated from C3-KO mice display several mitochondrial defects reflected by an impaired ability to adapt to oxidative stress. In contrast, C5-KO-derived BM cells show a high level of adaptation to this challenge. To support this data, C3-KO BM lin- cells were highly responsive to eATP stimulation, which correlates with enhanced levels of reactive oxygen species (ROS) generation and more efficient activation of intracellular Nlrp3 inflammasome. We conclude that the enhanced sensitivity of C3-KO mice cells to oxidative stress and better activation of the Nox2-ROS-Nlrp3 inflammasome signaling axis explains the molecular level differences in trafficking between C3- and C5-deficient HSPCs.

RACK1 and NEK7 mediate GSDMD-dependent macrophage pyroptosis upon Streptococcus suis infection

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that induces an NLRP3-dependent cytokine storm. NLRP3 inflammasome activation triggers not only an inflammatory response but also pyroptosis. However, the exact mechanism underlying S. suis-induced macrophage pyroptosis is not clear. Our results showed that SS2 induced the expression of pyroptosis-associated factors, including lactate dehydrogenase (LDH) release, propidium iodide (PI) uptake and GSDMD-N expression, as well as NLRP3 inflammasome activation and IL-1β secretion. However, GSDMD deficiency and NLRP3 inhibition using MCC950 attenuated the SS2-induced expression of pyroptosis-associated factors, suggesting that SS2 induces NLRP3-GSDMD-dependent pyroptosis. Furthermore, RACK1 knockdown also reduced the expression of pyroptosis-associated factors. In addition, RACK1 knockdown downregulated the expression of NLRP3 and Pro-IL-1β as well as the phosphorylation of P65. Surprisingly, the interaction between RACK1 and P65 was detected by co-immunoprecipitation, indicating that RACK1 induces macrophage pyroptosis by mediating the phosphorylation of P65 to promote the transcription of NLRP3 and pro-IL-1β. Similarly, NEK7 knockdown decreased the expression of pyroptosis-associated factors and ASC oligomerization. Moreover, the results of co-immunoprecipitation revealed the interaction of NEK7-RACK1-NLRP3 during SS2 infection, demonstrating that NEK7 mediates SS2-induced pyroptosis via the regulation of NLRP3 inflammasome assembly and activation. These results demonstrate the important role of RACK1 and NEK7 in SS2-induced pyroptosis. Our study provides new insight into SS2-induced cell death.

Taking AIM at Influenza: The Role of the AIM2 Inflammasome.

Influenza A viruses (IAV) are dynamic and highly mutable respiratory pathogens that present persistent public health challenges. Inflammasomes, as components of the innate immune system, play a crucial role in the early detection and response to infections. They react to viral pathogens by triggering inflammation to promote immune defences and initiate repair mechanisms. While a strong response is necessary for early viral control, overactivation of inflammasomes can precipitate harmful hyperinflammatory responses, a defining characteristic observed during severe influenza infections. The Absent in Melanoma 2 (AIM2) inflammasome, traditionally recognised for its role as a DNA sensor, has recently been implicated in the response to RNA viruses, like IAV. Paradoxically, AIM2 deficiency has been linked to both enhanced and reduced vulnerability to IAV infection. This review synthesises the current understanding of AIM2 inflammasome activation during IAV and explores its clinical implications. Understanding the nuances of AIM2's involvement could unveil novel therapeutic avenues for mitigating severe influenza outcomes.

The human disease-associated gene ZNFX1 controls inflammation through inhibition of the NLRP3 inflammasome.

Inherited deficiency of zinc finger NFX1-type containing 1 (ZNFX1), a dsRNA virus sensor, is associated with severe familial immunodeficiency, multisystem inflammatory disease, increased susceptibility to viruses, and early mortality. However, limited treatments for patients with pathological variants of ZNFX1 exist due to an incomplete understanding of the diseases resulting from ZNFX1 mutations. Here, we demonstrate that ZNFX1 specifically inhibits the activation of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome in response to NLRP3 activators both in vitro and in vivo. ZNFX1 retains NLRP3 in the cytoplasm and prevents its accumulation in the TGN38 + /TGN46+ vesicles in the resting state. Upon NLRP3 inflammasome activation, ZNFX1 is cleaved by caspase-1, establishing a feed-forward loop that promotes NLRP3 accumulation in the trans-Golgi network (TGN) and amplifies the activity of the downstream cascade. Expression of wild-type ZNFX1, but not of ZNFX1 with human pathogenic mutations, rescues the impairment of NLRP3 inflammasome inhibition. Our findings reveal a dual role of ZNFX1 in virus sensing and suppression of inflammation, which may become valuable for the development of treatments for ZNFX1 mutation-related diseases.

Gut microbiome and metabolites mediate the benefits of caloric restriction in mice after acute kidney injury

The role of gut microbiome in acute kidney injury (AKI) is increasing recognized. Caloric restriction (CR) has been shown to enhance the resistance to ischemia/reperfusion injury to the kidneys in rodents. Nonetheless, it is unknown whether intestinal microbiota mediated CR protection against ischemic/reperfusion-induced injury (IRI) in the kidneys. Herein, we showed that CR ameliorated IRI-elicited renal dysfunction, oxidative stress, apoptosis, and inflammation, along with enhanced intestinal barrier function. In addition, gut microbiota depletion blocked the favorable effects of CR in AKI mice. 16S rRNA and metabolomics analysis showed that CR enriched the gut commensal Parabacteroides goldsteinii (P. goldsteinii) and upregulated the level of serum metabolite dodecafluorpentan. Intestinal colonization of P. goldsteinii and oral administration of dodecafluorpentan showed the similar beneficial effects as CR in AKI mice. RNA sequencing and experimental data revealed that dodecafluorpentan protected against AKI-induced renal injury by antagonizing oxidative burst and NFκB-induced NLRP3 inflammasome activation. In addition, we screened and found that Hamaudol improved renal insufficiency by boosting the growth of P. goldsteinii. Our results shed light on the role of intestinal microbiota P. goldsteinii and serum metabolites dodecafluorpentan in CR benefits to AKI.

Design, Synthesis, and Bioevaluation of Novel NLRP3 Inhibitor with IBD Immunotherapy from the Virtual Screen.

NLRP3, a crucial member of the NLRP family, plays a pivotal role in immune regulation and inflammatory modulation. Here, we report a potent and specific NLRP3 inhibitor Z48 obtained though docking-based virtual screening and structure-activity relationship studies with an IC50 of 0.26 μM in THP-1 cells and 0.21 μM in mouse bone marrow-derived macrophages. Mechanistic studies indicated that Z48 could bind directly to the NLRP3 protein (KD = 1.05 μM), effectively blocking the assembly and activation of the NLRP3 inflammasome, consequently manifesting anti-inflammatory properties. Crucially, with acceptable mouse pharmacokinetic profiles, Z48 demonstrated notable therapeutic efficacy in a mouse model of DSS-induced ulcerative colitis, while displaying no significant therapeutic impact on NLRP3KO mice. In conclusion, this study provided a promising NLRP3 inflammasome inhibitor with novel molecular scaffold, poised for further development as a therapeutic candidate in the treatment of inflammatory bowel disease.

Antagonistic nanobodies implicate mechanism of GSDMD pore formation and potential therapeutic application

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.

Parthenolide ameliorates 3-nitropropionic acid-induced Huntington’s disease-like aberrations via modulating NLRP3 inflammasome, reducing microglial activation and inducing astrocyte shifting

BackgroundHuntington’s disease (HD) is a progressive neurodegenerative disease that causes motor, cognitive, and psychiatric abnormalities, with no satisfying disease-modifying therapy so far. 3-nitropropionic acid (3NP) induces behavioural deficits, together with biochemical and histological alterations in animals’ striata that mimic HD. The role of nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome in HD pathogenesis remains largely uncharacterized. Parthenolide (PTL), a naturally occurring nuclear factor kappa B (NF-κB) inhibitor, is also known to inhibit NLRP3 inflammasome. Whether PTL is beneficial in HD has not been established yet.AimThis study evaluated the possible neuroprotective effects of PTL against 3NP-induced behavioural abnormalities, striatal biochemical derangements, and histological aberrations.MethodsMale Wistar rats received PTL (0.5 mg/kg/day, i.p) for 3 weeks and 3NP (10 mg/kg/day, i.p) was administered alongside for the latter 2 weeks to induce HD. Finally, animals were subjected to open-field, Morris water maze and rotarod tests. Rat striata were examined histologically, striatal protein expression levels of glial fibrillary acidic protein (GFAP), cluster of differentiation 45 (CD45) and neuron-specific enolase (NSE) were evaluated immunohistochemically, while those of interleukin (IL)-1β, IL-18, ionized calcium-binding adapter molecule-1 (Iba1) and glutamate were determined by ELISA. Striatal nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein (Keap1), NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, S100 calcium-binding protein A10 (S100A10) and complement-3 (C3) were assessed by gene expression analysis.ResultsPTL improved motor, locomotor, cognitive and anxiety-like behaviours, restored neuronal integrity, upregulated Nrf2, and inhibited NLRP3 inflammasome, NF-κB and microglial activation. Additionally, PTL induced astrocyte shifting towards the neuroprotective A2 phenotype.ConclusionPTL exhibits neuroprotection against 3NP-induced HD, that might be ascribed, at least in part, to its modulatory effects on Keap1/Nrf2 and NF-κB/NLRP3 inflammasome signaling.

Short-term respiratory cadmium exposure partially activates pulmonary NLRP3 inflammasome by inducing ferroptosis in mice

Cadmium (Cd) is a common environmental metal. Previous studies indicated that long-term respiratory Cd exposure caused lung injury and airway inflammation. The purpose of this study was to evaluate whether short-term respiratory Cd exposure induces pulmonary ferroptosis and NLRP3 inflammasome activation. Adult C57BL/6J mice were exposed to Cd by inhaling CdCl2 aerosol (0, 10, or 100 ppm) for 5 days. Serum and lung Fe2+ contents were elevated in Cd-exposed mice. Oxidized AA metabolites, the major oxidized lipids during ferroptosis, were upregulated in Cd-exposed mouse lungs. Pulmonary MDA content and 4-HNE-positive cells were increased in Cd-exposed mice. ACSL4 and COX-2, two lipoxygenases, were upregulated in Cd-exposed mouse lungs. Further analyses found that phosphorylated NF-kB p65 was elevated in Cd-exposed mouse lungs. Innate immune receptor protein NLRP3 and adapter protein ASC were upregulated in Cd-exposed mouse lungs. Caspase-1 was activated and IL-1β and IL-18 were upregulated in Cd-exposed mouse lungs. Fer-1, a specific inhibitor of ferroptosis, attenuated Cd-induced elevation of pulmonary NLRP3 and ASC, caspase-1 activation, and IL-1β and IL-18 upregulation. Finally, mitoquinone (MitoQ), a mitochondria-target antioxidant, suppressed Cd-caused ferroptosis and NLRP3 inflammasome activation. Our results demonstrate that ferroptosis might partially mediate Cd-evoked activation of NLRP3 inflammasome in the lungs.

GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging.

Age-related susceptibility to sepsis and endotoxemia is poorly defined, although hyperactivation of the immune system and the expansion of the visceral adipose as an immunological reservoir are underlying features. Macrophages from older organisms exhibit substantial changes, including chronic NLRP3 inflammasome activation, genomic remodeling and a dysfunctional, amplified inflammatory response upon new exposure to pathogen. However, the mechanisms by which old macrophages maintain their inflammatory phenotype during endotoxemia remains elusive. We previously identified Gdf3 , a TGFβ superfamily cytokine, as a top-regulated gene by age and the NLRP3 inflammasome in adipose tissue macrophages (ATMs). Here, we demonstrate that endotoxemia increases inflammatory (CD11c + ) ATMs in a Gdf3- dependent manner in old mice. Lifelong systemic or myeloid-specific deletion of Gdf3 leads to reduced endotoxemia- induced inflammation, with decreased CD11c + ATMs and inflammatory cytokines, and protection from hypothermia. Moreover, acute blockade of Gdf3 using JQ1, a BRD4 inhibitor, phenocopies old mice with lifelong Gdf3- deficiency. We show that GDF3 promotes the inflammatory phenotype in ATMs by phosphorylating SMAD2/3. Mechanistically, the differential chromatin landscape of ATMs from old mice with or without myeloid-driven Gdf3 indicates that GDF3- SMAD2/3 signaling axis shifts the chromatin accessibility of ATMs towards an inflammatory state during aging. Furthermore, pharmaceutical inhibition of SMAD3 with a specific inhibitor of SMAD3 (SIS3) mimics Gdf3 deletion. SIS3 reduces endotoxemia-mediated inflammation with fewer CD11c + ATMs and less severe hypothermia in old, but not young mice, as well as reduced mortality. In human adipose tissue, age positively correlates with GDF3 level, while inflammation correlates with pSMAD2/3 level. Overall, these results highlight the importance of GDF3-SMAD2/3 axis in driving inflammation in older organisms and identify this signaling axis as a promising therapeutic target for mitigating endotoxemia-related inflammation in the aged.

Mitigation of lipopolysaccharide-induced intestinal injury in rats by Chimonanthus nitens Oliv. essential oil via suppression of mitochondrial fusion protein mitofusin 2 (MFN2)-mediated mitochondrial-associated endoplasmic reticulum membranes (MAMs) formation

Ethnopharmacological relevanceChimonanthus nitens Oliv. is a traditional Chinese medicine with anti-inflammatory and antioxidant properties that has commonly been used for colds, fevers, and other diseases. However, its role and specific mechanism in sepsis-associated intestinal injury have not been reported. Aim of the studyC. nitens Oliv. essential oil (CEO), an organic active compound extracted from the traditional Chinese medicine C. nitens Oliv. exhibits notable anti-inflammatory and antioxidant properties. Nevertheless, the therapeutic potential of CEO for septic intestinal injury remains undocumented. This study thus aims to elucidate the anti-inflammatory and antioxidant effects of CEO in the context of acute intestinal injury and to investigate its mechanisms of action in septic rats. Materials and methodsCell and animal models were established using LPS to investigate the impact of CEO on LPS-induced intestinal pathological injury and the secretion of inflammatory factor IL-1β. The effects of CEO on the expression of NLRP3, caspase-1, and MFN2, p-p65 protein were also examined, as well as its influence on oxidative stress injury and mitochondrial-associated endoplasmic reticulum membrane (MAM) formation. Generation of an MFN2 knockout IEC-6 cell line allowed comprehensive investigation of the protective mechanism of CEO. ResultsIn rat models, CEO reduced IL-1β secretion, inhibited caspase-1, ZO-1 expression and NF-κB p65 phosphorylation, while also decreasing malondialdehyde levels and enhancing superoxide dismutase activity in intestinal tissues. Cellular experiments demonstrated its ability to decrease IL-1β secretion; NLRP3, caspase-1, and MFN2 expression; NF-κB p65 phosphorylation; reactive oxygen species (ROS) production, and mitochondrial dysfunction. MFN2 knockdown enhanced these effects synergistically with CEO, indicating potential therapeutic synergy. Further, MFN2 knockdown significantly mitigated LPS-induced NLRP3 and caspase-1 expression, IL-1β secretion, ROS production, NF-κB p65 phosphorylation and MMP reduction in IEC-6 cells, while inhibiting MAM formation and NLRP3 localization on MAMs. Importantly, MFN2 downregulation and CEO synergistically reduced LPS-induced IL-1β secretion and ROS production while inhibiting MAM formation in IEC-6 cells, thus inhibiting NLRP3 inflammasome activation. ConclusionCEO mitigates inflammation and oxidative stress by inhibiting MAM formation and is thus a promising intervention for septic intestinal injury.

Tamarixetin Protects Chondrocytes against IL-1β-Induced Osteoarthritis Phenotype by Inhibiting NF-κB and Activating Nrf2 Signaling.

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage breakdown and chronic inflammation in joints. As the most prevalent form of arthritis, OA affects around 600 million people globally. Despite the increasing number of individuals with OA risk factors, such as aging and obesity, there is currently no effective cure for the disease. In this context, this study investigated the therapeutic effects of tamarixetin, a flavonoid with antioxidative and anti-inflammatory properties, against OA pathology and elucidated the underlying molecular mechanism. In interleukin-1β (IL-1β)-treated chondrocytes, tamarixetin inhibited the OA phenotypes, restoring cell viability and chondrogenic properties while reducing hypertrophic differentiation and dedifferentiation. Tamarixetin alleviated oxidative stress via the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway activation and inhibited mitogen-activated protein kinase and nuclear factor-κB (NF-κB). Furthermore, tamarixetin attenuated pyroptosis, a programmed cell death caused by excessive inflammation, by suppressing inflammasome activation. We confirmed that the chondroprotective effects of tamarixetin are mediated by the concurrent upregulation of Nrf2 signaling and downregulation of NF-κB signaling, which are key players in balancing antioxidative and inflammatory responses. Overall, our study demonstrated that tamarixetin possesses chondroprotective properties by alleviating IL-1β-induced cellular stress in chondrocytes, suggesting its therapeutic potential to relieve OA phenotype.

Pseudorabies Virus UL4 protein promotes the ASC-dependent inflammasome activation and pyroptosis to exacerbate inflammation.

Pseudorabies virus (PRV) infection causes systemic inflammatory responses and inflammatory damages in infected animals, which are associated with the activation of inflammasome and pyroptosis in infected tissues. Here, we identified a critical function of PRV non-structural protein UL4 that enhanced ASC-dependent inflammasome activation to promote pyroptosis. Whereas, the deficiency of viral UL4 was able to reduce ASC-dependent inflammasome activation and the occurrences of pyroptosis. Mechanistically, the 132-145 aa of UL4 permitted its translocation from the nucleus to the cytoplasm to interact with cytoplasmic ASC to promote the activation of NLRP3 and AIM2 inflammasome. Further research showed that UL4 promoted the phosphorylation levels of SYK and JNK to enhance the ASC phosphorylation, which led to the increase of ASC oligomerization, thus promoting the activation of NLRP3 and AIM2 inflammasome and enhanced GSDMD-mediated pyroptosis. In vivo experiments further showed that PRV UL4 (132DVAADAAAEAAAAE145) mutated strain (PRV-UL4mut) infection did not lead to a significant decrease in viral titers at 12 h. p. i, but it induced lower levels of IL-1β, IL-18, and GSDMD-NT, which led to an alleviated inflammatory infiltration and pathological damage in the lungs and brains, and a lower death rate compared with wild-type PRV strain infection. Taken together, our findings unravel that UL4 is an important viral regulator to manipulate the inflammasome signaling and pyroptosis of host cells to promote the pathogenicity of PRV, which might be further exploited as a new target for live attenuated vaccines or therapeutic strategies against pseudorabies in the future.

Use of Sodium-glucose cotransporter 2 (SGLT 2) inhibitor is associated with reduced emergency room visits and hospitalizations in patients with Chronic obstructive pulmonary disease (COPD) and type 2 Diabetes Mellitus

BackgroundThe Sodium-Glucose Cotransporter 2 inhibitors (SGLT2i) are a class of anti-diabetic medications that confer cardio-renal-metabolic (CRM) benefits. Emerging evidence also suggests that these agents provide better benefits for chronic pulmonary conditions, especially chronic obstructive pulmonary disease (COPD). Research questionWe aimed to assess the association between SGLT2i use and outcomes in patients with COPD and concomitant Type 2 Diabetes Mellitus (T2DM). Study design and methodsWe conducted a retrospective cohort study on adults with T2DM and COPD in a primary care clinic from January 01, 2019 to 01/01//2023. Patients were categorized into two groups based on SGLT2i use. We collected demographic information and outcomes such as emergency room (ER) visits, hospitalizations secondary to COPD exacerbation over the period of four years and time to hospitalization and ER visits. Chi-square analysis was used for categorical variables, whereas an unpaired t-test was used for continuous variables. Cox regression was performed to identify significant prognostic factors of hospitalization and ER visits. A Kaplan-Meir analysis was used to visualize the probability of non-hospitalization and the probability of not visiting the ER. Statistical significance was set at p-value <0.05. ResultsOf the 220 patients screened, 94 met the inclusion criteria, of which 20 patients (21.3 %) had SGLT2i use at admission, and 74 (78.7 %) did not. Baseline demographic information were well-matched between the two groups. SGLT2i use was associated with a significant reduction in ER visits (70 % vs. 97.3 %, p-0.001) and the number of hospitalizations (55 % vs 87.8 %, p-0.001). Further multivariate analysis showed lower hazards of hospitalization (adjusted HR-0.156; CI:0.073 to 0.331) and ER visits (HR)-0.232; CI:0.118 to 0.453) in patients on SGLT2i. InterpretationIn patients with T2DM with COPD, SGLT2i use was associated with reduced ER visits and hospitalizations related to COPD. This protective effect of SGLT2i could be explained by reduced systemic proinflammatory markers and increased anti-inflammatory markers via inhibition of Node like receptor protein 3(NLRP3) inflammasome activation in multiple tissues, including the lungs.

Inflammasome activation aggravates choroidal neovascularization.

Inflammasome activation is implicated in diseases of aberrant angiogenesis such as age-related macular degeneration (AMD), though its precise role in choroidal neovascularization (CNV), a characteristic pathology of advanced AMD, is ill-defined. Reports on inhibition of inflammasome constituents on CNV are variable and the precise role of inflammasome in mediating pathological angiogenesis is unclear. Historically, subretinal injection of inflammasome agonists alone has been used to investigate retinal pigmented epithelium (RPE) degeneration, while the laser photocoagulation model has been used to study pathological angiogenesis in a model of CNV. Here, we report that the simultaneous introduction of any of several disease-relevant inflammasome agonists (Alu or B2 RNA, Alu cDNA, or oligomerized amyloid β (1-40)) exacerbates laser-induced CNV. These activities were diminished or abrogated by genetic or pharmacological targeting of inflammasome signaling constituents including P2rx7, Nlrp3, caspase-1, caspase-11, and Myd88, as well as in myeloid-specific caspase-1 knockout mice. Alu RNA treatment induced inflammasome activation in macrophages within the CNV lesion, and increased accumulation of macrophages in an inflammasome-dependent manner. Finally, IL-1β neutralization prevented inflammasome agonist-induced chemotaxis, macrophage trafficking, and angiogenesis. Collectively, these observations support a model wherein inflammasome stimulation promotes and exacerbates CNV and may be a therapeutic target for diseases of angiogenesis such as neovascular AMD.

Remifentanil-induced inflammation in microglial cells: Activation of the PAK4-mediated NF-κB/NLRP3 pathway and onset of hyperalgesia

BackgroundThe perioperative use of remifentanil is associated with postoperative hyperalgesia, which can impair recovery and extend hospitalization. Recent studies have revealed that microglia-mediated activation of the NLRP3 inflammasome plays a critical role in opioid-induced hyperalgesia, with NF-κB acting as a pivotal activation point for NLRP3. Despite these findings, the specific molecular mechanisms underlying remifentanil-induced postoperative hyperalgesia remain unclear. This study aims to develop a model of remifentanil-induced hyperalgesia and investigate the molecular mechanisms, focusing on the NF-κB/NLRP3 pathway, using both in vitro and in vivo approaches. MethodWe established a remifentanil-induced hyperalgesia model and performed proteomic analysis to identify differential protein expression in the spinal cord tissue of rats. NLRP3 or PAK4 antagonists were administered intrathecally in vivo, and mechanical pain thresholds in the hind paws were measured using Von Frey testing. In vitro, we applied NLRP3 or PAK4 inhibitors or used lentivirus infection to silence PAK4, NF-κB, and NLRP3 genes. Protein expression was assessed through immunohistochemistry, immunofluorescence, and Western blotting. Additionally, ELISA was performed to measure IL-1β and IL-18 levels, and RT-qPCR was conducted to evaluate the transcription of target genes. ResultsProteomic analysis revealed that remifentanil upregulates PAK4 protein in spinal cord tissue two hours after the surgery. In addition, remifentanil induces morphological changes in the spinal cord dorsal horn, characterized by increased expression of PAK4, p-p65, NLRP3 and Iba-1 proteins, which in turn leads to elevated IL-1β and IL-18 levels and an inflammatory response. Intrathecal injection of NLRP3 or PAK4 inhibitors mitigates remifentanil-induced hyperalgesia and associated changes. In vitro, downregulation of PAK4 inhibits the increase in PAK4, p-p65, NLRP3 and Caspase-1 induced by LPS. Conversely, the downregulation of NLRP3 does not impact the levels of PAK4 and p-p65 proteins, aligning with the in vivo results and suggesting that PAK4 acts as an upstream signaling molecule of NLRP3. ConclusionRemifentanil can increase PAK4 expression in spinal cord dorsal horn cells by activating the NF-κB/NLRP3 pathway and mediating microglial activation, thereby contributing to postoperative hyperalgesia.

MiR-223-3p-loaded Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Ameliorate Mouse Myocardial Infarction Injury through Inhibiting NLRP3 Inflammasome Activation

MiR-223-3p-loaded Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Ameliorate Mouse Myocardial Infarction Injury through Inhibiting NLRP3 Inflammasome Activation

NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer's disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1β, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1β and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1β and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1β secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in full-body Nlrp3 knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective Gsdmd knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, Nlrp3 deficiency did not mitigate tauopathy or neurodegeneration in P301S mice in vivo, showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, Gsdmd deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective Gsdmd deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable ex vivo mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors.

Neonatal neutrophils exhibit reduced NLRP3 inflammasome activation

Abstract Newborns are at high risk to develop sepsis. This is linked to innate immune responses at birth which are not completely adapted to postnatal life. Neutrophils are key players of innate immunity and exhibit a marked ontogenetic regulation of their functionality. Here, we studied the NLRP3 inflammasome in neonatal neutrophils and found lower baseline expression of NLRP3, pro-caspase-1 and the K+-channel KV1.3 compared to adult neutrophils. Following stimulation with LPS/Nigericin, ASC oligomerization, caspase-1 activation and IL-1β release were significantly reduced in neonatal compared to adult neutrophils. Similarly, stimulation of neonatal neutrophils with E-selectin led to reduced NLRP3 inflammasome activation accompanied by diminished release of the alarmin S100A8/A9. Taken together, our results strongly indicate diminished NLRP3 inflammasome activation in neonatal neutrophils leading to a significant reduction of released IL-1β and S100A8/A9. These findings identify reduced neutrophil NLRP3 inflammasome activation as critical component contributing to the inherent susceptibility to infections in neonates.

Cell-based in vitro hemoassay for evaluation of NLRP3-inflammasome activity

Currently, gouty arthritis is considered as a polygenic multifactorial autoinflammatory disease caused by activation of the NOD (nucleotide-binding domain) -like protein receptor 3 inflammasome NLRP3. The two cytokines IL-1β and IL-18 are considered important biomarkers of NLRP3 inflammasome activation. However, usually the concentration of IL-1β in donor sera is extremely low and found to be at the limit of detection level (1-3 pg/ml), while the concentration of circulating cytokine IL-18 in the sera of individual donors varies greatly. This results in difficulty using these biomarkers in the diagnosis of autoinflammatory diseases. We hypothesized that the patient’s blood cells which were sensitized in vivo to the presence of specific factors characteristic of autoinflammatory diseases, in particular, gouty arthritis, would produce increased amounts of the inflammasome-regulated cytokines compared to blood cells obtained from healthy donors. A comparison of the IL-18 cytokine in healthy donors and patients with gouty arthritis was carried out using 2 methods: a) by traditional analysis of the level of serum circulated IL-18 and b) by using a cell-based Hemoassay in vitro developed at the research institute “Biotech” SamGMU. The comparative analysis demonstrated the advantages of using an in vitro cell-based Hemoassay to assess the IL-18 cytokine status of patients. Serum IL-18 values varied widely and showed no significant difference between donors and patients with gouty arthritis. Using the developed cell-based Hemoassay in vitro, significant quantitative differences in the production of the inflammatory cytokine IL-18 produced by blood cells of potentially healthy donors and patients with gouty arthritis were detected. Blood cells of individual patients, sensitized in vivo with specific factors characteristic of gouty arthritis, produce increased concentrations of IL-18 in the cell growth medium in vitro compared to cells from healthy donors. Thus, the in vitro cell-based Hemoassay can be used for a more accurate assessment of the cytokine status of patients.