Extracellular Caspase-1 induces hair stem cell migration in wounded and inflamed skin conditions.

The wound-healing process is a paradigm of the directed migration of various pools of stem cells from their niche to the site of injury where they replenish damaged cells. Two decades have elapsed since the observation that wounding activates multipotent hair follicle stem cells to infiltrate the epidermis, but the cues that coax these cells out of their niche remain unknown. Here, we report that Caspase-1, a protein classically known as an integral component of the cytosolic inflammasome, is secreted upon wounding and has a non-canonical role in the extracellular milieu. Through its caspase activation recruitment domain (CARD), Caspase-1 is sufficient to initiate the migration of hair follicle stem cells into the epidermis. Uncovering this novel function of Caspase-1 also facilitates a deeper understanding of the mechanistic basis of the epithelial hyperplasia found to accompany numerous inflammatory skin diseases.

Advances in the mechanism of inflammasomes activation in herpes virus infection.

Herpesviruses, prevalent DNA viruses with a double-stranded structure, establish enduring infections and play a part in various diseases. Despite their deployment of multiple tactics to evade the immune system, both localized and systemic inflammatory responses are triggered by the innate immune system's recognition of them. Recent progress has offered more profound understandings of the mechanisms behind the activation of the innate immune system by herpesviruses, specifically through inflammatory signaling. This process encompasses the initiation of an intracellular nucleoprotein complex, the inflammasome associated with inflammation.Following activation, proinflammatory cytokines such as IL-1ß and IL-18 are released by the inflammasome, concurrently instigating a programmed pathway for cell death. Despite the structural resemblances between herpesviruses, the distinctive methods of inflammatory activation and the ensuing outcomes in diseases linked to the virus exhibit variations.The objective of this review is to emphasize both the similarities and differences in the mechanisms of inflammatory activation among herpesviruses, elucidating their significance in diseases resulting from these viral infections.Additionally, it identifies areas requiring further research to comprehensively grasp the impact of this crucial innate immune signaling pathway on the pathogenesis of these prevalent viruses.

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1ß from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1ß result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

Amino Sugar-Enriched Fraction of Korean Red Ginseng Extract Induces the Priming Step of NLRP3 Inflammasome.

Intracellular protein complexes, known as inflammasomes, activate caspase-1 and induce the secretion of pro-inflammatory cytokines, namely interleukin (IL)-1ß and -18. Korean Red Ginseng extract (RGE) is a known immunomodulator and a potential candidate for the regulation of inflammasomes. The saponins, such as ginsenosides, of RGE inhibit inflammasome signaling, while non-saponin substances containing amino sugars promote the priming step, up-regulating inflammasome components (pro-IL-1ß, NLRP3, caspase-1, and Asc). In this study, the amino sugar-enriched fraction (ASEF), which increases only non-saponin components, including amino sugars, without changing the concentration of saponin substances, was used to investigate whether saponin or non-saponin components of RGE would have a greater impact on the priming step. When murine macrophages were treated with ASEF, the gene expression of inflammatory cytokines (IL-1α, TNFα, IL-6, and IL-10) increased. Additionally, ASEF induced the priming step but did not affect the inflammasome activation step, such as the secretion of IL-1ß, cleavage of caspase-1, and formation of Asc pyroptosome. Furthermore, the upregulation of gene expression of inflammasome components by ASEF was blocked by inhibitors of Toll-like receptor 4 signaling. Maltol, the main constituent of ASEF, promoted the priming step but inhibited the activation step of the inflammasome, while arginine, sugars, arginine-fructose-glucose, and fructose-arginine, the other main constituents of ASEF, had no effect on either step. Thus, certain amino sugars in RGE, excluding maltol, are believed to be the components that induce the priming step. The priming step that prepares the NLRP3 inflammasome for activation appears to be induced by amino sugars in RGE, thereby contributing to the immune-boosting effects of RGE.

Inflammasome-Related Genetic Polymorphisms as Severity Biomarkers of COVID-19.

The most critical forms of coronavirus disease 2019 (COVID-19) are associated with excessive activation of the inflammasome. Despite the COVID-19 impact on public health, we still do not fully understand the mechanisms by which the inflammatory response influences disease prognosis. Accordingly, we aimed to elucidate the role of polymorphisms in the key genes of the formation and signaling of the inflammasome as biomarkers of COVID-19 severity. For this purpose, a large and well-defined cohort of 377 COVID-19 patients with mild (n = 72), moderate (n = 84), severe (n = 100), and critical (n = 121) infections were included. A total of 24 polymorphisms located in inflammasome-related genes (NLRP3, NLRC4, NLRP1, CARD8, CASP1, IL1B, IL18, NFKB1, ATG16L1, and MIF) were genotyped in all of the patients and in the 192 healthy controls (HCs) (who were without COVID-19 at the time of and before the study) by RT-qPCR. Our results showed that patients with mild, moderate, severe, and critical COVID-19 presented similar allelic and genotypic distribution in all the variants studied. No statistically significant differences in the haplotypic distribution of NLRP3, NLRC4, NLRP1, CARD8, CASP1, IL1B, and ATG16L1 were observed between COVID-19 patients, who were stratified by disease severity. Each stratified group of patients presented a similar genetic distribution to the HCs. In conclusion, our results suggest that the inflammasome polymorphisms studied are not associated with the worsening of COVID-19.

The Golgi Apparatus: A Key Player in Innate Immunity.

The Golgi apparatus, long recognized for its roles in protein processing and vesicular trafficking, has recently been identified as a crucial contributor to innate immune signaling pathways. This review discusses our expanding understanding of the Golgi apparatus's involvement in initiating and activating these pathways. It highlights the significance of membrane connections between the Golgi and other organelles, such as the endoplasmic reticulum, mitochondria, endosomes, and autophagosomes. These connections are vital for the efficient transmission of innate immune signals and the activation of effector responses. Furthermore, the article delves into the Golgi apparatus's roles in key immune pathways, including the inflammasome-mediated activation of caspase-1, the cGAS-STING pathway, and TLR/RLR signaling. Overall, this review aims to provide insights into the multifunctional nature of the Golgi apparatus and its impact on innate immunity.

Mechanism of sodium butyrate, a metabolite of gut microbiota, regulating cardiac fibroblast transdifferentiation via the NLRP3/Caspase-1 pyroptosis pathway.

BACKGROUND: Cardiac fibroblasts (CFs) are activated after initial injury, and then differentiate into myofibroblasts (MFs), which play a pivotal role as the primary mediator cells in pathological remodeling. Sodium butyrate (NaB), being a metabolite of gut microbiota, exhibits anti-inflammatory property in local therapies on sites other than the intestine. Thus, this study aimed to probe the mechanism by which NaB regulates CFs transdifferentiation through the NLRP3/Caspase-1 pyroptosis pathway. METHODS: CFs were cultured in vitro and induced into MFs by TGFß1. CFs were identified by immunofluorescence labelling technique of vimentin and α-SMA, followed by treatment with NaB or NLRP3 inflammasome inhibitor (CY-09) and its activator [nigericin sodium salt (NSS)]. The expression levels of α-SMA, GSDMD-N/NLRP3/cleaved Caspase-1 proteins, and inflammatory factors IL-1ß/IL-18/IL-6/IL-10 were determined using immunofluorescence, Western blot and ELISA. Cell proliferation and migration were evaluated using the CCK-8 assay and the cell scratch test, respectively. RESULTS: Following the induction of TGFß1, CFs exhibited increased expression levels of α-SMA proteins and IL-6/IL-10, as well as cell proliferative and migratory abilities. TGFß1 induced CFs to differentiate into MFs, while NaB inhibited this differentiation. NaB inactivated the NLRP3/Caspase-1 pyroptosis pathway. CY-09 demonstrated inhibitory effects on the NLRP3/Caspase-1 pyroptosis pathway, leading to a reduction in TGFß1-induced CFs transdifferentiation. NSS activated the NLRP3/Caspase-1 pyroptosis pathway, and thus partially counteracting the inhibitory effect of intestinal microbiota metabolite NaB on CFs transdifferentiation. CONCLUSION: NaB, a metabolite of the gut microbiota, inhibited the activation of the NLRP3/Caspase-1 pyroptosis pathway in TGFß1-induced CFs, repressed the transdifferentiation of CFs into MFs.

Optogenetically controlled inflammasome activation demonstrates two phases of cell swelling during pyroptosis.

Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1ß and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.

Hope for vascular cognitive impairment: Ac-YVAD-cmk as a novel treatment against white matter rarefaction.

Vascular cognitive impairment (VCI) is the second leading cause of dementia with limited treatment options, characterised by cerebral hypoperfusion-induced white matter rarefaction (WMR). Subcortical VCI is the most common form of VCI, but the underlying reasons for region susceptibility remain elusive. Recent studies employing the bilateral cortical artery stenosis (BCAS) method demonstrate that various inflammasomes regulate white matter injury and blood-brain barrier dysfunction but whether caspase-1 inhibition will be beneficial remains unclear. To address this, we performed BCAS on C57/BL6 mice to study the effects of Ac-YVAD-cmk, a caspase-1 inhibitor, on the subcortical and cortical regions. Cerebral blood flow (CBF), WMR, neuroinflammation and the expression of tight junction-related proteins associated with blood-brain barrier integrity were assessed 15 days post BCAS. We observed that Ac-YVAD-cmk restored CBF, attenuated BCAS-induced WMR and restored subcortical myelin expression. Within the subcortical region, BCAS activated the NLRP3/caspase-1/interleukin-1beta axis only within the subcortical region, which was attenuated by Ac-YVAD-cmk. Although we observed that BCAS induced significant increases in VCAM-1 expression in both brain regions that were attenuated with Ac-YVAD-cmk, only ZO-1 and occludin were observed to be significantly altered in the subcortical region. Here we show that caspase-1 may contribute to subcortical regional susceptibility in a mouse model of VCI. In addition, our results support further investigations into the potential of Ac-YVAD-cmk as a novel treatment strategy against subcortical VCI and other conditions exhibiting cerebral hypoperfusion-induced WMR.

L-AP Alleviates Liver Injury in Septic Mice by Inhibiting Macrophage Activation via Suppressing NF-κB and NLRP3 Inflammasome/Caspase-1 Signal Pathways.

Liver injury and progressive liver failure are severe life-threatening complications in sepsis, further worsening the disease and leading to death. Macrophages and their mediated inflammatory cytokine storm are critical regulators in the occurrence and progression of liver injury in sepsis, for which effective treatments are still lacking. l-Ascorbic acid 6-palmitate (L-AP), a food additive, can inhibit neuroinflammation by modulating the phenotype of the microglia, but its pharmacological action in septic liver damage has not been fully explored. We aimed to investigate L-AP's antisepticemia action and the possible pharmacological mechanisms in attenuating septic liver damage by modulating macrophage function. We observed that L-AP treatment significantly increased survival in cecal ligation and puncture-induced WT mice and attenuated hepatic inflammatory injury, including the histopathology of the liver tissues, hepatocyte apoptosis, and the liver enzyme levels in plasma, which were comparable to NLRP3-deficiency in septic mice. L-AP supplementation significantly attenuated the excessive inflammatory response in hepatic tissues of septic mice in vivo and in cultured macrophages challenged by both LPS and ATP in vitro, by reducing the levels of NLRP3, pro-IL-1ß, and pro-IL-18 mRNA expression, as well as the levels of proteins for p-I-κB-α, p-NF-κB-p65, NLRP3, cleaved-caspase-1, IL-1ß, and IL-18. Additionally, it impaired the inflammasome ASC spot activation and reduced the inflammatory factor contents, including IL-1ß and IL-18 in plasma/cultured superannuants. It also prevented the infiltration/migration of macrophages and their M1-like inflammatory polarization while improving their M2-like polarization. Overall, our findings revealed that L-AP protected against sepsis by reducing macrophage activation and inflammatory cytokine production by suppressing their activation in NF-κB and NLRP3 inflammasome signal pathways in septic liver.

DNMT1 methylation of LncRNA-ANRIL causes myocardial fibrosis pyroptosis by interfering with the NLRP3/Caspase-1 pathway.

Myocardial fibrosis is a common pathological manifestation that occurs in various cardiac diseases. The present investigation aims to reveal how DNMT1/lncRNA-ANRIL/NLRP3 influences fibrosis and cardiac fibroblast pyroptosis. Here, we used ISO to induce myocardial fibrosis in mice, and LPS and ATP to induce myocardial fibroblast pyroptosis. The results showed that DNMT1, Caspase-1, and NLRP3 expression were significantly increased in fibrotic murine myocardium and pyroptotic cardiac fibroblasts, whereas LncRNA-ANRIL expression was decreased. DNMT1 overexpression decreased the level of LncRNA-ANRIL while increasing the levels of NLRP3 and Caspase-1. Contrarily, silencing DNMT1 increased the LncRNA-ANRIL and decreased the levels of NLRP3 and Caspase-1. Silencing LncRNA-ANRIL increased the levels of NLRP3 and Caspase-1. The present findings suggest that DNMT1 can methylate LncRNA-ANRIL during the development of myocardial fibrosis and CFs cell scorching, resulting in low LncRNA-ANRIL expression, thereby influencing myocardial fibrosis and cardiac fibroblast pyroptosis.

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

BACKGROUND: 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-1ß and IL-18, plays a key role in this process. While IL-1ß 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-1ß production, whereas activated by the Nlrp1b inflammasome in response to energy excess, mediates IL-18 production. However, this has not been formally tested. METHODS: 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. RESULTS: We found that IL-18 production in adipose tissue is independent of caspase-1 activity, regardless of the metabolic state, while Nlrp3-mediated IL-1ß 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. CONCLUSION: 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-1ß 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.

[Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells]. / åˆ†å­ä¼´ä¾£ä»‹å¯¼çš„è‡ªå™¬å¯¹èƒ†çº¢ç´ è¯±å¯¼çš„å°é¼ å°èƒ¶è´¨ç»†èƒžæŸä¼¤çš„å½±å“.

OBJECTIVES: To investigate the effect of chaperone-mediated autophagy (CMA) on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin (UCB). METHODS: The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide), QX77 group (treated with 20 µmol/L QX77 for 24 hours), UCB group (treated with 40 µmol/L UCB for 24 hours), and UCB+QX77 group (treated with both 20 µmol/L QX77 and 40 µmol/L UCB for 24 hours). (2) For the cell transfection experiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencing control+UCB group (treated with 40 µmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volume of dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 µmol/L UCB for 24 hours). The cell viability was assessed using the modified MTT method. The expression levels of p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence. RESULTS: Compared to the UCB group, the cell viability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as the mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α decreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65 and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2A silencing control+UCB group, the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as increased mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α (P<0.05). CONCLUSIONS: CMA is inhibited in UCB-induced BV2 cell damage, and activating CMA may reduce p65 and NLRP3 protein levels, suppress inflammatory responses, and counteract bilirubin neurotoxicity.

Type I interferon regulates interleukin-1beta and IL-18 production and secretion in human macrophages.

Inflammasomes are immune complexes whose activation leads to the release of pro-inflammatory cytokines IL-18 and IL-1ß. Type I IFNs play a role in fighting infection and stimulate the expression of IFN-stimulated genes (ISGs) involved in inflammation. Despite the importance of these cytokines in inflammation, the regulation of inflammasomes by type I IFNs remains poorly understood. Here, we analysed RNA-sequencing data from patients with monogenic interferonopathies and found an up-regulation of several inflammasome-related genes. To investigate the effect of type I IFN on the inflammasome, we treated human monocyte-derived macrophages with IFN-α and observed an increase in CASP1 and GSDMD mRNA levels over time, whereas IL1B and NLRP3 were not directly correlated to IFN-α exposure time. IFN-α treatment reduced the release of mature IL-1ß and IL-18, but not caspase-1, in response to ATP-mediated NLRP3 inflammasome activation, suggesting regulation occurs at cytokine expression levels and not the inflammasome itself. However, more studies are required to investigate how regulation by IFN-α occurs and impacts NLRP3 and other inflammasomes at both transcriptional and post-translational levels.

[Chlorogenic acid alleviates acute kidney injury in septic mice by inhibiting NLRP3 inflammasomes and the caspase-1 canonical pyroptosis pathway].

OBJECTIVE: To investigate the role of caspase-1-medicated canonical pyroptosis pathway in chlorogenic acid (CGA) treatment of acute kidney injury (AKI) in mice. METHOD: Twenty-four C57Bl/6J mice were randomized into sham-operated group, cecal ligation and puncture (CLP) group, CLP+dexamethasone group (CLP+DXM group), and CLP+CGA group (n=6) and subjected to either sham operation (laparotomy only) or CLP. After modeling the mice received intravenous infusion of 10 mg/kg normal saline (in sham and CLP groups), 1 µg/kg dexamethasone or 15 mg/kg of chlorogenic acid for 6 h delivered using an intravenous pump. Eight hours after the infusion, renal morphology and histology, renal cell apoptosis, and the renal function parameters such as urea nitrogen (BUN), creatinine (Scr), and kidney injury molecule 1 (KIM-1) were compared among the 4 groups; the 7-day survival rates of the mice were recorded, and the expressions of NLRP3 inflammasomes and key proteins of the caspase-1 pathway in the renal tissue were detected. RESULTS: CGA treatment significantly improved the 7-day survival rate, reduced renal pathologies of the septic mice (P < 0.05), and lowered the levels of BUN, Scr, KIM-1, NLRP3 inflammasome and expressions of key proteins of the caspase-1 pathway. CONCLUSION: CGA alleviates AKI in mice with CLP-induced sepsis by inhibiting NLRP3 inflammasomes and the caspase-1 signaling pathway.

Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages.

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336ΔfliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co-transcription. The deletion mutant ΔfliCΔdinJ increased cell death and IL-1ß secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1ß secretion in cells infected with ΔfliCΔdinJ compared to cells infected with ΔfliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3-/- and Nlrc4-/- mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1ß and IL-18 in vivo, contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.

Pepsin-mediated inflammation in laryngopharyngeal reflux via the ROS/NLRP3/IL-1ß signaling pathway.

BACKGROUND: Laryngopharyngeal reflux (LPR) is one of the most common disorders in otorhinolaryngology, affecting up to 10% of outpatients visiting otolaryngology departments. In addition, 50% of hoarseness cases are related to LPR. Pepsin reflux-induced aseptic inflammation is a major trigger of LPR; however, the underlying mechanisms are unclear. The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has become an important bridge between stimulation and sterile inflammation and is activated by intracellular reactive oxygen species (ROS) in response to danger signals, leading to an inflammatory cascade. In this study, we aimed to determine whether pepsin causes LPR-associated inflammatory injury via mediating inflammasome activation and explore the potential mechanism. METHODS: We evaluated NLRP3 inflammasome expression and ROS in the laryngeal mucosa using immunofluorescence and immunohistochemistry. Laryngeal epithelial cells were exposed to pepsin and analyzed using flow cytometry, western blotting, and real-time quantitative PCR to determine ROS, NLRP3, and pro-inflammatorycytokine levels. RESULTS: Pepsin expression was positively correlated with ROS as well as caspase-1 and IL-1ß levels in laryngeal tissues. Intracellular ROS levels were elevated by increased pepsin concentrations, which were attenuated by apocynin (APO)-a ROS inhibitor-in vitro. Furthermore, pepsin significantly induced the mRNA and protein expression of thioredoxin-interacting protein, NLRP3, caspase-1, and IL-1ß in a dose-dependent manner. APO and the NLRP3 inhibitor, MCC950, inhibited NLRP3 inflammasome formation and suppressed laryngeal epithelial cell damage. CONCLUSION: Our findings verified that pepsin could regulate the NLRP3/IL-1ß signaling pathway through ROS activation and further induce inflammatory injury in LPR. Targeting the ROS/NLRP3 inflammasome signaling pathway may help treat patients with LPR disease.

FOXQ1 inhibits the progression of osteoarthritis by regulating pyroptosis.

BACKGROUND: Osteoarthritis (OA) is the most common age-related joint disease, and the NLRP3-induced pyroptosis has been demonstrated in its progression. The upstream molecules or specific mechanisms controlling NLRP3 and pyroptosis in OA remain unclear. METHODS: Transcriptome sequencing was performed in the OA mice model, and the expression levels of differentially expressed genes were assessed by qRT-PCR. The cell model was constructed by IL-1ß-induced ATDC5 cells. The cell proliferation was examined using CCK-8 assay, and apoptosis was tested using flow cytometry. Western blot was used in protein inspection, and ELISA was used in inflammatory response evaluation. RESULTS: Compared with the control group, there were 229 up-regulated and 32 down-regulated genes in model group. We detected that FOXQ1 was down-regulated in the OA mice model, improved proliferation, and restrained apoptosis of chondrocytes. Over-expression of FOXQ1 could inhibit pyroptosis-related proteins and inflammatory cytokines, containing NLRP3, Caspase-1, GSDMD, IL-6, IL-18, and TNF-α, and in contrast, FOXQ1 silencing exerted the opposite trend. CONCLUSIONS: FOXQ1 may inhibit OA progression via down-regulating NLRP3-induced pyroptosis in the present study.

DDIT3/CHOP promotes LPS/ATP-induced pyroptosis in osteoblasts via mitophagy inhibition.

Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.

Fluoride induces pyroptosis via IL-17A-mediated caspase-1/11-dependent pathways and Bifidobacterium intervention in testis.

Fluoride, a ubiquitous environmental pollutant, poses a significant public health threat. Our previous study revealed a correlation between fluoride-induced testicular pyroptosis and male reproductive dysfunction. However, the underlying mechanism remains unclear. Wild-type and interleukin 17A knockout mice were exposed to sodium fluoride (100 mg/L) in deionized drinking water for 18 weeks. Bifidobacterium intervention (1 × 109 CFU/mL, 0.2 mL/day, administered via gavage) commenced in the 10th week. Sperm quality, testicular morphology, key pyroptosis markers, spermatogenesis key genes, IL-17A signaling pathway, and pyroptosis pathway related genes were determined. The results showed that fluoride reduced sperm quality, damaged testicular morphology, affected spermatogenesis, elevated IL-17A levels, and induced testicular pyroptosis. Bifidobacterium intervention alleviated adverse reproductive outcomes. Fluoride-activated testicular pyroptosis through both typical and atypical pathways, with IL-17A involvement. Bifidobacterium supplementation attenuated pyroptosis by downregulating IL-17A, inhibiting NLRP3 and PYRIN-mediated caspase-1 and caspase-11 dependent pathways in testis, thereby alleviating fluoride-induced male reproductive damage. In summary, this study uncovers the mechanism underlying fluorine-induced testicular pyroptosis and illustrates the novel protecting feature of Bifidobacterium against fluoride-induced harm to male reproduction, along with its potential regulatory mechanism. These results provide fresh perspectives on treating male reproductive dysfunction resulting from fluoride or other environmental toxins.