Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

Staphylococcus Aureus Membrane Vesicles Kill Tumor Cells Through a Caspase-1-Dependent Pyroptosis Pathway.

Introduction: Nanosized outer membrane vesicles (OMVs) from Gram-negative bacteria have attracted increasing interest because of their antitumor activity. However, the antitumor effects of MVs isolated from Gram-positive bacteria have rarely been investigated. Methods: MVs of Staphylococcus aureus USA300 were prepared and their antitumor efficacy was evaluated using tumor-bearing mouse models. A gene knock-in assay was performed to generate luciferase Antares2-MVs for bioluminescent detection. Cell counting kit-8 and lactic dehydrogenase release assays were used to detect the toxicity of the MVs against tumor cells in vitro. Active caspase-1 and gasdermin D (GSDMD) levels were determined using Western blot, and the tumor inhibition ability of MVs was determined in B16F10 cells treated with a caspase-1 inhibitor. Results: The vesicular particles of S. aureus USA300 MVs were 55.23 ± 8.17 nm in diameter, and 5 µg of MVs remarkably inhibited the growth of B16F10 melanoma in C57BL/6 mice and CT26 colon adenocarcinoma in BALB/c mice. The bioluminescent signals correlated well with the concentrations of the engineered Antares2-MVs (R2 = 0.999), and the sensitivity for bioluminescence imaging was 4 × 10-3 µg. Antares2-MVs can directly target tumor tissues in vivo, and 20 µg/mL Antares2-MVs considerably reduced the growth of B16F10 and CT26 tumor cells, but not non-carcinomatous bEnd.3 cells. MV treatment substantially increased the level of active caspase-1, which processes GSDMD to trigger pyroptosis in tumor cells. Blocking caspase-1 activation with VX-765 significantly protected tumor cells from MV killing in vitro and in vivo. Conclusion: S. aureus MVs can kill tumor cells by activating the pyroptosis pathway, and the induction of pyroptosis in tumor cells is a promising strategy for cancer treatment.

[Chronic intermittent hypoxia activates NLRP1 inflammasome and causes liver injury].

Objective To investigate the liver injury induced by chronic intermittent hypoxia (CIH) activation of NOD-like receptor pyrin domain containing protein 1 (NLRP1) inflammasome. Methods C57BL/6 male mice were randomly divided into control group and CIH group. Mice in CIH group were put into CIH chamber for molding (8 hours a day for 4 weeks). After 4 weeks of molding, liver tissue cells was observed by HE staining, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected by kit. The levels of reactive oxygen species (ROS) in liver tissue were detected by dihydroethidine (DHE). The expression and localization of NLRP1, apoptosis speck-like protein containing a caspase activation and recruiting domain (ASC) and caspase-1 were detected by immunohistochemical staining. The protein expressions of NLRP1, ASC, caspase-1, interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were detected by Western blot analysis. The serum levels of IL-1ß and TNF-α were detected by ELISA. Results Compared with the control group, the CIH group exhibited significant pathological changes in hepatocytes. Hepatocytes showed signs of rupture and necrosis, accompanied by inflammatory cell aggregation. Furthermore, the levels of ALT, AST, ROS, IL-1ß and TNF-α were elevated, along with increased protein expressions of NLRP1, ASC, caspase-1, IL-1ß and TNF-α. Conclusion CIH causes liver injury by activating NLRP1 inflammasome.

Interaction of obesity and proteins associated with the NLRP3 inflammasome following mild traumatic brain injury.

The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome has been associated with worse outcomes from severe traumatic brain injury (TBI). The NLRP3 inflammasome is also strongly associated with other pro-inflammatory conditions, such as obesity. Little is known about the potential effect of mild TBI (mTBI) on the NLRP3 inflammasome and the extent to which modifying factors, such as obesity, may augment the inflammatory response to mTBI. The purpose of this study was to evaluate the association of NLRP3 inflammasome proteins with obese body mass index (BMI ≥ 30) within 24 h of mTBI after presenting to a level 1 trauma center emergency department. This is a secondary analysis of prospectively enrolled patients with mTBI who presented to the emergency department of one U.S. Level 1 trauma center from 2013 to 2018 (n = 243). A series of regression models were built to evaluate the association of NLRP3 proteins obtained from blood plasma within 24 h of injury and BMI as well as the potential interaction effect of higher BMI with NLRP3 proteins (n = 243). A logistic regression model revealed a significant association between IL-18 (p < 0.001) in mTBI patients with obese BMI compared to mTBI patients with non-obese BMI (< 30). Moderation analyses revealed statistically significant interaction effects between apoptotic speck-like protein (ASC), caspase-1, IL-18, IL-1ß and obese BMI which worsened symptom burden, quality of life, and physical function at 2 weeks and 6 months post-injury. Higher acute concentrations of IL-1ß in the overall cohort predicted higher symptoms at 6-months and worse physical function at 2-weeks and 6-months. Higher acute concentrations of IL-18 in the overall cohort predicted worse physical function at 6-months. In this single center mTBI cohort, obese BMI interacted with higher acute concentrations of NLRP3 inflammasome proteins and worsened short- and long-term clinical outcomes.

Bioinformatics analysis of PANoptosis regulators in the diagnosis and subtyping of steroid-induced osteonecrosis of the femoral head.

In this study, we aimed to investigate the involvement of PANoptosis, a form of regulated cell death, in the development of steroid-induced osteonecrosis of the femoral head (SONFH). The underlying pathogenesis of PANoptosis in SONFH remains unclear. To address this, we employed bioinformatics approaches to analyze the key genes associated with PANoptosis. Our analysis was based on the GSE123568 dataset, allowing us to investigate both the expression profiles of PANoptosis-related genes (PRGs) and the immune profiles in SONFHallowing us to investigate the expression profiles of PRGs as well as the immune profiles in SONFH. We conducted cluster classification based on PRGs and assessed immune cell infiltration. Additionally, we used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific hub genes. Furthermore, we developed an optimal machine learning model to identify the key predictive genes responsible for SONFH progression. We also constructed a nomogram model with high predictive accuracy for assessing risk factors in SONFH patients, and validated the model using external data (area under the curve; AUC = 1.000). Furthermore, we identified potential drug targets for SONFH through the Coremine medical database. Using the optimal machine learning model, we found that 2 PRGs, CASP1 and MLKL, were significantly correlated with the key predictive genes and exhibited higher expression levels in SONFH. Our analysis revealed the existence of 2 distinct PANoptosis molecular subtypes (C1 and C2) within SONFH. Importantly, we observed significant variations in the distribution of immune cells across these subtypes, with C2 displaying higher levels of immune cell infiltration. Gene set variation analysis indicated that C2 was closely associated with multiple immune responses. In conclusion, our study sheds light on the intricate relationship between PANoptosis and SONFH. We successfully developed a risk predictive model for SONFH patients and different SONFH subtypes. These findings enhance our understanding of the pathogenesis of SONFH and offer potential insights into therapeutic strategies.

The ameliorative effect of vinpocetine against gentamicin-induced uterine-injury in rats involves the inflammasome/caspase-1/IL-1ß pathway.

BACKGROUND: There is limited data regarding the hazardous effect of gentamicin (GM) on the uterus and whether or not vinpocetine (Vinpo) ameliorates it. The present study aimed to identify the possible protective effect of Vinpo in GM-induced uterine injury in rats. METHODS: Female rats were assorted in control-group, Vinpo-group, GM-group, and Vinpo plus GM group. Serum and uterine GM concentration were measured. Uterine oxidative stress parameters besides inflammatory and apoptotic biomarkers were evaluated. Uterine histopathological examination and interlukin-1beta (IL-1ß) immune-histochemical study were detected. RESULTS: GM significantly increased uterine oxidative stress, inflammatory and apoptotic biomarkers. Histopathological picture of uterine damage and increased IL-1ß immunoexpression were detected. Vinpo significantly ameliorated the distributed GM concentration, oxidative stress, inflammatory and apoptotic biomarkers with a prompt improvement in histopathological picture and a decrease in IL-1ß immunoexpression. CONCLUSION: Vinpo protective effect against GM-induced uterine injury involves modulation of inflammasome/caspase-1/IL-1ß signaling pathway.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

Lentinus edodes mycelium polysaccharide inhibits AGEs-induced HUVECs pyroptosis by regulating LncRNA MALAT1/miR-199b/mTOR axis and NLRP3/Caspase-1/GSDMD pathway.

A novel Lentinus edodes mycelia polysaccharide (LMP) prepared in our laboratory has been identified to be effective in inhibiting the damage of islet ß cells induced by glucose toxicity. However, whether it can effectively alleviate the pyroptosis of human umbilical vein endothelial cells (HUVECs) induced by advanced glycation end products (AGEs) remains unclear. Bioinformatics and cell biology techniques were used to explore the mechanism of LMP inhibiting AGEs-induced HUVECs damage. The results indicated that AGEs significantly increased the expression of LncRNA MALAT1, decreased cell viability to 79.67 %, increased intracellular ROS level to 248.19 % compared with the control group, which further led to cell membrane rupture. The release of LDH in cellular supernatant was increased to 149.42 %, and the rate of propidium iodide staining positive cells increased to 277.19 %, indicating the cell pyroptosis occurred. However, the above trend was effectively retrieved after the treatment with LMP. LMP effectively decreased the expression of LncRNA MALAT1 and mTOR, promoted the expression of miR-199b, inhibited AGEs-induced HUVECs pyroptosis by regulating the NLRP3/Caspase-1/GSDMD pathway. LncRNA MALAT1 might be a new target for LMP to inhibit AGEs-induced HUVECs pyroptosis. This study manifested the role of LMP in improving diabetes angiopathy and broadens the application of polysaccharide.

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.

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.

Unveiling the role of PD-L1 in vascular endothelial dysfunction: Insights into the mtros/NLRP3/caspase-1 mediated pyroptotic pathway.

BACKGROUND: Programmed death ligand-1(PD-L1) has been postulated to play a crucial role in the regulation of barrier functions of the vascular endothelium, yet how this novel molecule mediates dysfunction in endothelial cells (ECs) during acute lung injury (ALI) remains largely unknown. METHODS: PD-L1 siRNA and plasmids were synthesized and applied respectively to down- or up-regulate PD-L1 expression in human lung microvascular endothelial cells (HMVECs). RNA sequencing was used to explore the differentially expressed genes following PD-L1 overexpression. The expression levels of tight junction proteins (ZO-1 and occludin) and the signaling pathways of NLRP-3/caspase-1/pyroptosis were analyzed. A mouse model of indirect ALI was established through hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), enabling further investigation into the effects of intravenous delivery of PD-L1 siRNA. RESULTS: A total of 1502 differentially expressed genes were identified, comprising 532 down-regulated and 970 up-regulated genes in ECs exhibiting PD-L1overexpression. Enrichment of PD-L1-correlated genes were observed in the NOD-like receptor signaling pathway and the TNF signaling pathway. Western blot assays confirmed that PD-L1 overexpression elevated the expression of NLRP3, cleaved-caspase-1, ASC and GSDMD, and concurrently diminished the expression of ZO-1 and occludin. This overexpression also enhanced mitochondrial oxidative phosphorylation and mitochondrial reactive oxygen species (mtROS) production. Interestingly, mitigating mitochondrial dysfunction with mitoQ partially countered the adverse effects of PD-L1 on the functionality of ECs. Furthermore, intravenous administration of PD-L1 siRNA effectively inhibited the activation of the NLRP3 inflammasome and pyroptosis in pulmonary ECs, subsequently ameliorating lung injury in HEM/CLP mice. CONCLUSION: PD-L1-mediated activation of the inflammasome contributes significantly to the disruption of tight junction and induction of pyroptosis in ECs, where oxidative stress associated with mitochondrial dysfunction serves as a pivotal mechanism underpinning these effects.

Heat stress induces IL-1ß and IL-18 overproduction via ROS-activated NLRP3 inflammasome: implication in neuroinflammation in mice with heat stroke.

Heat stroke induced cerebral damage via neuroinflammation. This study aimed to approach whether heat stress would promote NOD-like receptor protein 3 (NLRP3) inflammasome via reactive oxygen species (ROS). The mice were randomly divided into the sham group, the heat stress group, and the heat stress + TEMPOL (ROS scavenger) group. And the NLRP3 -/- mice were applied and divided into the NLRP3 -/-  + sham group and the NLRP3 -/-  + heat stress group. Furthermore, the BV2 cells were divided into four groups following the intervention measures: the heat stress + TEMPOL group, the heat stress + Z-VAD-FMK (caspase-1 inhibitor) group, the heat stress group, and the control group. ROS levels were examined. The expression levels of NLRP3, caspase-1, IL-1ß, and IL-18 were detected by western blotting and double immunofluorescence. We found that heat stress attack induced excessive ROS in microglia and subsequently activated NLRP3 inflammasome in both mice and BV2 cells. When ROS scavenged, the expression level of NLRP3 was downregulated. Furthermore, with NLRP3 inflammasome activation, the expression levels of caspase-1, IL-1ß, and IL-18 were increased. In NLRP3 -/- mice, however, the caspase-1, IL-1ß, and IL-18 were significantly declined. Further experiments showed that pretreatment of caspase-1 inhibitor decreased the expression levels of IL-1ß and IL-18. These results suggest that heat stress attack caused neuroinflammation via excessive ROS activating the NLRP3 inflammasome in microglia cells.

Caspase-1 deletion reveals pyroptosis participates in neural damage induced by cerebral ischemia/reperfusion in tMCAO model mice.

Pyroptosis, a form of programmed cell death, drives inflammation in the context of cerebral ischemia/reperfusion. The molecular mechanism of pyroptosis underlying ischemia/reperfusion, however, is not fully understood. The transient middle cerebral artery occlusion was applied to wild-type and caspase-1 knockout mice. 2,3,5-Triphenyltetrazolium chloride-staining and immunohistochemistry were used to identify the ischemic region, and western blot and immunofluorescence for the examination of neuronal pyroptosis. The expression of inflammatory factors and the behavioral function assessments were further conducted to examine the effects of caspase-1 knockout on protection against ischemia/reperfusion injury. Ischemia/reperfusion injury increased pyroptosis-related signals represented by the overexpression of pyroptosis-related proteins including caspase-1 and gasdermin D (GSDMD). Meanwhile, the number of GSDMD positive neurons increased in penumbra by immunofluorescence staining. Compared with wild-type mice, those with caspase-1 knockout exhibited decreased levels of pyroptosis-related proteins following ischemia/reperfusion. Furthermore, ischemia/reperfusion attack-induced brain infarction, cerebral edema, inflammatory factors, and neurological outcomes were partially improved in caspase-1 knockout mice. The data indicate that pyroptosis participates in ischemia/reperfusion induced-damage, and the caspase-1 might be involved, it provides some new insights into the molecular mechanism of ischemia.

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.

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.

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.

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.

Penehyclidine hydrochloride alleviates lung ischemia-reperfusion injury by inhibiting pyroptosis.

OBJECTIVE: The aim of this research was to examine how penehyclidine hydrochloride (PHC) impacts the occurrence of pyroptosis in lung tissue cells within a rat model of lung ischemia-reperfusion injury. METHODS: Twenty-four Sprague Dawley (SD) rats, weighing 250 g to 270 g, were randomly distributed into three distinct groups as outlined below: a sham operation group (S group), a control group (C group), and a test group (PHC group). Rats in the PHC group received a preliminary intravenous injection of PHC at a dose of 3 mg/kg. At the conclusion of the experiment, lung tissue and blood samples were collected and properly stored for subsequent analysis. The levels of malondialdehyde, superoxide dismutase, and myeloperoxidase in the lung tissue, as well as IL-18 and IL-1ß in the blood serum, were assessed using an Elisa kit. Pyroptosis-related proteins, including Caspase1 p20, GSDMD-N, and NLRP3, were detected through the western blot method. Additionally, the dry-to-wet ratio (D/W) of the lung tissue and the findings from the blood gas analysis were also documented. RESULTS: In contrast to the control group, the PHC group showed enhancements in oxygenation metrics, reductions in oxidative stress and inflammatory reactions, and a decrease in lung injury. Additionally, the PHC group exhibited lowered levels of pyroptosis-associated proteins, including the N-terminal segment of gasdermin D (GSDMD-N), caspase-1p20, and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3). CONCLUSION: Pre-administration of PHC has the potential to mitigate lung ischemia-reperfusion injuries by suppressing the pyroptosis of lung tissue cells, diminishing inflammatory reactions, and enhancing lung function. The primary mechanism behind anti-pyroptotic effect of PHC appears to involve the inhibition of oxidative stress.