Absent In Melanoma 2 Inflammasome Activation Research Articles

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.

Disrupted mitochondrial transcription factor A expression promotes mitochondrial dysfunction and enhances ocular surface inflammation by activating the absent in melanoma 2 inflammasome

PurposeSevere dry eye disease causes ocular surface damage, which is highly associated with mitochondrial dysfunction. Mitochondrial transcription factor A (TFAM) is essential for packaging mitochondrial DNA (mtDNA) and is crucial for maintaining mitochondrial function. Herein, we aimed to explore the effect of a decreased TFAM expression on ocular surface damage. MethodsFemale C57BL/6 mice were induced ocular surface injury by topical administrating benzalkonium chloride (BAC). Immortalized human corneal epithelial cells (HCECs) were stimulated by tert-butyl hydroperoxide (t-BHP) to create oxidative stress damage. HCECs with TFAM knockdown were established. RNA sequencing was employed to analyze the whole-genome expression. Mitochondrial changes were measured by transmission electron microscopy, Seahorse metabolic flux analysis, mitochondrial membrane potential, and mtDNA copy number. TFAM expression and inflammatory cytokines were determined using RT-qPCR, immunohistochemistry, immunofluorescence, and immunoblotting. ResultsIn both the corneas of BAC-treated mice and t-BHP-induced HCECs, we observed impaired TFAM expression, accompanied by mitochondrial structure and function defects. TFAM downregulation in HCECs suppressed mitochondrial respiratory capacity, reduced mtDNA content, induced mtDNA leakage into the cytoplasm, and led to inflammation. RNA sequencing revealed the absent in melanoma 2 (AIM2) inflammasome was activated in the corneas of BAC-treated mice. The AIM2 inflammasome activation was confirmed in TFAM knockdown HCECs. TFAM knockdown in t-BHP-stimulated HCECs aggravated mitochondrial dysfunction and the AIM2 inflammasome activation, thereby further triggering the secretion of inflammatory factors such as interleukin (IL) -1β and IL-18. ConclusionsTFAM reduction impaired mitochondrial function, activated AIM2 inflammasome and promoted ocular surface inflammation, revealing an underlying molecular mechanism for ocular surface disorders.

Bruceine A alleviates alcoholic liver disease by inhibiting AIM2 inflammasome activation via activating FXR

BackgroundAlcoholic liver disease (ALD), a public health challenge worldwide caused by long-term persistent drinking, is life-threatening with minimal approved therapies. Hepatic steatosis accompanied by inflammation is an initial and inevitable stage in the complex progression of simple alcoholic liver injury to more severe liver diseases such as hepatitis, liver fibrosis, cirrhosis and liver cancer. PurposeWe aimed to identify the therapeutic role of Bruceine A (BA) in ALD whilst attempting to explore whether its protective effects depend specifically on the farnesoid X receptor (FXR). MethodsAutodock was applied to detect the affinity between BA and FXR. Lieber-DeCarli liquid diet with 5 % ethanol (v/v) was adopted to establish the mouse ALD model. The lentivirus mediating FXR (LV-FXR) was injected into mice via the tail vein to establish FXR-overexpressed mice. FXR silencing or overexpression plasmids were transfected into AML-12 cells prior to ethanol stimulation. Quantitative real-time PCR, Western blotting and immunofluorescence assays were employed to determine the expression of related genes. We subjected liver sections to H&E and Oil Red O staining to evaluate the liver histological injury and the deposition of lipid droplets. ResultsBA significantly reduced body weight and liver-to-body weight ratios as well as biochemical indexes in mice. Ethanol-induced liver damage and lipid accumulation could be alleviated by BA treatment. BA bound to FXR by two hydrogen bonds. There was a positive correlation between BA administration and FXR expression. BA inhibited the expression of lipid synthesis genes and enhanced the expression of lipid metabolism genes by activating FXR, thus alleviating steatosis in ALD. Moreover, BA exerted an ameliorative effect against inflammation by inhibiting the activation of absent in melanoma 2 (AIM2) inflammasome by activating FXR. FXR overexpression possessed the ability to counter the accumulation of lipid and the activation of AIM2 inflammasome caused by ethanol. FXR deficiency exacerbated ethanol-induced liver steatosis and inflammation. The hepatoprotective effect of BA could be disrupted by FXR antagonist guggulsterone (GS) in vivo and FXR siRNA in vitro. ConclusionBA alleviated alcoholic liver disease by inhibiting AIM2 inflammasome activation through an FXR-dependent mechanism. This study may potentially represent a new therapeutic approach for ALD.

Abstract 3018: Expression Of The Aim2 Inflammasome In Skeletal Is Modulated By Surgical Bypass And Is A Target To Reduce Ischemic Myopathy In Pad

Introduction: Peripheral arterial disease (PAD) causes skeletal muscle myopathy and impairment, yet relevant pathways are understudied. We reported a new role for the absent in melanoma 2 (AIM2) inflammasome in PAD myocyte function. We hypothesize that AIM2 is an important mediator of cell death pathways in PAD muscle and is reduced by revascularization. Methods: Myocytes were obtained commercially or from ischemic and perfused PAD muscle using cell sorting. mRNA libraries underwent next generation sequencing (NGS) and differential gene expression (DGE) mapped to the GRCh368 human genome. Ingenuity Pathway Analysis (IPA) identified relevant biological pathways filtered by both fold change (fc) >2 and false discovery rate of p< 0.05. Sections were procured from human gastrocnemius before and after vascular bypass and analyzed for AIM2 with immunofluorescence (IF) and western blot (WB). Inflammatory caspase-1 activity was measured +/- siRNA against AIM2. Statistical analysis included t- test and ANOVA + post-hoc analysis. P<0.05 was considered significant. Results: DGE and IPA on isolated myocytes highlighted Neutrophil Degranulation and Pathogen Induced Cytokine Storm Signaling Pathways to be significantly altered in ischemic muscle cells (Fig1, p < 1xE-30 each, N=4) emphasizing AIM2 inflammasome activation (CASP1, fc 30.73; NLRP3/ASC/CASP1, fc 27.23; PYCARD fc 40.63; IL1B fc 63.49). AIM2 protein was high in PAD by IF and WB. Caspase-1 activity was attenuated in the presence of AIM2 inhibition (N=3; ****p<0.0001 in PAD). AIM2 expression was significantly reduced after surgery for PAD (p<0.05, N=6/group, t-test). Conclusion: AIM2 inflammasome expression in PAD induces pyroptotic caspase activity and is attenuated by surgical bypass, directly demonstrating a unique biologic benefit of muscle revascularization in PAD. Targeting AIM2 pathways either pharmacologically or by surgery may help to reduce myopathic damage in ischemic muscle from PAD.

Mitochondrial damage and clearance in retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a devastating eye disease that causes permanent vision loss in the central part of the retina, known as the macula. Patients with such severe visual loss face a reduced quality of life and are at a 1.5 times greater risk of death compared to the general population. Currently, there is no cure for or effective treatment for dry AMD. There are several mechanisms thought to underlie the disease, for example, ageing-associated chronic oxidative stress, mitochondrial damage, harmful protein aggregation and inflammation. As a way of gaining a better understanding of the molecular mechanisms behind AMD and thus developing new therapies, we have created a peroxisome proliferator-activated receptor gamma coactivator 1-alpha and nuclear factor erythroid 2-related factor 2 (PGC1α/NFE2L2) double-knockout (dKO) mouse model that mimics many of the clinical features of dry AMD, including elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in retinal pigment epithelial cells (RPE). In addition, a human RPE cell-based model was established to examine the impact of non-functional intracellular clearance systems on inflammasome activation. In this study, we found that there was a disturbance in the autolysosomal machinery responsible for clearing mitochondria in the RPE cells of one-year-old PGC1α/NFE2L2-deficient mice. The confocal immunohistochemical analysis revealed an increase in autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as multiple mitophagy markers such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN), along with signs of damaged mitochondria. However, no increase in autolysosome formation was detected, nor was there a colocalization of the lysosomal marker LAMP2 or the mitochondrial marker, ATP synthase β. There was an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells, together with autofluorescent aggregates. Additionally, we observed an increase in the numbers of Toll-like receptors 3 and 9, while those of NOD-like receptor 3 were decreased in PGC1α/NFE2L2 dKO retinal specimens compared to wild-type animals. There was a trend towards increased complement component C5a and increased involvement of the serine protease enzyme, thrombin, in enhancing the terminal pathway producing C5a, independent of C3. The levels of primary acute phase C-reactive protein and receptor for advanced glycation end products were also increased in the PGC1α/NFE2L2 dKO retina. Furthermore, selective proteasome inhibition with epoxomicin promoted both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial-mediated oxidative stress, leading to the release of mitochondrial DNA to the cytosol, resulting in potassium efflux-dependent activation of the absent in melanoma 2 (AIM2) inflammasome and the subsequent secretion of interleukin-1β in ARPE-19 cells. In conclusion, the data suggest that there is at least a relative decrease in mitophagy, increases in the amounts of C5 and thrombin and decreased C3 levels in this dry AMD-like model. Moreover, selective proteasome inhibition evoked mitochondrial damage and AIM2 inflammasome activation in ARPE-19 cells.

FXR overexpression prevents hepatic steatosis through inhibiting AIM2 inflammasome activation in alcoholic liver disease.

Alcoholic liver disease (ALD), a metabolic liver disease caused by excessive alcohol consumption, has attracted increasing attention due to its high prevalence and mortality. Up to date, there is no effective and feasible treatment method for ALD. This study was to investigate whether Farnesoid X receptor (FXR, NR1H4) can alleviate ALD and whether this effect is mediated by inhibiting absent in melanoma 2 (AIM2) inflammasome activation. The difference in FXR expression between normal subjects and ALD patients was analyzed using the Gene Expression Omnibus (GEO) database. Lieber-DeCarli liquid diet with 5% ethanol (v/v) (EtOH) was adopted to establish the mouse ALD model. Liver histopathological changes and the accumulation of lipid droplets were assessed by H&E and Oil Red O staining. Quantitative real-time PCR, Western blotting analysis and immunofluorescence staining were utilized to evaluate the expression levels of related genes and proteins. DCFH-DA staining was adopted to visualize reactive oxidative species (ROS). FXR was distinctly downregulated in liver tissues of patients with steatosis compared to normal livers using the GEO database, and in ethanol-induced AML-12 cellular steatosis model. FXR overexpression ameliorated hepatic lipid metabolism disorder and steatosis induced by ethanol by inhibiting the expression of genes involved in lipid synthesis and inducing the expression of genes responsible for lipid metabolism. Besides, FXR overexpression inhibited ethanol-induced AIM2 inflammasome activation and alleviated oxidative stress and ROS production during ethanol-induced hepatic steatosis. However, when FXR was knocked down, the results were completely opposite. FXR attenuated lipid metabolism disorders and lipid degeneration in alcohol-caused liver injury and alleviated oxidative stress and inflammation by inhibiting AIM2 inflammasome activation.

The Comparative Effects of Schwann Cells and Wharton's Jelly Mesenchymal Stem Cells on the AIM2 Inflammasome Activity in an Experimental Model of Spinal Cord Injury

Inflammasome activation and the consequent release of pro-inflammatory cytokines play a crucial role in the development of sensory/motor deficits following spinal cord injury (SCI). Immunomodulatory activities are exhibited by Schwann cells (SCs) and Wharton's jelly mesenchymal stem cells (WJ-MSCs). In this study, we aimed to compare the effectiveness of these two cell sources in modulating the absent in melanoma 2 (AIM2) inflammasome complex in rats with SCI. The Basso, Beattie, Bresnahan (BBB) test, Nissl staining, and Luxol fast blue (LFB) staining were performed to evaluate locomotor function, neuronal survival, and myelination, respectively. Real-time polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) were employed to analyze the gene and protein expressions of inflammasome components, including AIM2, ASC, caspase-1, interleukin-1β (IL-1β), and IL-18. Both gene and protein expressions of all evaluated factors were decreased after SC or WJ-MSC treatment, with a more pronounced effect observed in the SCs group (P < 0.05). Additionally, SCs promoted neuronal survival and myelination. Moreover, the administration of 3 × 105 cells resulted in motor recovery improvement in both treatment groups (P < 0.05). Although not statistically significant, these effects were more prominent in the SC-treated animals. In conclusion, SC therapy demonstrated greater efficacy in targeting AIM2 inflammasome activation and the associated inflammatory pathway in SCI experiments compared to WJ-MSCs.

Tangeretin Attenuates Cerebral Ischemia-Reperfusion-Induced Neuronal Pyroptosis by Inhibiting AIM2 Inflammasome Activation via Regulating NRF2.

Pyroptosis is closely involved in the pathopoiesis of cerebral ischemia and reperfusion (I/R) injury which seriously dangers human's life. Studies report that tangeretin (TANG), which is enriched in the peel of Citrus reticulata, has neuroprotective effects. Here, we explored whether absent in melanoma 2 (AIM2) inflammasome-mediated pyroptosis is involved in the cerebral I/R injury and the protective mechanism of TANG against cerebral I/R injury. In this study, we found that TANG treatment effectively alleviated I/R-induced brain injury and inhibited neuronal pyroptosis in anin vivomice model with middle cerebral artery occlusion/reperfusion (MCAO/R) injury andin an in vitro hippocampal HT22 cell model with oxygen-glucose deprivation and reoxygenation (OGD/R) injury. Furthermore, we found TANG inhibited cerebral I/R-induced neuronal AIM2 inflammasome activation in vivo and in vitro via regulating nuclear factor E2-related factor 2 (NRF2). Moreover, administration of ML385, a chemical inhibitor of NRF2, notably blocked the neuroprotective effects of TANG against cerebral I/R injury. In conclusion, TANG attenuates cerebral I/R-induced neuronal pyroptosis by inhibiting AIM2 inflammasome activation via regulating NRF2. These findings indicate TANG is a potential therapeutic agent for cerebral I/R injury.

Roxadustat (FG-4592) protects against ischaemia-induced acute kidney injury via improving CD73 and decreasing AIM2 inflammasome activation.

Hypoxia-mediated inflammation plays a crucial role in renal ischaemia-reperfusion (IR)-induced acute kidney injury (AKI) and may influence renal graft survival, with no available pharmacological treatments. Here we investigate the protective effects and mechanism of roxadustat (FG-4592), a hypoxia-inducible factor stabilizer, against renal IR injury. The protein expression levels of CD73 and AIM2 inflammasome complex were examined in kidney biopsy specimens of AKI and post-renal transplantation (PRT) patients. The effects of FG-4592 on CD73 and absent in melanoma 2 (AIM2) inflammasome components were examined in IR mice (right nephrectomy, followed by 30min of unilateral renal ischaemia and reperfusion for 24h), and some of the model mice received intraperitoneal administrations of adenosine 5'-(α,β-methylene)diphosphate sodium salt, which is an inhibitor of CD73. The function of FG-4592 was also investigated in vitro with HK-2 cells. In the AKI and PRT patients, the protein expression of AIM2 complex [AIM2-apoptosis-associated speck-like protein (ASC)-cleaved caspase-1) increased and the activation of CD73 signalling pathway was detected as well. The pretreatment of FG-4592 improved the creatinine elevation and renal tubular injuries induced by ischaemia. What's more, the administration of FG-4592 significantly enhanced CD73 synthesis in mouse kidney but suppressed the activation of the AIM2 inflammasome [decreased AIM2, ASC, caspase-1, interleukin (IL)-1β and IL-18 levels]. Notably, the renoprotection of FG-4592 and the inhibition of AIM2 were abolished by the CD73 inhibitor. FG-4592-conveyed protection against AKI might be mediated by the induction of CD73 and the suppression of the AIM2 inflammasome, which may provide a novel therapeutic method for the treatment of AKI.

Overexpression of NAG-1/GDF15 prevents hepatic steatosis through inhibiting oxidative stress-mediated dsDNA release and AIM2 inflammasome activation

Mitochondrial dysfunction and oxidative stress-mediated inflammasome activation play critical roles in the pathogenesis of the non-alcoholic fatty liver disease (NAFLD). Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1), or growth differentiation factor-15 (GDF15), is associated with many biological processes and diseases, including NAFLD. However, the role of NAG-1/GDF15 in regulating oxidative stress and whether this process is associated with absent in melanoma 2 (AIM2) inflammasome activation in NAFLD are unknown. In this study, we revealed that NAG-1/GDF15 is significantly downregulated in liver tissues of patients with steatosis compared to normal livers using the Gene Expression Omnibus (GEO) database, and in free fatty acids (FFA, oleic acid/palmitic acid, 2:1)-induced HepG2 and Huh-7 cellular steatosis models. Overexpression of NAG-1/GDF15 in transgenic (Tg) mice significantly alleviated HFD-induced obesity and hepatic steatosis, improved lipid homeostasis, enhanced fatty acid β-oxidation and lipolysis, inhibited fatty acid synthesis and uptake, and inhibited AIM2 inflammasome activation and the secretion of IL-18 and IL-1β, as compared to their wild-type (WT) littermates without reducing food intake. Furthermore, NAG-1/GDF15 overexpression attenuated FFA-induced triglyceride (TG) accumulation, lipid metabolism deregulation, and AIM2 inflammasome activation in hepatic steatotic cells, while knockdown of NAG-1/GDF15 demonstrated opposite effects. Moreover, NAG-1/GDF15 overexpression inhibited HFD- and FFA-induced oxidative stress and mitochondrial damage which in turn reduced double-strand DNA (dsDNA) release into the cytosol, while NAG-1/GDF15 siRNA showed opposite effects. The reduced ROS production and dsDNA release may be responsible for attenuated AIM2 activation by NAG-1/GDF15 upon fatty acid overload. In conclusion, our results provide evidence that other than regulating lipid homeostasis, NAG-1/GDF15 protects against hepatic steatosis through a novel mechanism via suppressing oxidative stress, mitochondrial damage, dsDNA release, and AIM2 inflammasome activation.

The AIM2 inflammasome is activated in astrocytes during the late phase of EAE.

Inflammasomes are a class of innate immune signaling platforms that activate in response to an array of cellular damage and pathogens. Inflammasomes promote inflammation under many circumstances to enhance immunity against pathogens and inflammatory responses through their effector cytokines, IL-1β and IL-18. Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune conditions influenced by inflammasomes. Despite work investigating inflammasomes during EAE, little remains known concerning the role of inflammasomes in the central nervous system (CNS) during the disease. Here, we used multiple genetically modified mouse models to monitor activated inflammasomes in situ based on oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC) in the spinal cord. Using inflammasome reporter mice, we found heightened inflammasome activation in astrocytes after the disease peak. In contrast, microglia and CNS-infiltrated myeloid cells had few activated inflammasomes in the CNS during EAE. Astrocyte inflammasome activation during EAE was dependent on absent in melanoma 2 (AIM2), but low IL-1β release and no significant signs of cell death were found. Thus, the AIM2 inflammasome activation in astrocytes may have a distinct role from traditional inflammasome-mediated inflammation.

Calpain Inhibitor Calpeptin Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury via Suppressing AIM2 Inflammasome and Upregulating Klotho Protein.

Renal ischemia/reperfusion injury is a major contributor of acute kidney injury (AKI), leading to renal cell necrosis, apoptosis, and inflammation. Calpains, a family of Ca2+-dependent cysteine proteases, play a pivotal role in the pathogenesis of renal diseases. Several studies have reported calpain inhibitors showing remarkable reno-protective effects against proteinuria and α-klotho deficiency-induced renal aging symptoms, particularly against glomerulus injury. However, little is known about the role of the calpain inhibitor calpeptin in acute kidney injury. The present study aims to investigate the potential mechanism of downregulation of Calpain 1 and 2 activity by calpeptin in the ischemia/reperfusion (IR)-induced AKI model. Firstly, we observed that the contents of Calpain 1 and 2 were significantly increased in the renal biopsy of clinical AKI patients, especially in the diseased tubules space. To investigate the impacts of calpain activity inhibition, we further pretreated with calpeptin in both the IR mouse model and in the HK-2 cells hypoxia model. We found that the calpain inhibitor calpeptin improved renal functional deterioration, attenuated pathological structure damage, and decreased tubular cell apoptosis in the IR injury-induced AKI mice model. Mechanistically, calpeptin significantly suppressed the AIM2 (absent in melanoma 2) and NLRP3 (NOD-like receptor protein 3) inflammasome signaling pathways and increased Klotho protein levels. Furthermore, immunofluorescence assays demonstrated that the application of calpeptin effectively inhibited Calpain 1 activation and gasdermin D (GSDMD) cleavage in the renal tubules of IR mice. Taken together, our both in vivo and in vitro experiments suggest that calpeptin conveyed reno-protection in AKI might be mediated by the inhibition of AIM2 inflammasome activation and upregulation of Klotho protein. As such, we provide new evidence that Calpain 1 and 2 activation may be closely associated with the pathogenesis of clinical AKI. The calpain-mediated AIM2 inflammasome signaling pathway and distinct interaction between calpain and Klotho may provide a potential novel preventative and therapeutic target for acute kidney injury.

Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

BackgroundNeuroinflammation is thought to be a cause of Alzheimer’s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD.MethodsBV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms.ResultsAs a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation.ConclusionsThese results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.

USP21 Deubiquitinase Regulates AIM2 Inflammasome Activation.

Innate immune sensing of cytosolic DNA via absent in melanoma 2 (AIM2) is a key mechanism leading to inflammatory responses. As aberrant immune responses by dysregulated AIM2 are associated with autoinflammatory diseases, activation of the AIM2 inflammasome should be tightly controlled. In this study, we discovered that ubiquitination and deubiquitination of AIM2 are critical events that regulate AIM2 inflammasome activation. In resting human macrophage cells, AIM2 is constitutively ubiquitinated and undergoes proteasomal degradation to avoid autoinflammation. Upon DNA stimulation, USP21 binds to AIM2 and deubiquitinates it, thereby increasing its protein stability. In addition to the role of USP21 in regulating AIM2 turnover, we uncovered that USP21-mediated deubiquitination of AIM2 is required for the assembly of the AIM2 inflammasome. Depletion of USP21 does not affect the DNA-binding ability of AIM2 but inhibits the formation of the AIM2-ASC complex. Our findings establish that fine-tuning of AIM2 by the ubiquitin system is important for regulating AIM2 inflammasome activation.

AIM2-driven inflammasome activation in heart failure.

Interleukin-1β (IL-1β) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 β are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1β. Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.

Inflammasome components and ADAMTS4 in premature rupture of membranes.

Inflammation may be responsible for the development of premature rupture of membranes (PROM) including preterm PROM (PPROM) and mature PROM (MPROM). A total of four classic receptor proteins have been confirmed to assemble inflammasomes: NLR family pyrin domain containing (NLRP)1, NLRP3 and NLR family CARD‑domain containing4(NLRC4) and absent in melanoma2(AIM2). The activation and expression of these receptor‑modulated inflammasomes in placenta and fetal membrane of PROM pregnancies requires investigation. In addition, a disintegrin and metalloproteinase with thrombospondin motifs4(ADAMTS4) is a risk factor for PROM, but whether its expression is associated with inflammasome activation remains to be elucidated. In the present study, the placenta and fetal membrane tissues of patients who had suffered PPROM and MPROM and healthy pregnancies were investigated. Reverse transcription‑quantitative PCR was used to determine the mRNA expression of inflammasomes and ADAMTS4. Western blotting, immunohistochemistry and ELISA were used to investigate the protein expression levels of inflammasomes and ADAMTS4. The results demonstrated that all four inflammasomes were elevated in placenta and fetal membrane of PPROMs as were mRNA and protein expression levels of IL‑18 and IL‑1β (compared with controls). A further increase of inflammasomes and interleukins was observed in MPROMs compared with controls. Similar results were also observed in ADAMTS4 expression in PPROM and MPROM groups. However, immunohistochemistry results revealed no significant difference of inflammasome receptor expression in PPROMs compared with controls. Finally, a general positive correlation between ADAMTS4 and all four inflammasome receptors in placenta and fetal membrane of PPROMs and MPROMs was observed. The present study revealed that NLRP1, NLRP3, AIM2 and NLRC4 inflammasome activation in PROM was increased. Promoted ADAMTS4 level was further observed in PROM group and was significantly correlated with inflammasome expression. Inhibition of inflammasome activation may provide a therapeutic target for clinical PROM treatment.

AIM2-driven inflammasome activation in chronic heart failure

Abstract Background Inflammation and cytokine release have been implicated in the pathogenesis of chronic heart failure (CHF). Of particular interest, Canakinumab, a monoclonal antibody against interleukin-1b (IL-1β), had provided benefit against cardiovascular events, suggesting that blockade of IL-1β secretion and signaling might be a promising new therapeutic target. Although, recent studies have provided evidence that inflammasome activation is the main contributor to IL-1β maturation, the role of inflammasome activation in CHF remains unknown. Objective Therefore, we aimed to assess inflammasome activation in myocardial samples from end-stage failing hearts. Methods Inflammasome activation was assessed by immunoblotting in left ventricular myocardial specimens harvested from patients with end-stage CHF. Furthermore, immunoblot measurements were also performed on translational animal models of CHF (e.g. rat models of permanent coronary artery ligation and transverse aortic constriction). Left ventricular monocyte and macrophage infiltration was detected by immunohistochemistry. To investigate the molecular background of inflammasome activation, a series of cell culture experiments were performed on AC16 human cardiomyocytes and THP-1 human monocytic cell lines. Results Out of the 4 major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human CHF while the NLRP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change. A similar expression pattern in AIM2 and NLRC4 was also noted in CHF animal models. Furthermore, robust infiltration of Iba1+ monocytes/macrophages was observed in human failing hearts as well as in different animal models of CHF. In vitro AIM2 inflammasome activation, as induced by transfection with double-stranded DNA [poly(deoxyadenylic-deoxythymidylic)] was reduced significantly by the pharmacological blockade of pannexin-1 channels. Conclusions AIM2 and NLRC4 inflammasome activation might contribute to chronic inflammation in CHF. Our findings suggest that pannexin-1 channels might be a promising novel target to reduce inflammasome activation. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): NVKP_16-1-2016-0017

Circulatory CD4-Positive T-Lymphocyte Imbalance Mediated by Homocysteine-Induced AIM2 and NLRP1 Inflammasome Upregulation and Activation Is Associated with Human Abdominal Aortic Aneurysm

Introduction: CD4-positive T lymphocytes (CD4 cells) play a significant role in human abdominal aortic aneurysm (AAA). However, we know little about the role of the different CD4 subtypes. Objective: We aimed to discover the circulatory CD4 phenotypic marker profile and the roles of the newly found T helper cell 9 (Th9) and follicular helper T cells (Tfh) and that of inflammasomes in CD4 cells from AAA patients. Methods: We extracted CD4 cells from 30 AAA patients and 21 age-matched controls. Phenotype-specific transcription factors (TFs) and inflammasomes were analyzed with qRT-PCR. Results: Th17-, Th1-, Th9-, and Tfh-specific TFs and inflammasome components NLRP1 (NLR family pyrin domain-containing 1), NLRP3, NLRC4, AIM2 (absent in melanoma 2), PYCARD (apoptosis speck-like protein containing a CARD), and CASP1 (caspase 1) were upregulated, and T regulatory cell- and Th2-specific TFs were downregulated in the patients’ peripheral blood CD4 cells. Homocysteine was involved in Tfh and Th17 imbalance by AIM2 and NLRP1 inflammasome upregulation. Blood total cholesterol level correlated positively with NLRP1 expression, and blood low-density lipoprotein level correlated negatively with FOXP3 expression. Conclusions: Inflammasome-induced CD4 cell imbalance was involved in AAA. We thought that AAA might be a consequence of synergism between systemic immune imbalance and local autoimmunity.

Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida

Abstract The DNA sensor absent in melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1b and IL-18 and pyroptosis. AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida–infected bone marrow–derived macrophages. Furthermore, caspase-1 activation was compromised in bone marrow–derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome.

The hepatoprotective effects of XCHD and MgIG against methotrexate-induced liver injury and inflammation in rats through suppressing the activation of AIM2 inflammasomes

BackgroundRecent studies have shown that drug-induced liver injury may be related to the immune response activated by drugs. A cytosolic dsDNA inflammasome called absent in melanoma 2 (AIM2) was found to be associated with aseptic inflammation. The present study aimed to explore the effects of on the liver injury and inflammation in methotrexate (Mtx)-induced rats. MethodsSprague Dawley (SD) rats were selected and classified into 4 groups randomly, includes control group, Mtx group, Mtx-Xiaochaihu decoction (XCHD) group and Mtx-magnesium isoglycyrrhizinate (MgIG) group. Light microscopy was used to examine histological specimens after hematoxylin-eosin (HE) staining. The AST levels in liver tissue and blood serum ALT in the rats were assessed with enzyme linked immunosorbent assay (ELISA). Then AIM2 expression and inflammatory factors, including caspase-1, IL-18, and IL-1β, in the liver biopsy specimens of rats were detected by immunohistochemistry. Furthermore, the correlation between inflammatory and AIM2 expression factors was comprehensively analyzed. ResultsFunctional and structural hepatotoxicity can be caused by the exposure to Mtx, which was supported by the improved biochemical marker levels and the worse histopathological changes in liver tissue. Compared with the Mtx group, the levels of liver enzymes ALT and AST, histological deterioration in the liver tissues were effectively decreased by XCHD and MgIG treatment, respectively. In addition, the expression of AIM2, caspase-1 and IL-1β was observably higher in the Mtx group, which was apparently inhibited in the Mtx-XCHD and Mtx-MgIG groups. There was no obvious change in IL-18 expression among four groups. AIM2 expression were positively associated with the severity of liver inflammation and had a higher relevance with caspase-1 expression. ConclusionsAIM2 inflammasome in hepatocytes has a significant effect on the development of Mtx-induced liver injury, which can be ameliorated by both XCHD and MgIG treatment. The latent mechanism and potential signal pathway require further study.