Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in interorgan communication in diverse pathological processes. ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of the alveolar macrophage (AM) markers CD11b, CD11c, and CD206 as well as ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction of inspired oxygen therapy (HO2⩾30%) had increased concentrations of AM-derived EV-ASC compared with infants on lower fraction of inspired oxygen (LO2<30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier, and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death, with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain cross-talk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

The Inflammasome Adaptor Protein ASC in Plasma as a Biomarker of Early Cognitive Changes.

Dementia is a group of symptoms including memory loss, language difficulties, and other types of cognitive and functional impairments that affects 57 million people worldwide, with the incidence expected to double by 2040. Therefore, there is an unmet need to develop reliable biomarkers to diagnose early brain impairments so that emerging interventions can be applied before brain degeneration. Here, we performed biomarker analyses for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and amyloid-ß 42/40 (Aß42/40) ratio in the plasma of older adults. Participants had blood drawn at baseline and underwent two annual clinical and cognitive evaluations. The groups tested either cognitively normal on both evaluations (NN), cognitively normal year 1 but cognitively impaired year 2 (NI), or cognitively impaired on both evaluations (II). ASC was elevated in the plasma of the NI group compared to the NN and II groups. Additionally, Aß42 was increased in the plasma in the NI and II groups compared to the NN group. Importantly, the area under the curve (AUC) for ASC in participants older than 70 years old in NN vs. NI groups was 0.81, indicating that ASC is a promising plasma biomarker for early detection of cognitive decline.

Serum Caspase-1 as an Independent Prognostic Factor in Traumatic Brain Injured Patients.

BACKGROUND: The objectives of this study were to assess the association between serum caspase 1 levels and known clinical and radiological prognostic factors and determine whether caspase 1was a more powerful predictor of outcome after traumatic brain injury (TBI) than clinical indices alone, to determine the association between the serum levels of caspase 1 and the 6-month outcome, and to evaluate if there is any association between caspase 1 with clinical and radiological variables. METHODS: This prospective and observational study was conducted in a university hospital and included patients with TBI who required hospital admission. Serum samples were collected at hospital admission and 24 h after TBI. Caspase 1 levels were determined by enzyme-linked immunosorbent assay. Receiver operating characteristic curves were obtained to test the potential of caspase 1 to predict mortality (Glasgow Outcome Scale Extended score of 1) and unfavorable outcome (Glasgow Outcome Scale Extended scores of 1-4). Multivariate logistic regression was used to assess the effect of serum caspase 1 levels, adjusted by known clinical and radiological prognostic indices, on the outcome. RESULTS: One hundred thirty-two patients and 33 healthy controls were included. We obtained 6-month outcome in 118 patients. On admission, the mean serum levels of caspase 1 were higher in patients with TBI compared with controls (157.9 vs. 108.5 pg/mL; p < 0.05) but not at 24 h after TBI. Serum caspase 1 levels on admission were higher in patients with unfavorable outcomes (189.5 vs. 144.1 pg/mL; p = 0.009). Similarly, serum caspase 1 levels on admission were higher in patients who died vs. patients who survived (213.6 vs. 146.8 pg/mL; p = 0.03). A logistic regression model showed that the serum caspase 1 level on admission was an independent predictor of 6-month unfavorable outcomes (odds ratio 1.05; 95% confidence interval 1-1.11; p = 0.05). Caspase 1 levels were higher in patients with severe TBI compared with those with moderate TBI, those with mild TBI, and healthy controls (p < 0.001). We did not find any correlation between caspase 1 and the radiological variables studied. CONCLUSIONS: In this cohort of patients with TBI, we show that serum caspase 1 protein levels on admission are an independent prognostic factor after TBI. Serum caspase 1 levels on admission are higher in patients who will present unfavorable outcomes 6 months after TBI. Caspase 1 levels on admission are associated with the injury severity determined by the Glasgow Coma Scale.

Tumor necrosis factor-alpha and interferon-gamma induce inflammasome-mediated corneal endothelial cell death.

PURPOSE: Chronic corneal endothelial cell (CEC) loss results in corneal edema and vision loss in conditions such as pseudophakic bullous keratopathy (PBK), Fuchs' dystrophy, and corneal graft failure. Low CEC density has been associated with an elevation of intraocular pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (INF)-γ. These cytokines are capable of triggering pyroptosis, a programmed cell death mechanism mediated by the inflammasome, prompting the activation of the pro-inflammatory cytokine interleukin (IL)-1ß, the perpetuation of inflammation, and subsequent damage of corneal endothelial tissue. Therefore, the purpose of this study was to determine the deleterious contribution of the inflammasome and pyroptosis to CEC loss. METHODS: CECs from human donor corneas were treated ex vivo with TNF-α and IFN-γ for 48 h. Levels of caspase-1 and IL-1ß were then assayed by ELISA, and the expression of caspase-1 and gasdermin-D (GSDM-D) were confirmed by immunofluorescence. Endothelial cell damage was analyzed by a lactate dehydrogenase (LDH) release assay, and oxidative stress was determined by measuring the levels of reactive oxygen species (ROS) in the culture media. RESULTS: Inflammasome activation and oxidative stress were elevated in CECs following exposure to TNF-α and IFN-γ, which resulted in cell death by pyroptosis as determined by LDH release which was inhibited by the caspase-1 inhibitor Ac-YVAD-cmk. CONCLUSION: CEC death is induced by the pro-inflammatory cytokines TNF-α and IFN-γ, which contribute to inflammasome activation. Moreover, the inflammasome is a promising therapeutic target for the treatment of chronic CEC loss.

Role of inflammasomes in multiple sclerosis and their potential as therapeutic targets.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS), and it remains the most common immune-mediated disorder affecting the CNS. While the cause of MS is unclear, the underlying pathomechanisms are thought to be either destruction by autoimmune T cells or dysfunction of myelin-producing cells. Recent advances have indicated that inflammasomes contribute the etiology of MS. Inflammasomes are multiprotein complexes of the innate immune response involved in the processing of caspase-1, the activation of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18 as well as the cell death-mediated mechanism of pyroptosis and the activation of the adaptive immune response. Here we review the literature to date on the role of different inflammasome signaling pathways in the pathogenesis of MS and how these pathways may be targeted to reduce deleterious inflammatory processes and improve outcomes in this patient population.

IC100: a novel anti-ASC monoclonal antibody improves functional outcomes in an animal model of multiple sclerosis.

BACKGROUND: The inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is involved in immune signaling by bridging the interactions between inflammasome sensors and caspase-1. Strong experimental evidence has shown that ASC-/- mice are protected from disease progression in animal models of multiple sclerosis (MS), suggesting that targeting inflammasome activation via ASC inhibition may be a promising therapeutic strategy in MS. Thus, the goal of our study is to test the efficacy of IC100, a novel humanized antibody targeting ASC, in preventing and/or suppressing disease in the experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: We employed the EAE model of MS where disease was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55). Mice were treated with vehicle or increasing doses of IC100 (10, 30, and 45 mg/kg) and clinical disease course was evaluated up to 35 days post EAE induction. Immune cell infiltration into the spinal cord and microglia responses were assessed. RESULTS: We show that IC100 treatment reduced the severity of EAE when compared to vehicle-treated controls. At a dose of 30 mg/kg, IC100 significantly reduced the number of CD4+ and CD8+ T cells and CD11b+MHCII+ activated myeloid cells entering the spinal cord from the periphery, and reduced the number of total and activated microglia. CONCLUSIONS: These data indicate that IC100 suppresses the immune-inflammatory response that drives EAE development and progression, thereby identifying ASC as a promising target for the treatment of MS as well as other neurological diseases with a neuroinflammatory component.

Oligodendrocytes modulate the immune-inflammatory response in EAE via TNFR2 signaling.

The pleotropic cytokine tumor necrosis factor (TNF) is involved in the pathophysiology of multiple sclerosis (MS). In various models of MS, including experimental autoimmune encephalomyelitis (EAE), the membrane-bound form of TNF (tmTNF), which signals primarily via TNFR2, mediates protective and reparative effects, whereas the soluble form (solTNF), which signals primarily via TNFR1, promotes pro-inflammatory and detrimental functions. In this study, we investigated the role of TNFR2 expressed in oligodendrocytes in the early phase of EAE pathogenesis. We demonstrated that mice with specific ablation of oligodendroglial TNFR2 displayed early onset and higher peak of motor dysfunction when subjected to EAE, in advance of which accelerated infiltration of immune cells was observed as early as 10 days post EAE induction. The immune cell influx was preceded by microglial activation and increased blood brain barrier permeability. Lack of oligodendroglial TNFR2 accelerated the expression of inflammatory cytokines as well as expression and activation of the inflammasome. Gene expression profiling of oligodendrocytes sorted from the spinal cord 14 days post EAE induction showed robust upregulation of inflammatory genes, some of which were elevated in cells lacking TNFR2 compared to controls. Together, our data demonstrate that oligodendrocytes are directly involved in inflammation and immune modulation in CNS disease and this function is regulated, at least in part, by TNFR2.

ASC, IL-18 and Galectin-3 as Biomarkers of Non-Alcoholic Steatohepatitis: A Proof of Concept Study.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease that is growing in prevalence. Symptoms of NASH become apparent when the disease has progressed significantly. Thus, there is a need to identify biomarkers of NASH in order to detect the disease earlier and to monitor disease severity. The inflammasome has been shown to play a role in liver diseases. Here, we performed a proof of concept study of biomarker analyses (cut-off points, positive and negative predictive values, receiver operating characteristic (ROC) curves, and likelihood ratios) on the serum of patients with NASH and healthy controls on apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), interleukin (IL)-18, Galectin-3 (Gal-3), and C-reactive protein (CRP). ASC, IL-18, and Gal-3 were elevated in the serum of NASH patients when compared to controls. The area under the curve (AUC) for ASC was the highest (0.7317) with an accuracy of 68%, followed by IL-18 (0.7036) with an accuracy of 66% and Gal-3 (0.6891) with an accuracy of 61%. Moreover, we then fit a stepwise multivariate logistic regression model using ASC, IL-18, and Gal-3 to determine the probability of patients having a NASH diagnosis, which resulted in an AUC of 0.71 and an accuracy of 79%, indicating that combining these biomarkers increases their diagnostic potential for NASH. These results indicate that ASC, IL-18, and Gal-3 are reliable biomarkers of NASH and that combining these analytes increases the biomarker potential of these proteins.

The Inflammasome Adaptor Protein ASC in Mild Cognitive Impairment and Alzheimer's Disease.

Mild cognitive impairment (MCI) is characterized by memory loss in the absence of dementia and is considered the translational stage between normal aging and early Alzheimer's disease (AD). Patients with MCI have a greater risk of advancing to AD. Thus, identifying early markers of MCI has the potential to increase the therapeutic window to treat and manage the disease. Protein levels of the inflammasome signaling proteins apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and interleukin (IL)-18 were analyzed in the serum of patients with MCI, AD and healthy age-matched donors as possible biomarkers, as well as levels of soluble amyloid precursor proteins α/ß (sAPP α/ß) and neurofilament light (NfL). Cut-off points and positive and negative predictive values, as well as receiver operator characteristic (ROC) curves, likelihood ratios and accuracy were determined for these proteins. Although the levels of ASC were higher in MCI and AD than in age-matched controls, protein levels of ASC were higher in MCI than in AD cases. For control vs. MCI, the area under the curve (AUC) for ASC was 0.974, with a cut-off point of 264.9 pg/mL. These data were comparable to the AUC for sAPP α and ß of 0.9687 and 0.9068, respectively, as well as 0.7734 for NfL. Moreover, similar results were obtained for control vs. AD and MCI vs. AD. These results indicate that ASC is a promising biomarker of MCI and AD.

Caspase-1 Inhibition Attenuates Hyperoxia-induced Lung and Brain Injury in Neonatal Mice.

Hyperoxia plays a key role in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants. Infants with BPD often have brain injury that leads to long-term neurodevelopmental impairment, but the underlying mechanisms that control BPD-induced neurodevelopmental impairment remain unclear. Our previous studies have shown that hyperoxia-induced BPD in rodents is associated with lung inflammasome activation. Here, we tested the hypothesis that hyperoxia-induced lung and brain injury is mediated by inflammasome activation, and that inhibition of caspase-1, a key component of the inflammasome, attenuates hyperoxia-induced lung and brain injury in neonatal mice. C57/BL6 mouse pups were randomized to receive daily intraperitoneal injections of Ac-YVAD-CMK, an irreversible caspase-1 inhibitor, or placebo during exposure to room air or hyperoxia (85% O2) for 10 days. We found that hyperoxia activated the NLRP1 inflammasome, increased production of mature IL-1ß, and upregulated expression of p30 gasdermin-D (GSDMD), the active form of GSDMD that is responsible for the programmed cell death mechanism of pyroptosis in both lung and brain tissue. Importantly, we show that inhibition of caspase-1 decreased IL-1ß activation and p30 GSDMD expression, and improved alveolar and vascular development in hyperoxia-exposed lungs. Moreover, caspase-1 inhibition also promoted cell proliferation in the subgranular zone and subventricular zone of hyperoxia-exposed brains, resulting in lessened atrophy of these zones. Thus, the inflammasome plays a critical role in hyperoxia-induced neonatal lung and brain injury, and targeting this pathway may be beneficial for the prevention of lung and brain injury in preterm infants.

The role of microglial inflammasome activation in pyroptotic cell death following penetrating traumatic brain injury.

BACKGROUND: Traumatic brain injury remains a significant cause of death and disability in the USA. Currently, there are no effective therapies to mitigate disability except for surgical interventions necessitating a need for continued research into uncovering novel therapeutic targets. In a recent study, we used a rodent model of penetrating traumatic brain injury known as penetrating ballistic-like brain injury (PBBI) to examine the role of innate immunity in post-traumatic secondary injury mechanisms. We previously reported that the inflammasome, a multiprotein complex composed of apoptosis-associated speck-like protein containing card and caspase-1, plays a role in secondary cell death mechanisms after PBBI, including inflammatory cell death (pyroptosis). METHODS: In the current study, we used flow cytometry analysis to evaluate activated microglia and CD11b-positive leukocytes after PBBI and assessed inflammasome activation and pyroptosis of specific cellular populations. Sprague-Dawley male rats underwent PBBI or sham-operated procedures and ipsilateral cortical regions processed for flow cytometry and cellular analysis. Flow cytometry results were compared using one-way ANOVA followed by Tukey's multiple comparisons. RESULTS: At 48 h following PBBI, there was an increase in activated microglia and infiltrating leukocytes compared to sham controls that were associated with increased caspase-1 activity. Using a florescent probe to identify caspase-1 activity and a fluorescent assay to determine cell viability, evidence for pyroptosis in CD11b+ cells was also determined. Finally, while post-traumatic treatment with an anti-ASC antibody had no effect on the number of activated microglia and infiltrating leukocytes, antibody treatment decreased caspase-1 activity in both resident microglia and infiltrating leukocytes and reduced pyroptotic CD11b+ cell death. CONCLUSIONS: These results provide evidence for inflammasome activation in microglia and infiltrating leukocytes after penetrating traumatic brain injury and a role for pyroptotic cell death in the pathophysiology. In addition to inhibiting neuronal cell death, therapeutic treatments targeting inflammasome activation may also provide beneficial effects by reducing the potentially detrimental consequences of activated microglia and infiltrating CD11b+ leukocytes following penetrating traumatic brain injury.

Nicotine Alters Estrogen Receptor-Beta-Regulated Inflammasome Activity and Exacerbates Ischemic Brain Damage in Female Rats.

Smoking is a preventable risk factor for stroke and smoking-derived nicotine exacerbates post-ischemic damage via inhibition of estrogen receptor beta (ER-β) signaling in the brain of female rats. ER-β regulates inflammasome activation in the brain. Therefore, we hypothesized that chronic nicotine exposure activates the inflammasome in the brain, thus exacerbating ischemic brain damage in female rats. To test this hypothesis, adult female Sprague-Dawley rats (6⁻7 months old) were exposed to nicotine (4.5 mg/kg/day) or saline for 16 days. Subsequently, brain tissue was collected for immunoblot analysis. In addition, another set of rats underwent transient middle cerebral artery occlusion (tMCAO; 90 min) with or without nicotine exposure. One month after tMCAO, histopathological analysis revealed a significant increase in infarct volume in the nicotine-treated group (64.24 ± 7.3 mm³; mean ± SEM; n = 6) compared to the saline-treated group (37.12 ± 7.37 mm³; n = 7, p < 0.05). Immunoblot analysis indicated that nicotine increased cortical protein levels of caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC) and pro-inflammatory cytokines interleukin (IL)-1β by 88% (p < 0.05), 48% (p < 0.05) and 149% (p < 0.05), respectively, when compared to the saline-treated group. Next, using an in vitro model of ischemia in organotypic slice cultures, we tested the hypothesis that inhibition of nicotine-induced inflammasome activation improves post-ischemic neuronal survival. Accordingly, slices were exposed to nicotine (100 ng/mL; 14⁻16 days) or saline, followed by treatment with the inflammasome inhibitor isoliquiritigenin (ILG; 24 h) prior to oxygen-glucose deprivation (OGD; 45 min). Quantification of neuronal death demonstrated that inflammasome inhibition significantly decreased nicotine-induced ischemic neuronal death. Overall, this study shows that chronic nicotine exposure exacerbates ischemic brain damage via activation of the inflammasome in the brain of female rats.

Estrogen receptor beta signaling alters cellular inflammasomes activity after global cerebral ischemia in reproductively senescence female rats.

Periodic treatments with estrogen receptor subtype-ß (ER-ß) agonist reduce post-ischemic hippocampal injury in ovariectomized rats. However, the underlying mechanism of how ER-ß agonists protect the brain remains unknown. Global cerebral ischemia activates the innate immune response, and a key component of the innate immune response is the inflammasome. This study tests the hypothesis that ER-ß regulates inflammasome activation in the hippocampus, thus reducing ischemic hippocampal damage in reproductively senescent female rats that received periodic ER-ß agonist treatments. First, we determined the effect of hippocampal ER-ß silencing on the expression of the inflammasome proteins caspase 1, apoptosis-associated speck-like protein containing a CARD (ASC), and interleukin (IL)-1ß. Silencing of ER-ß attenuated 17ß-estradiol mediated decrease in caspase 1, ASC, and IL-1ß. Next, we tested the hypothesis that periodic ER-ß agonist treatment reduces inflammasome activation and ischemic damage in reproductively senescent female rats. Periodic ER-ß agonist treatments significantly decreased inflammasome activation and increased post-ischemic live neuronal counts by 32% (p < 0.05) as compared to the vehicle-treated, reproductively senescent rats. Current findings demonstrated that ER-ß activation regulates inflammasome activation and protects the brain from global ischemic damage in reproductively senescent female rats. Further investigation on the role of a periodic ER-ß agonist regimen to reduce the innate immune response in the brain could help reduce the incidence and the impact of global cerebral ischemia in post-menopausal women. We propose that estrogen receptor subtype-ß (ER-ß) activation regulates inflammasome activation and protects the brain from global ischemic damage in reproductively senescent female rats.

Exosome-mediated inflammasome signaling after central nervous system injury.

Neuroinflammation is a response against harmful effects of diverse stimuli and participates in the pathogenesis of brain and spinal cord injury (SCI). The innate immune response plays a role in neuroinflammation following CNS injury via activation of multiprotein complexes termed inflammasomes that regulate the activation of caspase 1 and the processing of the pro-inflammatory cytokines IL-1ß and IL-18. We report here that the expression of components of the nucleotide-binding and oligomerization domain (NOD)-like receptor protein-1 (NLRP-1) inflammasome, apoptosis speck-like protein containing a caspase recruitment domain (ASC), and caspase 1 are significantly elevated in spinal cord motor neurons and cortical neurons after CNS trauma. Moreover, NLRP1 inflammasome proteins are present in exosomes derived from CSF of SCI and traumatic brain-injured patients following trauma. To investigate whether exosomes could be used to therapeutically block inflammasome activation in the CNS, exosomes were isolated from embryonic cortical neuronal cultures and loaded with short-interfering RNA (siRNA) against ASC and administered to spinal cord-injured animals. Neuronal-derived exosomes crossed the injured blood-spinal cord barrier, and delivered their cargo in vivo, resulting in knockdown of ASC protein levels by approximately 76% when compared to SCI rats treated with scrambled siRNA. Surprisingly, siRNA silencing of ASC also led to a significant decrease in caspase 1 activation and processing of IL-1ß after SCI. These findings indicate that exosome-mediated siRNA delivery may be a strong candidate to block inflammasome activation following CNS injury. We propose the following signaling cascade for inflammasome activation in peripheral tissues after CNS injury: CNS trauma induces inflammasome activation in the nervous system and secretion of exosomes containing inflammasome protein cargo into cerebral spinal fluid. The inflammasome containing exosomes then fuse with target cells to activate the innate immune response in peripheral tissues. We suggest that these findings may be used to develop new therapeutics to treat the devastating inflammation and cell destruction evoked by CNS injuries. IL-1ß and IL-18 = pro-inflammatory cytokines.

The inflammasome pyrin contributes to pertussis toxin-induced IL-1ß synthesis, neutrophil intravascular crawling and autoimmune encephalomyelitis.

Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1ß synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1ß into its active form. In turn, IL-1ß stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.

RIG-1 receptor expression in the pathology of Alzheimer's disease.

BACKGROUND: Neuroinflammation plays a critical role in the pathogenesis of Alzheimer's disease (AD) and involves activation of the innate immune response via recognition of diverse stimuli by pattern recognition receptors (PRRs). The inflammatory inducers and precise innate signaling pathway contributing to AD pathology remain largely undefined. RESULTS: In the present study we analyzed expression levels of innate immune proteins in temporal and occipital cortices from preclinical (no cognitive impairment, NCI, N = 22) to mild cognitive impairment (MCI, N = 20) associated with AD pathology (N = 20) and AD patients (N = 23). We found that retinoic acid-inducible gene-I (RIG-1) is significantly elevated in the temporal cortex and plasma in patients with MCI. In addition, primary human astrocytes stimulated with the RIG-1 ligand 5'ppp RNA showed increased expression of amyloid precursor protein (APP) and amyloid-ß (Aß), supporting the idea that RIG-1 is involved in the pathology of MCI associated with early progression to AD. CONCLUSION: These findings suggest that RIG-1 may play a critical role in incipient AD.

Inflammasome Proteins Are Reliable Biomarkers of the Inflammatory Response in Aneurysmal Subarachnoid Hemorrhage.

Aneurysmal subarachnoid hemorrhage (aSAH) is caused by abnormal blood vessel dilation and subsequent rupture, resulting in blood pooling in the subarachnoid space. This neurological insult results in the activation of the inflammasome, a multiprotein complex that processes pro-inflammatory interleukin (IL)-1 cytokines leading to morbidity and mortality. Moreover, increases in inflammasome proteins are associated with clinical deterioration in many neurological diseases. Limited studies have investigated inflammasome protein expression following aSAH. Reliable markers of the inflammatory response associated with aSAH may allow for earlier detection of patients at risk for complications and aid in the identification of novel pharmacologic targets. Here, we investigated whether inflammasome signaling proteins may serve as potential biomarkers of the inflammatory response in aSAH. Serum and cerebrospinal fluid (CSF) from fifteen aSAH subjects and healthy age-matched controls and hydrocephalus (CSF) no-aneurysm controls were evaluated for levels of inflammasome signaling proteins and downstream pro-inflammatory cytokines. Protein measurements were carried out using Simple Plex and Single-Molecule Array (Simoa) technology. The area under the curve (AUC) was calculated using receiver operating characteristics (ROCs) to obtain information on biomarker reliability, specificity, sensitivity, cut-off points, and likelihood ratio. In addition, a Spearman r correlation matrix was performed to determine the correlation between inflammasome protein levels and clinical outcome measures. aSAH subjects demonstrated elevated caspase-1, apoptosis-associated speck-like protein with a caspase recruiting domain (ASC), IL-18 and IL-1ß levels in serum, and CSF when compared to controls. Each of these proteins was found to be a promising biomarker of inflammation in aSAH in the CSF. In addition, ASC, caspase-1, and IL-1ß were found to be promising biomarkers of inflammation in aSAH in serum. Furthermore, we found that elevated levels of inflammasome proteins in serum are useful to predict worse functional outcomes following aSAH. Thus, the determination of inflammasome protein levels in CSF and serum in aSAH may be utilized as reliable biomarkers of inflammation in aSAH and used clinically to monitor patient outcomes.

Extracellular vesicles mediate inflammasome signaling in the brain and heart of Alzheimer's disease mice.

Introduction: Alzheimer's disease (AD) is an inflammatory neurodegenerative disease characterized by memory loss and cognitive impairment that worsens over time. AD is associated with many comorbidities, including cardiovascular disease that are associated with poorer outcomes. Comorbidities, especially heart disease and stroke, play a significant role in the demise of AD patients. Thus, it is important to understand how comorbidities are linked to AD. We have previously shown that extracellular vesicle (EV)-mediated inflammasome signaling plays an important role in the pathogenesis of brain injury and acute lung injury after traumatic brain injury. Methods: We analyzed the cortical, hippocampal, ventricular, and atrial protein lysates from APP/PS1 mice and their respective controls for inflammasome signaling activation. Additionally, we analyzed serum-derived EV for size, concentration, and content of inflammasome proteins as well as the EV marker CD63. Finally, we performed conditioned media experiments of EV from AD patients and healthy age-matched controls delivered to cardiovascular cells in culture to assess EV-induced inflammation. Results: We show a significant increase in Pyrin, NLRP1, caspase-1, and ASC in the brain cortex whereas caspase-8, ASC, and IL-1ß were significantly elevated in the heart ventricles of AD mice when compared to controls. We did not find significant differences in the size or concentration of EV between groups, but there was a significant increase of caspase-1 and IL-1ß in EV from AD mice compared to controls. In addition, conditioned media experiments of serum-derived EV from AD patients and age-matched controls delivered to cardiovascular cells in culture resulted in inflammasome activation, and significant increases in TNF-α and IL-2. Conclusion: These results indicate that EV-mediated inflammasome signaling in the heart may play a role in the development of cardiovascular diseases in AD patients.

Increased inflammasome protein expression identified in microglia from postmortem brains with schizophrenia.

Schizophrenia (SCZ) is a complex psychiatric disorder that involves an inflammatory response thought to be characterized by microglial activation. The inflammasome complex may play critical roles in the pathomechanism of neuroinflammation but how this relates to SCZ remains unclear. In this study, we performed an immunohistochemical (IHC) analysis to compare the expression of inflammasome proteins in brain tissue from donors with SCZ (n = 16) and non-psychiatric donors (NP; n = 13) isolated from the superior frontal cortex (SFC), superior temporal cortex, and anterior cingulate cortex brain regions. To assess changes in the cell populations that express key inflammasome proteins, we performed IHC analyses of apoptosis-associated speck-like protein containing a CARD (ASC), nod-like receptor protein 3 (NLRP3), and interleukin (IL)-18 to determine if these proteins are expressed in microglia, astrocytes, oligodendrocytes, or neurons. Inflammasome proteins were expressed mainly in microglia from SCZ and NP brains. Increased numbers of microglia were present in the SFC of SCZ brains and exhibited higher inflammasome protein expression of ASC, NLRP3, and IL-18 compared to NPs. These findings suggest that increased inflammasome signaling may contribute to the pathology underlying SCZ.