NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined.MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP).ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord.ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.     

Inflammasome activation and IL-1β/IL-18 processing are influenced by distinct pathways in microglia

Microglia are important innate immune effectors against invading CNS pathogens, such as Staphylococcus aureus (S. aureus), a common etiological agent of brain abscesses typified by widespread inflammation and necrosis. The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing following exposure to both pathogen- and danger-associated molecular patterns. Although previous studies from our laboratory have established that IL-1β is a major cytokine product of S. aureus-activated microglia and is pivotal for eliciting protective anti-bacterial immunity during brain abscess development, the molecular machinery responsible for cytokine release remains to be determined. Therefore, the functional role of the NLRP3 inflammasome and its adaptor protein apoptosis-associated speck-like protein (ASC) in eliciting IL-1β and IL-18 release was examined in primary microglia. Interestingly, we found that IL-1β, but not IL-18 production, was significantly attenuated in both NLRP3 and ASC knockout microglia following exposure to live S. aureus. NLRP3 inflammasome activation was partially dependent on autocrine/paracrine ATP release and α- and γ-hemolysins produced by live bacteria. A cathepsin B inhibitor attenuated IL-β release from NLRP3 and ASC knockout microglia, demonstrating the existence of alternative inflammasome-independent mechanisms for IL-1β processing. In contrast, microglial IL-18 secretion occurred independently of cathepsin B and inflammasome action. Collectively, these results demonstrate that microglial IL-1β processing is regulated by multiple pathways and diverges from mechanisms utilized for IL-18 cleavage. Understanding the molecular events that regulate IL-1β production is important for modulating this potent proinflammatory cytokine during CNS disease.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

The NLRP3 Inflammasome in Alzheimer’s Disease

Innate immunity and inflammatory response plays an important role in the pathogenesis of Alzheimer’s disease (AD). As the major resident immune cells in the brain, microglial cells constantly survey the microenvironment and are activated by and recruited to senile plaques. Subsequently, they can phagocytose amyloid-β (Aβ) and secrete pro-inflammatory cytokines that influence the surrounding brain tissue. Recently, a wealth of information linking the microglia-specific activation of NLRP3 inflammasome to AD pathogenesis has emerged. We review here the activation mechanisms of NLRP3 inflammasome in microglia and several downstream effects in the brain, demonstrating that toxic Aβ peptide can light a fire in NLRP3 inflammasome and eventually induce AD pathology and tissue damage. More importantly, it has been demonstrated that inhibition of NLRP3 could largely protect from memory loss and decrease Aβ deposition in AD transgenic mouse model. So, we further discuss the recent advances and challenges in targeting NLRP3 inflammasome for AD therapy.     

TRPV1 channel mediates NLRP3 inflammasome-dependent neuroinflammation in microglia

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease in the central nervous system (CNS). The NLRP3 inflammasome is considered an important regulator of immunity and inflammation, both of which play a critical role in MS. However, the underlying mechanism of NLRP3 inflammasome activation is not fully understood. Here we identified that the TRPV1 (transient receptor potential vanilloid type 1) channel in microglia, as a Ca²⁺ influx-regulating channel, played an important role in NLRP3 inflammasome activation. Deletion or pharmacological blockade of TRPV1 inhibited NLRP3 inflammasome activation in microglia in vitro. Further research revealed that TRPV1 channel regulated ATP-induced NLRP3 inflammasome activation through mediating Ca²⁺ influx and phosphorylation of phosphatase PP2A in microglia. In addition, TRPV1 deletion could alleviate mice experimental autoimmune encephalomyelitis (EAE) and reduce neuroinflammation by inhibiting NLRP3 inflammasome activation. These data suggested that the TRPV1 channel in microglia can regulate NLRP3 inflammasome activation and consequently mediate neuroinflammation. Meanwhile, our study indicated that TRPV1–Ca²⁺–PP2A pathway may be a novel regulator of NLRP3 inflammasome activation, pointing to TRPV1 as a potential target for CNS inflammatory diseases.Subject terms: Neuroimmunology, Neuroimmunology     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

PKR knockout in the 5xFAD model of Alzheimer's disease reveals beneficial effects on spatial memory and brain lesions

Brain lesions in Alzheimer's disease (AD) include amyloid plaques made of Aβ peptides and neurofibrillary tangles composed of hyperphosphorylated tau protein with synaptic and neuronal loss and neuroinflammation. Aβ oligomers can trigger tau phosphorylation and neuronal alterations through activation of neuronal kinases leading to progressive cognitive decline. PKR is a ubiquitous pro‐apoptotic serine/threonine kinase, and levels of activated PKR are increased in AD brains and AD CSF. In addition, PKR regulates negatively memory formation in mice. To assess the role of PKR in an AD in vivo model, we crossed 5xFAD transgenic mice with PKR knockout (PKRKO) mice and we explored the contribution of PKR on cognition and brain lesions in the 5xFAD mouse model of AD as well as in neuron–microglia co‐cultures exposed to the innate immunity activator lipopolysaccharide (LPS). Nine‐month‐old double‐mutant mice revealed significantly improved memory consolidation with the new object location test, starmaze test, and elevated plus maze test as compared to 5xFAD mice. Brain amyloid accumulation and BACE1 levels were statistically decreased in double‐mutant mice. Apoptosis, neurodegeneration markers, and synaptic alterations were significantly reduced in double‐mutant mice as well as neuroinflammation markers such as microglial load and brain cytokine levels. Using cocultures, we found that PKR in neurons was essential for LPS microglia‐induced neuronal death. Our results demonstrate the clear involvement of PKR in abnormal spatial memory and brain lesions in the 5xFAD model and underline its interest as a target for neuroprotection in AD.     

Epoxyeicosatrienoic acids inhibit the activation of NLRP3 inflammasome in murine macrophages

Epoxyeicosatrienoic acids (EETs) derived from arachidonic acid exert anti-inflammation effects. We have reported that blocking the degradation of EETs with a soluble epoxide hydrolase (sEH) inhibitor protects mice from lipopolysaccharide (LPS)-induced acute lung injury (ALI). The underlying mechanisms remain essential questions. In this study, we investigated the effects of EETs on the activation of nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in murine macrophages. In an LPS-induced ALI murine model, we found that sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl), TPPU, profoundly attenuated the pathological injury and inhibited the activation of the NLRP3 inflammasome, characterized by the reduction of the protein expression of NLRP3, ASC, pro-caspase-1, interleukin precursor (pro-IL-1β), and IL-1β p17 in the lungs of LPS-treated mice. In vitro, primary peritoneal macrophages from C57BL/6 were primed with LPS and activated with exogenous adenosine triphosphate (ATP). TPPU treatment remarkably reduced the expression of NLRP3 inflammasome-related molecules and blocked the activation of NLRP3 inflammasome. Importantly, four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) inhibited the activation of NLRP3 inflammasome induced by LPS + ATP or LPS + nigericin in macrophages in various degree. While the inhibitory effect of 5,6-EET was the weakest. Mechanismly, EETs profoundly decreased the content of reactive oxygen species (ROS) and restored the calcium overload in macrophages receiving LPS + ATP stimulation. In conclusion, this study suggests that EETs inhibit the activation of the NLRP3 inflammasome by suppressing calcium overload and ROS production in macrophages, contributing to the therapeutic potency to ALI.     

Frataxin Deficiency Promotes Excess Microglial DNA Damage and Inflammation that Is Rescued by PJ34

An inherited deficiency in the frataxin protein causes neurodegeneration of the dorsal root ganglia and Friedreich''s ataxia (FA). Frataxin deficiency leads to oxidative stress and inflammatory changes in cell and animal models; however, the cause of the inflammatory changes, and especially what causes brain microglial activation is unclear. Here we investigated: 1) the mechanism by which frataxin deficiency activates microglia, 2) whether a brain-localized inflammatory stimulus provokes a greater microglial response in FA animal models, and 3) whether an anti-inflammatory treatment improves their condition. Intracerebroventricular administration of LPS induced higher amounts of microglial activation in the FA mouse model vs controls. We also observed an increase in oxidative damage in the form of 8-oxoguanine (8-oxo-G) and the DNA repair proteins MUTYH and PARP-1 in cerebellar microglia of FA mutant mice. We hypothesized that frataxin deficiency increases DNA damage and DNA repair genes specifically in microglia, activating them. siRNA-mediated frataxin knockdown in microglial BV2 cells clearly elevated DNA damage and the expression of DNA repair genes MUTYH and PARP-1. Frataxin knockdown also induced a higher level of PARP-1 in MEF cells, and this was suppressed in MUTYH-/- knockout cells. Administration of the PARP-1 inhibitor PJ34 attenuated the microglial activation induced by intracerebroventricular injection of LPS. The combined administration of LPS and angiotensin II provoke an even stronger activation of microglia and neurobehavioral impairment. PJ34 treatment attenuated the neurobehavioral impairments in FA mice. These results suggest that the DNA repair proteins MUTYH and PARP-1 may form a pathway regulating microglial activation initiated by DNA damage, and inhibition of microglial PARP-1 induction could be an important therapeutic target in Friedreich''s ataxia.     

Verapamil inhibits early acute liver failure through suppressing the NLRP3 inflammasome pathway

Acute liver failure (ALF) is a rare disease characterized by the sudden onset of serious hepatic injury, as manifested by a profound liver dysfunction and hepatic encephalopathy in patients without prior liver disease. In this paper, we aim to investigate whether verapamil, an antagonist of TXNIP, inhibits early ALF through suppressing the NLRP3 inflammasome pathway. Firstly, an ALF mouse model was induced by lipopolysaccharide (LPS)/D-galactosamine (GalN) treatment. The optimal concentration of verapamil in treating early ALF mice was determined followed by investigation on its mechanism in LPS/GalN-induced liver injury. Western blot analysis and co-immunoprecipitation were performed to determine the activation of the TXNIP/NLRP3 inflammasome pathway. Subsequently, overexpression of NLRP3 in mouse liver was induced by transfection with AAV-NRLP3 in vivo and in vitro to identity whether verapamil inhibited early ALF through suppressing the activation of NLRP3 inflammasome. We found that ALF was induced by LPS/GalN in mice but was alleviated by verapamil through a mechanism that correlated with suppression of the NLRP3 inflammasome pathway. Oxidative stress and inflammatory response were induced by intraperitoneal injection of LPS/GalN, but alleviated with injection of verapamil. Overexpression of NLRP3 via AAV in mouse liver in vivo and in vitro reduced the therapeutic effect of verapamil on LPS/GalN-induced ALF. Taken together, the TXNIP antagonist verapamil could inhibit activation of the NLRP3 inflammasome, inflammatory responses and oxidative stress to alleviate LPS/GalN-induced ALF.     

DJ-1 inhibits microglial activation and protects dopaminergic neurons in vitro and in vivo through interacting with microglial p65

Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by progressive neurodegeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). DJ-1 acts essential roles in neuronal protection and anti-neuroinflammatory response, and its loss of function is tightly associated with a familial recessive form of PD. However, the molecular mechanism of DJ-1 involved in neuroinflammation is largely unclear. Here, we found that wild-type DJ-1, rather than the pathogenic L166P mutant DJ-1, directly binds to the subunit p65 of nuclear factor-κB (NF-κB) in the cytoplasm, and loss of DJ-1 promotes p65 nuclear translocation by facilitating the dissociation between p65 and NF-κB inhibitor α (IκBα). DJ-1 knockout (DJ-1^−/−) mice exhibit more microglial activation compared with wild-type littermate controls, especially in response to lipopolysaccharide (LPS) treatment. In cellular models, knockdown of DJ-1 significantly upregulates the gene expression and increases the release of LPS-treated inflammatory cytokines in primary microglia and BV2 cells. Furthermore, DJ-1 deficiency in microglia significantly enhances the neuronal toxicity in response to LPS stimulus. In addition, pharmacological blockage of NF-κB nuclear translocation by SN-50 prevents microglial activation and alleviates the damage of DA neurons induced by microglial DJ-1 deficiency in vivo and in vitro. Thus, our data illustrate a novel mechanism by which DJ-1 facilitates the interaction between IκBα and p65 by binding to p65 in microglia, and thus repressing microglial activation and exhibiting the protection of DA neurons from neuroinflammation-mediated injury in PD.Subject terms: Cell death in the nervous system, Parkinson''s disease     

Mitochondrial apoptosis is dispensable for NLRP3 inflammasome activation but non‐apoptotic caspase‐8 is required for inflammasome priming

A current paradigm proposes that mitochondrial damage is a critical determinant of NLRP3 inflammasome activation. Here, we genetically assess whether mitochondrial signalling represents a unified mechanism to explain how NLRP3 is activated by divergent stimuli. Neither co‐deletion of the essential executioners of mitochondrial apoptosis BAK and BAX, nor removal of the mitochondrial permeability transition pore component cyclophilin D, nor loss of the mitophagy regulator Parkin, nor deficiency in MAVS affects NLRP3 inflammasome function. In contrast, caspase‐8, a caspase essential for death‐receptor‐mediated apoptosis, is required for efficient Toll‐like‐receptor‐induced inflammasome priming and cytokine production. Collectively, these results demonstrate that mitochondrial apoptosis is not required for NLRP3 activation, and highlight an important non‐apoptotic role for caspase‐8 in regulating inflammasome activation and pro‐inflammatory cytokine levels.     

Repression of TXNIP–NLRP3 axis restores intestinal barrier function via inhibition of myeloperoxidase activity and oxidative stress in nonalcoholic steatohepatitis

Dysfunction of the intestinal barrier function occurs in hepatic injury, but the specific mechanisms responsible are largely unknown. Recently, NOD-like receptor 3 (NLRP3) inflammasome functions in impairing endothelial barrier function. In this study, we test the hypothesis that TXNIP–NLRP3 axis repression prevents against intestinal barrier function disruption in nonalcoholic steatohepatitis (NASH). First, lipopolysaccharide (LPS)-induced alterations in expression of ZO-1 and occludin, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) level, and transepithelial electric resistance (TEER) in intestinal epithelial cells (IECs) isolated from C57BL/6 wild-type (WT) and TXNIP^−/− mice were evaluated. The underlying regulatory mechanisms of TXNIP knockout in vivo were investigated with the detection of expressions of TXNIP, NLRP3 and ZO-1, and occludin, the interaction of TXNIP–NLRP3, MPO activity, ROS level, permeability of intestinal mucosa, levels of inflammatory factors in serum, and LPS concentration. We identified that TXNIP knockout promoted ZO-1 and occludin expression, yet reduced MPO activity, ROS level, and cell permeability in IECs, indicating restored the intestinal barrier function. However, LPS upregulated TXNIP and NLRP3 expression, as well as contributed to the interaction between TXNIP and NLRP3 in vitro. Furthermore, TXNIP was significantly upregulated in the intestinal mucosa of NASH mice and its knockout repaired the intestinal barrier disrupt, inhibited expression of inflammatory factors, and reduced LPS concentration as well as hepatic injury in vivo. Taken together, our findings demonstrated that inhibited the activation of the TXNIP–NLRP3 axis reduced MPO activity and oxidative stress and thus restoring the intestinal barrier function in NASH. TXNIP–NLRP3 axis may be a promising therapeutic strategy for the NASH treatment.     

Tripartite-motif protein 30 negatively regulates NLRP3 inflammasome activation by modulating reactive oxygen species production

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is critical for caspase-1 activation and the proteolytic processing of pro-IL-1β. However, the mechanism that regulates NLRP3 inflammasome activation remains unclear. In this paper, we demonstrate that tripartite-motif protein 30 (TRIM30) negatively regulates NLRP3 inflammasome activation. After stimulation with ATP, an agonist of the NLRP3 inflammasome, knockdown of TRIM30 enhanced caspase-1 activation and increased production of IL-1β in both J774 cells and bone marrow-derived macrophages. Similarly with ATP, knockdown of TRIM30 increased caspase-1 activation and IL-1β production triggered by other NLRP3 inflammasome agonists, including nigericin, monosodium urate, and silica. Production of reactive oxygen species was increased in TRIM30 knockdown cells, and its increase was required for enhanced NLRP3 inflammasome activation, because antioxidant treatment blocked excess IL-1β production. Conversely, overexpression of TRIM30 attenuated reactive oxygen species production and NLRP3 inflammasome activation. Finally, in a crystal-induced NLRP3 inflammasome-dependent peritonitis model, monosodium urate-induced neutrophil flux and IL-1β production was reduced significantly in TRIM30 transgenic mice as compared with that in their nontransgenic littermates. Taken together, our results indicate that TRIM30 is a negative regulator of NLRP3 inflammasome activation and provide insights into the role of TRIM30 in maintaining inflammatory responses.     

NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer’s Disease

The activation of the NLRP3 inflammasome signaling pathway plays an important role in the neuroinflammation in Alzheimer’s disease (AD). In this study, we investigated the effects of JC-124, a rationally designed NLRP3 inflammasome inhibitor, on AD-related deficits in CRND8 APP transgenic mice (TgCRND8). We first demonstrated increased formation and activation of NLRP3 inflammasome in TgCRND8 mice compared to non-transgenic littermate controls, which was inhibited by the treatment with JC-124. Importantly, JC-124 treatment led to decreased levels of Aβ deposition and decreased levels of soluble and insoluble Aβ_1–42 in the brain of CRND8 mice which was accompanied by reduced β-cleavage of APP, reduced activation of microglia but enhanced astrocytosis. Oxidative stress was decreased and synaptophysin was increased in the CRND8 mice after JC-124 treatment, demonstrating a neuroprotective effect. Overall, these data demonstrated beneficial effects of JC-124 as a specific NLRP3 inflammasome inhibitor in AD mouse model and supported the further development of NLRP3 inflammasome inhibitors as a viable option for AD therapeutics.     

Deubiquitination of NLRP3 by BRCC3 Critically Regulates Inflammasome Activity

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Highlights? Inhibition of deubiquitination blocks inflammasome activation ? Inhibition of deubiquitination induces NLRP3 polyubiquitination ? Deubiquitination of NLRP3 regulates the activation of inflammasome ? The deubiquitinating enzyme BRCC3 deubiquitinates the LRR domain of NLRP3     

Peripheral inflammatory mechanisms modulate microglial activation in response to mild impairment of oxidative metabolism

Thiamine deficiency (TD) models the selective neurodegeneration that accompanies the mild impairment of oxidative metabolism, which is observed in a variety of neurodegenerative diseases. Several markers of inflammation accompany neuronal death in TD and in these diseases. Studies in the submedial thalamic nucleus (SmTN), the region most sensitive to TD, have begun to define the temporal response of inflammation, immune response and neurodegeneration. Our previous studies show that the immune response is involved in TD-induced neurodegeneration. The current experiments tested the roles of other inflammatory cascades in TD-induced neuronal death. Deletion of genes for CD4, or CD8 (the co-receptors for T-cells), IFN-gamma (the cytokine produced by T-cell), or NADPH oxidase (the inflammation related oxidase) were tested. None protected against neuronal death in late stages of TD. On the other hand, deletion of the genes for CD4, CD8 and IFN-gamma increased the microglial activation, and deletion of the gene for NADPH oxidase decreased microglial activation when compared to control mice. In wild type mice, TD caused hypertrophy of CD68 positive microglia without increasing the number of microglia. However, TD induced hypertrophy and proliferation of CD68-positive microglia in the CD4 (97%), CD8 (57%) or IFN-gamma (96%) genetic knockout mice. In the genetic knockout mice for NADPH oxidase, the microglial activation was 65% less than the wild type mice. The results demonstrate that mice deficient in specific T cells (CD4-/-, CD8-/-) or activated T cell product, (IFN-gamma-/-) have increased microglia activation, but mice deficient in NADPH oxidase have decreased microglial activation. However, at the time point tested, the deletions were not neuroprotective. The results suggest that inflammatory responses play a role in TD-induced pathological changes in the brain, and the inflammation appears to be a late event that reflects a response to neuronal damage, which may spread the damage to other brain regions.