Jack Rivers-Auty

Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer’s disease in rodent models

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase-1 (COX-1) and COX-2 enzymes. The NLRP3 inflammasome is a multi-protein complex responsible for the processing of the proinflammatory cytokine interleukin-1β and is implicated in many inflammatory diseases. Here we show that several clinically approved and widely used NSAIDs of the fenamate class are effective and selective inhibitors of the NLRP3 inflammasome via inhibition of the volume-regulated anion channel in macrophages, independently of COX enzymes. Flufenamic acid and mefenamic acid are efficacious in NLRP3-dependent rodent models of inflammation in air pouch and peritoneum. We also show therapeutic effects of fenamates using a model of amyloid beta induced memory loss and a transgenic mouse model of Alzheimers disease. These data suggest that fenamate NSAIDs could be repurposed as NLRP3 inflammasome inhibitors and Alzheimers disease therapeutics.

The effect of CSE gene deletion in caerulein-induced acute pancreatitis in the mouse

Hydrogen sulfide (H2S) has been reported to be involved in the signaling of the inflammatory response; however, there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine whether endogenously synthesized H2S via cystathionine-γ-lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein-induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild-type (WT) and CSE knockout (KO) mice following 10 hourly administrations of 50 μg/kg caerulein or saline control. From this, we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury compared with the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines, as well as reduced acinar cell NF-κB activation in the CSE KO mice compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide-synthesizing activity in caerulein-induced pancreatitis compared with the saline control. When comparing the two saline-treated control groups, we noted that the CSE KO mice showed significantly less pancreatic H2S-synthesizing activity relative to the WT mice. These results indicate that endogenous H2S generated by CSE plays a key proinflammatory role via NF-κB activation in caerulein-induced pancreatitis, and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.

Inflammasomes as therapeutic targets for Alzheimer's disease

Alzheimers disease is the most common form of progressive dementia, typified initially by short term memory deficits which develop into a dramatic global cognitive decline. The classical hall marks of Alzheimers disease include the accumulation of amyloid oligomers and fibrils, and the intracellular formation of neurofibrillary tangles of hyperphosphorylated tau. It is now clear that inflammation also plays a central role in the pathogenesis of the disease through a number of neurotoxic mechanisms. Microglia are the key immune regulators of the CNS which detect amyloidopathy through cell surface and cytosolic pattern recognition receptors (PRRs) and respond by initiating inflammation through the secretion of cytokines such as interleukin‐1β (IL‐1β). Inflammasomes, which regulate IL‐1β release, are formed following activation of cytosolic PRRs, and using genetic and pharmacological approaches, NLRP3 and NLRP1 inflammasomes have been found to be integral in pathogenic neuroinflammation in animal models of Alzheimers disease. Therefore, the inflammasomes are very promising novel pharmacological targets which merit further research in the continued endeavor for efficacious therapeutics for Alzheimers disease.

Boron-Based Inhibitors of the NLRP3 Inflammasome

Summary NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimers disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics.

Elevation of brain glucose and polyol-pathway intermediates with accompanying brain-copper deficiency in patients with Alzheimer’s disease: metabolic basis for dementia

Impairment of brain-glucose uptake and brain-copper regulation occurs in Alzheimer’s disease (AD). Here we sought to further elucidate the processes that cause neurodegeneration in AD by measuring levels of metabolites and metals in brain regions that undergo different degrees of damage. We employed mass spectrometry (MS) to measure metabolites and metals in seven post-mortem brain regions of nine AD patients and nine controls, and plasma-glucose and plasma-copper levels in an ante-mortem case-control study. Glucose, sorbitol and fructose were markedly elevated in all AD brain regions, whereas copper was correspondingly deficient throughout (all P < 0.0001). In the ante-mortem case-control study, by contrast, plasma-glucose and plasma-copper levels did not differ between patients and controls. There were pervasive defects in regulation of glucose and copper in AD brain but no evidence for corresponding systemic abnormalities in plasma. Elevation of brain glucose and deficient brain copper potentially contribute to the pathogenesis of neurodegeneration in AD.

Potassium efflux fires the canon: Potassium efflux as a common trigger for canonical and noncanonical NLRP3 pathways

Murine caspase‐11 and its human orthologues, caspase‐4 and caspase‐5, activate an inflammatory response following cytoplasmic recognition of cell wall constituents from Gram‐negative bacteria, such as LPS. This inflammatory response involves pyroptotic cell death and the concomitant release of IL‐1α, as well as the production of IL‐1β and IL‐18 through the noncanonical NLR family, pyrin domain containing 3 (NLRP3) pathway. This commentary discusses three papers in this issue of the European Journal of Immunology that advance our understanding of the roles of caspase‐11, ‐4, and ‐5 in the noncanonical pathway. By utilizing the new gene editing technique, clustered regularly interspaced short palindromic repeats (CRISPR), as well as sensitive cell imaging techniques, these papers establish that cytoplasmic LPS‐dependent IL‐1β production requires the NLRP3 inflammasome and that its activation is dependent on K+ efflux, whereas IL‐1α release and pyroptotic cell death pathways are NLRP3‐independent. These findings expand on previous research implicating K+ efflux as the principal trigger for NLRP3 activation and suggest that canonical and noncanonical NLRP3 pathways are not as dissimilar as first thought.

Inhibition of hydrogen sulfide production by gene silencing attenuates inflammatory activity of LPS-activated RAW264.7 cells

Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine γ-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.

Mitochondrial Abnormalities and Synaptic Loss Underlie Memory Deficits Seen in Mouse Models of Obesity and Alzheimer’s Disease

Obesity is associated with impaired memory in humans, and obesity induced by high-fat diets leads to cognitive deficits in rodents and in mouse models of Alzheimer’s disease (AD). However, it remains unclear how high-fat diets contribute to memory impairment. Therefore, we tested the effect of a high-fat diet on memory in male and female control non-transgenic (Non-Tg) and triple-transgenic AD (3xTgAD) mice and determined if a high-fat diet caused similar ultrastructural abnormalities to those observed in AD. Behavior was assessed in mice on control or high-fat diet at 4, 8, or 14 months of age and ultrastructural analysis at 8 months of age. A high-fat diet increased body weight, fat weight, and insulin levels with some differences in these metabolic responses observed between Non-Tg and 3xTgAD mice. In both sexes, high-fat feeding caused memory impairments in Non-Tg mice and accelerated memory deficits in 3xTgAD mice. In 3xTgAD mice, changes in hippocampal mitochondrial morphology were observed in capillaries and brain neuropil that were accompanied by a reduction in synapse number. A high-fat diet also caused mitochondria abnormalities and a reduction in synapse number in Non-Tg mice, but did not exacerbate the changes seen in 3xTgAD mice. Our data demonstrate that a high-fat diet affected memory in Non-Tg mice and produced similar impairments in mitochondrial morphology and synapse number comparable to those seen in AD mice, suggesting that the detrimental effects of a high-fat diet on memory might be due to changes in mitochondrial morphology leading to a reduction in synaptic number.

The cannabinoid CB2 receptor agonist GW405833 does not ameliorate brain damage induced by hypoxia-ischemia in rats

The cannabinoid CB2 receptor has been under investigation as a potential target for neuroprotection with the suppression of neuroinflammation as the proposed mechanism of action. Several studies have now reported that CB2 agonists are neuroprotective in models of cerebral ischemia. However, these studies have tended to measure brain infarctions in rodents 1-3 days after drug administration and have not assessed behavioral outcomes. As it has been shown that apparent protection soon after injury is not necessarily correlated with improved outcome after several weeks, we tested the CB2 selective agonist GW405833 in a model of cerebral hypoxia-ischemia, and assessed histological and behavioral outcomes 15 days after injury. Many putatively neuroprotective drugs have failed to translate from promising preclinical results to clinical success. We designed our experiments to not only stringently test CB2 mediated neuroprotection, but also to test several drug administration regimens to maximize the chance of detecting any therapeutic effect. However, GW405833 failed to provide neuroprotection in any of our experiments. These results challenge how far the results of earlier studies into CB2 mediated neuroprotection as measured at early time points may be extrapolated to later time points or to other models.

Salmonella typhimurium -induced IL-1 release from primary human monocytes requires NLRP3 and can occur in the absence of pyroptosis

Large molecular complexes known as inflammasomes regulate the release of IL-1β from immune cells in response to infection and injury. Salmonella typhimurium infection is reported to activate NLRP3 and NLRC4 inflammasomes which are subsequently involved in pyroptosis of the cell and pathogen clearance. However, the response to S. typhimurium in primary human monocytes has not been studied in detail. The aim of this study was to investigate the effect of S. typhimurium on inflammasomes in primary human monocytes. Much of the previous research in the field has been conducted in murine models and human THP-1 cells, which may not reflect the responses of primary human monocytes. Here, we report that inhibiting NLRP3 with the selective inhibitor MCC950, blocked release of IL-1β and the related cytokine IL-1α from primary human monocytes in response to S. typhimurium. Additionally, under these conditions S. typhimurium-induced IL-1 release occurred independently of pyroptosis. We propose that IL-1β release without pyroptosis may occur in early-recruited monocytes to regulate a maximal innate immune response to Salmonella infection, allowing a sustained inflammatory signal. This insight into the mechanisms involved in IL-1 release from primary human monocytes highlights major differences between immune cell types, and the defences they employ during bacterial infection.