Tom Schilling

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.

Amyloid-β-induced reactive oxygen species production and priming are differentially regulated by ion channels in microglia

Production of reactive oxygen species (ROS) by microglial cells and subsequent oxidative stress are strongly implicated in the pathogenesis of Alzheimers disease. Although it is recognized that amyloid‐β (Aβ) plays a major role in inducing and regulating microglial ROS production in Alzheimers disease, to date little is known about cellular mechanisms underlying Aβ‐stimulated ROS production. Here, we identified ion channels involved in Aβ‐induced microglial ROS production and in Aβ‐induced microglial priming. Acute stimulation of microglial cells with either fibrillar Aβ1–42 (fAβ1–42) or soluble Aβ1–42 (sAβ1–42) caused significant increases in microglial ROS production, which were abolished by inhibition of TRPV1 cation channels with 5‐iodo‐resiniferatoxin (I‐RTX), but were unaffected by inhibition of K+ channels with charybdotoxin (CTX). Furthermore, pretreatment with either fAβ1–42 or sAβ1–42 induced microglial priming, that is, increased ROS production upon secondary stimulation with the phorbol ester PMA. Microglial priming induced by fAβ1–42 or sAβ1–42 remained unaffected by TRPV1 channel inhibition with I‐RTX. However, sAβ1–42‐induced priming was inhibited by CTX and margatoxin, but not by TRAM‐34 or paxilline, indicating a role of Kv1.3 voltage‐gated K+ channels, but not of Ca2+‐activated K+ channels, in the priming process. In summary, our data suggest that in microglia Aβ‐induced ROS production and priming are differentially regulated by ion channels, and that TRPV1 cation channels and Kv1.3 K+ channels may provide potential therapeutic targets to reduce microglia‐induced oxidative stress in Alzheimers disease. J. Cell. Physiol. 226: 3295–3302, 2011.

Importance of the non-selective cation channel TRPV1 for microglial reactive oxygen species generation

Activated microglial cells generate reactive oxygen species (ROS), which have detrimental effects in neuroinflammatory and neurodegenerative diseases. In the present study, we have identified a novel mechanism involved in microglial NADPH oxidase-mediated ROS production. In PMA-stimulated microglia, ROS production was substantially reduced upon inhibition of the non-selective cation channel TRPV1 with La(3+), ruthenium red, capsazepine and 5-iodo-resinferatoxin. Furthermore, sustained membrane depolarization, a hallmark of NADPH oxidase activity in phagocytes, was found to induce non-selective cation/TRPV1 channel activity in microglia. Together, our data suggest that TRPV1 channels are involved in regulating NADPH oxidase-mediated ROS generation in microglia.

Non-selective cation channel activity is required for lysophosphatidylcholine-induced monocyte migration

Lysophosphatidylcholine (LPC) is a major atherogenic lipid which stimulates the recruitment of monocytes to atherosclerotic lesions. The physiological mechanisms underlying LPC‐induced monocyte migration are poorly understood. Here we demonstrate that LPC activates non‐selective cation channels, which are significantly involved in LPC‐induced chemotaxis of monocytes. External LPC elicited the activation of non‐selective cation currents in THP‐1 monocytes, which occurred in a G protein and phospholipase C‐independent manner. LPC‐activated currents were almost completely inhibited by Gd3+, La3+, and TRAM‐34. Furthermore, currents were partially reduced by either 2‐aminoethoxydiphenyl borate (2‐APB) or ruthenium red, while combined application of 2‐APB and ruthenium red abolished LPC‐activated currents. The 2‐APB‐sensitive current component was potentiated by flufenamic acid and Ca2+‐free extracellular solution, while the ruthenium red‐sensitive current component was abolished by capsazepine. This pharmacological profile suggests that LPC simultaneously activates TRPC6 and TRPV1 channels in monocytes. Furthermore, in the presence of Gd3+, La3+, TRAM‐34, 2‐APB, ruthenium red or capsazepine, LPC‐induced chemotaxis of monocytes was substantially inhibited, indicating that activation of both channel types is required for optimal migration of LPC‐stimulated monocytes. Thus, ion channel inhibition may represent a powerful strategy to attenuate the progression of atherosclerosis by reducing monocyte infiltration. J. Cell. Physiol. 221: 325–334, 2009.

TRPM7 regulates proliferation and polarisation of macrophages

ABSTRACT Ion channels play pivotal roles in regulating important functions of macrophages, such as cytokine and chemokine production, migration, proliferation, phagocytosis and others. In this study, we have identified the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) for the first time in macrophages. TRPM7 activity is differentially regulated in macrophages, i.e. current density in TRPM7 is significantly larger in anti-inflammatory M2-type macrophages than in untreated and in pro-inflammatory M1-type macrophages, whereas mRNA levels of TRPM7 remain unchanged upon cell polarisation. The specific TRPM7 inhibitors NS8593 and FTY720 abolish proliferation of macrophages induced by interleukin-4 (IL-4) and macrophage colony-stimulating factor (M-CSF), respectively, whereas proliferation arrest was not accompanied by induction of apoptosis or necrosis in macrophages. Furthermore, NS8593 and FTY720 prevented polarisation of macrophages towards the anti-inflammatory M2 phenotype. Inhibition of TRPM7 reduced IL-4-induced upregulation of arginase-1 (Arg1) mRNA levels and Arg1 activity, and abolished the inhibitory effects of IL-4 or M-CSF on LPS-induced TNF-&agr; production by macrophages. In summary, our data suggest a main role of TRPM7 in the regulation of macrophage proliferation and polarisation.

Stimulus-dependent requirement of ion channels for microglial NADPH oxidase-mediated production of reactive oxygen species.

Reactive oxygen species (ROS) produced by activated microglial cells play a pivotal role in the pathogenesis of neuro-degenerative and neuro-inflammatory diseases. Here we demonstrate that the pro-inflammatory lipid lysophosphatidylcholine (LPC) is capable of inducing microglial ROS production, which is mediated by the activity of NADPH oxidase. Inhibition of TRPV1 non-selective cation channels abolished ROS production in LPC-stimulated microglia, whereas inhibitors of K(+) channels, H(+) channels and Cl(-) channels had no significant effects. In contrast, activity of all four ion channel types was required for PMA-induced NADPH oxidase-mediated ROS generation, suggesting a differential, stimulus-dependent regulation of microglial ROS production by ion channel activity.

Lysophosphatidylcholine- and MCP-1-induced chemotaxis of monocytes requires potassium channel activity

One of the earliest cellular responses in atherogenesis is the focal recruitment of circulating monocytes, while the most important atherogenic chemoattractants are monocyte chemoattractant protein-1 (MCP-1) and lysophosphatidylcholine (LPC). Invading monocytes transform into activated macrophages and foam cells, which stimulate inflammatory processes and promote atherosclerosis. In this study, we have searched for common mechanisms involved in MCP-1- and LPC-stimulated monocyte migration. We have found that migration of THP-1 monocytes stimulated with MCP-1 was reduced upon inhibition of Gi/o proteins with pertussis toxin and upon inhibition of platelet activating factor receptors with BN52021, whereas LPC-stimulated monocyte chemotaxis remained unaffected by both inhibitors. Furthermore, Cl− channels were only required for MCP-1-induced chemotaxis. However, activity of voltage-gated K+ channels and of Ca2+-activated K+ channels was found to be involved in migration of monocytes stimulated with either MCP-1 or LPC. Inhibition of voltage-gated K+ channels with 4-aminopyridine or margatoxin partially inhibited MCP-1- and LPC-stimulated migration of monocytes. Blockade of Ca2+-activated K+ channels with TRAM-34 also partially reduced migration of MCP-1- and LPC-stimulated monocytes. Simultaneous inhibition of voltage-gated and Ca2+-activated K+ channels abolished MCP-1- and LPC-induced chemotaxis of monocytes. Thus, K+ channel inhibition may represent a novel powerful strategy to reduce monocyte infiltration and subsequent inflammation in atherosclerosis.

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.

Importance of lipid rafts for lysophosphatidylcholine-induced caspase-1 activation and reactive oxygen species generation

Lipid rafts play an important role in regulating cellular processes and functions. Here, we demonstrate that in microglia stimulated with the pro-inflammatory lipid lysophosphatidylcholine (LPC), caspase-1 activation and NADPH oxidase activity depend on intact lipid rafts. Disruption of lipid rafts with methyl-β-cyclodextrin, fumonisin B1 or nystatin prevented LPC-stimulated caspase-1 activation and reactive oxygen species (ROS) production, whereas LPC-induced Na(+) influx remained unaffected. Since ROS regulate caspase-1 activity in LPC-stimulated microglia, the effects of lipid raft-disrupting agents on caspase-1 activation can be related to their inhibition of NADPH oxidase-mediated ROS production.

Sodium dependence of lysophosphatidylcholine-induced caspase-1 activity and reactive oxygen species generation.

The proinflammatory cytokines interleukin (IL)-1β and IL-18 play pivotal roles in neuroinflammatory diseases. Caspase-1-mediated proteolytic cleavage is required to convert the premature, biologically inactive cytokines to their biologically active forms capable of promoting tissue inflammation. Although caspases have been recognized as potential therapeutic targets in inflammatory diseases, mechanisms regulating caspase-1 activation are not fully understood. Here we demonstrate that the proinflammatory lipid lysophosphatidylcholine (LPC) initiates microglial caspase-1 activation in a Na(+)-dependent manner. LPC-induced caspase-1 activity was almost completely inhibited upon omission of extracellular Na(+), but was unaffected by inhibition of Na(+)/K(+)-ATPase with ouabain or by inhibition of Na(+)/H(+) antiport with amiloride. Inhibition of caspase-1-mediated IL-1β processing by Na(+)-free medium led to reduced amounts of mature IL-1β released from LPC-stimulated microglia. Furthermore, LPC-induced production of reactive oxygen species (ROS) was abolished by Na(+)-free medium, indicating Na(+) dependence of NADPH oxidase activity in LPC-stimulated microglia. Since ROS production was found to be crucial to caspase-1 activation in LPC-stimulated microglia, the Na(+) dependence of caspase-1 can be related to the Na(+) dependence of NADPH oxidase. In summary, it is suggested that in LPC-activated microglia, Na(+) influx is required for the production of NADPH oxidase-mediated ROS, which subsequently stimulate caspase-1 activity.