Redox Regulation of Microglial Inflammatory Response: Fine Control of NLRP3 Inflammasome through Nrf2 and NOX4.

The role of inflammation and immunity in the pathomechanism of neurodegenerative diseases has become increasingly relevant within the past few years. In this context, the NOD-like receptor protein 3 (NLRP3) inflammasome plays a crucial role in the activation of inflammatory responses by promoting the maturation and secretion of pro-inflammatory cytokines such as interleukin-1ß and interleukin-18. We hypothesized that the interplay between nuclear factor erythroid 2-related factor 2 (Nrf2) and NADPH oxidase 4 (NOX4) may play a critical role in the activation of the NLRP3 inflammasome and subsequent inflammatory responses. After priming mixed glial cultures with lipopolysaccharide (LPS), cells were stimulated with ATP, showing a significant reduction of IL1-ß release in NOX4 and Nrf2 KO mice. Importantly, NOX4 inhibition using GKT136901 also reduced IL-1ß release, as in NOX4 KO mixed glial cultures. Moreover, we measured NOX4 and NLRP3 expression in wild-type mixed glial cultures following LPS treatment, observing that both increased after TLR4 activation, while 24 h treatment with tert-butylhydroquinone, a potent Nrf2 inducer, significantly reduced NLRP3 expression. LPS administration resulted in significant cognitive impairment compared to the control group. Indeed, LPS also modified the expression of NLRP3 and NOX4 in mouse hippocampus. However, mice treated with GKT136901 after LPS impairment showed a significantly improved discrimination index and recovered the expression of inflammatory genes to normal levels compared with wild-type animals. Hence, we here validate NOX4 as a key player in NLRP3 inflammasome activation, suggesting NOX4 pharmacological inhibition as a potent therapeutic approach in neurodegenerative diseases.

Randomized controlled trial on healthy volunteers of pharmacokinetic and antimicrobial activity of a novel hydrogel-containing chlorhexidine dressing to prevent catheter-related bloodstream infection.

Introduction: Catheter-related blood stream infection (CRBSI) is one of the most relevant complications associated to the use of intravascular catheters. In this context, chlorhexidine gluconate (CHG) releasing dressings have been developed to reduce the catheter colonization rate and the risk of infection. The aim of this study is to analyze the release rate of CHG and the antimicrobial activity of a novel CHG-releasing dressing, Oper film® protect CHG, and to compare these parameters to those of the dressing Tegaderm™ CHG in healthy volunteers. Methods: The study was performed in a cohort of 25 healthy volunteers. Two commercially available chlorhexidine-containing dressings were evaluated and compared in this study, Oper film® protect CHG and Tegaderm™ CHG. The release of CHG and the antimicrobial capacity was determined for one week. Results: HPLC analysis revealed that both dressings have an equivalent CHG release to the skin 2 days (Oper film® protect CHG, 321 µg/cm2; Tegaderm™ CHG, 279 µg/cm2) and 7 days (Oper film® protect CHG, 456 µg/cm2; Tegaderm™ CHG, 381 µg/cm2) after the placement of the products in the non-disinfected back of the subjects. On the other hand, Oper film® protect CHG and Tegaderm™ CHG similarly reduced colony forming units (CFU) in cultures obtained from the skin under the CHG-containing hydrogel compared to control cultures at both 2 days (control, 3.34 log10 cfu/cm2; Oper film® protect CHG, 0.64 log10 cfu/cm2; Tegaderm™ CHG, 0.7 log10 cfu/cm2) and 7 days (control, 3.95 log10 cfu/cm2; Oper film® protect CHG, 0.11 log10 cfu/cm2; Tegaderm™ CHG, 1 log10 cfu/cm2). Discussion: Data confirm that the recent commercially available dressing Oper film® protect CHG maintains the release of CHG and the antimicrobial activity during at least 7 days, and possesses equivalent drug release and antimicrobial action to Tegaderm™ CHG.

Activation of NLRP3 Is Required for a Functional and Beneficial Microglia Response after Brain Trauma.

Despite the numerous research studies on traumatic brain injury (TBI), many physiopathologic mechanisms remain unknown. TBI is a complex process, in which neuroinflammation and glial cells play an important role in exerting a functional immune and damage-repair response. The activation of the NLRP3 inflammasome is one of the first steps to initiate neuroinflammation and so its regulation is essential. Using a closed-head injury model and a pharmacological (MCC950; 3 mg/kg, pre- and post-injury) and genetical approach (NLRP3 knockout (KO) mice), we defined the transcriptional and behavioral profiles 24 h after TBI. Wild-type (WT) mice showed a strong pro-inflammatory response, with increased expression of inflammasome components, microglia and astrocytes markers, and cytokines. There was no difference in the IL1ß production between WT and KO, nor compensatory mechanisms of other inflammasomes. However, some microglia and astrocyte markers were overexpressed in KO mice, resulting in an exacerbated cytokine expression. Pretreatment with MCC950 replicated the behavioral and blood-brain barrier results observed in KO mice and its administration 1 h after the lesion improved the damage. These findings highlight the importance of NLRP3 time-dependent activation and its role in the fine regulation of glial response.

Synthesis and Pharmacological Evaluation of New N-Sulfonylureas as NLRP3 Inflammasome Inhibitors: Identification of a Hit Compound to Treat Gout.

NLRP3 is involved in the pathophysiology of several inflammatory diseases. Therefore, there is high current interest in the clinical development of new NLRP3 inflammasome small inhibitors to treat these diseases. Novel N-sulfonylureas were obtained by the replacement of the hexahydroindacene moiety of the previously described NLRP3 inhibitor MCC950. These new derivatives show moderate to high potency in inhibiting IL-1ß release in vitro. The greatest effect was observed for compound 4b, which was similar to MCC950. Moreover, compound 4b was able to reduce caspase-1 activation, oligomerization of ASC, and therefore, IL-1ß processing. Additional in silico predictions confirmed the safety profile of compound 4b, and in vitro studies in AML12 hepatic cells confirmed the absence of toxicological effects. Finally, we evaluated in vivo anti-inflammatory properties of compound 4b, which showed a significant anti-inflammatory effect and reduced mechanical hyperalgesia at 3 and 10 mg/kg (i.p.) in an in vivo mouse model of gout.

Time-dependent dual effect of NLRP3 inflammasome in brain ischaemia.

BACKGROUND: Inflammasomes are cytosolic multiprotein complexes which, upon assembly, activate the maturation and secretion of the inflammatory cytokines IL-1ß and IL-18. However, participation of the NLRP3 inflammasome in ischaemic stroke remains controversial. Our aims were to determine the role of NLRP3 in brain ischaemia, and explore the mechanism involved in the potential protective effect of the neurovascular unit. METHODS: WT and NLRP3 knock-out mice were subjected to ischaemia by middle cerebral artery occlusion (60 min) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. RESULTS: We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1ß and TNF-α), NLRP3 inflammasome components (NLRP3 and pro-caspase-1), protease expression (MMP9), and endothelial adhesion molecules (ICAM and VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junction proteins (ZO-1 and Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. CONCLUSIONS: These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischaemia with potential clinical translation.

Serum Amyloid A1/Toll-Like Receptor-4 Axis, an Important Link between Inflammation and Outcome of TBI Patients.

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide without any validated biomarker or set of biomarkers to help the diagnosis and evaluation of the evolution/prognosis of TBI patients. To achieve this aim, a deeper knowledge of the biochemical and pathophysiological processes triggered after the trauma is essential. Here, we identified the serum amyloid A1 protein-Toll-like receptor 4 (SAA1-TLR4) axis as an important link between inflammation and the outcome of TBI patients. Using serum and mRNA from white blood cells (WBC) of TBI patients, we found a positive correlation between serum SAA1 levels and injury severity, as well as with the 6-month outcome of TBI patients. SAA1 levels also correlate with the presence of TLR4 mRNA in WBC. In vitro, we found that SAA1 contributes to inflammation via TLR4 activation that releases inflammatory cytokines, which in turn increases SAA1 levels, establishing a positive proinflammatory loop. In vivo, post-TBI treatment with the TLR4-antagonist TAK242 reduces SAA1 levels, improves neurobehavioral outcome, and prevents blood-brain barrier disruption. Our data support further evaluation of (i) post-TBI treatment in the presence of TLR4 inhibition for limiting TBI-induced damage and (ii) SAA1-TLR4 as a biomarker of injury progression in TBI patients.

TLR4 pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury.

BACKGROUND AND PURPOSE: Activation of astrocytes contributes to synaptic remodelling, tissue repair and neuronal survival following traumatic brain injury (TBI). The mechanisms by which these cells interact to resident/infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI. EXPERIMENTAL APPROACH: To determine how functional astrocyte alterations induced by activation of TLR4 pathway in inflammatory cells regulate synapses and neurovascular integrity after TBI, we used pharmacology, genetic approaches, live calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioural methods. KEY RESULTS: Shortly after a TBI, there is a recruitment of excitable and reactive astrocytes mediated by TLR4 pathway activation with detrimental effects on post-synaptic density-95 (PSD-95)/vesicular glutamate transporter 1 (VGLUT1) synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4 pathway with resatorvid (TAK-242) partially reversed many of the observed effects. Synapses and BBB recovery after resatorvid administration were not observed in IP3 R2-/- mice, indicating that effects of TLR4 inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that TLR4-mediated signalling, most probably through microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.

Melatonin Reduces NLRP3 Inflammasome Activation by Increasing α7 nAChR-Mediated Autophagic Flux.

Microglia controls the immune system response in the brain. Specifically, the activation and dysregulation of the NLRP3 inflammasome is responsible for the initiation of the inflammatory process through IL-1ß and IL-18 release. In this work, we have focused on studying the effect of melatonin on the regulation of the NLRP3 inflammasome through α7 nicotinic receptor (nAChR) and its relationship with autophagy. For this purpose, we have used pharmacological and genetic approaches in lipopolysaccharide (LPS)-induced inflammation models in both in vitro and in vivo models. In the BV2 cell line, LPS inhibited autophagy, which increased NLRP3 protein levels. However, melatonin promoted an increase in the autophagic flux. Treatment of glial cultures from wild-type (WT) mice with LPS followed by extracellular adenosine triphosphate (ATP) produced the release of IL-1ß, which was reversed by melatonin pretreatment. In cultures from α7 nAChR knock-out (KO) mice, melatonin did not reduce IL-1ß release. Furthermore, melatonin decreased the expression of inflammasome components and reactive oxygen species (ROS) induced by LPS; co-incubation of melatonin with α-bungarotoxin (α-bgt) or luzindole abolished the anti-inflammatory and antioxidant effects. In vivo, melatonin reverted LPS-induced cognitive decline, reduced NLRP3 levels and promoted autophagic flux in the hippocampi of WT mice, whereas in α7 nAChR KO mice melatonin effect was not observed. These results suggest that melatonin may modulate the complex interplay between α7 nAChR and autophagy signaling.

In Vitro and In Silico ADME-Tox Profiling and Safety Significance of Multifunctional Monoamine Oxidase Inhibitors Targeting Neurodegenerative Diseases.

Herein we report in vitro metabolic stability in human liver microsomes (HLMs), interactions with cytochrome P450 isoenzymes (CYP3A4, CYP2D6, and CYP2C9), and cytotoxicity analyses on HEK-293, HepG2, Huh7, and WTIIB cell lines of our most recent multitarget directed ligands PF9601N, ASS234, and contilisant. Based on these results, we conclude that (1) PF9601N and contilisant are metabolically stable in the HLM assay, in contrast to the very unstable ASS234; (2) CYP3A4 activity was decreased by PF9601N at all the tested concentrations and by ASS234 and contilisant only at the highest concentration; CYP2D6 activity was reduced by ASS234 at 1, 10, and 25 µM and by PF9601N at 10 and 25 µM, whereas contilisant increased its activity at the same concentrations; CYP2C9 was inhibited by the three compounds; (3) contilisant did not affect cell viability in the widest range of concentrations: up to 10 µM on HEK-293 cells, up to 30 µM on Huh7 cells, up to 50 µM on HepG2 cells, and up to 30 or 100 µM on WTIIB cells. Based on these results, we selected contilisant as a metabolically stable and nontoxic lead compound for further studies in Alzheimer's disease therapy.

Melatonin's efficacy in stroke patients; a matter of dose? A systematic review.

There is a lack of effective therapies for stroke patients; its treatment is even more difficult considering the unexpected onset of the disease. In the last decade, melatonin has emerged as a promising neuroprotective agent which is able to cross the blood-brain-barrier (BBB) and with a low toxicity profile. The aim of this systematic review was to summarize and critically review clinical and pre-clinical evidence related to melatonin's effectiveness as a stroke treatment. Together with a comparative dose extrapolation with those used in the selected randomized controlled trials (RCTs), and based on these data to discuss whether the administered doses correlate with those advisable in human patients. To address this purpose, we performed a systematic review of the available literature. A total of 529 records were screened with the selecting of six full articles containing RCTs that met the inclusion/exclusion criteria. The evidence drawn from these six reports was analyzed to identify remaining gaps, treatment efficacy, and to suggest future directions. The primary outcome reported was the reduction of the oxidative response; the secondary outcome was the increase of the survival rate of the patients in the intervention groups. Calculations derived from animal studies revealed that the translational doses to humans were substantially higher than those employed in the RCTs. The findings of this systematic review revealed that there are insufficient RCTs to prove melatonin's value in stroke patients. Nevertheless, the evidence is promising, and further clinical research may support the benefits of melatonin in stroke patients, if the adequate dose is administered.

Early toll-like receptor 4 blockade reduces ROS and inflammation triggered by microglial pro-inflammatory phenotype in rodent and human brain ischaemia models.

BACKGROUND AND PURPOSE: Ischaemic stroke is a leading cause of death, disability, and a high unmet medical need. Post-reperfusion inflammation and an up-regulation of toll-like receptor 4 (TLR4), an upstream sensor of innate immunity, are associated with poor outcome in stroke patients. Here, we identified the therapeutic effect of targeting the LPS/TLR4 signal transduction pathway. EXPERIMENTAL APPROACH: We tested the effect of the TLR4 inhibitor, eritoran (E5564) in different in vitro ischaemia-related models: human organotypic cortex culture, rat organotypic hippocampal cultures, and primary mixed glia cultures. We explored the therapeutic window of E5564 in the transient middle cerebral artery occlusion model of cerebral ischaemia in mice. KEY RESULTS: In vivo, administration of E5564 1 and 4 hr post-ischaemia reduced the expression of different pro-inflammatory chemokines and cytokines, infarct volume, blood-brain barrier breakdown, and improved neuromotor function, an important clinically relevant outcome. In the human organotypic cortex culture, E5564 reduced the activation of microglia and ROS production evoked by LPS. CONCLUSION AND IMPLICATIONS: TLR4 signalling has a causal role in the inflammation associated with a poor post-stroke outcome. Importantly, its inhibition by eritoran (E5564) provides neuroprotection both in vitro and in vivo, including in human tissue, suggesting a promising new therapeutic approach for ischaemic stroke.

Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis-mediated cell death.

Acute kidney injury is a common complication of rhabdomyolysis. A better understanding of this syndrome may be useful to identify novel therapeutic targets because there is no specific treatment so far. Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death that is involved in renal injury. In this study, we investigated whether ferroptosis is associated with rhabdomyolysis-mediated renal damage, and we studied the therapeutic effect of curcumin, a powerful antioxidant with renoprotective properties. Induction of rhabdomyolysis in mice increased serum creatinine levels, endothelial damage, inflammatory chemokines, and cytokine expression, alteration of redox balance (increased lipid peroxidation and decreased antioxidant defenses), and tubular cell death. Treatment with curcumin initiated before or after rhabdomyolysis induction ameliorated all these pathologic and molecular alterations. Although apoptosis or receptor-interacting protein kinase (RIPK)3-mediated necroptosis were activated in rhabdomyolysis, our results suggest a key role of ferroptosis. Thus, treatment with ferrostatin 1, a ferroptosis inhibitor, improved renal function in glycerol-injected mice, whereas no beneficial effects were observed with the pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone or in RIPK3-deficient mice. In cultured renal tubular cells, myoglobin (Mb) induced ferroptosis-sensitive cell death that was also inhibited by curcumin. Mechanistic in vitro studies showed that curcumin reduced Mb-mediated inflammation and oxidative stress by inhibiting the TLR4/NF-κB axis and activating the cytoprotective enzyme heme oxygenase 1. Our findings are the first to demonstrate the involvement of ferroptosis in rhabdomyolysis-associated renal damage and its sensitivity to curcumin treatment. Therefore, curcumin may be a potential therapeutic approach for patients with this syndrome.-Guerrero-Hue, M., García-Caballero, C., Palomino-Antolín, A., Rubio-Navarro, A., Vázquez-Carballo, C., Herencia, C., Martín-Sanchez, D., Farré-Alins, V., Egea, J., Cannata, P., Praga, M., Ortiz, A., Egido, J., Sanz, A. B., Moreno, J. A. Curcumin reduces renal damage associated with rhabdomyolysis by decreasing ferroptosis-mediated cell death.

Multitarget-Directed Ligands Combining Cholinesterase and Monoamine Oxidase Inhibition with Histamine H3 R Antagonism for Neurodegenerative Diseases.

The therapy of complex neurodegenerative diseases requires the development of multitarget-directed drugs (MTDs). Novel indole derivatives with inhibitory activity towards acetyl/butyrylcholinesterases and monoamine oxidases A/B as well as the histamine H3 receptor (H3R) were obtained by optimization of the neuroprotectant ASS234 by incorporating generally accepted H3R pharmacophore motifs. These small-molecule hits demonstrated balanced activities at the targets, mostly in the nanomolar concentration range. Additional in vitro studies showed antioxidative neuroprotective effects as well as the ability to penetrate the blood-brain barrier. With this promising in vitro profile, contilisant (at 1 mg kg-1 i.p.) also significantly improved lipopolysaccharide-induced cognitive deficits.

Targeting Nrf2 in Protection Against Renal Disease.

BACKGROUND: Renal disease is a serious health problem, with increasing incidence and prevalence. Oxidative stress and inflammation play a key role in the pathogenesis and progression of renal disease. Therefore, therapeutic approaches to decrease oxidative stress should be of interest. OBJECTIVE: This review aims to provide a comprehensive and updated overview of the protective mechanisms mediated by Nrf2 (nuclear factor erythroid 2-related factor 2), a description of novel compounds that target Nrf2, its effectiveness to prevent renal disease and the on-going clinical trials for this pathological condition. METHODS: We undertook a structured search of bibliographic databases for peer-reviewed research in literature about Nrf2 activators and renal disease. RESULTS: The transcription factor Nrf2 is an emerging regulator of cellular resistance to oxidants and inflammation. Nrf2 controls the basal and induced expression of a couple of cytoprotective and antiinflammatory genes that regulate the physiological and pathophysiological outcomes of oxidant exposure. We have analyzed numerous findings showing that Nrf2 induction protects against oxidative stress and modulates inflammation in acute kidney injury and chronic kidney disease progression. However, few clinical trials have been performed in humans. Recent studies suggested that renoprotective effects of Nrf2 activation are observed at low doses, whereas harmful effects appear at higher concentrations. CONCLUSION: The findings of this review confirm that novel studies are necessary to address whether Nrf2-targeting may be a safe therapeutic approach to decrease renal disease progression in humans.

Melatonin protects against oxygen and glucose deprivation by decreasing extracellular glutamate and Nox-derived ROS in rat hippocampal slices.

Therapeutic interventions on pathological processes involved in the ischemic cascade, such as oxidative stress, neuroinflammation, excitotoxicity and/or apoptosis, are of urgent need for stroke treatment. Melatonin regulates a large number of physiological actions and its beneficial properties have been reported. The aim of this study was to investigate whether melatonin mediates neuroprotection in rat hippocampal slices subjected to oxygen-glucose-deprivation (OGD) and glutamate excitotoxicity. Thus, we describe here that melatonin significantly reduced the amount of lactate dehydrogenase released in the OGD-treated slices, reverted neuronal injury caused by OGD-reoxygenation in CA1 and CA3 hippocampal regions, restored the reduction of GSH content of the hippocampal slices induced by OGD, and diminished the oxidative stress produced in the reoxygenation period. Furthermore, melatonin afforded maximum protection against glutamate-induced toxicity and reversed the glutamate released almost basal levels, at 10 and 30µM concentration, respectively. Consequently, we propose that melatonin might strongly and positively influence the outcome of brain ischemia/reperfusion.