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 world-wide 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 patientsThis work was supported by grants from Fundación Mutua Madrileña and Fondo de Investigaciones Sanitarias (FIS) (ISCIII/FEDER) (Programa Miguel Servet CP14/00008; CPII19/00005; PI16/00735; PI19/00082) to JE, RYC2019-026870-I to JMR and PI18/01387 to A

Multitarget-directed ligands combining cholinesterase and monoamine oxidase inhibition with histamine H3R antagonism for neurodegenerative diseases

J.M.C. thanks MINECO (SAF2012-33304 and SAF2015-65586-R). J.M.C., F.L.M., and A.R. thank UCJC for grants 2015-12, 2014-35, and 2015-21, respectively. J.E. thanks the Fondo de Investigaciones Sanitarias (FIS) (ISCIII/FEDER) (Programa Miguel Servet: CP14/00008 and PI16/00735) and Fundación Mutua Madrileña. O.S. and J.J. thank MHCZ-DRO (UHHK 00179906) for support. R.R.R., H.S., and J.M.C. acknowledge the EU COST Actions CM1103 and CM15135. E.P. and H.S. thank the German Research Foundation (DFG; PRO 1405/2-2, PRO 1405/4-1, SFB 1039 A07, and INST208/664-1).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.PostprintPeer reviewe

Validación de biomarcadores y dianas terapéuticas de origen inflamatorio en traumatismo craneoencefálico

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología. Fecha de Lectura: 18-06-2021Esta tesis tiene embargado el acceso al texto completo hasta el 18-12-2022El traumatismo craneoencefálico (TCE) es una de las principales causas de mortalidad y discapacidad en el mundo, especialmente en la población adulta joven. En los últimos años, su incidencia ha crecido considerablemente. La complejidad de la lesión, sumado a la gran heterogeneidad y variabilidad entre los sujetos que lo padecen, complica conocer con exactitud los mecanismos fisiopatológicos subyacentes que se desencadenan tras el trauma craneal. Una de las principales consecuencias es la inflamación cerebral descontrolada, que sostenida en el tiempo puede conducir a neurodegeneración, por lo que la inflamación juega un papel crucial en la recuperación de las secuelas sufridas tras TCE. Actualmente no existe ningún biomarcador o conjunto de biomarcadores validado para conocer la evolución o pronóstico de los pacientes que han sufrido TCE. En la misma línea, tampoco se han instaurado tratamientos para limitar los mecanismos fisiopatológicos más allá de los focalizados en reducir la presión intracraneal. Por lo tanto, debido a la necesidad clínica existente, en esta Tesis Doctoral hemos validado biomarcadores de pronóstico y dianas terapéuticas de origen inflamatorio en TCE. En una cohorte de pacientes que han sufrido TCE se observó que la proteína amiloide sérica A1 (SAA1) analizada en el suero 3 días después de la lesión tiene un buen valor predictivo del pronóstico; no obstante, su combinación con S100B medida el primer día post-TCE mejoró considerablemente la capacidad pronóstica. En el modelo murino de trauma craneal se evaluó la expresión de SAA1, su contribución a la progresión de la lesión y la modulación de la inflamación con el antagonismo TLR4 como terapia farmacológica en TCE. Se detectó que el antagonismo TLR4 es capaz de reducir los niveles séricos y cerebrales de SAA1. Asimismo, esta estrategia terapéutica produjo una reducción de la activación de la glía, evitó la pérdida de sinapsis glutamatérgicas, mejoró la funcionalidad de la barrera hematoencefálica (BHE) y propició la recuperación de la función neuromotora de los ratones. Por otro lado, también se estudió la inhibición del inflamasoma NLRP3 para reducir el edema cerebral. En el modelo de TCE experimental se observó que la inhibición de NLRP3 evitó la ruptura de la BHE. Finalmente, en un modelo de lesión mecánica in vitro, se encontró que la inhibición del inflamasoma NLRP3 no actúa directamente sobre las células endoteliales para proteger la BHE. Teniendo en cuenta los resultados obtenidos a lo largo de esta Tesis Doctoral, proponemos la combinación de SAA1 y S100B como biomarcadores de pronóstico, y el antagonismo TLR4 y la inhibición del inflamasoma NLRP3 como prometedoras estrategias terapéuticas para evitar la progresión de la lesión en TCE.La investigación que se presenta ha sido financiada por un proyecto de la Fundación Mutua Madrileña y por los proyectos del Fondo de Investigaciones Sanitarias (FIS) (ISCIII/FEDER) PI16/00735 y PI19/00082 concedidos al director de la Tesis Doctoral, el Dr. Javier Egea

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-1 beta 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-1 beta and TNF-alpha), 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

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-1β 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-β release in NOX4 and Nrf2 KO mice. Importantly, NOX4 inhibition using GKT136901 also reduced IL-1β 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

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-1β 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-1β, which was reversed by melatonin pretreatment. In cultures from α7 nAChR knock-out (KO) mice, melatonin did not reduce IL-1β 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

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-1β 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-β release in NOX4 and Nrf2 KO mice. Importantly, NOX4 inhibition using GKT136901 also reduced IL-1β 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.European CommissionDepto. de Farmacología y ToxicologíaFac. de VeterinariaTRUEpubDescuento UC