Microglial NLRP3 Inflammasome Induces Excitatory Synaptic Loss Through IL-1ß-Enriched Microvesicle Release: Implications for Sepsis-Associated Encephalopathy.

Acute cerebral dysfunction is a pathological state common in severe infections and a pivotal determinant of long-term cognitive outcomes. Current evidence indicates that a loss of synaptic contacts orchestrated by microglial activation is central in sepsis-associated encephalopathy. However, the upstream signals that lead to microglial activation and the mechanism involved in microglial-mediated synapse dysfunction in sepsis are poorly understood. This study investigated the involvement of the NLRP3 inflammasome in microglial activation and synaptic loss related to sepsis. We demonstrated that septic insult using the cecal ligation and puncture (CLP) model induced the expression of NLRP3 inflammasome components in the brain, such as NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and IL-1ß. Immunostaining techniques revealed increased expression of the NLRP3 inflammasome in microglial cells in the hippocampus of septic mice. Meanwhile, an in vitro model of primary microglia stimulated with LPS exhibited an increase in mitochondrial reactive oxygen species (ROS) production, NLRP3 complex recruitment, and IL-1ß release. Pharmacological inhibition of NLRP3, caspase-1, and mitochondrial ROS all decreased IL-1ß secretion by microglial cells. Furthermore, we found that microglial NLRP3 activation is the main pathway for IL-1ß-enriched microvesicle (MV) release, which is caspase-1-dependent. MV released from LPS-activated microglia induced neurite suppression and excitatory synaptic loss in neuronal cultures. Moreover, microglial caspase-1 inhibition prevented neurite damage and attenuated synaptic deficits induced by the activated microglial MV. These results suggest that microglial NLRP3 inflammasome activation is the mechanism of IL-1ß-enriched MV release and potentially synaptic impairment in sepsis.

Atazanavir Is a Competitive Inhibitor of SARS-CoV-2 Mpro, Impairing Variants Replication In Vitro and In Vivo.

Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19); however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (Mpro) was inhibited by ATV, with Morrison's inhibitory constant (Ki) 1.5-fold higher than GC376 (a positive control) dependent of the catalytic water (H2Ocat) content. ATV was a competitive inhibitor, increasing the Mpro's Michaelis-Menten (Km) more than sixfold. Cell-based assays indicated that different lineages of SARS-CoV-2 is susceptible to ATV. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the Mpro/ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.

Cerebral Multimodal Monitoring in Sepsis: An Experimental Study.

Acute brain dysfunction is a complication of sepsis, and its pathophysiology remains poorly understood. We studied the brain metabolism in a resuscitated animal model of sepsis. Twelve anesthetized, mechanically ventilated, and invasively monitored pigs were allocated to a sham procedure (N = 5) or sepsis (N = 7). Sepsis was induced through fecal inoculation in the peritoneum. Fluid resuscitation was maintained during the entire study period. Animals were observed until spontaneous death or for a maximum of 24 h. In addition to global hemodynamic and laboratory assessment, intracranial pressure and cerebral microdialysis (MD) were evaluated at baseline, 6, 12, 18, and 24 h after sepsis induction. After euthanasia, the brain was rapidly removed and a fragment from the frontal cortex was analyzed for markers of neuroinflammation, metabolism, and neurotransmission. Septic animals developed a hyperdynamic state associated with increased arterial lactate. Cerebral microdialysis showed unchanged levels of lactate/pyruvate ratios and brain glucose between the groups. Brain/serum glucose ratios were increased in the septic animals during the study period despite a progressive decrease in serum glucose. Moreover, extracellular glutamine levels were elevated starting at 6 h after sepsis. Tissue analysis showed elevated glutamate, glutamine, and glutamine synthetase in the sepsis group. However, C-Fos, a marker of neuronal activity, was unchanged between groups. In this animal model of resuscitated sepsis, we found increased oxidative stress and alterations in neuroenergetics characterized by exacerbated activity of the glutamate/glutamine cycle and increased glucose utilization by the brain, however without any evidence of decompensated energy metabolism.

Statins prevent cognitive impairment after sepsis by reverting neuroinflammation, and microcirculatory/endothelial dysfunction.

Acute brain dysfunction is a frequent condition in sepsis patients and is associated with increased mortality and long-term neurocognitive consequences. Impaired memory and executive function are common findings in sepsis survivors. Although neuroinflammation and blood-brain barrier dysfunction have been associated with acute brain dysfunction and its consequences, no specific treatments are available that prevent cognitive impairment after sepsis. Experimental sepsis was induced in Swiss Webster mice by intraperitoneal injection of cecal material (5mg/kg, 500µL). Control groups (n=5/group each experiment) received 500µL of saline. Support therapy recover (saline 0.9%, 1mL and imipenem 30mg/kg) were applied (6, 24 and 48h post injection, n=5-10/group, each experiment), together or not with additive orally treatment with statins (atorvastatin/simvastatin 20mg/kg b.w.). Survival rate was monitored at 6, 24 and 48h. In a setting of experiments, animals were euthanized at 6 and 24h after induction for biochemical, immunohistochemistry and intravital analysis. Statins did not prevented mortality in septic mice, however survivors presented lower clinical score. At another setting of experiments, after 15days, mice survivors from fecal supernatant peritoneal sepsis presented cognitive dysfunction for contextual hippocampal and aversive amygdala-dependent memories, which was prevented by atorvastatin/simvastatin treatment. Systemic and brain tissue levels of proinflammatory cytokines/chemokines and activation of microglial were lower in septic mice treated with statins. Brain lipid peroxidation and myeloperoxidase levels were also reduced by statins treatment. Intravital examination of the brain vessels of septic animals revealed decreased functional capillary density and increased rolling and adhesion of leukocytes, and blood flow impairment, which were reversed by treatment with statins. In addition, treatment with statins restored the cholinergic vasodilator response due to sepsis. Taken together, these data demonstrated that statins reverse microvascular dysfunction and reduce neuroinflammation during sepsis, preventing the development of long-term cognitive decline.

Activated Microglia-Induced Deficits in Excitatory Synapses Through IL-1ß: Implications for Cognitive Impairment in Sepsis.

Recent clinical studies have shown that sepsis survivors may develop long-term cognitive impairments. The cellular and molecular mechanisms involved in these events are not well understood. This study investigated synaptic deficits in sepsis and the involvement of glial cells in this process. Septic animals showed memory impairment and reduced numbers of hippocampal and cortical excitatory synapses, identified by synaptophysin/PSD-95 co-localization, 9 days after disease onset. The behavioral deficits and synaptophysin/PSD-95 co-localization were rescued to normal levels within 30 days post-sepsis. Septic mice presented activation of microglia and reactive astrogliosis, which are hallmarks of brain injury and could be involved in the associated synaptic deficits. We treated neuronal cultures with conditioned medium derived from cultured astrocytes (ACM) and microglia (MCM) that were either non-stimulated or stimulated with lipopolysaccharide (LPS) to investigate the molecular mechanisms underlying synaptic deficits in sepsis. ACM and MCM increased the number of synapses between cortical neurons in vitro, and these effects were antagonized by LPS stimulation. LPS-MCM reduced the number of synapses by 50%, but LPS-ACM increased the number of synapses by 500%. Analysis of the composition of these conditioned media revealed increased levels of IL-1ß in LPS-MCM. Furthermore, inhibition of IL-1ß signaling through the addition of a soluble IL-1ß receptor antagonist (IL-1 Ra) fully prevented the synaptic deficit induced by LPS-MCM. These results suggest that sepsis induces a transient synaptic deficit associated with memory impairments mediated by IL-1ß secreted by activated microglia.

The role of Nox2-derived ROS in the development of cognitive impairment after sepsis.

BACKGROUND: Sepsis- associated encephalopathy (SAE) is an early and common feature of severe infections. Oxidative stress is one of the mechanisms associated with the pathophysiology of SAE. The goal of this study was to investigate the involvement of NADPH oxidase in neuroinflammation and in the long-term cognitive impairment of sepsis survivors. METHODS: Sepsis was induced in WT and gp91(phox) knockout mice (gp91(phox-/-)) by cecal ligation and puncture (CLP) to induce fecal peritonitis. We measured oxidative stress, Nox2 and Nox4 gene expression and neuroinflammation in the hippocampus at six hours, twenty-four hours and five days post-sepsis. Mice were also treated with apocynin, a NADPH oxidase inhibitor. Behavioral outcomes were evaluated 15 days after sepsis with the inhibitory avoidance test and the Morris water maze in control and apocynin-treated WT mice. RESULTS: Acute oxidative damage to the hippocampus was identified by increased 4-HNE expression in parallel with an increase in Nox2 gene expression after sepsis. Pharmacological inhibition of Nox2 with apocynin completely inhibited hippocampal oxidative stress in septic animals. Pharmacologic inhibition or the absence of Nox2 in gp91(phox-/-) mice prevented glial cell activation, one of the central mechanisms associated with SAE. Finally, treatment with apocynin and inhibition of hippocampal oxidative stress in the acute phase of sepsis prevented the development of long-term cognitive impairment. CONCLUSIONS: Our results demonstrate that Nox2 is the main source of reactive oxygen species (ROS) involved in the oxidative damage to the hippocampus in SAE and that Nox2-derived ROS are determining factors for cognitive impairments after sepsis. These findings highlight the importance of Nox2-derived ROS as a central mechanism in the development of neuroinflammation associated with SAE.

In vivo detection of free radicals in mouse septic encephalopathy using molecular MRI and immuno-spin trapping.

Free radicals are known to play a major role in sepsis. Combined immuno-spin trapping and molecular magnetic resonance imaging (MRI) was used to detect in vivo and in situ levels of free radicals in murine septic encephalopathy after cecal ligation and puncture (CLP). DMPO (5,5-dimethyl pyrroline N-oxide) was injected over 6h after CLP, before administration of an anti-DMPO probe (anti-DMPO antibody bound to albumin-gadolinium-diethylene triamine pentaacetic acid-biotin MRI targeting contrast agent). In vitro assessment of the anti-DMPO probe in oxidatively stressed mouse astrocytes significantly decreased T1 relaxation (p < 0.0001) compared to controls. MRI detected the presence of anti-DMPO adducts via a substantial decrease in %T1 change within the hippocampus, striatum, occipital, and medial cortex brain regions (p < 0.01 for all) in septic animals compared to shams, which was sustained for over 60 min (p < 0.05 for all). Fluorescently labeled streptavidin was used to target the anti-DMPO probe biotin, which was elevated in septic brain, liver, and lungs compared to sham. Ex vivo DMPO adducts (qualitative) and oxidative products, including 4-hydroxynonenal and 3-nitrotyrosine (quantitative, p < 0.05 for both), were elevated in septic brains compared to shams. This is the first study that has reported on the detection of in vivo and in situ levels of free radicals in murine septic encephalopathy.

Bioenergetics, mitochondrial dysfunction, and oxidative stress in the pathophysiology of septic encephalopathy.

Sepsis is a major cause of mortality and morbidity in intensive care units. Acute and long-term brain dysfunctions have been demonstrated both in experimental models and septic patients. Sepsis-associated encephalopathy is an early and frequent manifestation but is underdiagnosed, because of the absence of specific biomarkers and of confounding factors such as sedatives used in the intensive care unit. Sepsis-associated encephalopathy may have acute and long-term consequences including development of autonomic dysfunction, delirium, and cognitive impairment. The mechanisms of sepsis-associated encephalopathy involve mitochondrial and vascular dysfunctions, oxidative stress, neurotransmission disturbances, inflammation, and cell death. Here we review specific evidence that links bioenergetics, mitochondrial dysfunction, and oxidative stress in the setting of brain dysfunctions associated to sepsis.

Microglial activation induced by brain trauma is suppressed by post-injury treatment with a PARP inhibitor.

BACKGROUND: Traumatic brain injury (TBI) induces activation of microglia. Activated microglia can in turn increase secondary injury and impair recovery. This innate immune response requires hours to days to become fully manifest, thus providing a clinically relevant window of opportunity for therapeutic intervention. Microglial activation is regulated in part by poly(ADP-ribose) polymerase-1 (PARP-1). Inhibition of PARP-1 activity suppresses NF-kB-dependent gene transcription and thereby blocks several aspects of microglial activation. Here we evaluated the efficacy of a PARP inhibitor, INO-1001, in suppressing microglial activation after cortical impact in the rat. METHODS: Rats were subjected to controlled cortical impact and subsequently treated with 10 mg/kg of INO-1001 (or vehicle alone) beginning 20 - 24 hours after the TBI. Brains were harvested at several time points for histological evaluation of inflammation and neuronal survival, using markers for microglial activation (morphology and CD11b expression), astrocyte activation (GFAP), and neuronal survival (NeuN). Rats were also evaluated at 8 weeks after TBI using measures of forelimb dexterity: the sticky tape test, cylinder test, and vermicelli test. RESULTS: Peak microglial and astrocyte activation was observed 5 to 7 days after this injury. INO-1001 significantly reduced microglial activation in the peri-lesion cortex and ipsilateral hippocampus. No rebound inflammation was observed in rats that were treated with INO-1001 or vehicle for 12 days followed by 4 days without drug. The reduced inflammation was associated with increased neuronal survival in the peri-lesion cortex and improved performance on tests of forelimb dexterity conducted 8 weeks after TBI. CONCLUSIONS: Treatment with a PARP inhibitor for 12 days after TBI, with the first dose given as long as 20 hours after injury, can reduce inflammation and improve histological and functional outcomes.

Statins decrease neuroinflammation and prevent cognitive impairment after cerebral malaria.

Cerebral malaria (CM) is the most severe manifestation of Plasmodium falciparum infection in children and non-immune adults. Previous work has documented a persistent cognitive impairment in children who survive an episode of CM that is mimicked in animal models of the disease. Potential therapeutic interventions for this complication have not been investigated, and are urgently needed. HMG-CoA reductase inhibitors (statins) are widely prescribed for cardiovascular diseases. In addition to their effects on the inhibition of cholesterol synthesis, statins have pleiotropic immunomodulatory activities. Here we tested if statins would prevent cognitive impairment in a murine model of cerebral malaria. Six days after infection with Plasmodium berghei ANKA (PbA) mice displayed clear signs of CM and were treated with chloroquine, or chloroquine and lovastatin. Intravital examination of pial vessels of infected animals demonstrated a decrease in functional capillary density and an increase in rolling and adhesion of leukocytes to inflamed endothelium that were reversed by treatment with lovastatin. In addition, oedema, ICAM-1, and CD11b mRNA levels were reduced in lovastatin-treated PbA-infected mice brains. Moreover, HMOX-1 mRNA levels are enhanced in lovastatin-treated healthy and infected brains. Oxidative stress and key inflammatory chemokines and cytokines were reduced to non-infected control levels in animals treated with lovastatin. Fifteen days post-infection cognitive dysfunction was detected by a battery of cognition tests in animals rescued from CM by chloroquine treatment. In contrast, it was absent in animals treated with lovastatin and chloroquine. The outcome was similar in experimental bacterial sepsis, suggesting that statins have neuroprotective effects in severe infectious syndromes in addition to CM. Statin treatment prevents neuroinflammation and blood brain barrier dysfunction in experimental CM and related conditions that are associated with cognitive sequelae, and may be a valuable adjuvant therapeutic agent for prevention of cognitive impairment in patients surviving an episode of CM.

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni.

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.

Inhibition of heme aggregation by chloroquine reduces Schistosoma mansoni infection.

Adult Schistosoma mansoni digest large amounts of host hemoglobin and release potentially toxic heme inside their guts. We have previously demonstrated that free heme in S. mansoni is detoxified through aggregation, forming hemozoin (Hz). Possible mechanisms of heme aggregation and the effects of chloroquine (CLQ) on formation of Hz and on the viability of this parasite have now been investigated. Different fractions isolated from S. mansoni, such as crude whole-worm homogenates, total lipid extracts, and Hz itself promoted heme aggregation in vitro in a CLQ-sensitive manner. Treatment of S. mansoni-infected mice with CLQ led to remarkable decreases in total protein, Hz content, and viability of the worms, as well as in parasitemia and deposition of eggs in mouse livers. These results indicate that inhibition of formation of Hz in S. mansoni, by CLQ, led to an important decrease in the overall severity of experimental murine schistosomiasis. Taken together, the results presented here suggest that formation of Hz is a major mechanism of heme detoxification and a potential target for chemotherapy in S. mansoni.