Inflammation-induced alteration of astrocyte mitochondrial dynamics requires autophagy for mitochondrial network maintenance.

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

Gas6 and its receptors are implicated in sepsis as modulators of innate immunity

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P 0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6-/- mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF- was higher in Gas6-/- than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6-/- plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6-/- than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6-/- mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl-/-, Tyro3-/- and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6-/- mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6-/- macrophages produced more TNF- and IL-6 than WT macrophages. Cytokine release by Gas6-/- macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6-/- macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6-/- macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NF B activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6-/- mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NF B activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6-/- mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.

Host innate immune responses to microbial pathogens.

Sepsis is among the leading causes of death worldwide and its incidence is increasing. Defined as the host response to infection, sepsis is a clinical syndrome considered to be the expression of a dysregulated immune reaction induced by danger signals that may lead to organ failure and death. Remarkable progresses have been made in our understanding of the molecular basis of host defenses in recent years. The host defense response is initiated by innate immune sensors of danger signals designated under the collective name of pattern-recognition receptors. Members of the family of microbial sensors include the complement system, the Toll-like receptors, the nucleotide-binding oligomerization domainlike receptors, the RIG-I-like helicases and the C-type lectin receptors. Ligand-activated pattern-recognition receptors kick off a cascade of intracellular events resulting in the expression of co-stimulatory molecules and release of effector molecules playing a fundamental role in the initiation of the innate and adaptive immune responses. Fine tuning of proinflammatory and anti-inflammatory reactions is critical for keeping the innate immune response in check. Overwhelming or dysregulated responses induced by infectious stimuli may have dramatic consequences for the host as shown by the profound derangements observed in sepsis. Unfortunately, translational research approaches aimed at the development of therapies targeting newly identified innate immune pathways have not held their promises. Indeed, all recent clinical investigations of adjunctive anti-sepsis treatments had little, if any, impact on morbidity and all-cause mortality of sepsis. Dissecting the mechanisms underlying the transition from infection to sepsis is essential for solving the sepsis enigma. Important components of the puzzle have already been identified, but the hunt must go on in the laboratory and at the bedside.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC(12)-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC(12)-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC(12)-HSL influences host responses is unclear, and no mammalian receptors for 3OC(12)-HSL have been identified to date. Here, we report that 3OC(12)-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC(12)-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC(12)-HSL functioned as an agonist of PPARbeta/delta transcriptional activity and an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The proinflammatory effect of 3OC(12)-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC(12)-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC(12)-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

Characterization of pupil responses to blue and red light stimuli in autosomal dominant retinitis pigmentosa due to NR2E3 mutation.

PURPOSE: We characterized the pupil responses that reflect rod, cone, and melanopsin function in a genetically homogeneous cohort of patients with autosomal dominant retinitis pigmentosa (adRP). METHODS: Nine patients with Gly56Arg mutation of the NR2E3 gene and 12 control subjects were studied. Pupil and subjective visual responses to red and blue light flashes over a 7 log-unit range of intensities were recorded under dark and light adaptation. The pupil responses were plotted against stimulus intensity to obtain red-light and blue-light response curves. RESULTS: In the dark-adapted blue-light stimulus condition, patients showed significantly higher threshold intensities for visual perception and for a pupil response compared to controls (P = 0.02 and P = 0.006, respectively). The rod-dependent, blue-light pupil responses decreased with disease progression. In contrast, the cone-dependent pupil responses (light-adapted red-light stimulus condition) did not differ between patients and controls. The difference in the retinal sensitivity to blue and red stimuli was the most sensitive parameter to detect photoreceptor dysfunction. Unexpectedly, the melanopsin-mediated pupil response was decreased in patients (P = 0.02). CONCLUSIONS: Pupil responses of patients with NR2E3-associated adRP demonstrated reduced retinal sensitivity to dim blue light under dark adaptation, presumably reflecting decreased rod function. Rod-dependent pupil responses were quantifiable in all patients, including those with non-recordable scotopic electroretinogram, and correlated with the extent of clinical disease. Thus, the chromatic pupil light reflex can be used to monitor photoreceptor degeneration over a larger range of disease progression compared to standard electrophysiology.

The inflammasome: an integrated view.

An inflammasome is a multiprotein complex that serves as a platform for caspase-1 activation and caspase-1-dependent proteolytic maturation and secretion of interleukin-1β (IL-1β). Though a number of inflammasomes have been described, the NLRP3 inflammasome is the most extensively studied but also the most elusive. It is unique in that it responds to numerous physically and chemically diverse stimuli. The potent proinflammatory and pyrogenic activities of IL-1β necessitate that inflammasome activity is tightly controlled. To this end, a priming step is first required to induce the expression of both NLRP3 and proIL-1β. This event renders the cell competent for NLRP3 inflammasome activation and IL-1β secretion, and it is highly regulated by negative feedback loops. Despite the wide array of NLRP3 activators, the actual triggering of NLRP3 is controlled by integration a comparatively small number of signals that are common to nearly all activators. Minimally, these include potassium efflux, elevated levels of reactive oxygen species (ROS), and, for certain activators, lysosomal destabilization. Further investigation of how these and potentially other as yet uncharacterized signals are integrated by the NLRP3 inflammasome and the relevance of these biochemical events in vivo should provide new insight into the mechanisms of host defense and autoinflammatory conditions.

Dynamics of the recruitment of lateral amygdala neurons following successive fear learning to different conditional stimuli

In fear conditioning, an animal learns to associate an unconditioned stimulus (US), such as a shock, and a conditioned stimulus (CS), such as a tone, so that the presentation of the CS alone can trigger conditioned responses. Recent research on the lateral amygdala has shown that following cued fear conditioning, only a subset of higher-excitable neurons are recruited in the memory trace. Their selective deletion after fear conditioning results in a selective erasure of the fearful memory. I hypothesize that the recruitment of highly excitable neurons depends on responsiveness to stimuli, intrinsic excitability and local connectivity. In addition, I hypothesize that neurons recruited for an initial memory also participate in subsequent memories, and that changes in neuronal excitability affect secondary fear learning. To address these hypotheses, I will show that A) a rat can learn to associate two successive short-term fearful memories; B) neuronal populations in the LA are competitively recruited in the memory traces depending on individual neuronal advantages, as well as advantages granted by the local network. By performing two successive cued fear conditioning experiments, I found that rats were able to learn and extinguish the two successive short-term memories, when tested 1 hour after learning for each memory. These rats were equipped with a system of stable extracellular recordings that I developed, which allowed to monitor neuronal activity during fear learning. 233 individual putative pyramidal neurons could modulate their firing rate in response to the conditioned tone (conditioned neurons) and/or non- conditioned tones (generalizing neurons). Out of these recorded putative pyramidal neurons 86 (37%) neurons were conditioned to one or both tones. More precisely, one population of neurons encoded for a shared memory while another group of neurons likely encoded the memories' new features. Notably, in spite of a successful behavioral extinction, the firing rate of those conditioned neurons in response to the conditioned tone remained unchanged throughout memory testing. Furthermore, by analyzing the pre-conditioning characteristics of the conditioned neurons, I determined that it was possible to predict neuronal recruitment based on three factors: 1) initial sensitivity to auditory inputs, with tone-sensitive neurons being more easily recruited than tone- insensitive neurons; 2) baseline excitability levels, with more highly excitable neurons being more likely to become conditioned; and 3) the number of afferent connections received from local neurons, with neurons destined to become conditioned receiving more connections than non-conditioned neurons. - En conditionnement de la peur, un animal apprend à associer un stimulus inconditionnel (SI), tel un choc électrique, et un stimulus conditionné (SC), comme un son, de sorte que la présentation du SC seul suffit pour déclencher des réflexes conditionnés. Des recherches récentes sur l'amygdale latérale (AL) ont montré que, suite au conditionnement à la peur, seul un sous-ensemble de neurones plus excitables sont recrutés pour constituer la trace mnésique. Pour apprendre à associer deux sons au même SI, je fais l'hypothèse que les neurones entrent en compétition afin d'être sélectionnés lors du recrutement pour coder la trace mnésique. Ce recrutement dépendrait d'un part à une activation facilité des neurones ainsi qu'une activation facilité de réseaux de neurones locaux. En outre, je fais l'hypothèse que l'activation de ces réseaux de l'AL, en soi, est suffisante pour induire une mémoire effrayante. Pour répondre à ces hypothèses, je vais montrer que A) selon un processus de mémoire à court terme, un rat peut apprendre à associer deux mémoires effrayantes apprises successivement; B) des populations neuronales dans l'AL sont compétitivement recrutées dans les traces mnésiques en fonction des avantages neuronaux individuels, ainsi que les avantages consentis par le réseau local. En effectuant deux expériences successives de conditionnement à la peur, des rats étaient capables d'apprendre, ainsi que de subir un processus d'extinction, pour les deux souvenirs effrayants. La mesure de l'efficacité du conditionnement à la peur a été effectuée 1 heure après l'apprentissage pour chaque souvenir. Ces rats ont été équipés d'un système d'enregistrements extracellulaires stables que j'ai développé, ce qui a permis de suivre l'activité neuronale pendant l'apprentissage de la peur. 233 neurones pyramidaux individuels pouvaient moduler leur taux d'activité en réponse au son conditionné (neurones conditionnés) et/ou au son non conditionné (neurones généralisant). Sur les 233 neurones pyramidaux putatifs enregistrés 86 (37%) d'entre eux ont été conditionnés à un ou deux tons. Plus précisément, une population de neurones code conjointement pour un souvenir partagé, alors qu'un groupe de neurones différent code pour de nouvelles caractéristiques de nouveaux souvenirs. En particulier, en dépit d'une extinction du comportement réussie, le taux de décharge de ces neurones conditionné en réponse à la tonalité conditionnée est resté inchangée tout au long de la mesure d'apprentissage. En outre, en analysant les caractéristiques de pré-conditionnement des neurones conditionnés, j'ai déterminé qu'il était possible de prévoir le recrutement neuronal basé sur trois facteurs : 1) la sensibilité initiale aux entrées auditives, avec les neurones sensibles aux sons étant plus facilement recrutés que les neurones ne répondant pas aux stimuli auditifs; 2) les niveaux d'excitabilité des neurones, avec les neurones plus facilement excitables étant plus susceptibles d'être conditionnés au son ; et 3) le nombre de connexions reçues, puisque les neurones conditionné reçoivent plus de connexions que les neurones non-conditionnés. Enfin, nous avons constaté qu'il était possible de remplacer de façon satisfaisante le SI lors d'un conditionnement à la peur par des injections bilatérales de bicuculline, un antagoniste des récepteurs de l'acide y-Aminobutirique.

Enhanced Proinflammatory Cytokine Response to Bacterial Lipopolysaccharide in the Adult Male Rat after either Neonatal or Prepubertal Ablation of Biological Testosterone Activity.

A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 μg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Aversive Stimuli and Functional Networks of Fear Learning in PTSD : 734

Background: One characteristic of post traumatic stress disorder is an inability to adapt to a safe environment i.e. to change behavior when predictions of adverse outcomes are not met. Recent studies have also indicated that PTSD patients have altered pain processing, with hyperactivation of the putamen and insula to aversive stimuli (Geuze et al, 2007). The present study examined neuronal responses to aversive and predicted aversive events. Methods: Twenty-four trauma exposed non-PTSD controls and nineteen subjects with PTSD underwent fMRI imaging during a partial reinforcement fear conditioning paradigm, with a mild electric shock as the unconditioned stimuli (UCS). Three conditions were analyzed: actual presentations of the UCS, events when a UCS was expected, but omitted (CS+), and events when the UCS was neither expected nor delivered (CS-). Results: The UCS evoked significant alterations in the pain matrix consisting of the brainstem, the midbrain, the thalamus, the insula, the anterior and middle cingulate and the contralateral somatosensory cortex. PTSD subjects displayed bilaterally elevated putamen activity to the electric shock, as compared to controls. In trials when USC was expected, but omitted, significant activations were observed in the brainstem, the midbrain, the anterior insula and the anterior cingulate. PTSD subjects displayed similar activations, but also elevated activations in the amygdala and the posterior insula. Conclusions: These results indicate altered fear and safety learning in PTSD, and neuronal activations are further explored in terms of functional connectivity using psychophysiological interaction analyses.

Targeting inflammasomes in rheumatic diseases.

Inflammasomes are key inducers of inflammation in response to exogenous and endogenous stimuli, because they regulate the processing and secretion of the proinflammatory cytokines IL-1β and IL-18. Thus, inflammasomes have a crucial role in host defence against infection, but they can also be involved in inflammatory diseases. Indeed, the NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome has been shown to play a part in several inflammatory rheumatic disorders, although the mechanisms involved are better elucidated in some of these diseases than in others. In particular, the pathogenesis of cryopyrin-associated periodic syndromes and microcrystal-induced arthritides is thought to be dependent on activation of the NLRP3 inflammasome, and IL-1 inhibition has shown efficacy as a therapeutic strategy in both groups of conditions. In this Review, we describe the current understanding of the mechanisms that trigger the inflammasome, and consider the relevance of the inflammasome to a variety of rheumatic diseases. In addition, we discuss the current therapies targeting this molecular complex, as well as future therapeutic prospects.