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Sleep is crucial for healthy cognition, including memory. The two main phases of sleep, REM (rapid eye movement) and non-REM sleep, are associated with characteristic electrophysiological patterns that are recorded using surface and intracranial electrodes. These patterns include sharp-wave ripples, cortical slow oscillations, delta waves, and spindles during non-REM sleep and theta oscillations during REM sleep. They reflect the precisely timed activity of underlying neural circuits. Here, we review how these electrical signatures have been guiding our understanding of the circuits and processes sustaining memory consolidation during sleep, focusing on hippocampal theta oscillations and sharp-wave ripples and how they coordinate with cortical patterns. Finally, we highlight how these brain patterns could also sustain sleep-dependent homeostatic processes and evoke several potential future directions for research on the memory function of sleep., [Figure: see text].

International audience; Aspergillus fumigatus is a human-pathogenic mold that extracts nutrients from the environment or from host tissues by secreting hydrolytic enzymes. The ability of A. fumigatus to adjust secretion levels in proportion to demand relies on the assistance of the unfolded protein response (UPR), an adaptive stress response pathway that regulates the unique protein-folding environment of the endoplasmic reticulum (ER). The P5-type ATPase Spf1 has recently been implicated in a novel mechanism of ER homeostasis that involves correcting errors in ER-membrane protein targeting. However, the contribution of this protein to the biology of A. fumigatus is unknown. Here, we employed a gene knockout and RNA sequencing strategy to determine the functional role of the A. fumigatus gene coding for the orthologous P5 ATPase SpfA. The data reveal that the spfA gene is induced by ER stress in a UPR-dependent manner. In the absence of spfA, the A. fumigatus transcriptome shifts toward a profile of altered redox and lipid balance, in addition to a signature of ER stress that includes srcA, encoding a second P-type ATPase in the ER. A ΔspfA deletion mutant showed increased sensitivity to ER stress, oxidative stress, and antifungal drugs that target the cell wall or plasma membrane. The combined loss of spfA and srcA exacerbated these phenotypes and attenuated virulence in two animal infection models. These findings demonstrate that the ER-resident ATPases SpfA and SrcA act jointly to support diverse adaptive functions of the ER that are necessary for fitness in the host environment.IMPORTANCE The fungal UPR is an adaptive signaling pathway in the ER that buffers fluctuations in ER stress but also serves as a virulence regulatory hub in species of pathogenic fungi that rely on secretory pathway homeostasis for pathogenicity. This study demonstrates that the gene encoding the ER-localized P5-type ATPase SpfA is a downstream target of the UPR in the pathogenic mold A. fumigatus and that it works together with a second ER-localized P-type ATPase, SrcA, to support ER homeostasis, oxidative stress resistance, susceptibility to antifungal drugs, and virulence of A. fumigatus.

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART., Author summary HIV-1 infects immune cells by binding CD4 and a coreceptor, CCR5 or CXCR4. CXCR4-using viruses are thought to accelerate depletion of CD4 T lymphocytes (CD4TL) and AIDS development. This paradigm, while often true, is however not seen in all patients, suggesting heterogeneity in the virulence of viruses. Here, we show that CXCR4-using viruses can be discriminated on the basis of their resistance to the anti-HIV-1 effect of the CXCR4 chemokine CXCL12. Resistant (RES) viruses are found in patients with low CD4TL levels. They are featured by unique properties of their envelope glycoprotein and the way they use CXCR4. Our data also indicate that RES viruses could contribute to CD4TL depletion. They more effectively kill bystander activated effector memory CD4TL, a subset of CD4TL enriched in HIV+ patients, and enter CD4TL subsets surrounded by CXCL12. Resistance could therefore enhance the ability of viruses to target naive cells and their precursors in lymphoid organs where CXCL12 is present, further hindering CD4TL renewal. Beyond presenting a novel view of CXCR4-using HIV-1 pathogenesis, this work also opens new therapeutic perspectives and increases our knowledge of the still debated role of CXCL12 in HIV-1 transmission.

International audience; Group 3 innate lymphoid cells (ILC3) have been detected in both murine and human decidual tissues where they are thought to play a relevant role in the induction and maintenance of pregnancy. However, limited information exists on the molecular mechanisms that regulate these cells, including immune checkpoints. Here, we show that ILC3 express the inhibitory checkpoints programmed cell death (PD-1) and T cell immunoglobulin and mucin domain containing protein 3 (TIM-3) during the first trimester of pregnancy and that these receptors could regulate production of cytokines, including IL-22, IL-8, and TNF-α, induced by IL-23. We also show that the intermediate extravillous trophoblast (iEVT) expresses high levels of the PD-1-ligand PD-L1, suggesting that PD-1/PD-L1 interaction may regulate ILC3 function at the feto-maternal interface. Our present data provide the first evidence that human decidual ILC3 express a functional PD-1. It is possible that an altered expression or function of PD-1 may break the immune-tolerance resulting in pregnancy failure.

International audience; Pulmonary arterial hypertension (PAH) is a severe cardiovascular disease that is caused by the progressive occlusion of the distal pulmonary arteries, eventually leading to right heart failure and death. Almost 40% of patients with PAH are iron deficient. Although widely studied, the mechanisms linking between PAH and iron deficiency remain unclear. Here we review the mechanisms regulating iron homeostasis and the preclinical and clinical data available on iron deficiency in PAH. Then we discuss the potential implications of iron deficiency on the development and management of PAH.

Iron has a fundamental role for cell physiology and especially in retina as a cofactor of many pathways of the visual transduction. A tightly regulated homeostasis avoids the accumulation of prooxidant and proinflammatory free iron. A dysfunction of iron retinal homeostasis is associated with many genetic or age-related degenerative diseases such as age-related macular degeneration (AMD). Here, we describe various mechanisms reported during AMD, enhanced by iron accumulation and its homeostasis dysregulation. We have investigated a local treatment with transferrin, the natural iron carrier, to control these pathological pathways and iron dysfunction, without side effects. Iron has a central role in pathogenesis of AMD and is a target for futures therapies.La dégénérescence maculaire liée à l’âge: La piste du fer.En raison de l’intense activité physiologique de la fonction visuelle, l’homéostasie du fer dans la rétine y est contrôlée localement. Sous l’effet de sa dérégulation (qui a des origines génétiques, environnementales, ou due au vieillissement), le fer libre s’accumule et devient, par ses propriétés oxydantes et inflammatoires, toxique, comme cela est observé au cours de la dégénérescence maculaire liée à l’âge (DMLA). Le rétablissement d’un métabolisme du fer équilibré est donc une possibilité thérapeutique. Néanmoins, la toxicité oculaire des chélateurs chimiques oriente les recherches vers des chélateurs biologiques naturels. Nos travaux montrent que la transferrine, le transporteur du fer, préserve la rétine des mécanismes associés à la DMLA.

International audience; Iron has a fundamental role for cell physiology and especially in retina as a cofactor of many pathways of the visual transduction. A tightly regulated homeostasis avoids the accumulation of prooxidant and proinflammatory free iron. A dysfunction of iron retinal homeostasis is associated with many genetic or age-related degenerative diseases such as age-related macular degeneration (AMD). Here, we describe various mechanisms reported during AMD, enhanced by iron accumulation and its homeostasis dysregulation. We have investigated a local treatment with transferrin, the natural iron carrier, to control these pathological pathways and iron dysfunction, without side effects. Iron has a central role in pathogenesis of AMD and is a target for futures therapies.

International audience; Innate lymphoid cell (ILC) subsets at barrier surfaces contribute to maintain tissue homeostasis and appropriate responses to infection. ILCs respond to environmental factors produced by non-hematopoietic cells within tissues, but also circulating cytokines or dietary compounds which allow them to adapt to organ milieu. Among these extrinsic signals, evidence is emerging that sex steroid hormones may act in a cell-intrinsic manner to regulate the development, maintenance in tissues and effector functions of specific subsets of ILCs. Understanding the nature and molecular mechanisms of sex steroid hormone actions on ILCs is important to unravel the cause of sexual disparity in human diseases and could lead to new drug development for the treatment of chronic inflammatory diseases or cancers. This review discusses the recent development in our understanding of the cell-intrinsic actions of sex steroid hormones on ILCs and their consequences on tissue-specific immunity with a particular focus on group 2 innate lymphoid cells and NK cells.

International audience; With the aging of the general population, there is an increasing need for bone defect repair, prompting the development of reliable alternatives to autologous bone grafting, without the usually associated major drawbacks (i.e., limited volume and severe postoperative pain). Given the crucial role that miRNAs appear to have in bone tissue physiopathology, exploring their potential has recently garnered increased interest. In this review, we first describe the involvement of miRNAs in bone metabolism, and then focus on their potential therapeutic applications (as bone biomarkers and molecular targets). We also highlight the as yet unsolved biological (i.e., off-target effects) and technological (i.e., specific delivering) challenges associated with their use.

International audience; Magnesium (Mg2+) is critical for a number of biological processes and 25% body Mg2+ is located in the skeletal muscle. Mg2+ transport and homeostasis systems (MgTHs) regulate intracellular Mg2+ concentration and muscle MgTHs are thus related to whole body Mg2+ homeostasis. Nonetheless, few studies have investigated the regulation of muscle MgTHs under (patho)physiological conditions. Herein, we assessed the relationship between the expression of MgTHs genes (Trpm6, Trpm7, Magt1, Mrs2, Cnnm1-4, Slc41a1-3) and relevant pathways in human sarcopenia, which is one of the most dramatic physiologic changes affecting the human body. Transcriptomic data were compared between young adult (YO, 22 y, n = 11) and old (EL, 73 y, n = 13) men from the PROOF cohort. MgTH mRNA levels did not change with aging, with the exception of a slight decrease for Slc41a3. Nevertheless, interindividual variations of mRNA levels revealed strong correlations between MgTHs in the YO group, while few were maintained in the EL muscle. Moreover, in the YO muscle, different clusters of MgTH mRNAs strongly correlated with divers physiological (BMI, blood pressure) and muscle characteristics (intramyocellular droplets, capillarization); however, most correlations changed or disappeared in the EL muscle. Further investigations of the whole transcriptome identified several sets of mRNAs correlated with defined MgTHs. There again was a sharp difference between YO and EL muscles, as the number of mRNAs correlated with MgTHs strongly decreased with aging. Gene ontology analyses of these sets of correlated mRNAs revealed 6 biological processes common to YO and EL, 3 specific to the YO (RNA processing, translation, respiration), and 2 (regulation of catabolic process, Wnt signaling) to the EL muscle. Overall, these observations lead to questions about potential resilience to muscle Mg2+ homeostasis in the elderly.

International audience; Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract 1-4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (T reg) cells 4-8 , and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease 9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain T reg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce T reg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88-and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining T reg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn's disease, and this correlated with lower frequencies of T reg cells. Our results reveal a previously unappreciated pathway in which a microbiota-and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine. To determine whether IL-2 is constitutively required for the maintenance of T reg cells and immunological homeostasis in the intestine, we administered isotype-control or anti-IL-2 neutralizing antibodies every other day to adult mice for two weeks. Within this short time period, the neutralization of IL-2 promoted an enlargement of the spleen and mesenteric lymph nodes, and caused significant reductions of T reg cells and significant increases in the proliferation of CD4 + T cells throughout the gastrointestinal tract and associated lymphoid tissues, including the mesenteric lymph nodes, large intestine and small intestine (Extended Data Fig. 1a-g). Blockade of IL-2 resulted in significantly enhanced IFNγ production by CD4 + T cells in both the small and large intestine, as well as increased IL-17A production in the large intestine (Extended Data Fig. 1h-k). Previous studies have suggested that CD4 + T cells are the dominant cellular source of IL-2 1,2. Therefore, we generated mice with a lineage-specific deletion of IL-2 in T cells by crossing IL-2-floxed mice 10 with Lck cre mice. Lck cre Il2 f/f mice exhibited a complete loss of IL-2 protein staining in T cells, and we observed a significant reduction in the number of T reg cells, and an increase in CD4 + T cell proliferation and effector function in the mesenteric lymph nodes and large intestine (Extended Data Fig. 2a-g). By contrast, deletion of

International audience; Polymyalgia rheumatica (PMR) is a frequent rheumatic condition among people over 50 years of age. Despite its frequent association with giant cell arteritis (GCA), PMR can be isolated. Its pathophysiology is poorly understood. Nevertheless, many studies are ongoing; 98 studies are currently referenced in ClinicalTrials.org involving several conventional and targeted therapies. In this review, we synthetize the current knowledge about PMR pathophysiology according to genetic and immunogenetic, immunologic, antibody and aging data. Immunogenetic data are mainly related to the HLA system and the association between the HLA-DRB1 and PMR. Few studies are also about immunogenetics of proinflammatory interleukins (i.e. IL-6). The decrease of CD8+Tcells and the strong increase of IL-6 where the main elements of PMR's pathophysiology until the recent years. The disturbance of B cell homeostasis, the search for IL-6 secretion by the innate immune system, the role of aging, are new elements revealed by recent studies. Aging might be a key element to consider as PMR occurs in patients over 50 years of age. Aging may act by the increased susceptibility to infections, by immunological modifications or hormonal disturbances. The role of the cellular infiltration around the joints remains a crucial question. Only a handful of studies described this infiltration. Finally, this review reveals the gaps in available data and suggests new leads and in-depth studies for further research on PMR pathophysiology.

International audience; Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.

Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP) is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment., Author summary Nucleotide-based second messengers play central functions in bacterial physiology and host-pathogen interactions. Among these signalling nucleotides, cyclic-di-AMP (c-di-AMP) synthesis was originally assumed to be essential for bacterial growth. In this study, we confirmed that the only di-adenylate cyclase enzyme in the opportunistic pathogen Streptococcus agalactiae is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable by accumulating spontaneous mutations in genes involved in osmotic regulation. We identified that c-di-AMP binds directly to four proteins necessary to maintain osmotic homeostasis, including three osmolyte transporters and the BusR transcriptional factor. We demonstrated that BusR negatively controls the expression of the busAB operon and that it is the main component leading to growth inhibition in the absence of c-di-AMP synthesis if osmoprotectants are present in the environment. Overall, c-di-AMP is essential to maintain osmotic homeostasis by coordinating osmolyte uptake and thus bacteria have developed specific mechanisms to keep c-di-AMP as the central regulator of osmotic homeostasis.

International audience; MicroRNAs (miRNAs) are evolutionarily conserved small regulatory RNAs that participate in the adjustment of many, if not all, fundamental biological processes. Molecular mechanisms involved in miRNA biogenesis and mode of action have been elucidated in the past two decades. Similar to many cellular pathways, miRNA processing and function can be globally or specifically regulated at several levels and by numerous proteins and RNAs. Given their role as fine-tuning molecules, it is essential for miRNA expression to be tightly regulated in order to maintain cellular homeostasis. Here, we review our current knowledge of the first step of their maturation occurring in the nucleus and how it can be specifically and dynamically modulated.

International audience; It is well known that pendrin, an apical Cl-/HCO3-exchanger in type B intercalated cells, is modulated by chronic acid-base disturbances and electrolyte intake. To study this adaptation further at the acute level, we analyzed urinary exosomes from individuals subjected to oral acute acid, alkali, and NaCl loading. Acute oral NH4Cl loading (n = 8) elicited systemic acidemia with a drop in urinary pH and an increase in urinary NH4 excretion. Nadir urinary pH was achieved 5 h after NH4Cl loading. Exosomal pendrin abundance was dramatically decreased at 3 h after acid loading. In contrast, after acute equimolar oral NaHCO3 loading (n = 8), urinary and venous blood pH rose rapidly with a significant attenuation of urinary NH4 excretion. Alkali loading caused rapid upregulation of exosomal pendrin abundance at 1 h and normalized within 3 h of treatment. Equimolar NaCl loading (n = 6) did not alter urinary or venous blood pH or urinary NH4 excretion. However, pendrin abundance in urinary exosomes was significantly reduced at 2 h of NaCl ingestion with lowest levels observed at 4 h after treatment. In patients with inherited distal renal tubular acidosis (dRTA), pendrin abundance in urinary exosomes was greatly reduced and did not change upon oral NH4Cl loading. In summary, pendrin can be detected and quantified in human urinary exosomes by immunoblotting. Acid, alkali, and NaCl loadings cause acute changes in pendrin abundance in urinary exosomes within a few hours. Our data suggest that exosomal pendrin is a promising urinary biomarker for acute acid-base and volume status changes in humans.

International audience; Microbial pathogens possess a diversity of weapons that disrupt host homeostasis and immune defenses, thus resulting in the establishment of infection. The best-characterized system mediating bacterial protein delivery into target eukaryotic cells is the type III secretion system (T3SS) expressed by Gram-negative bacteria, including the human enteric pathogens Shigella, Salmonella, Yersinia, and enteropathogenic/enterohemorragic Escherichia coli (EPEC/EHEC). The emerging global view is that these T3SS-bearing pathogens share similarities in their ability to target key cellular pathways such as the cell cytoskeleton, trafficking, cell death/survival, and the NF-κB and MAPK signaling pathways. In particular, multiple host proteins are targeted in a given pathway, and different T3SS effectors from various pathogens share functional similarities.

International audience; Imbalance in the homeostasis of K+ ions has been reported to contribute to the pathogenesis of neurodegenerative diseases. 7-ketocholesterol (7KC), 24S-hydroxycholesterol (24S-OHC), and tetracosanoic acid (C24:0), often found at increased levels in patients with Alzheimer's disease, Multiple Sclerosis and X-ALD, are able to trigger numerous nerve cell dysfunctions. We therefore studied the impact of 7KC, 24S-OHC, and C24:0 on 158N murine oligodendrocytes, and determined their impact on K+ homeostasis. The effects of 7KC, 24S-OHC and C24:0 on lipid membrane organization and membrane potential were examined with merocyanine 540 (MC540) and bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2(3)), respectively. The intracellular concentration of K+ ([K+]i) was measured by flame photometry and the ratiometric approach using the PBFI-AM fluorescence indicator. To determine the relationships between [K+]i and lipotoxicity, 158N cells were pre-treated with a universal Kv channels blocker, 4-aminopyridine (4-AP), without or with 7KC, 24S-OHC or C24:0. Cell adhesion, cell growth, mitochondrial depolarization, cytoplasmic membrane integrity, the presence of SubG1 and the morphological aspect of the nuclei were determined with various microscopy, flow cytometry and biochemistry methods. 7KC, 24S-OHC and C24:0 induced changes in lipid content and polarization of the cytoplasmic membrane. These events were associated with increased [K+]i. Blocking Kv channels with 4-AP exacerbated 7KC-, 24S-OHC- and C24:0-induced cell dysfunction. 4-AP exacerbated loss of cell adhesion and cell growth inhibition, amplified mitochondrial depolarization and cytoplasmic membrane damage, and increased the percentage of SubG1 cells. The positive correlation between [K+]i and cell death supports the potential involvement of K+ in 7KC-, 24S-OHC-, and C24:0-induced cytotoxicity.

International audience; The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.

International audience; Microglia are the resident macrophages of the central nervous system (CNS). Gene expression profiling has identified Sall1, which encodes a transcriptional regulator, as a microglial signature gene. We found that Sall1 was expressed by microglia but not by other members of the mononuclear phagocyte system or by other CNS-resident cells. Using Sall1 for microglia-specific gene targeting, we found that the cytokine receptor CSF1R was involved in the maintenance of adult microglia and that the receptor for the cytokine TGF-β suppressed activation of microglia. We then used the microglia-specific expression of Sall1 to inducibly inactivate the murine Sall1 locus in vivo, which resulted in the conversion of microglia from resting tissue macrophages into inflammatory phagocytes, leading to altered neurogenesis and disturbed tissue homeostasis. Collectively, our results show that transcriptional regulation by Sall1 maintains microglial identity and physiological properties in the CNS and allows microglia-specific manipulation in vivo.

Excitability differs among muscle fibers and undergoes continuous changes during development and growth, yet the neuromuscular synapse maintains a remarkable fidelity of execution. Here we show in two evolutionarily distant vertebrates (Xenopus laevis cell culture and mouse nerve-muscle ex-vivo) that the skeletal muscle cell constantly senses, through two identified calcium signals, synaptic events and their efficacy in eliciting spikes. These sensors trigger retrograde signal(s) that control presynaptic neurotransmitter release, resulting in synaptic potentiation or depression. In the absence of spikes, synaptic events trigger potentiation. Once the synapse is sufficiently strong to initiate spiking, the occurrence of these spikes activates a negative retrograde feedback. These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability. DOI: http://dx.doi.org/10.7554/eLife.12190.001, eLife digest Nerve cells communicate with each other, and with targets such as muscle cells, at junctions called synapses. The nerve cell before the synapses releases a chemical called a neurotransmitter, which binds to receptors on the cell after the synapses. However, the first cell cannot determine by itself whether it is releasing the correct amount of neurotransmitter to activate its partner. For this, it requires feedback from the second cell. This feedback is particularly important at synapses between nerve cells and muscle cells, which are known as neuromuscular junctions. The likelihood that a given amount of transmitter will activate a muscle cell can vary with age and after exercise. Muscle cells must therefore be able to instruct their nerve cell partners to increase or decrease neurotransmitter release to accommodate these changes. Ouanounou et al. have now identified the mechanism by which muscle cells determine whether nerve cells are releasing an appropriate amount of neurotransmitter. Experiments in two distantly related animals – mice and embryos from a frog called Xenopus – revealed that muscle cells use two calcium-based signals. The first is the flow of calcium ions into the muscle cell in response to binding of neurotransmitter to receptors at the synapses: this tells the muscle cell how active the nerve cell is. The second is the release of calcium ions from internal stores inside the muscle cell: this occurs whenever neurotransmitter release is sufficient to activate the muscle cell. In response to the first calcium signal, the muscle cell sends positive feedback to the neuron, telling it to increase neurotransmitter release further. In response to the second signal, the muscle cell sends negative feedback to reduce neurotransmitter release. Thus, when neurotransmitter release is not enough to activate the muscle, positive feedback dominates and neurotransmitter release increases. However, when the muscle is activated, the two types of feedback act in balance to maintain efficient communication across the synapse. The next steps are to identify the cell signaling cascades that are mobilized by the two calcium signals, including the specific molecule (or molecules) that regulate neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.12190.002

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase ( tdo-2 ), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l -tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.

Development of Myeloid Immune Cells As leukocytes develop to maturity, they proceed through an array of phenotypically distinct intermediates. For T and B lymphocyte populations, the different developmental stages, anatomical locations, and cell signals required for progression are well established. However, until recently, much less has been known about how development proceeds in the myeloid lineage, which includes monocytes, macrophages, and dendritic cells. Geissmann et al. (p. 656 ) review our current understanding of myeloid lineage development and describe the developmental pathways and cues that drive differentiation.

Objective— Bexarotene (Targretin) is a clinically used antitumoral agent which exerts its action through binding to and activation of the retinoid-X-receptor (RXR). The most frequent side-effect of bexarotene administration is an increase in plasma triglycerides, an independent risk factor of cardiovascular disease. The molecular mechanism behind this hypertriglyceridemia remains poorly understood. Methods and Results— Using wild-type and LXRα/β-deficient mice, we show here that bexarotene induces hypertriglyceridemia and activates hepatic LXR-target genes of lipogenesis in an LXR-dependent manner, hence exerting a permissive effect on RXR/LXR heterodimers. Interestingly, RNA analysis and Chromatin Immunoprecipitation assays performed in the liver reveal that the in vivo permissive effect of bexarotene on the RXR/LXR heterodimer is restricted to lipogenic genes without modulation of genes controlling cholesterol homeostasis. Conclusion— These findings demonstrate that the hypertriglyceridemic action of bexarotene occurs via the RXR/LXR heterodimer and show that RXR heterodimers can act with a selective permissivity on target genes of specific metabolic pathways in the liver.

International audience; BACKGROUND AND PURPOSE: Obstructive sleep apnea syndrome (OSAS) is an independent risk factor for stroke. Impairment of cerebral autoregulation may play a potential role in the pre-disposition to stroke of OSAS patients. In this study, we aimed to assess dynamic cerebral autoregulation (DCA) during wakefulness in OSAS patients and a group of matched controls. METHODS: Patients and controls were examined in the morning after an overnight complete polysomnography. Mean cerebral blood flow velocity (CBFV) in the middle cerebral artery and mean arterial blood pressure (ABP) were continuously recorded using transcranial Doppler and Finapres. DCA was assessed using the Mx autoregulatory index. Mx is a moving correlation coefficient between mean CBFV and mean ABP. More positive value of Mx indicates worse autoregulation. RESULTS: Eleven OSAS patients (mean age +/- SD; 52.6 +/- 7.9) and 9 controls (mean age +/- SD; 49.1 +/- 5.3) were enrolled. The mean apnea-hypopnea index (AHI) in the OSAS group was of 22.7 +/- 11.6. No significant difference was found between the two groups as for age, body mass index, mean ABP and endtidal CO(2) pressure. Cerebral autoregulation was impaired in OSAS patients compared with controls (Mx index: 0.414 +/- 0.138 vs. 0.233 +/- 0.100; P = 0.009). The severity of autoregulation impairment correlated to the severity of the sleep respiratory disturbance measured by the AHI (P = 0.003). CONCLUSION: Cerebral autoregulation is impaired in patients with OSAS during wakefulness. Impairment of cerebral autoregulation is correlated with the severity of OSAS.

International audience; Disruption of iron homeostasis at the levels of intestinal absorption or erythropoiesis contributes to cadmium toxicity. Cellular iron homeostasis in metazoans is maintained by the iron regulatory proteins (IRPs) that regulate the synthesis of proteins involved in the transport, use, and storage of iron. The effect of cadmium intoxication on this regulatory system has been investigated in a cellular model of human epithelium. Cadmium exposure of HeLa cells did not activate the IRPs; rather, the amount of these proteins relative to that of housekeeping proteins decreased. Accordingly, the transferrin receptor mRNA level decreased upon cadmium insult. In a more integrated investigation, separate groups of mice had free access to different doses of cadmium in drinking water for 3 weeks. Cadmium accumulated in all analyzed organs, but its concentration in mouse tissues did not correlate with changes of the activity of the IRPs. The intoxicated mice did not show any sign of anemia, indicating that iron homeostasis was not immediately disrupted after the onset of cadmium accumulation. These data establish that cadmium destabilizes IRPs in mammalian cells, but that iron imbalance is not an early event of cadmium intoxication.

International audience; The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.

International audience; Apelin is a neuropeptide that co-localizes with vasopressin (AVP) in magnocellular neurons and is involved in body fluid homeostasis. Osmotic stimuli have opposite effects on the regulation of apelin and AVP secretion in animal models, but whether this is true in humans is unknown. This study investigated the relationship among osmolality, apelin, and AVP in 10 healthy men after infusion of hypertonic saline or loading with water to increase and decrease plasma osmolality, respectively. Increasing plasma osmolality was accompanied by a parallel, linear increase in plasma AVP concentration and by a decrease in plasma apelin concentration. In contrast, decreasing plasma osmolality by water loading reduced plasma AVP concentration and rapidly increased plasma apelin concentration. These findings suggest that regulation of apelin secretion contributes to the maintenance of body fluid homeostasis.

International audience; Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

Renal sodium retention in experimental liver cirrhosis originates from the distal nephron sensitive to aldosterone. The aims of this study were to (1) determine the exact site of sodium retention along the aldosterone-sensitive distal nephron, and (2) to evaluate the role of aldosterone and mineralocorticoid receptor activation in this process. Liver cirrhosis was induced by bile duct ligation in either adrenal-intact or corticosteroid-clamped mice. Corticosteroid-clamp was achieved through adrenalectomy and corticosteroid supplementation with aldosterone and dexamethasone via osmotic minipumps. 24-hours renal sodium balance was evaluated in metabolic cages. Activity and expression of sodium- and potassium-dependent adenosine triphosphatase were determined in microdissected segments of nephron. Within 4-5 weeks, cirrhosis induced sodium retention in adrenal-intact mice and formation of ascites in 50% of mice. At that time, sodium- and potassium-dependent adenosine triphosphatase activity increased specifically in cortical collecting ducts. Hyperaldosteronemia was indicated by increases in urinary aldosterone excretion and in sgk1 (serum- and glucocorticoid-regulated kinase 1) mRNA expression in collecting ducts. Corticosteroid-clamp prevented induction of sgk1 but not cirrhosis-induced sodium retention, formation of ascites and stimulation of sodium- and potassium-dependent adenosine triphosphatase activity and expression (mRNA and protein) in collecting duct. These findings demonstrate that sodium retention in cirrhosis is independent of hyperaldosteronemia and of the activation of mineralocorticoid receptor. Conclusion: Bile duct ligation in mice induces cirrhosis which, within 4-5 weeks, leads to the induction of sodium- and potassium-dependent adenosine triphosphatase in cortical collecting ducts, to renal sodium retention and to the formation of ascites. Sodium retention, ascites formation and induction of sodium- and potassium-dependent adenosine triphosphatase are independent of the activation of mineralocorticoid receptors by either aldosterone or glucocorticoids. (HEPATOLOGY 2007;46:173–179.)

International audience; In bacteria-host interactions, competition for iron is critical for the outcome of the infection. As a result of its redox properties, this metal is essential for the growth and proliferation of most living organisms, including pathogenic bacteria. This metal is also potentially toxic, making the precise maintenance of iron homeostasis necessary for survival. Iron acquisition and storage control is mediated in most bacteria by the global ferric uptake regulator (Fur) and iron-responsive small regulatory non-coding RNAs (RyhB in the model organism Escherichia coli). While the role of these regulators in iron homeostasis is well documented in both pathogenic and non-pathogenic bacteria, many recent studies also demonstrate that these regulators are involved in the virulence of pathogenic bacteria. By sensing iron availability in the environment, Fur and RyhB are able to regulate, either directly or indirectly via other transcriptional regulators or modulation of intracellular iron concentration, many virulence determinants of pathogenic bacteria. Iron is thus both a nutritional and regulatory element, allowing bacteria to adapt to various host environments by adjusting expression of virulence factors. In this review, we present evidences that Fur and RyhB are the major regulators of this adaptation, as they are involved in diverse functions ranging from iron homeostasis to regulation of virulence by mediating key pathogen responses such as invasion of eukaryotic cells, toxin production, motility, quorum sensing, stress resistance or biofilm formation. Therefore, Fur and RyhB play a major role in regulating an adaptative response during bacterial infections, making them important targets in the fight against pathogenic bacteria.

International audience; Kupffer cells, the phagocytes of fetal origin that line the liver sinusoids, are key contributors of host defense against enteroinvasive bacteria. Here, we found that infection by Listeria monocytogenes induced the early necroptotic death of Kupffer cells, which was followed by monocyte recruitment and an anti-bacterial type 1 inflammatory response. Kupffer cell death also triggered a type 2 response that involved the hepatocyte-derived alarmin interleukin-33 (IL-33) and basophil-derived interleukin-4 (IL-4). This led to the alternative activation of the monocyte-derived macrophages recruited to the liver, which thereby replaced ablated Kupffer cells and restored liver homeostasis. Kupffer cell death is therefore a key signal orchestrating type 1 microbicidal inflammation and type-2-mediated liver repair upon infection. This indicates that beyond the classical dichotomy of type 1 and type 2 responses, these responses can develop sequentially in the context of a bacterial infection and act interdependently, orchestrating liver immune responses and return to homeostasis, respectively.

We previously showed that pancreatic β-cells express neuronal nitric oxide synthase (nNOS) that controls insulin secretion through two catalytic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now provide evidence that the endogenous protein inhibitor of nNOS (PIN) is expressed in rat pancreatic islets and INS-1 cells. Double-immunofluorescence studies showed a colocalization of PIN with both nNOS and myosin Va in insulin-secreting β-cells. Electron microscopy studies confirmed that PIN is mainly associated with insulin secretory granules and colocated with nNOS in the latter. In addition, PIN overexpression in INS-1 cells enhanced glucose-induced insulin secretion, which is only partly reversed by addition of an NO donor, sodium nitroprusside (SNP), and unaffected by the inhibitor of cytochrome c reductase activity, miconazole. In contrast, the pharmacological inhibitor of nNOS, Nω-nitro-l-arginine methyl ester, amplified glucose-induced insulin secretion, an effect insensitive to SNP but completely normalized by the addition of miconazole. Thus, PIN insulinotropic effect could be related to its colocalization with the actin-based molecular motor myosin Va and as such be implicated in the physiological regulation of glucose-induced insulin secretion at the level of the exocytotic machinery.

International audience; Bile acids (BAs), a group of structurally diverse molecules that are primarily synthesized in the liver from cholesterol, are the chief components of bile. Besides their well-established roles in dietary lipid absorption and cholesterol homeostasis, it has recently emerged that BAs are also signaling molecules, with systemic endocrine functions. BAs activate mitogen-activated protein kinase pathways, are ligands for the G-protein-coupled receptor TGR5, and activate nuclear hormone receptors such as farnesoid X receptor alpha. Through activation of these diverse signaling pathways, BAs can regulate their own enterohepatic circulation, but also triglyceride, cholesterol, energy, and glucose homeostasis. Thus, BA-controlled signaling pathways are promising novel drug targets to treat common metabolic diseases, such as obesity, type II diabetes, hyperlipidemia, and atherosclerosis.

International audience; With the aim of finding important mediators of muscle atrophy, we cloned SMHS1, a novel gene that was found to be upregulated in rat soleus muscle atrophied by restriction of activity. The SMHS1 amino acid sequence shares 65% similarity with RTP801-which is a cellular stress response protein regulated by HIF-1-but SMHS1 expression was demonstrated to be independent of HIF-1. SMHS1 was found to be mainly expressed in skeletal muscle, and comparisons of its expression in atrophied versus hypertrophied muscles and in oxidative versus glycolytic muscles suggested that SMHS1 contributes to the muscle energy metabolism phenotypes.

NCB5OR is a highly conserved NAD(P)H reductase that contains a cytochrome b5-like domain at the N terminus and a cytochrome b5 reductase-like domain at the C terminus. The enzyme is located in the endoplasmic reticulum (ER) and is widely expressed in organs and tissues. Targeted inactivation of this gene in mice has no impact on embryonic or fetal viability. At 4 weeks of age, Ncb5or -/- mice have normal blood glucose levels but impaired glucose tolerance. Isolated Ncb5or -/- islets have markedly impaired glucose- or arginine-stimulated insulin secretion. By 7 weeks of age, these mice develop severe hyperglycemia with markedly decreased serum insulin levels and nearly normal insulin tolerance. As the animals age, there is a progressive loss of beta cells in pancreatic islets, but there is no loss of alpha, delta, or PP cells. Electron microscopy reveals degranulation of beta cells and hypertrophic and hyperplastic mitochondria, some of which contain electron dense inclusions. Four-week-old Ncb5or -/- mice have enhanced sensitivity to the diabetogenic agent streptozotocin. NCB5OR appears to play a critical role in protecting pancreatic beta cells against oxidant stress.

TxeR, a sigma factor that directs Clostridium difficile RNA polymerase to recognize the promoters of two major toxin genes, was shown to stimulate its own synthesis. Whether expressed in C. difficile , Clostridium perfringens , or Escherichia coli , TxeR stimulated transcription of fusions of the txeR promoter region to reporter genes. As is the case for the tox genes, txeR expression was responsive to the cellular growth phase and the constituents of the medium. That is, the level of expression in broth culture was low during the exponential growth phase, but rapidly increased as cells approached the stationary phase. In the presence of excess glucose, expression from the txeR promoter was repressed. The results support a model for toxin gene expression in which synthesis of TxeR is induced by specific environmental signals. The increased level of TxeR then permits high-level expression of the toxin genes. The study of txeR gene regulation in C. difficile was made possible by introduction of a mobilizable, replicative plasmid via conjugation with E. coli .

We show that the lymphoid hyperplasia observed in IL-2Rα- and IL-2-deficient mice is due to the lack of a population of regulatory cells essential for CD4 T cell homeostasis. In chimeras reconstituted with bone marrow cells from IL-2Rα-deficient donors, restitution of a population of CD25+CD4+ T cells prevents the chaotic accumulation of lymphoid cells, and rescues the mice from autoimmune disease and death. The reintroduction of IL-2-producing cells in IL-2-deficient chimeras establishes a population of CD25+CD4+ T cells, and restores the peripheral lymphoid compartments to normal. The CD25+CD4+ T cells regulated selectively the number of naive CD4+ T cells transferred into T cell-deficient hosts. The CD25+CD4+/naive CD4 T cell ratio and the sequence of cell transfer determines the homeostatic plateau of CD4+ T cells. Overall, our findings demonstrate that IL-2Rα is an absolute requirement for the development of the regulatory CD25+CD4+ T cells that control peripheral CD4 T cell homeostasis, while IL-2 is required for establishing a sizeable population of these cells in the peripheral pools.

International audience; It is well known that the physiological impact imposed by events or behaviors displayed during the waking period determines the way organisms sleep. Among the situations known to affect sleep both in its duration and quality, stress has been widely studied and it is now admitted that its effects on sleep architecture depend on several factors specific to the stressor or the individual itself. Although numerous reports have highlighted the prominent role of the circadian cycle in the physiological, endocrine and behavioral consequences of restraint stress, a possible circadian influence in the effects of stress on the sleep-wake cycle has never been studied. Thus the present study was designed to compare the effects on sleep of a 1 h-lasting restraint stress applied at light onset to those observed after the same stressor was applied at light offset. We report that in both conditions stress induced a marked paradoxical sleep increase, whereas wakefulness displayed a moderate decrease and slow wave sleep a moderate augmentation. Although the effects of stress at lights on were of similar magnitude than those of stress at lights off, important differences in the sleep rebound latencies were observed: whatever the time of day the stress was applied, its effects on sleep always occurred during the dark period. This result thus shows that restraint stress could be efficiently used to study the interaction between the circadian and homeostatic components of sleep regulation.

International audience; The B cell-activating factor of the tumor-necrosis factor family (BAFF) plays a dominant role in the B cell homeostasis. By rescuing autoreactive B cells, excessive BAFF favors the development of autoimmune diseases. Given the numbers of variants of this B cell-specific cytokine, caution must be exercized when determining its serum level. Alternate splice isoforms, such as Δ3 BAFF and Δ4 BAFF, have been identified. They raise the possibility that their overproduction impact the synthesis of full-length BAFF.

NK cell differentiation mainly occurs in the bone marrow (BM) where a critical role in the regulation of developing lymphocyte distribution is played by members of the chemokine receptor family. In mouse, the chemokine receptor CX3CR1 identifies a late stage of NK cell development characterized by decreased effector functions and expression of the inhibitory receptor KLRG1. The role of CX3CR1 in the regulation of differentiation and positioning of NK cell subsets in the BM is not known. In this study, we found that CX3CR1 deficiency leads to accumulation of KLRG1+ NK cells in BM during steady-state conditions. The NK cell subset that expresses the receptor in wild-type mice was expanded in several tissues of CX3CR1-deficient mice, and NK cell degranulation in response to sensitive target cell stimulation was enhanced, suggesting a regulatory role of CX3CR1 in NK cell positioning and differentiation in BM. Indeed, the observed NK cell expansion was not due to altered turnover rate, whereas it was associated with preferential accumulation in the BM parenchyma. In addition, a role of CX3CR1 in NK cell trafficking from BM and spleen was evidenced also during inflammation, as CX3CR1-deficient NK cells were more prompt to exit the BM and did not decrease in spleen in response to polyinosinic-polycytidylic acid–promoted hepatitis. Overall, our results evidenced a relevant role of CX3CR1 in the regulation of NK cell subset exit from BM during homeostasis, and suggest that defect in the CX3CR1/CX3CL1 axis alters NK cell trafficking and functional response during inflammatory conditions.

Rationale: In atherosclerotic plaques, iron preferentially accumulates in macrophages where it can exert pro-oxidant activities. Objective: The objective of this study was, first, to better characterize the iron distribution and metabolism in macrophage subpopulations in human atherosclerotic plaques and, second, to determine whether iron homeostasis is under the control of nuclear receptors, such as the liver X receptors (LXRs). Methods and Results: Here we report that iron depots accumulate in human atherosclerotic plaque areas enriched in CD68 and mannose receptor (MR)-positive (CD68 + MR + ) alternative M2 macrophages. In vitro IL-4 polarization of human monocytes into M2 macrophages also resulted in a gene expression profile and phenotype favoring iron accumulation. However, M2 macrophages on iron exposure acquire a phenotype favoring iron release, through a strong increase in ferroportin expression, illustrated by a more avid oxidation of extracellular low-density lipoprotein by iron-loaded M2 macrophages. In line, in human atherosclerotic plaques, CD68 + MR + macrophages accumulate oxidized lipids, which activate LXRα and LXRβ, resulting in the induction of ABCA1, ABCG1, and apolipoprotein E expression. Moreover, in iron-loaded M2 macrophages, LXR activation induces nuclear factor erythroid 2-like 2 expression, thereby increasing ferroportin expression, which, together with a decrease of hepcidin mRNA levels, promotes iron export. Conclusions: These data identify a role for M2 macrophages in iron handling, a process regulated by LXR activation.

International audience; Prenatal stress impairs activity of the hypothalamo-pituitary-adrenal (HPA) axis in response to stress in adult offspring. So far, very few data are available on the effects of prenatal stress on circadian functioning of the HPA axis. Here, we studied the effects of prenatal stress on the circadian rhythm of corticosterone secretion in male and female adult rats. To evaluate the effects of prenatal stress on various regulatory components of corticosterone secretion, we also assessed the diurnal fluctuation of adrenocorticotropin, total and free corticosterone levels, and hippocampal corticosteroid receptors. Finally, in the search of possible maternal factors, we studied the effects of repeated restraint stress on the pattern of corticosterone secretion in pregnant female rats. Results demonstrate that prenatal stress induced higher levels of total and free corticosterone secretion at the end of the light period in both males and females, and hypercorticism over the entire diurnal cycle in females. No diurnal fluctuation of adrenocorticotropin was observed in any group studied. The effects of prenatal stress on corticosterone secretion could be mediated, at least in part, by a reduction in corticosteroid receptors at specific times of day. Results also show that prepartal stress alters the pattern of corticosterone secretion in pregnant females. Those data indicate that prenatally stressed rats exhibit an altered temporal functioning of the HPA axis, which, taken together with their abnormal response to stress, reinforces the idea of a general homeostatic dysfunction in those animals.

Although earlier, seminal studies demonstrated that the gut per se has the intrinsic ability to regulate the rates of iron absorption, the spotlight in the past decade has been placed on the systemic regulation of iron homeostasis by the hepatic hormone hepcidin and the molecular mechanisms that regulate its expression. Recently, however, attention has returned to the gut based on the finding that hypoxia inducible factor-2 (HIF-2α) regulates the expression of key genes that contribute to iron absorption. Here we review the current understanding of the molecular mechanisms that regulate iron homeostasis in the gut by focusing on the role of HIF-2 under physiological steady-state conditions and in the pathogenesis of iron-related diseases. We also discuss implications for adapting HIF-2–based therapeutic strategies in iron-related pathological conditions.

International audience; The nicotianamine synthase (NAS) enzymes catalyze the formation of nicotianamine (NA), a non-proteinogenic amino acid involved in iron homeostasis. We undertook the functional characterization of AtNAS4, the fourth member of the Arabidopsis thaliana NAS gene family. A mutant carrying a T-DNA insertion in AtNAS4 (atnas4), as well as lines overexpressing AtNAS4 both in the atnas4 and the wild-type genetic backgrounds, were used to decipher the role of AtNAS4 in NA synthesis, iron homeostasis and the plant response to iron deficiency or cadmium supply. We showed that AtNAS4 is an important source for NA. Whereas atnas4 had normal growth in iron-sufficient medium, it displayed a reduced accumulation of ferritins and exhibited a hypersensitivity to iron deficiency. This phenotype was rescued in the complemented lines. Under iron deficiency, atnas4 displayed a lower expression of the iron uptake-related genes IRT1 and FRO2 as well as a reduced ferric reductase activity. Atnas4 plants also showed an enhanced sensitivity to cadmium while the transgenic plants overexpressing AtNAS4 were more tolerant. Collectively, our data, together with recent studies, support the hypothesis that AtNAS4 displays an important role in iron distribution and is required for proper response to iron deficiency and to cadmium supply.

Objective— Endosomal signature phospholipid bis(monoacylglycero)phosphate (BMP) has been involved in the regulation of cellular cholesterol homeostasis. Accumulation of BMP is a hallmark of lipid storage disorders and was recently reported as a noticeable feature of oxidized low-density lipoprotein–laden macrophages. This study was designed to delineate the consequences of macrophage BMP accumulation on intracellular cholesterol distribution, metabolism, and efflux and to unravel the underlying molecular mechanisms. Approach and Results— We have developed an experimental design to specifically increase BMP content in RAW 264.7 macrophages. After BMP accumulation, cell cholesterol distribution was markedly altered, despite no change in low-density lipoprotein uptake and hydrolysis, cholesterol esterification, or total cell cholesterol content. The expression of cholesterol-regulated genes sterol regulatory element–binding protein 2 and hydroxymethylglutaryl-coenzyme A reductase was decreased by 40%, indicative of an increase of endoplasmic reticulum–associated cholesterol. Cholesterol delivery to plasma membrane was reduced as evidenced by the 20% decrease of efflux by cyclodextrin. Functionally, BMP accumulation reduced cholesterol efflux to both apolipoprotein A1 and high-density lipoprotein by 40% and correlated with a 40% decrease in mRNA contents of ATP-binding cassette transporter A1, ATP-binding cassette transporter G1, and liver-X receptor α and β. Foam cell formation induced by oxidized low-density lipoprotein exposure was exacerbated in BMP-enriched cells. Conclusions— The present work shows for the first time a strong functional link between BMP and cholesterol-regulating genes involved in both intracellular metabolism and efflux. We propose that accumulation of cellular BMP might contribute to the deregulation of cholesterol homeostasis in atheromatous macrophages.

International audience; Patrolling Ly6C(-) monocytes are blood-circulating cells that play a role in inflammation and in the defense against pathogens. Here, we show that similar to natural killer (NK) cells, patrolling monocytes express high levels of S1PR5, a G-coupled receptor for sphingosine-1 phosphate. We found that S1pr5(-/-) mice lack peripheral Ly6C(-) monocytes but have a normal number of these cells in the bone marrow (BM). Various lines of evidence exclude a direct contribution of S1PR5 in the survival of Ly6C(-) monocytes at the periphery. Rather, our data support a role for S1PR5 in the egress of Ly6C(-) monocytes from the BM. In particular, we observed a reduced frequency of patrolling monocytes in BM sinusoids of S1PR5 KO mice. Unexpectedly, S1P was not a chemoattractant for patrolling monocytes and had no significant effect on their viability in vitro. Moreover, the disruption of S1P gradients in vivo did not alter Ly6C(-) monocyte trafficking and viability. These data suggest that S1PR5 regulates the trafficking of monocytes via a mechanism independent of S1P gradients.

International audience; Iron (Fe) is an essential element for human nutrition. Given that plants represent a major dietary source of Fe worldwide, it is crucial to understand plant Fe homeostasis fully. A major breakthrough in the understanding of Fe sensing and signaling was the identification of several transcription factor cascades regulating Fe homeostasis. However, the mechanisms of activation of these cascades still remain to be elucidated. In this opinion, we focus on the possible roles of mitochondria and chloroplasts as cellular Fe sensing and signaling sites, offering a new perspective on the integrated regulation of Fe homeostasis and its interplay with cellular metabolism.

International audience; Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, one of the most common inherited small vessel diseases of the brain, is characterized by a progressive loss of vascular smooth muscle cells and extracellular matrix accumulation. The disease is caused by highly stereotyped mutations within the extracellular domain of the NOTCH3 receptor (Notch3(ECD)) that result in an odd number of cysteine residues. While CADASIL-associated NOTCH3 mutations differentially affect NOTCH3 receptor function and activity, they all are associated with early accumulation of Notch3(ECD)-containing aggregates in small vessels. We still lack mechanistic explanation to link NOTCH3 mutations with small vessel pathology. Herein, we hypothesized that excess Notch3(ECD) could recruit and sequester functionally important proteins within small vessels of the brain. We performed biochemical, nano-liquid chromatography-tandem mass spectrometry and immunohistochemical analyses, using cerebral and arterial tissue derived from patients with CADASIL and mouse models of CADASIL that exhibit vascular lesions in the end- and early-stage of the disease, respectively. Biochemical fractionation of brain and artery samples demonstrated that mutant Notch3(ECD) accumulates in disulphide cross-linked detergent-insoluble aggregates in mice and patients with CADASIL. Further proteomic and immunohistochemical analyses identified two functionally important extracellular matrix proteins, tissue inhibitor of metalloproteinases 3 (TIMP3) and vitronectin (VTN) that are sequestered into Notch3(ECD)-containing aggregates. Using cultured cells, we show that increased levels or aggregation of Notch3 enhances the formation of Notch3(ECD)-TIMP3 complex, promoting TIMP3 recruitment and accumulation. In turn, TIMP3 promotes complex formation including NOTCH3 and VTN. In vivo, brain vessels from mice and patients with CADASIL exhibit elevated levels of both insoluble cross-linked and soluble TIMP3 species. Moreover, reverse zymography assays show a significant elevation of TIMP3 activity in the brain vessels from mice and patients with CADASIL. Collectively, our findings lend support to a Notch3(ECD) cascade hypothesis in CADASIL disease pathology, which posits that aggregation/accumulation of Notch3(ECD) in the brain vessels is a central event, promoting the abnormal recruitment of functionally important extracellular matrix proteins that may ultimately cause multifactorial toxicity. Specifically, our results suggest a dysregulation of TIMP3 activity, which could contribute to mutant Notch3(ECD) toxicity by impairing extracellular matrix homeostasis in small vessels.

Lymphopenia induces T cells to undergo cell divisions as part of a homeostatic response mechanism. The clonal response to lymphopenia is extremely diverse, and it is unknown whether this heterogeneity represents distinct mechanisms of cell-cycle control or whether a common mechanism can account for the diversity. We addressed this question by combining in vivo and mathematical modeling of lymphopenia-induced proliferation (LIP) of two distinct T cell clonotypes. OT-I T cells undergo rapid LIP accompanied by differentiation that superficially resembles Ag-induced proliferation, whereas F5 T cells divide slowly and remain naive. Both F5 and OT-I LIP responses were most accurately described by a single stochastic division model where the rate of cell division was exponentially decreased with increasing cell numbers. The model successfully identified key biological parameters of the response and accurately predicted the homeostatic set point of each clone. Significantly, the model was successful in predicting interclonal competition between OT-I and F5 T cells, consistent with competition for the same resource(s) required for homeostatic proliferation. Our results show that diverse and heterogenous clonal T cell responses can be accounted for by a single common model of homeostasis.