Targeted lipidomic profiling of West Nile virus infection unveils alterations of sphingolipid metabolism in a mouse model and human patients

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of West Nile disease (WND) can curse with meningitis, encephalitis, or acute flaccid paralysis. A better understanding of the physiopathology associated with disease progression is mandatory to find biomarkers and effective therapies. In this scenario, blood derivatives (plasma and serum) constitute the more commonly used biofluids due to their ease of collection and high value for diagnostic purposes. Therefore, the potential impact of this virus in the circulating lipidome was addressed by combining the analysis of samples from experimentally infected mice and naturally WND patients. Our results unveil dynamic alterations in the lipidome that define specific metabolic fingerprints of different infection stages. Concomitant with neuroinvasion in mice, the lipid landscape was dominated by a metabolic reprogramming that resulted in significant elevations of circulating sphingolipids (ceramides, dihydroceramides and dihydrosphingomyelins), along with some phosphatidylethanolamines and triacylglycerols. Remarkably, patients suffering from WND also displayed an elevation of ceramides, dihydroceramides, and lactosylceramides in their sera. The dysregulation of sphingolipid metabolism by WNV may provide new therapeutic opportunities and supports the potential of certain lipids as novel peripheral biomarkers of WND progression

Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of “self-tagged” empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines

The Neutrophil Life Cycle.

Neutrophils are recognized as an essential part of the innate immune response, but an active debate still exists regarding the life cycle of these cells. Neutrophils first differentiate in the bone marrow through progenitor intermediaries before entering the blood, in a process that gauges the extramedullary pool size. Once believed to be directly eliminated in the marrow, liver, and spleen, neutrophils, after circulating for less than 1 day, are now known to redistribute into multiple tissues with poorly understood kinetics. In this review, we provide an update on the dynamic distribution of neutrophils across tissues in health and disease, and emphasize differences between humans and model organisms. We further highlight issues to be addressed to exploit the unique features of neutrophils in the clinic

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health.This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505)

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection.We thank all members of the Hidalgo Lab for discussion and insightful comments; J.M. Ligos, R. Nieto, and M. Viton for help with sorting and cytometric analyses; I. Ortega and E. Santos for animal husbandry; D. Rico, M.J. Gomez, C. Torroja, and F. Sanchez-Cabo for insightful comments and help with transcriptomic analyses; V. Labrador, E. Arza, A.M. Santos, and the Microscopy Unit of the CNIC for help with microscopy; S. Aznar-Benitah, U. Albrecht, Q.-J. Meng, B. Staels, and H. Duez for the generous gift of mice; J.A. Enriquez and J. Avila for scientific insights; and J.M. Garcia and A. Diez de la Cortina for art. This study was supported by Intramural grants from A* STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economia, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).S

Mechanisms of TSC-mediated Control of Synapse Assembly and Axon Guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development

Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection

Zika Virus: What Have We Learnt Since the Start of the Recent Epidemic?

Zika is a viral disease transmitted mainly by mosquitoes of the genus Aedes. In recent years, it has expanded geographically, changing from an endemic mosquito-borne disease across equatorial Asia and Africa, to an epidemic disease causing large outbreaks in several areas of the world. With the recent Zika virus (ZIKV) outbreaks in the Americas, the disease has become a focus of attention of public health agencies and of the international research community, especially due to an association with neurological disorders in adults and to the severe neurological and ophthalmological abnormalities found in fetuses and newborns of mothers exposed to ZIKV during pregnancy. A large number of studies have been published in the last 3 years, revealing the structure of the virus, how it is transmitted and how it affects human cells. Many different animal models have been developed, which recapitulate several features of ZIKV disease and its neurological consequences. Moreover, several vaccine candidates are now in active preclinical development, and three of them have already entered phase I clinical trials. Likewise, many different compounds targeting viral and cellular components are being tested in in vitro and in experimental animal models. This review aims to discuss the current state of this rapidly growing literature from a multidisciplinary perspective, as well as to present an overview of the public health response to Zika and of the perspectives for the prevention and treatment of this disease

Testing a global standard for quantifying species recovery and assessing conservation impact

Recognizing the imperative to evaluate species recovery and conservation impact, in 2012 the International Union for Conservation of Nature (IUCN) called for development of a “Green List of Species” (now the IUCN Green Status of Species). A draft Green Status framework for assessing species’ progress toward recovery, published in 2018, proposed 2 separate but interlinked components: a standardized method (i.e., measurement against benchmarks of species’ viability, functionality, and preimpact distribution) to determine current species recovery status (herein species recovery score) and application of that method to estimate past and potential future impacts of conservation based on 4 metrics (conservation legacy, conservation dependence, conservation gain, and recovery potential). We tested the framework with 181 species representing diverse taxa, life histories, biomes, and IUCN Red List categories (extinction risk). Based on the observed distribution of species’ recovery scores, we propose the following species recovery categories: fully recovered, slightly depleted, moderately depleted, largely depleted, critically depleted, extinct in the wild, and indeterminate. Fifty-nine percent of tested species were considered largely or critically depleted. Although there was a negative relationship between extinction risk and species recovery score, variation was considerable. Some species in lower risk categories were assessed as farther from recovery than those at higher risk. This emphasizes that species recovery is conceptually different from extinction risk and reinforces the utility of the IUCN Green Status of Species to more fully understand species conservation status. Although extinction risk did not predict conservation legacy, conservation dependence, or conservation gain, it was positively correlated with recovery potential. Only 1.7% of tested species were categorized as zero across all 4 of these conservation impact metrics, indicating that conservation has, or will, play a role in improving or maintaining species status for the vast majority of these species. Based on our results, we devised an updated assessment framework that introduces the option of using a dynamic baseline to assess future impacts of conservation over the short term to avoid misleading results which were generated in a small number of cases, and redefines short term as 10 years to better align with conservation planning. These changes are reflected in the IUCN Green Status of Species Standard