Environmental pollution promotes selection of microbial degradation pathways

Astonishing as it may seem, one organism's waste is often ideal food for another. Many waste products generated by human activities are routinely degraded by microorganisms under controlled conditions during waste-water treatment. Toxic pollutants resulting from inadvertent releases, such as oil spills, are also consumed by bacteria, the simplest organisms on Earth. Biodegradation of toxic or particularly persistent compounds, however, remains problematic. What has escaped the attention of many is that bacteria exposed to pollutants can adapt to them by mutating or acquiring degradative genes. These bacteria can proliferate in the environment as a result of the selection pressures created by pollutants. The positive outcome of selection pressure is that harmful compounds may eventually be broken down completely through biodegradation. The downside is that biodegradation may require extremely long periods of time. Although the adaptation process has been shown to be reproducible, it remains very difficult to predict.

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression.

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, α(V)β(3)-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.

Endoplasmic reticulum stress promotes inflammation through activation of the NLRP3 inflammasome

SummaryMulticellular organisms have evolved an immune system in order to cope with the constant threats they are facing. Foreign pathogens or endogenous danger signals released by injured or dying host cells can be readily detected through a set of germline- encoded pattern-recognition receptors. The NOD-like receptors are a cytoplasmic family of pattern-recognition receptors that have recently attracted considerable attention due to their ability to form inflammasomes, which are molecular complexes responsible for the activation of caspase-1 and the subsequent processing of the pro¬inflammatory cytokines IL-IB and 11-18 into their mature, bioactive form.In this study, we describe a novel pro-inflammatory signaling pathway, whereby the endoplasmic reticulum promotes inflammation through activation of the NLRP3 inflammasome. This was shown to be independent of the classical endoplasmic reticulum stress response pathway constituted by the effectors IREla, PERK and ATF6a. In keeping with other known NLRP3 activators, generation of reactive oxygen species and potassium efflux were required. We also provide evidence that calcium signaling is critical to this pathway, and possibly integrates signaling triggered by various NLRP3 inflammasome activators. Moreover, the mitochondrial channel VDAC1 was instrumental in mediating this response. We thus propose that the endoplasmic reticulum acts as an integrator of stress and is able to activate the mitochondria in a calcium-dependent manner in order to promote NLRP3 inflammasome activation in response to a wide range of activators.Given the role played by inflammation in the pathogenesis of atherosclerosis, we decided to investigate a possible role for the NLRP3 inflammasome in the progression of the disease. Using an ApoE mouse model, we find that deficiency in the NLRP3 inflammasome components NLRP3, ASC or Caspase-1 does not impair atherosclerosis progression, nor does it impact plaque stability. While previous studies have clearly shown a role for the interleukin-1 family of ligands in atherosclerosis, our results suggest that its contribution might be more complex than previously appreciated, and further research is thus warranted in this field.RésuméLes organismes multicellulaires ont développé un système immunitaire pour faire face aux menaces qui les entourent. Des pathogènes étrangers ou des signaux de danger relâchés par des cellules de l'hôte en détresse peuvent être rapidement détectés via un assemblage de récepteurs spécifiques qui sont présents dès la naissance. Certains membres de la famille de récepteurs NOD ont récemment attiré beaucoup d'attention au vu de leur capacité à former des inflammasomes, complexes moléculaires responsables de l'activation de la easpase-1 et de la maturation des cytokines pro-inflammatoires IL- 1β et IL-18 en leur forme bioactive.Dans cette étude, nous décrivons une nouvelle voie de signalisation pro-inflammatoire, par laquelle le réticulum endoplasmique induit l'inflammation via l'activation de l'inflammasome NLRP3. Cette voie est indépendante de la voie classique de réponse au stress du réticulum endoplasmique, qui comprend les effecteurs IRE1, PERK et ATF6. Comme pour d'autres activateurs de NLRP3, la génération de radicaux libres d'oxygène ainsi que Γ efflux de potassium sont requis. Nous montrons également que le calcium joue un rôle critique dans cette voie, et intègre possiblement la signalisation provoquée par divers activateurs de l'inflammasome NLRP3. De plus, le canal mitochondrial VDAC1 est essentiel dans cette réponse. Nous proposons donc que le réticulum endoplasmique agit comme un intégrateur de stress, activant la mitochondrie d'une façon calcium-dépendante pour promouvoir l'activation de l'inflammasome NLRP3 en réponse à divers activateurs.Au vu du rôle joué par l'inflammation dans la pathogenèse de l'athérosclérose, nous avons étudié un possible rôle pour l'inflammasome NLRP3 dans la progression de la maladie. Dans un modèle de souris ApoE, l'absence des composants de l'inflammasome NLRP3 que sont NLRP3, ASC et Caspase-1 n'influence pas la progression des plaques ni leur stabilité. Alors que d'autres études ont démontré un rôle pour les membres de la famille de l'interleukine-1 dans l'athérosclérose, nos résultats suggèrent que leur contribution pourrait être plus complexe que précédemment apprécié, et d'autres recherches dans ce domaine sont donc nécessaires.

PSD-95 promotes synaptogenesis and multiinnervated spine formation through nitric oxide signaling

Postsynaptic density 95 (PSD-95) is an important regulator of synaptic structure and plasticity. However, its contribution to synapse formation and organization remains unclear. Using a combined electron microscopic, genetic, and pharmacological approach, we uncover a new mechanism through which PSD-95 regulates synaptogenesis. We find that PSD-95 overexpression affected spine morphology but also promoted the formation of multiinnervated spines (MISs) contacted by up to seven presynaptic terminals. The formation of multiple contacts was specifically prevented by deletion of the PDZ(2) domain of PSD-95, which interacts with nitric oxide (NO) synthase (NOS). Similarly, PSD-95 overexpression combined with small interfering RNA-mediated down-regulation or the pharmacological blockade of NOS prevented axon differentiation into varicosities and multisynapse formation. Conversely, treatment of hippocampal slices with an NO donor or cyclic guanosine monophosphate analogue induced MISs. NOS blockade also reduced spine and synapse density in developing hippocampal cultures. These results indicate that the postsynaptic site, through an NOS-PSD-95 interaction and NO signaling, promotes synapse formation with nearby axons.

alphaENaC-mediated lithium absorption promotes nephrogenic diabetes insipidus.

Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.

Coevolutionary arms races: increased host immune defense promotes specialization by avian fleas.

We investigated the relationship between host defense and specialization by parasites in comparative analyses of bird fleas and T-cell mediated immune response of their avian hosts, showing that fleas with few main host species exploited hosts with weak or strong immune defenses, whereas flea species that parasitized a large number of host species only exploited hosts with weak immune responses. Hosts with strong immune responses were exploited by a larger number of flea species than hosts with weak responses. A path analysis model with an effect of T-cell response on the number of host species, or a model with host coloniality directly affecting host T-cell response, which in turn affected the number of host species used by fleas, best explained the data. Therefore, parasite specialization may have evolved in response to strong host defenses.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

The endocrine disruptor monoethyl-hexyl-phthalate is a selective peroxisome proliferator-activated receptor gamma modulator that promotes adipogenesis.

The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARgamma and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARgamma target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARgamma coregulators that includes Med1 and PGC-1alpha, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.

The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

PTEN downregulation in adipocytes promotes inflammation of adipose tissue

Background and aims: The phosphoinositide phosphatase PTEN is a potent tumor suppressor and a regulator of insulin sensitivity in peripheral tissues. In adipocytes, experimental alterations of PTEN expression modulate the sensitivity of these cells to insulin. However, virtually nothing is known about the pathophysiological regulation of endogenous PTEN in adipose tissue. Herein, we investigated in vivo and in vitro whether alterations of PTEN expression in adipocytes are associated with the metabolic syndrome and what are the functional outcomes of dysregulated PTEN expression/activity. Materials and methods: PTEN expression was examined in vivo in adipose tissue of rats and human with the metabolic syndrome. Metabolic factors mediating dysregulation of PTEN expression in adipocytes and the subsequent effects on the physiology of these cells were investigated in vitro using human CHUB-S7 preadipocytes. Results: We demonstrated that PTEN is downregulated, both at the mRNA and protein levels, in adipose tissue of diabetic/obese ZDF rats and in subcutaneous adipose tissue of obese human patients. PTEN downregulation correlated with degradation of IκBα and hyperactivation of NF-κB, a transcription factor previously described to modulate PTEN expression. The expression of SHIP2, another PtdIns(3,4,5)P3 phosphatase involved in the control of insulin sensitivity and the development of obesity, was not altered. In vitro analyses using differentiated human CHUB-S7 preadipocytes showed that PTEN downregulation is not triggered by high concentrations of glucose or fatty acids. In contrast, the pro-inflammatory cytokines IL-1α and TNFα, significantly downregulate PTEN expression. Consistent with the IL1α-dependent PTEN downregulation, long-term incubation of CHUB-S7 cells with IL-1α potentiates insulin-induced Akt and ERK1/2 signaling. We finally showed that PTEN downregulation in CHUB-S7 preadipocytes by PTEN siRNAs induced an increased secretion of the pro-inflammatory cytokines IL-1β, IL-6 and TNFα. Conclusion: Taken together, these data indicate that PTEN expression is downregulated in adipose tissue of obese/diabetic subjects, potentially via cytokine- mediated activation of the NF-κB pathway. PTEN downregulation in adipocytes might in turn worsen adipose tissue inflammation through a vicious circle by further stimulating the secretion of pro-inflammatory cytokines.