Pollution stress on aquatic microbial communities

Thousands of chemical compounds enter the natural environment but many have unknown effects and consequences, in particular at low concentrations. This thesis work contributes to our understanding of pollution effects by using bacteria as test organisms. Bacteria are important for this question because some of them degrade and transform pollutants into less harmful compounds, but secondly because they themselves can be inhibited in their reproduction by exposure to toxic compounds. When inhibitory effects occur this may change the composition of the microbial com¬munity in the long run, leading to altered or diminished ecosystem services by those communities. As a result chemicals of anthropogenic origin may accumulate and per¬sist in the environment, and finally, affect higher organisms as well. In addition to acquiring basic understanding of pollutant effects at low concentrations on bacterial communities an applied goal of this thesis work was to develop bacteria-based tests to screen new organic chemicals for toxicity and biodégradation. In the first part of this work we developed a flow cytometry-based assay on SYT09 plus ethidium-bromide or propidium-iodide stained cells of Pseudomonas ûuorescens exposed or not to a variety of pollutants under oligotrophic growth conditions. Flow cytometry (FC) allows fast and accurate counting of bacterial cells under simul¬taneous assessment of their physiological state, in particular in combination with different fluorescent dyes. Here we employed FC and fluorescent dyes to monitor the effect that pollutants may exert on Pseudomonas ûuorescens SV3. First we designed an oligotrophic growth test, which enabled us to follow population growth at low densities (104 - 10 7 cells per ml) using 0.1 mM sodium acetate as carbon source. Cells in the oligotrophic milieu were then exposed or not to a variety of common pollutants, such as 2-chlorobiphenyl (2CBP), naphthalene (NAH), 4-chlorophenol (4CP), tetradecane (TD), mercury chloride (HgCl2) or benzene, in different dosages. Exposed culture samples were stained with SYT09 (green fluorescent dye binding nucleic acids, generally staining all cells) in combination with propidium iodide (PI) or ethidium bromide (EB), both dyes being membrane integrity indicators. We ob- served that most of the tested compounds decreased population growth in a dosage- dependent manner. SYT09/PI or SYT09/EB staining then revealed that chemical exposure led to arisal of subpopulations of live and injured or dead cells. By modeling population growth on the total cell numbers in population or only the subpopulation of live cells we inferred that even in stressed populations live cells multiply at rates no different to unexposed controls. The net decrease in population growth would thus be a consequence of more and more cells being not able to multiply at all, rather than all cells multiplying at slower rates. In addition, the proportion of injured cells correlated to the compound dosage. We concluded that the oligotrophic test may be useful to asses toxicity of unknown chemicals on a variety of model bacteria. Mul¬tiple tests can be run in parallel and effects are rapidly measured within a period of 8 hours. Interestingly, in the same exposure tests with P. fluorescens SV3 we observed that some chemicals which did not lead to a reduction of net population growth rates did cause measurable effects on live cells. This was mainly observed in cells within the live subpopulation as an increase of the EB fluorescence signal. We showed that SYT09/EB is a more useful combination of dyes than SYT09/PI because PI fluorescence tend to increase only when cells are effectively dead, but not so much in live cells (less then twofold). In contrast, EB geometric mean fluorescence in live cells increased up to eightfold after exposure to toxic compounds. All compounds even at the lowest concentration caused a measurable increase in EB geometric mean fluorescence especially after 2 h incubation time. This effect was found to be transient for cells exposed to 2CBP and 4CP, but chronic for cells incubated with TD and NAH (ultimately leading to cell death). In order to understand the mechanism underlying the observed effects we used known membrane or energy uncouplers. The pattern of EB signal increase in chemical-exposed populations resembled mostly that of EDTA, although EB fluorescence in EDTA-treated or pasteurized cells was even higher than after exposure to the four test chemicals. We conclude that the ability of cells to efflux EB under equilibrium conditions is an appropriate measure for the potential of a chemical to exert toxicity. Since most bacterial species possess efflux systems for EB that all require cellular energy, our test should be more widely relevant to infer toxicity effects of chemical exposure on the physiological status of the bacterial cell. To better understand the effect of toxicant exposure on efflux defense systems, we studied 2-hydroxybiphenyl toxicity to Pseudomonas azeiaica HBP1. We showed that 2-HBP exerts toxicity even to P. azelaica HBP1, but only at concentrations higher than 0.5 mM. Above this concentration transient loss of membrane polarization and integrity occurred, which we conclude from staining of growing cells with fluorescent dyes. Cells finally recover and resume growth on 2HBP. The high resistance of P. azelaica HBP1 to 2-HBP was found to be the result of an efficient MexABOprM- type efflux pump system counteracting passive influx of this compound into the membrane and cellular interior. Mutants with disrupted mexA, mexB and oprM genes did no longer grow on 2-HBP at concentrations above 100 μΜ, whereas below this concentration we found 2-HBP-concentration dependent decrease of growth rate. The MexAB-OprM system in P. azeiaica HBP1 is indeed an efflux pump for ethidium bromide as well. By introducing gfp reporter fusions responsive to intracellular 2- HBP concentrations into HBP1 wild-type or the mutants we demonstrated that 2HBP enters into the cells in a similar way. In contrast, the reporter system in the wild-type cells does not react to 2-HBP at an outside concentration of 2.4 μΜ, whereas in mutant cells it does. This suggests that wild-type cells pump 2-HBP to the outside very effectively preventing accumulation of 2-HBP. 2HBP metabolism, therefore, is not efficient enough to lower the intracellular concentration and prevent toxicity. We conclude that P. azelaica HBP1 resistance to 2-HBP is mainly due to an efficient efflux system and that 2HBP in high concentrations exerts narcotic effects on the bacterial membrane. In the part of this thesis, we investigated the possibilities of bacteria to degrade pollutants at low concentrations (1 mg per L and below). As test components we used 2-hydroxybiphenyl, antibiotics and a variety of fragrances, many of which are known to be difficult to biodegrade. By using accurate counting of low numbers of bacterial cells we could demonstrate that specific growth on these compounds is possible. We demonstrated the accuracy of FC counting at low cell numbers (down to 103 bacterial cells per ml). Then we tested whether bacterial population growth could be specifically monitored at the expense of low substrate concentrations, us¬ing P. azelaica HBP1. A perfect relationship was found between growth rate, yield and 2-HBP concentrations in the range of 0.1 up to 5 mg per L. Mixing P. azelaica within sludge, however, suggested that growth yields in a mixed community can be much lower than in pure culture, perhaps because of loss of metabolic intermediates. We then isolated new strains from activated sludge using 2-HBP or antibiotics (Nal, AMP, SMX) at low concentrations (0.1-1 mg per L) as sole carbon and energy sub¬strate and PAO microdishes. The purified strains were then examined for growth on their respective substrate, which interestingly, showed that all strains can not with¬stand higher than 1 or 10 mg per L concentrations of target substrate. Thus, bacteria must exist that contribute to compound degradation at low pollutant concentrations but are inhibited at higher concentrations. Finally we tested whether specific biomass growth (in number of cells) at the expense of pollutants can also be detected with communities as starting material. Hereto, we focused on a number of fragrance chemicals and measured community biomass increase by flow cytometry cell counting on two distinct starter communities: (i) diluted Lake Geneva water, and dilute activated sludge from a wastewater treatment plant. We observed that most of the test compounds indeed resulted in significant biomass increase in the starter community compared to a no-carbon added control, but activated sludge and lake Geneva water strongly differed (almost mutually ex¬clusive) in their capacity to degrade the test chemicals. In two cases for activated sludge the same type of microbial community developed upon compound exposure, as concluded from transcription fragment length polymorphism analysis on community purified and PCR amplified 16S rRNA gene fragments. To properly test compound biodegradability it is thus important to use starter communities of different origin. We conclude that FC counting can be a valuable tool to screen chemicals for their biodegradability and toxicity. - Des milliers de produits chimiques sont libérés dans l'environnement mais beaucoup ont des effets inconnus, en particulier à basses concentrations. Ce travail de thèse contribue à notre comprehension des effets de la pollution en utilisant des bacteries comme des organismes-tests. Les bacteries sont importantes pour etudier cette ques¬tion car certaines d'entre elles peuvent degrader ou transformer les polluants, mais également parce qu'elles-mmes peuvent tre inhibees dans leur reproduction après avoit ete exposees à ces composes toxiques. Quand des effets inhibiteurs ont lieu, la composition de la communauté microbienne peut tre changee à long terme, ce qui mène à une reduction du service d'ecosystème offert par ces communautés. En consequence, après leur liberation dans l'environnement, les produits chimiques d'origine anthropogenique peuvent soit s'y accumuler et per¬sister, exerant ainsi des effets encore inconnus sur les organismes vivants. En plus d'acquérir des connaissances de base sur les effets des polluants à basses concentra¬tions sur les communautés microbiennes, un but applique de cette thèse était de développer des tests bases sur les bacteries afin d'identifier de nouveau composes pour leur toxicité ou leur biodégradation. Dans la première partie de ce travail, nous avons developpe un test base sur la cytometrie de flux (FC) sur des cellules de Pseudomonas fluorescens colorees par du bromure d'ethidium ou de l'iodure de propidium et exposees ou non à une palette de polluants sous des conditions de croissance oligotrophique. La cytometrie de flux est une technique qui connaît de nombreuses applications dans la microbiologie environ¬nementale. Cela est principalement du au fait qu'elle permet un comptage rapide et precis ainsi que l'évaluation de l'état physiologique, en particulier lorsqu'elle est combinée h des colorations fluorescentes. Ici, nous avons utilise la technique FC et des colorants fluorescents afin de mesurer l'effet que peuvent exercer certains pollu¬ants sur Pseudomonas ûuorescens SV3 . D'abord nous avons conu des tests oligo- trophiques qui nous permettent de suivre la croissance complète de cellules en culture h des densites faibles (104 -10 7 cellules par ml), sur de l'acetate de sodium à 0.1 mM, en presence ou absence de produits chimiques (2-chlorobiphenyl (2CBP), naphthalène (NAH), 4-chlorophenol (4CP), tetradecane (TD), chlorure de mercure(II) (HgCl2)) à différentes concentrations. Afin de montrer le devenir des bacteries tant au niveau de la cellule individuelle que celui de la population globale, après exposition à des series de composes chimiques, nous avons compte les cellules colorees avec du SYT09 (col¬orant fluorescent vert des acides nucléiques pour la discrimination des cellules par rapport au bruit de fond) en combinaison avec l'iodure de propidium (PI) ou le bromure d'ethidium (EB), indicateurs de l'intégrité de la membrane cellulaire avec FC. Nous avons observe que de nombreux composes testes avaient un effet sur la croissance bacterienne, resultant en une baisse du taux de reproduction de la pop¬ulation. En outre, la double coloration que nous avons utilisee dans cette etude SYT09/PI ou SYT09/EB a montre que les produits chimiques testes induisaient une reponse heterogène des cellules dans la population, divisant celle-ci en sous- populations "saine", "endommagee" ou "morte". Les nombres de cellules à partir du comptage et de la proportion de celles "saines" et "endommagees/mortes" ont ensuite ete utilises pour modeliser la croissance de P. ûuorescens SV3 exposee aux produits chimiques. La reduction nette dans la croissance de population est une consequence du fait que de plus en plus de cellules sont incapables de se reproduire, plutt que du fait d'une croissance plus lente de l'ensemble de la population. De plus, la proportion de cellules endommagees est correllee au dosage du compose chimique. Les résultats obtenus nous ont permis de conclure que le test oligotrophique que nous avons developpe peut tre utilise pour l'évaluation de la toxicité de produits chimiques sur différents modèles bacteriens. Des tests multiples peuvent tre lances en parallèle et les effets sont mesures en l'espace de huit heures. Par ailleurs, nous en déduisons que les produits chimiques exercént un effet sur la croissance des cellules de P. ûuorescens SV3, qui est heterogène parmi les cellules dans la population et depend du produit chimique. Il est intéressant de noter que dans les mmes tests d'exposition avec P. ûuorescens SV3, nous avons observe que certains composes qui n'ont pas conduit à une reduction du taux de la croissance nette de la population, ont cause des effets mesurables sur les cellule saines. Ceci a ete essentiellement observe dans la portion "saine" des cellules en tant qu'augmentation du signal de la fluorescence de 1ΈΒ. D'abord nous avons montre que SYT09/EB était une com¬binaison de colorants plus utile que celle de SYT09/PI parce que la fluorescence du PI a tendance à augmenter uniquement lorsque les cellules sont effectivement mortes, et non pas dans les cellules saines (moins de deux fois plus). Par opposi¬tion, la fluorescence moyenne de l'EB dans les cellules saines augmente jusqu'à huit fois plus après exposition aux composes toxiques. Tous les composes, mme aux plus basses concentrations, induisent une augmentation mesurable de la fluorescence moy¬enne de 1ΈΒ, plus particulièrement après deux heures d'incubation. Cet effet s'est revele tre transitoire pour les cellules exposees aux 2CNP et 4CP, mais est chro¬nique pour les cellules incubees avec le TD et le NAH (entranant la mort cellulaire). Afin de comprendre les mécanismes qui sous-tendent les effets observes, nous avons utilise des decoupleurs d'energie ou de membrane. L'augmentation du signal EB dans les populations causee par des produits chimiques ressemblait à celle exerce par le chelateur des ions divalents EDTA. Cependant, les intensités du signal EB des cellules exposees aux produits chimiques testees n'ont jamais atteint les valeurs des cellules traitees avec l'EDTA ou pasteurises. Nous en concluons que le test oli- gotrophique utilisant la coloration (SYT09/)EB des cellules exposees ou non à un produit chimique est utile afin d'evaluer l'effet toxique exerce par les polluants sur la physiologie bacterienne. Afin de mieux comprendre la reaction d'un système de defense par pompe à efflux après exposition à une toxine, nous avons étudié la toxicité du 2-hydroxybiphenyl (2-HBP) sur Pseudomonas azeiaica HBP1. Nous avons montre que le 2-HBP exerce une toxicité mme sur HBP1, mais uniquement à des concentrations supérieures à 0.5 mM. Au-dessus de cette concentration, des pertes transitoires d'intégrité et de polarization membranaire ont lieu, comme cela nous a ete montre par coloration des cellules en croissance. Les cellules sont finalement capables de se rétablir et de reprendre leur croissance sur 2-HBP. La forte resistance de P. azeiaica HBP1 h 2-HBP physiologie bacterienne s'est revele tre le résultat d'un système de pompe h efflux de type MexABOprM qui contre-balance l'influx passif de ce compose h travers la membrane. Nous avons montre, en construisant des mutants avec des insertions dans les gènes mexA, mexB and oprM et des fusions avec le gène rapporteur gfp, que l'altération de n'importe quelle partie du système d'efflux conduisait à accroître l'accumulation de 2-HBP dans la cellule, en comparaison avec la souche sauvage HBP1, provoquant une diminution de la resistance au 2-HBP ainsi qu'une baisse du taux de reproduction des cellules. Des systèmes d'efflux similaires sont répandus chez de nombreuses espèces bactériennes. Ils seraient responsables de la resistance aux produits chimiques tels que les colorants fluorescents (bromure d'ethidium) et des antibiotiques. Nous concluons que la resistance de P. azelaica HBP1 à 2-HBP est principalement due à un système d'efflux efficace et que 2-HBP, à des concentrations elevees, exerce un effet deletère sur la membrane bacterienne. En se basant sur le comptage des cellules avec la FC, nous avons developpe ensuite une methode pour evaluer la biodegradabilite de polluants tels que le 2-HBP ainsi que les antibiotiques (acide nalidixique (Nal), ampicilline (AMP) ou sulfamethoxazole (SMX)) à de faibles concentrations lmg par L et moins), par le suivi de la croissance spécifique sur le compose de cultures microbiennes pures et mixtes. En utilisant un comptage precis de faibles quantités de cellules nous avons pu demontrer que la croissance spécifique sur ces composes est possible. Nous avons pu illustrer la precision du comptage par cytometrie de flux à faible quantité de cellules (jusqu'à 10 3 cellules par ml). Ensuite, nous avons teste s'il était possible de suivre dynamiquement la croissance de la population de cellules sur faibles concentrations de substrats, en utilisant P. azelaica HBP1. Une relation parfaite a ete trouvee entre le taux de croissance, le rendement et les concentrations de 2-HBP (entre 0.1 et 5 mg par L). En mélangeant HBP1 à de la boue active, nous avons pu montrer que le rendement en communauté mixtes pouvait tre bien inférieur qu'en culture pure. Ceci étant peut tre le résultat d'une perte d'intermédiaires métaboliques. Nous avons ensuite isole de nouvelles souches à partir de la boue active en utilisant le 2-HBP ou des antibiotiques (Nal, AMP, SMX) h basses concentrations (0.1-1 mg par L) comme seules sources de carbone et d'energie. En combinaison avec ceci, nous avons également utilise des microplaques PAO. Les souches purifiees ont ensuite ete examinees pour leurs croissances sur leurs substrats respectifs. De faon intéressante, toutes ces souches ont montre qu'elles ne pouvaient pas survivre à des concentrations de substrats supérieures à 1 ou 10 mg par L. Ainsi, il existe des bacteries qui contribuent à la degradation de composes à basses concentrations de polluant mais sont inhibes lorsque ces concentrations deviennent plus hautes. Finalement, nous avons cherche à savoir s'il est possible de detecter une croissance spécifique à une biomasse au depend d'un polluant, en partant d'une communauté microbienne. Ainsi, nous nous sommes concentre sur certains composes et avons mesure l'augmentation de la biomasse d'une communauté grce à la cytometrie de flux. Nous avons compte deux communautés de depart distinctes: (i) une dilution d'eau du Lac Léman, et une dilution de boue active d'une station d'épuration. Nous avons observe que la plupart des composes testes ont entrane une augmentation de la biomasse de depart par rapport au control sans addition de source de carbone. Néanmoins, les échantillons du lac Léman et de la station d'épuration différaient largement (s'excluant mutuellement l'un l'autre) dans leur capacité à degrader les composes chimiques. Dans deux cas provenant de la station d'épuration, le mme type de communauté microbienne s'est developpe après exposition aux composes, comme l'a démontré l'analyse TRFLP sur les fragments d'ARN 16S purifie de la communauté et amplifie par PCR. Afin de tester correctement la biodegradabilite d'un compose, il est donc important d'utiliser des communautés de depart de différentes origines Nous en concluons que le comptage par cytometrie de flux peut tre un outil de grande utilité pour mettre en valeur la biodegradabillite et la toxicité des composes chimiques.

Examining chemical compound biodegradation at low concentrations through bacterial cell proliferation.

We show proof of principle for assessing compound biodegradation at 1-2 mg C per L by measuring microbial community growth over time with direct cell counting by flow cytometry. The concept is based on the assumption that the microbial community will increase in cell number through incorporation of carbon from the added test compound into new cells in the absence of (as much as possible) other assimilable carbon. We show on pure cultures of the bacterium Pseudomonas azelaica that specific population growth can be measured with as low as 0.1 mg 2-hydroxybiphenyl per L, whereas in mixed community 1 mg 2-hydroxybiphenyl per L still supported growth. Growth was also detected with a set of fragrance compounds dosed at 1-2 mg C per L into diluted activated sludge and freshwater lake communities at starting densities of 10(4) cells per ml. Yield approximations from the observed community growth was to some extent in agreement with standard OECD biodegradation test results for all, except one of the examined compounds.

Community-wide plasmid gene mobilization and selection.

Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host's chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions.

Peroxisome proliferator-activated receptor structures: ligand specificity, molecular switch and interactions with regulators.

Peroxisome proliferator-activated receptors (PPARs) compose a family of nuclear receptors that mediate the effects of lipidic ligands at the transcriptional level. In this review, we highlight advances in the understanding of the PPAR ligand binding domain (LBD) structure at the atomic level. The overall structure of PPARs LBD is described, and important protein ligand interactions are presented. Structure-activity relationships between isotypes structures and ligand specificity are addressed. It is shown that the numerous experimental three-dimensional structures available, together with in silico simulations, help understanding the role played by the activating function-2 (AF-2) in PPARs activation and its underlying molecular mechanism. The relation between the PPARs constitutive activity and the intrinsic stability of the active conformation is discussed. Finally, the interactions of PPARs LBD with co-activators or co-repressors, as well as with the retinoid X receptor (RXR) are described and considered in relation to PPARs activation.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Role of intracellular signaling pathways activated in fibroblasts in response to lipoproteins

Summary The best described physiological function of low-density lipoproteins (LDL) is to transport cholesterol to target tissues. LDL deliver their cholesterol cargo to cells following their interaction with the LDL receptor. LDL, when their vascular concentrations increase, have also been implicated in pathologies such as atherosclerosis. Among the cell types that are found in blood vessels, endothelial and smooth muscle cells have dominated cellular research on atherosclerotic mechanisms and LDL activation of signaling pathways, while very little is known about adventitial fibroblast activation caused by elevated lipoprotein levels. Since fibroblasts participate in wound repair and since it has recently been recognized that fibroblasts may play pivotal roles in vascular remodeling and repair of injury, we assessed whether lipoproteins affect fibroblast function. We have found that LDL specifically mediate the activation of a class of mitogen-activated protein kinases (MAPKs): the p38 MAPKs. The activation of this pathway in turn modulates cell shape by promoting lamellipodia formation and extensive cell spreading. This is of particular interest because it provides a mechanism by which LDL can promote wound healing or vessel wall remodeling as observed during the development of atherosclerosis. In order to understand the molecular mechanisms by which LDL induce p38 activation we searched for the component in the LDL particle responsible for the induction of this pathway. We found that cholesterol is the major component of lipoprotein particles that mediates their ability to stimulate the p38 MAPK pathway. Furthermore, we investigated the cellular mechanisms underlying the ability of LDL to induce cell shape changes and whether this could participate in wound repair. Our recent data demonstrates that the capacity of LDL to induce fibroblast spreading relies on their ability to stimulate IL-8 secretion, which in turn leads to accelerated wound healing. LDL-induced IL-8 production and subsequent wound closure are impaired upon inhibition of the p38 MAPK pathway indicating that the LDL-induced spreading and accelerated wound sealing rely on the ability of LDL to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Therefore, regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL-cholesterol levels, IL-8 production and extensive remodeling of the vessel wall. Résumé: La fonction physiologique des lipoprotéines à faible densité (LDL) la mieux décrite est celle du transport du cholestérol aux tissus cibles. Les LDL livrent leur cargaison de cholestérol aux cellules après leur interaction avec le récepteur au LDL. Une concentration vasculaire des LDL augmenté est également impliquée dans le développement de l'athérosclérose. Parmi les types de cellule présents dans les vaisseaux sanguins, les cellules endothéliales et les cellules du muscle lisse ont dominé la recherche cellulaire sur les mécanismes athérosclérotiques et sur l'activation par les LDL des voies de signalisation intracellulaire. A l'inverse peu de choses sont connues sur l'activation des fibroblastes de l'adventice par les lipoprotéines. Puisqu'il a été récemment reconnu que les fibroblastes peuvent jouer un rôle central dans la remodélisation vasculaire et la réparation tissulaire, nous avons étudié si les lipoprotéines affectent la fonction des fibroblastes. Nous avons constaté que les LDL activent spécifiquement une classe de protéines kinases: les p38 MAPK (mitogen-activated protein kinases). L'activation de cette voie module à son tour la forme de la cellule en favorisant la formation de lamellipodes et l'agrandissement des cellules. Cela a un intérêt particulier car il fournit un mécanisme par lequel les LDL peuvent promouvoir la cicatrisation ou la remodélisation des parois vasculaires comme observés lors du développement de l'athérosclérose. Pour comprendre les mécanismes moléculaires par lesquels les LDL provoquent l'activation des p38 MAPK, nous avons cherché à identifier les composants dans la particule de LDL responsables de l'induction de cette voie. Nous avons constaté que le cholestérol est l'élément principal des particules de lipoprotéine qui contrôle leur capacité à stimuler la voie des p38 MAPK. En outre, nous avons examiné les mécanismes cellulaires responsables de la capacité des LDL à induire des changements dans la forme des cellules. Nos données récentes démontrent que la capacité des LDL à induire l'agrandissement des cellules, ainsi que leur aptitude à favoriser la cicatrisation, reposant sur leur capacité à stimuler la sécrétiond'IL-8. La production d'IL-8 induite par les LDL est bloquée par l'inhibition de la voie p38 MAPK, ce qui indique que l'étalement des cellules induit par les LDL ainsi que l'accélération de la cicatrisation sont liés à la capacité des LDL à stimuler la sécrétion d'IL8 via l'activation des p38 MAPK. La régulation de la forme et de la migration des fibroblastes par les lipoprotéines peuvent donc participer au développement de l'athérosclérose qui est caractérisée par l'augmentation des niveaux de production de LDL-cholestérol et d'IL-8 ainsi que par une remodélisation augmentée de la paroi du vaisseau.

Expression of protease-activated receptors in arthritic synovial tissues

Clinical and experimental evidence suggests that synovial thrombin formation in arthritic joints is prominent and deleterious, leading to exacerbation of rheumatoid arthritis (RA). In this context, cellular effects of thrombin mediated by the protease-activated receptors (PARs) in arthritic joints may be of paramount significance. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin whereas PAR2 is activated by trypsin and few other proteases.We first explored PARs expression in RA synovial tissues. Synovial membranes from 11 RA patients were analyzed for PARs expression by RT-PCR and by immunohistology. PAR4 was found in all the biopsies, whereas the expression of PAR1, PAR 2 and PAR3 was more restricted (8/11, 5/11 and 3/11 respectively). In the arthritic synovial membrane of murine antigen-induced arthritis (AIA) we found coexpression of the four different PARs. Next, we explored the functional importance of PAR1 during AIA in vivo using PAR-1 deficient mice. The phenotype of PAR1-deficient mice (n = 22), based on the analysis of arthritis severity (as measured by 99 m tecnetium uptake, histological scoring and intra-articular fibrin measurements) was similar to that of wild-type mice (n = 24). In addition, the in vivo production of antibodies against mBSA was also similar. By contrast, the mBSA-induced in vitro lymph node cell proliferation was significantly decreased in PAR1-deficient mice as compared with controls. Accordingly, mBSA-induced production of interferon-γ by lymph node cells in culture was significantly decreased in PAR1-deficient mice as compared with controls, whereas opposite results were observed for production of IL-10.

Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics.

Advances in wound care are of great importance in clinical injury management. In this respect, the nuclear receptor peroxisome proliferator-activated receptor (PPAR)beta/delta occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPARbeta/delta expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-beta1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPARbeta/delta accelerate wound closure. The results reveal a dual role of TGF-beta1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPARbeta/delta expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-beta1 at wound sites.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T cell activation in vivo

Rapport de synthèse : Le récepteur activé par protéase de type 2 (PAR2) intervient dans l'inflammation dans divers modèles expérimentaux de maladies inflammatoires et auto-immunes, mais le mécanisme par lequel il exerce cette fonction reste mal compris. PAR2 est exprimé sur des cellules endothéliales et immunitaires et a été impliqué dans la différentiation des cellules dendritiques (DC). Avec leur rôle central dans la réponse immune, les DC pourraient jouer un rôle clef, l'activation de PAR2 à leur surface modulant la réponse immune. Des recherches précédentes ont montré que PAR2 a un effet dans le développement et la maturation des DC de moelle osseuse in vitro, ainsi que dans la promotion de la réponse immune en allergie. Dans cette étude, nous avons évalué l'impact in vivo de l'activation de PAR2 sur les DC et les cellules T dans des souris déficientes en PAR2 (KO) en utilisant un peptide agoniste spécifique du PAR2 (AP2). L'activation de PAR2 a augmenté la fréquence de DC matures dans les ganglions lymphatiques 24 heures après l'administration d'AP2 d'une manière significative. En outre, ces DC avaient une expression augmentée des molécules de co-stimulation CD86 et du complexe majeur d'histocompatibilité type 2 (MHC-II). 48 heures après l'injection d'AP2, nous avons également observé une élévation significative des lymphocytes T CD4+ et CD8+ activés, (CD44+CD62-) dans ces ganglions. Des changements dans le profil d'activation des DC et des cellules T n'ont pas été observés au niveau de a rate. L'influence de la signalisation de PAR2 sur le transport d'antigène aux ganglions lymphatiques inguinaux a été évaluée dans le contexte d'hypersensibilité retardée de type IV. Les souris KO sensibilisées par peinture de la peau avec fluorescéine isothyocyanate (FITC) afin d'induire une hypersensibilité retardée avaient un pourcentage diminué de DC FITC+ dans les ganglions lymphatiques 24 heures après l'application du FITC en comparaison avec les souris sauvages avec le même fond génétique (0.47% vs 0.95% des cellules ganglionnaires totales). En conclusion, ces résultats démontrent que la signalisation de PAR2 favorise et renforce la maturation et le transport d'antigène par des DC .vers les ganglions lymphatiques ainsi que l'activation ultérieure des lymphocytes T, et de ce fait fournissent une explication pour l'effet pro inflammatoire de PAR2 dans les modèles animaux d'inflammation. Une meilleure compréhension de ce mécanisme de modulation du système immun via PAR2 peut s'avérer particulièrement utile pour le développement des vaccins, ainsi que pour la découverte de nouvelles cibles thérapeutiques dans le contexte de l'allergie, l'auto-immunité, et les maladies inflammatoires.

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders.

Protective role of peroxisome proliferator-activated receptor-β/δ in septic shock.

Rationale: Peroxisome proliferator activated receptor (PPAR)-beta/delta is a transcription factor that belongs to the PPAR nuclear hormone receptor family, but the role of PPAR-beta/delta in sepsis is unknown. Objectives: We investigated the role of PPAR-beta/delta in murine models of LPS-induced organ injury and dysfunction and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. Methods: Wild-type (WT) and PPAR-beta/delta knockout (1(0) mice and C57BL/6 mice were subjected to LPS for 16 hours. C57BL/6 mice received the PPAR-beta/delta agonist GW0742 (0.03 mg/kg intravenously, 1 h after LPS) or GW0742 plus the PPAR-beta/delta antagonist GSK0660 (0.1 mg/kg intravenously, 30 min before LPS). CD-1 mice subjected to CLP received GW0742 or GW0742 plus GSK0660. Measurements and Main Results: In PPAR-beta/delta KO mice, endotoxemia exacerbated organ injury and dysfunction (cardiac, renal, and hepatic) and inflammation (lung) compared with WT mice. In C57BL/6 mice subjected to endotoxemia, GW0742 significantly (1) attenuated organ (cardiac and renal) dysfunction and inflammation (lung); (2) increased the phosphorylation of Akt and glycogen synthase kinase (GSK)-3 beta; (3) attenuated the increase in extracellular signal-regulated kinase (ERK)1/2 and signal transducer and activator of transcription (STAT)-3 phosphorylation; and (4) attenuated the activation of nuclear factor (NF)-kappa B and the expression of inducible nitric oxide synthase (iNOS). In CD-1 mice subjected to CLP, GW0742 improved 10-day survival. All the observed beneficial effects of GW0742 were attenuated by the PPAR-beta/delta antagonist GSK0660. Conclusions: PPAR-beta/delta protects against multiple organ injury and dysfunction, and inflammation caused by endotoxic shock and improves survival in polymicrobial sepsis by a mechanism that may involve activation of Akt and inhibition of GSK-3 beta and NF-kappa B.

Selective lysis of activated cells in oncorna virus infection.

The authors devised a cytotoxic assay based on cytofluorometric analysis of target surface markers in order to compare lysis exerted in vitro by cytotoxic T lymphocytes (CTLs) on different cell subsets in the context of a single lymphoid target cell population. Using this assay, the authors evaluated when oncorna virus-infected lymphocytes become a suitable target for virus-specific T cell effectors. A lymphocyte population from Moloney-murine leukaemia virus (M-MuLV)-infected (carrier) mice, in which the proliferation of selective V beta T-cell receptor (TCR) families was induced in response to Mlsa encoded antigens, was utilized as a target. The authors observed that a virus-specific T cell clone exerted lytic activity preferentially against activated cell subsets. Moreover, virus-specific CTLs generated in mixed leucocyte tumour cell cultures (MLTC) were also able to impair the concomitant anti-Mlsa response of lymphocytes from M-MuLV carrier mice. It was found that the proliferative status of oncorna virus-infected target cells played an important role in limiting the in vitro efficacy of the immune response, and it is speculated that this phenomenon might represent an in vivo escape mechanism from immunosurveillance.

Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo.

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vbeta8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.

Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that can be activated by various xenobiotics and natural fatty acids. These transcription factors primarily regulate genes involved in lipid metabolism and also play a role in adipocyte differentiation. We present the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPAR subtypes. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are coexpressed, with relative levels varying between them from one cell type to the other. PPAR-alpha is highly expressed in hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPAR-beta is expressed ubiquitously and often at higher levels than PPAR-alpha and -gamma. PPAR-gamma is expressed predominantly in adipose tissue and the immune system. Our results suggest new potential directions to investigate the functions of the different PPAR subtypes.

Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma.

Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds including fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates and thiazolidinediones. While thiazolidinediones and 15-deoxy-Delta12, 14-prostaglandin J2 have been shown to bind to PPARgamma, it has remained unclear whether other activators mediate their effects through direct interactions with the PPARs or via indirect mechanisms. Here, we describe a novel fibrate, designated GW2331, that is a high-affinity ligand for both PPARalpha and PPARgamma. Using GW2331 as a radioligand in competition binding assays, we show that certain mono- and polyunsaturated fatty acids bind directly to PPARalpha and PPARgamma at physiological concentrations, and that the eicosanoids 8(S)-hydroxyeicosatetraenoic acid and 15-deoxy-Delta12,14-prostaglandin J2 can function as subtype-selective ligands for PPARalpha and PPARgamma, respectively. These data provide evidence that PPARs serve as physiological sensors of lipid levels and suggest a molecular mechanism whereby dietary fatty acids can modulate lipid homeostasis.