Studies on IL-18 in HIV infection : effects of the cytokine on intestinal integrity, and platelets as a new source of the cytokine

L'interleukine IL-18 (IL-18), un membre de la famille de l’IL-1, est une cytokine pro-inflammatoire multifonctionnelle. Elle est produite par les monocytes, les macrophages, les cellules dendritiques, les cellules épithéliales, les kératinocytes et le cortex surrénal dans le corps humain. Cette cytokine est d'abord produite comme une protéine précurseure inactive, qui est par la suite clivée en une forme mature par la caspase-1 activée. La caspase, en elle-même, existe comme précurseur inactif dans les cellules humaines et requiert l'assemblage d'inflammasomes pour son activation. L'IL-18 pour joue un rôle clé dans la médiation des conditions inflammatoires. Notre laboratoire et d'autres ont montré que l'infection par le VIH est accompagnée d'une augmentation des taux circulants d'IL-18 avec une diminution des niveaux de son antagoniste, l'interleukine-18 binding protein (IL-18BP). Dans cette thèse, nous démontrons pour que l'IL-18 est également produite et sécrétée par les plaquettes humaines lors de leur activation. Les plaquettes contiennent des composants de l'inflammasome. Ils assemblent et activent la caspase-1, qui ensuite traite le précurseur de l'IL-18 dans sa forme mature au cours du processus d'activation des plaquettes. La cytokine est synthétisée de novo lors de l'activation des plaquettes. Contrairement à l'IL-18, les plaquettes expriment constitutivement l’IL-18BP, et la libèrent de manière constitutive, ainsi que lors de l'activation. L'IL-18 et l'IL-18BP sont colocalisés avec CD63, un marqueur pour les granules α des plaquettes. L'IL-18 libéré des plaquettes constitue la source principale de cette cytokine dans la circulation humaine chez les individus sains. Nous avons identifié des concentrations faibles de cette cytokine dans les lysats de plaquettes chez les individus infectés par le VIH par rapport à ceux en santé. D'autre part, les concentrations ont été augmentées dans le sérum et le plasma pauvre en plaquettes chez les individus infectés. Des résultats similaires ont été obtenus avec l'IL-18BP dans les lysats de plaquettes d'individus sains et infectés par le VIH. Cependant, des quantités plus faibles de cet antagoniste ont été trouvées dans le sérum et le plasma pauvre en plaquettes d'individus infectés par le VIH par rapport à ceux en santé. Nos résultats ont des implications importantes pour les maladies inflammatoires chroniques dans laquelle une activité accrue de l'IL-18 joue un rôle pathogène. Le VIH est également accompagné par une inflammation intestinale et une diminution de l'intégrité intestinale, mesurée par la réparation de la muqueuse, la régénération et la perméabilité. Cependant, on en sait peu sur la relation entre le niveau élevé de l'IL-18 associé à l'infection au VIH et la perméabilité intestinale: ceci n'a jamais été étudié. Dans cette thèse, nous démontrons le rôle du virus et sa protéine Tat à augmenter la production d'IL-18 chez deux lignées de cellules épithéliales intestinales (HT29 et Caco2) ainsi qu'une diminution de l'IL-18BP. L'IL-18 induit une hyperperméabilité de la barrière épithéliale en perturbant à la fois les jonctions serrées et adhérentes, et ce, en modulant l'expression et la distribution de l'occludine, de claudine-2 et de la bêta-caténine. Une désorganisation de l'actine F a également été observée dans les cellules lors de l'incubation avec l'IL-18. Les mêmes observations ont été faites avec la protéine Tat du VIH-1. Après une incubation prolongée, l'IL-18 a causé la mort des cellules intestinales et induit l'apoptose par l'activation de la caspase-1 et la caspase-3. Fait intéressant, les taux plasmatiques de lipopolysaccharides chez trois catégories différentes de patients au VIH (ART-naïf, ART-traitée et contrôleurs élite) sont en corrélation avec les niveaux plasmatiques de l'IL-18. Enfin, nous avons étudié la voie de signalisation à travers laquelle l'IL-18 induit une perméabilité intestinale accrue. En bref, nos études identifient les plaquettes comme une source importante d'IL-18, et leur activation lors d'une infection à VIH contribue à des concentrations accrues de cette cytokine. Le virus entraine également l'augmentation de la production de cytokines par les cellules épithéliales intestinales. L'activité biologique accrue de ces cytokines contribue à la pathogenèse du sida en augmentant la perméabilité intestinale et en causant la mort des cellules intestinales. L'IL-18 pourrait servir de cible moléculaire pour retarder la progression du sida et réduire l'inflammation chronique dans un stade précoce d'une infection à VIH.Interleukin IL-18 (IL-18), a member of the IL-1 family, is a multifunctional pro-inflammatory cytokine. It is known to be produced by monocytes, macrophages, dendritic cells, keratinocytes and the adrenal cortex in the human body. This cytokine is produced as an inactive precursor protein, which is cleaved into mature form by activated caspase-1. The caspase itself exists as an inactive precursor in human cells and requires inflammasomes assembly for its activation. IL-18 has been shown to play a key role in mediating different inflammatory conditions. Our laboratory and others have shown that HIV infection is accompanied with increased circulating levels of IL-18 along with decreased levels of its antagonist IL-18 Binding Protein (IL-18BP). In this thesis, we show that IL-18 is also produced and secreted by human platelets upon activation. The platelets also contain components of the inflammasome. They assemble and activate caspase-1 and process the precursor IL-18 into its mature form during the platelet activation process. The cytokine is synthesized in platelets de novo upon activation. Contrary to IL-18, the platelets constitutively express pre-formed IL-18BP, and release it constitutively as well as upon activation. Both IL-18 and IL-18BP colocalized with CD63, a marker for the platelet α granules. Platelet-released IL-18 constitutes the main source of this cytokine in the human circulation in healthy individuals. We found decreased amounts of this cytokine in the platelet lysates in HIV-infected individuals as compared to the healthy ones. On the other hand, its concentrations were increased in the serum and platelet-poor plasma in infected individuals. Similar findings were obtained with IL-18BP in platelet lysates from healthy and HIV-infected individuals. However, lower amounts of this IL-18 antagonist were found in the serum and platelet-poor plasma from HIV-infected individuals compared with the healthy ones. Our findings have important implications for chronic inflammatory disease conditions in which increased IL-18 activities play a pathogenic role. HIV is also accompanied by intestinal inflammation and decreased intestinal integrity as measured by mucosal repair, regeneration and permeability. However, little is known concerning the relation between high level of IL-18 associated to HIV infection and intestinal permeability. In this thesis, we demonstrate the role of HIV and its protein Tat in increasing IL-18 production in two intestinal epithelial cell lines (HT29 and Caco2) and decreasing IL-18BP. IL-18 induces epithelial barrier hyperpermeability by disrupting both tight and adherens Junctions by modulating expression and distribution of occludin, claudin-2 and beta-catenin. Disorganization of F-actin was also observed within the cells upon incubation with treated by IL-18. Upon prolonged incubation, IL-18 caused intestinal cells death and induced apoptosis by activating caspase-1 and caspase-3. Interestingly, the plasma levels of lipopolysaccharide in three different categories of HIV-infected patients (ART-naïve, ART-treated and Elite controllers) correlated with their IL-18 plasma levels. Finally we investigated the signaling pathway through which IL-18 induces increased intestinal permeability. Briefly, our studies identified platelets as an important source of IL-18, and their activation in HIV infection contributes to enhanced concentrations of the cytokine. The virus also induces increased production of the cytokine from intestinal epithelial cells. Increased biological activities of the cytokine contribute towards AIDS pathogenesis by increasing intestinal permeability and causing death of intestinal cells. The cytokine may serve as a molecular target for delaying AIDS progression and reducing low-grade chronic inflammation in HIV infection

Calcium Carbonate Suppresses Haem Toxicity Markers without Calcium Phosphate Side Effect on Colon Carcinogenesis

Red meat intake is associated with increased risk of colorectal cancer. We have previously shown that haemin, haemoglobin and red meat promote carcinogen-induced preneoplastic lesions, aberrant crypt foci, in the colon of rats. We have also shown that dietary calcium phosphate inhibits haemin-induced promotion, and normalizes faecal lipoperoxides and cytotoxicity. Unexpectedly, high-calcium phosphate control diet-fed rats had more preneoplastic lesions in the colon than low-calcium control diet-fed rats. The present study was designed to find a calcium supplementation with no adverse effect, by testing several doses and types of calcium salts. One in vitro study and two short-term studies in rats identified calcium carbonate as the most effective calcium salt to bind haem in vitro and to decrease faecal biomarkers previously associated with increased carcinogenesis: faecal water cytotoxicity, thiobarbituric acid reactive substances. A long term carcinogenesis study in dimethylhydrazine-injected rats demonstrated that a diet containing 100 µmol/g calcium carbonate did not promote aberrant crypt foci, in contrast with previously tested calcium phosphate diet. The results suggest that calcium carbonate, and not calcium phosphate, should be used to reduce haem-associated colorectal cancer risk in meat-eaters. They support the concept that the nature of the associated anion to a protective metal ion is important for chemoprevention

Freeze-Dried Ham Promotes Azoxymethane-Induced Mucin-Depleted Foci and Aberrant Crypt Foci in Rat Colon

Processed and red meat consumption is associated with the risk of colorectal cancer. Meta-analyses have suggested that the risk associated with processed meat is higher. Most processed meats are cured and cooked, which leads to formation of free nitrosyl heme. We speculated that free nitrosyl heme is more toxic than native myoglobin. The promoting effect of a freeze-dried, cooked, cured ham diet was looked for in a 100-day study. Colon carcinogenesis endpoints were aberrant crypt foci and mucin depleted foci (MDF). A second study (14 days) was designed 1) to compare the effect of ham, hemoglobin, and hemin; and 2) to test the effect of sodium chloride, nitrite, and phosphate in diet on early biomarkers associated with heme-induced promotion. In the 100-day study, control and ham-fed rats had 3.5 and 8.5 MDF/colon, respectively (P < 0.0001). Promotion was associated with cytotoxicity and lipid peroxidation. In the short-term study, cytotoxicity and lipid peroxidation of fecal water, and the urinary marker of lipid peroxidation, increased dramatically in ham- and hemin-fed rat. In contrast, the hemoglobin diet, sodium chloride, nitrite, phosphate diet had no effect. Freeze-dried cooked ham can promote colon carcinogenesis in a rodent model. Hemin, but not hemoglobin, mimicked ham effect on early biochemical markers associated with carcinogenesis

L’interleukine-21, une cytokine clé dans le contrôle du VIH et d’autres infections virales chroniques

La différenciation et les fonctions effectrices des cellules immunes sont principalement contrôlées par les cytokines. L’infection par les virus entraîne souvent des dysfonctionnements de la réponse immunitaire. Ces dérèglements se manifestent par une sécrétion altérée des cytokines et une dérégulation de leurs effets biologiques. Parmi celles-ci, on trouve l’interleukine-21 (IL-21), une cytokine récemment découverte qui exerce des effets multiples et pléïotropes sur les cellules immunes et joue un rôle indispensable dans le contrôle des infections virales aiguës et chroniques. Cette revue présente les connaissances actuelles concernant les effets biologiques de l’IL-21 sur la réponse immunitaire, et discute son rôle dans la mise en place de la réponse immune antivirale, et en particulier celle dirigée contre le VIH-1 (virus de l’immunodéficience humaine). L’IL-21 est présentée comme un nouvel agent thérapeutique et un « adjuvant » potentiellement utilisable dans des stratégies de vaccination contre les infections virales

Curcumin phytosomal softgel formulation: Development, optimization and physicochemical characterization

Curcumin, a naturally occurring lipophilic molecule can exert multiple and diverse bioactivities. However, its limited aqueous solubility and extensive presystemic metabolism restrict its bioavailability. Curcumin phytosomes were prepared by a simple solvent evaporation method where free flowing powder was obtained in addition to a newly developed semisolid formulation to increase curcumin content in softgels. Phytosomal powder was characterized in terms of drug content and zeta potential. Thirteen different softgel formulations were developed using oils such as Miglyol 812, castor oil and oleic acid, a hydrophilic vehicle such as PEG 400 and bioactive surfactants such as Cremophor EL and KLS P 124. Selected formulations were characterized in terms of curcumin in vitro dissolution. TEM analysis revealed good stability and a spherical, self-closed structure of curcumin phytosomes in complex formulations. Stability studies of chosen formulations prepared using the hydrophilic vehicle revealed a stable curcumin dissolution pattern. In contrast, a dramatic decrease in curcumin dissolution was observed in case of phytosomes formulated in oily vehicles

Curcumin phytosomal softgel formulation: Development, optimization and physicochemical characterization

Curcumin, a naturally occurring lipophilic molecule can exert multiple and diverse bioactivities. However, its limited aqueous solubility and extensive presystemic metabolism restricts its bioavailability. Curcuminphytosomeswere prepared by simple solvent evaporation method where free flowing powder was obtained in addition to a newly developed semisolid formulation to increase curcumin content in softgels. Phytosomal powder was characterized in terms of drug content and zeta-potential. Thirteen different softgel formulations were developed using oils such as Miglyol 812, castor oil and oleic acid, a hydrophilic vehicle as PEG 400 and bioactive surfactants such as Cremophor EL and KLS P 124. Selected formulations were characterized in terms of curcumin in vitro dissolution. TEM analysis revealed good stability and a spherical, self closed structure of curcumin phytosomes in complex formulations. Stability studies of chosen formulations prepared using the hydrophilic vehicle revealed a stable curcumin dissolution pattern. In contrast, a dramatic decrease in curcumin dissolution was observed in case of phytosomes formulated in oily vehicles

IL-18 and LPS activate caspase-1 and caspase-3 in HT-29 cells.

<p>The cell monolayers were treated with IL-18 (10 ng/ml) or with LPS (10 ng/ml) for 12 hours. Thereafter, the monolayers were washed with PBS, lysed and the activation of the caspases was determined on Western blots by using antibodies specific to the activated forms of the caspases. The Figure shows results from a representative of three independent experiments.</p