No description available.

Immune reconstitution following T-cell depletion consists of expansion of circulating T-cells or de novo synthesis of T-cells from the thymus. The IL-7/IL-7 receptor signalling pathway is critical for the maturation and differentiation of thymocytes before they leave the thymus as mature T-cells. Viral infections including HIV have been shown to decrease IL-7Ralpha (CD127) expression on circulating CD4+ and CD8+ T-cells. However little is known about the effects of HIV infection on CD127 expression and activity in thymocytes despite existing evidence of HIV infection of the thymus. Thymic function is altered in HIV infection leading to a dysregulation of the thymic epithelial network and reduced thymic output which may contribute, in part, to impaired immune reconstitution in progressive HIV disease. In vitro studies demonstrate that HIV infection interrupts thymopoiesis resulting in a developmental block in thymopoiesis similar to that seen in models of IL-7/IL-7R deficiencies suggesting a role for altered IL-7 signalling in HIV associated thymic dysfunction. Therefore we hypothesize that thymic dysfunction which occurs in HIV infection is due to reduced IL-7R and/or altered IL-7 signalling in thymocytes resulting in impaired de novo T-cell synthesis. In order to address this hypothesis an in vitro system for the functional study of human thymocytes has been optimized. The research conducted as part of this thesis assessed if in vitro HIV infection or if cytokines that are upregulated in the course of HIV infection altered CD127 expression on maturing thymocytes. It also evaluated if in vitro HIV infection disrupts thymocyte function at different stages of maturation and whether this disruption in function is due to impaired IL-7/IL-7R signalling. The host factors IL-7, TNF- and IL-4, which are upregulated in HIV infection, are found to downregulate CD127 expression on thymocytes. IL-4 pre-treatment of thymocytes reduced the ability of IL-7 to induce STAT-5 phosphorylation. Furthermore following in vitro HIV infection of thymocytes, CD127 expression of single positive CD8 thymocytes was decreased. In vitro HIV infection altered IL-7 activity as demonstrated by lower levels of Bcl-2 and phospho-STAT-5 expression in thymocytes following IL-7 stimulation. These accumulated results suggests that HIV may play a role in impaired thymic function by altering IL-7 responsiveness. Understanding the mechanisms of thymic dysfunction in HIV infection may provide some insight into therapies leading to immune reconstitution through increased thymic output.

Skeletal muscle secretes many signalisation proteins named myokines. These myokines act as hormones and induce metabolic changes throughout the whole body to facilitate adaptation to physical exercise. Interleukin-15 (IL-15) is highly expressed in skeletal muscle and appears to influence many metabolic parameters that are defective in metabolic pathologies such as insulin resistance. For instance, IL-15 increases glucose uptake in muscle and whole-body fatty acid oxidation and its overexpression in skeletal muscle in mice generates a very lean and active phenotype. However, there are discordant reports throughout scientific literature. The aim of the current study was to 1) characterize the metabolic effects of IL-15 in L6 myotubes to determine whether L6 is a good model to study IL-15 and 2) to determine whether IL-15 activates the AMPK signaling. L6 myotubes were exposed to different concentrations of IL-15 and different metabolic parameters were assayed namely; oxygen consumption, glucose uptake, fatty acid oxidation, Glucose transporter 4 (GLUT4) translocation, oxidative phosphorylation (OXPHOS) complexes protein expression, troponin T expression and Akt, AMPK and Acetyl-CoA Carboxylase (ACC) phosphorylation state. Acute IL-15 treatment increased glucose uptake without activating insulin signaling pathway or GLUT4 translocation. Furthermore, acute IL-15 treatment increased resting oxygen consumption rate (OCR) while chronic IL-15 treatment also increased mitochondrial spare capacity, suggesting an increased mitochondrial biogenesis. IL-15 induced ACC phosphorylation in a dose-dependent manner and tended to increase AMPK phosphorylation but it did not reach statistical significance. Lastly, IL-15 did not influence troponin T state. Altogether, the present study demonstrates that L6 myotubes do not express all the pro-oxidative qualities of IL-15 reported by scientific literature. Nonetheless, IL-15 induces certain pro-oxidative metabolic effect that could help people living with obesity and diabetes.

Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1β and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1β secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNβ mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage.

No description available.

Inflammation is the response of the body to injury or threats. Immune cells such as macrophages have a crucial role in controlling and regulating this process. The potent pro-inflammatory cytokines interleukin (IL)-1beta and IL-18 are synthesized by macrophages as inactive precursors which activation follows a unique mechanism involving the activation of caspase-1 by assembly of a macromolecular complex called the inflammasome. However, the assembly of the inflammasome is a double-edged sword. Although inflammasome activation is necessary for a normal inflammatory response, its malfunction can trigger and contribute to inflammatory disorders such as gout, arthritis or cryopirin-associated periodic syndromes (CAPS). The fine regulation of this mechanism and the cell death associated with it is key for the outcome of the inflammatory process. In this thesis we tackle three aspects of the mechanisms and consequences of inflammasome activation. First we studied the role of the deubiquitinases USP7 and USP47 in inflammasome activation. We showed how USP7 and USP47 activity is increased upon danger signals and how that is necessary for the assembly of the inflammasome. We also pointed how their inhibition dampens the deubiquitination of ASC using a BRET2 assay. Second we examined how the activity of IL-18 is controlled by the release of IL-18BP during inflammasome activation. We showed how IL-18BP release increased upon membrane permeabilization and pyroptosis. This release happens in other types of lytic cell (necrosis and necroptosis) death but not in apoptosis. Finally, we showed that this IL-18BP acute release dampens IL-18 signalling and IFN gamma production by PBMCs. These results demonstrate a novel mechanism by which lytic cell death could dampen IL-18-driven inflammation and highlights a key role for IL-18BP in inflammasome related diseases. Finally we studied the role of inflammasome in lung epithelial cells as a model to investigate lung infections. We found that lung epithelial cells lack NLRP3 inflammasome activity and components, but express caspase-4 and caspase-8 which could have a role in the release of IL-1 family of cytokines. To conclude we showed how lung epithelial release IL-18 upon Aspergillus fumigatus infection. Overall, this thesis enhances our understanding of the mechanisms that control IL-1beta and IL-18 activity by regulating inflammasome activation and by understanding the consequences of its activation.

Abstract Introduction: Current evidence suggests that the lungs of infants with the debilitating disorder, bronchopulmonary dysplasia (BPD), react to the challenges of extra-uterine adaptation with inappropriately aggressive inflammation. The reasons for this are not entirely clear and this study hypothesizes that a deficiency of interleukin (IL)-10, a potent anti-inflammatory mediator, leads to the functional and architectural changes characteristic of BPD. Aim: To characterize the behaviour of IL-10 and neutrophil apoptosis in the tracheal fluids (TF) of infants at risk of developing BPD. Method: TF from intubated infants of varying gestations at the Royal Hospital for Women, Randwick was spun and ILs 8, 10 and 16 were measured in the supernatant. The residual pellets of white cells were used to determine differential white cell counts and neutrophil apoptosis. Results: None of the 20 TF specimens from the extremely premature infants with BPD (n=11) had detectable IL-10, compared to 14/20(70%) of the specimens from preterm infants without BPD (n=20) and to 5/19 (26%) of the specimens from term infants (n=19). BPD infants also had a significantly lower number of apoptotic neutrophils during the 1st week of life. Premature infants with TF IL-10 &gt5pg/ml did not develop BPD. Levels of IL-8, a neutrophil chemotaxin, and white cell counts, while not differing significantly between the groups, increased considerably towards the end of the first week of life in the BPD group. IL-16, a chemotaxin for inflammatory CD4+ cells, was also detected in more BPD than non-BPD specimens (BPD: 16/46 (35%) v 1/30 (0.3%) non-BPD preterm and 2/7 (28%) term TF specimens). Conclusions: Extremely premature infants prone to BPD have decreased pulmonary anti-inflammatory activity as demonstrated by decreased IL-10 and apoptotic neutrophils in tracheal fluids. The lack of a counter-regulatory response to the inflammatory processes that are an inevitable consequence of extra-uterine adaptation may therefore place the extremely premature newborn infant at a considerable risk of developing BPD.

Bone metabolism is regulated by endocrine and paracrine signalling molecules influencing bone cells in the continuously remodelling bone tissue. These molecules include a variety of osteotropic stimulatory and inhibitory cytokines. Degradation of alveolar bone in periodontal disease is believed to be a result of local release of such osteotropic cytokines, although the relative importance of particular cytokines and their cellular origin is currently unknown. The aim of the present project was to study if, and how, pro-inflammatory cytokines in the interleukin-6 (IL-6) family of cytokines, and anti-inflammatory IL-4 and IL-13 type of cytokines, can affect osteoclast differentiation and bone resorption. Additionally, the objective was to study if gingival fibroblasts may influence alveolar bone resorption through secretion of IL-6 type cytokine release and if the secretion is regulated by pro-inflammatory as well as anti-inflammatory mediators such as IL-4 and IL-13. IL-6 in combination with its soluble receptor (sIL-6R) was found to stimulate mouse calvarial bone resorption. Similarly, two other IL-6 family members, leukemia inhibitory factor (LIF) and oncostatin M (OSM) were found to stimulate bone resorption. The stimulatory effect on bone resorption induced by the three cytokines was associated with increased expression of receptor activator of NF- κB ligand (RANKL), a cytokine which is essential in osteoclast formation and activation through binding to receptor activator of NF- κB (RANK) on osteoclastic cells. The interaction between RANKL and RANK can be inhibited by binding of the decoy receptor osteoprotegerin (OPG) to RANKL, and the expression of OPG was also regulated by IL-6, LIF and OSM (Paper I). The two related cytokines IL-4 and IL-13 were found to inhibit osteoclastogenesis and mouse calvarial bone resorption by mechanisms involving a decreased RANKL/OPG ratio in osteoblasts and decreased RANK expression in osteoclastic cells. The results further demonstrated that IL-4 and IL-13 exert their effects on both osteoblasts and osteoclasts by a mechanism involving the transcription factor signal transducer and activator of transcription 6 (STAT6) (Paper II). Constitutional expression of IL-6, LIF and another member of the IL-6 family of cytokines, IL-11, was demonstrated in human gingival fibroblasts. IL-6 type cytokine expression levels were found to be enhanced by IL-1β and tumour necrosis factor-α (TNF-α) (Paper III), whereas IL-4 and IL-13 inhibited IL 11 and LIF release from gingival fibroblasts (Paper IV). In conclusion, IL 6 type cytokines were found to be stimulators and IL-4 and IL-13 inhibitors of bone resorption in vitro via mechanisms involving RANK/RANKL/OPG interactions. Additionally, gingival fibroblasts were able to secrete several cytokines in the IL-6 family. Secretion was further enhanced by pro-inflammatory mediators and inhibited by IL-4 and IL- 13. These findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic cytokine production.

La stimulation de cellules T naïves nécessite du déclenchement de la signalisation par l'intermédiaire du récepteur d'antigène de cellule T (TCR) ainsi que l'activation simultanée des récepteurs de co-stimulation. Toutefois, les cellules T CD8+ naïves peuvent proliférer de façon antigène-indépendants suite à la stimulation synergique par certaines cytokines homéostatiques (IL-7 ou IL-15) et inflammatoires (IL-6 ou IL-21). Ces cellules pré-stimulées prolifèrent même à des faibles concentrations d'antigènes ou en présence d’agonistes du TCR. Ceci leur permet de sécréter des cytokines effectrices, d'être plus spécifiques à leur antigène et d’avoir une activité cytolytique plus importante. Les mécanismes déclenchés par les cellules T CD8+ permettant une sensibilité accrue à l'antigène suite à la "pré-stimulation aux cytokines" n'ont pas encore été élucidés. Nous avons utilisé trois différents modèles de souris transgéniques portant le TCR P14, PMEL ou 8.3-NOD sur les lymphocytes T CD8+ afin d’étudier les mécanismes moléculaires suite à la pré-stimulation aux cytokines. Les cellules T CD8+ portant le TCR transgénique amorcées avec les cytokines, possèdent une augmentation globale des protéines tyrosine-phosphorylés après stimulation du TCR par rapport aux cellules naïves. Cette augmentation de la phosphorylation de la protéine tyrosine a été associée à une augmentation de l'expression de CD8, et a été moins prononcé lorsque CD8 a également été réticulés avec le TCR. Ceci suggère que l'amorçage aux cytokines peut prédisposer le TCR et CD8 à colocaliser, ce qui renforcerait la phosphorylation des chaînes du TCR par la kinase Lck associée à CD8. Les lymphocytes T CD8+ amorcées aux cytokines présentent également des quantités accrues de radeaux lipidiques plasmatiques à la membrane, qui organisent la plate-forme de signalisation du TCR au cours de la stimulation antigénique. L’amorçage aux cytokines des lymphocytes T CD8+ a également augmenté la localisation de CD45, une phosphatase qui diminue l’inhibition automatique de la Lck dans les radeaux lipidiques. Cependant, l'amorçage aux cytokines n'a pas d'incidence sur la capacité des cellules CD8+ T pour former des conjugués avec les cellules présentatrices d'antigène puisées avec des peptides apparentés. En conclusion, ces résultats suggèrent que la composition et les fonctions des radeaux lipidiques peuvent moduler la sensibilité à l'antigène via le TCR lorsque les lymphocytes T CD8+ ont été pré-stimulés aux cytokines.

IL-12, an immunoregulatory cytokine, plays a key role in the development of cell-mediated immune responses. However, very little is known about the regulation and induction of the other members of this family, particularly IL-23 and IL-27. The regulation of these cytokines was studied in the human primary monocytes and monocyte-derived macrophages (MDMs) as they play a key role in innate and adaptive immune responses. THP-1 promonocytic cells were employed as a model system to confirm the results obtained with monocytes and MDMs. Two stimuli IFN-γ and LPS were used as both are strong inducers of IL-12 family cytokines. My results show that IFN-γ induced the production of IL-12/23p40 and IL-23p19 mRNA as well as IL-12p40 and IL-23 proteins in primary human monocytes isolated by positive selection. IFN-γ-induced IL-23 and IL-12/23p40 expression was positively regulated by the p38 mitogen-activated protein kinases (MAPK), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling. In contrast, IL-12 and IL-23 were negatively regulated by the Jak/STAT, phosphoinositide-3 kinase (PI3K) and the c-Jun-N-terminal kinase (JNK) MAPKs in IFN-γ-stimulated monocytes. LPS significantly stimulated IL-23p19 and IL-12/23p40 mRNA expression as well as IL-12/23p40 and IL-23 protein production in THP-1 cells, while IFN-γ stimulation alone did not affect IL-23 mRNA or protein levels. THP-1 cells were pre-treated with ERK, JNK or p38 MAPK inhibitors and then stimulated with LPS. LPS-induced IL-12p40 and IL-23 proteins were positively regulated by the p38 and JNK MAPKs and PI3K, whereas LPS-induced IL-23p19 mRNA expression was negatively regulated by these kinases. These results were confirmed using siRNA in LPS-stimulated THP-1 cells. My results also show that IFN-γ/LPS-induced IL-23 expression is not regulated through MAPK or PI3K signaling pathways in human MDMs. My results also show for the first time that IFN-γ alone without any second stimulus induced IL-27p28 gene expression and IL-27 protein production in human monocytic cells. I investigated the signalling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. IFN-γ-mediated IL-27 protein, but not IL-27p28 gene expression was positively regulated by JNK MAPK and PI3K, independent of JAK/STAT signaling in primary human monocytes. I also investigated the signalling pathways governing the regulation of IL-27 and its α subunit, IL-27p28 following stimulation with IFN-γ alone or IFN-γ-primed LPS-stimulated macrophages (IFN-γ/LPS) and THP-1 cells. A differential regulation of IL-27p28 and IL-27 in response to stimulation by either IFN-γ or IFN-γ/LPS was observed. IFN-γ- and IFN-γ/LPS induced IL-27 expression was positively regulated by the JNK, p38 MAPK and PI3K, independent of Jak/STAT signaling in human MDMs and THP-1 cells. Taken together, my results show that IL-23 induction is differentially regulated by different pathways in response to different stimuli, whereas IL-27 expression is regulated by JNK, p38 MAPK and PI3K regardless in the stimulus in human myeloid cells. These results may provide additional strategies aimed at targeting disease, autoimmune disorders and cancer.