Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Les maladies infectieuses dues aux bactéries Gram + sont causes de mortalité importante à travers le monde, et de récentes études ont mis en évidence le rôle pathologique de l’interféron de type I (I IFN) dans ces maladies. Les cellules dendritiques plasmacytoïdes (pDC) produisent des quantités importantes d’IFN de type I suite à la détection de virus. Des données récentes suggèrent que les pDC humaines pourraient également détecter des bactéries, mais les récepteurs impliqués restent inconnus. Au cours de ma thèse, j’ai caractérisé l’expression des récepteurs TLR2 / 1 par les pDC. Ces deux récepteurs permettent aux pDC de détecter les lipoprotéines bactériennes. Je montre que les pDC répondent aux bactéries Gram + (M. tuberculosis, S. aureus et L. monocytogenes) par la voie TLR2 / 1. Mon travail a montré que les pDC primaires humaines expriment TLR1 et TLR2 à la fois au niveau de l'ARNm et au niveau protéique. En réponse aux lipoprotéines bactériennes, la régulation des molécules costimulatrices par les pDCs est TLR1-dépendante tandis que la sécrétion d’I-IFN est TLR2-dépendante. De plus, TLR2 et TLR1 jouent des rôles distincts au cours du priming des cellules T CD4+ naïves par les pDCs, induisant une prolifération et différentiation en sous-populations Th1 / Th2 / Treg. Je démontre en outre que ces différences reposent sur les voies de signalisation distinctes de ces deux TLR. Ce travail de thèse pose ainsi les bases pour l’exploration du rôle des pDC dans les infections bactériennes humaines. / Infections by Gram+ bacteria are worldwide life-threatening diseases where new studies are highlighting the pathological role of Type I interferon (I IFN). Plasmacytoid dendritic cells (pDCs) are the main source of Type I IFN following viral sensing. Recent evidence suggests that human pDCs might sense bacteria. The receptors mediating bacterial sensing in pDCs are not known. During my thesis, I focused on the characterization of pDCs TLR2/1 receptors expression. These two receptors allow pDCs to sense Gram+ bacterial lipoproteins. My work showed that human primary pDCs express TLR1 and TLR2 at the mRNA and protein level. I show that pDCs respond to the Gram+ bacteria M. tuberculosis, S. aureus and L. monocytogenes through TLR2/1 pathway. In human primary pDC, I found that in response to bacterial lipoproteins up-regulation of costimulatory molecules is TLR1-dependent while IFN-I secretion is TLR2-dependent. TLR2 and TLR1 signalling play a different role in the pDCs priming of naïve CD4+ T-cells, inducing proliferation and differentiation to TH1/TH2/Treg subsets. I further demonstrate that these differences rely on the diverse signaling pathway activated by the two TLRs. This work provides the rationale to explore pDCs activity in human bacterial infection.

The innate immune system is at the interface between the host's immune system and the initial contact with HIV. Understanding the correlates of innate immune protection against Human Immunodeficiency Virus is an important goal for development of effective anti-HIV therapies or vaccines. Not all exposures to HIV end in infection. The innate immune system has been linked to the reduced susceptibility of HIV-exposed seronegative (HESN) female commercial sex workers in Kenya by a number of studies. This thesis is a comparison of Toll-like receptor (TLR) responses in different immune cells in peripheral blood mononuclear cells (PBMCs) from HESN and HIV negative (susceptible) female commercial sex workers (CSWs). This study tested the hypothesis that higher TLR8 responsiveness in PBMCs of HESN to ssRNA analogous to HIV's genetic material, would result in higher effector responses capable of making HIV target cells more refractory in vitro, compared to susceptible controls. The results showed that PBMCs of HESN were often hypo-responsive to TLR4 and TLR7 stimulations evidenced by often reduced cytokine responses to the corresponding ligands, but hyper-responsive to TLR8 following stimulation with ssRNA analogous to HIV's genetic material. The 'dichotomy' in TLR responsiveness of HESN PBMCs was associated with differential expression of cognate TLRs in PBMCs, and altered activation of TLR signalling pathways. The opposing pattern of TLR7 and TLR8 responsiveness corresponded to the ability of HIV to infect target cells in vitro; where pre-treatment of PBMCs with TLR7 enhanced HIV replication whereas TLR8 stimulation inhibited HIV replication. The differences in outcomes of the HIV infection assays were associated with distinct cytokine profiles, where TLR7 stimulation induced robust type I IFNs responses without proinflammatory TNF-α and IL-12 cytokine responses,while TLR8 stimulations produced type II IFN responses accompanied by robust proinflammatory responses in both groups. The cytokine milieu of HESN PBMCs prior to and following TLR4 and TLR8 stimulations was more tightly regulated, but was associated with higher activation of CD8+, NK cells, monocytes but not blood DCs. These results demonstrate that the lower activation or 'quiescent' state of HESN PBMCs did not limit the ability of their cells to recognize ssRNA analogous to HIV derived genetic material and mount potent responses capable of limiting HIV infection in vitro, supporting the overall hypothesis tested. This thesis contributes to the growing knowledge on the dichotomous outcomes between TLR7 and TLR8 treatments with respect to HIV infection that could be instrumental in the design of novel HIV inventions such as vaccines or microbicides. / May 2016

The commensal fungus, Candida albicans, can cause life-threatening infections in at risk individuals. C. albicans colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, C. albicans is not pathogenic nor does it cause epithelial cell damage in vitro; at high concentrations, C. albicans causes mucosal infections and kills epithelial cells in vitro. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to C. albicans. Using transcriptional profiling experiments and real time PCR, we found that wild-type C. albicans induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various C. albicans strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with C. albicans as epithelial cells themselves promote C. albicans stimulated damage.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Cysteine cathepsins B, L and S are endosomal/lysosomal proteases that participate in numerous physiological systems. Cathepsin expression and activity are altered during various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, neurodegenerative diseases and cancers. Early immune responses to microbial pathogens are mediated by pattern-recognition receptors, including Toll-like receptors (TLR). Signaling through TLR causes cell activation and release of inflammatory mediators, which can contribute to the severity of chronic inflammatory diseases. The impact of TLR cell activation on cathepsins B, L and S activities was investigated using live-cell enzymatic assays. Individual ligands of TLR4, TLR2 and TLR3 increased intracellular activities of the three cathepsins indicating the involvement of both MyD88-dependent and -independent pathways. To investigate the role of inflammatory cytokines in regulating these proteases, a lipopolysaccharide (LPS) non-responsive cell line was utilized. LPS non-responsive cells co-cultured with LPS responsive macrophages upregulated cathepsin activities. Furthermore, culture supernatants from LPS-stimulated macrophages increased cathepsin activities in LPS non-responsive cells, which could be reduced by neutralizing antibodies to TNF-α or IL-1β. These findings indicate cytokines regulate cathepsin activities during macrophage responses to TLR stimulation. Using LPS as a model for inflammation, the ability of the cannabinoids, delta9-tetrahydrocannabinol (THC), and CP55940 to suppress cysteine cathepsins during an inflammatory response was investigated. Cannabinoids, including the major psychoactive component of marijuana THC, modulate a variety of immune responses and have been proposed as possible therapeutics to control chronic inflammation. Cannabinoids may mediate their effects through receptor-dependent or independent mechanisms. Cannabinoid receptor subtype 1 (CB1) and receptor subtype 2 (CB2) have differential expression in leukocytes. Dose response studies showed that 1 nM THC was sufficient to inhibit cathepsin enhancement in LPS-stimulated cells. P388D1 macrophages expressed CB2 mRNA, but had no detectable CB1 mRNA indicating a role for the CB2 receptor. Utilizing a CB2-/- macrophage cell line, the role of CB2 receptor participation in THC inhibition of cysteine cathepsin upregulation was explored. THC did not affect cathepsin activity in LPS-stimulated cells lacking CB2 expression. These findings support the possibility of receptor selective agonists as therapeutic treatment during inflammatory diseases to prevent cathepsin involvement in pathological tissue destruction.

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...

8 Abstract Introduction: Celiac disease is indentified as the loss of oral tolerance to gluten, it is an organ-specific autoimmune disease in which both, adaptive and innate immunity participate. Monocytes are important part of immune system; they have many functions and express very diverse membrane receptors including Toll-like receptors (TLRs). TLRs are involved in the innate immune response, specifically TLR2 and TLR4 are crucial for recognition of bacterial components and TLR7 recognizes virus's ssRNA. Monocytes also produce prolaktin (PRL), which acts as a cytokine that modulates immune responses. To clarify the role of innate immunity and circulating monocytes in pathogenesis of celiac disease, we focused on changes in expression of selected Toll-like receptors (TLR2, TLR4, TLR7), prolactin, some pro- a anti-inflammatory cytokines (TNF-α, IL-6, IL-12, IL-10). We monitored the influence of the SNP - 1149 G/T on the expression of PRL mRNA. Another objective of this work was the introduction and optimization of in vitro methods for cultivation and stimulation of peripheral monocytes. Material and Methods: This pilot study includes 21 patients with celiac disease and 40 healthy controls. For determination of mRNA levels of the studied genes we isolated RNA from monocytes that were isolated by...

Introduction: Communication between neuroendocrinne and immune system is arranged by hormones and cytokines in endocrinne, paracrinne and autocrinne manner. One of the factors involved is also prolactin, a pituitary hormone and an immune cytokine. Sepsis is a system reaction to inflammation mediated by Th1 immune response, which is supported by prolactin as well. Primary protection against sepsis is mediated by innate immunity. Toll- like receptors distinguish molecules, which are connected with pathogens. Afterwards this identification of a specific pathogen toll-like receptors trigger immune reaction with the main goal of destroying this pathogen and also with the goal of renewing the balance of the organism. It is supposed that in the organism that is hardly attacked by a pathogen, the PRL, TLR2 and TLR4 gene expression is on the increase. We studied the levels of PRL, TLR2 and TLR4 mRNA production in circulating monocytes derived from septic patients. Simultaneously, the effect of PRL -1149 G/T SNP on physiological levels of PRL mRNA and its expression in the course of sepsis was evaluated. Materials and methods: As a source of monocytes, blood specimens from 43 septic patients and 40 healthy controls were used. The blood of septic patients was taken three times with some time difference and...