Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis that has a damaging economic and welfare impact on the livestock industry. Johne's disease in cattle is known to reduce milk yield and carcass value, making it of economic concern to both dairy and beef farmers. In addition, there is cause for concern regarding zoonotic transmission, as there is an unconfirmed but potential relationship between MAP infection and human Crohn's disease, which presents similar clinical symptoms. MAP is most often contracted by neonates through the faecal-oral route, but can also be spread through contact with contaminated milk and colostrum, as well as in utero. Once the host receives an oral dose, the bacteria traverse the gut epithelium and are phagocytosed by gut macrophages residing in the lamina propria and Peyer's patches. MAP are able to evade the macrophage response by resisting intracellular degradation within phagosomes. Infected macrophages respond to the infection by secreting several pro-inflammatory cytokines that drive the downstream immune response and granuloma formation. This work aimed to elucidate key early responses of bovine monocyte derived macrophages (MDM) to MAP infection, and determine the reliability of using the reference strain, K10 (which is likely to have undergone lab adaptation) to model the infection in vitro, by comparing the MDM response to K10 with the response to a recent clinical isolate, C49. At a multiplicity of infection of 5 (MOI 5), there was a significant decrease in K10 intracellular survival (~90%), compared to C49 intracellular survival, over a 24 hour infection time-course. This suggests that K10 may have lost some virulence mechanism through lab adaptation. Understanding the mechanisms of how MDM respond to these two strains could be informative for the design of targeted vaccines When further investigating the MDM response to both strains, it was found that, at MOI 5, MDM infected with K10 secreted higher levels of IL-1β and IL-10, compared to MDM infected with C49. Both cytokines are associated with mycobacterial infection and could perhaps indicate that MDM are more responsive to the K10 strain at early time-points. In addition, MDM infected with K10 produced significantly higher levels of reactive nitrogen species (RNS). RNS are antimicrobial products that can destroy invading pathogens, and have been shown to have bactericidal effects on MAP. The production of RNS could, therefore be a potential mechanism by which MDM are able to kill K10 more efficiently than C49. An additional aim of this project was to understand the importance of the route of phagocytosis in determining the outcome of MAP infection. MDM express several phagocytic receptors, including Fc receptors (FcRs), complement receptors (CR), Ctype lectin receptors and scavenger receptors. This project mainly focused on the role of the mannose receptor (MR) on bacterial uptake and downstream immune responses, as past studies have suggested that other species of mycobacteria such as M. tuberculosis, target the mannose receptor in order to regulate macrophage immune responses. Blocking the MR reduced intracellular survival for both strains of MAP; however, the mechanism by which the MR influences intracellular survival remains poorly understood The effect of opsonisation on MAP prior to uptake by phagocytic cells was also investigated, as presence of opsonins, such a complement proteins and antibody, can change the mechanism by which pathogens are phagocytosed. MAP were incubated in serum from either MAP- negative or MAP- positive cattle, prior to infection and the percentage uptake and survival assessed by performing colony counts. Opsonisation in serum from Johne's negative cattle resulted in marked increase in MAP uptake but not intracellular survival, whereas opsonisation in serum from Johne's positive cattle did not increase uptake but decreased the intracellular survival rate by 24 HPI. This finding highlights a potential protective role of antibody early in the infection process, and could significantly impact how the infection is modelled in future, as anti-MAP antibody may be present in contaminated milk at the point of infection. Taken together, the data presented in this thesis show that bacterial strain has a significant impact on MDM response to MAP infection, which may have important implications for the interpretation of previous studies and the design of future studies investigating host-pathogen interactions in the context of paratuberculosis. Additionally, this work has shown that RNS production and the mechanism of uptake can affect intracellular survival rates, and although this needs further investigation, the findings could have implications for the design of future vaccines.

The tumour necrosis factor (TNF) superfamily is a group of cytokines that orchestrate a variety of functions, both in the development of the architecture of immune organs and of the immune response. The mammalian TNF superfamily consists of 19 ligands and 29 receptors, whereas in the chicken only 10 ligands and 15 receptors are present. Chickens do not develop lymph nodes, possibly due to the absence of the lymphotoxin genes (TNF superfamily members) in their genome. New members of the chicken TNF superfamily have recently been identified in the genome, namely chicken receptor activator of NF-κB ligand (chRANKL), its signalling receptor, chRANK, and its decoy receptor, osteoprotegerin (chOPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells and mature dendritic cells (DC), respectively. OPG is expressed as a soluble protein from osteoblasts and DC, regulating the interaction between RANKL and RANK. To investigate the bioactivity of this triad of molecules, the extracellular soluble domains of chRANKL and chRANK and full-length chOPG were identified and cDNAs cloned. ChRANKL, chRANK and chOPG mRNA are ubiquitously expressed across non-lymphoid and lymphoid tissues and immune cells in the chicken. Similar to mammals, chRANK and chOPG mRNA expression levels are upregulated in mature bone marrow-derived DC (BMDC). ChRANKL transcription is regulated by Ca2+-mobilisation and is further enhanced by the activation of the protein kinase C pathway, as seen in mammals. The biological activities of chRANKL, chRANK and chOPG were investigated by the production of recombinant soluble fusion proteins. The extracellular, TNF-homology, domain of chRANKL (schRANKL) was sub-cloned into a modified pCI-neo vector expressing an in-frame isoleucine zipper to encourage trimer formation. FLAG-tagged schRANKL produced in COS-7 cells predominantly forms homotrimers and chOPG is expressed as homodimers, both signatures of their mammalian TNF superfamily orthologues. SchRANKL enhances the mRNA expression levels of pro-inflammatory cytokines in mature BMDC and BM-derived macrophages (BMDM). Pre-incubation with soluble chRANK-Fc or chOPG-Fc blocked the schRANKL-mediated increase in pro-inflammatory cytokine mRNA expression levels in BMDC. Expression of surface markers on BMDC and BMDM were not affected by schRANKL treatment. SchRANKL enhances the survival rates of BMDC and BMDM and can drive osteoclast differentiation from monocyte/macrophage progenitor cells. The chRANKL signalling receptor, chRANK, does not contain an intracellular catalytic domain but requires the binding of intracellular TNF receptor-associated factors (TRAF) to initiate signalling. TRAFs are a family of seven proteins (TRAF1-7) grouped due to their highly conserved RING domains, zinc finger domains, TRAF-N and TRAF-C domains. ChRANK possesses four of the five TRAF peptide-binding motifs found in mammalian RANK. The "missing" chRANK TRAF peptide-binding motif is TRAF6-specific, a vital protein for RANKL-mediated osteoclastogenesis. All seven members of the mammalian TRAF family are present in the chicken genome. To investigate the conservation of RANK-specific TRAF signalling proteins, chicken TRAF2 (chTRAF2), chTRAF5, chTRAF6 and a newly found member, chTRAF7, were identified and their cDNAs cloned. ChTRAF5, chTRAF6 and chTRAF7 had mRNA expression patterns, in non-lymphoid and lymphoid tissues and in a number of immune cells, similar to their orthologues in mammals. Interestingly, chTRAF2 has two variants, the full-length chTRAF2 and a novel isoform (chTRAF2S) lacking exon 4. ChTRAF2S lacks a portion of zinger finger one, all of zinc finger two and a portion of zinc finger three, producing a protein with a hybrid of zinc fingers 1 and 3 and intact zinc fingers 4 and 5. RT-PCR analyses indicated differential expression of both of the chTRAF2 isoforms in a number of non-lymphoid and lymphoid tissues, splenocyte subsets and in a kinetic study of ConA-stimulated splenocytes. ChTRAF2S is biologically active compared to chTRAF2, inducing higher levels of NF-κB activation. Co-transfections indicate that chTRAF2 may regulate chTRAF2S bioactivity as no synergistic effect was identified when cells were transfected with both isoforms. Knowledge gained from this study will help work to further dissect the interactions between chRANKL-expressing T cells and chRANK-expressing DC to drive Th1 immune responses and to understand how the chicken mounts an effective immune response while expressing a minimal essential repertoire of the TNF superfamily.

Inhibitors of apoptosis (IAPs) modulate cell death and play critical role in signal transduction that promotes inflammation. Recently, Smac mimetics, which are IAP antagonists, have attracted attention as novel cancer therapeutics. Cellular Inhibitor of Apoptosis 2 (cIAP2), a member of IAP family, positively affects both NF-κB and MAPK activation in response to many inflammatory stimuli. We observed that the lack of cIAP2 ablates LPS-induced neuroinflammation. Also, cIAP2-/- macrophages demonstrated diminished antigen presentation potential that could contribute to ablated immunity. In addition to these functions, we have previously reported that cIAP2 also regulates the activation of Interferon Regulatory Factor 1 (IRF1). Since IRF1-/- mice are resistant to experimental autoimmune encephalomyelitis (EAE), we hypothesized that cIAP2-/- mice will be protected from the disease. Surprisingly, induction of EAE in cIAP2-/- mice resulted in exaggerated infiltration of immune cells increased expression of proinflammatory cytokines and demyelination within CNS. We found that the lack of cIAP2 induces caspase-8 expression in microglia derived macrophages, contributing to their activation and polarization towards M1 phenotype, and exacerbates the symptoms of EAE. These findings suggest that cIAP2 limits neuroinflammation in the CNS and thus the use of Smac mimetics as chemotherapeutics needs to be reevaluated.

Bibliography: leaves 155-180.

Capítulo I: Para evaluar el efecto sinérgico de IL-12/IL-18 y M-CSF/GM-CSF en la diferenciación de los monocitos y su impacto en el estallido respiratorio y el metabolismo del colesterol de los monocitos derivados en macrófagos, los monocitos fueron diferenciados durante 7 días en presencia de la combinación de la granulocyte-macrophage colony-stimulating factor (GM-CSF) y macrophage colonystimulating factor (M-CSF) o la interleucina 12 (IL-12) e IL-18 para producir respectivamente la M / GM-Ф y IL12/IL18- Ф. Como control, los monocitos fueron diferenciados sólo con M-CSF, GM-CSF, IL-12 e IL-18 para producir respectivamente la M-Ф, GM-Ф, IL12-Ф y IL18-Ф. El análisis de muestras de cuatro donantes de monocitos demostró una diferencia en el metabolismo del colesterol y el estallido respiratorio de las subpoblaciones de macrófagos. Los M/GM-Ф y IL12/IL18-Ф estimulados producieron alto nivel de H2O2 y mieloperoxidasa, y generaron gran cantidad de HOCl en respuesta al PMA. Por el contrario, mostraron bajos niveles de enzima antioxidante catalasa. Además intensificaron la oxidación de LDL y acumularon de forma espontánea gran cantidad de colesterol cuando incubado con la lipoproteína de baja densidad nativa. Los resultados sugieren que las vías M-CSF/GM-CSF o IL12/IL18 podría ser algunas señales sinérgica crítica experimentado por los monocitos / macrófagos durante su diferenciación in-vivo, en el que su potencial aterogénico o su capacidad de oxidar el LDL aumenta. Capítulo II: Estudios recientes muestran que el rebote de la carga del VIH-1 después de largos períodos de interrupciones del tratamiento (IT), resulta en un estallido de biomarcadores de la enfermedad arterial coronaria (CAD). Hemos investigado si las interrupciones cortas inducen un estallido de estos biomarcadores, si los niveles de estos biomarcadores vuelven a la basal durante la reintroducción del tratamiento y si los estallidos eran relacionados con el número de interrupciones. Los biomarcadores de CAD CRP, CXCL8, dímero-D, MMP-9 y los lípidos plasmáticos fueron medidos a partir de muestras de plasma almacenadas de 21 sujetos con infección crónica por el VIH-1 sometidos en un estudio de evaluación de seis ciclos de "2 semanas de interrupción" / "4 semanas reintroducción" de la terapia antirretrovirales. Los sujetos fueron agrupados en aquellos con un rebote de la carga viral después de interrumpir el tratamiento y los que no sufrieron del rebote. Los niveles de CRP, MMP-9, CXCL8, dímero-D y los triglicéridos aumentaron significativamente después de cada IT en los pacientes con el rebote de la carga viral. Los cambios de incremento medio en los sujetos sin rebote de la carga viral eran muy bajos en comparación con la basal y sin interés clínico como los valores se mantuvieron entre los rangos plasmáticos normales. Ningún efecto tiempo se observó durante la IT a la excepción de la CRP. Todos los biomarcadores volvieron a los niveles basales después de cada reinicio del tratamiento. Los resultados sugieren que las IT antirretroviral de tan sólo dos semanas son asociadas con un estallido de relevancia clínica de los biomarcadores de CAD aguda, que indica la importancia de la adherencia al tratamiento. / We organized the thesis into two chapters: Chapter I: To evaluate the synergic effect of IL-12/IL-18 and M-CSF/GM-CSF on monocytes differentiation and their impact on the respiratory burst and cholesterol metabolism of monocytes-derived macrophages, monocytes were differentiated for 7 days in the presence of both granulocyte-macrophage colonystimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) or Interleukin-12 (IL- 12) and IL-18 to produce respectively M/GM-Ф and IL12/IL18-Ф. As control, monocytes were differentiated only with M-CSF, GM-CSF, IL-12 and IL-18 to produce respectively M-Ф, GM-Ф, IL12- Ф and IL18-Ф. Samples analyses of four monocytes donors demonstrated a differential in the cholesterol metabolism and respiratory burst of macrophage subpopulations. Stimulated M/GM-Ф and IL12/IL18-Ф produce high level of H2O2 and myeloperoxidase; and generate significant amount of HOCl in response to PMA. In contrast, they show low levels of anti-oxidant enzyme catalase. Moreover they intensify LDL oxidation and spontaneously accumulate significant amount of cholesterol when incubated with unmodified low-density lipoprotein. The results suggest that M-CSF/GM-CSF or IL12/IL18 pathways might be some critical synergic signals experienced by monocytes/macrophages during their differentiation in-vivo; in which their atherogenic potential or their capacity to oxidize LDL increase. Chapter II: Recent studies show that HIV-1 load rebound after long periods of treatment interruptions (TI), results in a burst of coronary artery disease (CAD) biomarkers. We investigate whether short interruptions induce a burst of these biomarkers, whether their levels return to the baseline during treatment resumption and if the burst were related to the number of interruptions. CAD biomarkers CRP, CXCL8, D-dimer, MMP-9 and plasma lipids were measured from stored plasma samples of 21 chronically HIV-1 infected subjects enrolled in a study evaluating six cycles of “2 weeks off” / “4 weeks on” antiretroviral therapy. Subjects were clustered into those with a viral load rebound after stopping treatment and those without. The levels of CRP, MMP-9, CXCL8, D-dimer and triglycerides rose significantly after each TI in subjects with viral load rebound. Changes of means increment in subjects without viral load rebound were too low relative to the baseline and without clinical interest as values stayed between the normal plasma ranges. No times effect was observed during TI except for CRP. All biomarkers return to baseline levels after each treatment resumption. The results suggest that antiretroviral TI as short as two weeks are associated with a clinically relevant burst of acute CAD biomarkers, that indicating the importance of adhering to treatment.

No description available.

Tuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.

Thèse de doctorat : Biologie cellulaire et moléculaire : Paris 12 : 2005. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 189 réf.

Thèse de doctorat : Sciences de la vie et de la santé : Paris 12 : 2004. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 305 réf.

Thèse doctorat : Biochimie : Villeurbanne, INSA : 2006. / Titre provenant de l'écran-titre. Bibliogr. p. 162-206. Notes bibliogr. , 2 p.