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Natural Killer (NK) cells are important in the elimination of virally-infected and cancer cells. NK cell recognise changes in surface molecules on cells, which can cause an NK cell to kill the target cell by either ligation of death receptors or perforin-mediated release of granzymes. Granzymes are serine proteases that cleave numerous cellular proteins that are important in co-ordinating cell death in the target cell. Induction of apoptosis involves dysregulation of cellular machinery including disruption of mitochondrial membrane potential, nuclear fragmentation and cell membrane permeability. Here I have analysed the differences in NK cell susceptibility of human cells transformed with either the adenovirus species A (Ad12) or adenovirus species C (AdS) El regions, as well as looking at the mechanisms of how a cancer cell could become resistant to NK cell-mediated killing.

Toll-like receptors (TLRs) fonn a vital part of the innate immune response to infection through the recognition of diverse molecular patterns leading to the generation of an inflammatory reaction. The resulting cytokines act on both tissue and immune cells to coordinate the response to infection. Cytokine networks also play an important role in the modulation of an increasing number of diseases which we now understand to have an inflammatory basis. TLR activation has been implicated in both chronic and acute diseases, and understanding and modulation of these responses may be central to th'e manageinent of inflammatory disease. This thesis investigates the impact of an enigmatic early response cytokine macrophage migration inhibitory factor on the TLR response of human cells. I have shown that the role ofMIF as an inflammatory cytokine is not clear-cut. Recombinant MIF failed to induce a significant inflammatory response from either a monocytic THP-l cell line or primary human monocytes. Furthennore, it failed to modulate the dexamethasone induced suppression of TLR induced cytokine release, a classically described activity of MIF. In keeping with these observations, anti-MIF antibodies did not modulate the LPS induced cytokine release of monocytes or monocytelHUVEC cocultures, which suggests that MIF may not act as a classical autocrine cytokine. I have demonstrated using a specific, cell-penneable MIF antagonist, known as ISO-I, and MIF siRNA, that MIF modulated specific arms of the TLR response leading to the activation of mitogen activated protein kinases (MAPKs) in monocytic cells. Cell-type spe~ific downstream effects on cytokine production were also seen. ISO-l potentiated LPS induced p38 phosphorylation and TNFa release from THP-I cells. Conversely, in primary monocytes, TNFa and CXCL8 production in response to LPS was significantly inhibited by both ISO-I and MIF siRNA, whilst TNFa but not CXCL8 production was maintained in response to TLR2 activation. LPS induced cytokine release from MDMs was unaffected by :NIIF inhibition. During the course of this thesis I have also observed differences in TLR2 induced inflammatory reactions in primary monocytes. This observation was explored further. These data suggest that whilst targeting MIF may be a useful therapeutic axis in disease, the roles of MIF are not straightforward. Further work will be needed to fully address the roles of this molecule in human biology.

Cellular responses to viral infection and non-self tissues depend on presentation of antigenic peptides by polymorphic MHC class I molecules. The regulatory mechanisms controlling MHC class I expression are fundamental to effective immune responses, and in the pig are not fully understood. In this study the cellular responses of SLA class I genes to cytokines were studied by measuring SLA class 1 expression at the cell surface and also by fusing the promoters of the classical genes SLA-1, -2 and -3 and non-classical genes SLA 6 and -7 independently to a luciferase reporter gene. Cell surface expression of SLA class I was measured on Max cells, Shimozuma cells and porcine aortic endothelial cells isolated from inbred pigs of the d/d haplotype and outbred pigs of an unknown haplotype. IFN-alpha and -gamma treatment increased SLA class I expression on d/d PAECs and Max cells but not on outbred PAECs or Shimozuma cells. Analysis of SLA class I promoter activity in Max cells showed constitutive activity of SLA-1, -2, -3, -6, -7 and MIC-2 promoters. In summary, classical SLA promoters were responsive to IFNs and co-expression of the transcription factors IRF-1, NF- KB p65 and CIITA. In contrast to their human counterparts, combined treatment with TNF-alpha and IFN-alpha/-gamma had no synergistic effect on classical SLA class I promoter activity. SLA-1 responded to TNF-alpha and co-expression of the transcription factors IRF-3, -7 and -9. Non-classical promoters were not induced by IFN-alpha or -gamma or CIITA. SLA-7 was responsive to TNF-alpha and co-expression of IRF-1 but not co-expression of NF-kappaB. Interestingly, basal expression of MIC-2 remained unaffected by cytokines or co-expression of transcription factors. These results suggest locus-specific responses of SLA class I genes to cytokines and transcription factors, which can be explained in part by sequence variation in three key SLA class I promoter motifs: ISRE, Enhancer A and SXY. Furthermore, this study has demonstrated that the porcine virus CSFV decreases SLA class I surface expression early during infection and that this effect correlates to decreased constitutive activity of SLA-1, -2 and -7 promoters.

The Gram-negative bacterium Photorhabdus luminescens is a pathogen of insects. It is able to secrete a variety of toxins and effectors against its host in order to escape its immune defences. The model insect Manduca sexta is able to mount a variety of humoral and cellular responses against pathogen attack. Ultimately these prove ineffective against P. luminescens. The pre-treatment of M. sexta with Escherichia coli provides protection against the pathogenesis of P. luminescens. Here, I use RNA interference and Fluorescence-assisted cell sorting techniques to investigate interactions between pathogen and host to further elucidate the roles of various host factors in mounting the immune response. I also investigate the nutrient requirements of the bacteria for pathogenesis. I show data that peptidoglycan recognition protein (PGRP) is essential for the up-regulation of antimicrobial peptides, an important immune defence. I also show that P. luminescens has a requirement for two types of iron during pathogenesis of M. sexta. And lastly I show that P. luminescens is able to avoid phagocytosis, another important immune defence.

Le porc est un modèle d’étude présentant à la fois une grande importance agronomique et un fort potentiel comme modèle biomédical pour différentes pathologies respiratoires. Pourtant, son système immunitaire pulmonaire est peu connu, limitant l’approfondissement de ces études. Notamment, les cellules dendritiques (DCs) et macrophages (Mθs) tissulaires, qui sont à l’interface entre immunités innée et acquise, n’étaient pas caractérisés. L’objectif de cette thèse était de décrire ces cellules dans le poumon porcin à l’état basal et durant des infections par différentes souches de virus Influenza A (VIA), un pathogène capable d’infecter le porc et l’Homme. Nous avons caractérisé pour la première fois les DCs conventionnelles (cDCs) et dérivées de monocytes (moDCs), ainsi que des macrophages dérivés de monocytes (moMθs) et les macrophages alvéolaires (AMs) porcins. Les cDC1 et cDC2 se sont révélées proches de leurs équivalentes murines et humaines de par leur phénotype et leurs capacités de migration et de présentation de l’antigène in vitro. Les moDCs sont, comme chez la souris, pro-inflammatoires et recrutées lors d’une infection par le VIA in vivo. Nous avons également étudié des cellules tissulaires fortement représentées dans le poumon porcin et dont le phénotype est très similaire à celui des AMs : les cellules AM-like. Ces dernières sont pro-inflammatoires lors d’infections in vitro par un virus H3N2 porcin ou une souche Lena du PRRSV. Nous avons enfin étudié ces différents types cellulaires dans le cadre d’infections par deux souches porcines du VIA, l’une du sous-type H3N2 et l’autre du sous-type H1N2, la première étant plus pathologique en élevage et induisant plus d’inflammation. Il se pourrait que les cellules AM-like aient un rôle important dans cette différence de pathogénicité. En conclusion, nous avons mis en place des moyens pour étudier plus précisément le rôle des DCs et Mθs dans le poumon porcin, et ainsi, de mieux comprendre l’inflammation et la réponse immunitaire pulmonaire. Ce travail ouvre des voies, en santés vétérinaire et biomédicale, qui étaient jusqu’à présent réservées au modèle murin. / Pig is a research model with a major agronomic importance and a great potential as a biomedical model for different respiratory pathologies. Yet, its pulmonary immune system is little known, limiting the deepening of these studies. In particular, tissular Dendritic Cells (DCs) and Macrophages (Mθs), which are at the interface between innate and adaptive immune systems, were not characterized. The aim of this thesis was to describe DCs and Mθs in the porcine lung at steady-state and during infections with different strains of influenza virus A (IAV), a pathogen capable of infecting pigs and humans. We characterized for the first time conventional DCs (cDCs) and monocyte derived (moDCs), as well as monocyte-derived macrophages (moMθs) and alveolar macrophages (AMs) in pig. The cDC1 and cDC2 were phenotypically close to their murine and human equivalents. They also had the same in vitro capabilities of migration and antigen presentation. The moDCs are, like murine ones, pro-inflammatory and are recruited during an IAV infection in vivo. We also found a highly represented interstitial population whose phenotype is very similar to that of AMs: AM-like cells. They are pro-inflammatory upon in vitro infection by a swine H3N2 strain or a Lena strain of PRRSV. Finally, we studied these different cell types through infection by two strains of porcine IAV, an H3N2 and an H1N2 strains, the first one being more pathological and inducing more inflammation. The AM-like cells might play an important role in this pathogenicity difference. In conclusion, we found a strategy to study more precisely the roles of DCs and Mθs in the porcine lung, and thus to better understand lung inflammation and immune response. This work opens pathways, in veterinary and biomedical health, which were previously reserved for mouse model.