La littérature indique que les miARN sont régulés à plusieurs niveaux de leur biogenèse et de leur activité. Cependant, il existe très peu d’information concernant la régulation de la stabilité des miARN. Le projet de thèse a consisté à étudier la dégradation spécifique d’un miRNA cellulaire (miR-27) induite par un transcrit viral (m169) au cours de l’infection par le cytomégalovirus murin (MCMV). Ce miARN est déstabilisé par un mécanisme moléculaire appelé ‘target-RNA directed miRNA degradation’ (TDMD). En suivant deux grands axes de recherche j’ai entrepris : premièrement l’étude et la caractérisation des déterminants moléculaires et des facteurs cellulaires impliqués dans le mécanisme de TDMD ; puis dans un second temps, la mise en place d’une approche protéomique permettant l’identification des partenaires de la protéine AGO2 potentiellement impliqué dans le TDMD dans des cellules infectées ou non par le MCMV. / Several regulatory mechanisms have been uncovered at every level of the biogenesis and the activity of miRNAs. However, there is less information about the regulation of the stability of miRNAs. The PhD project entailed the study of a process, which specifically enables the degradation of a cellular miRNA (miR-27) induced by a viral transcript (m169) during an infection by the mouse cytomegalovirus (MCMV). This miRNA is destabilized by a process called ‘target-RNA directed miRNA degradation’ (TDMD). I first undertook the study and the characterization of the molecular determinants and the cellular factors implicated in TDMD. Moreover, I started to set up a protocol in order to identify AGO2 partners of viral or host origin during MCMV infection, which would potentially be implicated in TDMD.

To study the interplay between spatial nuclear architecture and transcriptional activity during viral infections, I employed a genome-wide chromosome conformation capture approach (Hi-C) on infected murine and human cells and further enriched those libraries for genomic loci of interest and the viral genomes with biotinylated RNA baits. In parallel, I profiled newly transcribed RNA throughout the entire kinetic of murine cytomegalovirus (mCMV) infection in mice. Host genome rearrangement is a well-known phenomenon of mCMV infection but the underlying mechanisms are largely unknown. Furthermore, HPV infection can lead to cervical cancers in humans, with genomic instability and re-arrangements, leading to dysregulation of gene expression. Thus studying changes in genome architecture at early stages of HPV induced carcinogenesis can further our understanding on how certain integration events can provide a growth advantage. In this study, I identified clusters of genes characterized by distinct kinetic profiles upon CMV infection in the mouse, which were associated with distinct functional terms. ATAC-Seq uncovered proximal promoter regions (PPR) that showed an over-representation of specific transcription factor binding sites in each of the clusters. These correlated well with the annotated functions of the associated clusters. Further, I found that lytic mCMV infection is accompanied by local and global changes of chromosomal interactions in the host cell genome. Notably, chromatin properties, such as gene density, GC content and the association with the nuclear lamina, predict the structural dynamics upon infection and correlate well with transcriptional activity and changes thereof. High-resolution interaction profiles for TSSs of highly induced or repressed genes, suggest that in general, enhancer-promoter interactions already form in untreated cells; and these pre- existing DNA-structures are not significantly altered but function through transient activation or repression of enhancers. Finally, the viral genome showed a distinct pattern of open and closed chromatin late in infection. We found that the 7.2 kb viral intron displays the most open chromatin, and is highly enriched for chromosomal contacts with the host genome. Hi-C and capture Hi-C revealed that both short- (~50 kb) and long-range (~1 Mb) interactions occur during the early stages of HPV induced carcinogenesis between the host and the integrated HPV16 genomes. Integration and direct interactions between the viral genome and the host DNA were shown to be associated with changes in host gene expression. In addition, insertion of the virus can disrupt normal host architecture. In summary, this project pioneers the study of changes in nuclear architecture upon viral infection in man and mice. I uncover numerous structural features and changes of both the viral genomes and the infected host cellular genomes, and I demonstrate that these changes correlate with transcriptional activity.

MicroRNAs (miRNAs) have been identified as a key regulator in various biological processes and different diseases including cancer, heart disease, and viral infections. In the context of virus-host interactions, previous genome wide functional screen involving overexpression and inhibition of murine miRNAs in vitro identified several miRNAs that suppressed viral replication in diverse herpesviruses including herpes simplex virus 1 (HSV-1), murine cytomegalovirus (MCMV) and murine gamma herpesvirus 68 (MHV-68) (Santhakumar, D. et al, 2010). One of the top broad-spectrum anti-viral miRNAs, miR-542-5p, also suppressed human cytomegalovirus (HCMV) as well as a Semliki Forest virus (SFV) and two subtypes of influenza A virus (H1N1 & H3N2) in vitro. Following the previous study, this thesis focuses on generation of in vivo anti-viral efficacy data using miR-542-5p against two diverse viruses: MCMV and influenza (H1N1) in a pre-clinical model (mouse). One of the key challenges for generating in vivo efficacy data with miRNAs is the choice of delivery vehicle. To address this issue the first part of the project focused on optimising delivery conditions (dose, route of administration) for miR-542-5p mimic to target the lungs of mice (as both MCMV and H1N1 replicate in lungs). Initially, delivery was optimised using two cationic polymers: linear polyethylenimine (in vivo-JetPEI) and branched polyethylenimine (25KDa bPEI) that have been widely used previously to deliver nucleic acids in mouse. In parallel, two novel delivery systems were tested as an alternative delivery vehicle for miRNA mimics: biodegradable cationic lipids (Lifectin) and exosomes, natural vesicles produced by cells that can transport RNA. Results from in vivo delivery studies indicate that nebulisation of miR-542-5p mimic complexed with bPEI (25KDa) showed a more significant increase in the level of miRNA in the lung compared to other delivery systems and did not result in an immune response. Using bPEI as the delivery vehicle, the miR-542-5p mimic was administered to mouse lungs to test its anti-viral function against H1N1 and MCMV. Delivery of the miR-542-5p mimic resulted in 4.6 fold reduction of H1N1 virus titre in lungs (averaged across multiple experiments). The miR-542-5p mimic also had a 2 fold reduction in MCMV titre in the lungs. These data confirm the broad-spectrum anti-viral effect of miR-542-5p in mouse as observed in previous in vitro studies. Preliminary microarray analysis of genes regulated by miR-542-5p in vitro suggest this miRNA may target multiple genes required by diverse viruses during their life cycles and may modulate the PI3K-Akt signalling pathway.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The âMinha Casa Minha Vidaâ (MCMV), which aims to reduce the housing deficit, launched its second phase, with a bold goal to hire two million housing units by the year 2014. While this economic environment encouraged companies in the Construction Industry, they were not adequately prepared to absorb the new demands. To reach them, we need to implement innovations in traditional processes of design and construction of this type of housing, increasing productivity, but without loss of quality of the products generated. The projects related to housing construction have become increasingly complex, and has increased the number of projects needed to better execution of the work, causing serious problems of compatibility and rework. In view of the peculiar characteristics of the enterprises of Social Housing (HIS), the profit margins offered by these are greatly reduced, forcing companies to pursue lower production costs and execution time of compression, in order to minimize the incidence of fixed costs. It is worth noting that the initial stages of development, such as program and project are those that present the greatest opportunities for intervention and value. In some countries, especially the United States, these problems have been minimized through approaches such as Integrated Project Delivery (IPD) and the use of design tools such as the Building Information Modeling (BIM). In turn, the vast majority of projects in Brazil, especially the HIS, are not designed with this in mind integration. Within this context, the aim of this work is to propose guidelines for increasing the level of integrated management for design firms that operate in MCMV (range 0-3 minimum wages), based on the principles of IPD. This is a qualitative study with an exploratory-descriptive and research strategy used was the multiple case study, divided into four phases: literature review, exploratory stage, stage of conducting case studies and step analyzes and propositions . Therefore, we carried out a survey to contextualize the partial results of the program and analyze the role of each of the major players involved. There was a peculiarity in the state of CearÃ: the Sinduscon-Ce provides companies affiliated three types of architectural design, installations and structures (reference projects). We investigated how was the design process of these types and the level of integration between professionals. The main tool for data collection were semi-structured interviews with the designers, with the representative of Box and Sinduscon. Based on these data, we carried out a cross-sectional analysis of business and design, as well as a diagnosis of these assumptions with respect to the IPD. We also propose an adaptation of the principles of IPD directed to designers Finally, the main contribution of this work is to propose guidelines for increasing the level of integrated management between designers working in MCMV, relating them to the principles of IPD . / O Programa Minha Casa Minha Vida (MCMV), que tem por finalidade reduzir o dÃficit habitacional brasileiro, lanÃou sua segunda fase, com uma meta ousada de contratar dois milhÃes de unidades habitacionais atà o ano de 2014. Ao mesmo tempo em que este cenÃrio econÃmico incentivava as empresas da IndÃstria da ConstruÃÃo Civil, estas nÃo estavam adequadamente preparadas para absorver as novas demandas. Para alcanÃÃ-las, à preciso implementar inovaÃÃes nos processos tradicionais de projeto e construÃÃo deste tipo de moradia, aumentando a produtividade, porÃm sem prejuÃzo de qualidade dos produtos gerados. Os projetos ligados à construÃÃo habitacional tÃm se tornado cada vez mais complexos, bem como tem aumentado a quantidade de projetos necessÃrios a uma melhor execuÃÃo da obra, provocando sÃrios problemas de compatibilizaÃÃo e retrabalho. Em face das caracterÃsticas peculiares dos empreendimentos de HabitaÃÃo de Interesse Social (HIS), as margens de lucro proporcionadas por estes sÃo bastante reduzidas, forÃando as empresas a perseguirem menores custos de produÃÃo e a compressÃo dos prazos de execuÃÃo, como forma de minimizar a incidÃncia de custos fixos. Cabe ressaltar ainda que as etapas iniciais do empreendimento, tais como o programa e o projeto, sÃo as que apresentam as maiores oportunidades de intervenÃÃo e agregaÃÃo de valor. Em alguns paÃses, principalmente nos Estados Unidos, estes problemas tÃm sido minimizados atravÃs de abordagens como o Integrated Project Delivery (IPD) e do uso de ferramentas de projeto tais como o Building Information Modeling (BIM). Por sua vez, a grande maioria dos empreendimentos brasileiros, especialmente os de HIS, nÃo sÃo desenvolvidos dentro deste espÃrito de integraÃÃo. Dentro deste contexto, o objetivo deste trabalho à propor diretrizes para aumentar o nÃvel de gestÃo integrada em empresas de projeto que atuam no programa MCMV (faixa de 0-3 salÃrios mÃnimos), com base nos princÃpios do IPD. Trata-se de um estudo qualitativo, com carÃter exploratÃrio-descritivo, e a estratÃgia de pesquisa utilizada foi o estudo de caso mÃltiplo, dividida em quatro fases: pesquisa bibliogrÃfica, etapa exploratÃria, etapa de conduÃÃo dos estudos de caso e etapa de anÃlises e proposiÃÃes. Para tanto, realizou-se um levantamento para contextualizar os resultados parciais do referido programa e analisar o papel de cada um dos principais agentes envolvidos. Verificou-se uma particularidade no estado do CearÃ: o Sinduscon-Ce disponibiliza Ãs empresas filiadas trÃs tipologias de projeto de arquitetura, instalaÃÃes e estruturas (projetos de referÃncia). Investigou-se como se deu o processo de projeto destas tipologias e qual o nÃvel de integraÃÃo entre os profissionais. A principal ferramenta para a coleta de dados foram as entrevistas semiestruturadas com os projetistas, com representante da Caixa e do Sinduscon. Com base nesses dados, realizou-se uma anÃlise cruzada das empresas e projeto, assim como um diagnÃstico destas com relaÃÃo aos postulados do IPD. PropÃe-se ainda uma adaptaÃÃo dos princÃpios do IPD direcionada para os projetistas Por fim, a principal contribuiÃÃo deste trabalho à a proposiÃÃo de diretrizes para aumentar o nÃvel de gestÃo integrada entre os projetistas que atuam no programa MCMV, relacionando-as aos princÃpios do IPD.

The Tumor Necrosis Factor Receptor Superfamily (TNFR) is responsible in regulating a myriad of physiological function including the regulation of the immune system. Among the members include CD137 (4-1BB), an inducible costimulatory receptor known for its potent activation, proliferation, and survival effects on T cells. Stimulation of NK cells with agonistic α-CD137 antibodies are known to increase IFN-γ production and proliferation in NK cells as well as increase efficacy of anti-tumor responses. However, NK cell death has also been seen in certain circumstances, although the mechanism remains to be determined. In vitro stimulation of NK cells revealed that α-CD137 induced NK cell death occurs through both TNFR1 and TNFR2, although the action of TNF-α and TNF-ß remain uncertain. Death was independent of other cytotoxic mechanisms such as granzyme/perforin, Fas-Fas ligand, and TRAIL. During MCMV infection, α-CD137 induces NK cell death during the early phase of infection reducing viral resistance. This causes increased viral proliferation which drives NK cell proliferation, likely through Ly49H-m157 interactions, to high levels by day 4 of infection. The use of α-CD137 as a tumor therapeutic is promising with several applications undergoing clinical trials. However, my results raise concern of other effects including the depletion of NK cells. This may cause a temporary impairment in immune function against pathogenic infections and a compensatory reaction of NK cell proliferation, both of which may cause damage to the host. However, with proper co-stimulation or co-treatments, this impairment may be overcome and prevent adverse effects in patients.

Dans les organismes multicellulaires, la régulation de l’expression des gènes par les microARNs est un mécanisme essentiel pour le développement cellulaire et l’homéostasie. De plus, le rôle des microARNs a été démontré dans de nombreux processus immunitaires, tels que l’inflammation. Les virus évoluant conjointement avec leurs hôtes, ils ont appris à détourner la machinerie cellulaire pour leur propre bénéfice. Ainsi, des microARNs codés par certains génomes viraux ont été mis en évidence, mais leurs fonctions, ainsi que leurs cibles, restent encore largement inconnues. En utilisant une lignée de souris présentant une mutation hypomorphe pour le gène dicer, caractérisée par une diminution de la production des microARNs, et son hôte naturel, le cytomégalovirus murin, un virus membre de la famille des β-Herpesvirus, nous avons étudié le rôle potentiel des microARNs d’origine cellulaire et virale dans la pathogénèse de ce virus. Lors de l’infection aigüe, nos résultats montrent un rôle dominant et protecteur des microARNs cellulaires, comparé à celui des microARNs viraux, prédits pour être des facteurs de pathogénicité. / In multicellular organisms, gene expression regulation by microRNAs is an essential mechanism for cell development and homeostasis. Moreover, several immune-related processes, such as inflammation, have been demonstrated to require specific microRNAs. As viruses have coevolved with their host, they have learned to hijack the cellular defenses for their own benefit. Thus microRNAs-encoding genes were also recently discovered in the genome of Herpesviruses, but up to now, the function and the targets of most microRNAs of viral origin are still largely unknown. Using a hypomorphic mouse mutant line, characterized by a diminished production of microRNAs, and the Mouse Cytomegalovirus, a natural pathogen of mice which belongs to the family of β-Herpesviruses, we investigated the potential roles of microRNAs of both cellular and viral origin in the pathogenesis of this virus. Our results point toward a dominant role of cellular microRNAs as protective factors compared to virally-derived microRNAs which are usually predicted to carry pathogenic functions in acute infections.

Submitted by Puentes Torres Antônio (antoniopuentes@hotmail.com) on 2017-06-06T17:43:58Z No. of bitstreams: 1 TESE_VERSÃO PARA IMPRESSÃO.pdf: 12345807 bytes, checksum: 26ecfd9d51fc42cb60d781e3fc5a990b (MD5) / Approved for entry into archive by Vanessa Reis (vanessa.jamile@ufba.br) on 2017-06-16T15:21:19Z (GMT) No. of bitstreams: 1 TESE_VERSÃO PARA IMPRESSÃO.pdf: 12345807 bytes, checksum: 26ecfd9d51fc42cb60d781e3fc5a990b (MD5) / Made available in DSpace on 2017-06-16T15:21:19Z (GMT). No. of bitstreams: 1 TESE_VERSÃO PARA IMPRESSÃO.pdf: 12345807 bytes, checksum: 26ecfd9d51fc42cb60d781e3fc5a990b (MD5) / Essa tese apresenta uma análise do processo de produção do espaço associado aos residenciais do programa Minha Casa Minha Vida (MCMV) que foram construídos no bairro da Mangabeira, uma área periférica na cidade de Feira de Santana-Bahia, entre 2009-2014. As bases metodológica e teórica foram de Henri Lefebvre com o método regressivo-progressivo e a teoria “A produção do espaço”, segundo os quais se compreende o espaço como contido nas relações sociais e em uma dimensão tríplice: o espaço concebido, o espaço percebido e o espaço vivido. Para alcançar o objetivo central, realizou-se uma análise do espaço concebido, através do planejamento proposto pelo Governo Federal, em parceria com o Municipal e o capital imobiliário. Essa análise envolveu tanto a percepção da administração municipal, das construtoras como, e especialmente, a da população moradora dos residenciais do MCMV (o percebido), além das práticas espaciais destes últimos (o espaço vivido). Com isso, buscou-se compreender uma especificidade, a localização geográficas desses residenciais, e para tanto foi operacionalizado o conceito de periferia como norteador da pesquisa e do processo de periferização em Feira de Santana. Por meio do estudo de caso em Feira de Santana, a pesquisa permitiu a compreensão de aspectos das políticas públicas de habitação no país e na Bahia. Além de ter possibilitado o entendimento de que apesar dos residenciais do MCMV terem interferido no processo de produção do espaço, ao afetar e modificar as áreas nas quais foram construídos, quem de fato produziu esses espaços foram os moradores, quando se apropriaram e utilizaram os espaços e equipamentos previamente planejados tal qual suas necessidades e não pelo que sua função técnica determinava. / ABSTRACT This thesis presents an analysis about space production process associated with the housing Brazilian Program named Minha Casa Minha Vida (MCMV). Our study took place in the neighborhood of Mangabeira, a peripheral area in the city of Feira de Santana, Bahia, where the program built between 2009-2014 several housing units. The methodological and theoretical basis are Henri Lefebvre with his regressive-progressive method and his theory "The production of space", which includes the space as contained in social relations and hold a triple spatial dimension: the conceived, the perceived and the lived. To achieve the main objective, was conducted an analysis of the conceived space through the planned space proposed by the Federal Government, in partnership with the City and the real estate enterprises. We also analyze how the municipal administration, construction companies and, especially, the resident population of the residential MCMV houses see this space (perceived), without forgetting the spatial practices of the inhabitants producing the living space. We also operationalized the concept of periphery as a guiding research to understand the geographical location of these housing condominiums, as well to understand the peripherization process in Feira de Santana. The study allowed us to comprehend aspects of public housing policy in the country and in Bahia, through the case study in Feira de Santana. Moreover, despite the residential of MCMV have interfered in the space production process, affecting and modifying the areas in which they were built, who actually determine the production of these spaces were the inhabitants, when they appropriated and used the spaces and equipment previously planned due their needs and not because the technical or function determined by the planed proposal.

Murine cytomegalovirus (MCMV) encodes a non-coding RNA, m169, that inhibits the cellular miRNA, miR-27. Previous studies have shown that the overexpression of miR-27 in vitro suppresses replication of MCMV and degradation of miR-27 by m169 is important for the viral replication during the lytic stage of infection in vivo. To understand why the virus specifically targets this cellular miRNA for degradation, this thesis focuses on identification of cellular target genes of miR-27 that are involved in viral growth in the lytic infection. Microarray analysis was conducted to globally examine cellular genes differentially expressed following miR-27 overexpression or repression during MCMV infection. Data obtained from the microarray analysis were analysed in order to select potential targets of miR-27 for functional screening. Functional screening involved siRNA knockdown of individual genes followed by infection with a GFP reporter virus (GFP-MCMV) to assess the effects on viral growth. Knockdown of 5 out of 55 genes (Rpl18a, Lyar, Itga5, Mapkapk3 and Pik3r1) led to a significant reduction in GFP expression. Based on luciferase reporter assays, Mapkapk3 was validated as a direct target of miR-27 with a seed site interaction in its 3’UTR. Mutation of this site in the mRNA was shown to eliminate miR-27-mediated repression. Analysis of MAPKAPK3 protein levels upon infection demonstrates that the protein levels are higher in cells infected with wild type MCMV versus the m169 deletion virus (MCMV Δm169). This is in line with the difference in miR-27 levels in the two infections showing a decrease of miR-27 in wild type MCMV and unaltered levels in MCMV Δm169 infection. Mapkapk3 is a direct downstream target of p38 mitogen-activated protein (MAP) kinase within the p38 MAP kinase pathway, which has previously been shown to be an essential pathway for CMV replication. Expression levels of substrates of MAPKAPK3 including HSP27 and ATF1 were examined during infection to evaluate whether they are regulated by miR-27. The level of phosphorylation of HSP27 was shown to correlate with the levels of MAPKAPK3 during infection and was higher in cells infected with wild type MCMV versus MCMV Δm169. This suggests that MAPKAPK3 and its substrate, HSP27, are regulated by miR-27 during MCMV infection. This work provides an important foundation for further functional studies on the role of Mapkapk3 and its substrates in MCMV infection and its capacity to be dynamically regulated by miR-27. Based on the microarray analysis upon miR-27 overexpression, it was shown that miR-27 has an impact on the cell cycle, consistent with previous studies. Functional analysis of miR-27 in the cell cycle using miR-27 mimics and inhibitors demonstrated that the mimics cause an increase of cells in S phase at early time points (12 and 14 h), whereas the inhibition of miR-27 results in a significant reduction in the S phase population and accumulation of cells in G1 phase. Luciferase reporter assays confirmed that two genes known to be associated with the cell cycle are direct targets of miR-27: polycomb ring finger oncogene 1 (Bmi1) and caveolin 1 (Cav1). Knockdown of Bmi1 and Cav1 leads to a significant decrease in the number of cells in S phase and accumulation of cells in the G1 phase; however, this is the opposite result to that observed with the miR-27 mimics. These results suggest that the increase in cells in the S phase induced by miR-27 mimics is unlikely to be associated with targeting of Bmi1 and Cav1. Furthermore, knockdown of Bmi1 and Cav1 does not affect viral replication in vitro. Since miR-27 induces the transition of cells from the G1 to S phase, further studies are required to identify the miR-27 targets involved in this function. To identify direct targets of miR-27 through biochemical methods, one chapter of this thesis was devoted to developing CLASH datasets (cross-linking, ligation and sequencing of hybrid). This technique can directly map miRNA-mRNA interactions within the Argonaute protein (AGO). Initially, a NIH 3T3 stable cell line expressing AGO2 with a double affinity tag at the N terminus was generated. Analysis of the stable cell line revealed no significant alteration of miR-27 regulation or change in permissiveness to MCMV compared to wild type cells, making this amenable to further studies. Using the stable cell line, the CLASH protocol was carried out and preliminary data collected. In summary, this thesis identifies a direct target of miR-27, Mapkapk3, that is an important gene in MCMV replication that requires further investigation. Mapkapk3 is a substrate of p38 in the p38 MAP kinase pathway which is a signal transduction mediating numerous biological processes in response to cellular stresses including CMV infection. Furthermore, miR-27 overexpression was found to stimulate the G1/S transition of the cell cycle, and miR-27 inhibition had the opposite effect. Previous evidence has shown that MCMV and HCMV arrest the cell cycle in the G1 phase and inhibit host DNA synthesis to create an optimal condition for viral gene expression and DNA replication. Given that MCMV arrests host cells in the G1 phase, it is possible that degradation of miR-27 by MCMV contributes to this effect. Since miR-27 regulates both Mapkapk3 and the cell cycle, it seems likely that a number of targets and pathways underlie the antiviral properties of this miRNA.

Natural Killer (NK) cells, lymphocytes of the innate immune response, play a vital role in controlling infections and in tumor surveillance. NK cells provide protection by direct cytolysis of infected cells and by the production of pro-inflammatory cytokines such as, IFN-γ and TNF-α. Notably, NK cells have recently been identified to regulate the immune response by producing the anti-inflammatory cytokine IL-10. Several other cells can produce IL-10 during infections, however NK cell derived IL-10 can be critical in regulating immune response during early phases of infection and therefore protecting the host from excessive immunopathology. Although the regulatory role of NK cells seems to be plausible, the physiological relevance of NK cell mediated immune regulation during infections has not been demonstrated in detail. To investigate the immunoregulatory function of NK cells, I used Murine Cytomegalovirus (MCMV) infection induced by a high dose challenge and demonstrated that NK cells are a major IL-10 producer during acute stage of the infection. To elucidate the role of NK cell derived IL-10 during infections, I generated NK cell specific IL-10 knockout, NKp46iCre  Il-10flox/flox mice (NK-Il-10-/-) by crossing Il-10flox/flox mice with mice expressing Cre recombinase exclusively under the NK cell specific promoter, NKp46 (NKp46iCre knock-in mice). My results indicated that Cre mediated Il-10 genomic deletion occurred predominantly in NK cells but not in NKT, T and B cells. Enriched NK cells from NK-Il-10-/- mice failed to produce IL-10 upon ex vivo IL-2/IL-12 stimulation. Furthermore, histological analysis of the colon indicated that NK-Il-10-/- mice are free from aberrant inflammation. During sustained MCMV infection, significantly higher production of IFN-γ by CD8+ T cells of NK-Il-10-/- mice in salivary glands indicates that NK cell derived IL-10 contributes to the establishment of the immune suppressive environment in the organ. NK-Il-10-/- mice also demonstrated increased susceptibility to acute Listeria monocytogenes (LM) infection based on enhanced body weight loss. Taken together, I have successfully generated NK-Il-10-/- mice that lack the Il-10 gene exclusively in NK cells. The NK-Il-10-/- mouse can be used as an ideal model to dissect the immunoregulatory role of NK cells during various microbial infections and tumorogenesis.

No description available.