L'athérosclérose est un processus physiopathologique chronique impliqué dans la majorité des maladies cardio-vasculaires. Le développement des lésions d'athérosclérose est caractérisé par l'accumulation de lipides extra et intracellulaires dans la paroi artérielle à l'origine d'une forte réponse inflammatoire impliquant notamment les macrophages. Les macrophages sont considérés comme des acteurs clés dans le développement des plaques d'athérosclérose. En effet, de par leur capacité à métaboliser le cholestérol (captation, stockage, efflux), à réguler l'inflammation et à phagocyter les cellules apoptotiques, ils exercent des fonctions pro et/ou anti-athèrogènes qui peuvent être modulées à des fins thérapeutiques. Dans cette perspective, nous avons évalué le pouvoir thérapeutique des " macrophages protégés de l'apoptose " sur la progression des lésions d'athérosclérose constituées. Nous avons démontré que l'augmentation de la survie des macrophages permet de ralentir la progression des lésions, de stabiliser les lésions et de diminuer la cholestérolémie. Ces effets athéro-protecteurs sont attribués à l'augmentation des cellules de Kupffer et des monocytes Ly-6Clow en partie par leur capacité à produire de l'apolipoprotéine E. Nous montrons également que les cellules de Kupffer participent à la clairance des lipoprotéines pro-athérogènes. L'augmentation du pool d'apoE ainsi que l'augmentation des cellules de Kupffer permettent de diminuer la cholestérolémie et ainsi de diminuer la progression des lésions. / Atherosclerosis represents a chronic pathophysiological process implicated in the majority of cardiovascular diseases. The development of atherosclerotic lesions is characterized by an accumulation of extra and intracellular lipids in the arterial wall at the origin of a strong inflammatory response involving macrophages.Macrophages are considered key actors in the development of atherosclerotic plaques. Indeed, because of their ability to metabolize cholesterol (capture, storage, efflux), to regulate inflammation and to phagocyte apoptotic cells, they exert pro and/or anti-atherogenic functions that may be modulated therapeutically. In this context, we evaluated the therapeutic potential of macrophages protected against apoptosis, on the progression of established atherosclerotic lesions.We have demonstrated that increased macrophage survival can slow down the progression of established lesions, stabilize lesion and reduce cholesterol levels. These athero-protective effects are attributed to the increase in Kupffer cells and Ly-6Clow monocytes partly due to their ability to produce apolipoprotein E. We also show that Kupffer cells are involved in the clearance of pro-atherogenic lipoproteins. The increase in ApoE pool and in Kupffer cells reduces cholesterol levels and thus lesion progression.

L'obésité est aujourd’hui considérée comme une maladie inflammatoire chronique dite de « bas grade » principalement caractérisée par une augmentation de l’inflammation du tissu adipeux. Les adipocytes et les macrophages sont connus pour jouer un rôle clé dans l’établissement, la progression et le maintien de l'inflammation. Dans mon projet de thèse, nous nous sommes particulièrement intéressés aux mécanismes transcriptionnels impliqués dans l'inflammation chronique en décodant l'action du corégulateur transcriptionnel GPS2 (G protein pathway suppressor 2) dans les adipocytes et les macrophages du tissu adipeux. Dans un premiers temps, nous avons étudié la régulation et les actions de GPS2 (et ses partenaires SMRT et NCOR) dans le tissu adipeux humains de sujets obèses par rapport à des sujets minces. Dans cette première étude, nous avons identifié un mécanisme épigénomique qui participe à la régulation de la transcription des gènes inflammatoires dans les adipocytes lors de l’obésité. Nous avons démontré que la dérégulation de GPS2 contribuait à l'inflammation du tissu adipeux en permettant à la dérépression de certains gènes inflammatoires dont l’interleukine 6. Dans la deuxième étude, nous avons caractérisé les conséquences de l’invalidation de GPS2 dans le phénotype inflammatoire des macrophages ainsi que les conséquences in vivo sur la progression de l’insulino-résistance. Pour ceci, nous avons généré un modèle de souris où GPS2 a été spécifiquement invalidé dans les macrophages (GPS2-MacKO). De manière intéressante, les souris GPS2-MacKO, présentent une expression accrue des gènes impliqués dans la voie de signalisation des TLR et des chimiokines dans les macrophages isolés. Par conséquent, une augmentation significative de l'infiltration des macrophages dans le tissu adipeux est observée dans un contexte d’obésité induisant une altération de l’homéostasie glucidique. Par nos approches génomiques, transcriptomiques et épigénomiques, nous avons pu révéler les voies de signalisations spécifiquement contrôlées par GPS2. Ces travaux démontrent également l’importance des régulations épigénomiques dans l'inflammation métabolique du tissu adipeux durant l'obésité. / Obesity is now considered a chronic low-grade inflammatory disease with increased levels of inflammatory mediators both in circulation and adipose tissue. Among adipose tissue cell types, adipocytes and macrophages are known to play key roles in the progression of inflammation by establishing and maintaining it. In this PhD project, we particularly focus on the transcriptional mechanisms behind the chronic low-grade inflammation by deciphering the action of GPS2 in adipocytes and adipose tissue macrophages. We initially studied the gene regulation and the actions of GPS2 and its partners in adipose tissue and adipocytes of human obese subjects compared to lean subjects. In this first study we identified a novel regulatory pathway that participates in the transcriptional control of inflammation associated with obesity, both in adipose tissue and adipocytes. We have shown that GPS2 and SMRT were differentially expressed and regulated in obese adipocytes. In addition, this dysregulation contributes to inflammation of the adipose tissue by allowing the derepression of specific inflammatory genes. In a second study, in order to go further in the characterisation of the in vivo function of GPS2, we generated a mouse model were GPS2 was specifically invalidated in macrophages. Models of diet-induced obesity were applied in these experiments. Interestingly, GPS2-MacKO mice showed an increased expression of inflammatory genes both in adipose tissue and isolated ATMs (F4/80+ cells) associated with a significant increase of macrophages infiltration in the adipose tissue. Finally, we observed that GPS2-MacKO mice had impaired glucose metabolism as they presented high glucose intolerance as well as an important insulin resistance.

Salmonella enterica serovar Typhimurium is responsible for a variety of diseases in domestic animals and humans. The infection of mice causes similar disease progression to human typhoid fever, thus representing a model for this systemic disease. The ability of S. Typhimurium to reside in a macrophage phagosome is important for their survival and spread to different organs. The antimicrobial mechanisms in this compartment include reactive oxygen species, reactive nitrogen species and elevated copper levels. S. Typhimurium possesses two copper-exporting P1B-type ATPases, CopA and GolT, both of which contribute to copper resistance. A previous study has shown that copper export by CopA and GolT confers a survival advantage in resting macrophage phagosomes. In this study the role of copper resistance systems has been examined further. The reduced survival of ΔcopA/ΔgolT in macrophages is detected beyond 8 hours post infection and coincides with increased nitrite production by macrophages. We have established that ΔcopA/ΔgolT display some increased sensitivity to reactive nitrogen species. However, whilst treatment of macrophages with the iNOS inhibitor L-NMMA reduced macrophage bactericidal activity against wildtype S. Typhimurium, this was not the case for ΔcopA/ΔgolT. In contrast, survival of ΔcopA/ΔgolT was not impaired in macrophages treated with the copper-chelator BCS. Furthermore real-time PCR confirmed the expression of copA and golT is elevated during infection of macrophages treated with IFN-γ or L-NMMA, but is reduced during infection of BCS treated macrophages. This indicates that bactericidal activity in macrophages is associated with copper availability and this is unaffected by reactive nitrogen species released due to iNOS activity. In contrast to Escherichia coli Salmonella lacks a cus system associated with export across the outer membrane and hence the mechanism of copper export from the periplasm is not known. TolC was investigated as a potential outer membrane copper exporter based on clustering of TolC dependent systems to genes with sequence similarity to the S. typhimurium periplasmic copper chaperone CueP, across several bacteria. Mutation of tolC gave reduced copper tolerance and over-accumulation of copper at non-lethal concentrations under aerobic conditions. However TolC does not provide a role in copper tolerance or homeostasis under anaerobic conditions. TolC also does not provide tolerance or homeostasis to other divalent cations: Zn, Ni and Co. TolC therefore provides specific transport of copper under aerobic conditions in S. Typhimurium.

Orientador: Selma Giorgio / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:41:12Z (GMT). No. of bitstreams: 1 Cyrino_LarissaTavares_M.pdf: 2371187 bytes, checksum: 4a8ffd61bdea2150bc5ecd919622747a (MD5) Previous issue date: 2011 / Resumo: A autofagia é o principal mecanismo celular de degradação de proteínas e organelas citoplasmáticas. Este processo biológico vem sendo considerado importante no combate aos microrganismos durante processos infecciosos, eliminando-os e ativando respostas imunes inatas e adaptativas. A leishmaniose é um grupo de doenças causadas por protozoários do gênero Leishmania, que parasitam macrófagos e células dendríticas e é responsável pelo aparecimento de lesões cutâneas simples, lesões mucosas e leishmaniose visceral. O conhecimento dos fatores do hospedeiro e das espécies de Leishmania que influenciam a patogênese dos vários tipos de leishmaniose é limitado e pouco se sabe sobre o papel da autofagia nesta doença. O objetivo geral deste trabalho foi avaliar o processo autofágico durante a infecção de macrófagos com L. amazonensis. O monitoramento da autofagia por western blotting (detecção de LC3, proteína marcadora de autofagia), por microscopia eletrônica de transmissão, e pela detecção de atividade lisossomal (LysoTracker® Red) indicou que a infecção causada por L. amazonensis induz autofagia tanto em macrófagos de linhagem tumoral quanto em macrófagos primários de camundongos de diferentes linhagens. Nossos dados também indicam que o aumento do índice de infecção está relacionado ao aumento da autofagia nesses macrófagos e que a indução de autofagia por starvation (inanição) aumenta a porcentagem de infecção com L. amazonensis em macrófagos derivados de medula óssea de camundongos BALB/c. Por outro lado, a indução de autofagia não altera o índice de infecção de macrófagos derivados de medula óssea de camundongos C57BL/6, indicando que a linhagem de camundongos de origem dos macrófagos interfere no desfecho da infecção. Juntos, nossos resultados indicam que a autofagia apresenta um papel importante na infecção causada por L. amazonensis e abre novas perspectivas para o estudo desse processo biológico durante a infecção causada por outras espécies de Leishmania / Abstract: Autophagy is the primary mechanism of degradation of cellular proteins and organelles. This biological process has been considered important in fighting microorganisms during the infectious processes, eliminating them and activating innate and adaptive immune responses. Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania, which parasitize macrophages and dendritic cells, and it is responsible for the appearance of simple skin lesions, mucosal and visceral leishmaniasis, The knowledge of host factors and the Leishmania species that influence the pathogenesis of many types of leishmaniasis is limited and little is known about the role of autophagy in this disease. The general aim of this study was to evaluate the autophagic process during infection of macrophages with L. amazonensis. The monitoring of autophagy by western blotting (to detect LC3, a protein marker of autophagy), by Transmission Electron Microscopy, and by lysosomal activity detection (by using Lysotracker® Red) indicated that the infection caused by L. amazonensis induces autophagy in both tumor macrophage lineage and in primary macrophages from mice of different strains. Our data also indicate that the increase in the rate of infection is related to the increase of autophagy in these macrophages and that induction of autophagy by starvation increases the percentage of bone marrow-derived macrophages from BALB / c mice with L. amazonensis after three days of culture. On the other hand, the induction of autophagy does not change the infection rate of bone marrow -derived macrophages from C57BL / 6 mice, indicating that the mice strain interfere in the outcome of infection of macrophages. Together, our results indicate that autophagy has an important role in infection caused by L. amazonensis and creates new perspectives to the study of this biological processes during infection caused by other species of Leishmania / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Immunotherapy has revolutionized the treatment of certain cancers by facilitating the antitumor immune response and represents today one of the mainstays of cancer therapy. However, only a subset of patients responds to immunotherapy, which can also lead to serious complications. The tumor microenvironment is composed of multiple and complex cellular and molecular interactions providing to cancer cells not only a supportive framework but promoting also many steps of immunosuppression and tumor progression. To date, the mechanisms that drive the acquisition of these immunosuppressive features are still poorly defined. Tumor-associated macrophages can be highly represented in the tumor microenvironment where they are shaped and become key players in the innate and adaptive immune escape of the tumor cells.Heme oxygenase-1 is the rate-limiting enzyme that catabolizes heme into three major biologically active byproducts which display cytoprotective, antioxidant and immunomodulatory effects. We hypothesized that tumor-associated macrophages might suppress anti-tumor T-cell response through heme oxygenase-1 induction in the tumor microenvironment and macrophage polarization. We showed that heme oxygenase-1 is highly expressed in tumor-associated macrophages. By using a subcutaneous EG7-OVA lymphoma model on genetically engineered mice with a conditional deletion of heme oxygenase-1 in macrophages, our data show that myeloid-restricted heme oxygenase-1 deficiency improves the effect of a therapeutic antitumor immunization by enhancing tumor-infiltrating antitumor CD8+ T-cell proliferation and cytotoxicity and represses tumor growth. Our data suggest a major role of myeloid heme oxygenase-1 in the differentiation and the phenotypic, functional, transcriptional and epigenetic reprograming of tumor-associated macrophages. Myeloid HO-1 inhibition might be considered as a new myeloid HO-1-mediated immune checkpoint blockade. Targeting myeloid compartment could reprogram the tumor microenvironment and synergize with other cancer therapies. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

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Acute lower respiratory tract infections are a leading cause of morbidity and mortality world-wide. Streptococcus pneumoniae (pneumococcus) is the most common bacterial cause of community-acquired pneumonia. Recovery from pneumococcal pneumonia results in the formation of resident memory CD4+ T cells, which act on lung epithelial cells to accelerate immune responses. Alveolar macrophages (AMs) are tissue-resident macrophages localized in the air spaces, where they orchestrate the lung anti-microbial responses. We hypothesized that recovery from pneumococcal pneumonia results in remodeling of the pool of alveolar macrophages, which act in concordance with other immune cells to protect the lungs from future infections. Although AM numbers were unchanged in experienced lungs, their surface phenotype showed significant changes, most prominently an increased MHC-II and a decreased SiglecF. This experienced AM phenotype was regionally-localized and long-lasting. Experienced AMs also exhibited extensive remodeling on the metabolomics and transcriptional level. Experienced AMs demonstrated significant increases in phosphocreatine and its metabolite precursors. The transcriptional analyses also revealed extensive changes. At baseline, experienced AMs exhibited a significant reduction in cell cycle activity and mRNA processing compared to naïve mice. During acute pneumonia, experienced AMs exhibited significant increases in immune signaling and energy metabolism. Moreover, transcriptional data also revealed strong but imperfect enrichment of a signature previously associated with IFN𝛾 signaling and marrow-derived AMs. IFN𝛾 gain and loss of functions experiments corroborated transcriptional data and revealed an essential role for IFN𝛾 in directly driving the AM MHC-II remodeling. Several immune cells produced IFN𝛾, with neutrophils being the most prominent source after the 1st pneumococcal challenge but other cells predominating after the 2nd pneumococcal challenge. CD4+ T cell depletion studies demonstrated that AMs' experienced phenotype was independent of CD4+ T cells. In contrast to naïve mice, lineage-tracing studies demonstrated that marrow-derived AMs predominately constitute the experienced AM pool. Upon experience, both embryonic AMs and marrow-derived AMs demonstrated similar remodeling for both SiglecF and MHC-II on their surfaces. While all AM similarly remodeled independent of their origin, marrow-derived AMs in experienced lungs displayed some differences from their embryonic counterparts, being less phagocytic. In conclusion, recovery from pneumococcal pneumonia remodels the pool of alveolar macrophages to acquire adaptive characteristics. This remodeling involves a combination of recruitment of new cells and trained immunity via IFN𝛾 signaling.

Over the last 40-yr, the majority of research on glucans has focused on β-(1→3)-glucans. Recent studies indicate that β-(1→6)-glucans may be even more potent immune modulators than β-(1→3)-glucans. Mechanisms by which β-(1→6)-glucans are recognized and modulate immunity are unknown. In this study, we examined the interaction of purified water-soluble β-(1→6)-glucans with macrophage cell lines and primary peritoneal macrophages and the cellular and molecular consequences of this interaction. Our results indicate the existence of a specific β-(1→6)-glucan receptor that internalizes the glucan ligand via a clathrin-dependent mechanism. We show that the known β-(1→3)-glucans receptors are not responsible for β-(1→6)-glucan recognition and interaction. The receptor-ligand uptake/interaction has an apparent dissociation constant (KD) of ∼4-μM, and was associated with phosphorylation of ERK and JNK but not Iκ-α or p38. Our results indicate that macrophage interaction with β-(1→6)-glucans may lead to modulation of genes associated with anti-fungal immunity and recruitment/activation of neutrophils. In summary, we show that macrophages specifically bind and internalize β-(1→6)-glucans followed by activation of intracellular signaling and modulation of anti-fungal immune response-related gene regulation. Thus, we conclude that the interaction between innate immunity and β-(1→6)-glucans may play an important role in shaping the anti-fungal immune response.

The ability of fungal-derived β-glucan particles to induce leukocyte activation and the production of inflammatory mediators, such as tumor necrosis factor (TNF)-α, is a well characterized phenomenon. Although efforts have been made to understand how these carbohydrate polymers exert their immunomodulatory effects, the receptors involved in generating these responses are unknown. Here we show that Dectin-1 mediates the production of TNF-α in response to zymosan and live fungal pathogens, an activity that occurs at the cell surface and requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of Dectin-1 as well as Toll-like receptor (TLR)-2 and Myd88. This is the first demonstration that the inflammatory response to pathogens requires recognition by a specific receptor in addition to the TLRs. Furthermore, these studies implicate Dectin-1 in the production of TNF-α in response to fungi, a critical step required for the successful control of these pathogens.

Tim-3 and PD-1 are powerful immunoinhibitory molecules involved in immune tolerance, autoimmune responses, and antitumor or antiviral immune evasion. A current model for Tim-3 regulation during immune responses suggests a divergent function, such that Tim-3 acts synergistically with TLR signaling pathways in innate immune cells to promote inflammation, yet the same molecule terminates Th1 immunity in adaptive immune cells. To better understand how Tim-3 might be functioning in innate immune responses, we examined the kinetics of Tim-3 expression in human CD14 + M/M 4 in relation to expression of IL-12, a key cytokine in the transition of innate to adaptive immunity. Here, we show that Tim-3 is constitutively expressed on unstimulated peripheral blood CD14 + monocytes but decreases rapidly upon TLR stimulation. Conversely, IL-12 expression is low in these cells but increases rapidly in CD14 + M/M.J, in correlation with the decrease in Tim-3. Blocking Tim-3 signaling or silencing Tim-3 expression led to a significant increase in TLR-mediated IL-12 production, as well as a decrease in activation-induced up-regula-tion of the immunoinhibitor, PD-1; TNF-a production was not altered significantly, but IL-10 production was increased. These results suggest that Tim-3 has a role as a regulator of pro- and anti-inflammatory innate immune responses.