Choline is necessary for the synthesis of phosphatidylcholine (PC), the predominant phospholipid species and an important lipid intermediate. Macrophages, critical mediators of innate immunity, have been implicated in lipid dysregulation associated with metabolic disease. Despite the importance of choline in lipid metabolism, few studies have investigated the relationship between choline metabolism and inflammation. My research revealed that macrophage polarization increased choline metabolism and the expression of the choline transporter CTL1. In addition, choline deficient macrophages showed altered cytokine secretion, suggesting choline metabolism may play an important role in regulating the immune response. This study also describes the generation of a novel CTL1-/- mouse, which showed decreased choline uptake and incorporation into lipids. As an in vivo model for choline deficiency, CTL1-/- mice represent an important model for the future study of choline metabolism. Altogether, these findings suggest an important relationship exists between choline metabolism and inflammation.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.

L’accumulation de cellules spumeuses dans l’intima des artères est le point critique de l’initiation de l’athérosclérose. L’accumulation de cholestérol et l’activation des voies pro-inflammatoires sont responsables de l’acquisition par les macrophages de ce phénotype délétère. Les dérivés oxydés du cholestérol accumulés vont stimuler les récepteurs nucléaires LXR et incidemment l’efflux de cholestérol athéroprotecteur, tandis que la phagocytose de cholestérol cristallisé dans la lésion causera une perturbation du trafic vésiculaire qui activera l’inflammasome NLRP3, verrou de l’inflammation IL-1. Cette étude a pour but de décrypter ces voies dans le macrophage humain. La stimulation de l’efflux de cholestérol par un agoniste LXR dans le macrophage humain m’a est médiée par l’activation transcriptionnelle LXRα-dépendante d’ARL7 - qui permet le transport du cholestérol libre de la membrane des endosomes et lysosomes aux radeaux lipidiques – et du transporteur ABCA1 – l’exportant vers les HDL, lipoprotéines athéroprotectrices. L’activation du complexe multi-protéique NLRP3 dans le macrophage, et la sécrétion des cytokines délétères IL-1β et IL-18, est également réprimée par l’agoniste LXR. L’activation des cathepsines B et L par l’enzyme AEP est aussi nécessaire pour l’activation de NLRP3 par des cristaux. Inactiver l’AEP protège ainsi contre le développement de l’athérosclérose. Ces travaux ont démontré la nécessité des études chez l’humain pour la mise en évidence des mécanismes moléculaires et la nécessité d’intégrer les signalisations lipidiques et inflammatoires étroitement liées dans la cellule spumeuse. / Foam cell accumulation in arterial walls is the critical initiating event of atheroma plaque development. Macrophages acquire this phenotype by cholesterol ester accumulation and pro-inflammatory signaling pathways activation. Oxydized form of cholesterol activates LXR and subsequently atheroprotective cellular cholesterol efflux. In parallel, crystallized cholesterol phagocytosis will impair vesicular traffic, activating NLRP3 and deleterious IL-1 cytokines release. Here we describe further those pathways in human macrophages and to evidence new key factors in atherosclerosis development. First, stimulation of cellular cholesterol efflux to ApoA-I and HDL from human macrophage by LXR agonist is LXRα-dependent. Transcriptional activation of ARL7 increases cholesterol transport from endolysosomal membrane to efflux-prone plasmic membrane pools, lipid rafts. From there, cholesterol will be exported on ApoA-I containing lipoproteins by ABCA1 transporter, whose expression is stimulated by LXRα agonists as well. Cholesterol crystals phagocytose will lead to inflammasome NLRP3 activation, leading to pro-atherogenous IL-1β and IL-18 secretion. We first showed LXR agonist GW3965 role in repressing transcription of NLRP3 partners. Also, we evidenced the importance of cathepsin B and L maturation by asparagin endopeptidase (AEP) in human macrophages, and atheroprotective properties of AEP silencing. Overall, this work demonstrated the necessity of using human models to confirm murine data about molecular mechanisms. Also, it is important to integrate lipid homeostasis and inflammation signaling in foam cells, for there is a strong molecular link between both.

L’hématopoïèse est un processus actif, ordonné, et hautement régulé faisant intervenir des étapes de prolifération, de différenciation et d’apoptose et permettant la production de toutes les cellules sanguines matures à partir d’un nombre restreint de cellules souches hématopoïétiques. La dérégulation des mécanismes intervenant dans l’hématopoïèse induit le développement d’hémopathies, notamment de leucémies. De nombreux facteurs de transcription et microARN (miARN) ont été identifiés en tant que des régulateurs essentiels à l’établissement des différents lignages hématopoïétiques. Mon travail de thèse a porté sur l’étude du rôle des miARN dans la régulation de la différenciation macrophagique humaine. Nous avons reproduit le processus de différenciation macrophagique in vitro à partir de monocytes issus du sang périphérique traités avec du CSF-1 (colony stimulating factor-1). Suite à l’analyse du profil d’expression des miARN au cours du processus de différenciation, notre projet s’est orienté sur l’étude de miR-142-3p dont le taux d’expression diminue le plus fortement au cours de cette différenciation. Nous avons montré que miR-142-3p forme une boucle d’auto-régulation négative avec EGR2 (early growth response 2), un facteur de transcription connu pour réguler positivement la différenciation macrophagique. Cette boucle est essentielle au bon déroulement de la différenciation. Par ailleurs, nous avons observé une altération de cette boucle de régulation dans les monocytes de patients atteints d’une LMMC (leucémie myélomonocytaire chronique) suggérant que ce mécanisme puisse être impliqué dans la leucémogenèse. Au cours de ce projet, nous avons également initié une étude in vivo via l’utilisation du modèle que représente le Poisson-Zèbre. L’hématopoïèse du Poisson-Zèbre est très similaire à celle des mammifères que ce soit au niveau des populations hématopoïétiques ou des mécanismes de régulation impliqués. L’inhibition de l’expression du miR-142a-3p, homologue du miR-142-3p humain, se traduit par une absence de monocytes et de macrophages au niveau de l’ICM (intermediate cell mass), organe primaire de l’hématopoïèse, ainsi que par une diminution de l’expression de la myéloperoxydase, marqueur des granulocytes chez le Poisson-Zèbre. Ainsi, miR-142-3p semble être un inducteur de la formation des granulocytes et monocytes. / Hematopoiesis is an active process, orderly and highly regulated, involving proliferation, differentiation and apoptosis steps, and allowing the production of mature blood cells from a restricted number of hematopoietic stem cells. Deregulation of mechanisms involved in hematopoiesis leads to the development of leukemias. Many transcription factors and microRNAs (miRNAs) have been identified as essential regulators in the establishment of different hematopoietic lineages. My thesis investigated the role of miRNAs in the regulation of human macrophage differentiation. We examined macrophage differentiation in vitro, from peripheral blood monocytes treated with CSF-1 (colony stimulating factor-1). After the analysis of miRNAs expression profile, our project has focused on the study of miR-142-3p whose expression levels decreased most strongly during macrophage differentiation. We showed that miR-142-3p involved in a negative feedback loop with EGR2 (early growth response 2), a transcription factor known to favor macrophage differentiation. This molecular circuitry is necessary for the normal processus of differentiation. Furthermore, we observed an alteration of this regulation circuitry in monocytes of CMML (chronic myelomonocytic leukemia) patients, suggesting that this mechanism may be involved in leukemogenesis. During this project, we also initiated a study in vivo through the use of the zebrafish model. Zebrafish hematopoiesis is very similar to that in mammals both at the level of hematopoietic populations or regulatory mechanisms involved. The inhibition of miR-142a-3p expression, homolog of the human miR-142-3p, gave rise to an absence of monocytes and macrophages in ICM (intermediate cell mass), the primary organ of hematopoiesis and a decreased expression of myeloperoxidase, a marker of granulocytes in the zebrafish. Thus, miR-142-3p appears to be an inducer of granulocytes and monocytes formation.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.

A leishmaniose é causada por tripanossomatídeos do gênero Leishmania que infectam preferencialmente macrófagos. Vários factores influenciam a forma e a severidade da doença: a espécies de Leishmania e a resposta imune do hospedeiro. Considerando a importância da ativação dos macrófagos na infecção, o potencial papel de CD100 na modulação da ativação dos macrófagos e os nossos dados anteriores de que CD100 solúvel (sCD100) aumenta a infectividade pelo parasita, pretendemos caracterizar os efeitos do CD100 na infecção por Leishmania (L.) amazonensis. Descobrimos que ambos, promastigotas e amastigotas, são mais infecciosos na presença de sCD100 e que o receptor CD72 é o responsável pelo aumento da infecção. Experimentos in vitro indicaram índice de infecção similares entre macrófagos nocautes para CD100 e selvagens, mas curiosamente, os animais nocautes infectados desenvolveram lesões significativamente menores do que os selvagens, sugerindo que sCD100 presente em outras células pode influenciar a formação da lesão. / Leishmaniasis is caused by trypanosomes of the genus Leishmania that preferentially infect macrophages. Several factors influence the form and severity of the disease: the species of Leishmania and the host immune response. Considering the importance of the activation of macrophages in infection, potential role of CD100 in the modulation of macrophage activation and our previous data that CD100 soluble (sCD100) increase the infectivity of the parasite, we intend to characterize the effect of CD100 in infection with Leishmania (L.) amazonensis. We found that both promastigotes and amastigotes, are most infectious in the presence of sCD100 and the CD72 receptor is responsible for the increased infection. In vitro experiments indicated similar infection rate of macrophages to CD100 knockouts and wild type, but interestingly, the infected knockout animals developed significantly smaller lesions than wild type suggesting that sCD100 present in other cells may influence lesion formation.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2016-05-19T15:16:35Z No. of bitstreams: 1 Erika Elisabeth Evangelista.pdf: 1020129 bytes, checksum: dee500eb8c5a17c94acdc64c0d81e2de (MD5) / Made available in DSpace on 2016-05-19T15:16:35Z (GMT). No. of bitstreams: 1 Erika Elisabeth Evangelista.pdf: 1020129 bytes, checksum: dee500eb8c5a17c94acdc64c0d81e2de (MD5) Previous issue date: 2014-12-11 / Periodontal disease is an inflammatory disease affecting the supporting tissues of the teeth, leading to loss of periodontal ligament and bone. The treatment involves scaling and root planning, and in more advanced cases there is the need for surgery and sometimes antibiotics. Photodynamic therapy is a therapeutic antimicrobial resource to aid in modulation of the inflammatory response triggered by the light. One of the main cells is the macrophage inflammatory process which acts in reciprocity with T lymphocytes. Thus the aim of this study was to evaluate the effect of photodynamic therapy in the chemotaxis of macrophages and activated T lymphocytes in the periodontal tissue. For such, patients who have undergone previous periodontal treatment with good oral hygiene and periodontal pockets residual requiring surgery were allocated. PDT was applied on the following parameters: methylene blue (50ug / ml) was applied to the bottom of the pocket and after 5 minutes via transmucosal the photosensitizer was irradiated with diode laser 660 nm, P = 100 mW, t = 90 s per point 9 J/point; energy density: 22 J/cm2, power density: 250 mW/cm2. One week after PDT, the surgical removal of the pockets was performed and samples sent for routine histology and immunohistochemical detection of activated T lymphocytes (CD45RO +) and macrophages (CD68 +). The samples were photographed and positive cells counted with the aid of ImageJ software. Data were evaluated statistically and no significant difference between groups (Mann-Whitney p = 0.9). We conclude that PDT does not interfere with chemotaxis of macrophages and activated T lymphocytes in periodontal tissue after seven days. / A doença periodontal é uma doença inflamatória que afeta os tecidos de suporte dos dentes, levando à perda de ligamento periodontal e de osso. O tratamento consiste em raspagem e alisamento radicular e nos casos mais avançados há a necessidade de cirurgias e, às vezes antibioticoterapia. A terapia fotodinâmica é um recurso terapêutico antimicrobiano que auxilia a modulação da resposta inflamatória desencadeada pela luz. Uma das principais células do processo inflamatório é o macrófago, que age em reciprocidade com os linfócitos T. Assim o objetivo deste trabalho foi avaliar o efeito da terapia fotodinâmica na quimiotaxia de macrófagos e linfócitos T ativados no tecido periodontal. Para tal foi realizado um estudo clínico transversal no qual foram alocados pacientes que passaram por tratamento periodontal prévio com boa higiene oral e bolsas periodontais residuais com indicação cirúrgica. A PDT foi aplicada nos seguintes parâmetros: azul de metileno (50ug/mL) aplicado no fundo da bolsa e após 5 minutos foi irradiado via transmucosa com laser de diodo, 660 nm, P= 100 mW, t= 90 s por ponto, 9 J/ponto, densidade de energia: 22 J/cm2, densidade de potência: 250 mW/cm2. Uma semana após a PDT foi realizada remoção cirúrgica das bolsas e as amostras encaminhadas para processamento histológico de rotina e imunohistoquímica para detecção de linfócitos T ativados (CD45ro+) e macrófagos (CD68+). As amostras foram fotografadas e as células positivas contadas com auxílio do software ImageJ. Os dados foram avaliados estatisticamente e não houve diferença significativa entre os grupos (Mann-Whitney p= 0,9). Conclui-se que a PDT não interfere na quimiotaxia de macrófagos e linfócitos T ativados no tecido periodontal após 7 dias.

Submitted by Nadir Basilio (nadirsb@uninove.br) on 2018-06-19T18:27:35Z No. of bitstreams: 1 Terezinha Aparecida de Almeida.pdf: 739598 bytes, checksum: 0b0c7aabccbc5f5868d542f6e63d53f0 (MD5) / Made available in DSpace on 2018-06-19T18:27:35Z (GMT). No. of bitstreams: 1 Terezinha Aparecida de Almeida.pdf: 739598 bytes, checksum: 0b0c7aabccbc5f5868d542f6e63d53f0 (MD5) Previous issue date: 2015-12-16 / Interactions between the muscle and macrophages which invade it after an injury occurs are crucial to the evolution of repair in this tissue. On the other hand, low intensity laser (LLLT) has long been used in the treatment of such lesions, both in clinical and experimental settings; however, little is known about its effects on macrophages. The objective of this project was to evaluate the effect of irradiation with red and infrared laser on the activation state of M1 and M2a phenotypes of macrophages. For this purpose, J774 macrophage cultures were treated with polarizing agents (LPS and IFN-y or IL-4) for 24 hours and were then irradiated with 2 wavelengths of LLLT – 780 nm and 660 nm – in the same dosimetric parameters (70mW; 17.5J/cm2; 0.8J). After 24 hours of incubation, the activation state was checked by MTT technique. In each experimental situation, non-irradiated cells served as controls, and three independent experiments were performed. Laser irradiation with 780 nm proved able to decrease the activation of macrophages polarized for M1 phenotype and increase activation of M2a profile macrophages. Laser irradiation with 660 nm slightly intensified the state and activation of M1 macrophages, while significantly increasing the activation state of M2a profile macrophages. Although it is not possible to establish a relationship between the results of in vitro studies and future clinical outcomes, and the effects of laser irradiation on other macrophage phenotypes still need to be assessed, data from this study suggest that red and infrared laser could have different results when used in the different stages of muscle repair. / As interações entre o músculo e os macrófagos que o invadem após a ocorrência da lesão são determinantes para a evolução do reparo deste tecido. Por outro lado, o laser em baixa intensidade (LBI) tem sido muito utilizado no tratamento destas lesões no âmbito clínico e no experimental, mas pouco se conhece a respeito dos seus efeitos sobre os macrófagos. O objetivo deste projeto foi avaliar o efeito da irradiação com laser vermelho e infravermelho sobre o estado de ativação de macrófagos de fenótipo M1 e M2a. Para tanto, culturas de macrófagos J774 foram tratadas com agentes polarizadores (LPS e IFN-y ou IL-4) por 24h e então irradiadas com LBI em 2 comprimentos de onda 780nm e 660nm nos mesmos parâmetros dosimétricos (70mW; 17,5J/cm2; 0,8J). Após 24h de incubação, o estado de ativação foi verificado por meio da técnica MTT. Em cada situação experimental, células não irradiadas serviram como controle, sendo realizados três experimentos independentes. A irradiação com laser de 780 nm mostrou-se capaz de diminuir a ativação de macrófagos polarizados para fenótipo M1 e aumentar a ativação de macrófagos de perfil M2a. Já a irradiação com laser de 660 nm intensificou ligeiramente o estado e ativação dos macrófagos M1 e aumentou de maneira significante o estado de ativação de macrófagos de perfil M2a. Embora não se possa estabelecer uma relação entre resultados de estudos in vitro e desfechos clínicos futuros e ainda exista necessidade de avaliar os efeitos da irradiação laser nos outros fenótipos de macrófagos, os dados do presente estudo sugerem que os lasers vermelho e infravermelho pode trazer resultados diferentes quando utilizados nas diferentes fases do reparo muscular.

The age-related loss of skeletal muscle mass and function is accompanied by a decline in regenerative capacity. The processes that facilitate healthy muscle repair are complex, involving several phases of degradation and rebuilding of muscle tissue and the surrounding microenvironment. Specifically, myogenic progenitor cells known as satellite cells are the most influential in repairing damaged muscle tissue. Following injury, satellite cells become activated and migrate, proliferate and fuse with mature skeletal muscle fibers to restore homeostasis to the tissue. However, satellite cells do not act in isolation, a robust inflammatory response is necessary to facilitate successful and rapid healing. Macrophages are one of the first and most abundant immune cells to infiltrate damaged skeletal muscle tissue. Primarily, macrophages adapt to a proinflammatory state to clear the area of cellular debris, promote degradation of the extracellular matrix and stimulate satellite cell activation and proliferation. Afterwards, a timely transition to an anti-inflammatory state directs rebuilding of the extracellular matrix and terminal differentiation of satellite cells. Indeed, the inhibition of macrophage activity leads to impaired healing and loss of skeletal muscle function. Little is known regarding the behavior of macrophages in aged skeletal muscle following injury in humans. Thus, the objective of this dissertation is to investigate the age-related response of macrophages in human skeletal muscle, and their role in muscle repair.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22322号 / 医博第4563号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 椛島 健治, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM