A disfunção do sistema imune inato e a neuroinflamação tem sido cada vez mais reconhecidas como elementos importantes na fisiopatologia do transtorno bipolar (TB). Como componentes essenciais da imunidade inata, os macrófagos tem múltiplas funções tanto na inibição como na promoção da proliferação celular e na reparação tecidual, sendo a diversidade e a plasticidade características marcantes deste tipo celular. A polarização M1 clássica e a polarização alternativa M2 de macrófagos representam dois extremos de um estado dinâmico na mudança da ativação dos mesmos. Os macrófagos do tipo M1 sintetizam citocinas próinflamatórias que inibem a proliferação de células circundantes e danificam tecidos, enquanto os macrófagos do fenótipo M2 liberam citocinas antiinflamatórias que podem promover reparo tecidual. Um desequilíbrio da polarização M1-M2 dos macrófagos é frequentemente associado a várias doenças ou condições inflamatórias. O objetivo desta tese foi, além de revisar a importância da inflamação sistêmica na modulação da resposta inflamatória da microglia/macrófagos e consequentemente seu potencial envolvimento na fisiopatologia do TB, avaliar o perfil de polarização M1/M2 em cultura de macrófagos de sujeitos com TB comparados a indivíduos saudáveis. Monócitos foram isolados a partir de sangue periférico de dez sujeitos com TB e dez indivíduos saudáveis e diferenciados em macrófagos através da adição de fator estimulante de colônia de macrófagos (MCSF) ao meio de cultura. Para induzir a polarização M1 ou M2, as culturas foram incubadas com IFN-y e LPS ou IL-4 respectivamente. Após a incubação, recolheram-se os sobrenadantes e mediram-se as citocinas (IL-1β, IL-6, IL-10 e TNF-α) por ensaio multiplex. A secreção das citocinas IL-1β, TNF-α e IL-6 características do protótipo M1 e citocinas IL-10 do protótipo M2 foram semelhantes entre os pacientes e os controles. Utilizou-se a razão TNF-α / IL-10 do fenótipo M1 para refletir o estado inflamatório dos participantes. Não foi observada diferença entre os grupos (p=0,627). Duas hipóteses diferentes poderiam explicar esses resultados: todos os pacientes incluídos neste estudo representam um estágio inicial da doença como evidenciado pela pontuação FAST total inferior a 11. De acordo com o modelo de estadiamento em TB, as alterações biológicas (incluindo a inflamação) parecem estar relacionadas com os episódios de humor e progressão da doença. Juntamente com estudos anteriores, os nossos dados sugerem que os pacientes nos estágios iniciais ainda preservam a função do sistema imunológico sem apresentar um desequilíbrio a favor do perfil de macrófagos M1 como tem sido observado em pacientes no estágio tardio, destacando a relevância da intervenção precoce no TB. Ainda, estes pacientes estavam em tratamento com estabilizadores de humor e é plausível especular que esses fármacos exerçam efeitos sobre a polarização de macrófagos. Estudos futuros em pacientes drug-free são essenciais para avaliar esta questão. Em conclusão, nossos achados sugerem que os pacientes TB não apresentam desequilíbrio na polarização dos macrófagos em favor do fenótipo pró-inflamatório M1. O fato de todos estes pacientes estarem em estágios iniciais da doença reforça os efeitos protetores da intervenção precoce no TB na prevenção de alterações do sistema imune e, consequentemente, na progressão da doença. / Innate immune system dysfunction and neuroinflammation have been recognized as important elements in the pathophysiology of bipolar disorder (BD). As essential players of innate immunity, macrophages have multiple roles in inhibition and promotion of cell proliferation and tissue repair. The classical M1 polarization and the M2 alternative polarization of macrophages represent two extremes of a dynamic state in their change of activation. M1 macrophages synthesize proinflammatory cytokines that inhibit the proliferation of surrounding cells and damage tissues, whereas macrophages of the M2 phenotype release antiinflammatory cytokines that may promote tissue repair. An imbalance of the M1-M2 polarization of macrophages is often associated with various diseases or inflammatory conditions. The aim of this thesis was to review the importance of systemic inflammation in modulating the inflammatory response of microglia/ macrophages and consequently their potential involvement in the pathophysiology of BD, and also evaluate the M1/M2 polarization profile in macrophages of patients with BD compared to healthy individuals. Blood monocytes were obtained from ten BD patients and ten healthy controls. These cells were activated/polarized into the M1 (IFNγ + LPS) or M2(IL-4) phenotype. Supernatants were collected and the cytokines (IL-1β, IL-6, IL-10 and TNF-α) were measured by multiplex assay. Secretion of the IL- 1β, TNF-α, IL-6 and IL-10 were similar between patients and controls. The TNF-α/IL- 10 ratio of the M1 phenotype was used to reflect the inflammatory state of the participants. There was no difference between groups (p = 0.627). Two hypotheses could explain these results: all patients included in this study represent an early stage of disease as evidenced by the FAST score below 11. According to the BD staging model, biological changes (including inflammation) appear to be related to mood episodes and disease progression. Together with previous studies, our data suggest that patients in early stages of BD still preserve immune system function without presenting an imbalance in favor of M1 macrophages as has been observed in latestage patients, highlighting the relevance of early intervention. Moreover, these patients were under treatment with mood stabilizers and it is plausible to speculate that these drugs have effects on macrophage polarization. Future studies in drug-free patients are essential to assess this issue. In conclusion, our findings suggest that BD patients do not present imbalance in macrophage polarization in favor of the M1 proinflammatory phenotype. The fact that all these patients are in the early stages of the disease reinforces the protective effects of early intervention in BD to prevent changes in the immune system and, consequently, prevent the progression of the disease.

No description available.

A disfunção do sistema imune inato e a neuroinflamação tem sido cada vez mais reconhecidas como elementos importantes na fisiopatologia do transtorno bipolar (TB). Como componentes essenciais da imunidade inata, os macrófagos tem múltiplas funções tanto na inibição como na promoção da proliferação celular e na reparação tecidual, sendo a diversidade e a plasticidade características marcantes deste tipo celular. A polarização M1 clássica e a polarização alternativa M2 de macrófagos representam dois extremos de um estado dinâmico na mudança da ativação dos mesmos. Os macrófagos do tipo M1 sintetizam citocinas próinflamatórias que inibem a proliferação de células circundantes e danificam tecidos, enquanto os macrófagos do fenótipo M2 liberam citocinas antiinflamatórias que podem promover reparo tecidual. Um desequilíbrio da polarização M1-M2 dos macrófagos é frequentemente associado a várias doenças ou condições inflamatórias. O objetivo desta tese foi, além de revisar a importância da inflamação sistêmica na modulação da resposta inflamatória da microglia/macrófagos e consequentemente seu potencial envolvimento na fisiopatologia do TB, avaliar o perfil de polarização M1/M2 em cultura de macrófagos de sujeitos com TB comparados a indivíduos saudáveis. Monócitos foram isolados a partir de sangue periférico de dez sujeitos com TB e dez indivíduos saudáveis e diferenciados em macrófagos através da adição de fator estimulante de colônia de macrófagos (MCSF) ao meio de cultura. Para induzir a polarização M1 ou M2, as culturas foram incubadas com IFN-y e LPS ou IL-4 respectivamente. Após a incubação, recolheram-se os sobrenadantes e mediram-se as citocinas (IL-1β, IL-6, IL-10 e TNF-α) por ensaio multiplex. A secreção das citocinas IL-1β, TNF-α e IL-6 características do protótipo M1 e citocinas IL-10 do protótipo M2 foram semelhantes entre os pacientes e os controles. Utilizou-se a razão TNF-α / IL-10 do fenótipo M1 para refletir o estado inflamatório dos participantes. Não foi observada diferença entre os grupos (p=0,627). Duas hipóteses diferentes poderiam explicar esses resultados: todos os pacientes incluídos neste estudo representam um estágio inicial da doença como evidenciado pela pontuação FAST total inferior a 11. De acordo com o modelo de estadiamento em TB, as alterações biológicas (incluindo a inflamação) parecem estar relacionadas com os episódios de humor e progressão da doença. Juntamente com estudos anteriores, os nossos dados sugerem que os pacientes nos estágios iniciais ainda preservam a função do sistema imunológico sem apresentar um desequilíbrio a favor do perfil de macrófagos M1 como tem sido observado em pacientes no estágio tardio, destacando a relevância da intervenção precoce no TB. Ainda, estes pacientes estavam em tratamento com estabilizadores de humor e é plausível especular que esses fármacos exerçam efeitos sobre a polarização de macrófagos. Estudos futuros em pacientes drug-free são essenciais para avaliar esta questão. Em conclusão, nossos achados sugerem que os pacientes TB não apresentam desequilíbrio na polarização dos macrófagos em favor do fenótipo pró-inflamatório M1. O fato de todos estes pacientes estarem em estágios iniciais da doença reforça os efeitos protetores da intervenção precoce no TB na prevenção de alterações do sistema imune e, consequentemente, na progressão da doença. / Innate immune system dysfunction and neuroinflammation have been recognized as important elements in the pathophysiology of bipolar disorder (BD). As essential players of innate immunity, macrophages have multiple roles in inhibition and promotion of cell proliferation and tissue repair. The classical M1 polarization and the M2 alternative polarization of macrophages represent two extremes of a dynamic state in their change of activation. M1 macrophages synthesize proinflammatory cytokines that inhibit the proliferation of surrounding cells and damage tissues, whereas macrophages of the M2 phenotype release antiinflammatory cytokines that may promote tissue repair. An imbalance of the M1-M2 polarization of macrophages is often associated with various diseases or inflammatory conditions. The aim of this thesis was to review the importance of systemic inflammation in modulating the inflammatory response of microglia/ macrophages and consequently their potential involvement in the pathophysiology of BD, and also evaluate the M1/M2 polarization profile in macrophages of patients with BD compared to healthy individuals. Blood monocytes were obtained from ten BD patients and ten healthy controls. These cells were activated/polarized into the M1 (IFNγ + LPS) or M2(IL-4) phenotype. Supernatants were collected and the cytokines (IL-1β, IL-6, IL-10 and TNF-α) were measured by multiplex assay. Secretion of the IL- 1β, TNF-α, IL-6 and IL-10 were similar between patients and controls. The TNF-α/IL- 10 ratio of the M1 phenotype was used to reflect the inflammatory state of the participants. There was no difference between groups (p = 0.627). Two hypotheses could explain these results: all patients included in this study represent an early stage of disease as evidenced by the FAST score below 11. According to the BD staging model, biological changes (including inflammation) appear to be related to mood episodes and disease progression. Together with previous studies, our data suggest that patients in early stages of BD still preserve immune system function without presenting an imbalance in favor of M1 macrophages as has been observed in latestage patients, highlighting the relevance of early intervention. Moreover, these patients were under treatment with mood stabilizers and it is plausible to speculate that these drugs have effects on macrophage polarization. Future studies in drug-free patients are essential to assess this issue. In conclusion, our findings suggest that BD patients do not present imbalance in macrophage polarization in favor of the M1 proinflammatory phenotype. The fact that all these patients are in the early stages of the disease reinforces the protective effects of early intervention in BD to prevent changes in the immune system and, consequently, prevent the progression of the disease.

A disfunção do sistema imune inato e a neuroinflamação tem sido cada vez mais reconhecidas como elementos importantes na fisiopatologia do transtorno bipolar (TB). Como componentes essenciais da imunidade inata, os macrófagos tem múltiplas funções tanto na inibição como na promoção da proliferação celular e na reparação tecidual, sendo a diversidade e a plasticidade características marcantes deste tipo celular. A polarização M1 clássica e a polarização alternativa M2 de macrófagos representam dois extremos de um estado dinâmico na mudança da ativação dos mesmos. Os macrófagos do tipo M1 sintetizam citocinas próinflamatórias que inibem a proliferação de células circundantes e danificam tecidos, enquanto os macrófagos do fenótipo M2 liberam citocinas antiinflamatórias que podem promover reparo tecidual. Um desequilíbrio da polarização M1-M2 dos macrófagos é frequentemente associado a várias doenças ou condições inflamatórias. O objetivo desta tese foi, além de revisar a importância da inflamação sistêmica na modulação da resposta inflamatória da microglia/macrófagos e consequentemente seu potencial envolvimento na fisiopatologia do TB, avaliar o perfil de polarização M1/M2 em cultura de macrófagos de sujeitos com TB comparados a indivíduos saudáveis. Monócitos foram isolados a partir de sangue periférico de dez sujeitos com TB e dez indivíduos saudáveis e diferenciados em macrófagos através da adição de fator estimulante de colônia de macrófagos (MCSF) ao meio de cultura. Para induzir a polarização M1 ou M2, as culturas foram incubadas com IFN-y e LPS ou IL-4 respectivamente. Após a incubação, recolheram-se os sobrenadantes e mediram-se as citocinas (IL-1β, IL-6, IL-10 e TNF-α) por ensaio multiplex. A secreção das citocinas IL-1β, TNF-α e IL-6 características do protótipo M1 e citocinas IL-10 do protótipo M2 foram semelhantes entre os pacientes e os controles. Utilizou-se a razão TNF-α / IL-10 do fenótipo M1 para refletir o estado inflamatório dos participantes. Não foi observada diferença entre os grupos (p=0,627). Duas hipóteses diferentes poderiam explicar esses resultados: todos os pacientes incluídos neste estudo representam um estágio inicial da doença como evidenciado pela pontuação FAST total inferior a 11. De acordo com o modelo de estadiamento em TB, as alterações biológicas (incluindo a inflamação) parecem estar relacionadas com os episódios de humor e progressão da doença. Juntamente com estudos anteriores, os nossos dados sugerem que os pacientes nos estágios iniciais ainda preservam a função do sistema imunológico sem apresentar um desequilíbrio a favor do perfil de macrófagos M1 como tem sido observado em pacientes no estágio tardio, destacando a relevância da intervenção precoce no TB. Ainda, estes pacientes estavam em tratamento com estabilizadores de humor e é plausível especular que esses fármacos exerçam efeitos sobre a polarização de macrófagos. Estudos futuros em pacientes drug-free são essenciais para avaliar esta questão. Em conclusão, nossos achados sugerem que os pacientes TB não apresentam desequilíbrio na polarização dos macrófagos em favor do fenótipo pró-inflamatório M1. O fato de todos estes pacientes estarem em estágios iniciais da doença reforça os efeitos protetores da intervenção precoce no TB na prevenção de alterações do sistema imune e, consequentemente, na progressão da doença. / Innate immune system dysfunction and neuroinflammation have been recognized as important elements in the pathophysiology of bipolar disorder (BD). As essential players of innate immunity, macrophages have multiple roles in inhibition and promotion of cell proliferation and tissue repair. The classical M1 polarization and the M2 alternative polarization of macrophages represent two extremes of a dynamic state in their change of activation. M1 macrophages synthesize proinflammatory cytokines that inhibit the proliferation of surrounding cells and damage tissues, whereas macrophages of the M2 phenotype release antiinflammatory cytokines that may promote tissue repair. An imbalance of the M1-M2 polarization of macrophages is often associated with various diseases or inflammatory conditions. The aim of this thesis was to review the importance of systemic inflammation in modulating the inflammatory response of microglia/ macrophages and consequently their potential involvement in the pathophysiology of BD, and also evaluate the M1/M2 polarization profile in macrophages of patients with BD compared to healthy individuals. Blood monocytes were obtained from ten BD patients and ten healthy controls. These cells were activated/polarized into the M1 (IFNγ + LPS) or M2(IL-4) phenotype. Supernatants were collected and the cytokines (IL-1β, IL-6, IL-10 and TNF-α) were measured by multiplex assay. Secretion of the IL- 1β, TNF-α, IL-6 and IL-10 were similar between patients and controls. The TNF-α/IL- 10 ratio of the M1 phenotype was used to reflect the inflammatory state of the participants. There was no difference between groups (p = 0.627). Two hypotheses could explain these results: all patients included in this study represent an early stage of disease as evidenced by the FAST score below 11. According to the BD staging model, biological changes (including inflammation) appear to be related to mood episodes and disease progression. Together with previous studies, our data suggest that patients in early stages of BD still preserve immune system function without presenting an imbalance in favor of M1 macrophages as has been observed in latestage patients, highlighting the relevance of early intervention. Moreover, these patients were under treatment with mood stabilizers and it is plausible to speculate that these drugs have effects on macrophage polarization. Future studies in drug-free patients are essential to assess this issue. In conclusion, our findings suggest that BD patients do not present imbalance in macrophage polarization in favor of the M1 proinflammatory phenotype. The fact that all these patients are in the early stages of the disease reinforces the protective effects of early intervention in BD to prevent changes in the immune system and, consequently, prevent the progression of the disease.

Introduction: Increased activation of inflammatory pathways is associated with elevated metabolic stress, which leads to a constellation of metabolic pathologies like fatty liver, insulin resistance and atherosclerosis. Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a novel transcription co-factor that binds to and inhibits two main pro-inflammatory transcription factors, IRF2 and NFAT1. IRF2BP2 genetic variants are also linked to increased human serum cholesterol level in GWAS studies. Therefore, we hypothesized that IRF2BP2 may inhibit macrophage polarization to pro-inflammatory phenotype and considering the remarkable overlap between inflammatory and metabolic sensors, alter their metabolic function. We sought to determine if specific ablation IRF2BP2 in the mouse myeloid lineage (IRF2BP2MKO) leads to development of metabolic symptoms and alters the risk of atherosclerosis. Results: Our results indicate that IRF2BP2 ablation impairs macrophage polarization to the anti-inflammatory phenotype. IRF2BP2MKO bone marrow derived macrophages (BMDM) show increased oxidized LDL-cholesterol uptake and decreased cholesterol efflux. Also, mice with specific ablation of IRF2BP2 in macrophages are more susceptible to obesity, insulin resistance and hepatic steatosis compared to control mice, when fed high fat diet (HFD). However, LDLR-/- mice transplanted with IRF2BP2MKO bone marrow demonstrate similar extent of atherosclerotic lesions compared to LDLR-/- mice transplanted with control bone marrow, reflecting increased IRF2BP2MKO macrophage apoptosis. Conclusion: In conclusion, this is the first study to identify the metabolic and inflammatory functions of IRF2BP2 protein in macrophages, with important implications in metabolic syndrome and atherosclerosis.

Background: Pregnant individuals who participate in physical activity throughout gestation have been shown to experience a wide spectrum of health benefits, along with the fetus. In nonpregnant populations, PA influences the polarization state of tissue resident macrophages, resulting in increased regulatory and decreased inflammatory profiles. The effects of PA on placenta-resident macrophages, or Hofbauer cells (HBCs), remains unknown. My thesis aimed to explore this novel area. Methods: The first objective of my thesis was to identify any associations between gestational PA and HBC polarization. PA was objectively measured in both mid (24-28 weeks) and late (34-38 weeks) pregnancy using accelerometry. Immunofluorescent localization of the panmacrophage marker CD68 and the anti-inflammatory macrophage marker CD206 was used to assess polarization states. Protein and gene expression of CD68 and CD206 were assessed using Western blot and qPCR, respectively. The second objective was to explore the relationships between gestational PA, HBC polarization, and angiogenic factors in the placenta. Western blot measured the relative protein expression of FGF2 and SPRY2, and the localization of FGF2, SPRY2, and VEGF within HBCs was explored using immunofluorescent colocalization in term placenta tissue and primary HBC cultures. Results: While there were no differences in the absolute numbers of total or CD206+ HBCs, the proportion of CD206+ HBCs was elevated in active individuals. There were no significant differences in the gene expression of CD68 or CD206, nor in the gene expression of CD206; however, CD206 protein expression was observed to be lower in active participants. Both CD206+ and CD206- HBCs expressed VEGF. Active individuals had significantly higher low molecular weight-FGF2. There were no differences in the protein expression of SPRY2, total FGF2, or high molecular weight FGF2 based on PA. HBCs both in vitro and in vivo of all polarizations expressed VEGF, SPRY2, and FGF2, and were observed to create intracellular junctions and multi-nucleated giant cells. Conclusions: In conclusion, PA was associated with a higher proportion of CD206+ HBCs and reduced levels of CD206 protein. In combination with the lack of significant difference in CD206 mRNA based on PA levels, this suggests a potential effect mediated by PA on the transcriptional regulation of CD206. HBCs were seen to express SPRY2, VEGF, and FGF2, identifying them as potential players in angiogenesis regulation in the placenta. The elevated levels of low molecular weight FGF2 in active individuals suggests the PA may play a role in the modulation of placental angiogenesis. Future research should continue to explore the relationships between PA, HBC polarization, and angiogenesis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24834号 / 医博第5002号 / 新制||医||1067(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 上杉, 志成, 教授 金子, 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

Subclinical endotoxemia (low levels of circulating bacterial endotoxin) has been observed in patients suffering from chronic inflammatory diseases such as atherosclerosis, diabetes, and obesity. However, the link between this condition and chronic inflammation is poorly understood. Previous work from our lab has shown that chronic exposure to super-low doses of bacterial endotoxin (LPS) aggravates atherosclerosis resulting in increased plaque size and instability in a macrophage-dependent manner in a mouse model of atherosclerosis. Further, we showed that super-low dose LPS (SLD-LPS) treatment was able to inhibit lysosomal fusion in immortalized macrophages. However, this was done under more acute treatment conditions. The aim of this project was to examine the molecular mechanisms by which chronic SLD-LPS may polarize macrophages to a non-resolving pro-inflammatory state consistent with chronic inflammation. This was carried out in two projects, the first a more broad phenotypic paper showing the disruption in homeostasis by chronic SLD-LPS in immortalized macrophages, while the second uses primary bone marrow-derived mouse macrophages to identify specific molecular signaling pathways used by chronic SLD-LPS. Here we show that chronic SLD-LPS led to the novel upregulation of pro-inflammatory mediators p62 and ccl2 with simultaneous downregulation of homeostatic mediators Nrf2 and slc40a1 in immortalized wild-type mouse macrophages. Further we showed this effect was reversed using the homeostatic restorative agent sodium phenylbutyrate (4-PBA), a newly reported activity for this reagent in mouse macrophages. This indicated that a disruption in homeostasis, possibly involving autophagy, may be responsible for the non-resolving pro-inflammatory polarization of macrophages. Therefore, in our second project, we further explored the effect of chronic SLD-LPS treatment on the homeostatic arm of the response by focusing on the Nrf2 inhibitor Keap1. Here we show that chronic SLD-LPS results in an accumulation of Keap1 in mouse bone marrow-derived macrophages, an effect specific to chronic SLD-LPS, as high doses of LPS failed to induce Keap1. We suggest that this effect may be related to a disruption in lysosomal fusion as evidenced by accumulation of autophagy flux markers MLKL and p62. Further, we show that these effects are dependent on the non-traditional TLR4 adaptor TRAM, suggesting an alternative dose-dependent signaling pathway for LPS. Together this work identifies novel signaling mechanisms involved in non-resolving pro-inflammatory polarization of murine macrophages, providing new insight behind how chronic super-low dose LPS exposure may lead to chronic inflammation. / Doctor of Philosophy / Inflammation is the body's natural response to injury or insult and can be beneficial in certain contexts such as pathogen clearance. However, left un-checked, chronic inflammation can exacerbate or even lead to disease pathology, such as is the case with modern diseases such as atherosclerosis, obesity, diabetes, etc. Despite the high prevalence of these diseases, effective treatments and therapies are still lacking. Recently it was discovered that many patients suffering from chronic inflammatory diseases had low levels bacterial endotoxin (LPS) in their circulation, a condition referred to as subclinical endotoxemia. However, possible links between this condition and chronic inflammatory disease remain poorly understood. Using a mouse model of atherosclerosis, previous research from our lab showed that persistent exposure to super-low doses of bacterial endotoxin (similar to those observed in humans) lead to aggravated atherosclerosis with both increased plaque size and instability. Further, we showed that this effect was primarily mediated by pro-inflammatory polarized immune cells called macrophages, but the molecular mechanism behind this polarization is still unclear. Further research into these molecular mechanisms may provide better targets for the development of future chronic inflammatory disease treatments. Here using a combination of mouse cell line and primary cell cultures, we discuss how chronic exposure to super-low doses of bacterial endotoxin leads to the chronic non-resolving pro-inflammatory polarization of macrophage immune cells, with particular emphasis on the distinct molecular signaling mechanisms induced by chronic super-low dose LPS.