京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24140号 / 医科博第141号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 金子 新, 教授 椛島 健治, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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24785号 / 医博第4977号 / 新制||医||1066(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 金子 新, 教授 YOUSSEFIAN Shohab, 教授 阪上 優 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Macrophage generation of reactive nitrogen intermediates (RNI) represents a major effector mechanism in anti‐microbial immunity and non‐septic inflammatory reactions. The induction of macrophage RNI production has been demonstrated to require at least two signals which in microbial infections can be provided by interferon (IFN)‐γ and lipopolysaccharide (LPS). The current study demonstrates that, in the absence of LPS, T lymphocytes can provide cognate signal(s) which synergize with IFN‐γ in stimulating macrophage RNI production, as evidenced by the ability of plasma membranes from T cell clones to activate IFN‐γ‐primed macrophages. Although viable resting T cells can activate IFN‐γ‐primed macrophages by an interaction that is antigen specific, plasma membranes from resting T cells do not activate macrophages. Plasma membranes from T cells activated by immobilized anti‐CD3 were able to effectively induce RNI production in IFN‐γ‐primed macrophages. However, in contrast to the antigen‐specific interaction of macrophages with viable resting T cells, the activation of IFN‐γ‐primed macrophages by membranes from activated T cells does not display antigen specificity. Plasma membranes from activated T helper TH2 and from activated TH1 cells were equally effective in activating IFN‐γ‐primed macrophages, suggesting that the dominance of TH1 over TH2 cells in cell‐mediated responses involving macrophage effectors is not a reflection of differences in their ability to interact with macrophages but rather is a reflection of their different pattern of cytokine production. These results suggest that the T cell‐macrophage interaction involves reciprocal activation of both cells ‐ an antigen‐specific activation of the T cells which results in the acquisition of T cell membrane components involved in antigen‐nonspecific stimulation of the macrophages.

Background: Ischemic heart disease (IHD) is a leading cause of morbidity and mortality worldwide. Deprivation of oxygen/nutrients to the heart during IHD induces cardiac cell death resulting in an inflammatory response with the migration of neutrophils and macrophages, an essential process for cardiac repair. However, unresolved inflammation can cause adverse cardiac remodeling following myocardial ischemia/reperfusion (I/R) injury. Ubiquitin (UB) is an evolutionarily conserved protein. Previously, our lab has shown that exogenous UB treatment plays a cardioprotective role and significantly reduces infiltration of neutrophils and macrophages 3 days post-I/R injury in mice. Here, we investigated the role of exogenous UB in macrophage phenotype and function. It is hypothesized that exogenous UB modulates the phenotype and function of polarized M1 (pro-inflammatory), and/or M2 (anti-inflammatory) macrophages, thereby playing a cardioprotective role 3 days post-I/R. Methods: Isolated peritoneal macrophages (3 days post-thioglycolate injection) were pretreated with UB (20μg/mL) for 30 minutes followed by treatment with IFNγ (100U/mL), for M1 polarization, or IL-4 (20ng/mL), for M2 polarization for 72 hours. Polarized peritoneal macrophages were used to prepare cell lysates for western blot analyses, immunohistochemistry or migration. Plated and paraformaldehyde-fixed polarized peritoneal macrophages were stained with F4/80 to measure the cell spreading area. A scratch assay was used to measure polarized peritoneal macrophage migration. RAW264.7 macrophage cell line was used to assess polarized macrophage phagocytosis of pHrodo-conjugated E. coli bioparticles (100μg/mL). Results: Western blot analysis of polarized peritoneal macrophage lysates showed that UB treatment increased IFNγ-induced expression of iNOS (a marker of M1 phenotype). Treatment with UB decreased IFNγ induced-phosphorylation of STAT1 (transcription factor activated by IFNγ). IFNγ and IL-4 treatment decreased the expression of CXCR4 (receptor for UB). However, UB treatment had no effect on IFNγ and IL-4-mediated decrease in CXCR4 expression. Treatment with UB significantly reduced the cell spreading area of M1-polarized peritoneal macrophages. The migration of M1-polarized peritoneal macrophages was significantly increased with UB treatment. The migration of M2-polarized peritoneal macrophages was significantly greater compared to non-polarized control macrophages, and UB treatment had no effect on M2-polarized macrophage migration. In RAW264.7 cells, M1-polarized macrophages exhibited significantly increased phagocytosis of E. coli bioparticles vs. non-polarized control, and UB treatment enhanced M1-polarized macrophage phagocytosis. In contrast, UB treatment reduces the phagocytosis of E. coli bioparticles in M2-polarized macrophages. Conclusions: These findings indicate that treatment with exogenous UB can potentially alter the phenotype and function of M1- and M2-polarized macrophages.

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No description available.

The receptor tyrosine kinase, RON, is expressed on tissue-resident macrophages. RON functions by activating genes that promote wound repair and resolve inflammation, while repressing genes that perpetuate tissue damage and cell death. Chronic HIV infection is associated with dysregulated inflammation, and we hypothesize that diminished macrophage RON expression contributes to the development of end organ diseases including HIV-associated central nervous system (CNS) inflammation. We utilized CNS tissue from a SIV macaque model to examine the temporal regulation of RON in the brain during infection. Following prolonged SIV infection, RON expression was inversely correlated with the development of CNS disease: RON was highly expressed in animals that did not develop CNS lesions and lower in SIV infected macaques that demonstrated moderate to severe inflammatory lesions. Arginase-1 expression was low during late infection whereas expression of the inflammatory genes, IL-12 p40 and TNF &alpha, was elevated compared to uninfected animals. To validate a role for RON in regulating HIV, we infected human tonsillar tissue-resident macrophages. RON inhibited HIV replication in tissue-resident macrophages. Furthermore, HIV infection diminished RON in tonsil macrophages. We propose a model in which RON expression is decreased, genes that quell inflammation are repressed, and inflammatory mediators are induced to promote tissue inflammation following chronic HIV infection in the brain. The cyclin dependent kinase inhibitor p21 is a factor that, like RON, negatively regulates HIV transcription. Elevated expression of p21 in HIV+ elite controllers, or by ectopic expression in primary CD4+ T cells, resulted in reduced HIV expression. Furthermore, these elite controllers had increased binding of factors that negatively regulate transcription elongation at the HIV long terminal repeat. RON and p21 are examples of cellular factors that limit HIV transcription and contribute to HIV latency. Latently infected cells are not targeted by anti-retroviral therapy and permit rapid rebound of viremia following treatment interruption. Understanding intrinsic mechanisms that establish latency may provide targets for purging these HIV reservoirs or maintaining their transcriptionally silent state.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23801号 / 医博第4847号 / 新制||医||1058(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 小林 恭, 教授 竹内 理, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM