Macrophages are multifaceted innate immune cells, able to adapt their phenotype to respond to a myriad of conditions, engaging in tissue-specific functions and mediating either inflammatory or anti-inflammatory responses depending on the encountered stimuli. They conduct key roles in the orchestration of immune responses; from pathogen recognition through sterilising inflammation to resolution and repair. The Udalova laboratory has previously demonstrated that IRF5 promotes a pro- inflammatory macrophage phenotype, leading to the secretion of TNF, IL-12, and IL-23, enhancing Th1/Th17-mediated immune responses, and described the cooperation between IRF5 and the transcription factor RelA, which mediate the production of pro-inflammatory genes. The aim of this thesis is to further characterise the activity of IRF5 in macrophage inflammatory responses. I demonstrate that IRF5 not only regulates the transcription of cytokines and chemokines in response to bacterial stimuli, but also anti-microbial peptides, whilst simultaneously down-regulating homeostatic and resolving macrophage functions. My data also suggests that IRF5 plays a role in enforcing monocyte to macrophage differentiation by up-regulating the transcription of key macrophages markers and repressing dendritic cell identity genes. To further characterise the mechanisms of the inflammatory response mounted by macrophages I used an unbiased approach; combining twenty-three transcription factor ChIP-seq data sets with chromatin accessibility information from ATAC-seq, uncovering RUNX1 as a novel partner of IRF5 that binds co-operatively to clusters of enhancers, which control the transcription of pro-inflammatory genes in a signal-dependent manner. This is the first study demonstrating a critical role for RUNX1 in activity of inflammatory macrophages.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.

Adipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown if these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. The effect of HG on adipogenesis was assessed. Human subcutaneous abdominal preadipocytes were induced to differentiate in HG or NG conditions. HG did not affect adipogenesis. HG increased ChREBP-β mRNA and protein levels, and increased GLUT4 mRNA, in differentiated adipocytes. It did not change mRNA levels of ACC, SCD, and FAS. The increase in ChREBP-β mRNA was positively correlated with HG-induced increase in GLUT4 mRNA. The effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation was compared. Human monocyte-derived macrophages (MDM) were placed in NG or HG glucose for 24 hours to generate MacCM. HG-MacCM, but not NG-MacCM inhibited triacylglycerol accumulation and protein expression of PPARγ during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased MCP-1 and IL-6 and reduced adiponectin mRNA expression. HG increased phosphorylation of IKK-β and decreased protein expression of IκBα in MDMs. In addition, HG reduced protein expression of PPARγ in MDMs. The pro-inflammatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKβ inhibitor). My data demonstrate that HG-induced expression of ChREBP-β in adipocytes may be associated with increased GLUT4 mRNA. The anti-adipogenic and pro-inflammatory effects of HG-MacCM are more potent than NG-MacCM. This suggests the possibility that adipose tissue cellular remodeling in vivo may be altered with hyperglycemia.

Glioblastoma is the most common and aggressive type of adult brain tumour with a need for new treatments. CD47 has been shown to be overexpressed on some human cancers and to interact with macrophages but its role in glioblastoma has not yet been fully explored. Here, we identify a novel role for the CD47/SIRPα interaction between glioblastoma and macrophages in that it can stimulate glioblastoma proliferation in co-cultures. Blocking either CD47 or SIRPα resulted in decreased glioblastoma proliferation. Furthermore, we show that macrophage stimulated glioblastoma proliferation is not occurring through downstream signalling of SIRPα but likely through CD47 to the PI3Kβ pathway. Initial results of co-cultures using glioblastoma cells which express CD47 that is GPI linked to the membrane, and therefore cannot signal downstream, support these findings. The implication of this research is the possibility to develop new therapies targeted at CD47 to decrease glioblastoma proliferation.

Meningiomas are the most common primary brain tumors, yet few successful non-invasive treatment options are available for patients. Immunotherapy has provided new insights into treatments for solid tumors. The immune infiltrate of meningiomas has been broadly characterized, showing a significant monocytic cell content, but little is known about the phenotype and function of these myeloid cells within the tumor environment. As circulating monocytes differentiate into macrophages with highly plastic character within tissue, it remains to be seen how the macrophages in meningiomas are influencing the tumor. As many studies have described the presence of monocytic subpopulations within other solid tumors, we hypothesize that meningiomas contain two populations of myeloid cells: a pro-inflammatory macrophage-like population, and an immunosuppressive myeloid-derived suppressor cell (MDSC)-like population. We collected fresh tumor samples and processed them into a single-cell suspension. The cells were then stained with fluorescently labeled surface marker antibodies commonly found on macrophages and MDSCs. We used flow cytometry to quantify the myeloid populations, sorted the populations with a FACSAria™, and analyzed their gene expression profiles with NanoString® and TaqMan®. Two distinct myeloid populations were found in all analyzed tumor samples, varying in macrophage-like to MDSC-like ratios from tumor to tumor. Gene expression analysis of these populations confirmed the sorting strategy and provided new clues into the identity and function of the myeloid cell populations infiltrating meningiomas. NanoString® results confirmed a high HLA-DR gene expression in the HLA-DR+ sorted populations. The tumor HLA-DR+ population was found to have higher gene expression relative to the HLA-DR- population for chemoattractants such as IL8, CCL3, and CCL4. Compared to a healthy blood monocyte control, tumor myeloid cells expressed higher levels of the genes C3AR1, ROCK2, IL10, NOS2, IL18, and CSF2. Finally, qPCR analysis and NanoString® results showed high expression of the gene IL6 in the non-immune cell tumor cells (CD45-). The IL6 cytokine has been shown to induce MDSCs. These findings may have significant implications in identifying new targets in immunotherapy to stop tumor growth and increase survival outcomes. / 2016-11-03T00:00:00Z

Atherosclerosis is a chronic inflammatory and lipid disorder caused by the buildup of cholesterol-loaded cells of monocyte and muscle cell origin in the arterial intima. While the relationship between excess cholesterol and macrophage behavior is well observed, the molecular mechanisms linking the two remain unclear. Therefore, characterizing the pathways from changes in intracellular cholesterol to the resulting inflammatory output is key to understanding the behavioral changes observed in human macrophages in vitro. We identified that THP-1 macrophages acutely depleted of cholesterol increase the expression of JMJD3, an H3K27me3 demethylase. By using IL-10 as a marker for immune-modulating genes and TNF-α as a marker for pro-inflammatory genes, cholesterol-depleted THP-1 macrophages responded inconsistently to LPS and echinomycin, an inhibitor of HIF-1α, as determined by RT-qPCR and ELISA. Further studies investigating other regulators and outputs of macrophage behavior linked to cellular cholesterol modification are required.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18184号 / 医科博第49号 / 新制||医科||4(附属図書館) / 31042 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 松田 道行, 教授 岩井 一宏 / 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.

No description available.