Glioblastoma (GBM) is an aggressive brain cancer with devastating outcomes and few effective treatments. Although immunotherapy has shown promise in treating a variety of cancers, it is still unclear if and how it can be effectively used in GBM. Elucidating this will require a better understanding of the mechanistic role of immune cells and their interactions in the GBM tumor microenvironment. This thesis utilizes recent technological developments in cancer genomics and imaging to study the mechanisms underlying immunotherapy and the tumor microenvironment. First, we will provide background on our current understanding of GBM, its immune microenvironment, as well as modern sequencing and imaging methods. Second, we will present a longitudinal study of GBM patients before and after treatment with PD-1 immunotherapy. Only a small fraction of GBM patients respond to this type of therapy, so we perform genomic, transcriptomic, and spatial analyses to compare the molecular features of these rare responders versus non-responders. We show that clinical response to PD-1 immunotherapy in GBM is associated with specific molecular alterations and immune infiltration profiles that reflect the tumor’s clonal evolution during treatment. The most common infiltrating immune cells in GBM are macrophages, which are implicated in a wide variety of pro-tumor and anti-tumor roles. We then focus on this specific immune population by analyzing single-cell expression data from GBM tumors. We identify a novel macrophage subpopulation characterized by expression of the scavenger receptor MARCO, which drives tumor progression in GBM and is altered over the course of PD-1 immunotherapy. Next, we demonstrate that the methods we have developed for GBM are applicable to understanding the tumor microenvironments of other cancers as well. We analyze a cohort of melanoma cases to show that transcriptomic and imaging features can be combined to create a biomarker that stratifies patients into different risk groups. Finally, while most of the image analysis described so far has utilized histopathology, we include two appendices where we demonstrate new ways to process and analyze Magnetic Resonance Imaging (MRI) in GBM.

Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology). / Includes bibliography (leaves 107-151). / Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin (IL )-1, by monocytes/macrophages. Excessive and uncontrolled secretion of these compounds leads to multiple pathological conditions, such as septic shock. LPS receptors have been shown to be CD14, TLR4 and MD-2. LPS interaction with these receptors mediates many monocyte/macrophage functions. Even though only CD14 was demonstrated to bind to LPS, and TLR4/MD-2 were capable of transducing signals, data only show that LPS and CD 14 were in close proximity to TLR4 and no direct binding was reported. Quite recently, moesin, a member of the ERM family of proteins, has been also found to function as a receptor for LPS. We have shown that anti-moesin antibody inhibited the release of TNFa by LPS stimulated monocytes. Moesin was also found to be necessary for the detection of LPS, where homozygous knockout mice exhibited 3-fold reduction in neutrophil infiltrates in LPS injected sites when compared to their wild type controls. When moesin gene expression was completely suppressed with antisense oligonucleotides, there was a significant reduction of LPS-induced TNF-a secretion. LPS stimulation of mononuclear phagocytes activates several intracellular signaling pathways including the phosphorylation of IKBa, mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 / 2 (P44/42), p38. These signaling pathways in tum activate a variety of transcription factors including NF-KB, which coordinates the induction of several genes encoding inflammatory mediators. [TRUNCATED]

Necroptosis is a pathway of inflammatory cell death that is associated with several pathologies and is induced by ligation of surface TLR or cytokine receptors in macrophages. Many signaling pathways depend on endocytosis, a process mediated by GTPases such as dynamin. We evaluated the role of dynamin-dependent endocytosis in the necroptosis of macrophages using various dynamin inhibitors. Using flow cytometry, we confirmed that during necrosome signaling, various dynamin inhibitors (e.g. Dyngo 4a and Dynasore) blocked the internalization of TLR4, which also resulted in the inhibition of cytokine production. Despite the similar impact of Dynasore and Dyngo 4a on TLR4 endocytosis and cytokine production, only Dyngo 4a prevented TLR4-induced necroptosis of macrophages. Further studies indicated that Dyngo 4a was a potent stimulator of the p38 MAPK pathway, and activation of this pathway by Dyngo 4a was responsible for the inhibition of necroptosis of macrophages following TLR4 signaling. Thus, these studies reveal the previously unknown role of the p38 MAPK pathway in regulating the activation of necrosome signaling.

Endothelial cell dysfunction contributes to sepsis induced initiate immune response and the infiltration of immune cells into organs, resulting in organ injury. Heat shock protein A12B (HSPA12B) is predominantly expressed in endothelial cells. The present study investigated whether endothelial HSPA12B could regulate macrophage pro-inflammatory response during sepsis. Wild type (WT) and endothelial cell-specific HSPA12B deficient (HSPA12B–/–) mice were subjected to CLP sepsis. Mortality and cardiac function were monitored. Higher mortality, worsened cardiac dysfunction, and greater infiltrated macrophages in the myocardium and spleen were observed in HSPA12B–/– septic mice compared with the WT septic mice. The serum levels of TNF-α and IL-1β were higher and the levels of IL-10 were lower in HSPA12B–/– septic mice than in WT septic mice. Importantly, endothelial exosomes contain HSPA12B which can be uptaken by macrophages. Interestingly, endothelial exosomal HSPA12B significantly increases IL-10 levels and decreases TNF-α and IL-1β production in LPS-stimulated macrophages. Mechanistic studies show that endothelial exosomal HSPA12B downregulates NF-κB activation and nuclear translocation in LPS stimulated macrophages. These data suggest that endothelial HSPA12B plays a novel role in the regulation of macrophage pro-inflammatory response via exosomes during sepsis and that sepsis induced cardiomyopathy and mortality are associated with endothelial cell deficiency of HSPA12B.

Long noncoding RNAs (lncRNAs) are emerging as novel regulators of macrophage biology and related inflammatory cardiovascular diseases. However, studies focused on lncRNAs in human macrophage subtypes, particularly human-specific lncRNAs that are not conserved in rodents, are limited. Through deep RNA-seq of human monocyte-derived macrophages, we identified SIMALR (suppressor of inflammatory macrophage apoptosis lincRNA), a human macrophage-specific long intergenic noncoding RNA (lincRNA), to be highly induced in the nucleus of LPS/IFNγ-stimulated macrophages. Treatment of LPS/IFNγ-stimulated THP1 human macrophages with SIMALR antisense oligonucleotides induced apoptosis of inflammatory macrophages, as shown by increased Annexin V+ macrophages, and increased protein expression of cleaved PARP, caspase 9, and caspase 3. Differential expression analysis of RNA-seq data from SIMALR knockdown versus control in human macrophages revealed Netrin 1 (NTN1), a known regulator of macrophage apoptosis, to be one of the top downregulated genes. NTN1 knockdown in LPS/IFNγ-stimulated THP1 macrophages induced apoptosis. This apoptotic phenotype was attenuated by treating LPS/IFNγ-stimulated macrophages with recombinant human NTN1 after SIMALR knockdown. Furthermore, NTN1 promoter-luciferase reporter activity was increased in HEK293T cells treated with lentiviral overexpression of SIMALR. NTN1 promoter activity is known to require HIF1α and RNA immunoprecipitation (RIP) showed that SIMALR binds HIF1α, suggesting that SIMALR may modulate HIF1α binding at the NTN1 promoter to regulate apoptosis of macrophages. In human translational studies, SIMALR was found to be upregulated in macrophages in unstable human atherosclerotic plaques, suggesting a possible mechanistic link between inflammation and cardiovascular diseases. In addition to SIMALR, through deep RNA-seq of human monocyte-derived macrophages, we identified RP11-184M15.1, a human macrophage-specific lincRNA, to be highly induced in the cytoplasm of IL-4-stimulated macrophage. Preliminary data showed that treatment of IL-4-stimulated THP1 human macrophages with RP11-184M15.1 small interfering RNA (siRNA) repressed apoptosis of resolving macrophages, as shown by decreased Annexin V+ macrophages, and reduced protein expression of cleaved PARP. Biotinylated RP11-184M15.1 pulldown coupled with mass spectrometry indicated an interaction between RP11-184M15.1 and zinc finger RNA-binding protein (ZFR). RIP corroborated the proposed interaction between RP11-184M15.1 and ZFR. RNAInter revealed mRNAs predicted to interact with ZFR, and some of those genes (e.g., ALYREF, CCNYL1) were also differentially expressed in RNA-seq data of control versus RP11-184M15.1 knockdown in IL-4-stimulated THP1 macrophages. qPCR validated that ALYREF and CCNYL1 expression are reduced with RP11-184M15.1 knockdown. In contrast, with ZFR siRNA, ALYREF and CCNYL1 mRNA expressions were elevated. Thus, a hypothesis to be further tested is that RP11-184M15.1 interacts with ZFR to regulate mRNA stability in IL-4-stimulated macrophages. Nuclear RNA export factor 1 (NXF1) was also validated by RIP to interact with RP11-184M15.1. NXF1 is a known interacting partner of ALYREF in the transcription-export (TREX) complex. With RP11-184M15.1 knockdown, the protein level of ALYREF decreased, and Ingenuity Pathway Analysis (IPA) of RNA-seq data of control versus RP11-184M15.1 knockdown revealed that THO complex subunit 5 homolog (THOC5), another component of the TREX complex, may be an upstream regulator. In addition, past studies have revealed that ALYREF and NXF1 are involved in nuclear export of inflammatory mRNAs and proinflammatory macrophage phenotype, respectively. With RP11-184M15.1 knockdown, there was decreased expression of inflammatory macrophage-associated genes. It may be possible that RP11-184M15.1 functions in mRNA export, along with NXF1 and ALYREF. In human translational studies, RP11-184M15.1 was found to be upregulated in macrophages in unstable human atherosclerotic plaques. Further work is needed to better understand the functions and molecular mechanism of RP11-184M15.1. In summary, we found that SIMALR may interact with HIF1α to regulate macrophage apoptosis via NTN1. Our preliminary work also revealed that RP11-184M15.1 may regulate apoptosis, mRNA stability and mRNA export in anti-inflammatory macrophages. Both lincRNAs may be upregulated in unstable human atherosclerotic plaques. By studying SIMALR and RP11-184M15.1, we were able to illustrate the importance of interrogating the functions of human-specific lincRNAs despite the lack of rodent models, and demonstrated roles in macrophage inflammation that may be relevant to human cardiovascular disease.

© 2014, American Society for Microbiology. / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19630号 / 医科博第68号 / 新制||医科||5(附属図書館) / 32666 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 松田 道行, 教授 竹内 理, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21013号 / 医博第4359号 / 新制||医||1028(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 長船 健二, 教授 瀬原 淳子, 教授 小川 修 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree Master of Science in Medicine. Johannesburg, 1995 / HLA-linked haemochromatosis is the result of an inborn error of metabolism inherited as an autosomal recessive gene, closely linked to the HLA locus on chromosome six. In this condition iron absorption is inappropriately high leading to iron overload. Integral to the pathogenesis of this disorder and in contrast to other causes of iron overload, is the relatively modest accumulation of iron within cells of both the small intestine and the reticuloendothelial system and the excessive deposition of iron in parenchymal cells of the liver and other organs. This observation has led to the suggestion that the primary defect(s) could be present in either the gut, the liver, the reticuloendothelial system or all three. Abnormalities in iron uptake by cells, iron transport through and between cells and iron storage in cells have all been suggested as possible mechanisms responsible for the abnormal absorption and distribution of iron in haemochromatosis. Malfunction of the iron transport protein transferrin or its receptor could be responsible for abnormal distribution and iron loading while an abnormality of ferritin iron storage could explain why some cells appear to be unable to store iron and others are iron overloaded. / IT2018

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