L’athérosclérose est un important facteur de risque des maladies cardiovasculaires ischémiques. Cette pathologie est caractérisée par la formation de plaques lipidiques dans l’intima des vaisseaux sanguins. Le récepteur éboueur cluster de differentiation-36 (CD36) est impliqué dans l’internalisation et l’accumulation de lipoprotéines au sein des macrophages qui vont devenir des cellules spumeuses, ce qui est à l’origine de la formation de lésions athérosclérotiques. Notre hypothèse est que les azapeptides, dérivés du peptide de relâche de l’hormone de croissance 6 (GHRP-6), vont interférer avec l’internalisation des lipoprotéines par les macrophages et ainsi réduire la progression des lésions. Nous avons utilisé des souris déficientes en apolipoprotéine E (apoe-/-) qui ont été traitées quotidiennement par une injection sous-cutanée de 300 nmol/kg de MPE-001 ou de MPE-003. Les effets des azapeptides ont été étudiés sous deux régimes alimentaires, soit le maintien d’une diète enrichie en lipides et en cholestérol (HFHC) pendant la durée de l’étude, ou par une diète HFHC suivie d’une diète normale pendant la période de traitement. Nos résultats montrent que les azapeptides ont réduit la progression des lésions à différents sites aortiques et artériels et induit leur régression au niveau des sinus aortiques du coeur. Ces effets ont été associés à une diminution de médiateurs pro-inflammatoires au niveau plasmatique et à une augmentation des marqueurs caractéristiques des macrophages anti-inflammatoires (M2). Nos travaux ont montré que l’effet athéroprotecteur des azapeptides dépend de la présence du récepteur CD36. Ces résultats appuient le développement de ligands sélectifs du récepteur CD36 dans le traitement de l’athérosclérose. / Atherosclerosis is an important risk factor for the development of ischemic heart disease. This pathology is characterized by the formation of lipid plaques in the intima of the blood vessels. The receptor cluster of differentiation 36 (CD36) is involved in the internalization and retention of lipoproteins within macrophages that will become foam cells, which induce the formation of atherosclerotic lesions. Our hypothesis is that azapeptides, derived from growth hormone release peptide-6 (GHRP-6), will interfere with the internalization of lipoproteins by macrophages and thereby reduce the progression of lesions. We used apolipoprotein E-deficient mice (apoe-/-) which were treated daily with a subcutaneous injection of 300 nmol / kg of MPE-001 or MPE-003. The effects of azapeptides were studied under two diets regimen, the mice were either maintained under a diet enriched in fat and cholesterol (HFHC) for the duration of the study or were given a HFHC diet followed by a normal diet during the treatment period. Our results show that azapeptides reduced the progression of lesions at different aortic and arterial sites and induced their regression in the aortic sinuses of the heart. These effects have been associated with a decrease in pro-inflammatory mediators in plasma and an increase in markers characteristic of anti-inflammatory macrophages (M2). Our work has shown that the atheroprotective effect of azapeptides depends on the presence of the CD36 receptor. These results support the development of selective ligands for the CD36 receptor in the treatment of atherosclerosis.

Neutrophils and macrophages accumulate at sites of inflammation and cause chronic inflammation leading to various diseases. Therefore, to better understand chronic disease pathways it is important to investigate the properties of macrophage accumulation in inflamed tissues. The I-domain of the macrophage receptor integrin aDb2 plays a vital role in macrophage retention by binding to CEP (carboxyethyl pyrrole), a ligand available at inflammatory sites. This thesis mainly focuses on evaluating the binding site within integrin aDb2 that binds carboxyethyl pyrrole (CEP)-modified proteins. So, a recombinant plasmid construct containing the integrin I-domain was developed. Seven non-conserved amino acids were mutated by PCR-site-directed mutagenesis to create a mutant construct. After expressing in E. coli, the binding affinities of wild-type and mutant I-domains to CEP were analyzed using biolayer interferometry. It was found that a patch of seven positively charged amino acids contributes to the strong binding of the I domain to CEP.

Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury and requires a complex and highly organized wound healing processes for survival. Cortical bone stem cell (CBSC) therapy has been shown to attenuate the decline in cardiac function associated with MI in both mouse and swine models. However, the cellular changes brought about by CBSC treatment and their relationship to inflammation and the wound healing process are unknown. We observed that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably Macrophage Colony Stimulating Factor (M-CSF) and Transforming Growth Factor-b, but not IL-4. Macrophages treated with CBSC medium containing these factors polarized to a hybrid M2a/M2c phenotype characterized by increased CD206 expression but not CD206 and CD163 co-expression, increased efferocytic ability, increased IL-10, TGF-b and IL-1RA secretion, and increased mitochondrial respiration in the absence of IL-4. Media from these macrophages increased proliferation and decreased a-Smooth Muscle Actin expression in fibroblasts in vitro. In addition, CBSC therapy increased macrophages, CD4+ T-cells, and fibroblasts while decreasing myocyte, macrophage, and total apoptosis in an in vivo swine model of MI. From these data, we conclude that CBSCs are modulating the immune response to MI in favor of an anti-inflammatory reparative response, ultimately reducing cell death and altering fibroblast populations resulting in smaller scar and preserved cardiac geometry and function. / Biomedical Sciences

Obesity is a disease that increases risk of developing metabolic diseases including insulin resistance (IR), metabolic syndrome (MS), and type 2 diabetes (T2D). Adipose tissue expansion during obesity leads to immune cell infiltration, causing local inflammation and disruption of lipid homeostasis. There is an association between exposure to environmental chemicals, like p,p’-DDE, a metabolite of p,p’-DDT, and diagnosis of obesity, dyslipidemia, IR, and prevalence of MS and T2D. DDE accumulates in fatty tissues and has been shown to have immunomodulatory properties, affecting macrophage and T cell populations. Potential mechanisms were studied by which DDE could modulate adipocyte and immune cell function and facilitate an increased risk of obesity and immune dysregulation, potentially through cyclooxygenase-2 (COX-2). 3T3-L1 preadipocytes and J774A.1 macrophages were studied for the effects of DDE on adipogenesis and macrophage reactivity, respectively. 3T3-L1 cells were induced to differentiate to adipocytes using a sub-optimal differentiation cocktail with increasing concentrations of DDE (0.5uM-100uM). It was determined that DDE enhanced adipogenesis in a concentration dependent manner and the expression of adipogenic and lipogenic genes, indicating that DDE enhances adipogenesis. In J774A.1 cells, the ability of DDE or 10uM NS-398, a specific COX-2 inhibitor, to inhibit the production of the prostaglandins PGE2, PGD2, PGF2a, was assessed in vitro and in a cellree system. DDE or NS-398 followed by immune challenge reduced cellular PG secretion and reduced PG production in a cell free system, indicating that DDE may interfere with lipid mediator signaling. Additionally, DDE or NS-398 exposure altered gene expression in J774A.1 cells following M1 or M2 polarization stimulus. Lastly, male C57Bl mice were exposed to 2mg/kg DDE for 5 days and the macrophage population of the adipose stromal vascular fraction was analyzed by flow cytometry. Adipose from DDE treated animals contained approximately 40% F4/80+CD11b+ macrophages. These results indicate that DDE may alter the homeostasis of adipose tissue by both enhancing adipogenesis and altering the reactivity of the resident macrophage population in a manner that may contribute to adipose dysfunction. These data suggest a possible mechanism by which DDE exposure may contribute to adiposity and adipose tissue dysfunction commonly seen in metabolic disease.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24128号 / 医博第4868号 / 新制||医||1059(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 松田 秀一, 教授 杉田 昌彦, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Overall, the work presented in this thesis explored the impact of cigarette smoke on the immunopathogenesis of respiratory disease. This thesis highlighted the determinantal impact of cigarette smoke on (auto)antibody levels and pulmonary macrophage composition. Work completed by Steven P Cass 2016-2021. / Cigarette smoke is an insidious insult that is associated with a spectrum of respiratory diseases that range from cancer to obstructive diseases such as chronic obstructive pulmonary disease (COPD), to restrictive diseases such as idiopathic pulmonary fibrosis (IPF). In this thesis, we explore how cigarette smoke impacts immune components that contribute to respiratory disease. To begin, we assessed the impact of cigarette smoke on airway antibody and autoantibody levels. We assessed sputum, a non-invasive method to sample the lower airways, to directly assess the presence of antibodies and autoantibodies in COPD. Total immunoglobulin M (IgM), IgG and IgA were detectable in the sputum of subjects. Notably, in patients with mild to moderate COPD, current smoking status was associated with decreased IgM and IgG. Next, using a comprehensive autoantigen array, we measured matched sputum and serum autoantibodies in 224 individuals. Serum autoantibodies were more abundant than sputum autoantibodies and correlated strongly between two independent COPD cohorts. Overall, the autoantibody profile of a patient with COPD was the same as a control subject. A proportion of autoantibody specificities were differentially expressed in patients with COPD with anti-tissue autoantibodies weakly associated with measures of emphysema. Taken together, these data suggested chronic cigarette smoke exposure was associated with limited differential expression of autoantibodies, but these changes were not a reliable method to identify COPD status. In our third study, we assessed the impact of cigarette smoke exposure on the composition and function of pulmonary macrophage subpopulations. Macrophages perform a central role in respiratory host defence and are implicated in the pathobiology of several respiratory diseases. Using a mouse model of cigarette smoke exposure, we reported cigarette smoke-induced expansion of CD11b+ macrophage subpopulations including monocyte-derived alveolar macrophages and interstitial macrophages. The altered pulmonary macrophage composition following cigarette smoke exposure contributed to attenuated fibrogenesis in a model of bleomycin-induced lung injury. This study offered insight to pulmonary macrophage composition and function following cigarette smoke exposure. This thesis summarises the original contributions and work completed during the course of this Ph.D., aimed at understanding the impact of cigarette smoke exposure on immune components central to respiratory disease. In conclusion, these findings shed light on the presence of (auto)antibodies in patients with COPD and the composition of macrophage subpopulations following cigarette smoke exposure. / Thesis / Doctor of Philosophy (PhD) / Currently there are 1.3 billion people who use tobacco across the world. The most common method to consume tobacco is by smoking cigarettes. Cigarette smoking is well-known to cause disease; however, smoking rates are still increasing with more daily cigarette smokers in 2012 than there were in 1980. In this thesis, we explore the impact of cigarette smoke upon the immune system. We first assessed whether cigarette smoking impacts the levels of antibodies, proteins that are produced by the immune system to protect against foreign bodies, in healthy individuals, cigarette smokers without disease and patients with chronic obstructive pulmonary disease (COPD). We found that current smokers had decreased antibodies in the airways, thus predisposing cigarette smokers to increased damage. In our second study, we measured the presence of airway and blood autoantibodies. These are antibodies that target self and have the potential to inflict damage. We discovered that patients with COPD had minor changes in autoantibodies and these changes were weakly associated with emphysema. In our third study, we evaluated the impact of cigarette smoke on lung macrophages, cells that eat and destroy foreign bodies, in a mouse model of cigarette smoke exposure. Cigarette smoke increased the number of bone marrow-derived macrophages and this change in macrophage populations was associated with a reduced wound healing ability. Overall, these studies offer insight into how cigarette smoke impairs the function of the immune system and contributes to lung disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Pneumocystis pneumonia (PCP) is the leading opportunistic disease in immunocompromised individuals, particularly in AIDS patients. The alveolar macrophage (AM) is the major type of cell responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PCP due to increased intracellular polyamine levels. This study examined the mechanism of this polyamine mediated apoptosis and investigated an alternative therapy for PCP by targeting this mechanism. The elevated polyamine levels were determined to be caused by increased polyamine synthesis and uptake. Increased polyamine uptake was found to be AM-specific, and recruited inflammatory cells including monocytes, B cells, and CD8+ T cells were found to be a potential source of polyamines. The expression of the antizyme inhibitor (AZI), which regulates both polyamine synthesis and uptake, was found to be greatly up-regulated in AMs during PCP. AZI overexpression was confirmed to be the cause of increased polyamine synthesis and uptake and apoptosis of AMs during PCP by gene knockdown assays. Pneumocystis organisms and zymosan were found to induce AZI overexpression in AMs, suggesting that the β-glucan of the Pneumocystis cell wall is responsible for this AZI up-regulation. In addition, levels of mRNA, protein, and activity of polyamine oxidase (PAO) were also found to be increased in AMs during PCP, and its substrates N1-acetylspermidine and N1-acetylspermine were found to induce its up-regulation. These results indicate that the H2O2 generated during PAO-mediated polyamine catabolism caused AMs to undergo apoptosis. Since increased polyamine uptake was demonstrated to be a pathogenic mechanism of PCP in this study, the potential therapeutic activity of five putative polyamine transport inhibitors against PCP was tested. Results showed that compound 44-Ant-44 significantly decreased pulmonary inflammation, organism burden, and macrophage apoptosis, and prolonged the survival of rats with PCP. In summary, this study demonstrated that Pneumocystis organisms induce AZI overexpression, leading to increased polyamine synthesis, uptake, and apoptosis rate in AMs and that targeting polyamine transport is a viable therapeutic approach against PCP.

Masters of Science / The infants of women who smoke during pregnancy have a lower birth mass than those born of women who abstain. Animal studies reveal that reduced growth due to maternal nicotine exposure during gestation is accompanied by lung hypoplasia. Biochemical analysis suggests that these lungs contain more cells which implies that lung damage occurs. In this study we examined the in vivo effects of maternal nicotine exposure (lmg/Kg/day), the equivalent of 32 cigarettes per day, on the following parameters of fetal and neonatal Wistar rat lung:(i) the content and distribution of glycogen in fetal and neonatal lung (ii) the status of connective tissue in neonatal lung (iii) the cell composition of the alveoli in neonatal lung. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Fetal rat lungs of ages 17, 18, 19 and 20 days and neonatal lungs of 1, 7, 14 and 21 day old pups were used. Light microscope techniques and special stains were used to investigate glycogen, connective tissue, macrophage numbers and morphological status of the lungs. Transmission electron microscope (TEM) techniques were employed to investigate the characteristics and composition of the alveolus The results show clearly that maternal nicotine exposure elevates pulmonary alveolar macrophage numbers'(PAM's) and lung glycogen levels. The quantity of elastic fibres in 1 day old neonates was significantly reduced but no changes in the quantity of reticulin and collagen fibres was observed. As a result of this change in connective tissue status, emphysema-like lesions and alveolar collapse was evident in the lungs of nicotine-exposed pups. TEM investigations revealed that changes to the composition of alveoli occurred. These included increased numbers of type II pneumocytes with high numbers of lamellar bodies with degenerative changes. Thickening of the blood-air barrier was also observed. The effect of maternal nicotine exposure has been documented in this study. However, it has not been possible to pinpoint the mechanisms involved but explanations have been proposed. Further research is required to elucidate the mechanisms by which nicotine produces these effects. Information thus obtained could help prevent the harmful effects to the fetus and neonate caused by smoking during pregnancy.

No description available.

The presence of apoptotic cells in atherosclerotic lesions has been broadly reported in the past ten years. The majority of these apoptotic cells are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains to be elucidated. Elevated expression of Bax, one of the pivotal pro-apoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol treated mouse macrophages. In this study, we evaluated the influence of Bax deficiency on apoptosis in macrophage-like P388D1 cells by using small interfering RNA (siRNA) to suppress Bax expression, as well as in peritoneal macrophages isolated from Bax null mice (Bax-/-). Apoptotic activities in both cell types deficient for Bax were significantly reduced compared to that in control cells. To examine the effect of macrophage Bax deficiency on the development of atherosclerosis, fourteen 8-week-old male LDL-receptor null (LDLR-/-) mice were lethally irradiated and reconstituted with either wild type (WT) C57BL6 or Bax-/- bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax-/- group. However, quantitation of cross sections from proximal aortas revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax-/- group compared to the WT group. A 53% decrease in apoptotic macrophages in the Bax-/- group was found by TUNEL staining (P<0.05). In conclusion, Bax deficiency produces a reduction of apoptotic activity in macrophages and is associated with the accelerated atherosclerosis in LDLR null mice fed a Western diet. These results strongly support our hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.