Atherosclerosis is an inflammatory disease of the cardiovascular system. Discoidin domain receptor 1 is a receptor tyrosine kinase that binds collagens. Previous work in our lab has shown that deleting DDR1 in a mouse model results in attenuation of atherosclerosis, with fewer macrophages in the plaque. The aim of this study was to determine what changes in macrophage behaviour due to the lack of DDR1 was attenuating plaque development. In order to carry out experiments, primary mouse peritoneal macrophages were used. DDR1-deficient macrophages adhered significantly less to type IV collagen and fibronectin compared to DDR1-expressing cells. In addition, when plated on type IV collagen and fibronectin, DDR1-deficient macrophages produced decreased levels of MCP-1 protein, a cytokine known to be important in plaque development, particularly in leukocyte recruitment to plaque. These results suggest that DDR1 is an important mediator in macrophage adhesion to matrix and macrophage cytokine production

Lactobacillus plantarum is known to reduce the inflammatory response of macrophages in vitro and decrease inflammation in vivo during colitis. A predominance of M2, anti-inflammatory, macrophages correlates with a reduced severity of colitis. The purpose of this study was to determine if L. plantarum-conditioned media is able to produce an anti-inflammatory macrophage response during Salmonella-induced colitis while also reducing inflammation. Female C57BL/6 mice were infected with Salmonella after streptomycin pretreatment, then gavaged with L. plantarum-conditioned media (or a control) four hours prior to infection and twenty-four hours post-infection, they were sacrificed forty-eight hours post-infection. Samples of the intestines, blood, peritoneal exudate macrophages, spleen derived macrophages, and bone marrow-derived macrophages were collected. L. plantarum-conditioned media was found to limit inflammation in a dose related manner. Inflammation was measured by cecum histology, myeloperoxidase activity in the intestine, and monocyte chemoattractant protein-1 levels in the blood. IκB-α levels in the intestinal epithelium were measured by Western blot and degradation was reduced by L. plantarum-conditioned media treatment of Salmonella infected mice. Macrophages from L. plantarum-conditioned media treated Salmonella infected mice were found to have an M2 phenotype that was not found in any other treatment group. The phenotype markers arginase-1 and Ym-1 were found to be elevated in L. plantarum-conditioned media treated Salmonella infected mice by Western blot, while Src homology 2-containing inositol phosphatase-1 was reduced. Flow cytometry for the M2 markers CD206 and CD14 along with the M1 markers CD16 and CCR7 showed a similar M2 phenotype shift of macrophages from L. plantarum-conditioned media treated Salmonella infected mice. The cytokine profile of macrophages from L. plantarum-conditioned media treated Salmonella infected mice was anti-inflammatory with elevated IL-10 and decreased IL-6, IL-12, and TNF-α supporting the M2 phenotype. The protective effects of L. plantarum-conditioned media were found to be at least partially macrophage dependent in a macrophage transfer experiment. In vitro, L. plantarum-conditioned media was also found to produce M2 phenotype macrophages but have no effect on phagocytic or bactericidal function. In conclusion, L. plantarum-conditioned media provides a novel means of producing an anti-inflammatory immune response during Salmonella infection without compromising the host’s ability to combat infection. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-09-29 10:07:20.666

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.

Pulmonary intravascular macrophages (PIMs) promote lung inflammation and are found in ruminants, horses, pigs, cats, and dolphins, but not in primates, rats and mice. Rabbits are used to study mechanisms of lung inflammation in humans, but disagreement exists whether rabbits have PIMs. This study examined rabbits for PIMs, and their influence on endotoxin-induced lung inflammation.<p> Rabbits were treated with gadolinium chloride (10 mg/kg intravenous: Group GC, n=6) to produce apoptosis in PIMs, or with saline (Group SAL, n=6). Rabbits were euthanized 48 hours later. Light microscopic examination of epoxy-embedded rabbit lung sections revealed mononuclear phagocytes in alveolar septa. Transmission electron microscopy confirmed PIMs with lysosomes and close attachment to capillary endothelium. Light microscopic immuno-cytochemistry using rabbit anti-macrophage antibody (RAM-11) showed staining of septal and alveolar macrophages. There was no difference in number of RAM-11 positive septal cells between SAL and GC rabbits (P=0.2).<p> Rabbits were administered intravenous E.coli 0127:B8 endotoxin (100 Ýg/kg) 48 hours after GC (GC-LPS; n=5) or SAL treatment (SAL-LPS; n=6), and euthanized 24 hours later. Rabbits in both LPS treated groups were hypocalcaemic and exhibited compensated metabolic acidosis compared to SAL rabbits. Four rabbits died in the SAL-LPS group within 24 hours of the endotoxin treatment and were replaced. None died in the GC-LPS group (Chi-square comparison for survival P=0.063). Greater numbers of septal heterophils were found in groups SAL-LPS and GC-LPS compared to SAL and GC. TNFÑ protein in serum, and IL-1Ò and IL-6 mRNA in lung tissues were increased in SAL-LPS compared to SAL and GC rabbits. Lung tissues from SAL-LPS rabbits but not in GC-LPS showed moderate inflammation, but lung wet/dry ratios were not different. Lung tissue TNFÑ, IL-1Ònand IL-6 mRNA, myeloperoxidase activity, and serum TNFÑ were reduced in GC-LPS animals compared to SAL-LPS. Immuno-electron microscopy revealed TNFÑ in PIMs in normal and LPS-treated rabbits. Lung and liver tissue TNFÑ, IL-8 and MCP-1 protein concentrations were not different between groups. GC did not appear to reduce liver inflammation. These data show that rabbits have low numbers of PIMs. GC treatment induced apoptosis in PIMs and reduced endotoxin-induced lung inflammation and mortality.

Macrophages play a fundamental role in driving both inflammatory and immunosuppressive conditions of the oral mucosa. Periodontitis, a chronic inflammatory condition affecting the supporting structures of the teeth, is widely prevalent, affecting a large proportion of the global population, and has been linked to the development of systemic inflammatory diseases. Oral squamous cell carcinoma (OSCC) is placed sixth in the WHO rankings of cancer incidence worldwide, and despite continuing research into underlying mechanisms, incidence is on the rise. Aberrant macrophage function has been implicated in the pathogenesis of both diseases. On recruitment to sites of inflammation, macrophages become polarised within a spectrum of effector phenotypes depending on the factors they encounter in their microenvironment. These cells are highly plastic and continuously adapt their effector functions in response to locally derived stimuli. Mechanisms have been developed by pathogenic bacteria and transformed host tissues to exploit this plasticity and manipulate macrophage phenotype to facilitate disease progression. However, this plasticity is also available for therapeutic manipulation. The main objectives of this study therefore were to investigate the interactions between macrophages and pathogenic stimuli in the context of oral pathology with a view to identifying novel therapeutic targets. Firstly, a reproducible model of M1 and M2 macrophage polarisation using the THP-1 cell line was established to study their interactions with pathogenic stimuli. Treating the cells with combinations of PMA plus IFNγ or IL-4 for 24 hours led to two distinct populations of cells: PMA + IFNγ treated cells expressed higher levels of pro-inflammatory cytokines TNFα, IL-1β and IL-6, but lower levels of IL-10 and TGF-β, characteristic of M1 macrophages. PMA + IL-4 treated cells expressed lower levels of TNFα, IL-1β and IL-6 and higher levels of IL-10 and TGF-β, characteristic of M2 macrophages. As P. gingivalis LPS is present in the developing periodontal lesion, cytokine expression from macrophages exposed to LPS during polarisation was investigated. Exposure of macrophages to 1 μg/ml Pg LPS during polarisation led to a statistically significant down-regulation of inflammatory cytokines TNFα (10-, 4- and 5.5 –fold decrease in PMA, M1 and M2 cells, respectively) and IL-1β (1.9-, 2.0- and 1.5 –fold decrease in PMA, M1 and M2 macrophages, respectively) in response to subsequent stimulation with LPS. IL-6 production was not affected. The same pattern of cytokine down regulation was observed regardless of LPS species used, and in most cases, at a lower dose of 1 ng/ml LPS during polarisation. Finally, as macrophages recruited to the tumour environment will be influenced by tumour-secreted factors, the response of macrophages to LPS stimulation in the presence of OSCC conditioned media was examined. Contrariwise to polarisation with LPS, exposure of macrophages to OSCC produced factors during polarisation led to an amplification of IL-1β (13.8-, 2.3- and 8.8 –fold increase in PMA, M1 and M2 cells, respectively), and IL-6 (16.8-, 17.3- and 44.9 –fold increase in PMA, M1 and M2 cells, respectively), but not TNFα in response to LPS. Counter intuitively, these findings suggest that LPS manipulation of macrophage polarisation might result in a more M2 –like population of cells, whereas OSCC produced factors may result in a more M1- like population of cells. Viewed therapeutically, one short, single exposure of macrophages to LPS would up-regulate pro-inflammatory cytokines, whereas prolonged or chronic exposure would lead to the down-regulation of pro-inflammatory cytokines, therefore, LPS as a therapeutic modulator of macrophage function in an immunosuppressive (M2) environment to an inflammatory environment (M1) would only be viable as a single dose. For chronic inflammatory disease however, a repasted or prolonged exposure of macrophages to LPS skews macrophages to display a more M2-like cytokine profile and could dampen down detrimental pro-inflammatory cytokine production. The continued study of macrophage/ P. gingivalis interactions may shed light on pathogenic mechanisms not only in oral pathological conditions, but in a range of diseases.

No description available.

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.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Acute liver failure confers a high risk of death, with liver transplantation offering the only effective therapy for life-threatening cases. Hepatic macrophages are crucial for innate immune integrity and effective hepatocyte proliferation. The macrophage may therefore present a novel therapeutic target to enhance regeneration following acute liver injury. In this thesis I describe the development and use of mouse models of liver injury including partial hepatectomy, partial hepatectomy plus chronic liver injury and paracetamol intoxication. I show the development of liver function assays in these models including quantification of hepatic clearance of indocyanine green by fluorescent imaging and assessment of hepatic phagocytic capacity using fluorescent microbeads. I then describe macrophage based therapeutic interventions in mouse models of liver injury. Firstly the direct administration of bone marrow derived macrophages in partial hepatectomy plus chronic liver injury. I then tested the administration of macrophage colony stimulating factor in mouse models of partial hepatectomy, partial hepatectomy plus chronic liver injury and paracetamol intoxication, describing the phenotype and exploring mechanisms of action. Collaborating with others I assessed serum CSF1 levels in humans with liver injury due to partial hepatectomy or paracetamol intoxication. I show that in acute liver failure a high serum CSF1 level is predictive of survival, indicating a new mechanistic biomarker.