The invasion of trophoblast cells into the uterine wall is an essential component of normal human pregnancy. These trophoblast cells transform the uterine spiral arterioles into high-flow, low-resistance vessels that supply the placenta to support fetal growth and development. Inadequate trophoblast invasion and spiral arteriole remodelling may result in excessive placental pathology leading to pre-eclampsia and intra-uterine growth restriction (IUGR), which are major causes of maternal and fetal morbidity and mortality. These pregnancy complications have also been linked to an increased presence of pro-inflammatory cytokine-secreting (activated) macrophages at the fetal-maternal interface. In particular, increased production of tumour necrosis factor-alpha (TNF) by activated macrophages has been implicated as a causative factor mediating various pregnancy complications. Results from this thesis showed that macrophage-derived TNF decreased the invasiveness of trophoblast cells, primarily by affecting the urokinase system of plasminogen activators, a network of proteases that promotes cellular invasion. TNF also stimulated the production of macrophage chemoattractants by trophoblast cells, providing a putative mechanism for the aberrant recruitment and localization of macrophages in complicated pregnancies. Administration of lipopolysaccharide (LPS), a potent stimulator of macrophage activation and TNF production, to pregnant rats resulted in IUGR and fetal death correlating with significant placental pathology, including displaced endovascular trophoblast cells and increased fibrinoid and macrophage accumulation at the fetal-maternal interface. The immunoregulatory cytokine interleukin-10, which inhibited TNF production from macrophages after LPS-exposure, completely prevented the adverse effects of TNF in vitro and in vivo. Collectively, these findings show that the aberrant presence and localization of TNF-secreting macrophages may be involved in the etiology and pathophysiology of various pregnancy-related complications. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2008-09-29 16:32:00.845

Long thought to be irreversible, it is now clear that liver fibrogenesis is a dynamic process, with scar tissue capable of being remodelled as well as deposited. Macrophages have been shown to have a critical role in both liver fibrogenesis and fibrosis resolution. Whilst previous work has identified a Ly-6Chi hepatic macrophage population, derived from recruitment of inflammatory monocytes, as being the main pro-fibrogenic population, the nature and phenotype of the pro-resolution macrophage subset is unknown. In this thesis, I sought to identify and characterise this restorative hepatic macrophage. I established a reversible murine model of liver fibrosis using CCl4. At the time of initiation of fibrosis regression, Ly-6Clo CD11bhi F4/80int hepatic macrophages represented the most numerous macrophage population and the principal expresser of matrix degrading MMP enzymes. Depletion of this population in CD11b-diphtheria toxin (DTR) mice prevented fibrosis resolution. Subsequent, adoptive transfer and in situ labelling experiments, demonstrated that this restorative macrophage population derives from inflammatory monocytes, a common origin to the pro-fibrotic Ly-6Chi hepatic macrophage subset, indicating a switch in macrophage phenotype in situ to form the restorative phenotype. Characterisation of FACS-sorted restorative and pro-fibrogenic liver macrophage subsets using gene expression profiling demonstrated higher expression of pro-resolution genes and lower expression of pro-fibrotic genes in restorative macrophages, which also upregulated a number of genes involved in phagocytosis. Confocal microscopy confirmed that restorative macrophages showed evidence of prior phagocytosis. This could be replicated in vitro, where feeding macrophages with cellular debris resulted in matrix-degrading properties analogous to those seen in vivo, which was dependent on activation of the ERK signalling cascade. This effect was also demonstrated with the phagocytosis of liposomes in vitro. Finally, the administration of liposomes to CCl4-injured mice in vivo induced phagocytosis, causing an increase in hepatic restorative macrophage number and accelerating fibrosis regression. Hence, I have been able to identify and characterise the restorative hepatic macrophage and have utilised these data to develop a novel method to alter macrophage phenotype in vivo and accelerate the resolution of liver fibrosis and restoration of normal tissue architecture.

Injuries to the peripheral nervous system (PNS) are major and common source of disability, impairing the ability to move muscles and/or feel normal sensations, or resulting in painful neuropathies. Annually traumatic nerve injuries resulting from collisions, gunshot wounds, fractures, motor vehicle accidents, lacerations, and other forms of penetrating trauma, affected more than 250,000 patients just in the U.S. The clinical gold standard to bridge long non-healing nerve gaps is to use a nerve autograft- typically the patient’s own sural nerve. However, autografts are not ideal because of the need for secondary surgery to ‘source’ the nerve, loss of function at the donor site, lack of appropriate source nerve in diabetic patients, neuroma formation, and the need for multiple graft segments. Despite our best efforts, finding alternative ‘nerve bridges’ for peripheral nerve repair remains challenging – of the four nerve ‘tubes’ FDA approved for use in the clinic, none is typically used to bridge gaps longer than 10 mm due to poor outcomes. Hence, there is a compelling need to design alternatives that match or exceed the performance of autografts across critically sized nerve gaps. Here we demonstrate that early modulation of innate immune response at the site of peripheral nerve injury inside biomaterials-based conduit can favorably bias the endogenous regenerative potential after injury that obviates the need for the downstream modulation of multiple factors and has significant implications for the treatment of long peripheral nerve gaps. Moreover, our study strongly suggests that more than the extent of macrophage presence, their specific phenotype at the site of injury influence the regenerative outcomes. This research will advance our knowledge regarding peripheral nerve regeneration, and help developing technologies that are likely to improve clinical outcomes after peripheral nerve injury. The significant results presented here are complementary to a growing body of evidence showing the direct correlation between macrophage phenotype and the regeneration outcome of injured tissues.

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The biology of cells of the mononuclear phagocyte system has been studied extensively in the mouse. Studies of the pig as an experimental model have commonly been consigned to specialist animal science journals. This thesis considered some of the many ways that pigs may address the shortcomings of mice as models for the study of macrophage differentiation and activation in vitro, and the biology of sepsis and other pathologies in the living animal. Flow cytometry was used initially to phenotype cells from the porcine lung, peritoneal cavity, blood and bone marrow using the LPS receptor CD14 and the FC receptor CD16, markers frequently employed to differentiate human monocytes into subsets. The expression of SIRP-alpha (SWC3a, CD172a), which is present on all cells of myeloid origin, and the haemoglobin scavenger receptor, CD163 which has previously been used to study monocyte differentiation in the pig was also studied. The findings validated previous work where blood monocytes were divided into subsets on the expression of CD14 and CD163. Furthermore, like human and mouse, pig monocytes also exhibited variation in CD16 expression, having a subset which was CD14hiCD16lo and another which was CD14loCD16hi. A whole genome approach was then used to study the differences between the monocyte subsets in the pig, using monocytes sorted into two populations based on the expression of CD14 and CD163. The gene expression profiles obtained were then compared to publically available data from monocyte subsets in human and mouse. This thesis also investigated the expression of genes that are known to be differentially expressed between human and mouse. To do this gene expression in porcine bone marrow derived macrophages was analyzed across an LPS time course. Like human macrophages, pig macrophages did not induce nitric oxide nor any arginine metabolizing genes in response to LPS. Instead they responded with robust induction of indoleamine 2,3-dioxygenase (IDO) and other enzymes of the tryptophan metabolism pathway such as kynurenine hydroxylase, kynureninase and tryptophan-tRNA synthetase. The tryptophan metabolism pathway has been implicated in sepsis in man and the absence of this pathway in the mouse may be one of the reasons why an adequate rodent model of sepsis has not been developed. The IDO inhibitor 1-methyl-tryptophan (1-MT) has been used to treat mouse macrophages where it had a protective effect after LPS administration. Similar experiments on pig macrophages did not show the same protective effect and induction of key immune genes was increased after treatment with 1-MT suggesting IDO is involved in feedback control of the immune system. With the completion of the genome sequence and the characterisation of many key regulators and markers, the pig has emerged as a tractable model of human innate immunity and disease that should address the limited predictive value of rodents in preclinical studies. This project aimed to address the gap in our knowledge of the control of innate immunity in the pig and provided further evidence that the pig can function as an ideal model to study innate immunity.

Introduction: The uterine endometrium is a dynamic tissue that undergoes cycles of proliferation, differentiation breakdown and repair in response to fluctuations in the ovarian-derived sex steroids oestrogen (E) and progesterone (P4). During the P4- dominated secretory phase there is an influx of leukocytes which further increases during the menstrual phase. The second most populous leukocyte within the endometrium is the macrophage. Previous studies have postulated a role for the macrophage in breakdown and repair occurring during the menstrual phase associated with production of MMPs and both pro- and anti-inflammatory cytokines. However the mechanisms responsible for the impact of the endometrial subpopulation on endometrial function remain poorly understood. The specific aims of the studies described in this thesis were to examine: 1) the phenotype of the endometrial macrophage during the different phases of the menstrual cycle. 2) the relationship, if any, between the macrophage and endometrial vasculature and 3) whether platelet factor 4 may be one of the factors involved in the interplay between macrophages and other endometrial cells (endothelium, stroma). Methods/Results: Transmission electron microscopy (TEM) identified macrophages carrying out phagocytosis at all stages of the menstrual cycle. Single and double immunocytochemistry revealed that macrophages were proliferating (Ki67+, PH3+) during the late secretory and menstrual phases; TEM and immunohistochemistry identified macrophages in close proximity to the endometrial vasculature throughout the menstrual cycle. Macrophages were immunopositive for glucocorticoid receptor (GR). They expressed anti-angiogenic factors (ANG-2, THBS-1, TWEAK) during the second half of the menstrual cycle preceding endometrial breakdown and pro-angiogenic factors (ANG-1, CTGF, IL-8) during phases characterised by repair processes. When endometrial endothelial capillary networks were incubated in vitro with peripheral monocyte derived macrophages (PMDM) that were incubated in media promoting differentiation into proinflammatory or anti-inflammatory macrophages ‘breakdown’ of capillaries was observed. Further studies revealed media from cortisol(F)-exposed PMDM significantly upregulated expression of pro-angiogenic factors (CTGF, IL-8 and VEGFC) by human endometrial endothelial cells (HEEC) When human endometrial stromal cells (hESC) were incubated with media conditioned by P4-exposed PMDM they increased expression of the anti-angiogenic factor IL-12a and ANGPTL4, a pro-angiogenic factor reported to be upregulated by hypoxia. Notably expression of platelet factor 4 (PF4) by hESC was significantly upregulated by incubation with media from GM-CSF and IFNγ-treated, E-treated or P4-treated PMDM. Further research into the role of PF4 within the human endometrium found the protein increased as the cycle progressed from the proliferative to the menstrual phase, when levels were maximal. Expression was co-localised with CD68, the macrophage marker, throughout the menstrual cycle, as well as in other endometrial cell types. PF4 was significantly increased within HEECs after treatment with F and hESCs after experiencing “P4 withdrawal” (treatment of P4 and cAMP for 6 days then washed and incubated with treatment-free media for 48 hours). Both conditions occur locally within the endometrium during menstruation. Interestingly, PF4 was found to be chemotactic to macrophages that had been exposed to F. Addition of PF4 to HEEC capillary networks resulted in a significant breakdown of the network; PF4 was found to downregulate bFGF RNA expression within HEECs and hESCS. MMP-1 RNA expression was also downregulated by PF4 after P4 withdrawal within HEECs and hESCs. MMP-3 was downregulated by PF4 within F-primed hESCs. This regulation of bFGF indicates a role in anti-angiogenesis; inhibition of MMP-1 and MMP-3 in these culture conditions proposes a role for PF4 in the downregulation of MMPs at menstruation in order enable regrowth of the endometrium and the start of the next menstrual cycle. Conclusions: These data shed new insights into the importance of macrophages in regulating key events in endometrial tissue function during the normal cycle with strong evidence they play a key role in regulating the vasculature during the breakdown and repair of endometrium at menstruation. Notably, new evidence suggesting PF4 may control expression of genes encoding MMP-1 and MMP-3 during menstruation. Further study on the crosstalk between tissue resident populations of macrophages found within the endometrium and other endometrial cell types may provide novel targets for therapies for reproductive disorders associated with inflammation and aberrant angiogenesis including heavy menstrual bleeding and endometriosis.

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.

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