Studies of aortic plaque reveal the presence of tissue macrophages filled with cholesteryl esters. To study lipoprotein metabolism of in vivo, maturated human macrophages, I isolated cells from human peritoneal effluent. Population analysis using cytochemistry showed substantial numbers of acid-esterase positive monocytic cells, lymphocytes, leukocytes and erythrocytes. Substantial variation in cell populations existed among patients. Human peritoneal cells degraded low density lipoproteins (LDL) and acetylated LDL (AcLDL) by high affinity, receptor-mediated processes. AcLDL degradation saturated at 15 ug protein/ml and LDL degradation saturated at 11 ug protein/ml. Positive correlation of the percentages of monocytic cells with the degradation values (LDL, r =.710; AcLDL, r =.725) and a degradation assay using cells isolated by Lymphoprep showed that the monocytic cells substantially contributed to the degradation of LDL. AcLDL degradation was calcium independent and inhibited by fucoidin. LDL degradation was calcium dependent and very low density lipoprotein and apoE-containing high density lipoprotein (HDL) competed with LDL for receptor uptake; apoE-free HDL, AcLDL and fucoidin did not reduce LDL degradation. Both receptors were pronase-sensitive and degradation was dependent upon lysosomal activity. ACAT activity analysis showed that pre-incubation of cells with LDL or AcLDL stimulated ACAT activity. ACAT activity was greatest for cells preincubated using AcLDL and fresh medium was necessary to maintain the ACAT activity values beyond 24 hrs. LDL-stimulated ACAT activity declined as time was increased above 24 hrs. Flow cytometry analysis of a total cell population and the Lymphoprep-isolated cells revealed a heterogenous T cell population, the presence of monocyte/macrophages, suggested that some of the cells present were activated and confirmed cytochemistry analysis demonstrating that Lymphoprep concentrated the mononuclear cells. Human peritoneal macrophages formed foam cells when incubated in the presence of AcLDL or LDL for 72 hrs. The formation of foam cells in the presence of LDL was dependent upon cell exposure time to the medium. Foam cell formation in the presence of LDL was accompanied by dense vacuolization and in the demonstrated absence of the oxidation of LDL the oil red O stainable material collected outside the vacuoles.

No description available.

No description available.

No description available.

African trypanosomiasis remains a major health problem to both humans and animals due to lack of effective treatment or vaccine to control the disease. Animal trypanosomiasis is considered one of the most important diseases affecting livestock production and agricultural development in sub-Saharan Africa. Although the use of trypanocides remain the most important method for controlling the disease in animals, the mechanisms of action of these compounds are not completely known. The overall aim of this thesis is to decipher the molecular mechanisms involved in Trypanosoma congolense-induced cytokine production and how this is modulated by the trypanocide, Diminazene aceturate (Berenil). First, I investigated the molecular and biochemical mechanisms of action of Berenil to determine whether in addition to trypanolytic effect, it exerts a modulatory effect on the host immune system. Although it is known that T. congolense infection in mice is associated with increased production of pro-inflammatory cytokines by macrophages, the intracellular signaling pathways leading to the production of these cytokines remain unknown. Therefore, I investigated the innate receptors and intracellular signaling pathways that are involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. Next I further determined whether the inhibitory effect of Berenil on proinflammatory cytokine production in macrophages is specific to T. congolense. I found that Berenil treatment significantly reduced the immune activation and proinflammatory cytokine production in infected mice suggesting that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite. Next, I show that T. congolense induced pro-inflammatory cytokine production in macrophages is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) and signal transducer and activation of transcription (STAT) proteins in a TLR2-dependent manner. I further show that Berenil treatment downregulates T. congolense as well as LPS induced cytokine production by affecting the phosphorylation of MAPK and STAT proteins. Collectively, the results from this thesis provide novel insights into T. congolense-induced activation of the innate immune system and modulation of host immune response by Berenil. These findings are significant and could help in developing newer and better therapeutic strategies against the disease, in particular, and inflammatory responses in general. / October 2016

Macrophages are dynamic, plastic, and heterogeneous immune cells that play an important role in host immune defense against bacterial infection. Various bacterial pathogens, such as Neisseria meningitidis and Mycobacterium tuberculosis, can modulate host immune responses by interfering with macrophage differentiation and polarization. The focus of this thesis was to understand the role of macrophages in the pathogenesis of bacteria-induced diseases, which has important implications in the search for novel therapeutic strategies to control those infectious diseases. In Paper I, we found that NhhA, a conserved meningococcal outer membrane protein, can activate macrophages through both Toll-like receptor 4 (TLR4)-dependent and -independent pathways. In Paper II, we demonstrated that NhhA activates monocytes through TLR2 and triggers autocrine IL-10 and TNF production through the ERK and JNK pathways, which skew monocyte differentiation into CD200Rhi macrophages. These immune homeostatic macrophages are associated with nasopharyngeal carriage of meningococci. In Paper III, we examined the role of human CD46, a ubiquitous transmembrane protein, in regulating macrophage apoptosis, differentiation, and functional polarization. We revealed that macrophages expressing CD46 exhibit an M1 phenotype and are prone to generate proinflammatory cytokines, such as IL-6, TNF, and IL-12, upon lipopolysaccharide challenge or meningococcal infection. The important role of these macrophages in the development of septic shock was further confirmed by in vivo studies using a CD46 transgenic mouse disease model. M. tuberculosis, a gram-positive bacterium, remains an important cause of death in developing countries. In Paper IV, we reported that murine macrophages expressing human CD46 exhibit enhanced viability and bactericidal capacity and are prone to form granulomas following chronic mycobacterial infection. Increased understanding of host factor roles in the physiopathology of tuberculosis is critical for the design of effective vaccines and new drugs. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Recent studies have proposed that non-nitrogen-containing and nitrogen- containing bisphosphonate drugs inhibit osteoclastic bone resorption by different molecular mechanisms. The aim of this thesis was to investigate the molecular mechanisms of action of bisphosphonates in macrophages and osteoclasts and, in particular, the activation of caspase proteases and their role in apoptotic cell death. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates was found to involve the activation of caspase-3. By contrast, non-nitrogen- containing bisphosphonates did not cause caspase activation or J774 apoptosis, indicating that these bisphosphonates have different cellular effects. Further studies demonstrated that nitrogen-containing bisphosphonates induced J774 macrophage apoptosis by inhibiting the mevalonate pathway and preventing protein farnesylation and/or geranylgeranylation, since these compounds inhibited incorporation of [14 C] mevalonate into isoprenylated proteins, and addition of cell-permeable intermediates of the mevalonate pathway (FPP and GGPP) prevented bisphosphonate-induced apoptosis. Apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates or mevastatin (another inhibitor of the mevalonate pathway) was dependent on protein synthesis, since cycloheximide effectively prevented the activation of caspase-3 and prevented J774 cell apoptosis. Both nitrogen-containing bisphosphonates and non-nitrogen-containing bisphosphonates caused caspase-3 activation and apoptosis of rabbit and human osteoclasts in vitro. The active form of caspase-3 was detected in apoptotic osteoclasts by immunofluorescence staining, whilst caspase-3 activity was visualised in osteoclasts using a cell-permeable, fluorogenic substrate and detected in cell lysates using caspase-specific substrates. Bisphosphonate-induced osteoclast apoptosis involved loss of mitochondrial membrane potential and could be prevented by a specific inhibitor of caspase-3/-7. The ability of bisphosphonates to activate caspase-3 and cause apoptosis was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, suggesting that caspase activation and apoptosis in osteoclasts induced by bisphosphonates is the consequence of loss of geranylgeranylated proteins. Bisphosphonate-induced osteoclast apoptosis and inhibition of bone resorption in vitro was suppressed by RANK ligand. This did notappear to involve changes in Akt phosphorylation or increased expression of cIAP-1 or cIAP-2. These studies have helped to identify the molecular mechanisms of action of bisphosphonate drugs and have provided new insights into the involvement of caspases in osteoclast apoptosis.

Macrophages are key cells during inflammation and repair. Their activity is highly varied and requires precise regulation. The characterisation of cues coordinating macrophage functions has focussed on chemical and biological soluble mediators. Little is known about their responses to physical stimuli, in particular electric fields (EF) that are generated naturally in wounded tissue and infected tissue. Importantly, EFs are known to accelerate wound healing and limit infection but the mechanisms of this remain poorly understood. To address this gap in understanding, this study tested how key properties of human monocyte-derived macrophages are regulated by applied EFs equivalent to physiological EF strengths generated naturally. Using live-cell video microscopy, we show macrophage migration is directed anodally by EFs as low as 5 mV/mm and is EF-strength dependent, with effects peaking around 300 mV/mm. In contrast, monocytes, as macrophage-precursors, migrate in the opposite, cathodal direction. Strikingly, we show for the first time that EFs significantly enhance macrophage phagocytic uptake of a variety of targets, including carboxylate beads, apoptotic neutrophils and the nominal opportunist pathogen Candida albicans, all of which engage different classes of surface receptors. These EF-induced functional changes are accompanied by clustering of phagocytic receptors, enhanced PI3K and ERK activation, mobilization of intracellular calcium and actin polarization. EFs also selectively modulate cytokine production and augment effects of conventional polarising stimuli on cytokine secretion. Taken together, electrical signals have been identified as major contributors to the co-ordination and regulation of important human macrophage functions, including those essential for microbial clearance and healing. Our results open up a new area of research into effects of naturally occurring and clinically-applied EFs in conditions where macrophage activity is crucial.

Chemokine-induced macrophage recruitment into the vascular wall is an early pathological event in the progression of atherosclerosis. Macrophage activation and chemotaxis during cell recruitment are mediated by chemokine ligation of multiple G- protein coupled receptors. The Regulator of G-Protein Signalling-l (RGS-l) acts to down-regulate the response to sustained chemokine stimulation. Studies in this laboratory have shown Rgsl is up-regulated in atherosclerotic ApoE1- mice in association with atherosclerotic plaque progression and published findings have reported that RGS 1 is highly expressed in leukocytes. However an in vivo role for RGS-l in macrophage function or in atherosclerosis has not been investigated. This thesis aimed to address the hypothesis that RGS 1 has an important role in atherosclerosis and modulates the inflammatory response by controlling chemokine signalling and macrophage chemotaxis to atherosclerotic plaques. To investigate the role of RGS 1 in macrophage function and the development of atherosclerosis, Rgsrl- mice were characterised on the ApoE1- background. Flow cytometric analysis of leukocytes in blood, spleen and bone marrow indicated Rgsrl- ApoE1- mice had no significant differences in the numbers of monocytes or lymphocytes compared to ApoE1- mice. Rgsl was found to be highly expressed in macrophages from ApoE1- mice compared to B-Iymphocytes, where it has a non-redundant role, and other cells involved in plaque formation. Furthermore, Rgsl is up-regulated with monocyte- macrophage activation by innate stimuli. For the first time, RGS 1 'was shown to affect chemokine receptor signalling in macrophages in vitro. RgsrlApoE1- macrophages showed significantly enhanced chemotaxis to CCL2, CCL3 and CCLS and impaired homologous desensitisation to the chemokine CCLS in comparison to ApoE1- cells. To determine the role of RGS-l in leukocyte trafficking and atherosclerosis, a detailed atherosclerosis study was carried out. RgsrlApoE1- mice had significantly less lesion formation in the aortic roots at 9-weeks and in the aorta at 16-weeks on a chow diet in comparison to ApoE1- mice. This was accompanied with decreased macrophage content in the aortic root at 9-weeks. To further investigate aortic leukocyte recruitment, an Angiotensin IT-induced model of acute vascular inflammation was used. At 9 weeks of age, Rgsrl-ApoE1- mice had significantly less aortic CD4S+ leukocytes and cons' myeloid cells recruited to the aorta in comparison to ApoE1- mice. Collectively, these findings identify a new role for RGS-l in macrophage function and support a role for RGS-l in leukocyte recruitment and retention in the initial stages of atherosclerotic plaque formation. These results identify RGS 1 as a novel target for the treatment of acute vascular inflammation and early atherosclerosis.

Rainbow trout macrophages were stimulated with PMA to produce 02- and H2O2 as detected by the reduction of nitroblue tetrazolium (NBT) and the oxidation of phenol red respectively. Addition of DDC or nitroprusside, inhibitors of superoxide dismutase (SOD) increased O2-levels and decreased H2O2 levels, whereas addition of exogenous SOD had the reverse effect. Such data are indicative of a respiratory burst pathway in teleost macrophages comparable with that of mammals. Respiratory burst activity, acid phosphatase activity and RNA synthesis in rainbow trout macrophages which have been stimulated in vitro with the mitogen Concanavalin A (Con A) or in vivo by injection of formalin-fixed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA) was analysed. With Con A, in vitro stimulated head kidney (HK) or elicited macrophages had increased O2-production and RNA synthesis but no significant increases in H2O2 or acid phosphatase activity after 72h post-stimulation with Con A. In contrast, all functions were increased in in vivo stimulated macrophages compared with FIA-elicited peritoneal macrophages. In a bactericidal assay, Con A stimulated macrophages did not show an increase in killing of an avirulent strain of A. salmonicida (004) above control levels whereas in vivo stimulated macrophages not only displayed increased killing of the avirulent strain of bacteria but also acquired the ability to kill a virulent strain (048). Thus, Con A stimulated macrophages only possessed some of the features of activation whereas in vivo stimulated macrophages were activated as defined by the increased bactericidal activity. Peritoneal washes obtained in the collection of activated macrophages were able to increase NBT reduction in normal HK macrophages suggesting the presence of a soluble activating factor. Lymphokine (LK)-containing supernatants produced using either HK or blood derived leucocytes, by pulsing with 10ug/ml Con and 5ng/ml PMA, were able to increase O2- and H2O2 production, to enhance the killing of an avirulent strain of A. Salmonicida and conferred the ability to kill a virulent strain of A. salmonicida. The LK present in these supernatants was therefore designated a macrophage activating factor (MAF). The use of potential second signals to enhance the killing of bacteria by LK-treated macrophages, met with limited success. Only A. salmonicida (strain 004) LPS was able to produce a small increase in killing above LK-treatment. The MAF produced in this study was tested for antiviral/interferon (IFN) activity. The results showed that the supernatants did contain IFN activity. Attempts to semi-purify the MAF from antiviral activity showed the two activities to co-purify, indicating that both activities may be due to the same molecular species. The retention time of the MAF/IFN, coupled with the results of SDS-PAGE analysis showed the molecular weight of the moiety to be approximately 19K daltons. Both activities were sensitive to low pH (pH 2), high temperature (60oC) and trypsin, providing further evidence that the MAF and IFN activity produced in these studies may be due to the same molecular species, possibly akin to IFN- of higher vertebrates.