Pathogenic Gram-negative Yersinia species preferentially target and inactivate phagocytic cells of the innate immune defense by translocation of effector Yersinia outer proteins (Yops) into the cells via a type III secretion system. This indicates that inactivation and avoidance of the early innate immune response is an efficient way for Yersinia species to avoid elimination and to cause diseases ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to plague (Y. pestis). In this project, we aimed to study the interaction between enteropathogenic Y. pseudotuberculosis and phagocytic cells during early infection. In situ interaction studies on infected intestinal tissues showed that Y. pseudotuberculosis mainly interacts with dendritic cells (DCs) in lymphoid tissues of the intestine during initial infection. After massive recruitment of polymorphonuclear neutrophils (PMNs) to the infected tissues, wild-type (wt) bacteria also interacted with this phagocyte. In contrast to the wt, mutants lacking the anti-phagocytic effectors YopH and YopE are avirulent in mice and unable to spread systemically. Interestingly, our interaction assay showed that these mutants not only interacted with DCs, but also with PMNs during the initial stage of infection. Thus, indicating that Y. pseudotuberculosis can avoid interaction with PMNs during early infection and that this is Yop-dependent. In a phagocytosis assay Y. pseudotuberculosis was demonstrated to inhibit internalization by DCs in a YopE-dependent manner, while both YopH and YopE were shown to be involved in the blocking of phagocytosis by macrophages and PMNs. Thus, indicating that YopH has cell type-specific effects. To further investigate the role of DCs during initial stages of infection, a mouse DC depletion model (CD11c-DTRtg) was applied. However, the DTx-mediated depletion of DCs in CD11c-DTRtg mice induced neutrophilia and the model could not give a definite answer to whether DCs play an important role in either restricting or stimulating progression of Y. pseudotuberculosis infection. To investigate involvement of PMNs during early infection mice were injected with the depleting antibody α-Ly6G. In absence of PMNs wt, as well as yopH and yopE mutants became more virulent, which further supports the importance of these Yops for the ability of Y. pseudotuberculosis to disseminate from the initial infection sites in the intestine to cause systemic disease. In summary, our studies show that inhibiting internalization and maturation of DCs and avoiding phagocytosis by and interaction with macrophages and PMNs during the early stages of infection are important virulence strategies for Y. pseudotuberculosis to be able to colonize tissues, proliferate and disseminate systemically.

Chronic inflammation stimulates mammary tumourigenesis by disrupting signalling interactions between the epithelial ducts and the surrounding stromal microenvironment. Adipocyte enhancer-binding protein 1 (AEBP1) promotes mammary epithelial cell hyperplasia as a stromal factor that enhances activity of the proinflammatory transcription factor Nuclear Factor-?B (NF-?B) in macrophages. Aberrant NF-?B activity in macrophages elevates production of proinflammatory signals and the ligand sonic hedgehog (Shh), a significant contributor to tumourigenesis. In this study, Shh expression was elevated in macrophages isolated from transgenic mice (AEBP1TG) that overexpress AEBP1. Transient overexpression of AEBP1 in a macrophage cell line resulted in increased Shh expression. Furthermore, hedgehog target genes Gli1 and Bmi1 were up-regulated in mammary epithelium of AEBP1TG mice and HC11 mammary epithelial cells co-cultured with AEBP1TG macrophages. Growth of HC11 cells and mammary tumours was enhanced in response to AEBP1TG macrophages. These findings suggest that macrophage AEBP1 overexpression contributes to mammary hyperplasia through enhanced hedgehog signalling.

No description available.

No description available.

Severe influenza virus infection, including human infection with highly pathogenic H5N1 viruses is characterised by massive pulmonary inflammation, immunopathology and excessive cytokine production, a process in which macrophages may play a vital role. The aim of this project was to investigate the hypothesis that inhibition of inflammatory responses from infected macrophages, using either alternatively activated bone marrow derived macrophages (BMDMf), or IFNg receptor deficient (IFNgR-/-) mice may ameliorate the devastating immunopathology and inflammation routinely observed in highly pathogenic influenza virus infections. Infection of alternatively activated BMDMf resulted in enhanced positivity for viral proteins, compared with classically activated, inflammatory BMDMf. However, neither subset propagated the infection indicating that while infection is abortive in both classical and alternatively activated BMDMf, the latter may prove more efficient at removing infectious virus from the site of infection due to enhanced infectivity. However, influenza virus was capable of driving expression of proinflammatory mediators such as iNOS and TNFa from classical and alternatively activated BMDMf even in the absence of IFNg signalling. IFNgR-/- BMDMf demonstrated a reduced inflammatory response to infection compared to Sv129 counterparts, suggesting a potentially impaired inflammatory response in vivo. This was investigated by infection of IFNgR-/- mice, which resulted in ameliorated disease, lower viral titres and mild immunopathology, demonstrating that inhibition of IFNg signalling limits the severity of disease. Additionally, mRNA expression for key inflammatory mediators was reduced, demonstrating that inhibition of the overwhelming inflammatory response to influenza virus infection is beneficial to the host, resulting in protection from immunopathology and improved prognosis, without impairing viral clearance.

The molecular biology of macrophage activating and deactivating cytokines and their receptors was discussed. Comparison of IFN-γ amino acid sequences of several mammalian species reveals a low conservation of amino acids. The interaction of IFN-γ with its receptor system is complicated and coherent with the species specificity of IFN-γ. Identification by PCR of an IFN-γ-like gene in the trout genome was not possible. In contrast with IFN-γ, TGF-β is very conserved in its amino acid sequence. The PCR-amplification of a TGF-β fragment from amphibian, <I>Xenopus</I>, and rainbow trout cDNA libraries was possible. Two oligonucleotide primers were used in PCRs to amplify a 360 bp fragment of trout Mhc class II β chain. Using these two oligos, this 360 bp fragment could be amplified from trout spleen cDNA library and HK leucocytes. cDNA synthesized from RNA extracted from ConA/PMA stimulated HK leucocytes was used as template DNA in PCR, and a class II specific fragment was amplified. This class II fragment could not be amplified from HK macrophages treated with a MAF containing supernatant, although HK macrophages treated with a control supernatant did express class II molecules. This could suggest that priming or activation of trout macrophages results in a decreased expression of Mhc class II antigens. A novel method for analysing 5'nucleotidase activity of head kidney macrophages was optimised for use with cell monolayers, with respect to the effect of cell numbers, temperature and substrate concentration. Both lysed and whole cells could be used for determination of 5'nucleotidase activity. Maximal 5'nucleotidase activity was found in the range of 27°C to 33°C and using a substrate concentration of ≥ 1 μmol AMP ml^-1 for whole cells and ≥ 1.5 μmol AMP ml^-1 for lysed cells. 5'nucleotidase activity was also correlated with respiratory burst activity in cells treated with a variety of supernatants containing MAF activity. A significant inverse relationship between these two activities was found. MAF-treated cells were also found to lose 5'nucleotidase activity faster than control cells in the presence of cycloheximide, suggesting such cells may have a higher membrane turnover of this enzyme.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Obstetrics and Gynaecology, 1999. / Errata posted inside back end-paper (leaf 250). Bibliography: leaves 204-249.

Tissue-resident macrophages (M&Phi;) such as microglia, Kupffer and Langerhans cells derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident M&Phi; self-renew locally, independently of circulating adult monocytes and HSCs. In contrast, adult blood monocytes as well as infiltrating, gut and dermal M&Phi; derive from Myb-dependent HSCs and are less proliferative. These findings are derived from the mouse, using gene knock-outs and lineage tracing, but their applicability to human development has not been formally demonstrated. Here I use a human pluripotent stem cell (hPSC) differentiation model of hematopoiesis, capable of monocyte/M&Phi; production over prolonged periods of time, as a tool to investigate human mononuclear phagocyte ontogeny. Using a transcriptomic approach I showed that hiPSC-derived monocytes/M&Phi; (iPS-Mo/M&Phi;) produced early in differentiation (first weeks) are more proliferative and less immunologically mature than iPS-Mo/M&Phi; produced at a later time point. I therefore hypothesised either that iPS-Mo/M&Phi; only become fully mature after several weeks of differentiation or that there are two developmentally distinct waves of M&Phi; produced over time. By comparing the transcription profile of iPS-Mo/M&Phi;s to that of primary adult blood monocytes and fetal microglia I then showed that early and late iPS-Mo/M&Phi;s were transcriptionally closer to fetal microglia than to adult blood monocytes. To further investigate if iPS-Mo/M&Phi;s are indeed of the same developmental origin as MYB-independent M&Phi; such as microglia, I used a CRISPR-Cas9 knock-out strategy to show for the first time, that human iPS-Mo/M&Phi;s develop in a MYB-independent, RUNX1 and SPI1 (PU.1)-dependent fashion. This result makes human iPS-Mo/M&Phi;s developmentally related to, and a good model for, MYB-independent tissue-resident \Macros such as alveolar and kidney M&Phi;s, microglia, Kupffer and Langerhans cells. Interestingly, while MYB was not required for the generation of iPS-Mo/M&Phi;s, its knock-out resulted in an increase in iPS-Mo/M&Phi; production. To investigate this increase I developed two methods for quantifying the differentiation bottleneck occurring during hiPSC differentiation to iPS-Mo/M&Phi;s. Those techniques highlighted a potential increase in progenitor cell generation in MYB KO cells and thus lay foundation to improve our technical understanding of EB differentiation and will enable enhanced manipulation of the EB model.

Marek’s disease (MD) is a highly infectious economically important oncogenic viral disease of chickens. It is found throughout the world and is caused by an alphaherpesvirus, Marek’s disease virus (MDV). Though this disease can currently be successfully controlled by vaccination, the virus has continuously evolved to greater virulence over the last several decades. Hence, there is a need for alternative approaches to control MD. Selection and breeding of MD-resistant chickens presents an attractive option for prevention of this disease. MHC-congenic chicken inbred lines, 61 and 72, which are highly resistant and susceptible to MD, respectively, have been identified, but the cellular and genetic basis for these phenotypes is unknown. The overall aim of this study was to investigate the cellular basis of resistance to MD using an in vitro MDV infection model with the hypothesis that resistance is exerted by the innate immune cells. MDV is a highly cell-associated virus which makes in vitro studies difficult. In vivo, MDV infects APCs (antigen-presenting cells: macrophages and/or dendritic cells [DCs]), B cells and activated T cells. Though both B and T cells can be infected in vitro, co-culture infection models have not been described for APCs. Thus, the primary goal was to develop a model for infecting these cells with MDV in vitro and to characterise infected and uninfected cells. Developmental studies used APCs derived from outbred chickens. Chicken bone marrow cells were cultured with chCSF-1 (for macrophages) or chIL-4 and chCSF-2 (for DCs) for 4 days and then infected by the addition of chicken embryo fibroblasts (CEFs) infected with recombinant MDV expressing GFP. CEF preparations naturally contain a mixture of CEFs (92-98%) and macrophages (2-8%) and both appear to be infectable with MDV. Infected CEFs were therefore separated from infected macrophages by FACS before adding to the bone marrow-derived APCs. Infected and uninfected APCs were sorted by FACS using GFP expression and APC-specific mAb staining (KUL01 and anti-CD45). Characteristic virus-infected and uninfected APCs were revealed via examination with live cell confocal microscopy. The presence of herpesvirus specific immediate early (ICP4), early (pp38), late (gB) transcripts and MDV specific transcript, L-Meq, in infected APCs was confirmed by RT-PCR providing evidence for MDV replication. Hence, a new in vitro MDV infection model of APCs has been established. Using the infected macrophages to infect CEFs showed that the infection was productive. This model was then extended to infect APCs of lines 61 and 72. Flow cytometric analysis revealed that a higher percentage of macrophages were infected in the susceptible line (72) than in the resistant line (61). To analyse this in detail, RNA-Seq was carried out to identify differentially expressed (DE) genes between the two lines pre- and post-MDV infection. From these DE genes, potential candidate genes involved in MD resistance and susceptibility were identified. Functional analysis of DE genes support the hypothesis that resistance to MD is determined at the macrophage level of the resistant line (61) and the JAK-STAT signalling pathway is at least one anti-viral mechanism by which this signature is expressed.

The pathogenesis of systemic lupus erythematosus (SLE) and cardiovascular disease (CVD) are tightly linked, and CVD is one of the leading causes of death in lupus patients. There are many risk factors that increase the risk of CVD in SLE patients, including endothelial dysfunction, lipid dysregulation, and abnormal regulation of innate and adaptive immunity. We have previously investigated the role of interferon regulatory factor 5 (Irf5), on atherosclerosis in lupus mouse models. Irf5 has a pro-inflammatory function by activating macrophage and cytokine recruitment and is thus being considered as a potential therapeutic target for the treatment of SLE. We hypothesized that Irf5 deficiency would ameliorate lupus disease as well as improve cardiovascular disease in the Irf5-deficient mouse model. However, while lupus disease did improve in the mouse model, the atherosclerotic plaques were found to be significantly increased in size. This poses a challenge to our current understanding of Irf5, as well as adds complexity to an already difficult clinical problem. Therefore, our aim of this study is to characterize the cells within the atherosclerotic lesions to examine their inflammatory potential. The focus of this study is the infiltration of macrophages into the mouse aortic root as determined by immunohistochemistry staining. In a time-course study using apoE.Irf5-/- mice, we found that macrophages started to accumulate into aortic leaflets as early as two weeks after starting a Western diet. Macrophage infiltration into the site of leaflet attachment seemed to possibly be a precursor to atherosclerotic lesion formation and appeared as early as 4 weeks after starting Western diet. No apparent differences were found between Irf5 sufficient and Irf5 deficient mice at either two or four weeks on Western diet. In a bone marrow chimera study, we examined the effects of Irf5 from bone marrow- and non-bone marrow-derived cells on the accumulation of macrophages on aortic leaflets and in the tunica intima in the gld.apoE-/- mouse model of lupus and atherosclerosis. Macrophage accumulation did not correlate with differences in Irf5 production. However, the finding of macrophage accumulation on aortic leaflets suggests a role of macrophages in Libman-Sacks endocarditis, an inflammatory disease of the mitral and aortic valves seen in patients with lupus. Together, our results do not support nor refute a role of Irf5 in macrophage infiltration into the aortic root. More samples are needed, as are more methods of identifying macrophages and quantifying them. However, it is still likely that macrophages play a role in the pathogenesis of atherosclerotic lesion formation in a lupus mouse model, and it is an area of study worth exploring. In a time-course study using apoE.Irf5-/- mice, we found that macrophages started to accumulate into aortic leaflets as early as two weeks after starting a Western diet. Macrophage infiltration into the site of leaflet attachment seemed to possibly be a precursor to atherosclerotic lesion formation and appeared as early as 4 weeks after starting Western diet. No apparent differences were found between Irf5 sufficient and Irf5 deficient mice at either two or four weeks on Western diet. In a bone marrow chimera study, we examined the effects of Irf5 from bone marrow- and non-bone marrow-derived cells on the accumulation of macrophages on aortic leaflets and in the tunica intima in the gld.apoE-/- mouse model of lupus and atherosclerosis. Macrophage accumulation did not correlate with differences in Irf5 production. However, the finding of macrophage accumulation on aortic leaflets suggests a role of macrophages in Libman-Sacks endocarditis, an inflammatory disease of the mitral and aortic valves seen in patients with lupus. Together, our results do not support a role of Irf5 in macrophage infiltration into the aortic root. However, it is still likely that macrophages play a role in the pathogenesis of atherosclerotic lesion formation in a lupus mouse model, and it is an area of study worth exploring.