Identification of genes involved in macrophage activation and effector functions against intracellular pathogens

Includes bibliographical references.This dissertation addressed the hypothesis that macrophages have an alternative killing mechanism that is independent of superoxide and nitric oxide but dependent on IFN-γ, TNF and C/EBPβ. Since the mechanism and the genes involved in this alternative pathway are mostly unknown, the aim of this dissertation was to identify these macrophage effector genes and to functionally characterize their role during infection utilizing gene deficient mouse models. Since mice deficient for C/EBPβ (C/EBPβ-/-) expressed normal levels of IFN-y and TNF during Listeria monocytogenes infection, the macrophage effector genes involved in confinement and killing of L. monocytogenes were postulated to be downstream of C/EBPβ. Furthermore, C/EBPβ-/- mice are highly susceptible L. monocytogenes due to impaired listericidal activity. Comparison of the gene expression profiles of WT and C/EBPβ-/- macrophages infected with L. monocytogenes was postulated to increase the probability of identifying these effector genes, which would be differentially expressed between the two groups. Comparative gene expression profiling by DNA microarrays between L. monocytogenes in infected WT and C/EBPβ-/- macrophages, successfully identified 1268 genes to be differentially expressed between the two groups. A focussed functional clustering strategy reduced the number of candidate genes to 220. PKCδ was selected for further study since it was involved in humoral defense, immune signalling, production of superoxide, regulation of transcription and may be putatively transcriptionally regulated by C/EBPβ. Furthermore, PKCδ was indirectly shown to promote L. monocytogenes escape from the phagosome and to negatively regulate transcription activity of C/EBPβ. In addition, since PKCδ was un-regulated, as shown by microarray and confirmed by RT-PCR, in L. monocytogenes infected C/EBPβ-/- macrophages, it was therefore thought to play a detrimental role during L. monocytogenes. However, since this premise has never been investigated directly, the role PKCδ during innate immunity against L monocytogenes was examined using the PKCδ deficient (PKCδ-/-) mouse model. Data in this dissertation provides new insight into the role of PKCδ during innate immunity to L. monocytogenes. PKCδ-/- mice were highly susceptible to L. monocytogenes due to enhanced listerial escape and impaired listericidal activity. Despite full macrophage activation and production of nitric oxide, PKCδ-/- mice displayed uncontrolled bacterial growth and dissemination of L. monocytogenes, which led to early death of the mice. In contrast, PKCδ-/- mice were able to control Mycobacterium infection as well as WT mice, suggesting that the activity of PKCδ may be negatively regulated by L. monocytogenes. A systems biology approach generated the hypothesis that PKCδ may promote Rab5a activation, which together with localized release of superoxide into the phagosome and activation of C/EBPβ by PKCδ, resulted in the confinement of the L. monocytogenes within the phagosome. Alternatively, PKCδ may act in a separate pathway that confines L monocytogenes within the phagosome, by activating and/or synergizing with unidentified proteins to neutralize that activity of listerial LLO and PI-PLC. Data in this dissertation clearly demonstrates that PKCδ is critical for confinement of L monocytogenes within phagosomes and may be part of a listericidal mechanism that is independent or nitric oxide, superoxide and pro-inflammatory cytokines

OntoDas – a tool for facilitating the construction of complex queries to the Gene Ontology

<p>Abstract</p> <p>Background</p> <p>Ontologies such as the Gene Ontology can enable the construction of complex queries over biological information in a conceptual way, however existing systems to do this are too technical. Within the biological domain there is an increasing need for software that facilitates the flexible retrieval of information. OntoDas aims to fulfil this need by allowing the definition of queries by selecting valid ontology terms.</p> <p>Results</p> <p>OntoDas is a web-based tool that uses information visualisation techniques to provide an intuitive, interactive environment for constructing ontology-based queries against the Gene Ontology Database. Both a comprehensive use case and the interface itself were designed in a participatory manner by working with biologists to ensure that the interface matches the way biologists work. OntoDas was further tested with a separate group of biologists and refined based on their suggestions.</p> <p>Conclusion</p> <p>OntoDas provides a visual and intuitive means for constructing complex queries against the Gene Ontology. It was designed with the participation of biologists and compares favourably with similar tools. It is available at <url>http://ontodas.nbn.ac.za</url></p

The Role of B-cells and IgM Antibodies in Parasitemia, Anemia, and VSG Switching in Trypanosoma brucei–Infected Mice

African trypanosomes are extracellular parasitic protozoa, predominantly transmitted by the bite of the haematophagic tsetse fly. The main mechanism considered to mediate parasitemia control in a mammalian host is the continuous interaction between antibodies and the parasite surface, covered by variant-specific surface glycoproteins. Early experimental studies have shown that B-cell responses can be strongly protective but are limited by their VSG-specificity. We have used B-cell (µMT) and IgM-deficient (IgM−/−) mice to investigate the role of B-cells and IgM antibodies in parasitemia control and the in vivo induction of trypanosomiasis-associated anemia. These infection studies revealed that that the initial setting of peak levels of parasitemia in Trypanosoma brucei–infected µMT and IgM−/− mice occurred independent of the presence of B-cells. However, B-cells helped to periodically reduce circulating parasites levels and were required for long term survival, while IgM antibodies played only a limited role in this process. Infection-associated anemia, hypothesized to be mediated by B-cell responses, was induced during infection in µMT mice as well as in IgM−/− mice, and as such occurred independently from the infection-induced host antibody response. Antigenic variation, the main immune evasion mechanism of African trypanosomes, occurred independently from host antibody responses against the parasite's ever-changing antigenic glycoprotein coat. Collectively, these results demonstrated that in murine experimental T. brucei trypanosomiasis, B-cells were crucial for periodic peak parasitemia clearance, whereas parasite-induced IgM antibodies played only a limited role in the outcome of the infection

IL-4Rα-Independent Expression of Mannose Receptor and Ym1 by Macrophages Depends on their IL-10 Responsiveness

IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

Identification of proteins that interact with brain factor-1 and characterization of these interactions

Bibliography: leaves 110-124.abstractBrain Factor-1 (BF-1) is winged helix transcription factor that is essential for the development of the cerebral hemispheres and olfactory neuroepithelium. BF-1 is expressed in the telecephalic neuroepithelium, olfactory placode and the nasal half of the optic stalk and retina during embryogenesis (Hatini et al 1994; Lai and Tao 1992). In these tissues, BF-1 plays multiple roles in regional patterning and regulating cell proliferation and differentiation. In order to investigate how BF-1 controls these various processes, the aim of this thesis was to identify proteins which interact with BF-1 using the Yeast Two Hybrid System (Field and Song 1989) and to characterise these interactions

Localisation of mannose receptor and Ym1-expressing granuloma macrophages in close contact with <i>S. mansoni</i> eggs depends on their IL-4Rα signalling.

<p>Livers were collected at 8 weeks p.i. from <i>Il4ra^−/lox</i>, <i>LysM^creIl4ra^−/lox</i>, <i>iLck^creIl4ra^−/lox</i> and <i>Il4ra^−/−</i> mice and immunofluorescent stainings performed on cryosections. (A) Representative micrographs of MMR (panels v–viii), Ym1 (panels xiii–xvi), CD204 (panels xxi–xxiv) and iNOS (panels xxix–xxxii) stainings of liver cryosections as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000689#s2" target="_blank">Methods</a>. Stainings with secondary antibody (MMR, iNOS), streptavidin alone (Ym1) or isotype-control (CD204) is shown for each corresponding staining (MMR = i–iv, Ym1 = ix–xii, CD204 = xvii–xx, iNOS = xxv–xxviii). Note that MMR⁺ and Ym1⁺ cells are restricted to the periphery of <i>LysM^creIl4ra^−/lox</i> granulomas. Outlined regions represent the areas magnified in B. (B) Representative micrographs of liver cryosections stained with scavenger receptor (CD204) for macrophages detection (panels i–viii, green) or iNOS for classically activated macrophages detection (panels ix–xvi, green); and co-stained for MMR (panels i–iv and ix–xii, red) or Ym1 (panels v–viii and xiii–xvi, red). Note the low frequency of CD204⁺ macrophages co-expressing MMR⁺ or Ym1⁺ cells around the parasite eggs (panels ii and vi) but the high levels of iNOS⁺ cells (panels x and xiv) in <i>LysM^creIl4ra^−/lox</i> mice, suggesting these macrophages to be classically activated. White arrows indicate the parasite eggs. Original magnification: 400×. Data represent one of three independent experiments.</p