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    Diverse Streptococcus pneumoniae Strains Drive a Mucosal-Associated Invariant T-Cell Response Through Major Histocompatibility Complex class I-Related Molecule-Dependent and Cytokine-Driven Pathways.
    (Oxford University Press, 2018-03-15) Kurioka A; van Wilgenburg B; Javan RR; Hoyle R; van Tonder AJ; Harrold CL; Leng T; Howson LJ; Shepherd D; Cerundolo V; Brueggemann AB; Klenerman P
    Mucosal-associated invariant T (MAIT) cells represent an innate T-cell population that can recognize ligands generated by the microbial riboflavin synthesis pathway, presented via the major histocompatibility complex class I-related molecule (MR1). Streptococcus pneumoniae is a major human pathogen that is also associated with commensal carriage; thus, host control at the mucosal interface is critical. The recognition of pneumococci by MAIT cells has not been defined nor have the genomics and transcriptomics of the riboflavin operon. We observed robust recognition of pneumococci by MAIT cells, using both MR1-dependent and MR1-independent pathways. The pathway used was dependent on the antigen-presenting cell. The riboflavin operon was highly conserved across a range of 571 pneumococci from 39 countries, dating back to 1916, and different versions of the riboflavin operon were also identified in related Streptococcus species. These data indicate an important functional relationship between MAIT cells and pneumococci.
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    Transcriptional regulation in mouse macrophages : the role of enhancers in macrophage activation and infection
    (Massey University, 2018) Denisenko, Elena
    Macrophages are sentinel cells essential for tissue homeostasis and host defence. Owing to their plasticity, macrophages acquire a range of functional phenotypes in response to microenvironmental stimuli. Of those, M(IFN-γ) and M(IL-4/IL-13) macrophage activation states are well known for their opposing pro- and anti-inflammatory roles. Imbalance in these populations of macrophages has been implicated in progression of various diseases. Macrophages also comprise the first line of an organism’s defence against Mycobacterium tuberculosis, the causative agent of tuberculosis; interactions between the bacteria and host macrophages define the infection outcome. The area of mammalian transcriptional regulation progressed remarkably with recent advances in high-throughput technologies. Enhancers emerged as crucial regulatory DNA elements capable of activating transcription of target genes at distance in an orientation-independent manner. A recent discovery revealed that enhancers can be transcribed themselves into enhancer RNAs, or eRNAs. Enhancers were shown to be pervasive, yet the associated regulatory patterns remain largely unknown and require further research. In this thesis, we investigated in silico transcribed enhancers in mouse tissues and cell lines, with a particular focus on macrophages. We have performed a large-scale study to identify transcribed enhancers across multiple tissues and to characterise their properties. In macrophages, we have established the most accurate, to our knowledge, genome-wide catalogue of transcribed enhancers and enhancer-gene regulatory interactions. We have inferred enhancers that might drive transcriptional responses of protein-coding genes upon M(IFN-γ) and M(IL-4/IL-13) macrophage activation, and demonstrated stimuli specificity of regulatory associations. We have conducted the first to our knowledge study of the role of transcribed enhancers in macrophage response to Mycobacterium tuberculosis infection. Taken together, the present work provides new insights into genome-wide enhancer-mediated transcriptional control of macrophage protein-coding genes in different conditions. Given the increasing promise for enhancer and chromatin-directed therapy, this work paves the way for further studies towards hostdirected therapies and novel treatments for tuberculosis and immune diseases associated with macrophage dysfunction.
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    Deciphering the regulatory network of microRNAs in tuberculosis infected macrophages : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand
    (Massey University, 2017) Ho, Daniel Sik Wai
    Tuberculosis is an infectious disease that is caused by Mycobacterium tuberculosis (Mtb), an intracellular pathogen that uses macrophages as a host for replication. The outcome of the disease depends highly on Mtb’s strategies to circumvent the immune responses of macrophages. MicroRNAs (miRNAs) are small regulatory RNAs that influence gene functions post-transcriptionally. Recent studies indicate that miRNAs have prominent roles in cellular host-pathogen interactions. The aim of this study is to advance our understanding of the regulatory mechanisms that control key miRNAs in mouse M1 macrophages during Mtb infection using network analysis. The study began with a construction of a mouse miRNA-centric regulatory network model by combining a network of miRNA-controlling transcription factors (TFs) with a miRNA target network. The final network places miRNAs at the center of a comprehensive regulatory network of TFs, miRNAs and their targets. This network represents a useful resource for investigating miRNA functions and their control. Subsequently, we populated the network with CAGE-derived expression data for either Mtb-infected mouse M1 macrophages or non-infected controls. We used network analysis to determine key regulatory elements during the infection process. As a result, we identified a core set of TFs and miRNAs, which are likely critical regulatory elements during M1 macrophage host and Mtb interactions. Our results also demonstrate that among the core set of regulatory elements three highly activated miRNAs, mmu-mir-149, mmu-mir-449a, and mmu-mir-449b, work in unison with mmu-mir-155, the top-ranked miRNA. They co-regulate a set of downstream tuberculosis immune response related genes. Four top-ranked TFs, Fosl1, Bhlhe40, Egr1, and Egr2, were identified that they transcriptionally control this group of miRNAs. The TFs and miRNAs, together with their targets constitute a mmu-mir-155 regulatory sub-network. Our results also imply that Bhlhe40 is likely an important TF that modulates the activities of the mmu-mir-155 regulatory sub-network. Bhlhe40 and the mmu-mir-155 regulatory sub-network may be exploited by Mtb to manipulate the host immune defense for advancing survival interests. The findings of this study provide new insights into the host immune regulatory mechanisms of activated macrophages that are essential to control tuberculosis.
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    Cytological studies of ovine alveolar macrophages : interaction with Mycoplasma ovipneumoniae in vitro : this thesis is presented in partial fulfilment (30%) of the requirements for the degree of Master of Philosophy in Veterinary Pathology at Massey University
    (Massey University, 1981) Al-Kaissi, Ayad
    The attachment between Mycoplasma ovipneumoniae organisms and ovine alveolar macrophages was studied in culture for a 24 hour period and antibody-mediated phagocytosis of M. ovipneumoniae organisms was observed by both scanning and transmission electron microscopy. Mycoplasma ovipneumoniae organisms have the ability to attach to the alveolar macrophage membrane without inducing phagocytosis although they stimulated mitotic division in early cultured cells. The addition of specific antibody to the mycoplasma-macrophage cultures provoked phagocytosis of surface attached and surrounding M. ovipneumoniae organisms. Alveolar macrophages stimulated by specific antibody showed rapid and extensive spreading on the glass coverslip and prominent membrane ruffling and filopodia. Many exterior openings and fine cytoplasmic pits were also evident which may represent pinocytotic vesicle formation sites. With transmission electron microscopy M. ovipneumoniae organisms were observed surrounded by macrophage filopodia 2 hours after the addition of specific antibody and numerous micro-organisms were seen within phagocytic vacuoles. Some of the intracellular M. ovipneumoniae organisms appeared normal while others appeared partially or completely degraded. Twenty four hours after the addition of specific antibody, intracellular M. ovipneumoniae organisms had been digested. A new procedure for collection of alveolar macrophages was developed. The procedure provides an alternative to other methods and may be particularly useful for collecting alveolar macrophages from the lungs of large animal species such as sheep and cattle. Acetone was used to dehydrate macrophages for SEM with excellent results. In conclusion, it was found that the addition of specific antibody to an M. ovipneumoniae -macrophage culture stimulated phagocytosis of these micro-organisms. This suggests that if sheep gain high titres to M. ovipneumoniae, their alveolar macrophages will be able to destroy inhaled M. ovipneumoniae organisms quickly and effectively; a possibility which should be tested further in vivo.