Investigation of miR-652 in Host Immunity Against Intracellular Bacterial Pathogens

Stevens, Maxwell T.

Investigation of miR-652 in Host Immunity Against Intracellular Bacterial Pathogens

Stevens, Maxwell T.

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Publication Type:: Thesis
Issue Date:: 2022

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Field	Value	Language
dc.contributor.author	Stevens, Maxwell T.
dc.date.accessioned	2023-07-14T03:30:56Z
dc.date.available	2023-07-14T03:30:56Z
dc.date.issued	2022
dc.identifier.uri	http://hdl.handle.net/10453/171507
dc.description	University of Technology Sydney. Faculty of Science.	en_US.UTF-8
dc.description.abstract	Tuberculosis (TB) is caused by the bacterial pathogen Mycobacterium tuberculosis and is responsible for 1.5 million deaths each year. Recent literature indicated the microRNA hsa-miR-652-3p (miR-652) was downregulated in plasma of Chinese TB patients. This thesis investigated the activities of miR-652 during in vitro and in vivo infections with intracellular bacterial pathogens, focusing on macrophage responses to infection. My initial study aimed to characterise the phenotypic differences between murine alveolar (AMJ2-C11) and peritoneal (IC-21) macrophage cell lines during in vitro mycobacterial infections. Both cell lines were able to control M. bovis BCG and M. tuberculosis H37Rv bacterial loads. However, AMJ2-C11 cells exhibited a more inflammatory phenotype, with significantly increased cytokine release and increased expression of inflammatory cell surface markers. These data suggest while tissue origin can influence macrophage phenotype, cells retain plasticity and can respond to diverse pathogens. Chapter 4 investigated the impact of miR-652 on the immune response to M. tuberculosis. Macrophages from miR-652-/- C57BL/6 mice were able to control bacterial growth over 6 days in vitro, though IL-6, TNF, MIP-1α, and KC expression was significantly lower than in wild type macrophages. miR-652-/- mice infected aerogenically with M. tuberculosis controlled the bacterial load in the lungs and spleen equal to wild type mice. Early CD8+ effector T cell numbers were elevated in the miR-652-/- lung and lymph node, suggesting miR-652 may impact on T cell differentiation. Chapter 5 investigated this in a CD8+ T cell-focused model; low-dose intraperitoneal Listeria monocytogenes infection, 2000 CFU/mouse. miR-652-/- mice exhibited increased weight loss and early morbidity, indicating deficiency in the innate immune response. miR-652-/- livers displayed necrotic lesions with intense neutrophil and macrophage recruitment, though bacterial load was uncontrolled in these mice. Primary miR-652-/- peritoneal macrophages were infected with L. monocytogenes in vitro, and proteomics highlighted dysregulation of the lysosome and pentose phosphate pathways. Also downregulated was the in silico-predicted miR-652 target CAPZB. Transfection experiments indicated miR-652 does not target a predicted sequence in the CAPZB 3’UTR. CAPZB mRNA and protein were unaffected by transfection with a miR-652 mimic. This thesis demonstrates miR-652 plays clear roles in the proper innate immune response to acute infection with intracellular bacterial pathogens. Pathways impacted in miR-652-/- macrophages position miR-652 as an important immune regulator. Analyses of validated target genes underscores the promise for a therapeutic miR-652 mimic in chronic TB. Additional holistic research is needed to evaluate the impacts of miR-652 as a therapeutic miRNA.	en_US.UTF-8
dc.format	Thesis (PhD)
dc.language.iso	en_US	en_US.UTF-8
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/171507/2/02whole.pdf
dc.rights	info:eu-repo/semantics/openAccess
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	© 2022 Maxwell T. Stevens
dc.rights	au.edu.uts.lib/cph
dc.title	Investigation of miR-652 in Host Immunity Against Intracellular Bacterial Pathogens	en_US.UTF-8
dc.type	Thesis
utslib.copyright.status	open_access	*

Abstract:

Tuberculosis (TB) is caused by the bacterial pathogen Mycobacterium tuberculosis and is responsible for 1.5 million deaths each year. Recent literature indicated the microRNA hsa-miR-652-3p (miR-652) was downregulated in plasma of Chinese TB patients. This thesis investigated the activities of miR-652 during in vitro and in vivo infections with intracellular bacterial pathogens, focusing on macrophage responses to infection. My initial study aimed to characterise the phenotypic differences between murine alveolar (AMJ2-C11) and peritoneal (IC-21) macrophage cell lines during in vitro mycobacterial infections. Both cell lines were able to control M. bovis BCG and M. tuberculosis H37Rv bacterial loads. However, AMJ2-C11 cells exhibited a more inflammatory phenotype, with significantly increased cytokine release and increased expression of inflammatory cell surface markers. These data suggest while tissue origin can influence macrophage phenotype, cells retain plasticity and can respond to diverse pathogens. Chapter 4 investigated the impact of miR-652 on the immune response to M. tuberculosis. Macrophages from miR-652-/- C57BL/6 mice were able to control bacterial growth over 6 days in vitro, though IL-6, TNF, MIP-1α, and KC expression was significantly lower than in wild type macrophages. miR-652-/- mice infected aerogenically with M. tuberculosis controlled the bacterial load in the lungs and spleen equal to wild type mice. Early CD8+ effector T cell numbers were elevated in the miR-652-/- lung and lymph node, suggesting miR-652 may impact on T cell differentiation. Chapter 5 investigated this in a CD8+ T cell-focused model; low-dose intraperitoneal Listeria monocytogenes infection, 2000 CFU/mouse. miR-652-/- mice exhibited increased weight loss and early morbidity, indicating deficiency in the innate immune response. miR-652-/- livers displayed necrotic lesions with intense neutrophil and macrophage recruitment, though bacterial load was uncontrolled in these mice. Primary miR-652-/- peritoneal macrophages were infected with L. monocytogenes in vitro, and proteomics highlighted dysregulation of the lysosome and pentose phosphate pathways. Also downregulated was the in silico-predicted miR-652 target CAPZB. Transfection experiments indicated miR-652 does not target a predicted sequence in the CAPZB 3’UTR. CAPZB mRNA and protein were unaffected by transfection with a miR-652 mimic. This thesis demonstrates miR-652 plays clear roles in the proper innate immune response to acute infection with intracellular bacterial pathogens. Pathways impacted in miR-652-/- macrophages position miR-652 as an important immune regulator. Analyses of validated target genes underscores the promise for a therapeutic miR-652 mimic in chronic TB. Additional holistic research is needed to evaluate the impacts of miR-652 as a therapeutic miRNA.

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