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

Abstract

Tuberculosis (TB) is an infectious respiratory disease caused by the bacterial pathogen Mycobacterium tuberculosis. Each year, 1.5 million deaths are attributable to TB and survivors are prone to increased all-cause mortality, due to excessive TB-associated pulmonary inflammation. Recent literature indicated the microRNA hsa-miR-652-3p (miR-652) was downregulated in plasma of Chinese TB patients, and further decreased in patients who failed to clear the bacteria after antibiotic therapy. This thesis investigated the activities of miR-652 during in vitro and in vivoinfections with intracellular bacterial pathogens, with a special focus on the macrophage response 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, in order to illustrate the influence of tissue origin on macrophage function. Both cell lines were able to control M. bovis BCG and M. tuberculosisH37Rv bacterial loads. However, AMJ2-C11 cells exhibited a more inflammatory phenotype, with significantly increased cytokine release and nitric oxide generation. Additionally, expression of inflammatory cell surface markers was increased on AMJ2-C11 cells relative to IC-21 cells. These data suggest that whilst tissue origin can influence macrophage phenotype, cell plasticity ensures diverse macrophages can respond to invading pathogens.

Chapter 4 investigated the impact of miR-652 on the murine immune response to M. tuberculosis. Bone marrow 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 their wild type counterparts. Western blot results indicated AKT and mTOR activation was attenuated in miR-652-/- macrophages. miR-652-/- mice infected aerogenically with M. tuberculosiswere able to control the bacterial load in the lungs and spleen equal to wild type mice over 13 weeks. Leukocyte populations were comparable between mouse strains, however, early CD8+ effector T cell numbers were elevated in the lung and lymph node miR-652-/- mice, suggesting miR-652 may have some impact on T cell differentiation during bacterial infection.

Chapter 5 investigated this question in a CD8+ T cell-focused infection model; intraperitoneal Listeria monocytogenes infection. miR-652-/- mice were highly susceptible to a low-dose infection of 2000 CFU/mouse, exhibiting significantly increased weight loss and high morbidity. The early onset of morbidity indicated a deficiency in the innate immune response. Highly necrotic liver lesions in miR-652-/- mice displayed intense recruitment of neutrophils and macrophages, but bacterial load was uncontrolled in these mice. To investigate the antimicrobial phenotype of miR-652-/- macrophages, primary peritoneal macrophages were infected with L. monocytogenes in vitro. A proteomic analysis highlighted dysregulation of key immune pathways, including the lysosome pathway and the pentose phosphate pathway. Also downregulated was the in silico-predicted miR-652 target CAPZB. Transfection experiments using luciferase reporter constructs indicated miR-652 does not target a predicted sequence in the CAPZB 3’UTR. Further, CAPZB mRNA and protein were unaffected by transfection with a miR-652 mimic in IC-21 mouse peritoneal macrophage cells, indicating CAPZB expression is unaffected by miR-652.

This thesis demonstrates miR-652 plays clear roles in the proper innate immune response to acute infection with an intracellular bacterial pathogen. The pathways impacted in miR-652-/- macrophages position miR-652 as an important regulator of immune function, potentially regulating inflammation and cell metabolism. Hostdirected therapies possess amazing potential as a complement to existing antimicrobial drugs. microRNA-based therapeutics for infectious diseases are progressing well through clinical trials. Analysis of the genes validated as targets for miR-652 underscores the promise for a miR-652 mimic as a therapeutic in chronic TB, particular when administered with a cell-targeted delivery mechanism. Additional holistic research is needed to evaluate the impacts of miR-652 in macrophages to realise the potential of miR-652 as a therapeutic miRNA.