Novel macrophage microbicidal responses against gram-positive bacteria

Abstract

Antimicrobial resistance is a major global health threat, and there is growing interest in how modulation of the host immune response can enhance pathogen killing and reduce reliance on antimicrobials. One target cell is the macrophage; a key innate immune cell that possesses a range of microbicidal mechanisms and can combine responses for optimal pathogen killing. Streptococcus pneumoniae and Staphylococcus aureus are important gram-positive pathogens that represent differing intracellular burdens for the macrophage. A key macrophage microbicidal mechanism relevant to the killing of these pathogens is production of reactive oxygen species (ROS). While NADPH oxidase-derived ROS is an early response to infection, mitochondrial ROS (mROS) production is a later response and is enhanced during infection by alterations in mitochondrial dynamics. ROS and mROS can combine with other macrophage responses to facilitate pathogen killing, therefore the significance and potential for such interplay with other host defence mechanisms to enhance macrophage killing of pathogens such as S. pneumoniae and S. aureus is the focus of this thesis, with specific attention to mitochondrial-associated responses and the microbicidal and immunomodulatory host defence peptide cathelicidin.

The data presented in this thesis show that expression of the CAMP gene, encoding cathelicidin, was upregulated by vitamin D in macrophages, was synergistically enhanced by bacterial infection or phenylbutyrate and was impaired by pro-inflammatory cytokines. Cathelicidin directly killed extracellular S. pneumoniae and contributed to early macrophage killing of intracellular S. aureus when bacterial burden was high. Mitochondrial adaptations to S. pneumoniae were more prevalent in macrophages during later stages of bacterial challenge and included increased mitochondrial fission and increased mROS production.

Mitochondrial adaptations to S. aureus, which stresses macrophage microbicidal responses to a greater extent than S. pneumoniae, were observed during early stages of bacterial challenge. The regulators of canonical fission, dynamin-related protein 1 (Drp1) and mitochondrial fission factor (Mff), failed to influence overall levels of fission in the initial response to S. aureus. In contrast, Drp1 regulated localisation of mROS to intracellular S. aureus in a subset of macrophages, suggesting roles in mROS delivery to bacterial-containing phagolysosomes. In regard to mechanisms of mROS production, I have provided evidence that reverse electron transport (RET) occurs as an early response to S. pneumoniae challenge, but not late S. pneumoniae, or S. aureus challenge. S. aureus enhanced mROS production in macrophages, and while NADPH oxidase-derived ROS was the greater contributor to early killing of S. aureus, mROS also contributed to killing. Cathelicidin enhanced microbicidal responses against S. aureus particularly when NADPH oxidase-derived ROS generation was impaired, but also appeared to function as a brake on alterations in mitochondrial dynamics and mROS production in the presence of bacteria, therefore potentially regulating mitochondrial homeostasis. Results in this thesis demonstrate that macrophages use ROS, alterations in mitochondrial dynamics and mROS, and cathelicidin to combat S. pneumoniae and S. aureus infections with pathogen-dependent kinetics. Macrophages adapt responses to different pathogens to ensure a multi-layered immune response to clear pathogens. The work in this thesis provides greater insight into macrophage microbicidal responses to S. pneumoniae and S. aureus infection and could inform future therapeutic strategies to enhance macrophage microbicidal responses.