Approaches for identifying macrophage microbicidal mechanisms

Abstract

Host defence is usually successful in preventing pathogen exposure and colonisation from progressing to invasive disease. However, efforts to identify specific host microbicidal responses that can be therapeutically targeted to treat bacterial disease (host-directed therapy) have not readily translated into clinical utility. Streptococcus pneumoniae is a medically-important pathogen of global relevance. Macrophage pauciinflammatory bacterial killing determines the outcome of pneumococcal infection so augmenting macrophage microbicidal responses is an attractive therapeutic strategy.

New approaches are needed to identify macrophage microbicidal mechanisms as investigational targets for host-directed therapies. In this thesis I test the hypothesis that a combined pathogen-perspective data-driven approach can achieve this. Serotype 1 pneumococci are associated with disease outbreaks and severe clinical phenotypes. They have undergone diversifying selection in distinct geographic lineages and represent putative adaptive variants that could have evolved to resist host microbicidal responses. Bacterial phylogenetic analysis and quantification of monocytederived macrophage (MDM) intracellular bacterial killing identified two pairs of closely related but differentially-killed serotype 1 pneumococci. MDM transcriptional responses associated with differential killing were identified and differentially expressed host genes of interest were validated by siRNA knockdown in MDM, revealing anti-pneumococcal host genes including ACOD1, P2RX7, GZMB and NCOA7. Bone marrow-derived macrophages (BMDM) from ACOD1-/- mice exhibited defective intracellular killing of pneumococci. Itaconate, the product of ACOD1 enzymatic activity, was directly microbicidal against pneumococci at physiologically-relevant concentrations and enhanced macrophage intracellular bacterial killing. Chemical modification of P2RX7 signalling altered MDM intracellular killing of pneumococci. Clemastine, a licenced antihistamine drug with off-target P2RX7 potentiation, enhanced MDM and wild type murine BMDM intracellular killing of pneumococci. This effect was specific to clemastine, hostdirected and P2RX7-dependent. These effects of ACOD1/itaconate and clemastine extended to Streptococcus pyogenes and Streptococcus agalactiae but not Staphylococcus aureus.

Meta-analysis by information content was used to rank host factors involved in S. pneumoniae infection using existing evidence by integrating and prioritising results from genome-scale pathogen-host studies identified by a systematic literature review. The output of this data-driven analysis supported investigation of NAMPT (also differentially expressed in the RNAseq experiments). siRNA knockdown of NAMPT impaired MDM intracellular killing of pneumococci, and the NAMPT activator P7C3-A20 enhanced intracellular bacterial killing by MDM and wild type murine BMDM.

I have applied two complementary approaches to identify macrophage microbicidal mechanisms: insight from bacterial immune-adaptive pathogen variants and prioritisation of host factors through integration of existing datasets. This has led to the identification of mechanisms of host defence against pneumococci and investigational therapeutic targets for host-directed therapies to enhance host microbicidal responses.