Macrophages are key cellular mediators of innate immunity. They recognize pathogens through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), Nod-like receptors (NLRs), C-type lectin receptors (CLRs) and the HIN200 family of receptors. These are stimulated by specific pathogen-associated molecular patterns (PAMPs), which trigger the activation of antimicrobial mechanisms. Several differences between human and mouse macrophages have been identified. These likely reflect, at least in part, the co-evolution of innate immune response genes in the face of rapidly evolving pathogens. A key hypothesis of this project is that species differences are important for pathogen control. Recent work performed in this laboratory by Schroder et al. showed that about 24% of LPS-regulated orthologous genes in human and mouse macrophages were divergently regulated between the two species. These included several genes encoding ion channels and transporters including the zinc transporter SLC39A8, which was a major focus of this study.
LPS up regulated SLC39A8 mRNA expression in a panel of human macrophage populations but not in several mouse macrophage cell types. Human-specific SLC39A8 mRNA regulation was also observed in response to infection with Salmonella enterica serovar Typhimurium. Expression analysis of the other SLC39A family members revealed that SLC39A8 up-regulation was selective, with minimal regulation of other family members in human macrophages as a response to infection. A polyclonal antibody against SLC39A8 was generated and data suggests that this effect also occurs at the protein level. Ectopically expressed V5 tagged SLC39A8 was localized to the cell membrane in HEK293 cells. Attempts at localising ectopically or endogenously expressed SLC39A8 in macrophages were unsuccessful. siRNA knock down of SLC39A8 mRNA expression in human macrophages reduced intramacrophage survival of Salmonella, while survival of non-pathogenic E. coli was increased by 24h post infection. This suggests that SLC39A8 is involved in the antimicrobial response and that Salmonella has co-opted this transporter to promote intracellular survival. This effect on human macrophage pathways was selective since classic pro-inflammatory cytokines such as IL-6 and TNFα remained unaffected.
LPS treatment and Salmonella infection resulted in the localization of zinc to punctate intracellular vesicles, which did not co-localize with Salmonella nor were they localised to lysosomes or LAMP1+ vesicles. Interestingly, zinc did co-localise with copper following LPS or Salmonella stimulation. In addition ICPMS and flow cytometry analysis of intracellular zinc concentration in LPS stimulated human macrophages showed no significant change in the intracellular zinc concentration, whereas copper was increased by ~2 fold. Exogenous zinc treatment of human macrophages infected with S. Typhimurium resulted in a decreased intracellular bacterial load, thus indicating an antimicrobial effect of zinc. Interestingly a greater effect of exogenous zinc was seen on a non-pathogenic E. coli strain MG1655. Exogenous zinc was found not to have any effect on the production of reactive oxygen species (ROS), either in the first hour (oxidative burst) or 24h (likely mitochondrial ROS) after Salmonella infection.
The second part of this PhD project dealt with the study of a novel intracellular pathogen, uropathogenic E. coli (UPEC), which is the major causative agent of urinary tract infections (UTI). Until recently, E. coli spp. were considered to be extracellular pathogens. Recent studies have shown that E. coli can survive intracellularly in epithelial cells of the gut and the urinary bladder. This project examined the ability of four different UPEC strains (UTI89, 83972, VR50 and CFT073) to survive intracellularly in human and mouse macrophages. Of the four strains, UTI89 and VR50 survived in mouse macrophages but only UTI89 survived robustly in human macrophages. This correlates with clinical data implicating UTI89 in chronic UTI in humans. UTI89 localized to Lamp1+ vesicles in mouse macrophages similar to what has been observed in bladder epithelial cells. This study therefore suggests that some UPEC strains can effectively subvert macrophage antimicrobial mechanisms and species differences impact on intramacrophage survival of some UPEC strains. In addition a pilot project to analyse the host-pathogen transcriptome of murine bone marrow derived macrophages infected with UTI89, VR50 and 83972 was initiated and some novel candidate bacterial genes were identified.
In summary, the findings from this thesis implicate zinc and the zinc transporter SLC39A8 in human macrophage antimicrobial responses and also suggest that macrophages may represent a cellular reservoir enabling the survival of some UPEC strains.