Sphingosine-1-phosphate receptor 1 signalling in bacterial-macrophage interactions: exploring novel anti-bacterial strategies using immunomodulatory therapeutics

Abstract

Sphingosine-1-Phosphate (S1P) signalling via the Sphingosine-1- posphate Receptors (S1PRs) 1-5 profoundly influences the host-immune response to infection. In this study, the effect of S1PR1 signalling on the interactions between a murine macrophage-like cell line (J774A.1) and the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa is assessed. Intracellular persistence of S. aureus within live J774 cells was observed to 96 hrs post-infection. Unexpectedly, using a novel gentamicin protection assay, intracellular survival of P. aeruginosa within live J774 cells was observed to 24 hrs post-infection. This represents the first demonstration of prolonged P. aeruginosa persistence within a macrophage cell line. Interestingly, treatment of macrophages infected with either S. aureus or P. aeruginosa with an S1PR1 agonist (CYM-5442), produced fundamentally different outcomes. Treatment of S. aureus-infected macrophages with CYM5442 resulted in enhanced killing of S. aureus by the macrophage. Via 3D immunofluorescence microscopy and a novel image-analysis method, it was found that CYM-5442 treatment induced an increased colocalisation between S. aureus and mitochondria. Accompanying this was an increased exposure of internalised S. aureus to mitochondrial-derived reactive oxygen species (mROS). This increased mROS exposure is expected to contribute to S1PR1- mediated S. aureus killing. By contrast, CYM-5442 treatment of P. aeruginosa-infected macrophages increased both intracellular P. aeruginosa survival and host-cell lysis. Strikingly, CYM-5442 treatment promoted the formation of a macrophage bleb-niche in which motile, replicating P. aeruginosa were 3 observed. Collectively, this work highlights the influential and complex role of S1PR1 signalling in regulating macrophage-microbe interactions.