THE USE OF NITRIC OXIDE FREE RADICAL SENSITIVE FLUOROPHORES TO DETECT MACROPHAGE PHAGOLYSOSOME ACTIVITY

Abstract

Macrophages are phagocytes which facilitate innate immunity via phagocytosis, averting antagonistic effects resulting from bacterial infections. This is a strictly choreographed event initiated by bacterial-macrophage interactions between pathogen associated molecular patterns and toll-like receptors in macrophages. Consequently, the pathogen is ingested by the macrophage through a vacuole which matures to obtain an arsenal of antimicrobial properties including nitric oxide free-radicals (NO∙). Inducible nitric oxide synthase is an enzyme accountable for NO∙ production upon stimulation. This study utilized opportunistic pathogens Staphylococcus epidermidis, Serratia marcescens, an assemblage composed of the two species and a Lipopolysaccharide positive control to challenge the murine-macrophage J774 Cell-line. Phagolysosome activity was assessed using NO∙ sensitive fluorophore, DAF-FMDA. Fluorescence activity was measured for 300 seconds using a Nikon Eclipse TE200 fluorescence microscope and DXM1200F camera. In all treatments, maximal fluorophore activity was attained within 20 seconds; level of fluorophore activity was dependent on the treatment. S. epidermidis and the bacterial assemblage initiated relatively high activities (RFU = 73.48 ± 3.52 SD; RFU = 56.66 ± 4.74 respectively), comparable to the positive control (RFU = 71.66 ± 0.90). S. marcescens induction of fluorophore activity occurred, but to a lesser extent (RFU = 48.72 ± 3.36), over 20 seconds. The current study suggests the Gram positive S. epidermidis incites relatively high levels of NO∙ synthesis similar to the positive control which was primed with commercial Lipopolysaccharide in challenged macrophages while the NO∙ levels induced by Gram negative S. marcescens were inferior. The response to the bacterial assemblage largely mimicked the response to S. epidermidis alone suggesting macrophages preferentially phagocytosed this species. This study suggests that immune responses by macrophages depend on the bacterial species and therefore does not follow a consistent pattern, particularly in short term NO∙ synthesis.