Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation

NAHUEL RODRIGUEZ-RODRIGUES; LUIS A CASTILLO; LANDONI V.I; DAIANA MARTIRE-GRECO; M. AYELÉN MILILLO,; PAULA BARRIONUEVO; GABRIELA CRISTINA FERNANDEZ

frontiers in cellular and infection microbiology

Frontiers Media SA

Año: 2017 vol. 7

2235-2988

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial hostdefense. They sense pathogens through recognition of pathogen-associated molecularpatterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors(TLR). The aim of this study was to investigate whether PMN sense bacterial viability andexplore which viability factor could be involved in this phenomenon. For this purpose,different functions were evaluated in isolated human PMN using live Escherichia coli(Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable toinduce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surfaceexpression, chemotaxis, reactive oxygen species (ROS) generation and neutrophilextracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNAhas been recognized as a PAMP associated to bacterial viability by macrophages anddendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as astimulus for PMN. pRNA triggered similar responses to those observed with live bacteria.No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria.Moreover, the supernatant of dead bacteria was able to induce PMN activation, andthis was associated with the presence of pRNA in this supernatant, which is released inthe killing process. The induction of bactericidal functions (ROS and NETosis) by pRNAwere abolished when the supernatant of dead bacteria or isolated pRNA were treatedwith RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generationand NETosis, and priming was required for the induction of pRNA-induced ROS inwhole blood. However, responses related tomovement and degranulation (FSC increase,CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested withRNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion,our results indicate that PMN sense live bacteria through recognition of pRNA, and thissensing triggers potent bactericidal mechanisms.