DNA-based fluorescent probes of NOS2 activity in live brains

When a pathogen is engulfed by an immune cell, it is trapped in a transient organelle called the phagosome. The host cell recognizes the pathogen through receptors called Toll-like receptors (TLRs) that each recognize specific pathogen-associated molecular pattern (PAMP). When a TLR recognizes its cognate PAMP, it activates inducible nitric oxide synthase (NOS2), filling the phagosome with NO and destroying the pathogen. By developing a fluorescent reporter that directly images PAMP-triggered NOS2 activity in real time, we show that single-stranded RNA acts as a PAMP in zebrafish by activating its cognate TLR. Such a technology can help identify new PAMPs, pinpoint ligands for TLRs of unknown function, and suggest how PAMPs might synergize to help bugs evade the phagosomal deathtrap.Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP−TLR interactions in diverse organisms.