Induced negative regulation decodes PAMP quality and quantity to generate bacteria-specific inflammatory responses

Toll-like receptors (TLRs) expressed on macrophages are ligated in various combinations by diverse pathogen-associated molecular patterns (PAMPs), but how mixtures of signals through these receptors are integrated to mediate distinct biological responses to different pathogens remains unknown. By stimulating macrophages using a wide matrix of TLR2/TLR4 concentration combinations, we identified conditions resulting in non-linear responses. Transcriptomic and RNAi analyses identified negative regulators induced in a TLR-specific manner that regulated cytokine production dynamics and chemokine milieus across the combinatorial matrix. TLR4 ligand concentration explained much of the variation in production of many chemokines. However, combinations of sub-maximal amounts of either single ligand produced a zone of non-linear responses with sustained production of select inflammatory mediators, due to lack of negative regulation induced only at higher individual ligand concentrations. A similar sustained response was observed for Gram-positive bacteria, as compared to a panel of Gram-negative species, which express high amounts of the TLR4 ligand LPS. The novel TLR4-specific negative regulators we describe thus aid the innate immune system in discriminating Gram-negative from Gram-positive species, supporting qualitatively distinct inflammatory responses to these classes of bacteria. This research was supported by the Intramural Research Program of the NIH, NIAID