Dual ligand stimulation of RAW 264.7 cells uncovers feedback mechanisms that regulate TLR-mediated gene expression

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-γ, 2-methyl-thio-ATP (2MA), PGE₂, and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducioly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE₂ and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-γ and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-γ-induced changes in gene expression at 1 h, IFN-γ signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-γ- response genes by augmenting STAT1 activity and by activating NF-κB, which synergizes with IFN-γ-induced transcriptional factors; 2) IFN-γ attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokiae-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-γ and inhibiting IL-10 gene expression; 4) PGE₂ and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.