Dual Ligand Stimulation of RAW 264.7 Cells Uncovers Feedback Mechanisms That Regulate TLR-Mediated Gene Expression

Abstract

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 reproducibly 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 cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-β 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.

Francisella tularensisInduces Ubiquitin-Dependent Major Histocompatibility Complex Class II Degradation in Activated Macrophages

“…PGE 2 has been well documented to act in an autocrine manner, acting back on the producer cell to induce the secretion of nonprostaglandin factors such as matrix metalloproteinase-9 (60) and IL-6 (27), which are involved in multiple types of infection (3,37,44). Using gene chip analysis, Zhu et al have reported a number of macrophage genes that are induced after stimulation by PGE 2 (61). Only one of the induced genes (i.e., IL-1␤) is associated with downregulation of MHC class II expression, although via CIITA-mediated inhibition of class II transcription (48,61).…”

“…Using gene chip analysis, Zhu et al have reported a number of macrophage genes that are induced after stimulation by PGE 2 (61). Only one of the induced genes (i.e., IL-1␤) is associated with downregulation of MHC class II expression, although via CIITA-mediated inhibition of class II transcription (48,61). Interestingly, like the factor responsible for class II downregulation by F. tularensis infection supernatants, IL-1␤ is Ͼ10 kDa and resistant to PK treatment (25).…”

Toll-like receptor signal transduction

“…On a global front, the analysis of high throughput experimental data has demonstrated more detailed understanding of large scale gene expressions in innate immune system (Ricciardi-Castagnoli and Granucci, 2002;Zhu et al, 2004Zhu et al, , 2006Hirotani et al, 2005;Nilsson et al, 2006). Despite this, we are yet to have a comprehensive view of TLR signaling events due to the lack of understanding in the interplay between the dynamics of gene regulatory networks and signaling pathways.…”

Rapid Temporal Dynamics of Transcription, Protein Synthesis, and Secretion during Macrophage Activation

“…For instance, Ramsey et al combined mRNA expression analysis using microarrays with motif scanning of transcription factor binding sites, inferring a network of asso-ciations between transcription factor genes and clusters of co-expressed target genes (8). Similarly, transcriptome analyses revealed crosstalk between pathways downstream of TLR4 (9) and among the three transcription factors NF-B, C/EBPdelta, and ATF3 that discriminates between transient and persistent TLR4-induced signals (10).…”