IL-10 Rapidly Inhibits Transcription of Secondary Response Genes in LPS-Stimulated Macrophages

IL-10 is an anti-inflammatory cytokine that is essential for maintaining intestinal homeostasis. Here we examined the kinetics of IL-10-mediated suppression following LPS-stimulation of bone marrow-derived macrophages (BMDM). Following a delay, mRNA for the secondary response gene Il12b briefly increased after LPS stimulation, but then quickly leveled off at a steady state, concordant with the time that IL-10 was first detected in the culture medium. Treatment with actinomycin D in this steady-state phase demonstrated that the Il12b message was highly stable, indicating that IL-10 was not influencing mRNA stability. Using RT-PCR primers directed at pre-mRNA as a marker for active transcription, we found that production of Il12b pre-mRNA was transient, suggesting that IL-10 was inhibiting transcription of Il12b.  Studies in Il10^-/- BMDM confirmed that both Il12b mRNA and pre-mRNA were induced at significantly higher levels in the absence of IL-10. Remarkably, addition of IL-10 to LPS-stimulated Il10^-/- BMDM that were actively transcribing Il12b led to the rapid inhibition of transcription, as well as loss of RNA Pol II association with the Il12b promoter. Interestingly, while at early time points following LPS stimulation IL-10 had little effect on transcription of the primary response gene Cxcl2, it did inhibit transcription and association of RNA Pol II with the Cxcl2 promoter at later time points. Thus, IL-10 rapidly inhibits the transcription of target genes during the secondary phase of the response to LPS. We hypothesize that IL-10 interferes with the function of an inducible transcriptional regulator involved in directing the secondary response to LPS.