IL-10 primarly targets transcription of LPS-induced genes in human polymorphonuclear neutrophils

The mechanisms through which the anti-inflammatory cytokine IL-10 modulates the production of multiple mediators from activated immune cells have been extensively investigated over the last years, nevertheless several feature of IL-10 signalling are still controversial. In the present study, we analyzed the mechanism of IL-10-mediated modulation of cytokine expression in primary human neutrophils, given the crucial role that these cells play in directing and shaping the subsequent inflammatory and immune responses. We have recently shown that, differently from mononuclear cells, responsiveness of human neutrophils to IL-10 is strictly dependent on the levels of IL-10R1 expression, which is undetectable in freshly isolated neutrophils and upregulated upon exposure to LPS. Based on this knowledge, IL-10 effects on cytokine and chemokine expression has been investigated on neutrophils that have been “pre-conditioned” by LPS for few hours, and were readily responsive to this cytokine. The same analysis has been conducted in parallel on human monocytes purified from the same donors and immediately challanged with LPS with or without IL-10. Under these circumstances, we found that IL-10 targets LPS-induced gene expression at the level of transcription, both in human neutrophils and monocytes. Indeed, by using “Primary Transcript” RT Q-PCR, we were able to estimate that the accumulation of newly transcribed genes triggered by LPS was inhibited (in the case of TNF-and CXCL8) or enhanced (in the case of IL-1ra and SOCS-3) by IL-10. We then addresses the controversial question of the role of new protein synthesis in IL-10 anti-inflammatory effects. In the presence of cycloheximide IL-10 effects on LPS-induced genes were no longer observed in monocytes. In contrast, in neutrophils IL-10 continued to modulate the expression of LPS-induced genes independently of the presence of a protein synthesis inhibitor, indicating that IL-10 anti-inflammatory effects are mediated through a direct action of STAT3 on the transcriptional rate of the target genes. Moreover, in contrast to studies proposing that IL-10 inhibitory activity proceed via inhibition of NF-kB activation, we show that in neutrophils a different mechanism should be envisioned. In fact, IL-10 did not modify LPS-activated NF-kB nuclear translocation nor it modified LPS-induced transcription of IkB an early NF-kB-target gene