IL-10 and miRNAs as modulators of gene expression in lipopolysaccharide-activated neutropils and monocytes

The inflammatory process consists of a coordinated, sequential and self-limiting release of different mediators that orchestrate and control the chronological phases of leukocytes recruitment, activation, and clearance. In this contest, it is well known that the appropriate balance between pro- and anti-inflammatory cytokines production and action is essential for successful innate immune response that clears infectious pathogens but limits tissue damage and autoimmunity. Based on these knowledges, it is evident that the characterization of the pathways and checkpoints that regulate cytokine gene expression during physiologic inflammation is of particular importance, since it will yield insights into fundamental events occurring in disorders characterized by deregulated inflammation. The amplitude and the duration of the inflammatory response are fine-tuned by Interleukin (IL)-10, a potent antinflammatory cytokine produced, among others, by myeloid cells in response to Toll-like receptor (TLR) activation and, in turn, very effective at suppressing TLR-induced gene expression and inflammatory cytokine production. Although IL-10 antinflammatory properties have been thoroughly described, the molecular mechanisms governing IL-10 production and actions on cells of the innate immune system have not been fully elucidated yet. The purpose of the thesis is to provide a comprehensive characterization of the molecular mechanisms through which IL-10 modulates cytokine gene expression in human neutrophils and monocytes exposed to lipopolysaccharide (LPS). In particular, the study is focused on two main goals: (A) characterization of the mechanisms through which IL-10 modulates cytokine gene expression at the transcriptional level; (B) analysis of the role of IL-10 on post-transcriptional regulation of neutrophils and monocytes gene expression, via modulation of miRNAs. (A) Our results demonstrate that, in neutrophils and monocytes stimulated with LPS, IL-10 primarily targets the transcription of TNF-α, CXCL8 and IL-1ra genes, as revealed by Primary Transcript (PT) real-time RT-PCR. In addition, we show that the transcriptional repression of TNF-α and CXCL8 gene expression induced by IL-10 consists of two distinct phases: an early one, occurring rapidly and in a protein synthesis-independent manner, followed by a second phase, more delayed and dependent on protein synthesis. (B) In the second part of the thesis we addressed the question whether IL-10 could regulate LPS-induced cytokine expression also by using post-transcriptional mechanisms, in particular through the action of miRNAs. We initially characterized the profile of miRNAs induced in response to LPS. Subsequently, the effect of IL-10 on LPS-induced miRNA expression was characterized. This analysis, performed on a large scale, revealed a previously unrecognized ability of IL-10 to modulate the expression of miRNA induced in response to LPS. We specifically characterized the induction and the regulatory functions of LPS-induced miR-9 and miR-187, being the only miRNAs respectively down- or up-regulated by IL-10 in both cell types. Finally, NFKB1/p105/p50 was identified in silico and experimentally as a miR-9 target. In summary, the present work demonstrated that IL-10 can modulate the expression of both cytokines and miRNAs at the transcriptional level. In turn, , IL-10-regulated miRNAs could post-transcriptionally influence the expression of specific target genes involved in the regulation of transcription. Collectively these results suggest that IL-10 is able to influence the final output of neutrophils and monocytes gene expression by acting directly at the level of transcription and indirectly, via miRNA, at a post-transcriptional level.