REGULATORY ROLE OF MICRORNAS ON INFLAMMATORY SIGNALING PATHWAYS

Innate immunity is the primary defense mechanism that recognizes and respond to invading infectious microbes or their components, known as pathogen-associated molecular patterns (PAMPs), or to danger signals that comes from self components, known as danger-associated molecular patterns (DAMPs). These molecules are recognized in our body by the so called pattern recognition receptors (PRRs), whose activation quickly gives rise to an important cascade of events known as acute inflammatory response. Within few hours, pro-inflammatory cytokines, such as TNFα, IL-1β and IL6, and chemokine, such as CCL2, CCL3 or CXCL8, are released in the blood stream, and innate immune cellular components are induced to combat the pathogens to the site of injury. Circulating neutrophils and monocytes are implicated as essential players in defense against a range of microbial pathogens. The acute inflammation process is a double-edge sword. Normally, it terminated once triggering insult is eliminated, the infection is cleared and damage tissue is repaired. Misregulation of one or more step from initiation to resolution can significantly contribute to the pathogenesis of autoimmune, chronic inflammatory or infectious diseases. For this reason, the inflammatory response itself and its termination phase are active and highly regulated processes involving several key regulatory mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs recently emerged as powerful posttranscriptional regulators in various biological processes. A growing number of evidence suggests that the development and function of cells in the immune system is also subject to regulation by miRNAs, and in 2006 the first evidence on their potential involvement in inflammation control was provided by Taganov and colleagues, who reported a unique set of microRNAs (miR-146a, miR-155 and miR-132) overexpressed in the THP-1 monocytic cell line after TLR4 agonist engagement, and postulated that miR-146a may operate a negative feedback loop acting on TRAF6 and IRAK1, two keys adaptors in TLR4 signaling pathway. Moving from these information, this thesis project has characterized the complete microRNA expression profile induced by TLR4 activation in freshly purified human blood monocytes and neutrophils. Beyond the aforementioned miR-155, miR-146a and miR-132, a new set of molecules were first described as LPS-responsive miRNAs in monocytes, including mi-9, miR-187, and the miR-99b~7e~125a miR cluster. This study also identified miR-9 as the only microRNA also up-regulated in neutrophils in a MyD88- and NF-κB-dependent manner and highlighted a new feedback regulatory loop acting at the NF-kB level, as miR-9 was demonstrated to directly target NFKB1 mRNA and post-transcriptionally modulating its expression. Inflammatory response triggers an important number of events that bring not only to propagation (producing pro-inflammatory mediators such as TNFα or IL-1β) but also to resolution. Two fundamental anti-inflammatory mediators whose release is induced by inflammation itself are IL-10 and glucocorticoids (GC), that act in a autocrine/paracrine or systemic manner, respectively. Our work has revealed that miR-187 and miR-99b~7e~125a miR cluster induction by LPS resulted by an IL-10-dependent loop. Bioinformatic tools and cellular and biochemical assays allowed us to uncovering the function of these new IL-10-dependent miRNAs in the modulation of several proinflammatory cytokines and chemokines (TNFα, IL-6, CCL3, CXCL8) acting at different steps of the signaling pathway that bring to their production. Indeed, both the transcription factor IκBζ and the signalling complex TLR4/CD14 were found to be targets of miR-187 and miR-99b~7e~125a miR cluster, respectively. These results underline a new mechanism for IL-10 in inducing pro-inflammatory genes silencing. In parallel, GC were shown to induce expression of miR-511 in a synchronized fashion with its host gene, the mannose receptor MRC1 (also known as CD206). Bioinformatic tools highlighted a relevant number of molecules involved in TGFβ signalling among miR-511 predicted target genes, and miR-511 was shown to block MAPK cascade and gene expression induced upon TGFβ stimulation. Taken together, these results suggest that miRNA represent an emerging mechanism active in leukocytes to dampen inflammation and avoid exacerbated inflammatory mediators release by a multitargeting strategy affecting several key signalling pathways and transcription factors.