Modulation of lipopolysaccharide-induced gene transcription by IL-10: focus on transcription factor and chromatin modifications

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. 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 inflammatory cytokine gene expression in human monocytes and neutrophils exposed to lipopolysaccharide (LPS). In particular, the study is focused on two main goals: (A) characterization of the molecular mechanisms through which IL-10 potentiate the transcription of the LPS-induced anti-inflammatory mediator, the Interleukin 1 receptor antagonist (IL-1ra); (B) characterization of the molecular mechanisms through which IL-10 inhibits the LPS-induced transcription of two pro-inflammatory chemokine and cytokine, CXCL8 and TNF-a respectively. (A) Our results show that in monocytes and neutrophils while NF-kBp65 and NF-kBp50 proteins accumulate into the nuclei and bind to the promoter of IkBa during LPS stimulation, they are not recruited to the kB sites of the IL-1ra promoter. However, in the presence of LPS plus IL-10, which we found to induce chromatin acetylation, recruitment of both NF-kBp65 and NF-kBp50 to the IL-1ra promoter efficiently occurs in a STAT3-dependent manner. Accordingly, a failure of IL-10 to promote NF-kBp65 recruitment to the IL-1ra promoter, and consequently to potentiate LPS-induced IL-1ra transcription, was observed in neutrophils from hyper IgE syndrome patients, who carry a non-functional STAT3. Our study uncovers a gene-specific targeting of chromatin structure as a previously undescribed mechanisms used by IL-10 to modulate LPS-induced IL-1ra expression in human monocytes and neutrophils. The relevance of such mechanism is supported by the observations made in HIES patients, in whom the defective IL-10-induced activation of STAT3 is responsible for an overly activated innate immune responses. (B) In the second part of the thesis we addressed the question how does IL-10 inhibit LPS-induced pro-inflammatory gene transcription, focusing the study on CXCL8 and TNF-a. Using quantitative chromatin immunoprecipitation (ChIP) analysis, we characterized the molecular events triggered by IL-10 at the level of CXCL8 and/or TNF-a gene promoters. Our data show that IL-10 triggers cell type specific mechanisms to inhibit LPS-induced CXCL8 and TNF-a transcription. In fact, IL-10 inhibits LPS-induced recruitment of Pol II to the CXCL8 and TNF-a promoter in neutrophils, but not in monocytes. Reduction of Pol II recruitment to the CXCL8 and TNF-a promoters in neutrophils correlates with a reduction of the levels of LPS-induced c-fos recruitment to the same promoters, thus suggesting a cause-effect relationship between these two events. Conversely, in monocytes, IL-10 reduces the recruitment of CDK9 induced by LPS and, as a consequence, inhibits LPS-induced activation (i.e. ser-2-phosphorylation) of promoter-bound Pol II. Inhibition of CDK9 recruitment by IL-10 proceeds in a gene specific manner. In fact, in the case of CXCL8, IL-10, via the recruitment and/or activation of an histone deacethylase, reduces the levels of acetylated histone H4. This results into the inhibition of Brd4 and, subsequently, of the Pol II activating Cyclin kinase 9 recruitment to the CXCL8 promoter. In the case of TNF-a, the decrease in promoter-bound CDK9 is likely to be dependent on IL-10-mediated reduction of LPS-induced association of c-Jun to the TNF-a promoter. Collectively, our data unravel novel and cell type-specific mechanisms utilized by IL-10 to inhibit LPS-induced CXCL8 and TNF-a transcription.