Epigenetic Mechanisms in Monocyte-Associated Differentation and Inflammation Processes

Abstract

[eng] My doctoral thesis focused on the study of the epigenetic mechanisms involved in different MO-associated differentiation and inflammation processes. I described the molecular sequential events that lead to changes in the pattern of DNA methylation, as well as their significance in the context of inflammation. Terminal MO differentiation to dendritic cells (DC) and macrophages (MAC) can be recapitulated in vitro by the addition of external factors to the culture media. In humans, MO are differentiated to DC by the addition of GM-CSF and IL-4, and to MAC by only the addition of GM-CSF. In order to be functional, those cellular types could be maturated by the addition of bacterial lipopolysaccharide (LPS) which triggers TL4 signalling. I performed high-throughput DNA methylation in MO-to-DC and MO-to-MAC differentiation and in DC and MAC maturation. Extensive loss of DNA methylation occurs in both MO-to-MAC and MO-to-DC differentiation processes, whereas only few genes vary their methylation status following LPS treatment (maturation level). More interestingly, genes that are essential for cell identity were differentially methylated between MO-to-DC and MO-to-MAC differentiation, in turn indicating a possible role of methylation in cell fate determination. Since DC and MAC differentiation differ from each other by the addition of IL-4 in the former, I tracked the JAK3-STAT6 pathway downstream to IL-4. I demonstrated that the JAK3/STAT6 axis is the main pathway responsible for orchestrating changes in genes that are exclusively DNA demethylated in DC, via a mechanism dependent of Ten-Eleven Translocation 2 (TET2), an enzyme involved in active DNA methylation. A dual role for JA3/STAT6 was observed: the JAK3/STAT6 pathway was also in charge of preventing DNA demethylation of those MAC-specific genes during MO-to-DC differentiation as a mechanism in DC to prevent gene expression of genes from another lineage (MAC lineage). To our knowledge, this is the first time that the cytokine-mediated downstream sequence of events leading to direct gene-specific demethylation in differentiation of innate immune cells are described. Despite the fact that MO-to-DC and MO-to-MAC differentiation are mainly associated with DNA demethylation, I identified the acquisition of de novo DNA methylation in a few genes in both MO differentiation processes. Analysis of the dynamics of methylation and expression changes of these genes revealed that loss of expression is rapid and associated with loss of H3K4me3 and H3K36me3, whereas gains of DNA methylation are progressive and partially concomitant with increases in H3K9me3 and H3K27me3. Inhibition of DNMT3b, which is an enzyme involved in de novo DNA methylation, does not have any effects on gene expression nor on the loss of H3K4me3. On the other hand, DNMT3b inhibition produces impairment on DNA hypermethylation and a loss of H3K27me3. Most importantly, DNMT3b inhibition causes a loss of specific, surface DC markers, which highlight a role of de novo DNA methylation in gene expression stabilization needed for a proper phenotype acquisition. Our data give rise to a novel perspective on the functional relevance and mechanisms of the acquisition of DNA methylation in myeloid cell differentiation. A detailed analysis of DNA demethylated genes in MO-to-MAC differentiation revealed the presence of a subset of genes related to inflammasome function, which is a cellular complex activated during inflammation and involved in IL-1β secretion, and a type of cellular death called pyroptosis. I demonstrated that DNA demethylation of several inflammasome-related genes takes place during MO-to-MAC differentiation and MO activation, and that in both cases, DNA methylation is controlled by the Nuclear Factor Kappa B (NF-kB) pathway. This DNA demethylation occurs through an active mechanism mediated by TET2 and is crucial for the transcriptional control of inflammasome-related genes as well as for inflammasome activity. Interestingly, my study shows higher rates of DNA demethylation in MOs from patients with autoinflammatory diseases (Cryopyrin Associated Periodic Syndrome (CAPS) and Familial Mediterranean Fever(FMF)) than in control samples. Overall, these results reveal a potential, novel mechanism for the activation of inflammasome function by DNA demethylation. Moreover, I identify for the first time alterations on DNA methylation in patients with autoinflammatory diseases.

[cat] En aquesta tesi s’han estudiat els mecanismes epigenètics implicats en diferents processos d’inflamació i diferenciació a partir de monòcits (MO). És possible recapitular la diferenciació terminal de MO a cèl·lules dendrítiques (DC) i macròfags (MAC) mitjançant l’addició de factors externs al medi de cultiu. En humans, els MO són diferenciats a DC per l’addició de GM-CSF i IL-4, i a MAC si sols afegim GM-CSF. En la meua tesi he descrit com la diferenciació a MAC i a DC, va associada amb una desmetilació global del DNA. Així, he vist com gens claus per a la identitat de les cèl·lules DC/MAC estan regulats per la desmetilació del DNA. Com que la citoquina IL-4, és el factor diferencial entre la diferenciació a DC i MAC, vam rastrejar la via de senyalització JAK3-STAT6 que és troba just per davall de IL-4. Això ens va permetre veure com JAK3/STAT6 és la via principal implicada amb la desmetilació dels gens exclusivament desmetilats i expressats en DC, via un mecanisme dependent de IL-4. Fins on nosaltres sabem, aquesta és la primera vegada en la que es descriuen els esdeveniments seqüencials que porten a una desmetilació específica del DNA durant la diferenciació de cèl·lules del sistema innat. Malgrat que majoritàriament vam observar una desmetilació durant els diferents processos de diferenciació a partir de MO, vam ser capaços d’identificar com per alguns gens silenciats durant el procés de diferenciació és produïa un augment de novo de la metilació. A la meua tesis he demostrat que, en aquest cas, la metilació no estava associada a la repressió de l’expressió gènica, sinó que estava lligada a l'estabilització de l'expressió gènica, donant així una nova perspectiva de la funció de la metilació del DNA en aquest context. L’inflamasoma és un complex proteic implicat entre altres coses, en la secreció de citoquines inflamatòries com la IL-1β, i que es trova alterat en pacients amb sindromes autoinflamatòris. Al nostre estudi, vam veure que, durant la diferenciació de MO a MAC gens associats a l’activació de l’inflamasoma es trobaven regulats per metilació. Aquesta desmetilació és produïa via un mecanisme actiu dependent de l’acció del Factor Nuclear Kappa B (NF- κB). A més a més, vam veure com pacients amb enfermetats autoinflamatòries presenten alteracions amb el patró de metilació, sent el primer estudi que descriu alteracions epigenètics en aquests pacients.