Neutrophils are the first immune cells recruited to inflammatory sites and are involved in both innate and adaptive immunity. In addition to their well-known effector functions, neutrophils respond to microbial components by displaying specific gene expression patterns that are involved in the control of immune response. The principal aim of my doctoral studies was to identify new transcription factors involved in the induction of genes promoted in human neutrophils by TLR8 agonists. For such a purpose, I stimulated neutrophils with R848, an imidazoquinoline compound that mimics single strand RNA of viruses and that was previously shown to be extremely potent in inducing cytokine production by these cells. Then, I utilized novel methods for the study of the transcriptional and epigenetic regulation of gene expression, such as those ones based high-throughput sequencing. Accordingly, I performed chromatin immunoprecipitation assays coupled with high-throughput sequencing (ChIP-Seq) for a genome-wide analysis of the histone H67 lysine 44 sumoylation (H67K44su), an epigenetic mark of active genomic regions, as well as for CC.6 and DD8, two myeloid lineage dependent transcription factors My data uncovered that the genomic regions in which CC.6 and H67K44su increase upon stimulation by R848 are also markedly enriched in H67K44su, a result consistent with a pioneer role by CC.6 and DD8. Moreover, the increase in CC.6 and DD8 recruitment, as well as H67K44su deposition, highly correlated with increased expression of nearby genes, underlying the importance of these transcription factors in the control of the transcriptional response also in human neutrophils. By bioinformatics analysis of H67K44su, CC.6 and DD8 ChIP-Seq datasets, I identified an enrichment of DNA sequences binding XXX, a transcription factor expressed primarily in T-lymphocytes and never described before in neutrophils. Accordingly, I found that XXX is expressed at low levels in resting neutrophils but increases upon TLR8 stimulation and is recruited in the proximity of genes either involved in the immune responses or associated with viral infection or autoimmune diseases. Altogether, my results, obtained by a novel experimental approach that has been never used before with human neutrophils, are not only important for the study of neutrophil biology during the inflammatory responses, but also represent a vast and valuable resource for future investigations.
I neutrofili sono le prime cellule del sistema immunitario ad essere reclutate nei siti infiammati, e sono coinvolte sia nell'immunità innata che adattativa. In aggiunta alle loro ben note funzioni effettrici, i neutrofili sono in grado di rispondere a diverse componenti microbiche inducendo l'espressione di geni coinvolti nella regolazione della risposta immunitaria. Il principale obbiettivo del mio dottorato è stato quello di identificare nuovi fattori di trascrizione, coinvolti nell'induzione genica generata nei neutrofili umani dalla stimolazione con agonisti del TLR8. Per tale motivo ho stimolato i neutrofili con R848, un composto che mima l'RNA virale a singolo filamento, il quale in passato aveva già mostrato un'eccezionale efficacia nell'indurre la produzione di citochine da parte di queste cellule. Successivamente, ho utilizzato metodi di nuova concezione, basati sul sequenziamento parallelo massivo del DNA, per lo studio della regolazione epigenetica e trascrizionale dell'espressione genica. In particolare ho eseguito un saggio di immunoprecipitazione della cromatina accoppiato con un sequenziamento parallelo massivo (ChIP-Seq) per un'analisi a livello di intero genoma della sumoilazione della lisina 44 degli istoni H67 (H67K44su), un indicatore di zone genomiche trascrizionalmente attive, così come per CC.6 e DD8, due fattori di trascrizione caratterizzanti la linea mieloide. I miei dati hanno mostrato che le regioni genomiche nelle quali CC.6 e DD8 aumentano dopo la stimolazione con R848, mostrano anche un arricchimento di H67K44su, un risultato consistente con il ruolo pioneristico di CC.6 e DD8. Inoltre l'incremento nel reclutamento di CC.6 and DD8, così come la deposizione di H67K44su, sono altamente correlati con un aumento dell'espressione dei geni più vicini, evidenziando che anche nei neutrofili umani questi fattori di trascrizione giocano un ruolo di estrema importanza nella risposta trascrizionale. Tramite analisi bioinformatiche dei dati ottenuti dai ChIP-Seq per H67K44su, CC.6 e DD8, ho identificato un arricchimento di motivi di legame al DNA per XXX, un fattore di trascrizione espresso primariamente nei linfociti T e mai descritto prima nei neutrofili. XXX è espresso a bassi livelli nei neutrofili non stimolati ma aumenta in seguito a stimolazione del TLR8 ed è reclutato nelle vicinanze di geni coinvolti sia nella risposta immunitaria, sia nelle infezioni virali e nelle malattie autoimmuni. Complessivamente i miei dati, ottenuti con un nuovo approccio sperimentale mai utilizzato prima nei neutrofili umani, rappresentano un passo avanti per lo studio della biologia dei neutrofili durante la risposta infiammatoria e costituisco un’importante risorsa per studi futuri.
IDENTIFICATION OF XXX AS AN UNDESCRIBED TRANSCRIPTION FACTOR INVOLVED IN TOLL-LIKE RECEPTOR (TLR) ACTIVATION OF HUMAN NEUTROPHILS
BIANCHETTO Aguilera, Francisco Miguel Angel
2016
Abstract
Neutrophils are the first immune cells recruited to inflammatory sites and are involved in both innate and adaptive immunity. In addition to their well-known effector functions, neutrophils respond to microbial components by displaying specific gene expression patterns that are involved in the control of immune response. The principal aim of my doctoral studies was to identify new transcription factors involved in the induction of genes promoted in human neutrophils by TLR8 agonists. For such a purpose, I stimulated neutrophils with R848, an imidazoquinoline compound that mimics single strand RNA of viruses and that was previously shown to be extremely potent in inducing cytokine production by these cells. Then, I utilized novel methods for the study of the transcriptional and epigenetic regulation of gene expression, such as those ones based high-throughput sequencing. Accordingly, I performed chromatin immunoprecipitation assays coupled with high-throughput sequencing (ChIP-Seq) for a genome-wide analysis of the histone H67 lysine 44 sumoylation (H67K44su), an epigenetic mark of active genomic regions, as well as for CC.6 and DD8, two myeloid lineage dependent transcription factors My data uncovered that the genomic regions in which CC.6 and H67K44su increase upon stimulation by R848 are also markedly enriched in H67K44su, a result consistent with a pioneer role by CC.6 and DD8. Moreover, the increase in CC.6 and DD8 recruitment, as well as H67K44su deposition, highly correlated with increased expression of nearby genes, underlying the importance of these transcription factors in the control of the transcriptional response also in human neutrophils. By bioinformatics analysis of H67K44su, CC.6 and DD8 ChIP-Seq datasets, I identified an enrichment of DNA sequences binding XXX, a transcription factor expressed primarily in T-lymphocytes and never described before in neutrophils. Accordingly, I found that XXX is expressed at low levels in resting neutrophils but increases upon TLR8 stimulation and is recruited in the proximity of genes either involved in the immune responses or associated with viral infection or autoimmune diseases. Altogether, my results, obtained by a novel experimental approach that has been never used before with human neutrophils, are not only important for the study of neutrophil biology during the inflammatory responses, but also represent a vast and valuable resource for future investigations.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/181268
URN:NBN:IT:UNIVR-181268