Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils

How IL-6 expression is regulated in human neutrophils has remained unclear. Here the authors show, using highly purified neutrophils, that TLR8 or TLR4 signalling activates latent enhancers and cooperates with autocrine TNFα to induce IL-6 transcription

Cell-specific epigenetic landscapes drive differential cytokine expression in human neutrophils and monocytes

I neutrofili polimorfonucleati fungono da prima linea di difesa nella risposta immunitaria innata dell\u2019ospite contro microorganismi patogeni. Inoltre, numerose ricerche condotte negli ultimi 20 anni hanno permesso di capire quanto i neutrofili siano cellule versatili, in grado di svolgere anche ruoli importanti nel collegamento tra la risposta immunitaria innata e quella adattativa. Quest\u2019ultima funzione avviene in parte grazie alla capacit\ue0 dei neutrofili di sintetizzare de novo e in seguito rilasciare una grande variet\ue0 di citochine. In letteratura non \ue8 presente un generale accordo sulla capacit\ue0 da parte dei neutrofili di produrre le citochine IL-6 (ad azione sia pro- che anti-infiammatoria) e IL-10 (ad azione esclusivamente anti-infiammatoria), entrambe mediatori molto importanti per un funzionamento bilanciato del sistema immunitario. Di conseguenza l\u2019obiettivo del nostro studio \ue8 stato quello di chiarire, per mezzo di analisi a livello della struttura della cromatina, se neutrofili umani isolati al massimo grado di purezza, esprimano o meno IL-6 in risposta a vari stimoli, inclusi agonisti dei recettori TLR. Come ulteriore obiettivo ci siamo inoltre proposti di verificare se l\u2019incapacit\ue0 dei neutrofili di produrre IL-10, come da noi precedentemente osservato, avesse possibili spiegazioni sempre a livello epigenetico. Una visione completa di come l\u2019espressione di IL-6 e IL-10 sia regolata a livello molecolare in tipi cellulari specifici, ha ovvie implicazioni per una migliore comprensione della patogenesi di malattie infiammatorie, cos\uec come per eventuali strategie immunoterapiche. Tra i risultati ottenuti all'interno dell'attivit\ue0 di ricerca sviluppata durante il corso di dottorato, \ue8 stato osservato che neutrofili umani isolati al massimo livello di purezza sono in grado di produrre IL-6 in risposta ad agonisti del TLR8 e, in maniera meno efficiente, TLR4. E\u2019 interessante notare che in neutrofili e monociti autologhi sono state riscontrate cinetiche di espressione e rilascio di IL-6 molto pi\uf9 ritardate nei neutrofili rispetto ai monociti. La spiegazione della ritardata espressione di IL-6 \ue8 stata individuata nella conformazione chiusa e inattiva della cromatina nel locus genomico di IL-6 in neutrofili non stimolati. In monociti autologhi invece il locus di IL-6 \ue8 risultato essere pi\uf9 pronto per un rapido rimodellamento della cromatina e quindi della trascrizione. Tale differenza \ue8 stata indicata dalla mancanza di diverse modificazioni degli istoni, tra le quali H3K4me1, H3K27Ac e H4Ac, sul locus di IL-6 nei neutrofili ma non nei monociti, come identificato mediante immunoprecipitazione della cromatina (ChIP). Tuttavia, nei neutrofili, la cromatina sul locus di IL-6 viene riorganizzata in seguito a stimolazione con R848, come indicato dal reclutamento di PU.1, C/EBP\u3b2, NF-\u3baB e dall\u2019acquisizione di livelli significativi di modificazioni degli istoni (H3K27Ac, H4Ac, H3K4me1 e H3K4me3) in enhancers indotti de novo o nel promotore. Nel presente studio di dottorato \ue8 stato inoltre osservato che l\u2019espressione di IL-6 in neutrofili e monociti \ue8 regolata mediante l\u2019uso da parte dei due tipi cellulari di diversi enhancers. Infatti, tra i vari enhancers indotti de novo, ne \ue8 stato individuato uno posizionato 14 kb a monte del sito d'inizio della trascrizione (TSS) peculiare per i neutrofili mentre un altro, posizionato 64 kb a monte del TSS, \ue8 invece risultato essere un sito regolatorio pi\uf9 specifico per i monociti. Inoltre, in neutrofili stimolati a tempi lunghi con R848, \ue8 stato osservato un ruolo di amplificazione dell\u2019espressione di IL-6 da parte del TNF\u3b1 endogeno, esercitato da questa citochina mediante la modificazione della cromatina nel locus di IL-6 e la prolungata induzione dell\u2019espressione proteica di IkBz, portando quindi ad un aumentato legame di fattori di trascrizione nelle regioni genomiche regolatorie di IL-6. Come il locus di IL-6, anche il locus di IL-10 \ue8 stato confermato essere chiuso/inattivo in neutrofili non stimolati, come indicato mediante saggi di ChIP dalla mancanza delle modificazioni degli istoni H3K4me3, H3K27Ac, H4Ac nei neutrofili e contrariamente a quanto osservato nei monociti. Diversamente dal locus di IL-6 per\uf2 il locus di IL-10 in neutrofili umani, isolati sia da donatori sani che da pazienti affetti da melanoma, \ue8 stato dimostrato mantenere un\u2019organizzazione della cromatina inattiva anche in seguito a stimolazione con LPS, SAA o Pam3CSK4, come provato mediante ChIP dalla mancata induzione delle modificazioni degli istoni e dal reclutamento di fattori di trascrizione. Gli eventi sopracitati sono invece stati osservati sia in monociti autologhi che in neutrofili murini. Nel complesso, i risultati ottenuti all'interno dell'attivit\ue0 di ricerca sviluppata durante il corso di dottorato hanno permesso di chiarire le controversie presenti in letteratura a riguardo della capacit\ue0 dei neutrofili umani di sintetizzare IL-6 e IL-10 e scoprire che, anche in queste cellule, l\u2019espressione delle citochine pu\uf2 essere regolata in maniera sostanziale a livello epigenetico.Polymorphonuclear neutrophils typically function at the frontline of innate defense in host responses to foreign microorganisms. In addition, extensive research performed in the last 20 years has contributed to recognize neutrophils as versatile cells, also displaying an important role in linking the innate and adaptive arms of the immune response. The latter function occurs, in part, by virtue of the capacity of neutrophils to de novo synthesize and release a vast repertoire of cytokines. There is not a general consensus in the literature as to whether human neutrophils produce pro- or anti-inflammatory IL-6 or anti-inflammatory IL-10, both cytokines being highly important for the balanced functioning of the immune system. Consequently, the objective of the study was to clarify, by studies at the level of chromatin structure, whether highly pure human neutrophils express IL-6 in response to various stimuli, including TLR agonists, and whether we could confirm, at chromatin level, our previous observation about neutrophils\u2019 incapacity to produce IL-10. A complete understanding of how IL-6 and IL-10 expression is regulated at molecular level in specific cell types has obvious implications for a better comprehension of inflammatory disease pathogenesis, as well as for immunotherapeutic strategies. As a result of the doctoral study, it was observed that highly pure human neutrophils genuinely produce IL-6 in response to agonists for TLR8 and (less efficiently) TLR4. Interestingly, the kinetics of IL-6 expression and release differed between neutrophils and autologous monocytes, being both significantly delayed in neutrophils. The delayed expression of IL-6 was explained by closed/inactive conformation of chromatin in the entire IL-6 locus in resting neutrophils, while it appeared already poised for transcription and more prone for rapid chromatin remodeling in autologous monocytes, indicated by the lack of H3K4me1, H3K27Ac and H4Ac marks at the IL-6 locus in neutrophils while being readily present in monocytes, as discovered by chromatin immunoprecipitation (ChIP). Remarkably, IL-6 locus in neutrophils underwent chromatin reorganization upon R848-treatment, indicated by recruitment of PU.1, C/EBP\u3b2, NF-\u3baB and acquisition of significant levels of histone marks (H3K27Ac, H4Ac, H3K4me and H3K4me3) at de novo induced enhancers and at the promoter. Furthermore, in the current doctoral study it was discovered that IL-6 expression in neutrophils and monocytes was regulated by cell type-specific enhancer patterns. In fact, amongst others, de novo enhancer at 14kb upstream of transcription start site (TSS) was induced in neutrophils, but not in monocytes. Instead, an enhancer at 64kb upstream of TSS likely represented more prominent regulatory site in monocytes than in neutrophils. Furthermore, endogenous TNF\u3b1 was found to amplify IL-6 expression at later time points in R848-treated neutrophils by modulating the chromatin landscape at the IL-6 locus and prolonging the induction of I\u3baB\u3b6 antigenic expression, leading to enhanced transcription factor binding. The locus of IL-10, similarly to the locus of IL-6, was confirmed to be closed/inactive in resting neutrophils, indicated by the lack of H3K4me3, H3K27Ac, H4Ac marks at IL-10 locus, by ChIP, in neutrophils, contrary to monocytes. Interestingly, IL-10 locus, differently from IL-6 locus, retained inert chromatin organization even after upon LPS, SAA, or Pam3CSK4 stimulation, both in neutrophils from healthy donors and melanoma patients in contrary to autologous monocytes and murine neutrophils, indicated by the absence of induction of histone modifications and TF recruitment by ChIP. Altogether, the results of this doctoral study clarify controversial literature on the ability of human neutrophils to generate IL-6 and IL-10 and uncover that cytokine expression in these cells can be prominently regulated at epigenetic level

Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modifica-tion status at their genomic locus. We analyzedposttranslational modifications of histones associatedwith genes that are active, repressed, or poised for tran-scriptional activation, including H3K4me3, H4Ac,H3K27Ac, and H3K4me1 marks. Differently from au-tologous IL-10–producing monocytes, none of the marksunder evaluation was detected at the locus of rest-ing or activated neutrophils from healthy subjects ormelanoma patients. By contrast, increased H3K4me3,H4Ac, H3K4me1, and H3K27Ac levels were detectedat syntenic regions of the locus in mouse neutro-phils. Altogether, data demonstrate that human neutro-phils, differently from either monocytes or mouseneutrophils, cannot switch on the gene becauseits locus is in an inactive state, likely reflecting a neutro-phil-specific developmental outcome. Implicitly, dataalso definitively settle a currently unsolved issue on thecapacity of human neutrophils to produce IL-10

Cutting Edge: An Inactive Chromatin Configuration at the IL-10 Locus in Human Neutrophils

To identify the molecular basis of IL-10 expression in human phagocytes, we evaluated the chromatin modifica-tion status at their genomic locus. We analyzedposttranslational modifications of histones associatedwith genes that are active, repressed, or poised for tran-scriptional activation, including H3K4me3, H4Ac,H3K27Ac, and H3K4me1 marks. Differently from au-tologous IL-10–producing monocytes, none of the marksunder evaluation was detected at the locus of rest-ing or activated neutrophils from healthy subjects ormelanoma patients. By contrast, increased H3K4me3,H4Ac, H3K4me1, and H3K27Ac levels were detectedat syntenic regions of the locus in mouse neutro-phils. Altogether, data demonstrate that human neutro-phils, differently from either monocytes or mouseneutrophils, cannot switch on the gene becauseits locus is in an inactive state, likely reflecting a neutro-phil-specific developmental outcome. Implicitly, dataalso definitively settle a currently unsolved issue on thecapacity of human neutrophils to produce IL-10

Beneficis de l’activitat física moderada aquàtica i terrestre, tant per a la dona gestant com per al fetus, durant l’embaràs

[cat] L’activitat física durant l’embaràs té molts beneficis, tant per a la dona gestant com per al fetus. En el procés de gestació el cos de la dona experimenta una sèrie de canvis anatòmics, fisiològics i emocionals. Davant aquestes modificacions es planteja el dubte de quin tipus d’activitat física és més adequada per a les embarassades i per als fetus. S’ha realitzat una revisió sistemàtica dels darrers 10 anys, identificant els millors articles de la literatura que aporten evidència científica respecte a aquest tema. L’objectiu d’aquest treball de fi de grau és identificar quins beneficis aporta la realització d’activitat física moderada durant l’embaràs, tant per a la dona gestant com per al fetus, i analitzar quin tipus d’exercici físic és més adequat durant la gestació, el que es desenvolupa en medi aquàtic o en medi terrestre. Es pot afirmar que l’activitat física moderada en medi aquàtic és més beneficiosa per a la dona embarassada i per al fetus. Entre els principals beneficis que aporta la realització d’activitat física durant l’embaràs trobem la prevenció d’augment de pes de l’embarassada, la prevenció de la diabetis mellitus gestacional, disminució de la lumbàlgia, prevenció de la depressió postpart, prevenció dels trastorns del son, ajuda a combatre la fatiga causada per l’embaràs, disminució de l’edema, prevenció de la preclàmpsia, augmenta la força dels músculs del sòl pelvià evitant així els trastorns que hi ha associats com la incontinència urinària, disminució del nombre d’episiotomies i de cesàries, entre d’altres

Angiopoietin-2 Promotes Inflammatory Activation in Monocytes of Systemic Sclerosis Patients

Angiopoietin-2 (Ang-2), a ligand of the tyrosine kinase receptor Tie2, is essential for vascular development and blood vessel stability and is also involved in monocyte activation. Here, we examined the role of Ang-2 on monocyte activation in patients with systemic sclerosis (SSc). Ang-2 levels were measured in serum and skin of healthy controls (HCs) and SSc patients by ELISA and array profiling, respectively. mRNA expression of ANG2 was analyzed in monocytes, dermal fibroblasts, and human pulmonary arterial endothelial cells (HPAECs) by quantitative PCR. Monocytes were stimulated with Ang-2, or with serum from SSc patients in the presence of a Tie2 inhibitor or an anti-Ang2 neutralizing antibody. Interleukin (IL)-6 and IL-8 production was analyzed by ELISA. Ang-2 levels were elevated in the serum and skin of SSc patients compared to HCs. Importantly, serum Ang-2 levels correlated with clinical disease parameters, such as skin involvement. Lipopolysaccharide (LPS) LPS, R848, and interferon alpha2a (IFN-α) stimulation up-regulated the mRNA expression of ANG2 in monocytes, dermal fibroblasts, and HPAECs. Finally, Ang-2 induced the production of IL-6 and IL-8 in monocytes of SSc patients, while the inhibition of Tie2 or the neutralization of Ang-2 reduced the production of both cytokines in HC monocytes stimulated with the serum of SSc patients. Therefore, Ang-2 induces inflammatory activation of SSc monocytes and neutralization of Ang-2 might be a promising therapeutic target in the treatment of SSc

Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α

Objective. SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production.Methods. Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1 alpha and HIF-2 alpha gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays.Results. CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P = 0.0079) leading to stabilization of HIF-2 alpha (P = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs.Conclusion. TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2 alpha pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4