Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBP beta, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor kappa B (NF-kappa B) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation

SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease

Human neutrophils activated by TLR8 agonists, with or without IFN\u3b3, synthesize and release EBI3, but not IL-12, IL-27, IL-35, or IL-39

The IL-12 family of cytokines plays crucial functions in innate and adaptive immunity. These cytokines include heterodimers sharing distinct \u3b1 (IL-12A, IL-23A, and IL-27A) with two \u3b2 (IL-12B and Epstein-Barr virus induced gene 3 [EBI3]) chains, respectively, IL-12 (IL-12B plus IL-12A) and IL-23 (IL-12B plus IL-23A) sharing IL-12B, IL-27 (EBI3 plus IL-27A), IL-35 (EBI3 plus IL-12A), and IL-39 (EBI3 plus IL-23A) sharing EBI3. In this context, we have recently reported that highly pure neutrophils incubated with TLR8 agonists produce functional IL-23. Previously, we showed that neutrophils incubated with LPS plus IFN\u3b3 for 20 h produce IL-12. Herein, we investigated whether highly pure, TLR8-activated, neutrophils produce EBI3, and in turn IL-27, IL-35, and IL-39, the IL-12 members containing it. We report that neutrophils incubated with TLR8 ligands, TNF\u3b1 and, to a lesser extent, LPS, produce and release remarkable amounts of EBI3, but not IL-27A, consequently excluding the possibility for an IL-27 production. We also report a series of unsuccessful experiments performed to investigate whether neutrophil-derived EBI3 associates with IL-23A to form IL-39. Furthermore, we show that neutrophils incubated with IFN\u3b3 in combination with either TLR8 or TLR4 ligands express/produce neither IL-12, nor IL-35, due to the inability of IFN\u3b3, contrary to previous findings, to activate IL12A transcription. Even IL-27 was undetectable in supernatants harvested from IFN\u3b3 plus R848-treated neutrophils, although they were found to accumulate IL27A transcripts. Finally, by immunohistochemistry experiments, EBI3-positive neutrophils were found in discrete pathologies only, including diverticulitis, cholecystitis, Gorham disease, and Bartonella Henselae infection, implying a specific role of neutrophil-derived EBI3 in vivo

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23

Human neutrophils contribute to the regulation of inflammation via the generation of a range of cytokines that affect all elements of the immune system. Here, we investigated their ability to express some of the members of the IL‐12 family after incubation with TLR8 agonists. Highly pure human neutrophils were thus incubated for up to 48 h with or without R848, or other TLR8 agonists, to then measure the expression levels of transcripts and proteins for IL‐12 family member subunits by RNA‐seq, reverse transcription quantitative PCR, and ELISA. We show a TLR8‐mediated inducible expression of IL‐12B and IL‐23A, but not IL‐12A, mRNA, which occurs via chromatin remodeling (as assessed by ChIP‐seq), and subsequent production of IL‐23 and IL‐12B, but no IL‐12, proteins. Induction of IL‐23 requires endogenous TNF‐α, as both mRNA and protein levels were blocked in TLR8‐activated neutrophils via a TNF‐α‐neutralizing Ab. We also show that supernatants from TLR8‐activated neutrophils, but not autologous monocytes, induce the differentiation of Th17 cells from naïve T cells in an IL‐23‐dependent fashion. This study unequivocally demonstrates that highly pure human neutrophils express and produce IL‐23, further supporting the key roles played by these cells in the important IL‐17/IL‐23 network and Th17 responses

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

El desafío ambiental

La ciudad sostenible. Dependencia ecológica y relaciones regionales. Un estudio de caso en el área metropolitana de Medellín, Colombia. Luis Carlos Agudelo Patiño; Universidad Nacional de Colombia, Sede Medellín, Facultad de Arquitectura, Escuela de Planeación Urbano-Regional, Grupo de Investigación Dinámicas Urbano Regionales, Medellín, 2010, 197 págs., il

Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs

IDENTIFICATION OF XXX AS AN UNDESCRIBED TRANSCRIPTION FACTOR INVOLVED IN TOLL-LIKE RECEPTOR (TLR) ACTIVATION OF HUMAN NEUTROPHILS

I neutrofili sono le prime cellule del sistema immunitario ad essere reclutate nei siti infiammati, e sono coinvolte sia nell'immunit\ue0 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 \ue8 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 gi\ue0 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, cos\uec 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, cos\uec come la deposizione di H67K44su, sono altamente correlati con un aumento dell'espressione dei geni pi\uf9 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 \ue8 espresso a bassi livelli nei neutrofili non stimolati ma aumenta in seguito a stimolazione del TLR8 ed \ue8 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\u2019importante risorsa per studi futuri.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

Human dendritic cell subset 4 (DC4) correlates to a subset of CD14dim/-CD16++monocytes

Human dendritic cell subset 4 (DC4) correlates to a subset of CD14dim/-CD16++ monocytes

Cytokine production by human neutrophils: Revisiting the "dark side of the moon"

Polymorphonuclear neutrophils are the most numerous leucocytes present in human blood, and function as crucial players in innate immune responses. Neutrophils are indispensable for the defence towards microbes, as they effectively counter them by releasing toxic enzymes, by synthetizing reactive oxygen species and by producing inflammatory mediators. Interestingly, recent findings have highlighted an important role of neutrophils also as promoters of the resolution of inflammation process, indicating that their biological functions go well beyond simple pathogen killing. Consistently, data from the last decades have highlighted that neutrophils may even contribute to the development of adaptive immunity by performing previously unanticipated functions, including the capacity to extend their survival, directly interact with other leucocytes or cell types, and produce and release a variety of cytokines. In this article, we will summarize the main features of, as well as emphasize some important concepts on, the production of cytokines by human neutrophils