Editorial: Regulation of Soluble Immune Mediators by Non-Coding RNAs

Non-coding RNAs (ncRNAs), defined as transcripts that do not encode proteins, are known since long time for their role in translation (i.e. transfer RNAs, ribosomal RNAs) and in splicing events (i.e. small nuclear and small nucleolar RNAs). However, only recently, the revolutionary advances in deep sequencing technology brought to light several new classes of ncRNA, classified according to their length into \u201cshort\u201d ncRNAs (<200 nucleotides, that includes piwi-associated RNAs, endogenous short-interfering RNAs, microRNAs, Y-RNAs and others), and \u201clong\u201d ncRNAs (lncRNAs, >200 nucleotides) (1). Cytokines are crucial soluble messengers of the immune system that regulate and sustain inflammation and immunity. Cytokine expression is tightly regulated, reflecting the need of the immune system to tailor the magnitude and duration of its responses to induce pathogen clearance, but not tissue damage. Thus, understanding cytokine regulation is crucial to gain insight and eventually manipulate undesired immune responses. In this Research Topic, 53 authors contributed 11 articles touching on many of the combined roles of ncRNAs on the production of cytokines and their consequential effect on cytokine-related functional outputs, as well as inflammatory/autoimmune pathologies

Implementation of a combined bioinformatics and experimental approach to address lncRNA mechanism of action: The example of NRIR

n this study, we demonstrate the benefit of applying combined strategies to analyze lncRNA action based on bioinformatics and experimental information. This strategy was developed to identify the molecular function of negative regulator of interferon response (NRIR), a type I interferon-stimulated gene (ISG), that we have previously demonstrated to be involved in the upregulation of a subset of ISGs in LPS-stimulated human monocytes. In this study, we provide experimental evidence that NRIR is localized in cellular nuclei, enriched on the chromatin fraction, and upregulates ISGs acting at the transcriptional level. In silico analysis of secondary structures identified distinct NRIR structural domains, comprising putative DNA- and protein-binding regions. In parallel, the presence of a putative DNA-binding domain in NRIR and the five putative NRIR- binding sites in the promoter of NRIR-target genes support the function of NRIR as a transcriptional regulator of its target genes. By use of integrated experimental/bioinformatics approaches, comprising database and literature mining together with in silico analysis of putative NRIR-binding proteins, we identified a list of eight transcription factors (TFs) shared by the majority of NRIR-target genes and simultaneously able to bind TF binding sites enriched in the NRIR-target gene promoters. Among these TFs, the predicted NRIR:STAT interactions were experimentally validated by RIP assay

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Identification of a miR-146b-FasL axis in the development of neutropenia in T large granular lymphocyte leukemia

T Large Granular Lymphocytes leukemia is characterized by the expansion of several Large Granular Lymphocyte clones, among which a subset of Large Granular Lymphocytes showing constitutively activated STAT3, a specific CD8+/CD4- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNA has not been evaluated in T-Large Granular Lymphocyte Leukemia patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-Large Granular Lymphocyte Leukemia through an altered expression of miRNAs. The expression level of 756 mature miRNAs was assessed on purified T-LGLs by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-Large Granular Lymphocytes. Remarkably, CD8 T-Large Granular Lymphocytes exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of FasL, that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-Large Granular Lymphocytes occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-Large Granular Lymphocytes lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-Large Granular Lymphocyte Leukemia

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

Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis

Infection remains a major cause of morbidity, mortality and technique failure in patients with end stage kidney failure who receive peritoneal dialysis (PD). Recent research suggests that the early inflammatory response at the site of infection carries diagnostically relevant information, suggesting that organ and pathogen-specific “immune fingerprints” may guide targeted treatment decisions and allow patient stratification and risk prediction at the point of care. Here, we recorded microRNA profiles in the PD effluent of patients presenting with symptoms of acute peritonitis and show that elevated peritoneal miR-223 and reduced miR-31 levels were useful predictors of bacterial infection. Cell culture experiments indicated that miR-223 was predominantly produced by infiltrating immune cells (neutrophils, monocytes), while miR-31 was mainly derived from the local tissue (mesothelial cells, fibroblasts). miR-223 was found to be functionally stabilised in PD effluent from peritonitis patients, with a proportion likely to be incorporated into neutrophil-derived exosomes. Our study demonstrates that microRNAs are useful biomarkers of bacterial infection in PD-related peritonitis and have the potential to contribute to disease-specific immune fingerprints. Exosome-encapsulated microRNAs may have a functional role in intercellular communication between immune cells responding to the infection and the local tissue, to help clear the infection, resolve the inflammation and restore homeostasis

Multi-Step Regulation of the TLR4 Pathway by the miR-125a~99b~let-7e Cluster

An appropriate immune response requires a tight balance between pro- and anti-inflammatory mechanisms. IL-10 is induced at late time-points during acute inflammatory conditions triggered by TLR-dependent recognition of infectious agents and is involved in setting this balance, operating as a negative regulator of the TLR-dependent signaling pathway. We identified miR-125a~99b~let-7e as an evolutionary conserved microRNA cluster late-induced in human monocytes exposed to the TLR4 agonist LPS as an effect of this IL-10-dependent regulatory loop. We demonstrated that microRNAs generated by this cluster perform a pervasive regulation of the TLR signaling pathway by direct targeting receptors (TLR4, CD14), signaling molecules (IRAK1), and effector cytokines (TNFα, IL-6, CCL3, CCL7, CXCL8). Modulation of miR-125a~99b~let-7e cluster influenced the production of proinflammatory cytokines in response to LPS and the IL-10-mediated tolerance to LPS, thus identifying this gene as a previously unrecognized major regulatory element of the inflammatory response and endotoxin tolerance

Histone modifications underlie monocyte dysregulation in patients with systemic sclerosis, underlining the treatment potential of epigenetic targeting.

Background and objective S ystemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. Methods C hromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFNresponsive gene expression. Results 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFN\u3b1 induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. Conclusion SS c monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc

The Long Non-coding RNA NRIR Drives IFN-Response in Monocytes: Implication for Systemic Sclerosis

TLR4 activation initiates a signaling cascade leading to the production of type I IFNs and of the downstream IFN-stimulated genes (ISGs). Recently, a number of IFN-induced long non-coding RNAs (lncRNAs) that feed-back regulate the IFN response have been identified. Dysregulation of this process, collectively known as the “Interferon (IFN) Response,” represents a common molecular basis in the development of autoimmune and autoinflammatory disorders. Concurrently, alteration of lncRNA profile has been described in several type I IFN-driven autoimmune diseases. In particular, both TLR activation and the upregulation of ISGs in peripheral blood mononuclear cells have been identified as possible contributors to the pathogenesis of systemic sclerosis (SSc), a connective tissue disease characterized by vascular abnormalities, immune activation, and fibrosis. However, hitherto, a potential link between specific lncRNA and the presence of a type I IFN signature remains unclear in SSc. In this study, we identified, by RNA sequencing, a group of lncRNAs related to the IFN and anti-viral response consistently modulated in a type I IFN-dependent manner in human monocytes in response to TLR4 activation by LPS. Remarkably, these lncRNAs were concurrently upregulated in a total of 46 SSc patients in different stages of their disease as compared to 18 healthy controls enrolled in this study. Among these lncRNAs, Negative Regulator of the IFN Response (NRIR) was found significantly upregulated in vivo in SSc monocytes, strongly correlating with the IFN score of SSc patients. Weighted Gene Co-expression Network Analysis showed that NRIR-specific modules, identified in the two datasets, were enriched in “type I IFN” and “viral response” biological processes. Protein coding genes common to the two distinct NRIR modules were selected as putative NRIR target genes. Fifteen in silico-predicted NRIR target genes were experimentally validated in NRIR-silenced monocytes. Remarkably, induction of CXCL10 and CXCL11, two IFN-related chemokines associated with SSc pathogenesis, was reduced in NRIR-knockdown monocytes, while their plasmatic level was increased in SSc patients. Collectively, our data show that NRIR affects the expression of ISGs and that dysregulation of NRIR in SSc monocytes may account, at least in part, for the type I IFN signature present in SSc patients