Characterization of Long Non-Coding RNAs in Systemic Sclerosis Monocytes: A Potential Role for PSMB8-AS1 in Altered Cytokine Secretion.

Systemic sclerosis (SSc) is a chronic autoimmune disease mainly affecting the connective tissue. In SSc patients, monocytes are increased in circulation, infiltrate affected tissues, and show a pro-inflammatory activation status, including the so-called interferon (IFN) signature. We previously demonstrated that the dysregulation of the IFN response in SSc monocytes is sustained by altered epigenetic factors as well as by upregulation of the long non-coding RNA (lncRNA) NRIR. Considering the enormously diverse molecular functions of lncRNAs in immune regulation, the present study investigated the genome-wide profile of lncRNAs in SSc monocytes, with the aim to further unravel their possible role in monocyte dysregulation and disease pathogenesis. Transcriptomic data from two independent cohorts of SSc patients identified 886 lncRNAs with an altered expression in SSc monocytes. Differentially expressed lncRNAs were correlated with neighboring protein coding genes implicated in the regulation of IFN responses and apoptotic signaling in SSc monocytes. In parallel, gene co-expression network analysis identified the lncRNA PSMB8-AS1 as a top-ranking hub gene in co-expression modules implicated in cell activation and response to viral and external stimuli. Functional characterization of PSMB8-AS1 in monocytes demonstrated that this lncRNA is involved in the secretion of IL-6 and TNFα, two pivotal pro-inflammatory cytokines altered in the circulation of SSc patients and associated with fibrosis and disease severity. Collectively, our data showed that lncRNAs are linked to monocyte dysregulation in SSc, and highlight their potential contribution to disease pathogenesis.

Effects of Antithrombotic Agents on Ophthalmological Outcomes, Cardiovascular Risk, and Mortality in Hypertensive Patients with Retinal Vein Occlusion: An Exploratory Retrospective Study.

Background and objectives: Because few data are available, the aim of this study is to analyze the effects of antithrombotic agents (ATAs) on visual function and long-term risk of cardiovascular events and mortality in hypertensive patients with retinal vein occlusion (RVO). Materials and methods: Hypertensive patients with RVO were consecutively selected from 2008 to 2012 and followed for a median of 8.7 years. Ophthalmologists evaluated and treated RVO complications, and best-corrected visual acuity (BCVA) was checked at each visit during the first one year of follow-up. Survival analysis was conducted on the rate of the composite endpoint of all-cause deaths or non-fatal cardiovascular events. Results: Retrospectively, we collected data from 80 patients (age 68 ± 12 years, 39 males). Central and branch RVO was present in 41 and 39 patients, respectively, and 56 patients started ATAs (50 antiplatelet drugs, 6 warfarin, and 2 low-molecular weight heparin). Average BCVA of the cohort did not change significantly during one-year of follow-up. The only predictor of BCVA was the baseline BCVA value. There was a reduction in proportion and severity of macular edema and an increase in the cumulative proportion of retinal vein patency reestablishment during the follow-up, independent of treatment. ATAs had no effects on one-year BCVA, intraocular complications, or the composite endpoint rate. Conclusions: In this exploratory study, ATAs had no effect on BCVA during the first one year of follow-up and on the composite endpoint during the long-term follow-up. Further prospective studies need to be conducted with an accurate standardization of the intraocular and antithrombotic treatment to define the positive or negative role of ATAs in hypertensive patients with RVO.

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

Tlarge granular lymphocyte leukemia (T-LGLL) 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 miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (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-LGLs. Remarkably, CD8 T-LGLs 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 Fas ligand (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-LGLs 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-LGLs 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-LGLL.

A tree from waste: Decontaminated dredged sediments for growing forest tree seedlings.

The sediments dredged from a waterway and decontaminated through a phytoremediation process have been used as substrates alternatively to the traditional forest nursery substrate for pot productions of holm oak (Quercus ilex L.) planting stocks. The substrates, made by mixing decontaminated sediments to agricultural soil at different degrees, were tested in order to evaluate their suitability as growth substrates. The experiment was carried out at the nursery of the Department of Agricultural, Food and Forestry Systems of the University of Florence (Italy). The experimental design consisted of four randomized blocks with six pots as replicates for each of the following treatments: 100% sediments, 66% sediments, 33% sediments, 100% agronomic soil and 100% traditional peat based substrate. In each pot, one holm oak acorn was seeded. Germination and both physiological and morphological traits of the seedlings were analysed during and at the end of the first growing season. Holm oak grown in phytoremediated sediments at higher concentrations showed germination levels comparable to those in the traditional substrate, and survival capacity (especially in 66% sediments) slightly higher than in 100% soil. Physiological performance of seedlings resembled that on the traditional substrate which required the addition of fertilizer, at least for the first growing season. Seedlings grown in mixed substrates with higher sediment concentrations occasionally showed better photosynthetic capacity with improved connectivity between the units of the photosystem II. At the end of the first growing season, height as well as the number of growth flushes of the seedlings grown in sole sediment or soil-sediment substrates were similar to what generally is observed for forest nursery stock of Quercus spp.. Regarding the root-system articulation and growth in depth, results in the mixed substrates were comparable to those for seedlings grown in the traditional forest nursery media, and higher than seedlings grown in 100% agronomic soil. According to our results, the reclamation of dredged sediments can provide appropriate nursery substrate for germination beds for forestry species.

IL-10-induced microRNA-187 negatively regulates TNF-α, IL-6, and IL-12p40 production in TLR4-stimulated monocytes.

IL-10 is a potent anti-inflammatory molecule that, in phagocytes, negatively targets cytokine expression at transcriptional and posttranscriptional levels. Posttranscriptional checkpoints also represent the specific target of a recently discovered, evolutionary conserved class of small silencing RNAs known as "microRNAs" (miRNAs), which display the peculiar function of negatively regulating mRNA processing, stability, and translation. In this study, we report that activation of primary human monocytes up-regulates the expression of miR-187 both in vitro and in vivo. Accordingly, we identify miR-187 as an IL-10-dependent miRNA playing a role in IL-10-mediated suppression of TNF-α, IL-6, and the p40 subunit of IL-12 (IL-12p40) produced by primary human monocytes following activation of Toll-like receptor 4 (TLR4). Ectopic expression of miR-187 consistently and selectively reduces TNFα, IL-6, and IL-12p40 produced by LPS-activated monocytes. Conversely, the production of LPS-induced TNF-α, IL-6, and IL-12p40 is increased significantly when miR-187 expression is silenced. Our data demonstrate that miR-187 directly targets TNF-α mRNA stability and translation and indirectly decreases IL-6 and IL-12p40 expression via down-modulation of IκBζ, a master regulator of the transcription of these latter two cytokines. These results uncover an miRNA-mediated pathway controlling cytokine expression and demonstrate a central role of miR-187 in the physiological regulation of IL-10-driven anti-inflammatory responses.

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.

Cultivation Using Coir Substrate and P or K Enriched Fertilizer Provides Higher Resistance to Drought in Ecologically Diverse Quercus Species.

Nursery cultivation practices can be modified to increase resistance to water stress in forest seedlings following field establishment, which may be increasingly important under climate change. We evaluated the morphological (survival, growth) and physiological (chlorophyll fluorescence, leaf water potential) responses to water stress for three ecologically diverse Quercus species (Q. robur, Q. pubescens, and Q. ilex) with varying traits resulting from the combination of growing media (peat, coir) and fertilization (standard, P-enriched, K-enriched). For all species under water stress, seedlings grown in coir had generally higher growth than those grown in peat. Seedlings fertilized with P performed better, particularly for survival; conversely, K fertilization resulted in inconsistent findings. Such results could be explained by a combination of factors. P fertilization resulted in higher P accumulation in seedlings, while no K accumulation was observed in K fertilized seedlings. As expected, the more drought-sensitive species, Q. robur, showed the worst response, while Q. pubescens had a drought resistance equal or better to Q. ilex despite being classified as intermediate in drought resistance in Mediterranean environments.

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

In 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.

Impact of Sex on Circulating Leukocytes Composition in COPD Patients.

PURPOSE: Chronic obstructive pulmonary disease is characterized by chronic inflammatory response both at the lung site and at the systemic level. Abnormalities in circulating leukocytes have been reported to occur in COPD patients and have been often shown to correlate with the decline in lung function. COPD affects men and women at a virtually comparable rate, even though distinct sex specific symptoms, progression and therapeutic implications have been described. Nonetheless, these sex-associated differences have not been analyzed in terms of circulating leukocytes. To assess the impact of sex on the changes of circulating immune cells in COPD patients. PATIENTS AND METHODS: Blood samples were collected from 50 COPD patients (31 males, 19 females) and 63 age and sex-matched controls (35 males, 28 females) enrolled in this pilot study. Complete blood cell count and multi-parametric flow cytometry analysis were performed to characterize the leukocyte populations and subsets. RESULTS: Male COPD patients are distinguished from controls by a significant increase in white blood cell counts, neutrophil total and differential counts, and neutrophil-to-lymphocyte ratio. Conversely, a generalized leukocyte decrease discriminated female COPD patients from the related controls. The impact of sex is further remarked by a decrease in adaptive immune cell subpopulations in males as opposed to a consistent increase of innate immune cell types in females correlating with disease severity. CONCLUSION: These data indicate that the definition of specific changes of circulating leukocytes to be used as reliable biomarkers of the disease severity cannot be accomplished irrespectively of sex.

Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture.

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60-80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype-phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

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.

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.