Dysregulation in type I IFN and IFN-stimulated genes (ISGs) induced by monocytes is one of the key features of systemic sclerosis (SSc) pathogenesis. Abnormalities in microRNA (miRNA) expression are related to excessive IFN production, however the role of miRNA remains largely elusive in SSc monocytes. This study explores global miRNA-mRNA profiling of SSc monocytes and functional attenuation of IFN and ISGs by specific miR-NAs. Global sequencing of mRNA (mRNA-seq) and miRNA (miRNA-seq) samples were performed simultaneously on healthy controls and SSc monocytes. Following computational analysis, selected miRNAs-mRNA candidates were validated, correlated with clinical parameters, and tested by functional assays. Transcriptomics data and qPCR analysis confirmed IFN signature in SSc but not in rheumatoid arthritis monocytes. Based on miRNA-seq analysis, five miRNAs were selected for further validation. Only the expression patterns of miRNA-26a-2-3p and miRNA-485-3p were confirmed and negatively correlated with clinical parameters. Exogenous delivery of miRNA-26a-2-3p to TLR-stimulated monocytic THP-1 cells specifically inhibited ISGs but not inflammasome activity in functional assays. In conclusion, our miRNA-mRNA co-sequencing and functional analysis identify miRNA-26a-2-3p as a new candidate, which is predicated to negatively regulate ISGs. This implies that reduced expression of miRNA-26a-2-3 may be involved in pathogenic IFN signature in SSc monocytes.
Background: Congenital neutropenia (CN) is a heterogeneous group of rare inborn genetic defects with different gene mutations and different patterns of inheritance. Mutations in the HCLS1 associated Protein X-1 (HAX1) make up the largest cohort of autosomal recessive CN in Europe. There are two HAX1 splice isoforms. Mutations affecting one isoform (p.W44X) only can be found in consanguineous families of Turkic or Arabic origin while mutations affecting both isoforms were found in the original pedigree from Sweden (p.Q190X) and in Japanese pts (p.R86X). Independent of the mutation, HAX1 pts benefit from G-CSF treatment, which improved the life expectancy and quality of life significantly. However, a significant risk of secondary leukemia has been unmasked for pts with HAX1 mutations (comparable to pts with ELANE mutations).Aims: Here we report on the genotype -phenotype correlation and outcome of pts revealing different HAX1 mutations. Methods: We identified 57 pts with HAX1 mutations within the CN cohort documented by the European Branch of the SCNIR since 1994. Results: HAX1 mutations have been identified in 57 (25 female; 32 male) of 501 pts with CN. The median age is 9.49 years (range 0.28-38.69 years), with 664.83 pt years under observation. 45 of 57 pts reveal homozygous mutations in one transcript variant (p.W44X), in 1pt with an additional heterozygous ELANE mutation. In 2 of 57 pts, both isoforms are affected (p.Q190X). 5 of 57 pts have mutations at other sites of the HAX1 gene and in 5 pts the position was not reported. At diagnosis, all HAX1 pts presented with severe neutropenia and a maturation arrest at the promyelocyte/myelocyte stage in the bone marrow. All pts received long-term G-CSF treatment with a median dose of 4.25 µg/kg/day compared to 4.61 µg/kg/day for the total CN cohort of the SCNIR Europe. Median ANC increased from 160 to 1775/µL during G-CSF treatment. Additional clinical features included cardiac defects in 4 pts, hypogonadism in 5 female pts and growth retardation in 6 pts,
BackgroundMonocytes play an important role in autoimmune disease including rheumatoid arthritis (RA) or systemic sclerosis (SSc). Monocytes are the first immune cells which migrate from blood to the site of inflammation leading to tissue destruction due to enhanced proinflammatory cytokines secretion. It has been shown that abnormalities in microRNA (miRNA) expression are related to inflammatory cytokines production by T and B cells in several rheumatic diseases, however miRNAs have never been fully analysed in monocytes population. The aim of this study was to evaluate a global miRNAs profiling of monocytes from RA and SSc patients which are predicted to target proinflammatory genes.Materials and methodsTotal RNAs from CD14+ monocytes of 12 RA, 10 SSc patients and 10 HC were isolated. Thirty-two samples were barcoded using Small-RNA-Library-Set, pooled and sequenced on Illumina HiSeq 2000 platform. Expression of circulating miRNA-5196 was also measured in sera of 12 RA patients before and after 6 month TNF-α therapy and correlated (Spearman analysis) with disease activity score (DAS) 28.ResultsBased on next generation sequencing (NGS) we received 10 million reads from each sample. Following computational analysis we selected 14 specific miRNA candidates which are predicted to target inflammatory mediators out of 188 and 171 significantly changed miRNAs in monocytes of RA and SSc patients, respectively. Interestingly, miRNA-10–5p (targeting IRAK4) was 3.1-fold increased in SSc compared to RA (p<0.001) and 7.8-fold (p<0.001) upregulated compared to HC monocytes. Additional validation of miRNA candidates using qPCR and correlation with clinical data will be performed. Furthermore, the level of circulating miRNA-5196 was significantly elevated (p<0.001, 4.7-fold) in RA compared to HC sera. There was a positive correlation between delta DAS28 following 6 months therapy and delta miRNA-5196 expression (p=0.03).ConclusionsOur results identify new miRNA candidates which are predicated to regulate inflammatory genes in RA and SSc monocytes whereas miRNA-10–5p could be used as a biomarker exclusively characterising SSc. Also circulating miRNA-5196 can be used as a good predictor of anti-TNF-α treatment in RA patients.Supported by 2015/16/S/NZ6/00041 from National Science Centre, Poland and EMBO ST Fellowship.
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