Genome-wide analysis of human neutrophils mirnome identified miR-23a as a critical regulator of the apoptotic Fas antigen expression

Over the past decade, genome-wide studies have made it clear that the mammalian genome is pervasively transcribed and this led to the identification of non-protein coding RNA molecules. Only 2% of the mammalian genome accounts for protein-coding sequences, so that the majority is transcribed as noncoding RNA (ncRNA). ncRNAs appear to be highly conserved and are considered to be an important component for genetic regulation via epigenetic mechanisms. The regulatory ncRNAs can be classified into long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). miRNAs increasingly recognized to play a pivotal role in both physiological and pathological conditions, including mammalian development, cardiovascular, neurodegenerative and metabolic diseases, cancer and immune disorders. Despite the evidence for the importance of miRNAs in immune cell function and development, very limited information is available regarding how miRNAs regulate neutrophils development, lifespan and functions. Based on these premises, the purpose of this study was to provide a comprehensive analysis of microRNAs expression profile and to identify their role in primary human neutrophils under resting and stimulatory conditions. To achieve this goal we performed whole transcriptome analysis and characterized the pri-miRNome of resting and LPS-stimulated neutrophils. In parallel, the same analysis was performed also on autologous monocytes, in order to get insight into cell-specific miRNA profile. The results of this study showed that TLR4 engagement triggers the transcription of 37 pri-miRNAs. Additionally, comparison of LPS-induced pri-miRNA expression profile of neutrophils with that of autologous monocytes identified subsets of pri-miRNAs modulated by LPS in both cell type in a cell-specific manner. LPS induced the expression of twelve pri-miRNAs in neutrophils, among which we further validated the expression of the mature forms of the miR23a cluster members. The members of this cluster have been described to play important roles in various biological and pathological processes, but their role has never been previously identified in resting and/or activated neutrophils. Upon LPS stimulation neutrophils upregulate only the mature forms of miR-23a-5p and miR-27a-5p. In silico target prediction indicated Fas, a death receptor mediating cell apoptosis, among the miR-23a predicted target genes. Herein we provide several experimental proofs demonstrating that LPS reduces neutrophil Fas-induced apoptosis in a miR-23a-dependent manner. In fact, and consistent with a prolonged neutrophils\u2019 half-life, in the presence of LPS a parallel upregulation of miR-23a and down-regulation of Fas membrane protein, but not Fas mRNA, expression was detected, suggesting involvement of a post-transcriptional regulation for Fas. Both Fas mRNA and miR-23a were detected in Ago immunoprecipitates only in LPS-stimulated neutrophils, thus indicating that LPS promotes miR-mediated post-transcriptional silencing of FAS. Finally, neutrophils overexpressing miR-23a reduced Fas membrane receptor. Taken together these data demonstrated that LPS stimulation affects neutrophil apoptosis via increasing miR-23a, which in return decreases the Fas expression on neutrophil surface. Collectively, we have described the expression profile of pri-miRNAs in resting and LPS-activated human neutrophils and their autologous monocytes, resulting in identification of cell type specific pri-miRNAs. The information from this whole transcriptome study led us to link the LPS to neutrophil apoptosis via miRNA modulation. Our data could be substantially used for further investigation on the role of miRNAs in neutrophils biology

Blockade of \u3b14 integrins reduces leukocyte-endothelial interactions in cerebral vessels and improves memory in a mouse model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline associated with the deposition of amyloid-beta (A beta) plaques, hyperphosphorylation of tau protein, and neuronal loss. Vascular inflammation and leukocyte trafficking may contribute to AD pathogenesis, and a better understanding of these inflammation mechanisms could therefore facilitate the development of new AD therapies. Here we show that T cells extravasate in the proximity of cerebral VCAM-1(+) vessels in 3xTg-AD transgenic mice, which develop both A beta and tau pathologies. The counter-ligand of VCAM-1-alpha 4 beta 1 integrin, also known as very late antigen-4 (VLA-4) - was more abundant on circulating CD4(+) T cells and was also expressed by a significant proportion of blood CD8(+) T cells and neutrophils in AD mice. Intravital microscopy of the brain microcirculation revealed that alpha 4 integrins control leukocyte-endothelial interactions in AD mice. Therapeutic targeting of VLA-4 using antibodies that specifically block alpha 4 integrins improved the memory of 3xTg-AD mice compared to an isotype control. These antibodies also reduced neuropathological hallmarks of AD, including microgliosis, A beta load and tau hyperphosphorylation. Our results demonstrate that alpha 4 integrin-dependent leukocyte trafficking promotes cognitive impairment and AD neuropathology, suggesting that the blockade of alpha 4 integrins may offer a new therapeutic strategy in AD

MicroRNA profiling in human neutrophils during activation

The purpose of our study was to describe the expression profiles of primary and mature forms of microRNAs (pri-miRs and miRs) in freshly isolated human polymorphonuclear neutrophils (PMN) as well as cultured cells under resting and stimulatory conditions. Human PMNs were purified from buffy coats and cultured in presence or absence of LPS for 1.5 and 4 hours. RNA-seq analysis was conducted using the human transcriptome as the reference (Ensembl 77). DESeq2 was used for differential transcript expression analysis. Pri-miRs and miRs differentially expressed were validated by RT-qPCR and Individual TaqMan miRNA Assay, respectively. miRanda and TargetScan miRNA target prediction algorithms were used to identify the predicted miRs target genes. The FatiGO tool of the Babelomics was used to identify significant over-representation of functional annotation for the predicted miR target genes. Our data show that 381 pri-miRs are constitutively expressed in circulating PMNs. Culturing PMN modulates the expression of 59 pri-miRs, which the modulation is not affected by the presence of LPS. The expression of 13 pri-miRs was increased in LPS-stimulated PMNs as compared to 4h cultured cells. The increase in expression of the mature forms of 3 pri-miRs (miR-23a, miR24-2 and miR27a) was validated. Based on the evidence that the main function of a miR is destabilizing its target mRNA, the mRNA subset with decrease in their expression upon LPS-stimulation was extracted from RNAseq data. Subsequently these mRNAs were analyzed by target prediction algorithms. We found 425 mRNA transcripts to be potential targets of the 3 miRs with increase expression pattern. Moreover GO term analysis revealed that kinase activity was over-represented among targeted mRNAs. In conclusion, this study provides the first comprehensive miR expression profile of resting and LPS-activated human PMN, which serves as a reference for further research on PMN related abnormalities

Additional file 1: of Cancer-related CD15/FUT4 overexpression decreases benefit to agents targeting EGFR or VEGF acting as a novel RAF-MEK-ERK kinase downstream regulator in metastatic colorectal cancer

Supplementary Tables S1-S4. (DOC 121 kb