Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress.

Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools.

Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming.

The responses of CD8+ T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8+ T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8+ T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.

Abnormal DNA Methylation Induced by Hyperglycemia Reduces CXCR 4 Gene Expression in CD 34+ Stem Cells.

Background CD 34+ stem/progenitor cells are involved in vascular homeostasis and in neovascularization of ischemic tissues. The number of circulating CD 34+ stem cells is a predictive biomarker of adverse cardiovascular outcomes in diabetic patients. Here, we provide evidence that hyperglycemia can be "memorized" by the stem cells through epigenetic changes that contribute to onset and maintenance of their dysfunction in diabetes mellitus. Methods and Results Cord-blood-derived CD 34+ stem cells exposed to high glucose displayed increased reactive oxygen species production, overexpression of p66shc gene, and downregulation of antioxidant genes catalase and manganese superoxide dismutase when compared with normoglycemic cells. This altered oxidative state was associated with impaired migration ability toward stromal-cell-derived factor 1 alpha and reduced protein and mRNA expression of the C-X-C chemokine receptor type 4 ( CXCR 4) receptor. The methylation analysis by bisulfite Sanger sequencing of the CXCR 4 promoter revealed a significant increase in DNA methylation density in high-glucose CD 34+ stem cells that negatively correlated with mRNA expression (Pearson r=-0.76; P=0.004). Consistently, we found, by chromatin immunoprecipitation assay, a more transcriptionally inactive chromatin conformation and reduced RNA polymerase II engagement on the CXCR 4 promoter. Notably, alteration of CXCR 4 DNA methylation, as well as transcriptional and functional defects, persisted in high-glucose CD 34+ stem cells despite recovery in normoglycemic conditions. Importantly, such an epigenetic modification was thoroughly confirmed in bone marrow CD 34+ stem cells isolated from sternal biopsies of diabetic patients undergoing coronary bypass surgery. Conclusions CD 34+ stem cells "memorize" the hyperglycemic environment in the form of epigenetic modifications that collude to alter CXCR 4 receptor expression and migration.

Evaluating the methylation status of CXCR4 promoter: A cost-effective and sensitive two-step PCR method.

The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region.

Methylation profiling by bisulfite sequencing analysis of the mtDNA Non-Coding Region in replicative and senescent Endothelial Cells.

The regulation and function of Mitochondrial DNA (mtDNA) cytosine methylation (5 mC) are largely unexplored. Mitochondria, Endothelial Cell (EC) senescence, and cardiovascular dysfunction are closely related. We extensively investigated the mtDNA Non-Coding Region (NCR) methylation pattern and its variations in EC replicative senescence. We observed previously undescribed 5 mC clusters and a biased distribution of 5 mC among DNA sites and throughout the NCR. The methylation pattern in senescent EC showed non-random variations, including the hypo-methylation of mtDNA replication regulatory sites. Additional experiments opened to a possible role for 5 mC in D-loop formation, rather than in mitochondrial gene expression.

The mitochondrial lncRNA ASncmtRNA-2 is induced in aging and replicative senescence in Endothelial Cells.

Age-associated cardiovascular diseases are at least partially ascribable to vascular cell senescence. Replicative senescence (RS) and stress-induced premature senescence (SIPS) are provoked respectively by endogenous (telomere erosion) and exogenous (H2O2, UV) stimuli resulting in cell cycle arrest in G1 and G2 phases. In both scenarios, mitochondria-derived ROS are important players in senescence initiation. We aimed to define whether a mtDNA-transcribed long-non-coding-RNA (lncRNA), ASncmtRNA-2, has a role in vascular aging and senescence. Aortas of old mice, characterized by increased senescence, showed an increment in ASncmtRNA-2 expression. In vitro analysis of Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC) established that ASncmtRNA-2 is induced in EC, but not in VSMC, during RS. Surprisingly, ASncmtRNA-2 is not upregulated in two different EC SIPS scenarios, treated with H2O2 and UV. The p16 gene displayed similar ASncmtRNA-2 expression patterns, suggesting a possible co-regulation of the two genes. Interestingly, the expression of two miRNAs, hsa-miR-4485 and hsa-miR-1973, with perfect homology to the double strand region of ASncmtRNA-2 and originating at least in part from a mitochondrial transcript, was induced in RS, opening to the possibility that this lncRNA functions as a non-canonical precursor of these miRNAs. Cell cycle analysis of EC transiently over-expressing ASncmtRNA-2 revealed an accumulation of EC in the G2/M phase, but not in the G1 phase. We propose that ASncmtRNA-2 in EC might be involved in the RS establishment by participating in the cell cycle arrest in G2/M phase, possibly through the production of hsa-miR-4485 and hsa-miR-1973. This article is part of a Special Issue entitled: Mitochondria.