A conserved long noncoding RNA, GAPLINC, modulates the immune response during endotoxic shock.

Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes. Although lncRNAs have been identified in a variety of immune cells and implicated in immune response, the biological function and mechanism of the majority remain unexplored, especially in sepsis. Here, we identify a role for a lncRNA-gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC)-previously characterized for its role in cancer, now in the context of innate immunity, macrophages, and LPS-induced endotoxic shock. Transcriptome analysis of macrophages from humans and mice reveals that GAPLINC is a conserved lncRNA that is highly expressed following macrophage differentiation. Upon inflammatory activation, GAPLINC is rapidly down-regulated. Macrophages depleted of GAPLINC display enhanced expression of inflammatory genes at baseline, while overexpression of GAPLINC suppresses this response. Consistent with GAPLINC-depleted cells, Gaplinc knockout mice display enhanced basal levels of inflammatory genes and show resistance to LPS-induced endotoxic shock. Mechanistically, survival is linked to increased levels of nuclear NF-κB in Gaplinc knockout mice that drives basal expression of target genes typically only activated following inflammatory stimulation. We show that this activation of immune response genes prior to LPS challenge leads to decreased blood clot formation, which protects Gaplinc knockout mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock

A conserved long noncoding RNA, GAPLINC, modulates the immune response during endotoxic shock.

Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes. Although lncRNAs have been identified in a variety of immune cells and implicated in immune response, the biological function and mechanism of the majority remain unexplored, especially in sepsis. Here, we identify a role for a lncRNA-gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC)-previously characterized for its role in cancer, now in the context of innate immunity, macrophages, and LPS-induced endotoxic shock. Transcriptome analysis of macrophages from humans and mice reveals that GAPLINC is a conserved lncRNA that is highly expressed following macrophage differentiation. Upon inflammatory activation, GAPLINC is rapidly down-regulated. Macrophages depleted of GAPLINC display enhanced expression of inflammatory genes at baseline, while overexpression of GAPLINC suppresses this response. Consistent with GAPLINC-depleted cells, Gaplinc knockout mice display enhanced basal levels of inflammatory genes and show resistance to LPS-induced endotoxic shock. Mechanistically, survival is linked to increased levels of nuclear NF-κB in Gaplinc knockout mice that drives basal expression of target genes typically only activated following inflammatory stimulation. We show that this activation of immune response genes prior to LPS challenge leads to decreased blood clot formation, which protects Gaplinc knockout mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock

Few Single Nucleotide Variations in Exomes of Human Cord Blood Induced Pluripotent Stem Cells

<div><p>The effect of the cellular reprogramming process <i>per se</i> on mutation load remains unclear. To address this issue, we performed whole exome sequencing analysis of induced pluripotent stem cells (iPSCs) reprogrammed from human cord blood (CB) CD34⁺ cells. Cells from a single donor and improved lentiviral vectors for high-efficiency (2–14%) reprogramming were used to examine the effects of three different combinations of reprogramming factors: OCT4 and SOX2 (OS), OS and ZSCAN4 (OSZ), OS and MYC and KLF4 (OSMK). Five clones from each group were subject to whole exome sequencing analysis. We identified 14, 11, and 9 single nucleotide variations (SNVs), in exomes, including untranslated regions (UTR), in the five clones of OSMK, OS, and OSZ iPSC lines. Only 8, 7, and 4 of these, respectively, were protein-coding mutations. An average of 1.3 coding mutations per CB iPSC line is remarkably lower than previous studies using fibroblasts and low-efficiency reprogramming approaches. These data demonstrate that point nucleotide mutations during cord blood reprogramming are negligible and that the inclusion of genome stabilizers like ZSCAN4 during reprogramming may further decrease reprogramming-associated mutations. Our findings provide evidence that CB is a superior source of cells for iPSC banking.</p> </div

Efficient generation of iPSCs from cord blood CD34⁺ cells.

<p>(<b>A</b>) Efficient reprogramming of cord blood with lentiviral vectors. Three different combinations of reprogramming factors were used: OCT4 and SOX2 (OS), OS+ZSCAN4 (OSZ), and OS+MYC and KLF4 (OSMK). After transduction, 2500–5000 cells were seeded in 6-well plates. iPSC colonies were counted 2 weeks later and reprogramming efficiencies were calculated accordingly. (<b>B</b>) iPSCs express pluripotency makers OCT4, NANOG and SSEA4. Representative pictures for each group (OS iPSC lines, OSZ iPSC lines, and MK iPSC lines (OSMK)) are presented (200×). Confocal imaging did not identify any differences in expression of pluripotency makers among 15 iPSC lines.</p

Coding mutations in each of the 15 CB iPSC lines.

<p>Number of coding SNVs (<b>A</b>) and nonsynonymous coding SNVs (<b>B</b>) in each line compared to the parent cord blood cells. MK: iPSC lines generated with OCT4, SOX2, MYC and KLF; OS: iPSC lines generated with OCT4 and SOX2; Z: iPSC lines generated with OCT4, SOX2; and ZSCAN4. The use of OSZ appears to decrease the coding SNV load in iPSC lines compared to the OSMK control (<i>P</i>>0.05) (<b>A</b>), while OSZ iPSC lines harbor significant fewer number of nonsynonymous coding SNVs relative to the OSMK control (<i>P</i><0.05) (<b>B</b>).</p

Summary of the exome sequencing data and the identified single nucleotide variants.

<p>The numbers of heterozygous variants are those that have a minimum of 5× coverage. The dbSNP percentage represents the portion of identified variants present in the Single Nucleotide Polymorphism Database.</p

Genes found to be mutated in exomes of 15 CB iPSC lines.

<p>The full details of each SNV including reads of SNV and wildtype alleles are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059908#pone.0059908.s005" target="_blank">Table S1</a>.</p><p>MK: iPSC lines generated with OCT4, SOX2, MYC and KLF4; OS: iPSC lines generated with OCT4 and SOX2; Z: iPSC lines generated with OCT4, SOX2 and ZSCAN4.</p><p>CDS: coding sequence; UTR: untranslated region; Downstream: SNV is at downstream of 5′UTR; S: synonymous coding mutation; NS: nonsynonymous coding mutation.</p

Regulatory polymorphisms modulate the expression of HLA class II molecules and promote autoimmunity.

Targeted sequencing of sixteen SLE risk loci among 1349 Caucasian cases and controls produced a comprehensive dataset of the variations causing susceptibility to systemic lupus erythematosus (SLE). Two independent disease association signals in the HLA-D region identified two regulatory regions containing 3562 polymorphisms that modified thirty-seven transcription factor binding sites. These extensive functional variations are a new and potent facet of HLA polymorphism. Variations modifying the consensus binding motifs of IRF4 and CTCF in the XL9 regulatory complex modified the transcription of HLA-DRB1, HLA-DQA1 and HLA-DQB1 in a chromosome-specific manner, resulting in a 2.5-fold increase in the surface expression of HLA-DR and DQ molecules on dendritic cells with SLE risk genotypes, which increases to >4-fold after stimulation. Similar analyses of fifteen other SLE risk loci identified 1206 functional variants tightly linked with disease-associated SNPs and demonstrated that common disease alleles contain multiple causal variants modulating multiple immune system genes