Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes

Hematopoietic stem and progenitor cells (HSPCs) are multipotent stem cells that have been harnessed as a curative therapy for patients with hematological malignancies. Notably, the discovery that HSPCs are endowed with immunoregulatory properties suggests that HSPC-based therapeutic approaches may be used to treat autoimmune diseases. Indeed, infusion with HSPCs has shown promising results in the treatment of type 1 diabetes (T1D) and remains the only “experimental therapy” that has achieved a satisfactory rate of remission (nearly 60%) in T1D. Patients with newly diagnosed T1D have been successfully reverted to normoglycemia by administration of autologous HSPCs in association with a non-myeloablative immunosuppressive regimen. However, this approach is hampered by a high incidence of adverse effects linked to immunosuppression. Herein, we report that while the use of autologous HSPCs is capable of improving C-peptide production in patients with T1D, ex vivo modulation of HSPCs with prostaglandins (PGs) increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. Surprisingly, CXCR4 was upregulated as well, which could enhance HSPC trafficking toward the inflamed pancreatic zone. When tested in murine and human in vitro autoimmune assays, PG-modulated HSPCs were shown to abrogate the autoreactive T cell response. The use of PG-modulated HSPCs may thus provide an attractive and novel treatment of autoimmune diabetes

N6-isopentenyladenosine and analogs activate the NRF2-mediated antioxidant response

N6-isopentenyladenosine (i6A), a naturally occurring modified nucleoside, inhibits the proliferation of human tumor cell lines in vitro, but its mechanism of action remains unclear. Treatment of MCF7 human breast adenocarcinoma cells with i6A or with three synthetic analogs (allyl6A, benzyl6A, and butyl6A) inhibited growth and altered gene expression. About 60% of the genes that were differentially expressed in response to i6A treatment were also modulated by the analogs, and pathway enrichment analysis identified the NRF2-mediated oxidative stress response as being significantly modulated by all four compounds. Luciferase reporter gene assays in transfected MCF7 cells confirmed that i6A activates the transcription factor NRF2. Assays for cellular production of reactive oxygen species indicated that i6A and analogs had antioxidant effects, reducing basal levels and inhibiting the H2O2- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced production in MCF7 or dHL-60 (HL-60 cells induced to differentiate along the neutrophilic lineage) cell lines, respectively. In vivo, topical application of i6A or benzyl6A to mouse ears prior to TPA stimulation lessened the inflammatory response and significantly reduced the number of infiltrating neutrophils. These results suggest that i6A and analogs trigger a cellular response against oxidative stress and open the possibility of i6A and benzyl6A being used as topical anti-inflammatory drugs

Mouse Pulmonary Adenoma Susceptibility 1 Locus Is an Expression QTL Modulating Kras-4A

Pulmonary adenoma susceptibility 1 (Pas1) is the major locus responsible for lung tumor susceptibility in mice; among the six genes mapping in this locus, Kras is considered the best candidate for Pas1 function although how it determines tumor susceptibility remains unknown. In an (A/J×C57BL/6)F4 intercross population treated with urethane to induce lung tumors, Pas1 not only modulated tumor susceptibility (LOD score = 48, 69% of phenotypic variance explained) but also acted, in lung tumor tissue, as an expression quantitative trait locus (QTL) for Kras-4A, one of two alternatively spliced Kras transcripts, but not Kras-4B. Additionally, Kras-4A showed differential allelic expression in lung tumor tissue of (A/J×C57BL/6)F4 heterozygous mice, with significantly higher expression from the A/J-derived allele; these results suggest that cis-acting elements control Kras-4A expression. In normal lung tissue from untreated mice of the same cross, Kras-4A levels were also highly linked to the Pas1 locus (LOD score = 23.2, 62% of phenotypic variance explained) and preferentially generated from the A/J-derived allele, indicating that Pas1 is an expression QTL in normal lung tissue as well. Overall, the present findings shed new light on the genetic mechanism by which Pas1 modulates the susceptibility to lung tumorigenesis, through the fine control of Kras isoform levels. © 2014 Dassano et al

<i>Kras</i>-4A expression is highly controlled by <i>Pas1</i> locus in normal lung.

<p>(a) Genetic linkage analysis of expression levels (square root transformed) of two <i>Kras</i> transcripts in 111 untreated ABF4 mice showed that the <i>Kras</i>-4A isoform was strongly linked to the <i>Pas1</i> locus (LOD score = 23.2) whereas the <i>Kras</i>-4B isoform showed a weaker linkage (LOD score = 4.1). Horizontal line at LOD = 2.13 marks the threshold for significance. Tick marks show the position, in a recombinational map, of the nine genotyped markers spanning from chromosome 6 position 96.7 Mb to 148.3 Mb. (b, c) Expression levels of <i>Kras</i>-4A and -4B in normal lung tissue, by genotype for the 37-bp insertion mutation common to both isoforms. The A/J-derived susceptible allele is negative (-) for the insertion whereas the C57BL/6 allele is positive (ins); 30 mice were -/-, 52 ins/-, and 29 ins/ins. (b) For <i>Kras</i>-4A, <i>P</i> = 9.7×10⁻¹⁴, ANOVA. Tukey's test for multiple comparisons, *<i>P</i> = 0.01, ***<i>P</i> = 7.4×10⁻¹⁴ vs. -/-. (c) For <i>Kras</i>-4B, <i>P</i> = 9.7×10⁻⁵, ANOVA. Tukey's test for multiple comparisons, **<i>P</i> = 1.5×10⁻⁴ vs. -/-. Values are means and SE.</p

Differential allelic expression of <i>Kras</i>-4A isoform indicates the existence of functional polymorphisms in <i>Kras</i> gene.

<p>Allelic ratios were determined by pyrosequencing for rs30022167 and rs29968550, which map in a region of the <i>Kras</i> 3′-UTR common to both isoforms, on genomic DNA and cDNA from normal lung tissue (n = 20) and lung tumor specimens (n = 15) from ABF4 heterozygous mice. Values are mean and SE; *** P<0.001, two-sided Welch's t test (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004307#pgen-1004307-t001" target="_blank"><b>Table 1</b></a> for complete data).</p

<i>Kras</i>-4A levels in lung tumors from 80 urethane-treated ABF4 mice are controlled by <i>Pas1</i> locus.

<p>(a) Expression QTL analysis of <i>Kras</i> transcripts showed that square-root-transformed levels of <i>Kras</i>-4A linked to the <i>Pas1</i> locus (LOD = 4.5). No significant linkage was observed for the <i>Kras</i>-4B mRNA isoform. Tick marks show the position of the genotyped markers in a recombinational map. Horizontal line at LOD = 2.55 marks the 95% threshold for significance. (b) Relative expression levels of the <i>Kras</i>-4A isoform according to rs6265387 genotype. Mice homozygous for the A/J-derived susceptible allele (GG, n = 37) had higher levels than either heterozygous animals (AG, n = 34) or mice homozygous for the C57BL/6 resistant allele (AA, n = 9). ***<i>P</i><0.001, **<i>P</i><0.01 vs. GG mice, ANOVA followed by Tukey's test for multiple comparisons. Values are means and SE.</p

Lung tumor multiplicity in 183 urethane-treated male ABF4 mice is controlled by the <i>Pa</i>s<i>1</i> locus.

<p>(a) LOD score plot for chromosome 6 on which a quantitative trait locus (QTL) for lung tumor multiplicity (square root transformed values) mapped to the telomeric region. The QTL peak (LOD score = 48, phenotypic variance explained = 69%) overlapped with the <i>Pas1</i> locus. Tick marks show the position of 37 genotyped markers, including rs6265387 at the QTL peak. Horizontal line indicates the 95% LOD threshold. (b) Number of lung tumors per animal, grouped according to genotype at rs6265387. Mice homozygous for the A/J-derived allele (GG; n = 59) had more tumors than either heterozygous animals (n = 82) or mice homozygous for the C57BL/6-derived allele (AA; n = 42). ***<i>P</i><1.0×10⁻⁶ versus the A/J-derived allele, ANOVA followed by Tukey's test for multiple comparisons. The line within each box represents the median; upper and lower edges of each box are 75^th and 25^th percentiles, respectively; upper and lower bars indicate the highest and lowest values less than one interquartile range from the extremes of the box.</p

<i>Kras</i>-4A is expressed at higher levels in susceptible (A/J) than resistant (C57BL/6) strains.

<p>Expression levels of <i>Kras</i>-4A (a) and <i>Kras</i>-4B (b) isoforms were measured by qPCR in normal lung of 11 A/J and 11 C57BL/6 mice. * <i>P</i> = 7.1×10⁻⁵, ANOVA. The line within each box represents the median of square root transformed values; upper and lower edges of each box are 75^th and 25^th percentiles, respectively; upper and lower bars indicate the highest and lowest values less than one interquartile range from the extremes of the box.</p