Enhanced natural killer activity and production of pro-inflammatory cytokines in mice selected for high acute inflammatory response (AIRmax)

Strains of mice with maximal and minimal acute inflammatory responsiveness (AIRmax and AIRmin, respectively) were developed through selective breeding based on their high- or low-acute inflammatory responsiveness. Previous reports have shown that AIRmax mice are more resistant to the development of a variety of tumours than AIRmin mice, including spontaneous metastasis of murine melanoma. Natural killer activity is involved in immunosurveillance against tumour development, so we analysed the number and activity of natural killer cells (CD49b+), T-lymphocyte subsets and in vitro cytokine production by spleen cells of normal AIRmax and AIRmin mice. Analysis of lymphocyte subsets by flow cytometry showed that AIRmax mice had a higher relative number of CD49b+ cells than AIRmin mice, as well as cytolytic activity against Yac.1 target cells. The number of CD3+ CD8+ cells was also higher in AIRmax mice. These findings were associated with the ability of spleen cells from AIRmax mice in vitro to produce higher levels of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin-12p40 and interferon-γ but not the anti-inflammatory interleukin-10. Taken together, our data suggest that the selective breeding to achieve the AIRmax and AIRmin strains was able to polarize the genes associated with cytotoxic activity, which can be responsible for the antitumour resistance observed in AIRmax mice

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

Genetic Predisposition to Hepatocarcinogenesis in Inbred and Outbred Mouse Lines Selected for High or Low Inflammatory Response

AIRmax and AIRmin mouse strains phenotypically selected for high and low acute inflammatory responsiveness (AIR) are, respectively, susceptible or resistant to developing hepatocellular carcinoma (HCC) induced by the chemical carcinogens urethane and diethylnitrosamine (DEN). Early production of TNF-α, IL-1β, and IL-6 in the liver after DEN treatment correlated with tumor development in AIRmax mice. Transcriptome analysis of livers from untreated AIRmax and AIRmin mice showed specific gene expression profiles in each line, which might play a role in their differential susceptibility to HCC. Linkage analysis with SNP markers in F2 (AIRmax×AIRmin) intercross mice revealed two quantitative trait loci (QTL) in chromosomes 2 and 9, which are significantly associated with the number and progression of urethane-induced liver tumors. An independent linkage analysis with an intercross population from A/J and C57BL/6J inbred mice mapped regions in chromosomes 1 and 7 associated with the progression of urethane-induced liver tumors, evidencing the heterogeneity of HCC genetic control

<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

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 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

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