Bank1 and NF-kappaB as key regulators in anti-nucleolar antibody development

<div><p>Systemic autoimmune rheumatic disorders (SARD) represent important causes of morbidity and mortality in humans. The mechanisms triggering autoimmune responses are complex and involve a network of genetic factors. Mercury-induced autoimmunity (HgIA) in mice is an established model to study the mechanisms of the development of antinuclear antibodies (ANA), which is a hallmark in the diagnosis of SARD. A.SW mice with HgIA show a significantly higher titer of antinucleolar antibodies (ANoA) than the B10.S mice, although both share the same MHC class II (H-2). We applied a genome-wide association study (GWAS) to their Hg-exposed F2 offspring to investigate the non-MHC genes involved in the development of ANoA. Quantitative trait locus (QTL) analysis showed a peak logarithm of odds ratio (LOD) score of 3.05 on chromosome 3. Microsatellites were used for haplotyping, and fine mapping was conducted with next generation sequencing. The candidate genes <i>Bank1</i> (B-cell scaffold protein with ankyrin repeats 1) and <i>Nfkb1</i> (nuclear factor kappa B subunit 1) were identified by additional QTL analysis. Expression of the <i>Bank1</i> and <i>Nfkb1</i> genes and their downstream target genes involved in the intracellular pathway (<i>Tlr9</i>, <i>Il6</i>, <i>Tnf</i>) was investigated in mercury-exposed A.SW and B10.S mice by real-time PCR. <i>Bank1</i> showed significantly lower gene expression in the A.SW strain after Hg-exposure, whereas the B10.S strain showed no significant difference. <i>Nfkb1</i>, <i>Tlr9</i>, <i>Il6</i> and <i>Tnf</i> had significantly higher gene expression in the A.SW strain after Hg-exposure, while the B10.S strain showed no difference. This study supports the roles of <i>Bank1 (</i>produced mainly in B-cells) and <i>Nfkb1</i> (produced in most immune cells) as key regulators of ANoA development in HgIA.</p></div

Gene expression of <i>Il6</i> and <i>Tnf</i>.

<p>Experimental design same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>. Total RNA expression of <i>Il6</i> in <b>A</b>) A.SW and <b>B</b>) B10.S mice followed by <i>Tnf</i> expression in <b>C)</b> A.SW and <b>D)</b> B10.S mice. Figure represents fold difference (y-axis) and presented as median ± interquartile range for each group (Kruskal Wallis, Dunn's multiple comparisons test). Significant difference (* p < 0.05, ** p < 0.01) between groups in each strain. Endogenous controls and statistical analysis same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>.</p

Serum antinucleolar antibodies (ANoA).

<p>Serum IgG ANoA in F2 mice control (n = 14) and F2 mice exposed to 4 mg HgCl₂/L (n = 129) after 6-week exposure. A) ANoA assessed by indirect immunofluorescence using HEp2 cells. Arrows show strong clumpy staining of the nucleoli. B) Y-axis represents the ANoA titer (0–20,480). Graph is presented as the median ± interquartile range, ****p = < 0.0001 (Mann–Whitney test).</p

Genome-wide scan and effect plot.

<p><b>A)</b> A genome-wide scan (n = 129) on autosomes was performed to identify quantitative trait loci associated with anti-nucleolar antibodies (ANoA) in mice exposed to mercury. Logarithm of odds (LOD) scores (y-axis) demonstrate curves over the murine autosomal chromosomes. X-axis demonstrates SNP markers on 19 autosomes. Lines represents association between genetic position and phenotype, serum antinucleolar antibodies. Arrow indicates the top peak on chromosome 3. <b>B)</b> Effect of different alleles in F2 offspring at peak marker rs3670168 on chromosome 3. Allele effects in the F2 offspring (X-axis), homozygous for A.SW (AA) or B10.S (BB) or heterozygous (AB) for ANoA titer (y-axis). The plot displays the mean ± SEM. **p < 0.01 (Mann-Whitney test).</p

Fine mapping and QTL.

<p><b>A</b>) Haplotypes associated with ANoA in the range 128 110 214–136 217 610 bp on chromosome 3, with 11 genes containing differences in SNPs between background strains A (for A.SW) and C57BL/6 (for B10.S). <b>B</b>) Fine mapping exons in 11 genes within the haplotype region performed with NGS to identify QTL associated with ANoA in 30 F2 mice homozygous for A.SW on rs3670168. LOD scores of 2.44 for <i>Nfkb1</i> and 2.46 and 2.47 for <i>Bank1</i>.</p

Dose of Hg exposure and number of mice in each study.

<p>Dose of Hg exposure and number of mice in each study.</p

Relative total RNA expression of splice variants of Bank1.

<p>Relative total RNA expression of splice variants of <i>Bank1</i> in spleens. Experimental design is same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>. Figure represents relative RNA expression (y-axis) of full length and Δ2 splice variants in <b>A)</b> A.SW and <b>B)</b> B10.S. Empty bars represents full-length (FL) splice variant and filled bar represents short length (Δ2) splice variant. Figure presented as median ± interquartile range for each group (Kruskal Wallis, Dunn's multiple comparisons test). Significant difference (** p < 0.01) between groups in each strain.</p

Gene expression of <i>Tlr9</i>.

<p>Experimental design same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>. Total RNA expression of <i>Tlr9</i> in female <b>A</b>) A.SW and <b>B</b>) B10.S. Figure represents fold difference (y-axis) and presented as median ± interquartile range for each group (Kruskal Wallis, Dunn's multiple comparisons test). Significant difference (* p < 0.05) between groups in each strain. Endogenous controls and statistical analysis same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>.</p

Gene expression of <i>Nfkb1</i>.

<p>Experimental design same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>. Total RNA expression of <i>Nfkb1</i> in <b>A</b>) A.SW and <b>B</b>) B10.S mice. Figure represents fold difference (y-axis) and presented as median ± interquartile range for each group (Kruskal Wallis, Dunn's multiple comparisons test). Significant difference (* p < 0.05, ** p < 0.01) between groups in each strain. Endogenous controls and statistical analysis same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>.</p

Additional file 1 of Designing a tool ensuring older patients the right medication at the right time after discharge from hospital– the first step in a participatory design process

Supplementary Material