Paraoxonase‑3 Is Depleted from the High-Density Lipoproteins of Autoimmune Disease Patients with Subclinical Atherosclerosis

Patients with autoimmune diseases have a significantly increased risk of developing cardiovascular disease. In disease, high-density lipoprotein (HDL) particles lose their anti-inflammatory and antioxidant properties and become dysfunctional. The purpose of this study was to test the hypothesis that alterations in the HDL proteomic profile are associated with subclinical atherosclerosis and HDL dysfunction in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and type 1 diabetes. Targeted proteomics was used to quantify the relative abundance of 18 proteins in HDL from SLE patients with and without atherosclerotic plaque detectable by carotid ultrasound. Changes in the proteomic profile were compared against the in vitro ability of HDL to protect against lipid oxidation. The same proteins were quantified in HDL from patients with type 1 diabetes with or without coronary artery calcification as determined by computed tomography. In each population, paraoxonase-3 (PON3), a potent antioxidant protein, was depleted from the HDL of patients with subclinical atherosclerosis. PON3 expression in HDL was positively correlated with HDL antioxidant function. These results suggest that PON3 may be an important protein in preventing atherosclerosis and highlight the importance of antioxidant proteins in the prevention of atherosclerosis in vivo

The SLE-risk G allele of rs3853839 confers elevated TLR7 expression through slower mRNA degradation.

<p>(A) Association of rs3853839 genotypes with <i>TLR7/8</i> transcript levels in EA normal PBMCs. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (B) Association of rs3853839 genotypes with TLR7 protein levels in normal PBMCs. FACS histograms show the log MFI values plotted against the cell counts for PBMCs in individuals carrying G or C allele, compared with isotype control. Results are from one representative pair (GG or G vs. CC or C) of 7 in each gender group. MFI of TLR7 expression in PBMCs is graphically depicted. Each symbol represents an individual and horizontal lines indicate mean ± SEM values. (C) Verification of the G allele conferring elevated expression of a luciferase reporter <i>in vitro</i>. <i>TLR7</i> 3′UTR segment bearing G or C allele of rs3853839 was cloned into the psiCHECK-2 reporter vector and luciferase activity was determined after 24 hours of transfection. Relative luciferase activity is Renilla/Firefly luciferase ratio. Data show the mean ± SEM and are representative of cumulative data from four independent experiments. (D) The kinetics of the G/C allele ratio in <i>TLR7</i> transcripts from PBMCs of healthy heterozygous individuals (n = 7) in the absence or presence of actinomycin D (ActD). The G/C allele ratio obtained in <i>TLR7</i> transcripts was normalized to that measured from gDNA of the same sample. Data are expressed as mean ± SEM at each time point and representative of cumulative data from two independent experiments with seven healthy donors. ^*<i>P</i><0.05. (E) Summary of the G/C allele ratio in <i>TLR7</i> transcripts 4 hours after the addition of ActD or vehicle control (n = 7). Comparisons are between ActD and vehicle control cultures; <i>P</i> = 0.04; paired <i>t</i> test. FACS, Fluorescence-activated cell sorter; MFI, mean fluorescence intensity.</p

Association of rs3853839 with SLE in multiple ancestries.

*<p>AS: Previously published data in population of Eastern Asian descent (5).</p><p>Abbreviation: AS, Eastern Asian; AA, African American; EA, European American; HS, Amerindian/Hispanics; OR, odds ratio; CI, confidence interval.</p

Allelic associations of SNPs in the <i>TLR7-TLR8</i> region with SLE.

<p>(A) The genomic structure of the <i>TLR7-TLR8</i> region and the location of all studied SNPs are indicated. (B) Association signals (−log₁₀<i>P</i>) are plotted against the position of each SNP (based on GRch37/hg19) in European Americans (EA), African Americans (AA), and Hispanics (HS). Genotyped and imputed SNPs are depicted with circles and triangles, respectively. The diamond identifies the <i>TLR7</i> 3′UTR SNP rs3853839. SNPs are highlighted using different colors according to their LD strength (r²) with rs3853839. (C) A trans-ancestral meta-analysis is conducted on 40 genotyped SNPs (circles) and 14 imputed SNPs (triangles) that are shared by the three ancestries (SNPs listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003336#pgen.1003336.s006" target="_blank">Table S1</a>) using fixed and random model, respectively. The dashed line represents the significance level of 5×10⁻⁸.</p

The SLE-risk G allele of rs3853839 displays reduced transcript modulation by miR-3148.

<p>(A) TargetScan's predicted miR-3148-binding site in <i>TLR7</i> 3′UTR. The C allele, rather than G allele of rs3853839 corresponds to the second base of this seed region. (B) Inverse correlation of miR-3148 and <i>TLR7</i> transcript levels in PBMCs from 16 patients with SLE (solid circles) and 21 controls (open diamonds). (C) HEK-293 cells were cotransfected with empty reporter vector (EV), luciferase constructs driven by <i>TLR7</i> 3′UTR segment containing either C or G allele of rs3853839 and increasing concentrations (1, 6, 12, and 48 nM) of miR-3148 or nontarget control (NC) mimics. Luciferase activity was determined 24 hours after transfection. Normalized luciferase activity is the Renilla/Firefly ratio of miR-3148-treated reporter vector compared with the same NC-treated reporter vector. Data show the mean ± SEM and are representative of cumulative data from three independent experiments. <i>P</i> = 0.0003 over all miR-3148-treated C-allele vector groups, and not significant over all miR-3148-treated G-allele or empty vector groups (ANOVA test). <i>P*</i> = 0.02, <i>P**</i><0.0001 (Student's <i>t</i> test) for the comparison of indicated groups.</p

SNPs of the <i>IL10</i> gene cluster associated with SLE in European Americans.

<p>(A) Association of 19 genotyped SNPs with SLE in EA (red), AA (yellow), AS (blue) and HS (green). Allelic <i>P</i> value (−log₁₀<i>P</i>) of each SNP was plotted against its genomic position. (B) Association of 19 genotyped and an additional 109 imputed SNPs with SLE in EA. Genotyped and imputed SNPs were indicated as circles and triangles, respectively. Based on its pairwised LD strength with rs3024505, each SNP was highlighted as red (r²>0.9) or grey (r²<0.9). (C) Genomic structure of the <i>IL10</i> gene cluster and the location of each SNP. (D) Haplotypic analysis in EA. Haplotypes were constructed using four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> (rs3024505, rs3024495, rs3024493 and rs3122605), three previously reported SLE-associated SNPs (rs1800872, rs1800871 and rs1800896 in the promoter of <i>IL10</i>) and rs1518111 (the T allele associated with Bechet's disease). Risk alleles of four SLE-associated SNPs shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003870#pgen-1003870-g001" target="_blank">Figure 1B</a> were bolded and italicized.</p

Dose-dependent association of rs3122605 risk G-allele with elevated levels of <i>IL10</i> mRNA and protein.

<p><i>IL10</i> mRNA (A) and protein levels (B) were measured in PBMCs and plasma from EA SLE patients and healthy controls, respectively. Each symbol represents an individual and horizontal lines indicate mean ± SEM values.</p