Tissue-specific RNA-Seq in human evoked inflammation identifies blood and adipose LincRNA signatures of cardiometabolic diseases

Abstract

Objective: Inappropriate transcriptional activation of innate immunity is a pathological feature of several cardiometabolic disorders, but little is known about inflammatory modulation of long intergenic noncoding RNAs (lincRNAs) in disease-relevant human tissues.

Approach and results: We applied deep RNA sequencing (>500 million filtered reads per sample) to blood and adipose during low-dose experimental endotoxemia (lipopolysaccharide) in a healthy human, with targeted replication in separate individuals undergoing endotoxemia (n=6), to identify inflammatory lincRNAs. A subset of these lincRNAs was examined for expression in adipocytes and monocytes, modulation in adipose of obese humans, and overlap with genome-wide association study signals for inflammatory and cardiometabolic traits. Of a stringent set of 4284 lincRNAs, ≈11% to 22% were expressed with 201 and 56 lincRNAs modulated by lipopolysaccharide in blood or adipose, respectively. Tissue-specific expression of a subset of 6 lipopolysaccharide-lincRNAs was replicated with lipopolysaccharide modulation confirmed for all 3 expressed in blood and 2 of 4 expressed in adipose. The broader generalizability of findings in blood of subject A was confirmed by RNA sequencing in 7 additional subjects. We confirmed adipocytes and monocytes as potential cell-sources of selective lipopolysaccharide-regulated lincRNAs, and 2 of these, linc-DMRT2 (P=0.002) and linc-TP53I13 (P=0.01), were suppressed in adipose of obese humans. Finally, we provide examples of lipopolysaccharide-modulated lincRNAs that overlap single nucleotide polymorphisms that are associated with cardiometabolic traits.

Conclusions: Our findings provide novel insights into tissue-level, inflammatory transcriptome regulation in cardiometabolic diseases. These are complementary to more usual approaches limited to interrogation of DNA variations.

Trial registration: ClinicalTrials.gov NCT00953667.

Keywords: RNA sequence; genomics; lincRNA.

Figures

Figure 1. Overview of study design and results

Discovery (subject A) and replication subjects (B–G) were selected from those participants in the Genetics of Evoked-response to Niacin and Endotoxemia (GENE) study with above median inflammatory cytokine responses during endotoxemia (Table 1). Deep RNA-sequencing generated >500 million (“500M”) reads per sample. *Of six LPS-regulated lincRNAs selected for follow-up, three were expressed in blood and four expressed in adipose. Quantitative real-time PCR (qRT-PCR) assays were used for validation (subject A), replication (subjects B-G), human cell studies and a pilot case-control study of obesity (the four adipose lincRNAs were tested). †All LPS-regulated lincRNAs (49 in blood and 56 in adipose) were tested relative to expressed non-LPS modulated lincRNAs (182 blood and 413 adipose) for enrichment with SNPs that have genome-wide significant association with inflammatory and cardio-metabolic traits in the NHGRI GWAS catalogue (http://www.genome.gov/gwastudies). Linc-CEP110-13, induced by LPS, overlaps a SNP (rs10115586) that associates with N-Glycosylation of circulating IgG. Linc-VWF, a LPS-induced lincRNA, contained a SNP (rs1558324) associated with mean platelet volume, a predictor of cardiovascular disease.

Figure 2

Venn diagram for expression, tissue overlap and LPS-modulation of (A) lincRNAs and (B) protein-coding genes in adipose (yellow, left) and blood (blue, right) of subject A.

Figure 3. Validation and replication for LPS modulated lincRNAs

Data for (A) blood lincRNAs, linc-ARFIP1-4 and linc-CMC1-2 as well as (B) adipose lincRNAs, linc-DMRT2 and linc-POTED8 are shown. (i) Genome browser views of RNA-seq data before and after LPS in subject A. (ii) Quantitative real-time PCR (qRT-PCR) validation of lincRNA in tissue of subject A. (iii) qRT-PCR replication of lincRNA in tissues of subject B-G (subject G blood sample failed). For (ii) and (iii) expression data are presented as bar-graphs of relative fold-change compared with pre-LPS. The delta CT’s represent the median cycle threshold for lincRNAs relative to B-actin mRNA as the reference in each sample.

Figure 4. Translational studies of selective LPS-modulated lincRNAs

(A) Human cells; expression of linc-DMRT2, linc-SLC16A7, linc-TP53I1, linc-POTED8, linc-ARFIP1-4, linc-CMC1-2 (by qRT-PCR), in adipocytes, primary human monocytes, macrophages, M1-type macrophages (M1), and M-2 type macrophages (M2). LincRNA expression data are presented as bar graphs of the mean (SD) fold-change in in monocytes, macrophages and M1 or M2 macrophages relative to adipocyte expression; the delta CT’s represent the median cycle threshold for lincRNAs relative to B-actin mRNA as the reference in each sample; (B) Adipose of lean vs. obese subjects; expression (by qRT-PCR) of linc-DMRT2, linc-SLC16A7, linc-TP53I1, linc-POTED8 in subcutaneous adipose of obese (n=12) compared to lean (n=12) humans. LincRNA expression data are presented as bar graphs of the mean (SD) fold-change in obese vs. lean adipose tissue. Linc-DMRT2 (P=0.002) and linc-TP53I1 (P=0.01) expression are suppressed in obesity (Mann Whitney U non-parametric test on the delta CTs). ND=not detected. NA=not assessed.

Figure 5. (A) Genome browser view and (B) linkage disequilibrium plot for the chromosome 12 linc-VWF and VWF regions in European Ancestry

Highlighted linc-VWF SNPs (rs1558324, rs7342306) have genome-wide association with platelet traits in Gieger et al.