The SLE transcriptome exhibits evidence of chronic endotoxin exposure and has widespread dysregulation of non-coding and coding RNAs

Abstract

Background: Gene expression studies of peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE) have demonstrated a type I interferon signature and increased expression of inflammatory cytokine genes. Studies of patients with Aicardi Goutières syndrome, commonly cited as a single gene model for SLE, have suggested that accumulation of non-coding RNAs may drive some of the pathologic gene expression, however, no RNA sequencing studies of SLE patients have been performed. This study was designed to define altered expression of coding and non-coding RNAs and to detect globally altered RNA processing in SLE.

Methods: Purified monocytes from eight healthy age/gender matched controls and nine SLE patients (with low-moderate disease activity and lack of biologic drug use or immune suppressive treatment) were studied using RNA-seq. Quantitative RT-PCR was used to validate findings. Serum levels of endotoxin were measured by ELISA.

Results: We found that SLE patients had diminished expression of most endogenous retroviruses and small nucleolar RNAs, but exhibited increased expression of pri-miRNAs. Splicing patterns and polyadenylation were significantly altered. In addition, SLE monocytes expressed novel transcripts, an effect that was replicated by LPS treatment of control monocytes. We further identified increased circulating endotoxin in SLE patients.

Conclusions: Monocytes from SLE patients exhibit globally dysregulated gene expression. The transcriptome is not simply altered by the transcriptional activation of a set of genes, but is qualitatively different in SLE. The identification of novel loci, inducible by LPS, suggests that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism.

Figure 1. Comparison of SLE and control transcriptomes.

The Venn diagrams demonstrate the overlap of actively transcribed A) coding genes, B) antisense transcripts, C) novel loci, and D) novel isoforms in the two sample groups. Eight control and nine SLE libraries were used.

Figure 2. Differential expression of RNA classes.

A) The average transcriptional changes of five RNA classes in SLE. B) The average transcriptional change of four sub-classes of small RNAs in SLE. C) The average transcriptional change of 17 subclasses of repetitive elements in SLE. The eight control and nine SLE libraries were used for this analysis. Error bars indicate standard deviation.

Figure 3. PCR validation in new samples.

A) Ten novel loci (70, 107, 122, 142, 171, 172, 282. 721, 772, and 775) were amplified using 11 controls (8 new controls and 3 controls used for the RNA-seq libraries) and 11 new SLE patients. Transcript levels were normalized to β-actin. In each case, the differential expression between SLE and controls was statistically significant with p<0.05, according to the Mann-Whitney test. The cross bars indicate mean and standard error. Primers and locations are given in Methods S1. B) There was a good agreement of expression fold changes in SLE between the RNA-seq and qRT-PCR experiments with p<0.05 (*), p<0.01 (**) or p<0.001(***). All but two of 22 tested novel loci were upregulated in both SLE patient cohorts.

Figure 4. LPS stimulation of monocytes.

A) MonoMac 6 cells were stimulated for 16 hours as indicated and qRT-PCR was performed for the novel loci. Only LPS (1 µg/ml) stimulation consistently upregulated these loci. TNF-α (10 ng/ml) led to upregulation of 16/25 (fold change>1.5), αIFN (100 U/ml) led to upregulation of 9/25 (fold change>1.5), γIFN (10 ng/ml) led to upregulation of 5/25 (fold change>1.5), and LPS led to upregulation of 22/25 (fold change>1.5). Three experiments with duplciates or triplicates were averaged. The error bars indicate standard deviation. B) Human primary monocytes were stimulated as above and the abundance of the novel transcripts quantitated by qRT-PCR. TNF-α led to upregulation of 9/25 (fold change>1.5), αIFN led to upregulation of 7/25 (fold change>1.5), γIFN led to upregulation of 9/25 (fold change>1.5), and LPS led to upregulation of 23/25 (fold change>1.5). Pre-treatment of cells with the p38 inhibitor, SB203580 led to markedly diminished induction of expression by LPS. Three experiments with duplciates or triplicates were averaged. The error bars indicate standard deviation. C) The average fold change for the aggregated novel upregulated loci in SLE were calculated for MonoMac 6 cells and D) healthy human primary monocytes stimulated by LPS, TNF-α, αIFN,or γIFN. Locus 896 was down-regulated and locus 477 was not changed in the orignal SLE samples and were included as controls. The error bars indicate standard deviation in C and D.

Figure 5. SLE patient circulating endotoxin levels.

Circulating endotoxin was quantitated using the Limulus assay. 99 female SLE patients and 112 female Red Cross blood donors were anlayzed. SLE patients had significantly more endotoxin on average than controls (P<0.0001). The cross bars indicate the mean and standard error.