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. 2022 Nov 16:13:999155.
doi: 10.3389/fimmu.2022.999155. eCollection 2022.

Diagnostic and therapeutic potential of RNASET2 in Crohn's disease: Disease-risk polymorphism modulates allelic-imbalance in expression and circulating protein levels and recombinant-RNASET2 attenuates pro-inflammatory cytokine secretion

Eva Biener-Ramanujan  1 , Florian Rosier  1 , Simon G Coetzee  2 , Dermot D P McGovern  1 , Dennis Hazelett  2 , Stephan R Targan  1 , Rivkah Gonsky  1
Affiliations

Diagnostic and therapeutic potential of RNASET2 in Crohn's disease: Disease-risk polymorphism modulates allelic-imbalance in expression and circulating protein levels and recombinant-RNASET2 attenuates pro-inflammatory cytokine secretion

Eva Biener-Ramanujan et al. Front Immunol. .

Abstract

Ribonuclease T2 gene (RNASET2) variants are associated in genome wide association studies (GWAS) with risk for several autoimmune diseases, including Crohn's disease (CD). In T cells, a functional and biological relationship exists between TNFSF15-mediated enhancement of IFN-γ production, mucosal inflammation and RNASET2. Disease risk variants are associated with decreased mRNA expression and clinical characteristics of severe CD; however, functional classifications of variants and underlying molecular mechanisms contributing to pathogenesis remain largely unknown. In this study we demonstrate that allelic imbalance of RNASET2 disease risk variant rs2149092 is associated with transcriptional and post-transcriptional mechanisms regulating transcription factor binding, promoter-transactivation and allele-specific expression. RNASET2 mRNA expression decreases in response to multiple modes of T cell activation and recovers following elimination of activator. In CD patients with severe disease necessitating surgical intervention, preoperative circulating RNASET2 protein levels were decreased compared to non-IBD subjects and rebounded post-operatively following removal of the inflamed region, with levels associated with allelic carriage. Furthermore, overexpression or treatment with recombinant RNASET2 significantly reduced IFN-γ secretion. These findings reveal that RNASET2 cis- and trans-acting variation contributed regulatory complexity and determined expression and provide a basis for linking genetic variation with CD pathobiology. These data may ultimately identify RNASET2 as an effective therapeutic target in a subset of CD patients with severe disease.

Keywords: Crohn’s disease; allelic imbalance; cytokine; gene regulation; human; inflammation.

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Conflict of interest statement

ST – is a stockholder and a consultant for Prometheus Biosciences; DM - is a stockholder and a consultant for Prometheus Biosciences Cedars-Sinai - has financial interests in Prometheus Biosciences, Inc., a company which has access to the data and specimens in Cedars-Sinai’s MIRIAD Biobank (including the data and specimens used in this study) and seeks to develop commercial products. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Cis-Regulatory SNP, rs2149092, on RNASET2 promoter is predicted to alter ETS TF binding. (A) StateHub modeling of RNASET2 in proximity of SNP rs12149092 (B) DNA topography analysis demonstrating that SNP rs2149092 (C>T) is predicted to alter DNA propeller twist configuration at the ETS1 binding site. (C) Heat map and Pearson’s correlation coefficients (PCC) comparing region containing rs2149092 C and T SNP to consensus ETS1 binding site.
Figure 2
Figure 2
Allele specific nucleo-protein binding to RNASET2 promoter regions. (A) Sequence of rs1819333 and rs2149092 oligonucleotide probes. ETS1 TFBS are underlined. (B–D) EMSA analysis using nuclear extract from CD4+ T cells binding to (B) rs1819333 (C) rs2149092 or (D) rs2149092 supershifted complexes in the presence of ETS1-specific antibody. (E) ETS1 Chip-seq data from human purified T cells (FANTOM project). Unlabeled oligo competitors (comp): rs2149092 C or T SNP. Representative of 2-4 experiments.
Figure 3
Figure 3
RNASET2 transcript downregulation is a hallmark of T cell activation. Expression of RNASET2 in CD4+ T cells activated with TL1A (upper panels) or TCR (lower panels). (A) Expression of RNASET2 is decreased in cells isolated from multiple donors (TL1A n=22, TCR n=9). (B) Concordant decrease in ELF2, ETS1 and RNASET2 (TL1A n=5, TCR n=6). Data show the mean ± SD. (C) Kinetics of RNASET2 expression following activation (TL1A n=8, TCR n=3). ns, not significant.
Figure 4
Figure 4
Functional analysis of RNASET2 promoter SNP variants. (A) Schematic illustration of luciferase reporter construct used to analyze functional contribution of risk and non-risk index rs1819333 and regulatory rs2149092 variants on RNASET2 promoter activity. Tick marks represent SNPs in LD with both rs1819333 tagging and rs2149092 regulatory variants. Transcription factors (TF) associated with both a putative SNP-dependent binding site and with RNASET2 eQTL risk carriage are listed. Boxed region directly upstream of rs1819333 depicts TF binding sites for ETS family transcriptional repressors. (B) EQTL ELF4 expression vs. rs1819333 allelic carriage. (C, D). CD4+ T cells were transfected with promoter-reporter vectors. (C) Resting (-4kb, n=16, -3.6kb, n=22) or (D) TL1A (-4kb, n=8, -3.6kb n=8) activated cells. Data show the mean ± SD ***P < 0.001, **P < 0.01; *P < 0.05.
Figure 5
Figure 5
TL1A activation inversely impacts nascent and pre-existing RNASET2 transcripts. (A) Increase in nascent (p=0.047) and decease in pre-existing (p=0.03) RNASET2 transcript from PBMC cells treated with TL1A. Levels are plotted as fold over expression in untreated cells (n=6). (B) Intracellular IFN-γ protein increases and RNASET2 protein decreases following TL1A stimulation. Flow cytometry analysis representative of three with similar results.
Figure 6
Figure 6
RNASET2 Risk variant dominates RNASET2 expression. (A) Diagram mapping the location and LD of rs1044059 on RNASET2 promoter. (B) Analysis in CD4+ T cells of total RNASET2 expression (left panel) or allele specific expression (right panel) with or without TL1A activation of (n=12). Allele specific expression in (C). Resting PBMC isolated from non-IBD (n=6), CD (n=9) or UC (n=8) subjects. Across all samples the allele specific ratio of risk vs. non-risk expression was ~ 2:1 with a p value <0.001.
Figure 7
Figure 7
RNASET2 risk variant is associated with decreased levels of circulating protein. Circulating RNASET2 protein levels associated with allelic carriage in (A) non-IBD subjects (n=84) or (B) CD patients at time of surgery (n=93 left panel) and up to one year post surgery (n=47 right panel). (C) Increase in circulating levels of RNASET2 following surgical removal of inflamed intestinal region. (D) Recovery of RNASET2 transcript levels in CD4+ T cells following removal of TL1A activation (n=6). Data show the mean ± SD. *P < 0.05.
Figure 8
Figure 8
Recombinant RNASET2 attenuates IFN-γ secretion in a dose dependent manner. CD4+ T cells were either (A) Transfected with RNASET2 overexpression vector (n=21) or (B) Treated with recombinant RNASET2 prior to TL1A stimulation (n=5). (C) Resting PBMC isolated from IBD subjects (CD n=2; UC n=3) treated with 400 ng/ml recombinant RNASET2 protein. IFN−γ secretion was measured after 24 hours. Bottom panels depict fold inhibition in IFN−γ secretion comparing overexpression vs. empty vector or recombinant RNASET2 treatment vs. no recombinant protein.
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