Cellular sensing of extracellular purine nucleosides triggers an innate IFN-β response

Abstract

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-β (IFN-β) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-β. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-β. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-β production.

Fig. 1. Loss of ADA2 triggers spontaneous IFNβ production.

(A) siRNA screen of 38 human disease genes in HUVEC (table S1), quantifying IRF3 nuclear translocation upon poly (dA:dT) DNA treatment (200 ng/ml for 3 hours). Averaged ranked z score for each gene-specific siRNA oligonucleotide pool is represented. STING, TREX1, and ADA2 scores are indicated in red (n = 3 technical replicates for n = 3 biological replicates, >200 single cells per technical replicate). (B) Western blot analysis of siControl, siADA2-, or siTREX-transfected HUVEC lysates (35 μg per lane). ImageJ quantification of the intensity ratio between phospho-IRF3/pan IRF3 and phospho-TBK1/TBK in unstimulated cells is shown in bar graphs. IB, immunoblot.; au, arbitrary units. (C) IFN-β mRNA levels, measured by qRT-PCR, in siControl, siADA2-, or siTREX1-transfected HUVEC upon treatment with vehicle or infection with hCMV [multiplicity of infection (MOI) = 1 for 3 hours] (n = 3 technical replicates). (D) Differential gene expression between siControl and siADA2-transfected HUVEC measured by polyadenylated poly (A⁺)–enriched RNA-seq (n = 3 biological replicates) and REACTOME pathway analysis of the ADA2-specific genes. Red and blue dots denote genes significantly up- or down-regulated >2-fold. (E) Expression levels of IRF3-driven or IFN-β–driven genes, measured by qRT-PCR, in siControl, siADA2-, or siTREX1-transfected HUVEC upon mock or hCMV infection (MOI = 1 for 3 hours). (F) Expression levels of IRF3-driven or IFN-β–driven genes, measured by qRT-PCR, in siControl or siADA2-transfected HUVEC treated with IFN-β–neutralizing antibody (10, 20, and 40 U/ml) (n = 3 technical replicates). All results were replicated in three independent experiments. Values are presented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001. MHC, major histocompatibility complex; ER, endoplasmic reticulum; IB, immunoblot.

Fig. 2. Extracellular ADA2 and dAdo are paracrine modulators of IFNβ.

(A) ADA2 mRNA levels, measured by qRT-PCR, in primary endothelial cells and U937 monocytic cells (n = 3 technical replicates). (B) Secreted ADA2 protein, measured by ELISA, in primary endothelial cells and U937 monocytic cells (n = 3 technical replicates). (C) Secreted ADA2 protein, assessed by Western blotting of serum-free cell-conditioned supernatants, from HUVEC and U937 monocytic cells. (D) ADA activity, measured by de novo conversion of 1 mM isotopically labeled dAdo to dIno in whole-cell lysates (intracellular) or cell-conditioned supernatants (extracellular) from siControl, siADA1-, or siADA2-transfected HUVEC. Values are normalized to cell number and protein concentration (n = 5 technical replicates). (E) Expression levels of IRF3-driven or IFN-β–driven genes, measured by qRT-PCR, and extracellular ADA activity, measured by de novo conversion of 1 mM isotopically labeled dAdo to dIno in cell-conditioned supernatants, from siControl or siADA2-transfected HUVEC supplemented with rADA1 or rADA2 and pretreated with vehicle or pentostatin (10 μM for 30 min). Activity values are normalized to cell number and protein concentration (n = 5 technical replicates). (F) IFN-β mRNA, measured by qRT-PCR, in U937 monocytic cells supplemented with adenosine (Ado) or deoxyadenosine (dAdo) (100 μM for 48 hours) (n = 3 technical replicates). (G) IFN-β mRNA levels, measured by qRT-PCR, in U937 cocultured in Transwell inserts with siControl or siADA2-treated HUVEC for 48 hours (n = 3 technical replicates). All results were replicated in three independent experiments. Values are presented as means ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. HDVEC, dermal microvascular endothelial cells; HBVEC, brain microvascular endothelial cells; HKVEC, kidney microvascular endothelial cells.

Fig. 3. Cellular nucleoside transport is required for induction of IFNβ upon depletion of ADA2.

(A) Schematic representation of extracellular Ado receptor signaling and dAdo uptake/intracellular metabolism. cAMP, cyclic adenosine 3′,5′-monophosphate; PKA, cAMP-dependent protein kinase. (B) Equilibrative nucleoside transporter (ENT) mRNA levels, measured by qRT-PCR, in HUVEC and U937 (n = 3 technical replicates). (C) Extracellular and intracellular levels of isotope-labeled dAdo, measured by LC-MS/MS (n = 5 biological replicates). (D) Differential gene expression in siControl, siADA2-, or siADA2/DPM-treated (40 μM for 48 hours) HUVEC, measured by polyA⁺ RNA-seq (n = 3 biological replicates). All results were replicated in three independent experiments. Values are presented as means ± SD. *P ≤ 0.05.

Fig. 4. Loss of ADA2 drives intracellular dAdo catabolism and accumulation of dIno.

(A) Intracellular metabolic pathways of purine degradation [ADA1 (protein); PNP (protein); and HGPRT (protein), hypoxanthine phosphoribosyltransferase] and purine salvage [ADK (protein), adenosine kinase; dCK (protein), deoxycytidine kinase; and 5′-NT, 5′-nucleotidase]. (B) Relative levels and concentrations of small-molecule polar metabolites measured by LC-MS/MS quantification of siControl or siADA2-transfected HUVEC (n = 5 biological replicates). (C) De novo accumulation of ¹⁵N-dAdo–labeled dIno measured by LC-MS/MS quantification of siControl or siADA2-treated HUVEC (n = 5 biological replicates). (D) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC transfected with siRNAs targeting ADA2 and ADA1, PNP, HGPRT (protein), or dCK. (D) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC supplemented with dIno (500 μM for 24 hours) or (E) pretreated with the PNP inhibitor 9-deazaguanine (100 μM for 30 min) before dIno supplementation (500 μM for 24 hours) (n = 3 technical replicates). (F) Flux analysis of the methionine/SAM cycle, measured by LC-MS/MS quantification of U-¹³C methionine–labeled methionine, SAM, and SAH, in siControl or siADA2-treated HUVEC (n = 5 biological replicates). (G) Methionine adenosyltransferase (MAT) enzyme activity, measured by LC-MS/MS quantification of SAM, in HUVEC lysates supplemented with nucleosides (n = 3 technical replicates). (H) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC pretreated with cycloleucine (MAT inhibitor, 20 mM for 30 min) or 5-azacytidine (DNMT inhibitor, 500 nM for 30 min) (n = 3 technical replicates). (I) De novo DNA methylation, measured by LC-MS/MS quantification of U-¹³C-methionine labeled 2′-deoxy-5-methylcytidine in siControl, siADA2-, or siDNMT1-treated HUVEC (n = 3 biological replicates). Values are represented as the percentage of total label incorporation. All results were replicated in three independent experiments. Values are presented as means ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. ADP, adenosine diphosphate; IMP, inosine monophosphate; THF, tetrahydrofolate; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase or methionine synthase.

Fig. 5. Induction of methylation-sensitive ERV triggers cytosolic dsRNA signaling and IFNβ production.

(A) IFN-β mRNA levels, measured by qRT-PCR, in HUVEC transfected with siRNAs targeting ADA2 and innate sensing molecules (n = 3 technical replicates). (B) Differential expression of LTR-containing ERV genes between siControl and siADA2-transfected HUVEC, measured by polydenylated poly(A⁺)–enriched RNA-seq (n = 3 biological replicates). Red and blue dots denote ERV significantly up- or down-regulated ≥1.5-fold. (C) mRNA levels of ERVFRD1 and ERVK28, measured by qRT-PCR, in HUVEC supplemented for 24 hours with dIno (500 μM) or pretreated for 30 min with the PNP inhibitor 9-deazaguanine (100 μM) before dIno supplementation for 24 hours (n = 3 technical replicates). (D) mRNA levels of ERV at 24 to 120 hours, measured by qRT-PCR, in siADA2- or siDNMT1-transfected HUVEC (n = 3 technical replicates). (E) mRNA levels of ISG and ERV, measured by qRT-PCR, in siADA2-transfected HUVEC treated with anti–IFN-β–neutralizing antibody (40 U/ml) (n = 3 technical replicates). (F) ERV, IFN-β, CCL5, and CXCL10 mRNA levels, measured by qRT-PCR, in HUVEC transfected with control plasmid or plasmids encoding ERVK28 transcript variants. Values are presented as means ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Fig. 6. Cellular sensing of extracellular purine nucleosides triggers an innate immune response.

Loss of extracellular ADA2 activity or an excess of extracellular purine nucleosides drives uptake of dAdo and intracellular catabolism by ADA1, yielding dIno, an immune-metabolite that directly inhibits MAT activity, the methionine cycle, and DNA methylation. Genomic hypomethylation drives up-regulation of ERV, dsRNA molecules that engage cytosolic dsRNA sensors RIG-I and MDA5 via the signaling adaptor MAVS.