Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response

Abstract

Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.

Keywords: Aicardi–Goutières syndrome; autoinflammation; cGAS‐STING; ribonuclease H2.

Figure 1. RNase H2 complex levels and enzymatic activity are reduced in Rnaseh2b ^A174T/A174T mouse and RNASEH2 B^{A 177T/A177T} AGS patient cells

1. A

   Targeted mutagenesis of the Rnaseh2b gene. Top: A 7‐kb region of the Rnaseh2b genomic locus; black boxes, exons 6 (ex6) and 7 (ex7). Middle: NotI/SalI restriction fragment of the targeting construct, comprising 4.5 kb of genomic DNA and a neomycin selection cassette (Neo) flanked by loxP sites (triangles). (Bottom) Targeted locus containing exon 7 with the c.520G>A mutation (ex7*). Red arrowheads, primers used to confirm correct targeting. Red bar, 400‐bp probe for Southern blotting.

2. B

   Southern blotting confirms successful targeting. Introduction of an additional EcoRI site results in a 4.1‐kb restriction fragment detectable by Southern for targeted ES cells (A174T/+) but not for parental DNA (+/+).

3. C

   Capillary sequencing for Rnaseh2b ^+/+, Rnaseh2b ^A174T/+ and Rnaseh2b ^A174T/A174T DNA confirmed the presence of the introduced missense mutation.

4. D

   Mouse genotyping by multiplex PCR. Top: A 221‐bp PCR product is present in wild‐type mice (+/+); the Rnaseh2b ^A174T allele (also) gives a 460‐bp product. Bottom: Position of forward (x) and reverse primers (y, z).

5. E

   Immunoblotting demonstrates depletion of all three RNase H2 protein subunits in Rnaseh2b ^A174T/A174T MEFs and RNASEH2B ^A177T/A177T LCLs. Representative of three independent experiments.

6. F

   Schematic showing enzyme activities attributed to RNase H1 and RNase H2 (DNA blue, RNA red).

7. G, H

   RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2b ^A174T/A174T MEFs and passage‐matched Rnaseh2b ^+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2‐specific substrate, double‐stranded DNA with a single‐embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars represent SEM. Enzymatic activity expressed relative to the average value of control MEFs. ***P < 0.001, two‐tailed t‐test (n = 3 Rnaseh2b ^A174T/A174T and n = 3 Rnaseh2b ^+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B‐A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars represent SEM. ***P < 0.001 versus either control, two‐tailed t‐test.

Source data are available online for this figure.

Figure 2. Increased ISG expression in tissues from Rnaseh2b ^A174T/A174T mice

1. Transcript levels of multiple ISGs are significantly elevated in heart.

2. Transcript levels of a subset of ISGs are significantly increased in kidney.

3. No ISG upregulation is evident in the brain.

Data information: ISG transcript levels determined by RT–qPCR normalised to transcript levels of the housekeeping gene HPRT (Oas1a was undetectable in brain). Each data point represents the mean of technical replicates of tissue RNA from a single mouse. n = 9 nine‐month‐old Rnaseh2b ^A174T/A174T mice and n = 4 age‐matched control wild‐type C57BL/6J mice. Horizontal line, mean; error bars, SEM. *P < 0.05, **P < 0.01, two‐tailed t‐test.

Figure 3. A proinflammatory response is detectable in vitro in Rnaseh2b ^−/− MEFs

Rnaseh2b ^−/− MEFs have significantly elevated ISG transcript levels and secrete the proinflammatory cytokines CXCL10 and CCL5.

1. Validation of Rnaseh2b ^−/− MEF lines. RER activity (DRD:DNA) is undetectable in a Rnaseh2b ^−/− line consistent with complete inactivation of the Rnaseh2b gene (Reijns et al, 2012). Mean of three independent experiments; error bars, SEM, ****P < 0.0001, two‐tailed t‐test. Rnaseh2b ^−/− and Rnaseh2b ^+/+ control MEFs on a C57BL/6 p53 ^−/− background.

2. ISG transcript levels are increased in Rnaseh2b ^−/− MEFs.

3. BeadArray transcript analysis (Illumina) detected induction of multiple ISGs, including the cytokines CXCL10 and CCL5, but not other cytokine transcripts in Rnaseh2b ^−/− MEFs. Plotted, average fold enrichment versus P‐value of significantly upregulated transcripts (P < 0.05, after multiple testing correction) comparing two Rnaseh2b ^−/− MEF lines versus 4 Rnaseh2b ^+/+ MEF lines. Seventeen out of 29 transcripts are ISGs.

4. Cxcl10 and Ccl5 transcripts (detected by RT–qPCR) are significantly elevated.

5. Increased CXCL10 and CCL5 protein (detected by ELISA) is secreted by Rnaseh2b ^−/− MEFs.

Data information: Data in (B, D, E) are mean from three experiments for six independent Rnaseh2b ^−/− MEF lines versus four independent Rnaseh2b ^+/+ MEF lines. Error bars, SEM. *P < 0.05, **P < 0.01, Mann–Whitney U‐test.

Figure 4. ISG induction in RNase H2 deficiency is dependent on the cGAS‐STING nucleic acid‐sensing pathway

1. A

   ISG activation and cytokine secretion in Rnaseh2b ^−/− MEFs is markedly impaired by cGAS or STING siRNA depletion. Upper left and lower panels: RT–qPCR of Ifit1, cGas and Sting transcripts, 48 h after siRNA targeting luciferase (control), cGAS or STING. Upper right panel: CCL5 is significantly reduced in culture supernatants 48 h after cGAS or STING depletion. Concentration of CCL5 (ELISA), normalised to luciferase siRNA control levels in each experiment. Mean from three independent experiments using one Rnaseh2b ^−/− MEF line; error bars, SEM. *P < 0.05, **P < 0.01, two‐tailed t‐test for RT–qPCR, one‐sample t‐test for CCL5 ELISA.

2. B–D

   ISG induction and cytokine secretion is abolished in Rnaseh2b ^−/− cGas ^−/− MEFs. cGas was targeted by CRISPR/Cas9 genome editing of a Rnaseh2b ^−/− MEF line to inactivate cGAS/STING signalling. In addition to sequence validation, functional inactivation of cGAS was confirmed in Rnaseh2b ^−/− cGas ^−/− CRISPR lines by the absence of CCL5 secretion in response to dsDNA (Appendix Fig S4). CCL5 (B) and CXCL10 (C) production, as well as ISG expression (D), was abrogated in Rnaseh2b ^−/− cGas ^−/− clones, assessed by ELISA and RT–qPCR, respectively. Four independent experiments, n = 2 Rnaseh2b ^−/− cGas ^−/− clones, n = 4 Rnaseh2b ^−/− cGas ^+/+ clones, error bars, SEM of each experiment; *P < 0.05, **P < 0.01, ***P < 0.001, two‐tailed t‐test.

3. E

   ISG induction in Rnaseh2b ^A174T/A174T mice is STING dependent. RT–qPCR of RNA extracted from hearts from Sting ^+/+ Rnaseh2b ^A174T/A174T (n = 4) and Sting ^−/− Rnaseh2b ^A174T/A174T (n = 6) 3‐month‐old mice. Each data point represents the mean of technical triplicates from one mouse. Horizontal line, mean; error bars, SEM. *P < 0.05, Mann–Whitney U‐test.

4. F

   Absence of STING does not significantly decrease basal ISG expression. RT–qPCR of RNA extracted from hearts from Sting ^+/+ (n = 3) and Sting ^−/− (n = 5) three‐month‐old mice. Each data point represents the mean of technical triplicates from one mouse. Horizontal line, mean; error bars, SEM. *P < 0.05, Mann–Whitney U‐test.

Figure 5. Cellular RER and not enzyme activity against RNA:DNA hybrids correlates with DNA damage and proinflammatory response

1. A

   Overexpression of RNase H1 in Rnaseh2b ^−/− cells restores RNase H activity against RNA:DNA hybrids to 81 ± 10% of wild‐type levels, while overexpression of RNASEH2B restores cellular enzyme activity for cleavage of both RNA:DNA and DRD:DNA substrates (RER). Rnaseh2b ^−/− MEFs were complemented with Rnaseh1 (+H1), Rnaseh2b (+H2B) or EGFP by retroviral infection. Mean of n = 3 independent experiments ± SEM.

2. B, C

   DNA damage is reduced to wild‐type levels by complementation with Rnaseh2b but not Rnaseh1, measured by 53BP1 foci formation in detergent‐extracted fixed cells. (B) Representative images (scale bar, 10 μm). (C) At least 150 cells were counted for each cell line in three independent experiments. Mean ± SEM, ****P < 0.0001 two‐tailed t‐test.

3. D–F

   CCL5 (D) and CXCL10 production (E), as well as ISG induction (F) in Rnaseh2b ^−/− MEFs are reduced close to wild‐type levels (Rnaseh2b ^+/+), by complementation with Rnaseh2b but not Rnaseh1. Mean of n = 6 independent experiments ± SEM for complemented cells; n = 3 independent experiments for Rnaseh2b ^−/− parental and Rnaseh2b ^+/+ controls cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 two‐tailed t‐test indicates significantly reduced expression compared to Rnaseh2b ^−/− parental cells.