IRF3 activates RB to authorize cGAS-STING-induced senescence and mitigate liver fibrosis

Abstract

Cytosolic double-stranded DNA surveillance by cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) signaling triggers cellular senescence, autophagy, biased mRNA translation, and interferon-mediated immune responses. However, detailed mechanisms and physiological relevance of STING-induced senescence are not fully understood. Here, we unexpectedly found that interferon regulatory factor 3 (IRF3), activated during innate DNA sensing, forms substantial endogenous complexes in the nucleus with retinoblastoma (RB), a key cell cycle regulator. The IRF3-RB interaction attenuates cyclin-dependent kinase 4/6 (CDK4/6)-mediated RB hyperphosphorylation that mobilizes RB to deactivate E2 family (E2F) transcription factors, thereby driving cells into senescence. STING-IRF3-RB signaling plays a notable role in hepatic stellate cells (HSCs) within various murine models, pushing activated HSCs toward senescence. Accordingly, IRF3 global knockout or conditional deletion in HSCs aggravated liver fibrosis, a process mitigated by the CDK4/6 inhibitor. These findings underscore a straightforward yet vital mechanism of cGAS-STING signaling in inducing cellular senescence and unveil its unexpected biology in limiting liver fibrosis.

Fig. 1.. IRF3 deletion attenuates cellular senescence phenotypes.

(A) Treatment of DNA damage inducer HU for 24 hours induced the activation of DNA sensing in DLD1 gut epithelial cells at day 1, revealed by the activating phosphorylation of TBK1 and IRF3. Phospho-IRF3, p16^INK4a, and p21^Cip1/Waf1 were accumulated over time (days 4 to 7). IB, immunoblotting. (B and C) DNA damage, triggered by treatment of HU or DOX, induced robust senescence phenotypes in DLD1 cells at day 6, revealed by SA-β-Gal staining (B), senescence marker p16^INK4a, and SASPs (C). Senescence phenotypes were markedly suppressed in DLD1 cells with KO of IRF3, which was largely restored by reintroduction of IRF3. (D) mRNA-seq assay was performed in HU-induced senescent WT or IRF3 KO DLD1 cells, revealing by the heatmap depicting relative mRNA levels. (E and F) The gene set enrichment analysis plot (E) and heatmap (F) showed that genes associated with senescence were enriched in HU-treated WT but not in IRF3 KO DLD1 cells. NES, normalized enrichment score. (G) Human embryonic fibroblasts MRC-5 at passages 28 and 35 were stained for SA-β-Gal and quantified to assess the replicative senescence in WT and IRF3-deficient human fibroblasts. (H) Primary MEFs at indicated days from Irf3^+/+ or Irf3^−/− mice were analyzed by SA-β-Gal staining to evaluate IRF3’s role in spontaneous immortalization. Scale bars, 100 μm. n = 3 independent biological repeats unless specified. *P < 0.05; **P < 0.01; ***P < 0.001, by analysis of variance (ANOVA) with Bonferroni correction. The statistics source data are provided in table S1, and the scanned films of each immunoblotting are provided in fig. S7.

Fig. 2.. IRF3 controls senescence through the p16^INK4a-RB pathway.

(A and B) Treatment of human IFN-α1b (1000 U/ml, every 24 hours) failed to restore senescence in HU-treated DLD1 cells with IRF3 deficiency, as evaluated by SA-β-Gal staining (A) and SASPs (B). n.s., not significant. (C and D) Four-day treatment of MS failed to restore senescence in HU-treated IRF3 KO DLD1 cells, as indicated by SA-β-Gal staining (C) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for senescence marker p21^Cip1/Waf1 and SASP factor IL-1α (D). (E and F) WT and IRF3 KO DLD1 cells were treated with 1 μM palbociclib, a Food and Drug Administration–approved specific inhibitor of CDK4/6, for 3 days and examined for SA-β-Gal staining (E) and qRT-PCR assays of SASPs (F). (G and H) HU-induced senescence of WT and RB KO DLD1 cells was examined by SA-β-Gal staining (G) and SASPs (H); RB KO DLD1 cells were generated by a CRISPR-mediated approach and verified by sequencing and immunoblotting. Scale bars, 100 μm. n = 3 independent biological repeats unless specified. *P < 0.05; ***P < 0.001, by analyses of variance (ANOVA) with Bonferroni correction.

Fig. 3.. IRF3 interacts directly with RB, a key regulator of cellular senescence.

(A) Mass spectrometry analysis of the IRF3 immunoprecipitations (IP) from DLD1 cells indicated an elevated interaction between IRF3 and endogenous RB at day 6 upon HU-induced cellular senescence. Flag-tagged IRF3 was stably reintroduced into IRF3 KO DLD1 cells in this setting. (B) The endogenous interaction of IRF3 and RB was evaluated by coimmunoprecipitation assay in HU/DOX-induced senescent DLD1 cells. (C) PLAs in DLD1 cells demonstrated in situ signals for the IRF3-RB complex during HU/DOX-induced senescence. Scale bars, 10 μm. (D) Coimmunoprecipitation assays were performed in transfected human embryonic kidney (HEK) 293 cells to evaluate the interactions between RB and WT/activated IRF3 or IRF7. IRF3 5D (S396D/S398D/S402D/S405D/S427D) and IRF7 7D (S475D/S476D/S477D/S479D/S483D/S484D/S487D) are phosphomimetics for activated IRF3 and IRF7, respectively. (E to G) Domain mapping assays by coimmunoprecipitation assays indicated an interaction between the interferon-activating domain (IAD) of IRF3 5D (E) and the RB-B (F), as depicted by a schematic of their interacting domains (G). (H and I) The coimmunoprecipitation assays in HEK293 cells revealed two IRF3 mutants defective to RB interaction (S221A and R255A/R262A/H263A) (H). The transcriptional potentials of IRF3 phosphomimetics and mutants (S221A and R78A/R86A, R255A/R262A/H263A) were individually analyzed by IRF3-responsive reporters of IFNβ and 5×interferon-stimulated response element (ISRE) (I). n = 3 independent biological repeats unless specified. ***P < 0.001, by analysis of variance (ANOVA) with Bonferroni correction.

Fig. 4.. IRF3 attenuates RB hyperphosphorylation.

(A) Coimmunoprecipitation assays showed an enhanced interaction between IRF3 5D and RB mutant T821/826A, the mutation RB at a constant state of hypophosphorylation. (B) Compared with WT cells, MRC-5 cells with IRF3 deficiency displayed a higher phospho-RB (T826) level, as measured in passage 34. (C) Immunoblotting showed an increase in phosphorylated RB in MEFs isolated from Irf3^−/− mice. (D) IRF3 deficiency in DLD1 cells prevented the DNA damage–induced senescence markers, including decreased RB phosphorylation at T826 and up-regulation of p16^INK4a and p21^Cip1/Waf1, which were restored by reintroducing IRF3 via a lentiviral delivery. (E) Flag-tagged IRF3 WT and mutants were stably reconstituted into IRF3 KO DLD1 cells via a lentiviral delivery. Differences in RB phosphorylation, p16^INK4a, and p21^Cip1/Waf1 protein levels upon HU-induced cellular senescence were measured by immunoblotting in cells harboring distinct IRF3 mutants. (F) Doxycycline-induced expression of STING R281Q, a constitutively active form of STING, reduced RB phosphorylation levels at T826 but increased p16^INK4a and p21^Cip1/Waf1 expression, as measured by immunoblotting. (G) Constitutively activation of STING promoted senescence entry, as revealed by SA-β-Gal staining and quantifications. Scale bars, 50 μm. (H) Induced expression of STING in A549 cells by a Tet-On system triggered IRF3 activation and attenuated phospho-RB at T826, a process blocked by CRISPR-mediated IRF3 KO. (I) IRF3 deficiency blocked STING-induced senescence in A549 cells, as assessed by SA-β-Gal staining. Scale bars, 100 μm. (J) A clone formation assay was performed in WT, STING Tet-On, and IRF3 KO A549 cells and observed on day 10. (K) IRF3 deficiency diminished STING-induced cell growth arrest, as assessed by Cell Counting Kit-8 assay from day 0 to day 4. OD, optical density. n = 3 independent biological repeats unless specified. ***P < 0.001, by analysis of variance (ANOVA) with Bonferroni correction.

Fig. 5.. IRF3 activates RB by attenuating CDK-induced RB phosphorylation.

(A) Flag-tagged IRF3 WT and mutants were stably reconstituted into IRF3 KO DLD1 cells. Coimmunoprecipitation assays evaluated the associations of endogenous RB and IRF3 mutants in HU/DOX-induced senescent DLD1 cells. (B) PLA assays were performed in IRF3 KO DLD1 cells stably expressed IRF3 or mutants, which revealed in situ cellular signals for the IRF3-RB complex with or without HU treatment. 4′,6-diamidino-2-phenylindole (DAPI) staining was used to visualize the cell nuclei. Scale bars, 10 μm. (C) Roles of IRF3 mutants on HU-induced cellular senescence were assessed by SA-β-Gal staining. Scale bars, 100 μm. (D) An in vitro kinase assay of CDK4/6 and RB was performed. RB proteins were expressed in HEK293 cells and pulled down using Myc-tag antibody and pretreated with λ-protein phosphatase (λPPase) to remove its phosphorylation. CDK4, CDK6, and cyclin D1 were expressed in HEK293 cells and pulled down using Flag-tag antibody, incubated with separately purified RB and IRF3 5D for kinase assay in the absence or presence of palbociclib, with adenosine 5′-triphosphate (ATP). (E) An in vitro kinase assay used RB and Smad3 as phosphorylation substrates, which were expressed in HEK293 cells and purified by Myc-tag antibody with λPPase for pretreatment. (F) The RB–cyclin D1 complex was detected by coimmunoprecipitation, by which IRF3 5D dissociated but not the IRF3 mutant defective for RB interaction. (G) The association of RB and E2F1 was enhanced in the presence of IRF3 5D, as revealed by coimmunoprecipitation assays. (H) Regular or senescent WT and IRF3 KO DLD1 cells were subjected to coimmunoprecipitation and immunoblotting, which revealed their diverse interactions of RB and E2F1. Besides, an endogenous complex of IRF3, E2F1, and RB was detected by coimmunoprecipitation in senescent DLD1 cells. n = 3 independent biological repeats unless specified. ***P < 0.001, by analysis of variance (ANOVA) with Bonferroni correction.

Fig. 6.. The IRF3-RB axis regulates senescence during liver fibrosis.

(A) The carbon tetrachloride (CCl₄; 0.5 mg/kg) was injected twice a week for 4 weeks to induce liver fibrosis in Irf3^+/+ and Irf3^−/− 8-week-old C57BL/6 mice. Mouse livers after CCl₄ treatment exhibited an accumulation of SA-β-Gal–positive cells, which were less in Irf3^−/− mice. Scale bars, 100 μm. (B) The bile duct of Irf3^+/+ and Irf3^−/− 8-week-old C57BL/6 mice was ligated to induce liver fibrosis. Palbociclib (100 mg/kg) was used on days 7, 8, and 9 after the surgery. Representative images and quantification of SA-β-Gal activity in the liver of Irf3^+/+ and Irf3^−/− mice showed the percentage of SA-β-Gal–positive cells. Senescence is raised after treatment with palbociclib in Irf3^−/− mice after BDL. Scale bars, 100 μm. (C) Representative Sirius Red–stained liver sections in sham/CCl₄ treatment revealing collagen deposition and bridging fibrosis. The degree of collagen deposition was increased in Irf3^−/− mice. Scale bars, 100 μm. (D) qRT-PCR assays of collagen mRNA level of livers were shown. (E) Representative Sirius Red–stained liver sections in BDL surgery and palbociclib treatment. Scale bars, 100 μm. (F) Serum ALT and AST levels markered the degree of liver injury. Palbociclib administration markedly reduced serum ALT and AST in Irf3^−/− mice. (G) Hepatic parenchymal cells (HCs) and primary hepatic NPCs were isolated from CCl₄-treated Irf3^+/+ or Irf3^−/− mouse livers. qRT-PCR assays of mRNA level of senescence makers p16^INK4a, p21^Cip1/Waf1, and SASPs such as CXCL1 and IL-6 were shown. (H) Primary HCs and NPCs were isolated from CCl₄-treated Irf3^+/+ or Irf3^−/− mouse livers. Cellular senescence and the activation of cGAS-STING-IRF3 signaling were analyzed by immunoblotting of phospho-RB (T826), p16^INK4a, p21^Cip1/Waf1, and phospho-IRF3 (S396) antibodies. (I) qRT-PCR analyzed cGAS, STING, and IRF3 expression in HCs and NPCs after CCl₄-induced liver fibrosis. (J) Immunofluorescence assay to costain α-SMA and p21^Cip1/Waf1 in WT and Irf3^−/− mouse livers during liver fibrosis. α-SMA–positive cells in Irf3^−/− mouse livers showed fewer p21^Cip1/Waf1 signals, which was reversed by palbociclib treatment. Scale bars, 20 μm. n = 5 independent biological repeats unless specified. *P < 0.05; **P < 0.01; ***P < 0.001, by analysis of variance (ANOVA) with Bonferroni correction.

Fig. 7.. Selective deletion of IRF3 in HSCs attenuates HSC senescence and promotes liver fibrosis.

(A) A flow chart of the CCl₄-induced hepatic fibrosis model in IRF3 conditional KO mice was shown. The mice with IRF3 conditional KO in HSC were established by tail vein injection of AAV8–GFAP (promoter)–EGFP–Cre twice at the indicated time to Irf3^flox/flox mice. (B) qRT-PCR assays of Cre mRNA and immunofluorescence of α-SMA and GFP indicated that upon AAV8-GFAP-EGFP-Cre injection in mouse livers, epidermal growth factor receptor–Cre was expressed and localized in HSCs. Scale bars, 20 μm. (C and D) RB phosphorylation and IRF3 activation were evaluated by immunoblotting in primary HSCs isolated from livers of CCl₄-treated WT or IRF3 conditional KO (Cre) mice. (C). SASPs and senescence marker p21^Cip1/Waf1 were evaluated by qRT-PCR assays (D). (E) Representative SA-β-Gal staining showed fewer SA-β-Gal positive signals from IRF3 conditional KO mice in liver sections. Scale bars, 100 μm. (F to I) Liver sections from CCl₄-treated control or IRF3 conditional KO (Cre) mice were evaluated by Sirius Red staining (F), qRT-PCR of collagens, and α-SMA (G), hematoxylin and eosin (H&E) staining (H), and qRT-PCR of tumor necrosis factor–α (TNF-α) and IL-1β, markers of inflammation (I). Scale bars, 100 μm. (J) This model illustrates the regulatory role of the STING-IRF3-RB axis in HSCs during liver fibrosis. The process initiates with the activation of cGAS-STING signaling, leading to the phosphorylation and nuclear translocation of IRF3. Inside the nucleus, IRF3 forms complexes with RB. This interaction inhibits the CDK4/6-cyclin-RB complex, resulting in the activation of RB. The activated RB then binds to and inhibits E2F transcription factors, effectively triggering cellular senescence in activated HSCs. Consequently, this pathway mitigates liver fibrosis by promoting senescence in HSCs. n = 5 independent biological repeats unless specified. **P < 0.01; ***P < 0.001, by analysis of variance (ANOVA) with Bonferroni correction.