STING promotes NLRP3 localization in ER and facilitates NLRP3 deubiquitination to activate the inflammasome upon HSV-1 infection

Abstract

One of the fundamental reactions of the innate immune responses to pathogen infection is the release of pro-inflammatory cytokines, including IL-1β, processed by the NLRP3 inflammasome. The stimulator of interferon genes (STING) has the essential roles in innate immune response against pathogen infections. Here we reveal a distinct mechanism by which STING regulates the NLRP3 inflammasome activation, IL-1β secretion, and inflammatory responses in human cell lines, mice primary cells, and mice. Interestingly, upon HSV-1 infection and cytosolic DNA stimulation, STING binds to NLRP3 and promotes the inflammasome activation through two approaches. First, STING recruits NLRP3 and facilitates NLRP3 localization in the endoplasmic reticulum, thereby facilitating the inflammasome formation. Second, STING interacts with NLRP3 and attenuates K48- and K63-linked polyubiquitination of NLRP3, thereby promoting the inflammasome activation. Collectively, we demonstrate that the cGAS-STING-NLRP3 signaling is essential for host defense against HSV-1 infection.

Fig 1. STING interacts with NLRP3 to facilitate the inflammasome activation.

(A–C) HEK293T cells were co-transfected with pFlag-STING and pHA-NLRP3 (A and B), or with pFlag-STING, pHA-NLRP3, pHA-ASC, and pHA-Casp-1 (C). (D) Diagrams of NLRP3, PYRIN, NACHT, and LRR. (E, F) HEK293T cells were co-transfected with pHA-STING and pFlag-NLRP3, pFlag-PYRIN, pFlag-NACHT, or pFlag-LRR (E), or with pFlag-STING and pHA-NLRP3, pHA-PYRIN, pHA-NACHT, or pHA-LRR (F). (G) Diagrams of STING and its truncated proteins. (H, I) HEK293T cells were co-transfected with pHA-NLRP3 and pFlag-STING, truncated proteins (H) or TM5-deleted STING(delTM5) (I). (J, K) HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Casp-1, and pro-IL-1β to generate a pro-IL-1β cleavage system (NACI), and transfected with pFlag-STING, truncated proteins (J), TM2-deleted STING(delTM2), TM5-deleted STING(delTM5) or TM2 and TM5-deleted STING(delTM2 and 5) (K). IL-1β in supernatants was determined by ELISA. (L) HEK293T cells were co-transfected with pFlag-ASC, pHA-NLRP3, or pFlag-STING. (A–C, E, F, H, I, and L) Cell lysates were subjected to co-immunoprecipitation (Co-IP) using IgG (Mouse) and anti-Flag antibody (C, F), IgG (Rabbit) and anti-HA antibody (L), anti-HA antibody (B), and anti-Flag antibody (A, E, H and I), and analyzed by immunoblotting using anti-HA or anti-Flag antibody (top) or subjected directly to Western blot using anti-Flag or anti-HA antibody (input) (bottom). (M) HEK293T cells were co-transfected with pFlag-ASC, pHA-NLRP3, and pMyc-STING. Pellets were subjected to ASC oligomerization assays (top) and lysates were prepared for Western blots (bottom). (N, O) HEK293T cells (N) or Hela cells (O) were co-transfected with pFlag-STING and/or pHA-NLRP3. Sub-cellular localization of Flag-STING (green), HA-NLRP3 (red), and DAPI (blue) were examined by confocal microscopy. Data shown are means ± SEMs; **p < 0.01, ***p < 0.0001; ns, no significance.

Fig 2. HSV-1 infection promotes the STING-NLRP3 interaction.

(A, B) TPA-differentiated THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for 4 h (A) or 2 h and 4 h (B). (C, D) HEK293T cells (C) and Hela cells (D) were co-transfected with pFlag-STING and pHA-NLRP3, and then infected with HSV-1 (MOI = 1) for 2–4 h. (E) TPA-differentiated THP-1 macrophages were transfected with HSV120 (3 μg/ml) by Lipo2000 for 4 h. The Lipo2000 was used as the control. (F, G) HEK293T cells (F) and Hela cells (G) were co-transfected with pFlag-STING and pHA-NLRP3 and then transfected with HSV120 (3 μg/ml) for 4 h. (H) Primary MEFs were primed with LPS (1 μg/ml) for 6 h, and then infected with HSV-1 (MOI = 1) for 4 h or transfected with HSV120 (3 μg/ml) for 4 h. (A–H) Cell lysates were subjected to Co-IP using IgG (Mouse) or anti-NLRP3 antibody (A), anti-STING antibody (B), IgG (Mouse) or anti-Flag antibody (C), anti-Flag antibody (D), IgG (Mouse) or anti-NLRP3 antibody (E), IgG (Mouse) or anti-Flag antibody (F), anti-Flag antibody (G), and IgG (Mouse) or anti-NLRP3 antibody (H), and then analyzed by immunoblotting using anti-NLRP3 and anti-STING antibody (top) or analyzed directly by immunoblotting using anti-NLRP3 and anti-STING antibody (as input) (bottom). Densitometry of the blots were measured by Image J.

Fig 3. HSV-1 infection induces IL-1β expression and secretion.

(A–K) TPA-differentiated THP-1 macrophages were treated with 2 μM Nigericin for 2 h, and infected with HSV-1 at MOI = 0.1, 0.2, 0.4 and 0.8 for 8 h (A, C, E and G), infected with HSV-1 at MOI = 0.8 for 2, 4, 6 and 8 h (B, D, F and H), or transfected with HSV120 (3 μg/ml) for 2, 4 and 6 h (I–K). (L–O) Primary MEFs were primed with LPS (1 μg/ml) for 6 h, and then treated with 5 mM ATP for 2 h or infected with HSV-1 (MOI = 1) (L and N) or transfected with HSV120 (3 μg/ml) (M, O) for 2, 4 and 6 h. (A, B, I, L and M) IL-1β protein was determined by ELISA. (C, D and J) Matured IL-1β (p17) and cleaved Casp-1 in supernatants (top) and pro-IL-1β production in lysates (bottom) were determined by Western-blot analyses. (E, F, G, H, K, N and O) IL-1β mRNA and GAPDH mRNA (E, F), HSV-1 ICP27 mRNA and GAPDH mRNA (G, H and N), and IFN-β mRNA and GAPDH mRNA (K, O) were quantified by RT-PCR. Data shown are means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.0001; ns, no significance.

Fig 4. The NLRP3 inflammasome is required for HSV-1-induced IL-1β activation.

(A–C) THP-1 cells stably expressing shRNAs targeting IFI16 or cGAS were generated and then treated with 2 μM Nigericin for 2 h or infected with HSV-1 at MOI = 0.8 for 8 h. (D–F) THP-1 cells stably expressing shRNAs targeting AIM2 were generated and then treated with 5 μg/ml poly(dA:dT) for 6 h or infected with HSV-1 at MOI = 0.8 for 8 h. (G–L) TPA-differentiated THP-1 macrophages were treated with 2 μM Nigericin for 2 h, infected with HSV-1 at MOI = 0.8 for 8 h, and then treated with Glybenclamide (25 μg/ml) (G, I and K) or Ac-YVAD-cmk (10 μg/ml) (H, J and L). (M–Q) THP-1 cells stably expressing shRNAs targeting NLRP3, ASC or Casp-1 were generated and then treated with 2 μM Nigericin for 2 h (M, N) or infected with HSV-1 at MOI = 0.8 for 8 h (O–Q). (R–T) Primary MEFs of C57BL/6 WT mice and C57BL/6 NLRP3^-/- mice were primed with LPS (1 μg/ml) for 6 h, and then treated with 5 mM ATP for 2 h or infected with HSV-1 (MOI = 1) for 6 h. NLRP3 and GAPDH protein in lysates were determined by Western blot (R). IL-1β levels were determined by ELISA (A, D, G, I, M and S). Matured IL-1β (p17) and cleaved Casp-1 (p22/p20) in supernatants (top) or pro-IL-1β and pro-Casp-1 in lysates (bottom) were determined by Western-blot (B, E, H, N and P). HSV-1 ICP27 mRNA and GAPDH mRNA were quantified by RT-PCR (C, F, K, L, Q and T). Data shown are means ± SEMs; ***p < 0.0001; ns, no significance.

Fig 5. STING recruits NLRP3 to the ER and promotes the inflammasome formation.

(A–D) Hela cells were co-transfected with pFlag-STING, TM5-deleted STING(delTM5) and pHA-NLRP3 (A), transfected with pFlag-NLRP3 and infected with HSV-1 (MOI = 1) for 4 h or transfected with HSV120 (3 μg/ml) for 4 h (B, C and D). Sub-cellular localization of HA-NLRP3 (green) (A and D), Flag-STING (red) or TM5-deleted STING(delTM5) (green) (A), ER Blue (blue) (A, D), Flag-NLRP3 (red) (B, C), Calnexin (green) (B), DAPI (blue) (B and C), and Calnexin (green) (C) were examined by confocal microscopy. (E) TPA-differentiated THP-1 macrophages were infected with mock or HSV-1 (MOI = 1) for 4 h. Sub-cellular localization of NLRP3 (green), Calnexin (red) and DAPI (blue) were examined by confocal microscopy. (F–H) Hela cells were co-transfected with pFlag-STING and pHA-NLRP3 (F). THP-1 macrophages were infected with HSV-1 (MOI = 1) for 4 h (G). Primary MEFs were primed with LPS (1 μg/ml) for 6 h and infected with HSV-1 (MOI = 1) for 4 h (H). HA-NLRP3, Flag-STING, STING, ERGIC p58, GM130 (cis Golgi), Calnexin (ER), IFI16, AIM2 and GAPDH in whole cell lysate (WCL) and purified ER were determined by Western-blot analyses. (I, J) Hela cells stably expressing sh-STNG were transfected with pFlag-NLRP3 and infected with HSV-1 (MOI = 1) for 4 h or transfected with HSV120 (3 μg/ml) for 4 h. Sub-cellular localization of Flag-NLRP3 (red) (I), Calnexin (green) (I), DAPI (blue) (I), HA-NLRP3 (red) (J) and ER blue (J) were examined by confocal microscopy. (K–M) Hela cells stably expressing sh-STING were transfected with pHA-NLRP3 and infected with HSV-1 (MOI = 1) for 4 h (K). THP-1 cells stably expressing sh-STING were differentiated to macrophages, and infected with HSV-1 (MOI = 1) for 4 h (L). Primary MEFs stably expressing sh-STING were primed with LPS (1 μg/ml) for 6 h, and infected with HSV-1 (MOI = 1) for 4 h (M). HA-NLRP3 (K), NLRP3 (L, M), STING (K–M), Calnexin (ER) (K–M) and GAPDH (K–M) in WCL and purified ER fraction were determined by Western-blot analyses. Densitometry of the blots were measured by Image J.

Fig 6. STING deubiquitinates NLRP3 to activate the NLRP3 inflammasome.

(A–E) Hela cells were co-transfected with pFlag-NLRP3, pHA-Ubiquitin, and pMyc-STING (A), with pFlag-NLRP3, pHA-Ubiquitin, pHA-Ubiquitin mutations (K48R), pHA-Ubiquitin mutations (K63R) and pMyc-STING (B), with pFlag-NLRP3, pHA-Ubiquitin, pHA-Ubiquitin mutations(K48O), pHA-Ubiquitin mutations(K63O), and pMyc-STING (C), with pFlag-NLRP3 and pHA-Ubiquitin and then infected with HSV-1(MOI = 1) for 2 and 4 h (D), and transfected with HSV120 (3 μg/ml) for 2 and 4 h (E). Cell lysates were prepared and subjected to denature-IP using anti-Flag antibody and then analyzed by immunoblotting using an anti-HA or anti-Flag antibody (top) or subjected directly to Western blot using an anti-Flag, anti-HA or anti-Myc antibody (as input) (bottom). (F–I) TPA-differentiated THP-1 macrophages were infected with HSV-1 (MOI = 1) for 2 and 4 h (F), and transfected with HSV120 (3 μg/ml) for 2, 4 and 6 h (G). Mice primary MEFs were infected with HSV-1 (MOI = 1) for 2 h (H). THP-1 cells stably expressing sh-NC or sh-STING were generated and differentiated to macrophages and then infected with HSV-1 (MOI = 1) for 4 h (I). Cell lysates were prepared and subjected to denature-IP using anti-NLRP3 antibody and then analyzed by immunoblotting using an anti-Ubiquitin, anti-K48-Ubiquitin, anti-K63-Ubiquitin or anti-NLRP3 antibody (top) (F) or using anti-NLRP3 antibody (G, H and I), or subjected directly to Western blot using an anti- Ubiquitin or anti-NLRP3 antibody (as input) (bottom).

Fig 7. STING is required for the NLRP3 inflammasome activation upon DNA virus infection.

(A–I) THP-1 cells stably expressing sh-NC or sh-STING were generated and differentiated to macrophages, and then infected with HSV-1 at MOI = 0.8 for 2, 4 and 8 h (A–C), transfected with DNA90 (3 μg/ml), HSV120 (3 μg/ml) or infected with HSV-1 at MOI = 0.8 for 8 h (D, E), and treated with 2 μM Nigericin for 2 h and infected with HSV-1 at MOI = 0.8 for 8 h (F–I). IL-1β levels were determined by ELISA (A, D and F). Matured IL-1β (p17) and cleaved Casp-1 (p22/p20) in supernatants or STING, pro-IL-1β and pro-Casp-1 in lysates were determined by Western-blot (B, C and G). HSV-1 ICP27 mRNA and GAPDH mRNA were quantified by RT-PCR (C, H). Pellets were subjected to ASC oligomerization assays (top) and lysates were prepared for Western blots (bottom) (I). (J–N) THP-1 cells stably expressing shRNA targeting STING was generated and differentiated to macrophages, then infected with SeV (MOI = 1) for 24 h, ZIKV (MOI = 1) for 24 h or HSV-1 (MOI = 0.8) for 8 h. IL-1β levels were determined by ELISA (J). Matured IL-1β (p17) and cleaved Casp-1 (p22/p20) in supernatants or STING, pro-IL-1β and pro-Casp-1 in lysates were determined by Western-blot (K). SeV P mRNA (L), ZIKV mRNA (M), HSV-1 ICP27 mRNA (N) and GAPDH mRNA were quantified by RT-PCR. Data shown are means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.0001; ns, no significance.

Fig 8. NLRP3 is critical for host defense against HSV-1 infection in mice.

(A, B) C57BL/6 WT mice (8-week-old, female, n = 9) and C57BL/6 NLRP3^-/- mice (8-week-old, female, n = 5) were infected i.p. with HSV-1 at 1×10⁷ pfu for 11 days. The survival rates (A) and body weights (B) of mice were evaluated. (C–G) WT mice (8-week-old, female) and NLRP3^-/- mice (8-week-old, female) were mock-infected i.p. with PBS (WT and NLRP3^-/- mice, n = 3) or infected i.p. with HSV-1 (WT mice, n = 4, NLRP3^-/- mice, n = 3) at 1×10⁷ pfu for 6 h. IL-1β in mice sera was determined by ELISA (C). IL-1β mRNA (D), IL-6 mRNA (E), TNF-α mRNA (F), HSV-1 UL30 mRNA (G) and GAPDH mRNA in mice blood were quantified by RT-PCR. (H–J) WT mice (8-week-old, female, n = 4) and NLRP3^-/- mice (8-week-old, female, n = 4) were infected i.p. with HSV-1 at 1×10⁷ pfu for 1 days. IL-1β mRNA (H), IL-6 mRNA (I) and GAPDH mRNA in mice lung were quantified by RT-PCR. HSV-1 viral titers were measured by plaque assays for mice lung (J). (K–M) WT mice (8-week-old, female, n = 7) and NLRP3^-/- mice (8-week-old, female, n = 7) were infected i.p. with HSV-1 at 1×10⁷ pfu for 4 days. IL-1β mRNA (K), IL-6 mRNA (L) and GAPDH mRNA in mice brain were quantified by RT-PCR. HSV-1 viral titers were measured by plaque assays for mice brain (M). (N) WT mice (8-week-old, female) and NLRP3^-/- mice (8-week-old, female) were infected i.p. with HSV-1 at 1×10⁷ pfu for 1 day. The lung tissues were stained with histological analysis (H&E or IL-1β). (O) WT mice (8-week-old, female) and NLRP3^-/- mice (8-week-old, female) were infected i.p. with HSV-1 at 1×10⁷ pfu for 4 days. The brain tissues were stained with histological analysis (H&E or IL-1β). Data shown are means ± SEMs; *p < 0.05, **p < 0.01, ***p < 0.0001; ns, no significance.

Fig 9. A proposed model for the regulation of NLRP3 inflammasome activation mediated by the cGAS-STING-NLRP3 pathway.

Left: In the mock infection, all these proteins are in the cytoplasm. NLRP3 contains an N-terminal pyrin domain (PYD), a NACHT-associated domain (NAD) and 7 C-terminal leucine rich repeats (LRR). ASC (the apoptosis-associated speck-like protein with CARD domain) has two domains (PYD and CARD). The effecter protein pro-Casp-1 has the three domains (CARD, p20 and p10). It has been demonstrated that the NLR protein NLRP3, together with the adaptor protein, apoptosis-associated speck-like protein with CARD domain (ASC), promotes the cleavage of the pro-Casp-1 to generate active subunits p20 and p10, which regulate the maturation of IL-1β.STING comprises 5 putative transmembrane (TM) regions, predominantly resides in the endoplasmic reticulum (ER) and regulates innate immune signaling processes. Right: After the infection by HSV-1, everything has changed. Herpes simplex virus type 1 (HSV-1) belongs to the family of Herpesviridae, which causes various mild clinical symptoms. HSV-1 genomic DNA then induce the cGAS-cGAMP-STING pathway for the IFN-I production. Upon binding of cytoplasmic viral DNA, GAS (cyclic GMP-AMP synthase) catalyzed the formation of the second messenger cGAMP, which is an endogenous second messenger by binding to STING and inducing STING’s dimerization. After that, HSV-1 infection also induced NLRP3 translocation to Endoplasmic Reticulum (ER) by STING through the interaction between STING and NLRP3. Then STING promotes the deubiquitination of NLRP3 in K48-linked and K63-linked polyubiquitin chains and initiates the activation of NLRP3 inflammasome, promotes the cleavage of the pro-Casp-1 to generate active subunits p20 and p10, which regulate the maturation of IL-1β, and IL-1β then secreted outside the cells.