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. 2018 Aug 8;4(8):eaas9784.
doi: 10.1126/sciadv.aas9784. eCollection 2018 Aug.

Kisspeptin/GPR54 signaling restricts antiviral innate immune response through regulating calcineurin phosphatase activity

Hongjun Huang  1 , Qingqing Xiong  1 , Ning Wang  1 , Ruoyu Chen  1 , Hua Ren  1 , Stefan Siwko  2 , Honghui Han  3 , Mingyao Liu  1   2 , Min Qian  1 , Bing Du  1
Affiliations

Kisspeptin/GPR54 signaling restricts antiviral innate immune response through regulating calcineurin phosphatase activity

Hongjun Huang et al. Sci Adv. .

Abstract

G protein-coupled receptor 54 (GPR54), the key receptor for the neuropeptide hormone kisspeptin, plays essential roles in regulating puberty development and cancer metastasis. However, its role in the antiviral innate immune response is unknown. We report that virus-induced type I interferon (IFN-I) production was significantly enhanced in Gpr54-deficient cells and mice and resulted in restricted viral replication. We found a marked increase of kisspeptin in mouse serum during viral infection, which, in turn, impaired IFN-I production and antiviral immunity through the GPR54/calcineurin axis. Mechanistically, kisspeptin/GPR54 signaling recruited calcineurin and increased its phosphatase activity to dephosphorylate and deactivate TANK [tumor necrosis factor receptor-associated factor (TRAF) family member-associated NF-κB activator]-binding kinase 1 (TBK1) in a Ca2+-dependent manner. Thus, our data reveal a kisspeptin/GPR54/calcineurin-mediated immune evasion pathway exploited by virus through the negative feedback loop of TBK1 signaling. These findings also provide insights into the function and cross-talk of kisspeptin, a known neuropeptide hormone, in antiviral innate immune response.

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Figures

Fig. 1
Fig. 1. GPR54 modulates IFN-I signaling.
(A) Enzyme-linked immunosorbent assay (ELISA) of IFN-β levels in supernatants of Gpr54+/+ and Gpr54−/− PEMs infected with VSV [multiplicity of infection (MOI), 1] for 12 hours. (B) Quantitative polymerase chain reaction (qPCR) analysis of Ifn-β and Ifn-α4 expression in Gpr54+/+ and Gpr54−/− PEMs infected with VSV (MOI, 1) for 8 hours. (C) qPCR analysis of Ifn-β expression in Gpr54+/+ and Gpr54−/− PEMs transfected with poly(I:C) (1 μg/ml) for 4 hours. (D) qPCR analysis of Ifn-β expression in Gpr54+/+ and Gpr54−/− PEMs infected with HSV-1 (MOI, 1) for 8 hours. (E) qPCR analysis of Ifn-β and Ifn-α4 expression in Gpr54+/+ and Gpr54−/− BMMs infected with VSV (MOI, 1) for 8 hours. (F) qPCR analysis of Ifn-β expression in Gpr54+/+ and Gpr54−/− BMMs infected with HSV-1 (MOI, 1) for 8 hours. (G) Immunoblot analysis of phosphorylated TBK1 and IRF3 or total proteins in lysates of Gpr54+/+ and Gpr54−/− PEMs infected with VSV (MOI, 1) for the indicated times. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (H) ELISA of IFN-β levels in supernatants of GPR54-overexpressing HEK-293T (293T) cells infected with VSV (MOI, 1) for 16 hours. Emv, empty vector. (I) qPCR analysis of IFN-β and IFN-α4 expression in GPR54-overexpressing HEK-293T cells infected with VSV (MOI, 1) for 12 hours. (J) ELISA of IFN-β levels in sera from Gpr54+/+ and Gpr54−/− mice given intraperitoneal injection of VSV [1 × 108 plaque-forming units (PFU) per mouse] for 24 hours. (K) qPCR analysis of Ifn-β expression in the liver, spleen, and lung from mice in (J). GAPDH was used as an internal control for qPCR. Data are representative of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001. nd, not detected.
Fig. 2
Fig. 2. GPR54 negatively regulates host defense against viruses.
(A) qPCR analysis of VSV RNA replicates in Gpr54+/+ and Gpr54−/− PEMs or BMMs infected with VSV (MOI, 1) for the indicated times or the indicated VSV MOI for 12 hours. (B) PEMs from Gpr54+/+ and Gpr54−/− mice were infected with VSV-GFP (MOI, 0.01) for 12 hours, and VSV-GFP was measured by fluorescence-activated cell sorting (FACS). SSC-H, side scatter-height. (C) qPCR analysis of HSV-UL-30 expression in Gpr54+/+ and Gpr54−/− PEMs or BMMs infected with HSV-1 (MOI, 1) for the indicated times. (D) qPCR analysis of HSV-1 genomic DNA replicates in Gpr54+/+ and Gpr54−/− PEMs or BMMs infected with HSV-1 (MOI, 1) for 24 hours. (E) qPCR analysis of NDV RNA replicates in Gpr54+/+ and Gpr54−/− PEMs infected with NDV (MOI, 1) for the indicated times. (F) qPCR analysis of VSV RNA replicates in HEK-293T cells transfected for 28 hours with different amounts (1 and 2 μg) of GPR54 plasmid and then infected with VSV (MOI, 1) for 12 hours. (G) qPCR analysis of VSV RNA replicates in the liver, spleen, and lung from Gpr54+/+ and Gpr54−/− mice infected with VSV (1 × 108 PFU per mouse) intraperitoneally for 24 hours (n = 5; mean ± SEM). (H) Hematoxylin and eosin staining of lung sections from mice in (G). Scale bars, 200 μm. (I) Survival of 8-week-old Gpr54+/+ and Gpr54−/− mice given intraperitoneal injection of VSV (1 × 108 PFU/g) (mean ± SEM). GAPDH was used as an internal control for qPCR. Data are representative of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 3
Fig. 3. Kisspeptin constrains virus-induced IFN-β production.
(A) qPCR analysis of Kiss1 and Gpr54 expression in the hypothalamus or pituitary gland from mice infected with VSV (1 × 108 PFU per mouse) intraperitoneally for the indicated times. (B) ELISA of kisspeptin1 levels in sera from mice in (A). (C) qPCR analysis of Ifn-β expression in PEMs pretreated with KP-10 (1 μM) for 6 hours and then infected with VSV (MOI, 1) or HSV-1 (MOI, 1) for 8 hours. DMSO, dimethyl sulfoxide. (D) qPCR analysis of VSV RNA replicates in (C) (left panel). (E) qPCR analysis of Ifn-β expression in PEMs pretreated with KP-10 (1 μM) for 6 hours and then stimulated with poly(I:C) (10 μg/ml) or LPS (100 ng/ml) for 4 hours. (F) qPCR analysis of VSV RNA replicates in PEMs pretreated with 2-APB (100 μM) for 1 hour and then infected with VSV (MOI, 1) for 8 hours. (G) qPCR analysis of Ifn-β expression in PEMs pretreated with 2-APB (100 μM) for 1 hour before treatment with KP-10 (1 μM) for 6 hours and then infected with VSV (MOI, 1) for 8 hours. (H) qPCR analysis of Ifn-β expression in Gpr54+/+ and Gpr54−/− PEMs pretreated with KP-10 (1 μM) for 6 hours and then infected with VSV (MOI, 1) for 8 hours. (I) qPCR analysis of Ifn-β expression in the liver, spleen, and lung from mice injected with KP-10 (50 μl of 1 mM KP-10) and VSV (1 × 108 PFU per mouse) for 12 hours and then treated with the same dosage of KP-10 again for 12 hours. (J) qPCR analysis of VSV RNA replicates in organs from mice in (I). GAPDH was used as an internal control for qPCR. Data are representative of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.
Fig. 4
Fig. 4. Calcineurin mediates GPR54-dependent reduction of IFN-I.
(A) HEK-293T cells were transfected with GFP-GPR54-C-ter (GFP-tagged C terminus of GPR54) for 36 hours, then GFP-GPR54-C-ter was immunoprecipitated with GFP antibody, and binding proteins were analyzed by MS. (B) HEK-293T cells were transfected with plasmids encoding GFP-GPR54-C-ter and Flag-CALNA for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to GFP or Flag tags. WCE, whole-cell extracts. (C) HEK-293T cells were transfected with plasmids encoding GFP-GPR54 and Flag-CALNA for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to GFP or Flag tags. (D) HEK-293T cells were transfected with plasmids encoding GFP-GPR54 and Flag-CALNA for 28 hours. The transfected cells were stimulated with KP-10 (1 μM) for 6 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to GFP or Flag tags. (E) GFP-GPR54-C-ter was cotransfected with Flag-CALNB or Flag-CALNC into HEK-293T cells for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to GFP or Flag tags. (F) Flag-CALNA was cotransfected with GFP-GPR54-C-ter or GFP-GPR54-C-ter PRR repeats deleted (GFP-GPR54-C-ter-delPRR) into HEK-293T cells for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to GFP or Flag tags. (G) hemagglutinin (HA)–CALNA was cotransfected with GFP-GPR54-C-ter or GFP-GPR54-C-ter-delPRR into HEK-293T cells for 28 hours. The supernatants of cell lysates were immunoprecipitated using a GFP antibody and then immunoblotted with antibodies to HA or GFP tags. (H) Sequence analysis of the PRR repeats of GPR54 indicates that Arg344 and Arg346 (in bold) are conserved among species. (I) Flag-CALNA was cotransfected with GFP-GPR54-C-ter or GFP-GPR54-C-ter Arg344 and Arg346 mutants (arginine mutated to alanine) into HEK-293T cells for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to GFP or Flag tags. (J) qPCR analysis of Ifn-β expression in PEMs transfected with Calna siRNA for 48 hours before stimulated with KP-10 (1 μM) for 6 hours and then infected with VSV (MOI, 1) for 8 hours. (K) qPCR analysis of IFN-β expression in GFP-GPR54– or GFP-GPR54-R344A-R346A–overexpressing HEK-293T cells infected with VSV (MOI, 1) for 12 hours. WT, wild type. (L and M) Gpr54+/+ and Gpr54−/− PEMs were pretreated with KP-10 (1 μM) for 6 hours, and the supernatants of cell lysates were immunoprecipitated using a CALNA antibody. The purified protein was used to measure CALNA activity by the standard pNpp method. GAPDH was used as an internal control for qPCR. Data are representative of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5
Fig. 5. Calcineurin negatively regulates IFN-I signaling.
(A) qPCR analysis of Ifn-β and Ifn-α4 expression in PEMs transfected with Calna siRNA for 48 hours and then infected with VSV (MOI, 1) for 8 hours. (B) qPCR analysis of Ifn-β expression in PEMs transfected with Calna siRNA for 48 hours and then transfected with poly(I:C) (1 μg/ml) for 4 hours. (C) qPCR analysis of Ifn-β expression in PEMs transfected with Calna siRNA for 48 hours and then infected with HSV-1 (MOI, 10) for 8 hours. (D) Immunoblot analysis of phosphorylated TBK1 and IRF3 or total proteins in lysates of PEMs transfected with Calna siRNA for 48 hours and then infected with VSV (MOI, 1) for the indicated times. (E) qPCR analysis of VSV RNA replicates in (A). (F) qPCR analysis of Ifn-β expression in PEMs pretreated with CsA (10 μg/ml) for 1 hour and then infected with VSV (MOI, 1) for 8 hours. (G) qPCR analysis of Ifn-β expression in PEMs treated with FK506 (10 μg/ml) or poly(I:C) (10 μg/ml) for 4 hours. (H) qPCR analysis of VSV RNA replicates in (F). GAPDH was used as an internal control for qPCR. Data are representative of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 6
Fig. 6. Calcineurin targets and deactivates TBK1 through dephosphorylation.
(A) Flag-CALNA was cotransfected with RIG-I(N) (RIG-N), STING, TBK1, IKKƐ, IRF3-5D, IRF7, or Emvs, together with IFN-β luciferase reporter, into HEK-293T cells for 28 hours. IFN-β luciferase activity was detected and normalized to Renilla luciferase activity. (B and C) HEK-293T cells were transfected with plasmids encoding Myc-TBK1 and Flag-CALNA for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads (B) or a TBK1 antibody (C) and then immunoblotted with antibodies to TBK1 or Flag tag. (D) Purified GST-CALNA protein was incubated with purified His-TBK1. After GST pull-down assay, the proteins were immunoblotted with antibodies to TBK1 or GST tag. (E) HEK-293T cells transfected with Myc-TBK1 and Flag-CALNA for 28 hours were infected with VSV (MOI, 1) for the indicated times. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to TBK1 or Flag tag. (F) PEMs were infected with VSV (MOI, 1) for 8 hours. The supernatants of cell lysates were immunoprecipitated using a CALNA antibody and then immunoblotted with antibodies to CALNA or TBK1. (G) Flag-TBK1 was cotransfected with Flag-CALNB or Flag-CALNC, together with the IFN-β luciferase reporter, into HEK-293T cells for 28 hours. IFN-β luciferase activity was detected and normalized to Renilla luciferase activity. (H) Myc-TBK1 was cotransfected with Flag-CALNB or Flag-CALNC for 28 hours. The supernatants of cell lysates were immunoprecipitated using M2 beads and then immunoblotted with antibodies to TBK1 or Flag tag. (I) Flag-TBK1 was cotransfected with Flag-CALNA, Flag-CALNA (H151A) or Emvs, together with IFN-β luciferase reporter, into HEK-293T cells for 28 hours. IFN-β luciferase activity was detected and normalized to Renilla luciferase activity. IB, immunoblotting. (J) HEK-293T cells were transfected with the indicated plasmids. The supernatants of cell lysates were immunoprecipitated using HA beads and then immunoblotted with the indicated antibodies. (K) Purified His-TBK1 from Escherichia coli was incubated with Flag-CALNA or Flag-CALNA (H151A), which had been expressed in HEK-293T cells. In vitro phosphatase assays were performed, and then, proteins were immunoblotted with the indicated antibodies. Data are representative of three independent experiments (mean ± SD). ***P < 0.001.
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