The PYHIN Protein p205 Regulates the Inflammasome by Controlling Asc Expression

Abstract

Members of the IFN-inducible PYHIN protein family, such as absent in melanoma-2 and IFN-γ-inducible protein (IFI)16, bind dsDNA and form caspase-1-activating inflammasomes that are important in immunity to cytosolic bacteria, DNA viruses, or HIV. IFI16 has also been shown to regulate transcription of type I IFNs during HSV infection. The role of other members of the PYHIN protein family in the regulation of immune responses is much less clear. In this study, we identified an immune-regulatory function for a member of the murine PYHIN protein family, p205 (also called Ifi205). Examination of immune responses induced by dsDNA and other microbial ligands in bone marrow-derived macrophages lacking p205 revealed that inflammasome activation by dsDNA, as well as ligands that engage the NLRP3 inflammasome, was severely compromised in these cells. Further analysis revealed that p205-knockdown cells showed reduced expression of apoptosis-associated speck-like molecule containing CARD domain (Asc) at the protein and RNA levels. p205 knockdown resulted in reduced binding of actively transcribing RNA polymerase II to the endogenous Asc gene, resulting in decreased transcription and processing of Asc pre-mRNA. Deletion of p205 in B16 melanoma cells using CRISPR/Cas9 showed a similar loss of Asc expression. Ectopic expression of p205 induced expression of an Asc promoter-luciferase reporter gene. Together, these findings suggest that p205 controls expression of Asc mRNA to regulate inflammasome responses. These findings expand on our understanding of immune-regulatory roles for the PYHIN protein family.

Figure 1. p205 is induced by LPS, Type I and II IFN and localizes to the nucleus

Primary BMDMs stimulated with (A) LPS (200 ng/ml), (B) Type-I IFN (100 U/ml) or (C) IFN-γ (20 ng/ml) at different time points (0, 2, 4, 8, 16, 24 h) were tested for p205 mRNA expression as well as other PYHIN genes, Mnda, p204 and Aim2. Levels of IL-6 or Cxcl10 mRNA were included as positive controls. Gene expression is reported relative to a combination of three housekeeping genes Gapdh, Hprt, β-actin. (D) Immunoblot analysis of the nuclear and cytoplasmic fractions of wild-type BMDM transduced with empty vector (EV) and wild-type BMDM overexpressing HA-tagged p205 using anti-HA antibody. Histone 3 and Aim2 were used as controls for nuclear and cytosolic extracts respectively. (E) Confocal microscopy of CFP-tagged p205 (green) in HEK 293T cells stained for nucleus using Acridine orange in first panel and Cholera Toxin B (CtxB) staining plasma membrane in second panel. Data is representative of two independent experiments. (F) Primary BMDM untreated (Ctl) or treated with LPS for 6h, type-I IFN for 16h or IFN-γ for 16h were separated into nuclear and cytosolic fractions and immunoblotted for endogenous p205 (n.s.- non-specific band). (G) Western blot analysis of endogenous p205 expression in the nuclear and cytosolic extracts of primary macrophages treated with type-I IFN as indicated. Usf2 and Gapdh were used as controls for nuclear and cytosolic fractions respectively (n.s.- non-specific band).

Figure 2. p205 knockdown using shRNA results in impaired inflammasomes activation

BMDM transduced with shRNA targeting either (A) 3′UTR or (B) CDS of p205 gene were inspected for expression of p205, Mnda and p204 mRNA relative to β-actin mRNA and normalized to expression in GFP shRNA BMDM. (C) Heatmap of PYHIN gene expression in p205 KD 3′UTR and KD CDS BMDM compared to control (Ctl) BMDM. The p205 knockdown BMDMs were primed with LPS (200ng/ml) for 3h and then stimulated with transfected pdAdT (1μg/ml for 6h), transfected ISD (3μM for 6h), Nigericin (10μM for 1h), ATP (5μM) or, stimulated alone with Sendai virus (SV; overnight) or poly I:C (overnight). Secreted (D) IFNβ and (E) IL-1β levels were assessed by ELISA. (F) GFP shRNA CTL, p205 KD 3′UTR and KD CDS were primed with LPS (200ng/ml) for 3h and then stimulated with pdAdT (1 μg/ml for 6h) or Nigericin (10 μM for 1h) and the supernatants and the lysates from the macrophages were immunoblotted for pro-IL1β (35 kD), cleaved form of IL-1β (p17), pro-caspase 1 (45kD) and the active subunit of caspase 1(p20).

Figure 3. Loss of p205 leads to a defect in Asc expression

(A) Levels of Nlrp3, Aim2, Asc and β-actin proteins were elucidated by Western blot in LPS stimulated (200 ng/ml) GFP shRNA CTL, p205 KD 3′UTR and KD CDS macrophages. (B) Immunoblot of p205, Asc and β-actin proteins in LPS-stimulated (200 ng/ml for 6h) p205 knockdown macrophages. (C) Levels of p205 and Asc mRNA (relative to β-actin and normalized to GFP shRNA BMDM) were detected by qPCR in the same cell lines. (D) Western blot analysis of Asc overexpression in the p205 knockdown BMDM either left untreated or treated with LPS treated (200 ng/ml for 3h). Asc reconstituted cell lines tested for IL-1β production (E) on stimulation with LPS (200 ng/ml for 3h) and pdAdT (1 μg/ml for 6h) or ATP (5 μM for 1h) (left) and (F) with overnight culture (stationary phase) or log phase culture of Salmonella sp. by ELISA. (G) Levels of caspase 1 and IL-1β processing in the Asc reconstituted cells were detected by Western Blot and (H) cell death was measured by amounts of LDH released with LPS (200 ng/ml for 3h) and pdAdT (1 μg, 2 μg, or 3 μg per ml for 6h) and/or ATP (5 μM for 1h) stimulation.

Figure 4. Reconstitution of p205 rescues Asc expression and inflammasome activation

(A) p205 and (B) Asc mRNA expression relative to β-actin and normalized to GFP shRNA BMDM were measured in BMDMs reconstituted with p205. (C) Western blot analysis of nuclear and cytoplasmic fractions of p205 reconstituted cell lines to detect p205-HA, Histone H3, Asc and Aim2. (D) p205 and Asc mRNA levels by qPCR relative to β-actin in p205- overexpressing BMDM. The rescued cells were tested for (E) IL1β production and (F) cell death with LPS (200ng/ml for 3h) and pdAdT (1 μg, 2 μg, or 3μg per ml for 6h) or ATP (5 μM for 1h) by ELISA and by measuring amount of LDH released respectively.

Figure 5. p205 modulates transcription and splicing from the endogenous Asc gene

(A) Schematic of the Asc gene locus and Chromatin-IP primers locations on the gene. (B) Recruitment of total RNA Pol II to endogenous Asc gene in GFP shRNA CTL, p205 KD 3′UTR or KD CDS macrophages. Recruitment of (C) Serine-5-P RNA Pol II and (D) Serine-2-P RNA Pol II on endogenous Asc gene. (E) Recruitment of total, Serine-5-P or Serine-2-P RNA Pol II to the Gapdh transcription start site. All values are represented as percent fraction of total input DNA. Data was calculated against the IgG isotype control and is representative of three independent experiments. (D) qRT-PCR analysis of p205, nascent or mature Asc mRNA expression (relative to Hprt and Gapdh, and normalized to EV CTL BMDM) in p205 knockdown macrophages.

Figure 6. p205 drives expression from an Asc promoter-luciferase reporter construct

HEK 293T cells were transfected with a mixture containing 10 ng of TK-Renilla luciferase with 1 ng of Asc promoter firefly luciferase reporter and increasing amounts of (A) p205-HA, p204-HA or Aim2-FLAG, or (B) with full length p205 or p205 deletion mutants as indicated. (C) Transfection of increasing concentrations of c/EBPβ, p65/RelA and p205 alone or, co-transfection of increasing concentrations of p205-HA with either c/EBPβ or p65/RelA with Asc promoter-reporter or, transfection of the Asc promoter-reporter with increasing concentrations of p205 with both c/EBPβ and p65/RelA, as indicated. All luciferase values were measured and normalized to Renilla values. Values are displayed as fold change over the Asc reporter construct alone. Data is representative of three independent experiments. (D) Co-immunoprecipitation and immunoblot of overexpressed HA-tagged p205 and c/EBPβ in HEK 293T cells using anti-HA and anti-c/EBPβ antibodies. (E) Co-immunoprecipitation and Western blot of endogenous p205 and c/EBPβ in LPS-stimulated BMDM using antibodies against p205 and c/EBPβ. Data is representative of two independent experiments (n.s.- non-specific band).

Figure 7. CRISPR KO of p205 leads to reduced Asc expression in B16 melanoma

(A) Expression of p205 mRNA in IFN-stimulated wild type (WT) and p205 knockout (KO) B16 melanoma cell lines relative to Hprt and normalized to non-treated (NT). (B) p205 and β-actin protein expression in WT and p205 KO cell lines stimulated with Sendai virus (n.s.- non-specific band) (C) Mature mRNA and pre-mRNA profile of Asc expression (relative to Hprt and normalized to WT) and (D) Western blot analysis of Asc and β-actin in WT and p205 KO B16 melanoma cell lines.