p204 Is Required for Canonical Lipopolysaccharide-induced TLR4 Signaling in Mice

Abstract

p204, a murine member of an interferon-inducible p200 family, was reported to recognize intracellular viral and bacterial DNAs, however, its role in the innate immunity in vivo remains unknown due to the lack of p204-deficient animal models. In this study we first generated the p204^-/- mice. Unexpectedly, p204 deficiency led to significant defect in extracellular LPS signaling in macrophages, as demonstrated by dramatic reductions of LPS-mediated IFN-β and pro-inflammatory cytokines. The serum levels of IFN-β and pro-inflammatory cytokines were also significantly reduced in p204^-/- mice following LPS challenge. In addition, p204^-/- mice were resistant to LPS-induced shock. LPS-activated NF-ĸB and IRF-3 pathways were all defective in p204-deficient macrophages. p204 binds to TLR4 through its Pyrin domain, and it is required for the dimerization of TLR4 following LPS-challenge. Collectively, p204 is a critical component of canonical LPS-TLR4 signaling pathway, and these studies also suggest that p204 could be a potential target to prevent and treat inflammatory and infectious diseases.

Keywords: IFN-β; Inflammatory responses; LPS; Macrophages; TLR4; p204.

Fig. 1

p204-deficiency suppresses IFN-β production in macrophages transfected with viral DNA sequences or treated with bacterial components. (A) Gene targeting strategy for the generation of p204^−/− mice, and confirmation of p204 deletion by PCR using two different pairs of primers, p204–1 (482 bp) and p204–2 (386 bp) in the BMDMs isolated from p204^−/− mice and by Western blot analysis in the indicated tissues of the p204^−/− mice. Black boxes and numbers represent exons and exon numbers, respectively. The mRNA expression and the release of IFN-β in WT and p204^−/− BMDMs transfected with indicated viral DNA sequences (B–C) or treated with indicated bacterial components (D–E). (F) Western blot analysis of targeted knock-down of p204 expression by CRISPR/Cas9 system in Raw264.7 macrophages. mRNA expression and release of IFN-β in WT and p204-deficient Raw264.7 macrophages transfected with indicated viral DNA sequences (G–H) or treated with indicated bacterial components (I–J). For the analysis of mRNA expression and release of IFN-β, BMDMs and Raw264.7 macrophages were transfected or treated for 6 h and 18 h, respectively. Bar graphs are presented as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01.

Fig. 2

Production of pro-inflammatory cytokines was suppressed in p204-deficient macrophages treated with LPS in vitro. mRNA expression (A–D) and release (E–H) of IFN-β, TNF-α, IL6 and IL-1β in WT and p204^−/− BMDMs treated with LPS (1 μg/ml). mRNA expression (I–L) and release (M–P) of IFN-β, TNF-α, IL6 and IL-1β in WT and p204-deficient Raw264.7 macrophages treated with LPS (1 μg/ml). BMDMs and Raw264.7 macrophages were treated with LPS for 6 h and the indicated time for the analysis of mRNA expression and release of cytokines, respectively. Bar graphs are presented as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01.

Fig. 3

Serum levels of pro-inflammatory cytokines were suppressed and survival rate was increased in p204^−/− mice injected with LPS in vivo. (A–D) Serum levels of IFN-β, TNF-α, IL-6 and IL-1β in WT and p204^−/− mice injected intraperitoneally with LPS (1 mg/kg bodyweight) for the indicated time. Plots are presented as the mean ± SD of three independent experiments. (E) Kaplan-Meier survival plot for WT and p204^−/− mice injected intraperitoneally with a lethal dose of LPS (30 mg/kg bodyweight) for the indicated time (n = 6). *p < 0.05 and **p < 0.01.

Fig. 4

p204 does not recognize intracellular LPS and is dispensable for the activation of inflammasome signaling pathways in macrophages. (A) IL-1β and (B) IL-18 released from the BMDMs isolated from WT, p204^−/−, CASP-11^−/− and Tlr4^−/− mice transfected with either vehicle or LPS (Serotype O111:B4, 2 μg/ml) for 18 h after Pam3CSK4 (1 μg/ml) priming for 6 h. (C) IL-1β released from the BMDMs isolated from WT and p204^−/− mice transfected with either vehicle or indicated LPSs (2 μg/ml) for 18 h after Pam3CSK4 (1 μg/ml) priming for 6 h. (D) LDH levels in the cell culture media of the BMDMs isolated from WT, p204^−/−, CASP-11^−/− and Tlr4^−/− mice transfected with either vehicle or LPS (Serotype O111:B4, 2 μg/ml) for 18 h after Pam3CSK4 (1 μg/ml) priming for 6 h. (E) LDH levels in the cell culture media of the BMDMs isolated from WT and p204^−/− mice transfected with either vehicle or indicated LPSs (2 μg/ml) for 18 h after Pam3CSK4 (1 μg/ml) priming for 6 h. (F) Western blot analysis of GFP and FLAG of the whole cell lysates (input) and streptavidin pull-downs of either GFP-p204 or FLAG-CASP-11-transfected HEK293T cell lysates incubated with the indicated amounts of Biotin-LPS. Bar graphs are presented as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01.

Fig. 5

NF-κB and IRF-3 signaling pathways are suppressed in p204-deficient macrophages treated with LPS. WT and p204-deficient Raw264.7 macrophages were treated with LPS (1 μg/ml) for the indicated time. (A) Phosphorylated forms of TBK1, PI3K/p85, AKT, and IKKα/β were determined by Western blot analysis in the whole lysates of the cells. (B) Activation of MAPK pathway in WT and p204-deficient Raw264.7 macrophages was determined by Western blotting. (C)Phosphorylated and total form of IκBα was determined by Western blot analysis in the cytoplasmic fraction (Cyto) of the cells. (D) Phosphorylated IRF-3 and NF-κB/p65 were determined by Western blot analysis in the nuclear fraction (Nuc) of the cells. GAPDH and lamin B were used for the internal control for the cytoplasmic and nuclear fraction, respectively. Luciferase reporter gene assay of (E) NF-κB promoter and (F) IRF-3 promoter activity in WT and p204-deficient Raw264.7 macrophages treated with LPS (1 μg/ml) for 24 h. (G) WT and p204−/− BMDMs were treated with LPS (1 μg/ml) for the indicated time, and phosphorylation of TBK1, PI3K/p85, AKT, and IKKα/β was determined by Western blotting. (H) Phosphorylation of MAPK pathways in WT and p204−/− BMDMs following LPS treatment at indicated time points was determined by Western blotting. Bar graphs are presented as the mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01.

Fig. 6

p204 binds to TLR4 and is required for LPS-triggered TLR4 dimerization. (A) p204 binds to TLR4 in overexpressed 293T cells. GFP control vector and p204-GFP expression plasmid were co-transfected with a combination of TLR4, MD2 and CD14 expressing constructs, followed by LPS challenge. The cell lysates were pulled down by GFP antibody, and probed with TLR4 antibody (upper panel) and the inputs were probed with GFP antibody (lower panel). (B) Endogenous p204 binds to TLR4 followed LPS stimulation. Raw cells were treated with LPS (1 μg/ml) for 2 h, and the cell lysates were used for immunoprecipitation with p204 antibody. The interaction between p204 and TLR4 was detected with TLR4 antibody. (C) p204 is required for TLR4 dimerization. BMDM isolated from WT and p204 KO mice were treated with LPS (1 μg/ml) for 2 h, then cell lysates were prepared. The TLR4 antibody was used for immunoprecipitation, and the samples were run on non-reducing gel to visualize the dimerization of TLR (upper panel), and p204 binds to TLR4 in WT, but not in p204−/− BMDMs, following LPS treatment (middle panel), and the input of TLR4 was comparable (lower panel). (D) Dimerization of membrane TLR4 is defective in p204−/− BMDMs. BMDMs from WT and p204−/− were stimulated with LPS (100 ng/ml) for 2 h. The TLR4-MD2 complex was stained with the specific antibody (MTS510). Percentage of dimerization of TLR4 was calculated based on previous report (Zanoni et al., 2016). (E) Structure of p204 serial deletion mutants. To determine the binding domain that mediates the interaction between p204 and TLR4, we generated serial p204 deletions from its C-terminus, and all the constructs were fused with a GFP tag. (F) The expression of p204 constructs in (E) was examined by western-blot. (G) Pyrin domain alone is sufficient for binding to TLR4. p204 serial constructs were co-transfected with TLR4-expression plasmid into 293 T cells, followed by LPS stimulation. The IP experiment was performed by using antibody against GFP, and samples were probed with TLR4 antibody. (H) Pyrin domain is required to mediate the interaction between p204 and TLR4. The expression constructs of GFP, p204-GFP, or p204-GFP lacking Pyrin domain (ΔPYD) were transfected into 293 T cells, and the cells were stimulated with LPS. The immunoprecipitation was performed with anti-GFP antibody, and the immunoprecipitated complexes were detected with anti-TLR4 antibody. (I–K) Pyrin domain also can restore the response to LPS in p204-deficient RAW cells. p204-deficient RAW cells were transiently transfected with GFP control vector, p204-GFP, and its serial deletion mutants. After 24 h after transfection, LPS (1 μg/ml) were added to the cell culture medium for additional 24 h. The levels of IFN-β (I), TNF-α (J), and IL-6 (K) were measured by ELISA. (L) RKR motif was replaced with AAA by site-directed mutagenesis in p204-GFP expressing plasmids. The p204-GFP and p204m-GFP plasmids were co-transfected with TLR4 expression plasmid into 293 T cells. After LPS treatment, The immunoprecipitation was performed with anti-GFP antibody, and the immunoprecipitated complexes were detected with anti-TLR4 antibody. (M) RKR motif is critical to restore LPS-triggered IFN-β production in p204-deficient RAW cells. RKR motif was replaced with AAA motif using site-directed mutagenesis in CD3-GFP expressing plasmid. GFP, p204-GFP, CD3-GFP, and CD3m-GFP were transiently transfected into p204-deficient RAW cells and stimulated with LPS for 24 h. The IFN-β levels were measured by ELISA.

Fig. 7

Illustration of p204 function in LPS/TLR4 signaling pathway. p204 is recruited to TLR4 receptor complex following LPS stimulation. p204, known to form a homodimer through its C-terminal HIN-200 domain, induces the dimerization of TLR4 through the binding of its N-terminal Pyrin domain to TLR4, followed by the activation of downstream IRF-3 and NF-ĸB signaling pathways and the release of IFN-β and pro-inflammatory cytokines, respectively. “PYD”: Pyrin domain; “A” and “B”: p204 C-terminal HIN-A and HIN-B domain respectively.