Zinc finger protein 64 promotes Toll-like receptor-triggered proinflammatory and type I interferon production in macrophages by enhancing p65 subunit activation

Abstract

The molecular mechanisms that fine-tune the Toll-like receptor (TLR)-triggered innate immune response need further investigation. As an important transcription factor, zinc finger proteins (ZFPs) play important roles in many cell functions, including development, differentiation, tumorigenesis, and functions of the immune system. However, the role of ZFP members in the innate immune responses remains unclear. Here we showed that the expression of C2H2-type ZFP, ZFP64, was significantly up-regulated in macrophages upon stimulation with TLR ligands, including LPS, CpG oligodeoxynucleotides, or poly(I:C). ZFP64 overexpression promoted TLR-triggered TNF-α, IL-6, and IFN-β production in macrophages. Coincidently, knockdown of ZFP64 expression significantly inhibited the production of the above cytokines. However, activation of MAPK and IRF3 was not responsible for the ZFP64-mediated promotion of cytokine production. Interestingly, ZFP64 significantly up-regulated TLR-induced NF-κB activation. ZFP64 could bind to the promoter of the TNF-α, IL-6, and IFN-β genes in macrophages only after TLR ligation. Furthermore, ZFP64 associated with the NF-κB p65 subunit upon LPS stimulation, and TLR-ligated macrophages showed a lower level of p65 recruitment to the TNF-α, IL-6, and IFN-β gene promoter in the absence of ZFP64. The data identify ZFP64 as a downstream positive regulator of TLR-initiated innate immune responses by associating with the NF-κB p65 subunit, enhancing p65 recruitment to the target gene promoters and increasing p65 activation and, thus, leading to the promotion of TLR-triggered proinflammatory cytokine and type I interferon production. Our findings add mechanistic insight into the efficient activation of the TLR innate response against invading pathogens.

Keywords: Inflammation; Innate Immunity; Macrophages; NF-κB (NF-κB); Toll-like Receptors (TLR); ZFP64; p65.

FIGURE 1.

TLR ligands up-regulate ZFP64 expression in macrophages. A and B, RT-PCR analysis of the ZFP64 mRNA expression level in different mouse tissues and immune cells. β-Actin was used as a control. NK, natural killer. C, mouse peritoneal macrophages were stimulated with LPS (100 ng/ml), CpG ODN (0.33 μm), or poly(I:C) (10 μg/ml) for the indicated times. The ZFP64 mRNA expression level was detected by Q-PCR. The results were presented as fold expression of ZFP64 mRNA to that of β-actin. Data are representative of three independent experiments with similar results (A and B), and data are shown as mean ± S.D. of three independent experiments (C).

FIGURE 2.

ZFP64 promotes TLR-triggered proinflammatory cytokine and type I interferon production in macrophages. A, RAW264.7 cells were transiently transfected with a ZFP64 expression plasmid (ZFP64-HA). Forty-eight hours later, the expression of HA-tagged ZFP64 was detected by Western blot analysis (top panel), or cells were stimulated with LPS (100 ng/ml), CpG ODN (0.3 μm), or poly(I:C) (10 μg/ml) for 6 h or left unstimulated (Med), and then the production of TNF-α, IL-6, and IFN-β in the supernatants was measured by ELISA (bottom panel). B, mouse peritoneal macrophages were transfected with control siRNA (si-Non) or ZFP64 siRNA (si-ZFP64). Forty-eight hours later, the efficiency of silencing was detected by Western blot analysis (top panel), or cells were stimulated with LPS (100 ng/ml), CpG ODN (0.3 μm), or Poly(I:C) (10 μg/ml) for 6 h, and the production of TNF-α, IL-6, and IFN-β in the supernatants was measured by ELISA (bottom panel). C, HEK293 cells were cotransfected with or without 50 ng MyD88- or TRIF-expressing plasmid; 40 ng TNF-α, IL-6, or IFN-β luciferase reporter plasmid; and 10 ng pTK-Renilla-luciferase together with 50, 100, or 150 ng of ZFP64-expressing plasmid. Total amounts of plasmid DNA were equalized using an empty control vector. After 24 h of culture, luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01.

FIGURE 3.

Increased activation of the TLR-triggered MAPK pathway is not required for ZFP64-mediated promotion of proinflammation cytokine production. A and B, mouse peritoneal macrophages were transfected with control siRNA (si-Non) or ZFP64 siRNA (si-ZFP64). After 48 h, cells were stimulated with LPS (100 ng/ml) or poly(I:C) (10 μg/ml) for the indicated times. Cells were lysed and subjected to Western blot analysis with the indicated antibodies. C, RAW264.7 cells were transfected with a ZFP64 expression plasmid (ZFP64-HA). After 48 h, cells were pretreated with PD98059 (10 nm) or dimethyl sulfoxide for 30 min and then stimulated with LPS (100 ng/ml) for 6 h. The production of TNF-α and IL-6 in the supernatants were measured by ELISA. Data are representative of three independent experiments with similar results (A and B) and are shown as mean ± S.D. of three independent experiments. *, p < 0.05.

FIGURE 4.

ZFP64 enhances TLR-triggered NF-κB activation. A and B, mouse peritoneal macrophages were silenced with ZFP64 siRNA (si-ZFP64) or control siRNA (si-Non) for 48 h and then stimulated with LPS (100 ng/ml) (A) or poly(I:C) (10 μg/ml) (B). Phosphorylated-IKBα (p-IKBα), p-p65, and p-IRF3 were detected by Western blot analysis. C and D, HEK293T cells were transfected with an NF-κB or IRF-3 reporter gene plasmid together with pTK-Renilla-luciferase and MyD88- or TRIF-expressing plasmid alone or with 50, 100, or 150 ng of ZFP64-expressing plasmid. After 24 h, NF-κB (C) or IRF3 (D) reporter activities in lysates were measured by luciferase assay. Total amounts of plasmid DNA were equalized using an empty control vector. After 24 h of culture, luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean ± S.D. (n = 6) of one typical result from three independent experiments with similar results. *, p < 0.05; ** p < 0.01. Ctrl, control.

FIGURE 5.

ZFP64 binds to the promoters of the TNF-α, IL-6, or IFN-β genes in TLR-activated macrophages. Mouse peritoneal macrophages were stimulated with LPS, CpG ODN, or poly(I:C) for 2 h or left unstimulated (Med), and then the recruitment of ZFP64 to the TNF-α, IL-6, and IFN-β gene promoters was detected with a ChIP assay and analyzed by Q-PCR. The value represents the enrichment fold of ZFP64 bound to the TNF-α, IL-6, and IFN-β promoter compared with control IgG precipitations. Data are shown as mean ± S.D. of three independent experiments. **, p < 0.01.

FIGURE 6.

ZFP64 associates with p65. A and B, HEK293T cells were cotransfected with a ZFP64-HA- and p65-expressing plasmid. After 48 h, cell lysates were immunoprecipitated (IP) with anti-HA (A) or anti-FLAG (B) antibody and then immunoblotted (IB) with anti-FLAG and anti-HA antibody. C, mouse peritoneal macrophages were stimulated with LPS for the indicated time. Cell lysates were immunoprecipitated with ZFP64 antibody and then immunoblotted with the indicated antibodies. Similar results were obtained in three independent experiments.

FIGURE 7.

ZFP64 enhances p65 recruitment to cytokine gene promoters. A, HEK293 cells were cotransfected with or without p65-expressing plasmid, TNF-α, IL-6, or IFN-β luciferase reporter plasmid and 10 ng of pTK-Renilla-luciferase together with 50, 100, or 150 ng of ZFP64-expressing plasmid. Total amounts of plasmid DNA were equalized using an empty control vector. After 24 h of culture, luciferase activity was measured and normalized by Renilla luciferase activity. Ctrl, control. B, mouse peritoneal macrophages were transfected with control siRNA (si-Non) or ZFP64 siRNA (si-ZFP64). After 48 h, cells were stimulated with LPS (100 ng/ml) for 2 h or left unstimulated (Med). The recruitment of p65 to the TNF-α, IL-6, or IFN-β gene promoters was detected with a ChIP assay and analyzed by Q-PCR. The value represents the enrichment fold of p65 bound to the TNF-α, IL-6, or IFN-β gene promoters compared with control IgG precipitations. Data are shown as mean ± S.D. of three independent experiments. **, p < 0.01.