Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-alpha and PGE2 - PubMed (original) (raw)

Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-alpha and PGE2

Yang Hu et al. J Leukoc Biol. 2008 Nov.

Abstract

Maturation of dendritic cells (DCs) by TLR ligands induces expression of IFN-beta and autocrine activation of IFN-inducible Stat1-dependent genes important for DC function. In this study, we analyzed the regulation of STAT signaling during maturation of human DCs by TNF-alpha and PGE2, which induced maturation of human DCs comparably with LPS but did not induce detectable IFN-beta production or Stat1 tyrosine phosphorylation. Consistent with these results, TNF-alpha and PGE2 did not induce Stat1 DNA binding to a standard Stat1-binding oligonucleotide. Instead, TNF-alpha and PGE2 increased Stat1 serine phosphorylation and Stat4 tyrosine phosphorylation and activated expression of the NF-kappaB and Stat1 target gene IFN regulatory factor 1 (IRF1), which contributes to IFN responses. TNF-alpha and PGE2 induced a complex that bound an oligonucleotide derived from the IRF1 promoter that contains a STAT-binding sequence embedded in a larger palindromic sequence, and this complex was recognized by Stat1 antibodies. These results suggest that TNF-alpha and PGE2 activate STAT-mediated components of human DC maturation by alternative pathways to the IFN-beta-mediated autocrine loop used by TLRs.

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Figures

Fig. 1.

Cell surface phenotype of human monocyte-derived, immature DCs (iDC) and mature DCs (mDC). DCs were generated as described in Materials and Methods, and the expression of CD14, CD40, CD25, CD80, CD86, and HLA-DR was analyzed by flow cytometry after 2 days of stimulation with 10 ng/ml LPS or TNF-α (25 ng/ml) + PGE2 (1 ng/ml). Open histograms = isotype control staining; shaded histograms = staining with corresponding antibodies. One representative experiment of seven is shown.

Fig. 2.

TNF-α + PGE2 do not detectably activate Stat1 or autocrine IFN-β production. Human monocyte-derived, immature DCs were stimulated with 10 ng/ml LPS, TNF-α (25 ng/ml), PGE2 (1 ng/ml), or TNF-α + PGE2 for indicated times. (A) Whole cell extracts were analyzed using immunoblotting with antibodies against tyrosine-phosphorylated (P-)Stat1 and Stat3, followed by probing the same filter with antibodies against Stat1 and tubulin. (B) mRNA levels of IFN-β, IP-10, and CD25 were measured by real-time PCR and normalized relative to GAPDH. (C) mRNA levels of ISG56 were measured by real-time PCR and normalized relative to GAPDH. (D) Cell extracts were subjected to EMSA using a radiolabeled hSIE oligonucleotide. Representative results from at least three experiments are shown.

Fig. 3.

TNF-α + PGE2 induce serine phosphorylation of STAT1 and tyrosine phosphorylation of STAT4. (A) Human monocyte-derived, immature DCs were stimulated with 10 ng/ml LPS, TNF-α (25 ng/ml), PGE2 (1 ng/ml), or TNF-α + PGE2 for indicated times. Whole cell extracts were analyzed using immunoblotting with antibodies against tyrosine-phosphorylated (Ptyr-) and serine-phosphorylated (Pser-) Stat1 and tyrosine-phosporylated Stat2, followed by probing the same filter with antibodies against Stat3. One representative experiment of three is shown. (B) Human monocyte-derived, immature DCs were stimulated with 10 ng/ml LPS, TNF-α (25 ng/ml), PGE2 (1 ng/ml), or TNF-α + PGE2 for 48 h. Whole cell extracts were analyzed using immunoblotting with antibodies against tyrosine-phosphorylated STAT4, STAT4, and STAT3. One representative experiment of three is shown.

Fig. 4.

TNF-α + PGE2-induced IRF1 expression is partially dependent on NF-κB but not on IFN-α/β. (A) mRNA levels of IRF1 in immature DC treated with LPS, TNF-α, PGE2, or TNF-α + PGE2 for indicated times were measured by real-time PCR and normalized relative to GAPDH. (B) mRNA levels of IRF1 in immature DC treated with TNF-α, PGE2, or TNF-α + PGE2, with or without blocking anti-IFNR-α/β antibodies, were measured by real-time PCR and normalized relative to GAPDH. (C) mRNA levels of IRF1 in immature DC treated with TNF-α, PGE2, or TNF-α + PGE2, with or without the IKK inhibitor Bay11, were measured by real-time PCR and normalized relative to GAPDH. (D) The expression of CD25, CD40, CD86, and HLA-DR was analyzed by flow cytometry after 48 h of stimulation with TNF-α (25 ng/ml) + PGE2 (1 ng/ml), with or without Bay11 treatment. Open histograms with dotted line = isotype control staining; shaded histograms = staining with corresponding antibodies without Bay11 treatment; and open histograms with solid line = staining with corresponding antibodies in immature DC treated with Bay11. One representative experiment of two is shown.

Fig. 5.

TNF-α + PGE2 induce a complex that binds to an IRF1 promoter GAS sequence. (A) Cell extracts were subjected to EMSA using the radiolabeled IRF1 oligonucleotide. (B) The sequence of IRF1 oligonucleotide used in EMSA assays. The core GAS sequence and the extended palindromic sequence are identified. (C) TNF-α + PGE2 mature DC extracts were subjected to EMSA using the radiolabeled IRF1 oligonucleotide and unlabeled wild-type (WT) or mutant oligonucleotides as competitors. (D) Cell extracts were subjected to EMSA using the radiolabeled IRF1 oligonucleotide. Stat1 or Stat5 antibodies were added to extracts for 1 h prior to addition of the radiolabeled probe to supershift Stat-containing complexes.

References

1. Banchereau J, Steinman R M. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. - PubMed
1. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol. 2001;1:135–145. - PubMed
1. Toshchakov V, Jones B W, Perera P Y, Thomas K, Cody M J, Zhang S, Williams B R, Major J, Hamilton T A, Fenton M J, Vogel S N. TLR4, but not TLR2, mediates IFN-β-induced STAT1α/β-dependent gene expression in macrophages. Nat Immunol. 2002;3:392–398. - PubMed
1. Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M, Takeda K, Akira S. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science. 2003;301:640–643. - PubMed
1. Yamamoto M, Sato S, Mori K, Hoshino K, Takeuchi O, Takeda K, Akira S. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling. J Immunol. 2002;169:6668–6672. - PubMed

Regulation of STAT pathways and IRF1 during human dendritic cell maturation by TNF-alpha and PGE2 - PubMed (original) (raw)