A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA–induced IL-12 production and Th cell differentiation
Kenji Sugimoto, … , Yasuhiro Shimada, Tetsuya Matsuguchi
Kenji Sugimoto, … , Yasuhiro Shimada, Tetsuya Matsuguchi
Published September 15, 2004
Citation Information: J Clin Invest. 2004;114(6):857-866. https://doi.org/10.1172/JCI20014.
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Article Immunology

A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA–induced IL-12 production and Th cell differentiation

Abstract

A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal–regulated kinase (ERK) activation and production of TNF-α and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2–independent manner. Peritoneal macrophages and bone marrow–derived DCs from Cot/Tpl2–/– mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2–/– macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2–/– macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter–binding protein (GA-12–binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2–/– mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA–guided vaccination.

Authors

Kenji Sugimoto, Mutsuhiro Ohata, Jun Miyoshi, Hiroyoshi Ishizaki, Naotake Tsuboi, Akio Masuda, Yasunobu Yoshikai, Masaya Takamoto, Kazuo Sugane, Seiichi Matsuo, Yasuhiro Shimada, Tetsuya Matsuguchi

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CpG-DNA–treated Cot/Tpl2–/– macrophages showed significantly increased I...
CpG-DNA–treated Cot/Tpl2–/– macrophages showed significantly increased IL-12 production. (A) WT (black bars) and Cot/Tpl2–/– (white bars) macrophages were stimulated with LPS or CpG-DNA for 24 hours. Levels of TNF-α, IL-12 p40, and IL-10 in culture supernatants were measured by ELISA. *P < 0.05 compared with WT. **P < 0.01 compared with WT. (B) WT and Cot/Tpl2–/– macrophages were pretreated with 1 μM U0126 and stimulated with 1 μM CpG-DNA for 8 hours. The level of IL-12 p40 in culture supernatants was measured by ELISA. Data are expressed as mean ± SD and are representative of 3 independent experiments. (C) WT and Cot/Tpl2–/– macrophages were treated for 2 hours with 1 μg/ml LPS or for 4 hours with 1 μM CpG-DNA. Total RNA extracted from peritoneal macrophages was subjected to Northern blot analysis and RNA protection assay (RPA). (D) WT and Cot/Tpl2–/– macrophages were unstimulated or stimulated with 1 μg/ml LPS or 1 μM CpG-DNA for the indicated times. Nuclear extracts were subjected to EMSA to evaluate DNA-binding activities of NF-κB or GAP-12. The same nuclear extracts were subjected to Western blot analyses with Ab’s against c-Maf and histone H1.