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The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors

Nature Immunology volume 8, pages 619–629 (2007)Cite this article

Abstract

Immunoreceptor tyrosine-based activation motifs (ITAMs) are crucial in antigen receptor signaling in acquired immunity. Although receptors associated with the ITAM-bearing adaptors FcRγ and DAP12 on myeloid cells have been suggested to activate innate immune responses, the mechanism coupling those receptors to 'downstream' signaling events is unclear. The CARMA1–Bcl-10–MALT1 complex is critical for the activation of transcription factor NF-κB in lymphocytes but has an unclear function in myeloid cells. Here we report that deletion of the gene encoding the Bcl-10 adaptor–binding partner CARD9 resulted in impaired myeloid cell activation of NF-κB signaling by several ITAM-associated receptors. Moreover, CARD9 was required for Toll-like receptor–induced activation of dendritic cells through the activation of mitogen-activated protein kinases. Although Bcl10−/− and Card9−/− mice had similar signaling impairment in myeloid cells, Card11−/− (CARMA1-deficient) myeloid cell responses were normal, and although Card11−/− lymphocytes were defective in antigen receptor–mediated activation, Card9−/− lymphocytes were not. Thus, the activation of lymphoid and myeloid cells through ITAM-associated receptors or Toll-like receptors is regulated by CARMA1–Bcl-10 and CARD9–Bcl-10, respectively.

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Figure 1: Normal activation of T and B lymphocytes in Card9−/− mice.
Figure 2: Impaired cytokine production by Card9−/− myeloid cells after stimulation through ITAM receptors.
Figure 3: CARD9 is required for both MyD88- and dectin-1-mediated cytokine responses.
Figure 4: Impaired ITAM-associated receptor–mediated NF-κB activation in Card9−/− DCs.
Figure 5: CARD9-dependent ITAM receptor–induced activation of DCs requires Bcl-10 but not CARMA1.
Figure 6: Impaired TLR response by Card9−/− DCs.
Figure 7: Impaired MAPK activation in TLR-stimulated Card9−/− DCs.
Figure 8: Card9−/− mice are more susceptible to L. monocytogenes.

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Acknowledgements

We thank N. Suzuki, S. Yamasaki, and T. Yokosuka for discussions; H. Arase (Osaka University) and N. Kanazawa (Wakayama Medical University) for reagents; S. Akira (Osaka University) for Myd88−/− mice and Tlr2−/−; X. Lin (University of Texas M.D. Anderson Cancer Center) for reagents, discussions and sharing unpublished data; and H. Yamaguchi and M. Ikari for secretarial assistance. Expression plasmids pCMVSPORT-IRAK1, pcDNA3-RIP2–myc and pRK6–myc-Bcl-10 were provided by T.W. Mak (Campbell Family Institute for Breast Cancer Research and Ontario Cancer Institue), N. Inohara (The University of Michigan Medical School) and X. Lin (University of Texas M.D. Anderson Cancer Center), respectively. Supported by a Grant-in-Aid for Priority Area Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the National Cancer Institute (R01 CA87064 to S.W.M.), Cancer Center (CORE grant CA21765), the Council of Scientific and Industrial Research (Government of India; S.K.P.), the Department of Biotechnology (Government of India) and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.

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Authors and Affiliations

  1. Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, 230-0045, Kanagawa, Japan

    Hiromitsu Hara, Chitose Ishihara, Arata Takeuchi, Takayuki Imanishi & Takashi Saito

  2. Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga, 849-8501, Saga, Japan

    Hiromitsu Hara & Hiroki Yoshida

  3. Department of Pathology and Oncology, St. Jude Children's Research Hospital, Memphis, 38105, Tennessee, USA

    Liquan Xue & Stephan W Morris

  4. Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, 980-8575, Miyagi, Japan

    Masanori Inui & Toshiyuki Takai

  5. Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305-8575, Ibaraki, Japan

    Akira Shibuya

  6. Center for Experimental Medicine, The Institute of Medical Science, The University of Tokyo, Minato-ku, 108-8639, Tokyo, Japan

    Shinobu Saijo & Yoichiro Iwakura

  7. Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, 192-0392, Tokyo, Japan

    Naohito Ohno

  8. Laboratory for Developmental Genetics, RIKEN Research Center for Allergy and Immunology, Yokohama, 230-0045, Kanagawa, Japan

    Haruhiko Koseki

  9. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, 1030, Austria

    Josef M Penninger

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Contributions

H.H. designed and did experiments and wrote the paper; C.I., A.T. and H.K. made knockout mice; T.I. did experiments; L.X., S.W.M., S.S. and Y.I. provided knockout mice; M.I., T.T., A.S. and N.O. provided antibodies or reagents; H.Y. and J.M.P. contributed to discussions; and T.S. designed experiments and wrote the paper.

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Correspondence to Hiromitsu Hara or Takashi Saito.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Gene targeting of Card9. (PDF 78 kb)

Supplementary Fig. 2

Normal development of lymphocytes, macrophages, and dendritic cells in Card9−/− mice. (PDF 143 kb)

Supplementary Fig. 3

Normal cytokine responses of BMDCs from Card9−/− mice upon stimulation by anti-CD40 or PMA plus calcium ionophore. (PDF 43 kb)

Supplementary Fig. 4

Expression profiles of Carma1 mRNA in immune cells. (PDF 53 kb)

Supplementary Fig. 5

Real-time RT-PCR analysis of Il6 mRNA from loxoribine-stimulated Card9−/− DCs. (PDF 42 kb)

Supplementary Fig. 6

TLR-induced cytokine response in Card9−/− macrophages. (PDF 71 kb)

Supplementary Fig. 7

CARD9 and RIP2 synergistically induce Bcl10 phosphorylation and MAPK activation. (PDF 64 kb)

Supplementary Fig. 8

Two types of 'CBM' complexes control activation signals through ITAM-receptors and TLRs in lymphoid and myeloid cells. (PDF 42 kb)

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Hara, H., Ishihara, C., Takeuchi, A. et al. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat Immunol 8, 619–629 (2007). https://doi.org/10.1038/ni1466

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