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CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation

Abstract

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors1. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor κB, activation protein 1 and interferon regulatory factors2. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists3,4,5,6. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca2+/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cγ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity7,8. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure9,10, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

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Figure 1: CD14-dependent Ca 2+ mobilization in dendritic cells following LPS treatment.
Figure 2: CD14-dependent activation of NFAT in BMDCs after LPS treatment.
Figure 3: Ca 2+ -NFAT-mediated regulation of dendritic cell death.
Figure 4: Ca 2+ mobilization and survival of macrophages after LPS treatment.

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Gene Expression Omnibus

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All microarray data are available from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession code GSE15759.

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Acknowledgements

We thank S. Akira, C. Kirschning and K. Miyake for mutant mice; M. Colonna for mutant mice and advice on PLC-γ2 activation; E. Serfling for mutant mice and advice on NFAT activation; A. Rao for the VIVIT peptide. We also thank Genopolis–Consorzio di Genomica Funzionale for microarray hybridization and data analysis and K. Mahnke for the anti-DEC205–OVA conjugate. This work was supported by grants from the CARIPLO Foundation, the European Commission 6th Framework Program (MUGEN and DC-THERA contracts), the European Commission 7th Framework Program (TOLERAGE and ENCITE contracts), the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the and the Italian Ministry of Education and Research (COFIN).

Author Contributions F.G. conceived and oversaw the project and wrote the paper; I.Z. conceived the research and conducted most of the experiments with R.O.; M.C., M.C., M.R., G.C., B.C. and A.Z. helped with calcium experiments; G.C. and F.M. helped with experiments on D1 cells; M.F. helped with quantitative real-time PCR; A.E.R. performed the EMSA experiments; P.R.-C. provided advice.

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Correspondence to Francesca Granucci.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S14, Supplementary Tables 1-2 and Supplementary References. (PDF 3307 kb)

Supplementary Movie 1

Movie BMDC_ATP shows Ca2+ mobilization in BMDCs after steady-state superfusion with ATP. (MOV 2391 kb)

Supplementary Movie 2

Movie BMDC_LPS shows Ca2+ mobilization in BMDCs after steady-state superfusion with LPS. (MOV 1823 kb)

Supplementary Movie 3

Movie DI_ATP shows Ca2+ mobilization in D1 cells after steady-state superfusion with ATP. (MOV 3312 kb)

Supplementary Movie 4

Movie DI_LPS shows Ca2+ mobilization in D1 cells after steady-state superfusion with LPS. (MOV 2042 kb)

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Zanoni, I., Ostuni, R., Capuano, G. et al. CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Nature 460, 264–268 (2009). https://doi.org/10.1038/nature08118

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