Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1β in salmonella-infected macrophages

Abstract

Gram-negative bacteria that replicate in the cytosol of mammalian macrophages can activate a signaling pathway leading to caspase-1 cleavage and secretion of interleukin 1β, a powerful host response factor. Ipaf, a cytosolic pattern-recognition receptor in the family of nucleotide-binding oligomerization domain–leucine-rich repeat proteins, is critical in such a response to salmonella infection, but the mechanism of how Ipaf is activated by the bacterium remains poorly understood. Here we demonstrate that salmonella strains either lacking flagellin or expressing mutant flagellin were deficient in activation of caspase-1 and in interleukin 1β secretion, although transcription factor NF-κB–dependent production of interleukin 6 or the chemokine MCP-1 was unimpaired. Delivery of flagellin to the macrophage cytosol induced Ipaf-dependent activation of caspase-1 that was independent of Toll-like receptor 5, required for recognition of extracellular flagellin. In macrophages made tolerant by previous exposure to lipopolysaccharide, which abrogates activation of NF-κB and mitogen-activated protein kinases, salmonella infection still activated caspase-1. Thus, detection of flagellin through Ipaf induces caspase-1 activation independently of Toll-like receptor 5 in salmonella-infected and lipopolysaccharide-tolerized macrophages.

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Figure 1: Exposure of bacterial components in the host cytosol is necessary and sufficient for secretion of IL-1β in macrophages.
Figure 2: Flagellin is required for IL-1β, but not IL-6 or MCP-1 secretion, in salmonella-infected macrophages.
Figure 3: Activation of caspase-1 in response to salmonella requires flagellin and Ipaf but is independent of TLR5.
Figure 4: The main salmonella flagellin gene required for IL-1β secretion, caspase-1 activation and early cell death in macrophages is fliC.
Figure 5: Delivery of flagellin to the cytosol is sufficient for caspase-1 activation in wild-type but not Ipaf-deficient macrophages.
Figure 6: Salmonella induces caspase-1 activation in the absence of activation of NF-κB, Erk and p38.
Figure 7: Salmonella potently induces IL-1β secretion that is dependent on flagellin and Ipaf in macrophages insensitive to TLR stimulation.

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Acknowledgements

We thank C. McDonald for critical review of the manuscript. Supported by the National Institutes of Health (R01 AI064748 and R01 AI063331; and 5/T32/HL007517 to T.-D.K.), Fondazione Italiana Ricerca sul Cancro (L.F.) and Fondation pour la Recherche Medicale (M.B.-M.).

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Correspondence to Gabriel Núñez.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Infection of macrophages with wild-type and mutant salmonella. (PDF 732 kb)

Supplementary Fig. 2

Generation of Ipaf KO mice. (PDF 1125 kb)

Supplementary Fig. 3

Detection of IL-1β (p17 subunit) in culture supernatants after infection with salmonella. (PDF 749 kb)

Supplementary Methods (PDF 41 kb)

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