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. 2006 Apr 17;203(4):1093-104.
doi: 10.1084/jem.20051659. Epub 2006 Apr 10.

Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection

Ari B Molofsky  1 , Brenda G Byrne, Natalie N Whitfield, Cressida A Madigan, Etsu T Fuse, Kazuhiro Tateda, Michele S Swanson
Affiliations

Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection

Ari B Molofsky et al. J Exp Med. .

Abstract

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1beta secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

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Figures

Figure 1.
Figure 1.
L. pneumophila flagellin contributed to macrophage death but not pore formation. (A and B) After centrifugation with twofold dilutions of the strains indicated, A/J mouse macrophages and microbes were incubated for 1 h, and viability was determined by Alamar blue reduction. Mean percent viable macrophages ± SEM (error bars) are shown that were pooled from three or more experiments in MOI bins of twofold dilutions; the middle value for each bin is indicated. To facilitate comparisons between strains, the WT and dotA values are displayed in both A and B. (C) To quantify red blood cell lysis after incubation for 1 h with the microbes at each MOI indicated, soluble hemoglobin was measured spectrophotometrically. E, exponential phase (nonmotile); PE, postexponential phase (motile); acid, bacteria washed with acid to remove flagella; pFlaA and pMobAB, complementation plasmids carried by strains and described previously (20).
Figure 2.
Figure 2.
Flagellin+L. pneumophila induced death by a mechanism independent of MyD88 but sensitive to Naip5. (A) To test whether TLR signaling induces death, the viability of C57BL/6 (solid lines, BL/6) or C57BL/6 myD88 −/− macrophages (dashed lines) was determined after infection for 1 h as shown and described in Fig. 1. (B) To test whether TLR signaling is required to restrict L. pneumophila growth, A/J, C57BL/6 (BL/6), or C57BL/6 myD88 −/− macrophages (myD88) were infected for the periods shown, and bacterial yield was determined by enumerating CFU. (C and D) To test whether Naip5 contributes to the host response, mean percent (± SEM) LDH released from naip5 A/J (B) or Naip5+ C57BL/6 (C) macrophages was quantified 1 h after the infections indicated in three or more experiments performed in triplicate, pooling results into MOI bins of twofold dilutions. E, exponential phase (nonmotile); PE, postexponential phase (motile); acid, bacteria washed with acid to remove flagella.
Figure 3.
Figure 3.
When present with a pore-forming activity, flagellin triggered macrophage death. (A) To test whether pore formation or substrates of the type IV secretion system are required for flagellin to stimulate death, LDH released by C57BL/6 cells incubated for 1 h with WT L. monocytogenes (Lm; constant MOI of 25) or dotA or flaA mutant L. pneumophila either alone or mixed (+) was quantified. To test whether cytosolic flagellin is toxic to macrophages, C57BL/6 macrophages were incubated with or without 1 ug/ml of the pore-forming toxin LLO for 2 h after an initial centrifugation with heat-treated or native CFPs (∼300 ng flagellin; B) or heat-treated flagellin (∼3 ng) that had been affinity purified and affixed to beads via a flagellin-specific monoclonal antibody (C). (D) To test whether macrophages responded to cytosolic flagellin from other microbes, C57BL/6 macrophages were incubated for 2 h without (black bars) or with 1 ug/ml LLO (gray bars) and 1.25 μg of either heated crude flagellin from L. pneumophila (Lp) or commercial preparations of S. typhimurium (St) or B. subtilis (Bs) flagellin. Means ± SD (error bars) of the percent total LDH released from permeable macrophages are shown, which were calculated from one experiment that is representative of at least two performed.
Figure 4.
Figure 4.
Pyroptosis was induced by pore-forming Flagellin+L. pneumophila. (A) After infection at an MOI of <1.0 for 1 h with WT L. pneumophila–expressing GFP, A/J macrophage permeability was analyzed by phase (left) and fluorescence microscopy (right). Arrows indicate single L. pneumophila; arrowheads indicate infected cells that have permeable membranes and phase dark condensed nuclei. (B) After infection with an MOI of 50–100 for 1 h as shown, mean percentages ± SD (error bars) of A/J macrophages that were permeable (black bars) or contained phase dark condensed nuclei (gray bars) were calculated from three or more independent experiments. (C) After infection at an MOI of <1.0 for 2 h as shown, mean percentages ± SD of A/J macrophages containing one bacterium that had phase dark nuclei was determined. (D) After infecting C57BL/6 macrophages for 1 h at an MOI of 30–60 as shown, mean percentages ± SD of LDH release was calculated from two to three experiments. (E and F) After infecting the macrophages shown for 1 h as indicated, secreted IL-1β was quantified. Results from one experiment representative of two to three others are shown. Where indicated, macrophages were treated for 1 h before and during the infection with 100 μM of inhibitors of caspase 1 (Ac-YVAD-cmk), pancaspases (Z-VAD-fmk), or caspase 3 (Ac-DEVD-cho). t test (*, P < 0.05) indicates significant differences ± caspase inhibitors. BMM, bone marrow–derived macrophage.
Figure 5.
Figure 5.
Naip5+ C57BL/6 macrophages restricted the growth of L. pneumophila that encode flagellin, in part, by degrading the intracellular progeny. Growth of the L. pneumophila strain shown in macrophages of permissive A/J mice (A) or restrictive C57BL/6 mice (B) was quantified in three or more experiments; representative data are shown. (C) The macrophages indicated were infected for 2 h with an MOI of <1 of WT or flaA mutant L. pneumophila, and at 24 or 48 h, the integrity of L. pneumophila and macrophages was analyzed by immunofluorescence (left) and phase-contrast (right) microscopy, respectively. BMM, bone marrow–derived macrophage.
Figure 6.
Figure 6.
Naip5+ C57BL/6 mice restricted the growth of L. pneumophila that encode flagellin. C57BL/6 and A/J mice were infected via the trachea with L. pneumophila Lp01 or its flaA-deficient mutant, and the lung bacterial burden was quantified 1, 2, and 3 d later. Mean CFUs ± SD (error bars) are shown, each calculated from five animals.
Figure 7.
Figure 7.
Model for the induction of a caspase 1– and Naip5-dependent mouse macrophage innate immune response to cytosolic L. pneumophila flagellin. NLR, NOD-like receptor.
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