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Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation
Barbara Wegiel, … , Miguel P. Soares, Leo E. Otterbein
Barbara Wegiel, … , Miguel P. Soares, Leo E. Otterbein
Published October 8, 2014
Citation Information: J Clin Invest. 2014;124(11):4926-4940. https://doi.org/10.1172/JCI72853.
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Research Article Immunology

Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

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Abstract

Microbial clearance by eukaryotes relies on complex and coordinated processes that remain poorly understood. The gasotransmitter carbon monoxide (CO) is generated by the stress-responsive enzyme heme oxygenase-1 (HO-1, encoded by Hmox1), which is highly induced in macrophages in response to bacterial infection. HO-1 deficiency results in inadequate pathogen clearance, exaggerated tissue damage, and increased mortality. Here, we determined that macrophage-generated CO promotes ATP production and release by bacteria, which then activates the Nacht, LRR, and PYD domains-containing protein 3 (NALP3) inflammasome, intensifying bacterial killing. Bacterial killing defects in HO-1–deficient murine macrophages were restored by administration of CO. Moreover, increased CO levels enhanced the bacterial clearance capacity of human macrophages and WT murine macrophages. CO-dependent bacterial clearance required the NALP3 inflammasome, as CO did not increase bacterial killing in macrophages isolated from NALP3-deficient or caspase-1–deficient mice. IL-1β cleavage and secretion were impaired in HO-1–deficient macrophages, and CO-dependent processing of IL-1β required the presence of bacteria-derived ATP. We found that bacteria remained viable to generate and release ATP in response to CO. The ATP then bound to macrophage nucleotide P2 receptors, resulting in activation of the NALP3/IL-1β inflammasome to amplify bacterial phagocytosis by macrophages. Taken together, our results indicate that macrophage-derived CO permits efficient and coordinated regulation of the host innate response to invading microbes.

Authors

Barbara Wegiel, Rasmus Larsen, David Gallo, Beek Yoke Chin, Clair Harris, Praveen Mannam, Elzbieta Kaczmarek, Patty J. Lee, Brian S. Zuckerbraun, Richard Flavell, Miguel P. Soares, Leo E. Otterbein

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Figure 4

NALP3 is targeted by CO to enhance IL-1β expression and mediate macrophage bacteria killing.

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NALP3 is targeted by CO to enhance IL-1β expression and mediate macropha...
(A) CFU in WT (air), Nalp3–/–, and Casp1–/– BMDM supernatants infected with E. faecalis ± CO. *P < 0.01 versus air-treated controls. (B) Quantitation and representative images (inset) of cells positive for lysozyme (red) and E. faecalis in BMDMs from WT (air), Casp1–/–, and Nalp3–/– mice. Nuclei are stained blue. BMDMs were treated with E. faecalis for 1 hour followed by 4 hours of CO (white bars) or air (black bars). **P < 0.001, CO versus air; #P < 0.05, Nalp3–/– versus WT; &P < 0.05, Casp1–/– versus WT. Original magnification, ×40. (C) Bacterial counts in BMDM treated with supernatants ± E. faecalis for 1 hour and then treated ± anti–IL-1β or IL-1β recombinant protein. CO (white bars) or air (black bars) was applied as above. *P < 0.001, CO versus air; **P < 0.01, CO versus air. Results represent mean ± SD of 2 to 4 independent experiments in triplicate. (D) Flow cytometry in BMDMs treated with E. faecalis for 1 hour or BMDMs with antibody against ILRa-APC. IgG-APC as negative control. Mean ± SD from 3 independent experiments. *P < 0.05, E. faecalis versus control (C) or IgG. (E) IL-1β levels in E. faecalis–treated BMDM supernatants as above for 4 hours followed by 1 hour treatment with cathepsin B inhibitor, cytochalasin B, or apyrase. Cells were then exposed to CO or air for 4 hours. Results are presented as percentage of control to account for vehicle effects and represent mean ± SD of 3 independent experiments in duplicate. *P < 0.05, comparing CO versus air in both the vehicle-treated control group and the cathepsin B inhibitor-treated group.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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