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Science 339 (6122): 975-978

Copyright © 2013 by the American Association for the Advancement of Science

Caspase-11 Protects Against Bacteria That Escape the Vacuole

Youssef Aachoui1,2,{dagger}, Irina A. Leaf3,{dagger}, Jon A. Hagar1,2,{dagger}, Mary F. Fontana4,*, Cristine G. Campos1, Daniel E. Zak3, Michael H. Tan4, Peggy A. Cotter1, Russell E. Vance4, Alan Aderem3, and Edward A. Miao1,2,{ddagger}

1 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
3 Seattle Biomedical Research Institute, Seattle, WA 98109, USA.
4 Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, Berkeley, CA 94720, USA.


Figure 1
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Fig. 1. Burkholderia detection and protection conferred by Casp1/11 is independent of all known canonical inflammasomes. LPS-primed BMMs were infected with B. pseudomallei (A and G) or B. thailandensis (B and F) for 4 hours. (A and B) IL-1β secretion was determined by enzyme-linked immunosorbent assay (ELISA), or (F and G) cytotoxicity was determined by lactate dehydrogenase (LDH) release assay. (C, D, and E) Survival curves of wild-type C57BL/6 or the indicated knockout mice infected with 2 x 107 colony-forming units (cfu) i.p. with B. thailandensis. Days p.i., days postinfection. Data are representative of at least three (A), (B), (F), and (G) or of two (D) and (E) experiments. (C) Data are pooled from three experiments. For number of mice in each panel, see table S2. Statistically significant differences with respect to controls are indicated (Student's t test or log-rank test for survival; *P ≤ 0.05).

 

Figure 2
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Fig. 2. Diverse cytosolic bacteria activate pyroptosis independent of NLRC4, NLRP3, and ASC. (A) LPS-primed BMMs were infected for 4 hours with either B. thailandensis or the indicated mutants, and cytotoxicity was determined. BMMs were infected for 8 hours with (B) S. typhimurium or S. typhimurium {triangleup}sifA or (D) S. typhimurium {triangleup}sifA or S. typhimurium {triangleup}sifA flgB, and cytotoxicity was determined. WT, wild type. LPS-primed BMMs were infected for 8 hours with (C) S. typhimurium or S. typhimurium {triangleup}sifA or (E) S. typhimurium {triangleup}sifA or S. typhimurium {triangleup}sifA flgB, and IL-1β secretion was determined. (F and G) Cytotoxicity in wild-type or Asc–/– BMMs infected for 4 hours with L. pneumophila, L. pneumophila {triangleup}flaA, or L. pneumophila {triangleup}sdhA {triangleup}flaA. Cytotoxicity was determined by LDH release and IL-1β secretion by ELISA. Data are representative of at least three experiments. Statistically significant differences with respect to controls are indicated (Student's t test; *P ≤ 0.05; n.s., P > 0.05).

 

Figure 3
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Fig. 3. Caspase-11 mediates pyroptosis after infection by cytosolic bacteria. Macrophage cytotoxicity and IL-1β secretion were determined after infection with S. typhimurium {Delta}sifA (8 hours), L. pneumophila {Delta}flaA {Delta}sdhA (4 hours), or B. thailandensis (4 hours). (A) C57BL/6, Casp1–/–Casp11–/–, Tlr4–/–, Trif–/–, and Myd88–/– BMMs infected with S. typhimurium {triangleup}sifA with or without IFN-{gamma} priming before infection. (B and C) Retroviral transduction was used to complement Casp1 or Casp11 in Casp1–/–Casp11–/– immortalized BMMs. Macrophages were primed with LPS (B) or IFN-{gamma} (C), and responses to B. thailandensis (B) or S. typhimurium {Delta}sifA (C) infection were examined. (D) Control or complemented Casp1–/–Casp11–/– BMMs infected with L. pneumophila {Delta}flaA {Delta}sdhA. (E and F) Retroviral transduction was used to introduce control or Casp11-targeting of shRNAmir into Nlrc4–/–Asc–/– immortalized BMMs. Macrophages were primed overnight with LPS (E) or IFN-{gamma} (F) and then infected as indicated. (G to I) C57BL/6, Casp1–/–Casp11–/–, and Casp11–/– BMMs infected with B. thailandensis (G), S. typhimurium {Delta}sifA (H), or L. pneumophila {Delta}flaA {Delta}sdhA (I). Data are representative of at least three (A to C), (E), (G), and (H) or of two (D), (F), and (I) independent experiments. Statistically significant differences with respect to controls are indicated (Student's t test; *P ≤ 0.05). nd, none detected.

 

Figure 4
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Fig. 4. Caspase-11 protects against cytosolic bacteria in vivo. (A and B) S. typhimurium or S. typhimurium {triangleup}sifA were injected i.p. into C57BL/6 (1000 cfu) or Casp1–/–Casp11–/– mice (250 cfu), and survival was monitored. (C to E) The indicated mice were infected with 5 x 104 cfu of both wild-type S. typhimurium and S. typhimurium {triangleup}sifA marked with ampicillin or kanamycin resistance, respectively. Bacterial loads from three to four mice per genotype were determined 48 hours later, and the competitive index was calculated [CI = log(S. typhimurium {triangleup}sifA/S. typhimurium)]. A CI of –1 corresponds to 10 cfu of S. typhimurium for every 1 cfu of S. typhimurium {triangleup}sifA. (F and G) C57BL/6, Casp1–/–Casp11–/–, or Casp11–/– mice were infected with (F) 2 x 107 cfu mouse passaged B. thailandensis i.p. or (G) 100 cfu B. pseudomallei i.n. (A), (B), (F), and (G) Data are pooled from two independent experiments. (C to E) Representative of two experiments. For the number of mice in each panel, see table S2. Statistically significant differences with respect to controls are indicated (Student's t test or log-rank test for survival; *P ≤ 0.05; n.s., P > 0.05).

 


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