Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2006 Jan 3;103(1):141-6.
doi: 10.1073/pnas.0504271103. Epub 2005 Dec 22.

Internalization and phagosome escape required for Francisella to induce human monocyte IL-1beta processing and release

Mikhail A Gavrilin  1 , Imad J Bouakl, Nina L Knatz, Michelle D Duncan, Mark W Hall, John S Gunn, Mark D Wewers
Affiliations
Comparative Study

Internalization and phagosome escape required for Francisella to induce human monocyte IL-1beta processing and release

Mikhail A Gavrilin et al. Proc Natl Acad Sci U S A. .

Abstract

Macrophage responses to Francisella infection have been characterized previously by subdued proinflammatory responses; however, these studies have generally focused on macrophage cell lines or monocyte-derived macrophages. Therefore, we studied the ability of fresh human blood monocytes to engulf and respond to Francisella by using the live vaccine strain variant and Francisella novicida. Because Francisella organisms have been reported to escape from the phagolysosome into the cytosol, we hypothesized that this escape may trigger the activation of caspase-1. Francisella tularensis variants were readily taken up by fresh human CD14(+) monocytes, inducing the release of IL-1beta, as well as IL-8, in a time- and dose-dependent fashion. Importantly, whereas live and dead Escherichia coli, F. novicida, and live vaccine strain, as well as the LPS of E. coli, were able to induce abundant IL-1beta mRNA synthesis and intracellular pro-IL-1beta production, only live Francisella induced enhanced IL-1beta processing and release (51 +/- 10 vs. 7.1 +/- 2.1 ng/ml, for F. novicida vs. E. coli LPS; P = 0.0032). Cytochalasin D blocked the Francisella internalization and the Francisella-induced monocyte IL-1beta processing and release but not that induced by the exogenous stimulus E. coli LPS. Also, killing bacteria did not block uptake but significantly diminished the IL-1beta processing and release that was induced by Francisella. Blocking bacterial escape from the phagosome into the cytosol also decreased IL-1beta but not IL-8 release. These findings demonstrate that Francisella organisms efficiently induce IL-1beta processing and release in fresh monocytes by means of a sensing system that requires the uptake of live bacteria capable of phagosome escape.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Comparison of F. novicida with F. tularensis–LVS for IL-1β and IL-8 release. Monocytes at 106 cells per ml in RPMI medium 1640 were cultured either alone or with F. novicida or F. tularensis–LVS at an moi of 100. Supernatants were harvested at 2, 8, and 24 h and analyzed for cytokines by immunoassay. Data for IL-1β and IL-8 are given as mean ± SEM for n = 9 independent donors. *, P < 0.01, for differences between the two organisms.
Fig. 2.
Fig. 2.
Requirement of live Francisella for induction of mature IL-1β release by human monocytes. (A) Monocytes were stimulated with live or killed F. tularensis–LVS (LVS), F. novicida (F.nov), or E. coli or the purified endotoxins of these organisms at 1 μg/ml and harvested at 8 h for mRNA. IL-1β mRNA was determined by real-time PCR expressed as RCN. (B) Supernatants of the samples from A were analyzed for IL-1β by immunoassay. (C) Monocyte lysates from the experiment were also analyzed for intracellular pro-IL-1β synthesis by immunoblotting using rabbit polyclonal antibody to IL-1β as detected by chemiluminescence. (D) Samples from A were also analyzed for IL-8 mRNA. (E) Supernatants of the samples from A were analyzed by immunoassay for IL-8. (F) To demonstrate the relative ability of mRNA to be translated and then subsequently released, the protein to mRNA ratio (ng/ml protein/RCN) is plotted for IL-1β and IL-8 for live F. novicida and for F. novicida killed by heating (95°C and 56°C) vs. PFM as compared with E. coli LPS. Experiments reflect an n = 4 for killed bacteria and bacterial endotoxin and n = 9 separate experiments for live F. tularensis.
Fig. 3.
Fig. 3.
Live and dead bacteria engulfed by monocytes. Transmission EM images of human monocytes infected with live (A) and heat-killed (B) F. novicida, both showing engulfment of the organisms. (C) E. coli can be seen within more well defined phagosomes. (D) cD inhibits F. novicida engulfment. The black arrow indicates F. novicida, the white arrow indicates mitochondria, the double arrow indicates E. coli, and the arrowhead indicates killed F. novicida.
Fig. 4.
Fig. 4.
Effect of Francisella internalization on IL-1β processing and release by monocytes. (A) IL-1β mRNA detection in monocytes by real-time PCR after stimulation with either F. novicida (F.nov) or E. coli LPS as affected by pretreatment with cD. (B) Effect of cD pretreatment on monocyte mature IL-1β release induced by either live F.nov or LPS. (Inset) LPS data enlarged. (C) Effect of cD on intracellular pro-IL-1β production as detected by immunoblotting of monocyte cell lysates prepared as described above. (D) Effect of cD on IL-8 mRNA. (E) Effect of cD on IL-8 release into media from monocytes stimulate as above. Results are the average of three experiments.
Fig. 5.
Fig. 5.
Preventing escape of Francisella from phagosome impairs IL-1β release. Monocytes (Mo) were incubated with F. novicida (F.nov) or mglA mutant in the presence or absence of 20 mM ammonium chloride (NH4Cl), which is a lysosomotropic agent that prevents bacteria from escaping the phagosome. At 8 h later, supernatant was collected and assayed for IL-1β and IL-8. *, P < 0.01; ***, P < 0.0001. (n = 5 for NH4Cl treatment and n = 11 for the rest of the experiments.)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akira, S. & Takeda, K. (2004) Nat. Rev. Immunol. 4, 499–511. - PubMed
    1. O'Connor, W., Jr., Harton, J. A., Zhu, X., Linhoff, M. W. & Ting, J. P. (2003) J. Immunol. 171, 6329–6333. - PubMed
    1. Harton, J. A., Linhoff, M. W., Zhang, J. & Ting, J. P. (2002) J. Immunol. 169, 4088–4093. - PubMed
    1. Anthony, L. D., Burke, R. D. & Nano, F. E. (1991) Infect. Immun. 59, 3291–3296. - PMC - PubMed
    1. Ellis, J., Oyston, P. C., Green, M. & Titball, R. W. (2002) Clin. Microbiol. Rev. 15, 631–646. - PMC - PubMed

Publication types

  NODES
INTERN 7
twitter 2