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. 2011;6(6):e20609.
doi: 10.1371/journal.pone.0020609. Epub 2011 Jun 16.

TLR2 Signaling Contributes to Rapid Inflammasome Activation During F. Novicida Infection

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Free PMC article

TLR2 Signaling Contributes to Rapid Inflammasome Activation During F. Novicida Infection

Crystal L Jones et al. PLoS One. .
Free PMC article

Abstract

Background: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria.

Methodology/significant findings: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection.

Conclusions/significance: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TLR2 is required for rapid inflammasome activation in response to F. novicida infection.
(A) Bone marrow-derived macrophages (BMDM) from wild-type and TLR2−/− C57BL/6 mice were infected with wild-type F. novicida at an MOI of 100∶1 and supernatants were collected at 6–14 h post-infection for quantification of macrophage death as measured by LDH release. (B) Macrophages were infected with wild-type (WT) and mglA mutant strains of F. novicida (MglA) at the indicated MOIs for 6.5 h and supernatants were collected for quantification of cell death, and levels of (C) IL-1β and (D) IL-18. Data are representative of five independent experiments. Error bars represent the standard deviation of triplicate samples. *** p≤0.0007, ** p<0.0085.
Figure 2
Figure 2. TLR2 contributes to rapid inflammasome assembly during infection with F. novicida.
Wild-type and TLR2−/− BMDM were infected with F. novicida at an MOI of 100∶1 for 6 h. (A) Macrophages were stained for the presence of caspase-1 (green), ASC (red), DNA (blue) and actin (purple) and visualized by fluorescence microscopy. Stars show ASC foci and arrows point to ASC and caspase-1 colocalized foci. Inflammasome assembly was quantified by counting the number of macrophages containing (B) ASC foci or (C) ASC-caspase-1 colocalized foci. Error bars represent the standard error of the mean (SEM). Data are representative of two independent experiments, in which at least 200 macrophages were counted for each sample. *** p = 0.0008, ** p <0.0040, * p = 0.0167.
Figure 3
Figure 3. TLR2 is required for rapid activation of caspase-1 during F. novicida infection.
BMDM were infected with wild-type (WT) and mglA mutant strains of F. novicida (MglA) at an MOI of 100∶1 and lysed at 6 h post-infection. The levels of procaspase-1, mature caspase-1 (p20 subunit) and ASC in infected macrophages were measured by Western blot. β-actin was used as a loading control. Data are representative of three independent experiments.
Figure 4
Figure 4. MyD88 is involved in rapid inflammasome activation in response to F. novicida infection.
BMDM from wild-type and MyD88−/− C57BL/6 mice were infected with wild-type (WT) and mglA mutant strains of F. novicida (MglA) at the indicated MOIs. At 6.5 h post-infection, (A) death of the infected macrophages was measured by LDH release and (B) the concentration of IL-18 in the supernatants was quantified by ELISA. Data are representative of four independent experiments. Error bars represent the standard deviation of triplicate samples. ** p<0.0090.
Figure 5
Figure 5. NF-κB activation contributes to rapid inflammasome activation during F. novicida infection.
Macrophages were left untreated or treated with 20 µM of CAPE (NF-κB inhibitor) for 2 h, then infected with F. novicida at an MOI of 100∶1. Supernatants were collected for measurement of cell death by quantifying LDH release (A) and IL-18 secretion (B) at 6.5 h post-infection. Data are representative of four independent experiments. Error bars represent the standard deviation of triplicate samples. *** p≤0.0004, ** p = 0.0074.
Figure 6
Figure 6. The P2X7 receptor is not required for F. novicida-dependent inflammasome activation.
BMDMs from wild-type, TLR2−/− and P2X7R−/− mice were infected with wild-type F. novicida at the indicated MOIs. At 7 h post-infection, host cell death was measured by quantifying the amount of LDH released into the supernatant. Data are representative of three independent experiments. Error bars represent the standard deviation of triplicate samples. *** p≤0.0003, * p = 0.0319.
Figure 7
Figure 7. TLR2 plays a role in inflammasome activation in vivo.
Wild-type and TLR2−/− mice were left uninfected (Un) or infected intraperitoneally with 2.4×106 CFU of wild-type F. novicida (F.n.) and livers were harvested at 4 h post-infection. (A) Cell death in the liver was visualized by TUNEL staining, and (B) the number of TUNEL positive cells per field was quantified. (C) The bacterial burden in the liver of infected mice was determined by counting colony-forming units. (D) Sections were stained for presence of active caspase-1 (p20) in the livers of uninfected and F. novicida-infected mice, and (E) the level of caspase-1 activation was quantified by measuring the total fluorescence intensity emitted by the FITC-labeled p20 subunit of capase-1. (F) The amount of IL-18 present in the serum of F. novicida-infected mice was measured by ELISA. Data are representative of three independent experiments. Error bars represent the standard deviation of triplicate samples. ** p = 0.0030, * p≤0.0370.

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