Article Figures & Data
Figures
- FIGURE 1.
Differential induction of proinflammatory cytokines by attenuated and virulent strains of F. tularensis. Primary hDC were infected at a multiplicity of infection of 50 with the indicated strains of F. tularensis. A, Intracellular bacteria were enumerated at the indicated times postinfection. *p < 0.01, compared with SchuS4-infected hDC. B, Supernatants were harvested from uninfected or F. tularensis-infected cultures at the indicated time points postinfection and analyzed for TNF-α, IL-6, and IL-12p40 by ELISA. hDC stimulated 24 h prior to harvest with ultrapure E. coli LPS (10 ng/ml) served as positive controls. *p < 0.01, compared with uninfected and SchuS4-infected hDC; **p < 0.001, compared with all samples. C, SchuS4- or mock-infected hDC cultures were stimulated 24 h postinfection with ultrapure E. coli LPS. Concentrations of TNF-α, IL-6, and IL-12p40 in culture supernatants were determined an additional 24 h after LPS treatment. *p < 0.01, compared with uninfected, LPS-treated samples. Error bars represent SEM. Each data point represents the mean of triplicate samples. Data in A are the mean of eight experiments; data in B and C are representative of three experiments of similar design.
- FIGURE 2.
Induction of IFN-β by virulent F. tularensis. A, hDC were infected with SchuS4. RNA was harvested at the indicated time points postinfection for analysis of host genes by qRT-PCR. The fold change of TNF-α, IL-1β, IL-10, TOLLIP, IFN-α, IFN-β, and IFN-γ, normalized to uninfected samples, are depicted. B, RNA from hDC infected with F. tularensis strain SchuS4 or LVS was harvested at the indicated time points postinfection for analysis of IFN-β by qRT-PCR. IFN-β transcript levels were normalized to those from uninfected hDC. The intracellular bacterial loads at the indicated time points are shown. *p < 0.01, compared with LVS-infected cells at each time point. C, hDC were infected with SchuS4 or LVS, and culture supernatants were assessed for IFN-β by ELISA 12 h postinfection. Uninfected hDC served as negative controls. *p < 0.05, compared with LVS and uninfected samples. Error bars represent SEM. Each data point represents the mean of triplicate samples. Data are representative of three experiments of similar design.
- FIGURE 3.
Requirements for SchuS4-mediated IFN-β production in hDC. One hour prior to infection, hDC were treated with cytochalasin D to inhibit phagocytosis (A) or BAF A to inhibit endosomal acidification (B). Eight hours postinfection, RNA was extracted for analysis by qRT-PCR. RNA collected from hDC treated with LPS served as a positive control. IFN-β transcript levels were normalized to those from mock-infected cells. *p < 0.01, compared with untreated, SchuS4-infected hDC; **p < 0.01, compared with hDC treated with LPS in the absence of inhibitors. C, hDC were exposed to SchuS4 that had been killed with 2% PFA (SchuS4+PFA). Eight hours postinfection, RNA was extracted for analysis of IFN-β transcripts by qRT-PCR. *p < 0.01, compared with PFA-killed SchuS4. Error bars represent SEM. Each data point represents the mean of triplicate samples. Data are representative of three experiments of similar design.
- FIGURE 4.
Endosomal escape and early cytosolic replication are not sufficient for SchuS4-mediated induction of IFN-β. hDC were infected with the indicated SchuS4 strains. A, Intracellular bacteria were enumerated at the indicated times points postinfection. B, Cytoplasmic bacteria were identified 3 h postinfection using a phagosomal-integrity assay and were enumerated by microscopy. C, RNA was harvested 8 h postinfection for assessment of IFN-β transcript by qRT-PCR. IFN-β transcript levels were normalized to those from uninfected hDC. *p < 0.05, compared with all other samples. Error bars represent SEM. Each data point represents the mean of triplicate samples. A and C represent the mean of five independent experiments, and data in B are representative of three experiments of similar design.
- FIGURE 5.
IFN-β is not correlated with activation of the inflammasome following F. tularensis infection of hDC. A and B, hDC were mock infected or infected with the indicated strains of F. tularensis or treated with E. coli LPS with or without pretreatment with rhIFN-β. A, Forty-eight hours postinfection, supernatants were collected and analyzed for IL-1β by ELISA. *p < 0.0001, compared with all other samples. B, Eight hours postinfection, intracellular pro–IL-1β was detected in hDC lysates by Western blotting. Blots were stripped and reprobed with anti–β-actin to demonstrate equal loading. C, hDC were mock infected or infected with F. tularensis SchuS4 or LVS. At the indicated time points postinfection, the number of hDC positive for activated caspase-1 was detected by flow cytometry using Green FLICA Caspase-1 assay kit. *p < 0.05, compared with all other samples; **p < 0.01, compared with all other samples. Error bars represent SEM. Each data point represents the mean of triplicate samples. Data are representative of three experiments of similar design.
- FIGURE 6.
SchuS4-induced IFN-β selectively inhibits IL-12p40. A, hDC were treated with PBS (−) or the indicated concentration of rhIFN-β for 16 h prior to exposure to E. coli LPS. Supernatants were harvested 24 h later and examined for IL-12p40 and TNF-α by ELISA. *p < 0.01, compared with cells treated with LPS alone. B, hDC were infected with SchuS4 in the presence of neutralizing polyclonal anti–IFN-β Ab or polyclonal rabbit IgG (isotype) control. Sixteen hours later, infected or mock-infected hDC were treated with ultrapure E. coli LPS and assessed for intracellular IL-12p40 and TNF-α by flow cytometry. Data were normalized by defining the percentage of cells in an uninfected culture of hDC that respond to LPS by production of cytokine as 100%. C, hDC were infected with SchuS4 in the presence of neutralizing monoclonal anti–IFN-β Ab or mouse IgG1 (isotype) control. Sixteen hours later, infected or mock-infected hDC were treated with ultrapure E. coli LPS. Sixteen hours later, culture supernatants were assessed for IL-12p40 and TNF-α by ELISA. Data were normalized by defining the concentration of cytokine secretion in an uninfected culture of hDC that respond to LPS by production of cytokine as 100%. D, hDC were infected with the indicated SchuS4 strains. Uninfected hDC served as negative controls. Supernatants were harvested 48 h postinfection and analyzed for IL-12p40 and TNF-α by ELISA. *p < 0.05, compared with all other samples. E, hDC were treated with rhIFN-β and infected with LVS. Twenty-four hours postinfection, supernatants were assessed for IL-12p40 by ELISA. Uninfected hDC served as negative controls. *p < 0.01, compared with untreated, LVS-infected hDC. F, hDC were primed with rhIFN-β, followed by infection with the designated strains of F. tularensis. Intracellular bacteria were enumerated at 3 and 24 h postinfection. *p < 0.001, compared with all other samples. Each data point represents the mean of triplicate samples. Error bars represent SEM. Data are representative of three experiments of similar design. ns, not significant.
Additional Files
Data Supplement
Files in this Data Supplement:
- Supplemental Figures 1-3 and Supplemental Table 1 (PDF, 205 Kb) -
Description:
Figure S1. SchuS4 infected hDC increase expression of IRF7.
Figure S2. SchuS4 and SchuS4 Δ0369c escape the endosome at a similar rate.
Figure S3. RNA from hDC infected with F. tularensis strain SchuS4 or SchuS4Δ0369c was harvested at the indicated time points after infection...
Supplemental Table 1. Gene Accession Numbers.
- Supplemental Figures 1-3 and Supplemental Table 1 (PDF, 205 Kb) -
Description:
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