Antioxidant Defenses of Francisella tularensis Modulate Macrophage Function and Production of Proinflammatory Cytokines

Abstract

Francisella tularensis, the causative agent of a fatal human disease known as tularemia, has been used in the bioweapon programs of several countries in the past, and now it is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress the host's innate immune responses. However, Francisella-encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate reactive oxygen species (ROS)/reactive nitrogen species as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens, and at low concentrations they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that the antioxidant defenses of F. tularensis maintain redox homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components that ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-κB signaling, and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth.

Keywords: bacterial pathogenesis; cytokine induction; immunosuppression; p38 MAPK; redox signaling.

FIGURE 1.

Live F. tularensis suppresses an ongoing inflammatory immune response induced by F. novicida. Primary BMDMs derived from C57BL/6 mice were infected with F. tularensis (Ft) LVS, F. novicida (Fn), UV-killed F. tularensis LVS (UV Ft LVS), UV-killed F. novicida (UV Fn), or a combination of these organisms at 100 m.o.i. (n = 3–5 biological replicates). The cell supernatants were collected at 24 h post-infection and analyzed for levels of TNF-α and IL-6. The data are representative of four independent experiments conducted with identical results. The data were analyzed by one-way ANOVA with Tukey-Kramer post-test, and a p value of 0.05 or less was considered significant.

FIGURE 2.

sodBC mutant of F. tularensis LVS has an impaired ability to suppress cytokine production. Raw264.7 macrophages were infected with F. novicida (Fn), wild type F. tularensis (Ft) LVS, or the sodBC mutants at 100 m.o.i. In co-infection experiments, 50 m.o.i. of each strain (total 100 m.o.i.) was used (n = 3 biological replicates). Cell supernatants were collected 24 h post-infection to measure TNF-α levels.

FIGURE 3.

sodBC mutant induces NADPH oxidase-dependent production of TNF-α and IL-6 in vivo. A, wild type C57BL/6 and phox^−/− mice were infected intranasally with 2 × 10³ cfu of the sodBC mutant or F. tularensis (Ft) LVS. On days 1, 5, and 7 post-infection, mice (n = 4 per group) were euthanized, and their excised lungs were homogenized. Bacterial burdens were quantified in their lungs. Bacterial counts are expressed as log₁₀ cfu. B, clear lung homogenates were used for quantification of TNF-α and IL-6 using cytometric bead array. The data are represented as mean ± S.D. The p values were determined using one-way ANOVA with Tukey-Kramer post-test, and a p value of 0.05 or less was considered significant. The data are representative of two independent experiments.

FIGURE 4.

Antioxidant enzymes of F. tularensis prevent accumulation of ROS in infected macrophages. Raw264.7 macrophages were infected with F. tularensis (Ft) LVS, sodBC mutant, or its transcomplement (sodBC+psodC) for 30 min and treated with redox-sensitive fluorescent dyes DCFDA (for total ROS, A) and DHE (for superoxide radicals, O₂^˙̄, B) and analyzed by flow cytometry. Hydrogen peroxide (100 μm) and paraquat (100 μm) were used as positive controls in A and B, respectively. The results are expressed as mean fluorescence intensity. The data are representative of 3–4 independent experiments and were analyzed by one-way ANOVA, and p values were calculated. A p value of 0.05 or less was considered significant. C, macrophages were infected with GFP-expressing bacteria and stained with Mitosox-Red to detect mROS 30 min post-infection. Arrowheads indicate infected macrophages. Magnification, ×63.

FIGURE 5.

Activation of MAPK signaling is observed in macrophages infected with the sodBC mutant. A, quantitation of phosphorylated p38, JNK, and ERK at the indicated times. ELISA was performed using Raw264.7 macrophages infected with F. tularensis (Ft) LVS, sodBC mutant, or the transcomplemented (sodBC+psodC) strain. The data are representative of two independent experiments and were analyzed using one-way ANOVA, and p values were calculated. B, Raw264.7 macrophages were infected with F. tularensis LVS, sodBC mutant, or the sodBC+psodC strain for 30 min, lysed, separated by SDS-PAGE, and immunoblotted with phosphorylated (P) and total p38, JNK, and ERK. β-Actin was used as a loading control. Lower panels show quantitation of P-p38, P-JNK, and P-ERK bands. Uninfected macrophages were used as controls. The data were analyzed using one-way ANOVA, and p values were calculated (n = 2–3 blots).

FIGURE 6.

Activation of MAPK signaling observed in macrophages infected with the sodBC mutant is dependent on NADPH oxidase-generated ROS. Quantitation of phosphorylated (Phospho) p38, JNK, and ERK at 30 min post-infection. ELISA was performed using wild type or phox^−/− BMDMs infected with F. tularensis (Ft) LVS, sodBC mutant, or the transcomplemented (sodBC+psodC) strain. The data are representative of two independent experiments (n = 3 replicates) and were analyzed using one-way ANOVA, and p values were calculated.

FIGURE 7.

ASK1 serves as a potential trigger for activation of MAPK signaling in macrophages infected with the sodBC mutant. BMDMs were infected with F. tularensis (Ft) LVS or sodBC mutant for 15 min, lysed, and separated by SDS-PAGE, and immunoblotted with phosphorylated (P)-ASK1 (Thr⁸⁴⁵) and total ASK1 antibodies, and bands were quantitated using ImageJ (right panel, n = 3 blots). The data were analyzed using one-way ANOVA, and p values were calculated.

FIGURE 8.

Infection of macrophages with the sodBC mutant results in an enhanced ROS-dependent activation of NF-κB. A, Raw264.7 macrophages were infected with F. tularensis LVS, sodBC mutant, or the sodBC+psodC strain for 20 min. The macrophages were either untreated or treated with 10 mm NAC. Immunofluorescence staining was performed to detect cellular localization of the p65 subunit of NF-κB (magnification ×63, red, p65; blue, nucleus). B, quantification of subcellular localization of p65 subunit is shown. At least 100 cells were counted manually in randomly chosen fields and expressed as percent macrophages with p65 staining. The data are representative of two independent experiments conducted. Lane 1, F. tularensis LVS; lane 2, sodBC mutant; lane 3, sodBC+psodC. C, BMDMs were infected with F. tularensis (Ft) LVS or the sodBC mutant in the absence or presence of ROS inhibitor NAC. The cell lysates were prepared at the indicated times and resolved by SDS-PAGE. Western blot analysis was performed using total IκB-α and phosphorylated (P)-IKB-α antibodies. β-Actin was used as a loading control. The data are representative of two independent experiments conducted. Arrows indicate cells with nuclear localization of p65 subunit of NF-κB.

FIGURE 9.

Infection of macrophages with sodBC mutant results in enhanced ROS-dependent autophagy. A, detection of autophagy in Raw264.7 macrophages infected with F. tularensis LVS, sodBC mutant, or the transcomplemented strain sodBC+psodC by immunofluorescence staining 24 h post-infection (magnification ×63, green, LC3; blue nucleus). B, quantitation of autophagosomes by counting the number of cells bearing LC3 punctae at 12 and 24 h post-infection. C, macrophages were treated with 3MA, an inhibitor of autophagy, and NAC, a ROS inhibitor and infected with F. tularensis LVS, sodBC mutant, or the transcomplemented strain sodBC+psodC. Quantification of autophagosomes was done by counting the cells showing LC3-positive autophagic vacuoles or the numbers of LC3 punctate dots. At least 200 cells were counted manually in randomly chosen fields for each treatment group. The data are representative of 2–3 independent experiments and were analyzed by one-way ANOVA, and p values were calculated.

FIGURE 10.

Targeting sodBC mutant though FcRγ results in an enhanced production of pro-inflammatory cytokines in HMDMs. HMDMs were infected with unopsonized F. tularensis (Ft) LVS or the sodBC mutant or opsonized with anti-F. tularensis LPS antibodies (5 μg/ml) (+IC) at an m.o.i. of 100 (A). HMDMs infected with opsonized F. tularensis LVS or the sodBC mutant (+IC) were also treated with ROS inhibitor apocynin (250 μm) (B). The culture supernatants collected at 24 h post-infection were analyzed for TNF-α and IL-6 levels. The data are representative of 2–3 independent experiments and were analyzed by one-way ANOVA, and p values were calculated.