Interaction of Interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular Burkholderia pseudomallei

Abstract

Burkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-γ) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-γ- and ceftazidime-mediated killing, which led to our hypothesis that IFN-γ-induced ROS interacted with ceftazidime to synergistically kill Burkholderia bacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-γ to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-γ induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 μg/ml. We investigated the exact role of IFN-γ-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated with Burkholderia bacteria in IFN-γ-treated macrophages, which led to our finding that IFN-γ-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-γ-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity against Burkholderia bacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria.

FIG 1

Ceftazidime and IFN-γ induce synergistic killing of intracellular bacteria in macrophages. Adherent RAW 264.7 cells (A) or primary bone marrow macrophages from C57BL/6 mice (B), BALB/c mice (C), or ICR mice (D) were infected with B. thailandensis and treated with ceftazidime (10 μg/ml for RAW 264.7 cells, 3 μg/ml for bone marrow macrophages), IFN-γ (10 ng/ml), or the combination of ceftazidime and IFN-γ for 18 h. Intracellular bacteria were then enumerated by plating macrophage lysates. Synergistic interactions between ceftazidime and IFN-γ were determined by two-way ANOVA (*, P < 0.01; **, P < 0.001; ***, P < 0.0001). Graphs are representative of data from two independent experiments with treatment groups run in triplicate.

FIG 2

ROS pathway inhibitors reverse IFN-γ and ceftazidime synergy. NAC (A) and GSH (D) reverse the intracellular killing effect of IFN-γ (10 ng/ml) and ceftazidime (10 μg/ml) combination therapy on B. thailandensis-infected macrophages in a dose-dependent manner. Statistical differences compared to the group treated with IFN-γ and ceftazidime were assessed by one-way ANOVA with Tukey's posttest (*, P < 0.05). (B) Mean fluorescent intensity of mBCl after treatment of uninfected RAW cells with NAC (20 mM) for 2 h (***, P = 0.0004); (C) intracellular ROS levels (as detected by carboxy-H2DCFDA) in RAW 264.7 cells after 10 h of treatment with ceftazidime and IFN-γ, with or without NAC (20 mM); (E and F) histogram overlays of intracellular ROS responses as measured with carboxy-H2DCFDA by flow cytometry; (E) IFN-γ-elicited ROS responses from uninfected RAW 264.7 macrophages, various hours after stimulation; (F) intracellular ROS responses of B. thailandensis-infected RAW 264.7 macrophages 18 h after treatment with ceftazidime (10 μg/ml) or IFN-γ (10 ng/ml). Results are representative of data from at least two independent experiments.

FIG 3

Glutathione depletion enhances antibiotic killing and induces intracellular ROS. (A) RAW 264.7 macrophages were infected with B. thailandensis and treated with ceftazidime (10 μg/ml), IFN-γ (10 ng/ml), or the combination of ceftazidime and IFN-γ for 18 h with or without BSO treatment (5 mM). In order to maximally deplete intracellular GSH, all BSO groups also received BSO treatment 18 to 24 h prior to infection. Intracellular bacteria were then enumerated by plating macrophage lysates. Significant differences between controls and BSO-treated groups were determined by two-way ANOVA (*, P < 0.05). (B) Histogram overlays of intracellular ROS after macrophage infection with B. thailandensis and treatment with BSO (5 mM) or the combination of IFN-γ and BSO for 12 h as measured by carboxy-H2DCFDA and flow cytometry; (C) mean fluorescent intensity of intracellular mBCl, indicating GSH content, after 18 h of treatment with 5 mM BSO (**, P = 0.0079). All data are representative of results from at least two independent experiments.

FIG 4

IFN-γ activation of macrophages kills intracellular bacteria. RAW 264.7 macrophages were preactivated with IFN-γ (10 ng/ml) for 18 h prior to infection with B. thailandensis. At the indicated times, lysates were plated to enumerate surviving intracellular bacteria. (A) Time course of intracellular killing due to preactivation with IFN-γ prior to infection. The difference in intracellular bacterial numbers at time t = 0 between untreated and preactivated macrophages was likely due to the time lapse between the initial start of infection and the end of the kanamycin treatment step. Significant differences compared to the untreated control were assessed at each time point by two-way ANOVA (***, P < 0.001). (B) Intracellular bacterial burden after 6 h of infection in macrophages pretreated with IFN-γ (as before) or with ceftazidime (10 μg/ml). Statistical differences were assessed by one-way ANOVA, a > b (P < 0.0001). (C) NAC (50 mM) or GSH (50 mM) were applied to macrophages for the last 3 h of the pretreatment regimen. Then all treatments were washed off and macrophages were infected with B. thailandensis. Intracellular bacterial burden was assessed 6 h after the end of the kanamycin step by plating lysates, a > b > c > d (P < 0.05). Data are representative of results from two independent experiments run in triplicate.

FIG 5

B. thailandensis fails to form actin tails inside IFN-γ-treated macrophages. RAW cells were infected with B. thailandensis and not treated (A) or treated with ceftazidime (10 μg/ml) (B), IFN-γ (10 ng/ml) (C), or ceftazidime and IFN-γ (D) for 12 h. Macrophages were then fixed, permeabilized, and stained with phalloidin to identify host cell actin (green), DAPI to stain nuclei (blue), and anti-Burkholderia serum followed by secondary antibody conjugated to Cy3 to identify B. thailandensis (red). Images were captured under ×40 magnification. Actin tails are seen as bright green protrusions from the red bacteria in panels A and B. Images are representative of data from three independent experiments.

FIG 6

Burkholderia bacteria inside IFN-γ-treated macrophages have higher proportions of LAMP-1⁺ colocalization than the untreated control. Quantitation of B. thailandensis colocalization with LAMP-1 antibody after 8 h of IFN-γ treatment of infected macrophages. Data are presented as the ratio of colocalized bacteria (LAMP-1⁺) to total bacteria per field of view. Data represent 10 fields of view for each treatment group (***, P < 0.0001), and results are representative of data from 3 independent experiments.

FIG 7

Ceftazidime controls primarily extracellular bacterial burden. (A) RAW 264.7 macrophages were infected with B. thailandensis and treated for 24 h with IFN-γ (10 ng/ml), ceftazidime (10 μg/ml), or the combination of IFN-γ and ceftazidime. Extracellular bacterial burden was assessed at 0, 6, 10, and 24 h posttreatment by plating serial dilutions of well supernatants. Significant differences were assessed at all time points by two-way repeated-measures ANOVA and compared to the untreated control (*, P < 0.05; ***, P < 0.001). (B and C) B. thailandensis was treated with ceftazidime (10 μg/ml), IFN-γ (10 ng/ml), or the combination of both treatments for 18 h in the absence of macrophages. Surviving bacteria were enumerated by plating dilutions of remaining bacteria in wells after 18 h of treatments. Significant differences were assessed by one-way ANOVA, a > b > c > d (P < 0.0001). All graphs are representative of results from two independent experiments with treatment groups run in triplicate.

FIG 8

Compartmentalized killing describes the mechanism of synergy between IFN-γ and ceftazidime. We propose that during the macrophage infection assay, there is a dynamic exchange between bacteria in the intracellular and extracellular compartments. Therefore, it is only with IFN-γ control of intracellular spread and replication, combined with ceftazidime control of extracellular bacterial burden, that synergy is achieved with low bacterial burden in the system as a whole.