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B Activation and Host Defense in Pseudomonas Pneumonia1
* Department of Veterans Affairs and Division of Allergy, Pulmonary and Critical Care,
Department of Cancer Biology,
Division of Infectious Diseases, and
Department of Biomedical Engineering, Vanderbilt University School of Medicine, Nashville, TN 37232;
¶ Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; and
|| Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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B and host defense against Pseudomonas aeruginosa pneumonia. Using mice with an NF-B-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P. aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-B. To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-B, we crossbred mice deficient in p47phox with NF-B reporter mice (p47phox-/-HLL). These p47phox-/-HLL mice were unable to activate NF-B to the same degree as HLL mice with intact NADPH oxidase following P. aeruginosa infection. In addition, lung TNF-
levels were significantly lower in p47phox-/-HLL mice compared with HLL mice. Bacterial clearance was impaired in p47phox-/-HLL mice. In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-B activation following treatment with P. aeruginosa. Additional studies with macrophages from p47phox-/- mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-B activation in this model. These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-B pathway. |
Introduction |
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Vital functions of phagocytic cells include microbicidal and signaling capabilities. Microbicidal effects are thought to be mediated primarily by the generation of reactive oxygen species (ROS)3 and reactive nitrogen species, although they may play a stronger signaling role than previously suspected. Intracellular signaling is critical in facilitating the shift from resting state to an activated state upon encounter with a microbe. Following exposure to bacterial components, activation of NF-B via Toll-like receptors (TLRs) is required in macrophages and other cell types to synthesize protein mediators that are critical for host defense (3, 4).
NADPH oxidase is a multicomponent enzyme localized in the plasma membrane of phagocytic leukocytes and is a major oxidant-generating enzyme. It accepts electrons from NADPH and donates these to molecular oxygen to produce superoxide (5). In recent years, it has been increasingly evident that ROS serve as second messengers and activate signaling pathways that result in a broad array of physiological responses that range from cell proliferation to gene expression and apoptosis (6, 7). It is possible that the free radicals generated by NADPH oxidase contribute to host defenses not only through their microbicidal action but also through modulation of redox-sensitive pathways in phagocytes.
NF-B is an important intracellular signaling pathway for both innate and acquired immunity. The NF-B family of transcription factors impacts host defense against infectious agents by inducing the expression of inflammatory genes. Target genes that are transcriptionally regulated by NF-B and play an important role in host defense include the following: 1) proinflammatory cytokines such as TNF- and IL-1
, 2) chemokines such as KC and macrophage-inflammatory protein (MIP)-2, and 3) enzymes such as inducible NO synthase and cyclooxygenase-2, and 4) -defensins (8, 9, 10). Activation of NF-B can be initiated by many stimuli including bacterial components such as LPS from Gram-negative bacteria and host-derived products such as TNF-, IL-1, and ROS (8, 9).
The purpose of the present study was to investigate regulation of the NF-B pathway in a model of Pseudomonas pneumonia, particularly the role of ROS generated by the NADPH oxidase system. In these studies, we used a transgenic mouse model to quantitatively evaluate NF-B-dependent transcriptional activity. We have developed a transgenic reporter mouse model that possesses the HIV long terminal repeat (LTR) driving the expression of Photinus luciferase cDNA (referred to as HIV-LTR/luciferase (HLL) mice) luciferase reporter mice (HLL) (11, 12). To examine the effects of NADPH oxidase products on NF-B activation in vivo, we crossbred p47phox-deficient mice with HLL mice (p47phox-/-HLL). We used in vivo bioluminescent imaging (BLI) to assess the levels of luciferase expression from the reporter transgenes or bacterial load in living animals (13, 14). Because the NADPH oxidase system functions primarily in phagocytes, and macrophages are important for host defense, we performed in vitro studies using bone marrow-derived macrophages (BMDM). In these studies, we investigated whether Pseudomonas interacts with TLR4 to mediate NF-B activation and whether NADPH oxidase impacts activation of NF-B via a TLR4-dependent pathway.
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Materials and Methods |
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HLL transgenic and p47phox-/-HLL mice express Photinus luciferase cDNA under the control of proximal 5' HIV-LTR promoter, and the mice are on C57B6/DBA background. Mice weighing 20–30 g were used (15). After sedation with ketamine/xylazine, mice were treated with intratracheal (IT) administration of P. aeruginosa (strain PA103) or XEN-5 (see strain descriptions below). Mouse tracheas were directly exposed by surgical resection, pierced with a 26-gauge needle, and injected with 100 µl of bacterial inoculum preparation diluted in sterile PBS. The neck wound was closed with sterile sutures under aseptic conditions.
Mice were asphyxiated with CO2. Lungs were removed; one lung was ground in 1 ml of reporter lysis buffer (Promega, Madison, WI) and stored at -20°C for luciferase assays, and the other lung was frozen at -70°C. Where indicated, tracheas were cannulated, and lungs were lavaged in situ with sterile pyrogen-free physiological saline that was instilled in four 1-ml aliquots and gently withdrawn with a 1-ml tuberculin syringe.
P. aeruginosa PA103
This strain was selected because it is a well-characterized and highly toxic strain. Bacteria from frozen stocks were streaked onto trypticase soy agar plates and grown in a dialysate of tripticase soy broth supplemented with 10 mM nitrilotriacetic acid (Sigma-Aldrich, St. Louis MO), 1% glycerol, and 100 mM monosodium glutamate at 33°C for 1–3 h in a shaking incubator. Cultures were centrifuged at 8500 x g for 5 min, and the bacterial pellet was washed twice in Ringers lactate and diluted into the appropriate number of CFU per milliliter in Ringers lactate solution determined by spectrophotometer. The concentration of bacteria was confirmed by serial dilutions plated on sheep blood agar.
P. aeruginosa XEN-5
To visualize the extent of the bacterial load in vivo, we used P. aeruginosa expressing the lux operon, luxCDABE (bioluminescence genes), from the nematode symbiont bacterium Photorhabdus luminescens (previously known as Xenorhabdus luminescens). This strain was obtained from Xenogen (Almeda, CA) (16).
In vivo measurement of luciferase gene expression by bioluminescence
HLL and p47phox-/-HLL mice were anesthetized, and the hair was removed over the chest and abdomen before imaging. Luciferin (150 mg/kg/mouse in 200 µl of isotonic saline) was administered by i.p. injection, and mice were imaged with an intensified charge-coupled device camera (model no. C2400-32; Hamamatsu, Bridgewater, NJ). For the duration of photon counting (3 min), mice were placed inside a light-tight box. Light emission from the mouse was detected as photon counts using the intensified charge-coupled device camera and customized image processing hardware and software (Hamamatsu) and expressed as photon counts. A digital false-color photon emission image of the mouse was generated, and photons were counted over a standard region of interest corresponding to the area of the chest overlying the midlung zone. Images were obtained before and following treatment with bacteria so that each mouse could be used as its own control (17). Bioluminescence of the XEN-5 bacteria was assessed similarly in separate mice that were not treated with luciferin before imaging. The five-gene operon from P. luminescens includes genes that encode enzymes for the biosynthesis of the substrate, decanal, hence exogenous addition of substrate is not required for light emission (18).
Measurement of luciferase activity in lung tissue
Luciferase activity was measured in postmortem tissue samples by adding 100 µl of freshly reconstituted luciferase assay buffer to 20 µl of the homogenated lung tissue that was ground in reporter lysis buffer (Promega). Luciferase activity was expressed as relative light units normalized for protein content, which was measured by Bradford assay (19).
Lung lavage total and differential cell counts
Lung lavage fluid was centrifuged at 400 x g for 10 min to separate cells from supernatant. Supernatant was saved separately and frozen at -70°C. The cell pellet was suspended in serum-free RPMI 1640 culture medium, and total cell counts were determined on a grid hemocytometer. Differential cell counts were determined by staining cytocentrifuge slides with a modified Wright stain (Diff-Quik; Baxter, McGraw Park, IL) and counting 400–600 cells in complete cross sections.
TNF- and MIP-2 ELISA
TNF- and MIP-2 levels were measured using a sandwich ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).
Extraction of nuclear proteins from tissue samples
Tissue nuclear proteins were extracted from whole-lung tissue by the method of Deryckere and Gannon (20). Briefly, 50–100 mg of tissue was mechanically homogenized in liquid nitrogen, to which 4 ml of buffer A (150 mM NaCl, 10 mM HEPES (pH 7.9), 0.6% (v/v) Nonidet P-40, 0.2 M EDTA, and 0.1 M PMSF) was added. The homogenate was transferred to a 15-ml Falcon tube and centrifuged at 850 x g in a tabletop centrifuge for 30 s to remove cellular debris. The supernatant was then transferred to a 50-ml Falcon tube and incubated on ice for 5 min before being centrifuged for 10 min at 3500 x g. Supernatant was collected as a cytoplasmic extract. The pellet was resuspended in 300 µl of buffer B (sterile water, 25% (v/v) glycerol, 20 mM HEPES (pH 7.9), 5 M NaCl, 1 M MgCl2, 0.2 M EDTA, 0.1 M phenylsulfonyl fluoride, 1 M DTT, 10 mg/ml benzamidine, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mg/ml aprotonin) and incubated on ice for 30 min. Following centrifugation at 14,000 rpm in an Eppendorf microcentrifuge for 2 min, the supernatant was collected as the nuclear extract and frozen at -70°C. Protein concentrations in nuclear extracts were determined by using Bradford assay (19).
Western blot for RelA in nuclear protein extracts
Twenty-five micrograms of protein from tissue homogenates were separated on a 10% acrylamide gel, transblotted, and immunodetected. Abs to RelA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Bacterial clearance studies
Before harvesting the lungs, the right ventricle was infused with 1 ml of sterile PBS to remove blood from the lung tissue. The lungs were removed aseptically and placed in 3 ml of sterile saline. Lungs were homogenized in a tissue homogenizer under sterile conditions. Serial dilutions of the homogenates were made, and 10 µl of each dilution was plated in soy base blood agar plates that were incubated for 18 h at 37°C, and then number of colonies was counted.
BMDM
After asphyxiation of mice with CO2, cellular material from femurs was aspirated and spun at 400 x g at 4°C for 5 min. Cells were then resuspended in DMEM with 10% FBS and 10% L929 cell-conditioned medium. Cells were allowed to mature into phenotypic macrophages by incubation in the presence of L929 cell-conditioned medium for 5 days. Cells were lifted, washed, counted, and replated before study.
Superoxide measurements
Superoxide production was measured from BMDM after treatment with P. aeruginosa using a commercially available Lumimax superoxide kit from Stratagene (La Jolla, CA).
Statistical analysis
Our statistical analyses were performed with GraphPad InStat, version 3.01 for Windows NT (GraphPad Software, San Diego, CA), using an unpaired t test and unpaired ANOVA.
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Results |
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B and neutrophilic influx in the lungs
We first performed experiments to assess NF-B activation after P. aeruginosa infection. HLL mice were treated by a single IT injection of PA103 P. aeruginosa in a dose of 1 x 106 CFU diluted in 100 µl of PBS. Control mice were treated with 100 µl of pyrogen-free PBS by IT injection. BLI was used to detect luciferase activity in vivo as a reflection of the intensity of NF-B activation in these reporter transgenic mice. Mice were treated with luciferin by the i.p. route and imaged at 24, 48, and 72 h postinfection. Control mice treated with PBS showed no increase in bioluminescent signal intensity, but mice treated with IT P. aeruginosa (106 CFU) showed increased photon emission from a standardized region of the thorax (photon counts (mean ± SEM): baseline, 950 ± 349; 24 h, 3908 ± 421; 48 h, 2961 ± 511; and 72 h, 2443 ± 384). Because peak luciferase activity occurred at 24 h, we harvested mice at this time point for dose-response experiments. Fig. 1 shows representative images of HLL mice treated with PBS or PA103 (105, 106, and 107 CFU) at 24 h. The computer software generates a false-color image that reflects the intensity of photon emission (blue is less intense, and white is most intense). To visualize the dimmer parts of the image, the brighter pixels in the images are displayed as white (thus appearing saturated); however, the detected light emission is well below the saturation limit of the camera. These images illustrate a dose-dependent increase in bioluminescence over the chest after treatment with PA103. In these studies, three of the seven mice treated with a dose of 1 x 107 CFU died. Hence, additional experiments were performed with a sublethal dose of 1 x 106 CFU.
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B and neutrophil influx into the lungs.
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B activation and impaired bacterial clearance from the lungs
To examine the effects of ROS from NADPH oxidase on NF-B activation in vivo, HLL mice were crossbred with p47phox-/- mice (p47phox-/-HLL). p47phox-/-HLL mice or HLL mice were treated with P. aeruginosa (106 CFU). Control animals from both groups were treated with pyrogen-free PBS. The p47phox-/-HLL mice showed lower luciferase activity at 24 h compared with the HLL mice as measured by BLI (Fig. 3A). The peak photonic counts were significantly lower in p47phox-/- mice (1400 ± 230; n = 6) compared with HLL mice (3250 ± 370; n = 6; p < 0.005). After imaging, mice were harvested at 24 h, and luciferase activity from lung homogenates confirmed that production of the NF-B-dependent reporter expression was lower in infected p47phox-/-HLL mice than HLL mice (Fig. 3B). Analysis of the cellularity of BAL showed no statistically significant difference in the total and neutrophil counts between the p47phox-/-HLL (34 x 105) and HLL mice (6 x 105), although the p47phox-/-HLL mice showed a trend toward higher neutrophil counts. MIP-2 was measured in the BAL, because it is an NF-B-dependent chemokine that is important for neutrophil recruitment. MIP-2 levels in BAL closely paralleled the neutrophil counts, and there was no statistically significant difference between the two groups of mice (data not shown).
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B activation was detected by performing Western blot analysis for RelA from lung nuclear protein extracts. HLL mice showed greater amounts of RelA in the nuclear extracts compared with the p47phox-/-HLL mice both at 12 and 24 h (Fig. 4, A and B). Analysis by densitometry of the RelA bands on Western blots confirmed that more RelA was translocated in HLL mice compared with the p47phox-/-HLL mice after treatment with PA103 (Fig. 4C).
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is an NF-B-dependent gene which has important functions in host defense. Hence, we measured the levels of TNF- in BAL and lungs from HLL and p47phox-/-HLL mice after administration of P. aeruginosa using ELISA. There were significantly higher levels of TNF- both in the BAL and lung homogenates of HLL mice compared with the p47phox-/-HLL mice (Fig. 5).
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B in macrophages infected with P. aeruginosa
To assess the relative contributions of TLR4 and redox signaling in phagocytes to NF-B activation in response to P. aeruginosa, we performed studies on BMDM from TLR4-deficient or p47phox-deficient mice. TLR4 transduces the signals that lead to the production of inflammatory mediators in response to LPS via activation of NF-B, but its role in mediating the host response to P. aeruginosa has not been clearly defined. To investigate the role of TLR4 in activation of NF-B, we used BMDM from C3H/HeJ mice that have a functional mutation of TLR4 gene. Bone marrow cells were isolated and cultured from C3H/HeJ or BALBc mice as described above. After 6 days in culture, cells were infected with P. aeruginosa (PA103) at a multiplicity of infection (MOI) of 1 for 4 h. Cells were then washed with PBS and incubated for 24 h. NF-B activation was detected by Western blot analysis for RelA from the nuclear extracts of the cells. RelA nuclear translocation was significantly impaired in mice with mutant TLR4 (Fig. 8).
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B activation, BMDM were isolated and cultured from p47phox-/-HLL or HLL mice. Identical studies to those described above were performed, and luciferase activity in cell lysates was measured to determine NF-B activation. Luciferase activity in untreated cells was similar for p47phox-/-HLL and HLL mice. After treatment with PA103, cells from HLL mice showed an 8-fold increase in luciferase activity, whereas cells from p47phox-/-HLL mice showed only a 2-fold increase over macrophages treated with PBS (Fig. 9). These data indicate that Pseudomonas activates NF-B primarily through TLR4 in phagocytes; however, the additional effects of redox signaling are necessary for maximal activation of NF-B.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and host defense against P. aeruginosa infection. We used p47phox-deficient mice with an NF-B luciferase reporter (p47phox-/-HLL), where luciferase activity serves as an in vivo detectable marker of NF-B activation. p47phox-/-HLL mice showed significantly reduced luciferase activity as compared with the nondeficient HLL transgenic reporter mice following Pseudomonas infection. In comparison, infected p47phox-/-HLL mice had higher numbers of viable bacteria in the lungs than HLL mice, indicating that impaired NF-B activation was not due to an insufficient inflammatory stimulus. Our correlative cell culture experiments, using BMDM from these mice, showed that NF-B activation in response to treatment with P. aeruginosa is transduced through the TLR4 receptor but is also dependent on functional NADPH oxidase.
The production of superoxide is initiated by NADPH oxidase, which becomes activated upon translocation of several cytosolic proteins (p40phox, p47phox, p67phox, and the Rho family of GTPases, Rac1 or -2) to the membrane-bound complex carrying cytochrome c (gp91phox, p22phox, and Rap1a). The assembled oxidase is then able to transfer electrons to oxygen by means of its electron-carrying prosthetic group (5). The respiratory burst generated by the NADPH oxidase is critical for defending the host against invading pathogens. These host defense functions are attributed largely to direct microbicidal action of the toxic intermediates such as H2O2 and OH ions. Reeves et al. (21) showed that the production of ROS in phagocytes also facilitates the activation of neutrophil elastase and cathepsin G by liberating these bound enzymes from the matrix. ROS have been shown to be involved in signaling by activation of transcription factors (NF-B, AP-1) and mitogen-activated protein kinases (6, 7, 22). A variety of in vitro studies have suggested that ROS act as second messengers and affect NF-B activation (15, 23, 24). In some cells, direct treatment with oxidants activates NF-B. In vitro treatment with antioxidants such as N-acetylcysteine or overexpression of endogenous antioxidants such as superoxide dismutase has been shown to block NF-B activation (25, 26). The binding of NF-B to its cognate DNA sequence is also influenced by the redox state (27). Similar studies investigating the role of ROS in activation of transcription factors in vivo are scant (28, 29, 30). In vivo, LPS induced NF-B activation, and acute lung inflammation can be inhibited by treatment with antioxidants such as N-acetylcysteine (30). In addition, we have shown that nuclear translocation of NF-B in the lungs is reduced in p47phox-/- mice compared with wild-type mice in models of lung inflammation induced by i.p. injection or aerosolization of Escherichia coli LPS (31). In a model of TNF--induced systemic inflammation, Fan et al. (32) showed that NF-B activation and ICAM-1 expression were significantly attenuated in lungs of p47phox-/- mice compared with wild-type mice. Together, these data indicate that ROS produced by NADPH oxidase are required for maximal activation of NF-B following a variety of inflammatory stimuli. The present study demonstrates that ROS are important for the maximal activation of NF-B in response to P. aeruginosa infection and thus may contribute to host defense via redox signaling.
Because NADPH oxidase affects both microbicidal activity and signaling capabilities of phagocytes, it is difficult to dissect out the relative contribution of each to the antimicrobial response. Nonetheless, we found that the levels of TNF- were significantly lower in p47phox-/- mice compared with wild type, possibly contributing to impaired host defenses in these animals. TNF- expression is NF-B dependent and signals activation of macrophage phagocytosis and microbicidal activity in culture. It also facilitates site-directed recruitment of phagocytic cells in vivo. Inhibition of TNF has been shown to result in decreased bacterial clearance and increased mortality in various animal models of infection (33, 34). Our studies demonstrate that NF-B-dependent production of TNF- is deficient in p47phox-/- mice. Thus, the altered host response in these mice in terms of decreased bacterial clearance may also be related to these deficiencies.
TLRs have been implicated in regulation of host responses to microbial products. TLR4 recognizes LPS from Gram-negative bacteria and mediates the innate immune response. The absence of NF-B activation in response to P. aeruginosa infection in phagocyte cultures from TLR4-deficient mice indicates a critical role for TLR4. In the present study, we showed that nuclear translocation of RelA is significantly reduced in both the TLR4-deficient and NADPH oxidase-deficient macrophages. Thus, our data suggest that an intact TLR4 signaling pathway and NADPH oxidase system are necessary for maximal activation of NF-B.
This study has important implications in understanding the role of NF-B and redox signaling in infection and host defense mechanisms. We have shown that P. aeruginosa infection is associated with intense activation of NF-B, and the inability of p47phox-/- mice to generate oxidative burst impairs their ability to activate NF-B following challenge with P. aeruginosa. Thus, ROS from NADPH oxidase contribute to host defenses through direct microbicidal action and through modulation of redox-sensitive signal transduction pathways.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ruxana T. Sadikot, Division of Allergy, Pulmonary and Critical Care, Vanderbilt University School of Medicine, T-1217 Medical Center North, Nashville, TN 37232-2650. E-mail address: ruxana.sadikot{at}Vanderbilt.edu
3 Abbreviations used in this paper: ROS, reactive oxygen species; TLR, Toll-like receptor; MIP, macrophage-inflammatory protein; LTR, long terminal repeat; HLL, HIV-LTR/luciferase; BLI, bioluminescent imaging; BMDM, bone marrow-derived macrophage; IT, intratracheal; BAL, bronchoalveolar lavage; MOI, multiplicity of infection.
Received for publication July 9, 2003. Accepted for publication November 19, 2003.
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