Dextromethorphan Attenuates NADPH Oxidase-Regulated Glycogen Synthase Kinase 3β and NF-κB Activation and Reduces Nitric Oxide Production in Group A Streptococcal Infection

ABSTRACT

Group A Streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), an antitussive drug, has been demonstrated to efficiently reduce inflammatory responses, thereby contributing to an increased survival rate of GAS-infected mice. However, the anti-inflammatory mechanisms underlying DM treatment in GAS infection remain unclear. DM is known to exert neuroprotective effects through an NADPH oxidase-dependent regulated process. In the present study, membrane translocation of NADPH oxidase subunit p47^phox and subsequent reactive oxygen species (ROS) generation induced by GAS infection were significantly inhibited via DM treatment in RAW264.7 murine macrophage cells. Further determination of proinflammatory mediators revealed that DM effectively suppressed inducible nitric oxide synthase (iNOS) expression and NO, tumor necrosis factor alpha, and interleukin-6 generation in GAS-infected RAW264.7 cells as well as in air-pouch-infiltrating cells from GAS/DM-treated mice. GAS infection caused AKT dephosphorylation, glycogen synthase kinase-3β (GSK-3β) activation, and subsequent NF-κB nuclear translocation, which were also markedly inhibited by treatment with DM and an NADPH oxidase inhibitor, diphenylene iodonium. These results suggest that DM attenuates GAS infection-induced overactive inflammation by inhibiting NADPH oxidase-mediated ROS production that leads to downregulation of the GSK-3β/NF-κB/NO signaling pathway.

INTRODUCTION

Group A Streptococcus (GAS) is an important Gram-positive human pathogen that causes diseases ranging from relatively mild superficial skin infection and pharyngeal infection to severe and life-threatening invasive diseases, such as necrotizing fasciitis and streptococcal toxic shock syndrome. Despite the availability of effective antimicrobial agents and prompt medical care, an increase in the incidence of severity, rapid onset, and high mortality rate of GAS disease has been noted worldwide (1, 2). In addition to virulence factors, host defense mechanisms have been shown to be involved in modulating disease severity in GAS infection. The excessive host responses contributing to GAS disease pathology may be caused by a number of factors, including superantigenic stimulation of T-cell responses, M1 protein-mediated heparin binding protein release, streptolysin O-mediated inflammatory reactions, and host polymorphism in the human leukocyte antigen complex (3). Macrophages and polymorphonuclear leukocytes (PMN) acting as vital effector cells of innate immunity exhibit professional bactericidal capacity through the recognition of toll-like receptors (TLRs) with GAS, followed by signal transduction and inflammatory mediator release (4, 5). Nevertheless, phagocyte-produced excessive reactive oxygen species (ROS), nitric oxide (NO), and proinflammatory cytokines may cause tissue damage and murine sepsis (6–10). Moreover, clinical studies also revealed a higher frequency of interleukin-6 (IL-6)- and tumor necrosis factor alpha (TNF-α)-producing cells in the circulation of severe invasive cases suffering from toxic shock and/or necrotizing fasciitis compared to nonsevere invasive cases (11–13). Accordingly, it is important to characterize more fully the role of host factors to provide efficacious and personalized treatments against GAS infection.

Dextromethorphan (DM), an over-the-counter antitussive drug, is one of the most widely used active ingredients as a cough suppressant in cold and cough medications, with high safety and efficacy at recommended doses (14). DM is reported to be neuroprotective against glutamate excitatory toxicity and degeneration of dopaminergic neurons through the antagonization of the N-methyl–d-aspartic acid (NMDA) receptor (15–17). The anti-inflammatory activity of DM has also been reported to improve endothelial function and reduce vascular oxidative stress and inflammation markers in habitual smokers (18). Additionally, in lipopolysaccharide (LPS)-induced endotoxic shock, DM can provide protection against hepatotoxicity by decreasing TNF-α and intracellular ROS production, as well as inflammation and cell death-related components (19). In a heat-induced acute lung inflammation and injury rat model, DM treatment significantly attenuated lung damage by decreasing neutrophil infiltration, proinflammatory cytokine secretion, and oxidative damage marker expression (20). Likewise, our previous study demonstrated that DM effectively enhanced bactericidal activity, decreased bacterial numbers at the infection site, and reduced overactive inflammatory responses, all of which may contribute to increasing the survival rate of GAS-infected mice (21).

NADP oxidase (NADPH oxidase) of the NOX family has been recognized as an important source of cellular ROS participating in host defense by killing invading pathogens (22–24). There are five isoforms of NADPH oxidase, including NOX1 to NOX5. NOX2 was first described in neutrophils and macrophages and is often referred to as phagocyte NADPH oxidase. NOX2 is composed of two membrane-bound subunits, gp91^phox and p22^phox, three cytosolic subunits, p47^phox, p67^phox and p40^phox, and a small GTPase, Rac. Upon stimulation, p47^phox is phosphorylated and translocated from cytosol to plasma membrane, followed by interaction with gp91^phox and p22^phox and triggering p67^phox and p40^phox recruitment to the membrane (25). NADPH oxidase-mediated ROS production serves as an important bactericidal mechanism of macrophages; however, excessive ROS production may also contribute to tissue damage (26). In GAS-induced septic shock, the elevation of inducible NO synthase (iNOS), NO, and ROS correlated with mortality, while reducing intracellular ROS levels by aminoethyl-isothiourea treatment significantly attenuated inflammatory responses and prolonged survival rates (10, 27).

DM exerts antioxidative and anti-inflammatory effects, potentially through inhibiting NADPH oxidase activation (17, 19, 28–30). We previously demonstrated that DM treatment offered protection in reducing GAS septic shock (21). To further explore the protective mechanism of DM after GAS infection, we show in the present study that DM treatment inhibits the membrane translocation of p47^phox and subsequent ROS production in GAS-infected macrophages. Administration of DM reduced GAS-evoked iNOS expression and NO, TNF-α, and IL-6 generation, which was caused by downregulation of GSK-3β/NF-κB signaling. Therefore, DM possesses an NADPH oxidase-dependent anti-inflammatory modulatory effect against GAS infection.

RESULTS

GAS infection induces NADPH oxidase activation.

The expression and activation of phagocytic NADPH oxidase is required for regulating bactericidal activity of macrophages against GAS (31). The generation of ROS is a consequence of the assembly of membrane-associated gp91^phox and gp22^phox with cytosolic components p67^phox and p47^phox (23, 25). To determine the activation of NADPH oxidase in GAS-infected murine macrophage RAW264.7 cells, we examined the membrane translocation of p47^phox by confocal microscopy. Results showed that p47^phox exhibited a more condensed distribution of fluorescent dots in GAS-infected cells but showed a diffuse pattern in mock-infected cells and cells at 0 h postinfection, indicating p47^phox condensation and membrane translocation after GAS infection (Fig. 1A, arrows). The initial condensation of p47^phox could be detected at 0.5 h postinfection (see Fig. S1 in the supplemental material). By double staining with a plasma membrane marker, cholera toxin subunit B (CT-B), we confirmed the colocalization of CT-B and p47^phox on the plasma membrane (Fig. 1B, arrows). The percentages of p47^phox-condensed cells in DAPI⁺ (4′,6-diamidino-2-phenylindole) CT-B⁺ cells from random fields of each slide were calculated. The quantification of p47^phox membrane translocation showed a significant elevation after GAS infection (Fig. 1C). Therefore, GAS effectively initiates NADPH oxidase activation.

FIG 1.

GAS infection induces the membrane translocation of p47^phox in RAW264.7 cells. (A) RAW264.7 cells were infected with GAS (MOI of 25) for 1 h. At 0 h and 2 h postinfection, cells were fixed and stained with anti-p47^phox antibody followed by Alexa Fluor 488-conjugated anti-rabbit IgG (green). Nuclei were stained with DAPI (blue). The cellular location of p47^phox subunit was determined using confocal microscopy. Arrows indicate membrane translocation of the p47^phox subunit. The scale bar is shown. (B) RAW264.7 cells were infected with GAS (MOI of 25) for 1 h. At 0 h and 2 h postinfection, cells were stained with plasma membrane affinity dye using Alexa Fluor 549-conjugated cholera toxin subunit B (CT-B; red) for 20 min and then fixed and stained with anti-p47^phox antibody followed by Alexa Fluor 488-conjugated anti-rabbit IgG staining (green). Nuclei were stained with DAPI (blue). The colocalization of p47^phox subunit and CT-B was determined using confocal microscopy. Arrows indicate the colocalization of p47^phox subunit and CT-B on plasma membrane (yellow). The scale bar is shown. (C) Membrane translocation of p47^phox subunit was quantified, and the percentages of p47^phox-condensed cells in approximately 150 DAPI⁺ CT-B⁺ cells are shown as the means ± standard deviations (SD) from triplicate cultures. ***, P < 0.001 compared with the mock control.

DM attenuates GAS-induced p47^phox membrane translocation.

The anti-inflammatory activity of DM has been reported to attenuate NADPH oxidase activation, thereby reducing the severity of disease (19, 29, 32). Our previous findings indicated that DM treatment significantly increased bactericidal activity and reduced overactive inflammatory responses against GAS sepsis (21). To examine the protective mechanisms underlying DM treatment in GAS infection, we found that in the presence of DM, p47^phox membrane translocation induced by GAS was reduced, as determined at 2 h postinfection (Fig. 2A), which was further quantified by the percentages of p47^phox-condensed cells in DAPI⁺ cells showing a significant inhibition (Fig. 2B). We further isolated membrane fractions and showed that the expression of p47^phox in membrane fractions was increased after 2 h postinfection, whereas the presence of DM markedly reduced the membrane trafficking of p47^phox (Fig. 2C). There were no significant changes in total p47^phox expression after GAS infection. Our results show that DM blocks GAS infection-mediated NADPH oxidase activation.

FIG 2.

DM treatment reduces p47^phox membrane translocation in GAS-infected cells. (A) RAW264.7 cells were infected with GAS (MOI of 25) for 1 h in the presence or absence of 10 μM DM. At 2 h postinfection, cells were fixed and stained with anti-p47^phox antibody followed by Alexa Fluor 488-conjugated anti-rabbit IgG (green). Nuclei were stained with DAPI (blue). The p47^phox membrane translocation was determined by confocal microscopy. Arrows indicate membrane translocation of the p47^phox subunit. The scale bar is shown. (B) The percentages of p47^phox-condensed cells in approximately 150 DAPI⁺ cells were quantified and are shown as the means ± SD from triplicate cultures. **, P < 0.01; ***, P < 0.001. (C) Membrane fractions were isolated from GAS-infected cells in the presence or absence of DM. The expression of the p47^phox subunit in membrane fractions was measured using Western blotting. Caveolin was used as the membrane fractional control. The ratios of p47^phox to caveolin are shown. Total protein levels of the p47^phox subunit and α-tubulin were also determined.

DM reduces ROS production in GAS infection.

ROS serves as a significant regulator in GAS-induced septic shock (27). Meanwhile, DM-mediated intracellular ROS reduction was reported in NADPH oxidase-dependent regulation to provide a neuroprotective effect in Parkinson's disease (28). We therefore speculated that DM has an effect on GAS-induced ROS generation. RAW264.7 cells were infected with GAS in the presence or absence of DM, followed by the measurement of ROS-producing cells using flow cytometry. Compared to ROS production in GAS-infected cells, the presence of DM significantly inhibited ROS production at 30 min postinfection (Fig. 3A and B), and the inhibition persisted until 2 h postinfection (Fig. S2). Tert-butyl hydroperoxide (tbHP) was used as a positive control for ROS generation (Fig. 3A and B). In addition, the generation of ROS in both GAS-infected human monocytic THP-1 cells and human microvascular endothelial cells (HMEC-1) was inhibited in the presence of DM (Fig. 3C and D), indicating that DM treatment effectively reduces GAS-mediated ROS production.

FIG 3.

ROS production in GAS-infected cells is attenuated with DM treatment. (A) RAW264.7 cells were infected with GAS (MOI of 25) for 1 h in the presence or absence of 10 μM DM. After infection, cells were further incubated with DCFDA (20 μM) and assayed by flow cytometry analysis at 30 min postinfection. tbHP (20 μM) was used as a positive control. (B) The percentages of ROS-producing cells were gated and quantified. In the presence or absence of DM, THP-1 cells (C) and HMEC-1 cells (D) were infected with GAS for 2 h, followed by ROS detection by DCFDA staining. Data are shown as the means ± SD from triplicate cultures. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

DM treatment attenuates iNOS/NO induction in GAS-infected cells.

In addition to ROS, the restriction of iNOS and subsequent NO production have been reported to effectively prevent GAS-induced sepsis (27). The expression of iNOS and NO in GAS-infected cells was determined. At 12 h postinfection, GAS-induced iNOS expression was blocked in the presence of DM and diphenyleneiodonium (DPI), a general inhibitor of NADPH oxidase (Fig. 4A). The measurement of NO at 24 h postinfection also revealed the blocking effects of DM and DPI on GAS-induced NO production (Fig. 4B). Phagocytes produce NO to increase bacterial killing capability (33, 34); however, NO production also correlates with severe GAS infection (35). We previously demonstrated the protective effects of DM against GAS-induced septic shock through increasing viability and bactericidal activity of air-pouch-infiltrating cells (21). To further verify that DM effectively regulates GAS-mediated iNOS expression, the air-pouch-infiltrating cells isolated from GAS-infected mice with or without DM administration were assayed. DM treatment markedly reduced the iNOS expression of infiltrating cells in GAS-infected mice at 48 h after bacterial inoculation (Fig. 4C). The quantified ratios of iNOS to β-actin showed that DM caused a significant reduction (Fig. 4D). In addition to bacterial killing capability, DM treatment was demonstrated to reduce GAS-induced systemic inflammation, as shown by decreased TNF-α and IL-6 in the sera as well as reduced organ damage (21). Similarly, the production of TNF-α and IL-6 in GAS-infected RAW264.7 cells was reduced by DM treatment (Fig. 4E). The present results indicate that DM modulates the inflammatory response by inhibiting NADPH oxidase activation and subsequent modulation of iNOS/NO and proinflammatory cytokine expression against GAS-induced septic shock.

FIG 4.

DM attenuates iNOS/NO, TNF-α, and IL-6 production after GAS infection. (A) In the presence or absence of DM and DPI, iNOS expression in RAW264.7 cells was analyzed at 12 h postinfection. GAPDH was used as an internal control. (B) At 24 h postinfection, the NO production was determined by Griess reagent assay. Nitrite concentrations were detected using sodium nitrite as a standard and are shown as the means ± SD from triplicate cultures. *, P < 0.05; **, P < 0.01. (C) The air-pouch-infiltrating cells were collected from mice inoculated with 3 × 10⁸ CFU of GAS for 48 h with or without DM treatment, followed by iNOS protein detection. The expression of β-actin was used as an internal control. (D) The ratios of iNOS to β-actin were quantified and are shown as the means ± SD from three mice. *, P < 0.05. (E) In the presence or absence of DM, the levels of TNF-α and IL-6 in GAS-infected RAW264.7 cell culture supernatants were measured at 12 h postinfection and are shown as the means ± SD from triplicate cultures. *, P < 0.05.

DM treatment attenuates GSK-3β-mediated NF-κB activation.

GAS-induced iNOS expression and NO and TNF-α production through GSK-3β-regulated NF-κB activation were previously demonstrated (36). Here, we showed GAS infection caused time-dependent dephosphorylation of AKT at serine-473 (an active phosphorylation site) and GSK-3β at serine-9 (an inhibitory phosphorylation site), indicating the inactivation of AKT and activation of GSK-3β as a consequence of GAS infection (Fig. 5A). However, heat-killed GAS did not cause a similar effect (Fig. S3). Treatment with DM and DPI reversed GAS infection-mediated dephosphorylation of AKT and GSK-3β, suggesting that DM and DPI induce the activation of AKT as well as the inactivation of GSK-3β (Fig. 5B). The attenuation of GSK-3β via DM and DPI in GAS infection might modulate subsequent NF-κB activation. By confocal microscopic observation, the nuclear translocation of NF-κB was blocked at 4 h postinfection in the presence of DM, DPI, and an ROS scavenger, N-acetyl-cysteine (NAC) (Fig. 5C). Compared to GAS infection alone, quantified results from GAS cotreated with DM, DPI, or NAC showed a significant reduction in the percentage of NF-κB nuclear translocation (Fig. 5D). While the expression of NF-κB in nuclear fractions was inhibited by DM and DPI at 4 h postinfection, GAS-triggered IκB degradation was reversed at 2 h postinfection (Fig. 5E). The treatments with DM, DPI, and NAC showed no significant effect on GAS infection in RAW264.7 cells and did not cause any cytotoxic effects (Fig. S4). Therefore, in GAS-infected cells, DM suppresses NF-κB-mediated iNOS expression and NO, TNF-α, and IL-6 production through inhibition of NADPH oxidase, ROS attenuation, and GSK-3β inactivation (Fig. 6).

FIG 5.

DM suppresses GAS infection-induced GSK-3β and NF-κB activation. (A) RAW264.7 cells were infected with GAS for 1 h. At the indicated time points after infection, the phosphorylation of AKT at serine-473 (pAKT) and GSK-3β at serine-9 (pGSK-3β) and protein expression of AKT, GSK-3β, and GAPDH were detected. (B) In the presence of DM and DPI, cells were infected with GAS for 1 h, followed by pAKT, pGSK-3β, total AKT, and GSK-3β determination at 2 h postinfection. GAPDH was used as an internal control. (C) In the presence or absence of 10 μM DM, 2.5 μM DPI, and 10 μM NAC, GAS-infected cells were fixed and stained with specific antibodies against NF-κB (green) at 4 h postinfection. The NF-κB nuclear translocation was measured using confocal microscopy. Nuclei were stained by DAPI (blue). The scale bar is shown. (D) Quantitative measurements of NF-κB nuclear translocation (percentages of total cells) were performed and are shown as the means ± SD from triplicate cultures. ***, P < 0.001. (E) In the presence or absence of DM and DPI, nuclear fractions were isolated from GAS-infected cells and the expression of NF-κB was determined at 4 h postinfection. PCNA was used as the internal control for nuclear fractions. The expression of IκB and α-tubulin in total cell lysate was measured at 2 h postinfection. The ratios of NF-κB to PCNA are shown.

FIG 6.

DM attenuates GAS infection-induced iNOS expression and NO, TNF-α, and IL-6 production by negatively regulating the NADPH oxidase/GSK-3β/NF-κB activation pathway. GAS infection effectively causes the membrane translocation of p47^phox subunit of NADPH oxidase to produce abundant ROS. The generation of ROS subsequently regulates AKT dephosphorylation, GSK-3β activation, and NF-κB-mediated iNOS/NO, TNF-α, and IL-6 expression. Treatment with DM markedly attenuates NADPH oxidase activation and ROS production in GAS-infected macrophages. In addition, NADPH oxidase-evoked GSK-3β/NF-κB activation is suppressed by DM treatment. Therefore, DM serves as an antioxidative and anti-inflammatory modulator to provide protection against GAS infection.

DISCUSSION

Despite intensive antimicrobial therapy, GAS infection within the deeper tissue and bloodstream is still associated with high mortality rates due to hyperinflammation, organ failure, and septic shock (1, 2). DM is a nonopioid morphinan derivative that has been used extensively and safely as a nonprescription antitussive. Recent studies indicate that DM exerts anti-inflammatory and immunomodulatory effects in many cell types (17, 19, 28, 29, 37). DM has been reported to provide neuroprotective and anti-inflammatory effects by inhibiting LPS-stimulated NF-κB activation and subsequent iNOS/NO and proinflammatory mediator production in BV2 mouse microglial cells (38). We have previously demonstrated that DM can efficiently increase bactericidal activity, reduce inflammation, and contribute to survival in GAS-infected mice (21). To clarify the protective mechanisms of DM in GAS infection, here we further reveal that GAS infection induces the inactivation of AKT and activation of the GSK-3β/NF-κB signaling pathway, thereby triggering downstream iNOS expression and NO, TNF-α, and IL-6 production in RAW264.7 cells. Importantly, DM treatment partly constrains GSK-3β-mediated NF-κB activation and subsequent inflammation via NADPH oxidase-dependent regulation (Fig. 6).

In addition to findings from our previous study on GAS-induced septic shock (21), DM has been shown to exert remarkable anti-inflammatory and antioxidative capability in models of Parkinson's disease, LPS-induced endotoxin shock, and atherosclerosis, as well as experimental autoimmune encephalomyelitis (19, 28, 29, 32). DM reduces NADPH oxidase p47^phox subunit membrane translocation and subsequent ROS production to deliver beneficial effects in alleviating neurotoxicity, atherosclerosis, and endotoxemia. Our present results suggest the efficacy of DM in protecting mice against GAS-elicited sepsis by NADPH oxidase-dependent regulation. GAS can initiate both MyD88-dependent and -independent pathways without the involvement of TLR2/4/9 to induce NF-κB activation (39, 40). Whether the blockage of DM on GAS-activated NADPH oxidase is associated with TLRs and MyD88 inactivation in macrophages remains to be investigated. In contrast to the transcriptional induction of the p47^phox gene, Ncf1, in GAS-infected macrophages as previously reported (31), we did not find any changes of protein expression after GAS infection. Our study showed that DM attenuated p47^phox membrane translocation to decrease ROS burden. The antioxidant capability of DM indicates substantial potential for DM to provide protection against GAS-induced septic shock via offsetting excessive ROS.

ROS generation via NADPH oxidase activation not only contributes to killing bacteria but also is regarded as an important proinflammatory regulator in activating NF-κB in many cells, including GAS-infected macrophages (10, 41, 42). ROS can regulate the modifications of IKK, IκB, MEKKs, and AKT to influence NF-κB activation (43). In GAS-infected RAW264.7 cells, we observed the dephosphorylation of AKT at serine-473 at 1 h postinfection, while IκB degradation and NF-κB nuclear translocation occurred at 4 h postinfection. The inactivation of AKT suggests the involvement of GSK-3β, which is a downstream kinase of AKT, leading to NF-κB activation in GAS infection. The results shown in Fig. 5 are in accordance with our previous findings (36), showing that GAS infection effectively causes GSK-3β-mediated NF-κB activation. Notably, treatment with DM and DPI block the dephosphorylation (i.e., inactivation) of AKT and also dephosphorylation (i.e., activation) of GSK-3β, as well as subsequent NF-κB activation. These results are the first to reveal AKT/GSK-3β as another potential target for ROS in influencing NF-κB signaling of macrophages during GAS infection.

In fact, the activation of GSK-3β is critical to initiate NF-κB-mediated inflammation, whereas the administration of GSK-3 inhibitors provides efficient protection against endotoxic shock (44), TLR2-induced peritonitis and arthritis (45), experimental colitis, and bacterial infections (46–48). As in GAS infection, GSK-3 inhibition significantly reduced serum TNF-α and improved the survival rate (36). In this study, we provide evidence showing that DM treatment markedly reverses AKT/GSK-3β dephosphorylation in GAS-infected macrophages, which thus decreases NF-κB activation and relieves subsequent iNOS/NO, TNF-α, and IL-6 generation. We also show the ability of DM to delay GSK-3β-initiated proinflammation, consistent with DM-associated protective effects against GAS-induced septic shock in vivo. Heat-inactivated Staphylococcus aureus has been demonstrated to induce NO production via GSK-3β activation (49). In our study, only live GAS and not heat-killed GAS can induce GSK-3β activation, which suggests that in addition to bacterial cell wall components, other virulence factors have effects on GSK-3β activation.

Previous reports showed that the presence of an ROS scavenger or NADPH oxidase inhibitor efficiently reduced GAS-induced cell death in epithelial cells and keratinocytes (42, 50). Since DM acts as a negative regulator of NADPH oxidase, DM may provide dual protective effects by inhibiting ROS-mediated inflammation and cell death to alleviate the severity of GAS disease. Indeed, the presence of DM effectively reduces GAS-induced NO, TNF-α, and IL-6 production, and those cytokines have been shown to be highly correlated with disease severity (11–13). Moreover, administration with DM significantly increases the viability of air-pouch-infiltrating cells, thereby enhancing bacterial elimination in GAS-infected mice (21).

In conclusion, we have demonstrated an important protective mechanism of DM in attenuating inflammatory responses by inhibiting NADPH oxidase-mediated ROS production followed by GSK-3β/NF-κB inactivation, leading to iNOS/NO, TNF-α, and IL-6 reduction in GAS-infected macrophages. Clinical therapy for bacterial infection usually involves treatment with antibiotics and prevention of systemic infection. Despite intensive antimicrobial therapy, the release of bacterial components from dead bacteria, such as peptidoglycans, lipoproteins, and LPS, may be responsible for systemic inflammation and contribute to organ failure and even sepsis. DM possesses the capability to reduce overactive inflammation. We therefore suggest that DM has potential as an adjunct agent for antimicrobial treatment.

MATERIALS AND METHODS

Bacteria.

GAS NZ131 (type M49, T14 strain) was a gift from D. R. Martin, New Zealand Communicable Disease Center, Porirua, New Zealand. A fresh colony was inoculated into tryptic soy broth containing 0.5% yeast extract (TSBY; Difco Laboratories, Detroit, MI) for 16 h at 37°C and then diluted (1:50) with fresh TSBY broth, followed by another 3 h of incubation at 37°C. The bacteria were harvested using centrifugation and then resuspended in sterile phosphate-buffered saline (PBS). The bacterial density was determined and the sample diluted with PBS to 10⁹ CFU/ml by measuring the absorbance at 600 nm with a spectrophotometer (Beckman Instruments, Somerset, NJ), plating serial dilutions of the samples on TSBY agar, and counting CFU after incubation overnight at 37°C.

Cell cultures and reagents.

RAW264.7 macrophage cells and THP-1 monocytic cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 (Gibco, Grand Island, NY), respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS), and maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The passage number of cells which were used for experiments was less than 15. The human microvascular endothelial cell line 1 (HMEC-1) was cultured in endothelial cell growth medium M200 (Cascade Biologics, Portland, OR) supplemented with 2% FBS, 1 mg/ml hydrocortisone, 10 ng/ml epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 mg/ml heparin, and antibiotics. Dextromethophan (DM), diphenylene iodonium (DPI), and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO).

Mice.

BALB/c breeder mice were obtained from The Jackson Laboratory, Bar Harbor, Maine, and maintained on standard laboratory food and water ad libitum in our medical college laboratory animal center. Their 8- to 10-week-old progeny were used for the air pouch infection model as described previously (51). Mice were injected subcutaneously with 2 ml of air to form an air pouch, and the bacterial suspension (0.3 ml) was then inoculated into the air pouch. DM (12.5 mg/kg of body weight) was injected intraperitoneally into mice 30 min before and 1, 12, and 24 h after GAS inoculation. Mice in the control group received an equal volume of saline. The air-pouch-infiltrating cells were collected by the injection of 1 ml PBS into the air pouch and aspiration of the exudates at 48 h postinfection. The animal use protocol was reviewed and performed in strict accordance with the Experimental Animal Committee of National Cheng Kung University.

GAS infection in cells.

RAW264.7 and THP-1 cells were seeded into 12-well culture plates at 10⁶ cells/ml, and HMEC-1 cells were seeded into 6-well plates at 2 × 10⁵ cells/ml and then incubated overnight. Bacteria were prepared at a multiplicity of infection (MOI) of 25:1 and mixed with cells, followed by 1,200-rpm centrifugation for 5 min. After 1 h of incubation, culture supernatants were replaced with fresh medium containing 10 μg/ml of penicillin and 50 μg/ml of gentamicin for further incubation at 37°C. At different hours postinfection, cells were harvested and analyzed.

Immunostaining.

Cells were seeded on glass coverslips in 12-well plates and infected with GAS as described above. For p47^phox staining, cells were stained with Alexa Fluor-549-conjugated cholera toxin subunit B (CT-B; Invitrogen, Camarillo, CA) at 37°C for 20 min. After washing, cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature and incubated with specific antibodies (Abs) against p47^phox overnight at 4°C, followed by Alexa Fluor-488-conjugated donkey anti-rabbit IgG (Invitrogen) staining for 1 h at room temperature and analysis using confocal microscopy (Olympus FV-1000; Japan). For the measurement of NF-κB nuclear translocation, fixed cells were stained with rabbit anti-NF-κB p65 (Cell Signaling Technology, Beverly, MA), followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Invitrogen) staining, and analyzed by confocal microscopy as well. DAPI (Calbiochem, San Diego, CA) was used for nuclear staining. All immunostaining studies were performed in at least two independent experiments. Image J software (NIH, Bethesda, MD) was used for image quantification analysis.

Immunoblotting.

To detect the indicated proteins, total cell lysates were extracted using a Triton X-100-based lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.5, 5 mM EDTA, 5 mM NaN₃, 10 mM NaF, and 10 mM sodium pyrophosphate) with a protease inhibitor mix and phosphatase inhibitor cocktail I (Sigma) and were centrifuged for 10 min at 13,300 rpm. Proteins were resolved using SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA). After blocking, blots were developed with a series of Abs as indicated. Abs specific for GSK-3β, phospho-GSK-3β (serine 9), AKT, phospho-AKT (serine 473), caveolin-1, NF-κB, and IκB were purchased from Cell Signaling Technology. Rabbit anti-p47^phox (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-iNOS (BD Biosciences, San Jose, CA) Abs were also used. Mouse Abs specific for proliferating cell nuclear antigen (PCNA; Dako Corporation, Carpinteria, CA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Millipore Corporation), and α-tubulin and β-actin (Santa Cruz Biotechnology) were used for internal controls. Finally, blots were hybridized with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology) and developed using an ECL Western blot detection kit (Millipore Corporation) according to the manufacturer's instructions. All immunoblotting studies were performed in at least two independent experiments. The band intensity was measured using Image J software.

Nuclear and membrane fractionations.

The nuclear fractions and membrane fractions of cells were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL) and a ProteoExtract native membrane protein extraction kit (Calbiochem), respectively, according to the manufacturer's instructions. In brief, 4 × 10⁶ cells were collected and subjected to subcellular fractionation immediately. After extraction, 20 μl of nuclear proteins or 80 μl of membrane proteins was analyzed by immunoblot assay.

ROS detection.

Intracellular ROS production was detected using a cellular reactive oxygen species detection assay kit (Abcam, Cambridge, MA), followed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA) analysis. In brief, cells were infected with GAS and then coincubated with 20 μM fluoroprobe carboxymethyl-H2-dichlorofluorescein diacetate (CM-H2DCFDA) for 30 min at 37°C in the dark. After washing, cells were collected and analyzed using flow cytometry with excitation set at 488 nm. The emission was detected with the FL-1 channel, followed by CellQuest Pro 4.0.2 software (BD Biosciences) analysis, and quantification was done using WinMDI 2.8 software (The Scripps Institute, La Jolla, CA).

NO and cytokine determination.

To detect NO production, nitrite (NO₂⁻) accumulation in the cell culture medium was used as an indicator of NO production by the Griess reaction. RAW264.7 cells were seeded into 96-well culture plates at 10⁵ cells/well and infected with GAS as described above. Briefly, supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% H₃PO₄) and incubated for 10 min at room temperature. The relative optical density of nitrite was measured at 540 nm, and the concentration was evaluated by using sodium nitrite as a standard. The concentrations of TNF-α and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.

Statistical analysis.

Comparisons between various treatments were performed by unpaired t test with GraphPad Prism, version 6.0 (La Jolla, CA). Statistical significance was set at a P value of <0.05.