Resveratrol ameliorates Serratia marcescens

Resveratrol ameliorates Serratia marcescens-induced acute pneumonia in rats Chia-Chen Lu,*,1 Hsin-Chih Lai,†,1 Shang-Chen Hsieh,‡,1 and Jan-Kan Chen*,2 *Department of Physiology, College of Medicine, and †Graduate Institute of Medical Biotechnology and Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Kweishan, Taiwan, Republic of China; and ‡Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, Taiwan, Republic of China

Abstract: Serratia marcescens is an important nosocomial pathogen, which has been especially problematic as a cause of hospital-acquired pneumonia in the past two decades. Treatment of S. marcescensrelated infections has been limited by emergence of multiple drug-resistant strains. Thus, the development of alternative agents for the prevention and treatment of Serratia infection is urgently needed. Resveratrol (RSV) is a compound with diverse biological effects including anti-cancer, anti-inflammation, anti-diabetes, and cancer chemoprevention. Whether RSV has in vivo prophylactic or therapeutic potential against infection remains uncharacterized. In the present study, we used a murine acute pneumonia model initiated by intratracheal application of S. marcescens to evaluate whether RSV possesses anti-infection properties. We showed that pretreatment with RSV for 3 days markedly increased alveolar macrophage infiltration, elevated NK cell activity, and decreased bacterial burden in the infected lung with a subsequent decrease in mortality. These effects were associated with significantly less-severe inflammatory phenotypes in lung tissue and bronchoalveolar lavage fluid, including reduced neutrophil infiltration of the lungs, reduced phagocytosis activity, and reduced secretion of cytokines such as TNF-␣, IL-1␤, and IL-6. To further characterize the underlying mechanism responsible for these effects of RSV, LPS derived from S. marcescens was used to induce acute pneumonia in rats, with or without RSV pretreatment. RSV was shown to ameliorate acute pneumonia via inhibition of the NF-␬B signaling pathway, including inhibition of I␬B␣ phosphorylation and subsequent NF-␬B activation. These findings suggest that RSV might be beneficial as a prophylactic treatment in patients at risk of an episode of S. marcescens-induced acute pneumonia. J. Leukoc. Biol. 83: 1028 –1037; 2008. Key Words: anti-inflammation 䡠 NF-␬B

different degrees [1]. During the infectious process, when faced with large numbers of infectious particles or virulent microbes, the alveolar macrophages (AMs) function to initiate and resolve pulmonary inflammation, which is particularly important in the preservation of normal lung function [2]. AMs synthesize and secrete a wide array of cytokines (including IL-1, IL-6, and TNF-␣) and chemokines (including IL-8) [3]. Using these cell-tocell signals, AMs initiate inflammatory responses and recruit activated neutrophils into the alveolar spaces for prevention of bacterial infection. Serratia marcescens is an important, opportunistic human pathogen, especially for immunocompromised patients [4]. During the recent 20 years, S. marcescens has been the cause of an increasing number of nosocomial infections. The most common types of these nosocomial infections are acute pneumonia in immunocompromised or mechanically ventilated patients and chronic pulmonary infection [5]. Treatment of S. marcescensinduced pneumonia is complicated further by the emerging resistance of many clinical strains to multiple antibiotics [5, 6]. Development of alternative drugs or compounds for prevention or even treatment of Serratia infection is thus essential. Resveratrol (RSV; trans-3,5,4⬘-trihydroxystilbene) is a major polyphenolic compound found in red wine. It has diverse biological activities including anti-oxidation, anti-inflammation, anti-fungal infection, anti-cancer, anti-mutagenesis, cardioprotection, and neuroprotection [7–10]. However, our review of the literatures revealed no studies evaluating the in vivo effect of a preventative effect of RSV on bacterial infection. Thus, little is known about the potential of RSV as a chemopreventive compound for bacterial infections. The aim of this study was to investigate the effect of RSV pretreatment on the physiopathology of acute pulmonary infection by bacteria. For this purpose, we used a rat model of acute pneumonia induced by instillation of S. marcescens. This model was used to examine whether RSV pretreatment could reduce the extent of S. marcescens lung infection and its associated mortality rate following bacterial challenge. Physiopathological changes in the lungs of these animals were studied. The effects

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These authors contributed equally to this work. Correspondence: Department of Physiology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, Taiwan, Republic of China, 333. E-mail: [email protected] Received September 24, 2007; revised November 21, 2007; accepted December 3, 2007. doi: 10.1189/jlb.0907647 2

INTRODUCTION Bacterial pneumonia is a respiratory infection characterized by cough with sputum production, high fever, and mortality of 1028

Journal of Leukocyte Biology Volume 83, April 2008

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of RSV on lung bacterial burden and host innate immunity were also evaluated. Our results showed that RSV pretreatment was associated with significantly less-severe, acute pneumonia symptoms and lung bacterial load. In addition, RSV-pretreated rats had markedly increased AM infiltration and elevated NK cell activity together with decreased bacterial burden in lung and a decreased mortality rate. These less-severe pneumonia infections were also associated with decreased TNF-␣, IL-1␤, and IL-6 levels in bronchoalveolar lavage (BAL) fluids. As complex interactions may occur between S. marcescens and animal hosts during the process of acute pneumonia, an acute pneumonia assay induced by LPS, derived from S. marcescens, was subsequently used to further investigate the mechanism responsible for the effect of RSV pretreatment. In these experiments, RSV pretreatment 30 min before LPS administration was associated with an anti-inflammatory effect, inhibition of I␬B␣ phosphorylation, inhibition of NF-␬B translocation, and subsequently, inhibition of NF-␬B binding efficacy onto the promoters of its target genes. Our findings in rats demonstrated that RSV pretreatment ameliorates acute pneumonia initiated by S. marcescens infection and suggested its strong potential to be developed as an agent for the prevention of lung infection.

saline (37°C; 2⫻3 ml). The lungs were perfused with saline, and the right lung was excised; special care was taken to exclude hilar tissues or proximal bronchi. The right lung was minced, suspended in 10 ml normal saline, and homogenized. Aliquots (10 ␮L) from each specimen were serially diluted and plated on LB agar plates containing tetracycline (12.5 ␮g ml–1) and incubated at 37°C. Bacterial colonies were counted at 24 h as CFU per lung and CFU per ml BAL fluid.

Histological examinations Lung tissues were fixed in 10% formalin for 24 h and embedded in paraffin; 4 ␮m sections were cut, stained with H&E, and processed for light microscopy.

Semiquantification of BAL fluid cellularity Cells were harvested from 100 ␮l BAL fluid after centrifugation at 400 rpm for 10 min in a Cytospin 4 (Shandon, UK) and were determined by a hemocytometer (Hausser Scientific, Horsham, PA, USA) analysis. The cells were stained with Diff-Quick stain (IMEB, Japan), and differential cell counts were performed on three random fields per slide with cell types identified using routine methods.

AM phagocytosis assay

Trans-RSV [trans-3,4,5-trihydroxylstilbene] and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). LPS (10 mg kg–1), RSV (0.5 mg kg–1), and dexamethasone (Dex; 0.5 mg kg–1) were dissolved in normal saline right before use.

Phagocytotic activity was assayed according to the protocols of Ojielo et al. [14] with minor modifications. Four hours after administration of S. marcescens, BAL fluid was collected, and alveolar leukocytes were isolated. Total leukocytes in BAL fluid were determined by hemocytometer counting. AMs were isolated from the lungs of uninfected and infected rats. AMs of 50,000 cells per well were plated in a 96-well plate and allowed to adhere for 1 h in serum-free medium. After incubation, medium was removed, followed by addition of 100 ␮l FITC-labeled S. typhimurium (107 CFU/100 ␮l) [15] and 200 ␮l crystal violet (0.8%). After centrifugation for 3 min, supernatant was removed before addition of 150 ␮l-sterilized PBS and 150 ␮l propidium iodide solutions. Phagocytosis efficacy was determined by flow cytometry (FACScan, Becton Dickinson, San Jose, CA, USA) analysis. The percentage of phagocytosis was recorded and phagocytic index (PI) was calculated as: PI ⫽ [(% positive⫻mean channel)/100%].

Animals and the bacterial strains

Cytotoxicity assay

Male Sprague Dawley rats, 6 – 8 weeks old (200 –250 g), were used in this study. Rats in a group of 12 were divided into four groups: Group 1, uninfected, saline-treated; Group 2, uninfected, RSV-treated; Group 3, infected, salinetreated; Group 4, infected, RSV-treated. Food and water were supplied ad libitum. RSV was intragastric-administered to the rats at 0.5 mg kg–1 body weight, three times a day. On Day 4, rats were challenged with S. marcescens CH-1, which was a clinically isolated, virulent strain obtained from Addenbrook Hospital (Cambridge, UK) [11]. FITC-labeled Salmonella typhimurium was obtained from the Department of Clinical Pathology, Chang Gung Memorial Hospital (Taoyuan, Taiwan).

Cytotoxicity assay was performed as described by Zhang et al. [16] with minor modifications. Cytotoxic activity of the spleen NK cells was assessed by the release of 51Cr radioactivity from 51Cr-labeled YAC-1 cells (1⫻105) [16], which were labeled with 200 ␮Ci sodium chromate for 1 h at 37°C and thoroughly rinsed after labeling. Effector (spleen tissue) and target (YAC-1) were coincubated for 4 h at an E:T of 20:1. Spontaneous release of 51Cr was determined by incubating the target cells with medium alone for the same period of time, and the release was consistently less than 10%. Maximum release was determined by adding Triton X-100 to a final concentration of 2%. The specific release was calculated by subtracting data of the spontaneous release from the experimental data.

MATERIALS AND METHODS Drugs and chemicals

Intratracheal (i.t.) inoculation of S. marcescens The protocols for lung infection were as Lyerly et al. [12] with minor modifications. Bacteria were cultivated in Luria-Bertani (LB) broth at 37°C with vigorous shaking (100 rpm) to reach an OD 600 nm of 0.9. Bacterial suspension was diluted to the desired concentration for rat inoculation. Rats were lightly anesthetized with halothane. The trachea were exposed in a sterile atmosphere, and 50 ␮l kg–1 S. marcescens inoculum (9⫻106 CFU) was administered i.t. using a sterile, 26-gauge needle.

Survival studies Rats given RSV or saline pretreatment and i.t.-inoculated with or without S. marcescens were closely monitored for 24 h, and the survival rate and mean survival time were calculated.

Quantification of lung and BAL fluid burden The experiment protocols were as Wang et al. [13]. Following i.t. inoculation, rats were killed at 24 h, and the thoracic cavity was exposed. The left lung was removed intact with the endotracheal tube in place and lavaged with 6 ml

LPS induction of lung inflammation and Dex treatment LPS-induced acute lung inflammation was performed as reported by Fan et al. [17]. Briefly, animals were i.t.-injected with LPS (10 mg kg–1 body weight) derived from S. marcescens by a 26-gauge needle. Thirty minutes before LPS injection, animals were orally fed with or without RSV or Dex at the desired doses by gastric intubation. Rats were divided into four groups (six animals per group): Group 1 rats were vehicle-treated (saline), Group 2 was LPS-treated, Group 3 was fed with RSV at 0.1, 0.25, or 0.5 mg kg–1 before LPS treatment, and Group 4 was fed with Dex at 0.1, 0.25, or 0.5 mg kg–1 before LPS treatment.

Cytokine measurement BAL fluid was centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was used for cytokine measurement. Concentrations of TNF-␣, IL-1␤, and IL-6 were determined by ELISA (Biosource, UK). ODs were read on a microplate reader at 450 nm (Bio-Rad, Hercules, CA, USA). Results were plotted against

Lu et al. Resveratrol attenuates acute pneumonia

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RESULTS RSV reduces mortality and lung bacterial burden

Fig. 1. Survival rate of rats infected with S. marcescens, with or without RSV pretreatment. Rats were pretreated with RSV or saline for 3 days and then inoculated i.t., with or without S. marcescens. Results shown are representative of three independent experiments (n⫽12).

the linear portion of the standard curves. All cytokine levels were expressed as Pg/ml BAL fluid.

Cytokine gene expression Two hours after LPS challenge, the right lung was excised, and total RNA was isolated. TNF-␣, IL-1␤, and IL-6 transcripts were determined by RT-nested PCR. Gene-specific oligonucleotide primers were designed according to the sequences published in the GenBank databases [18].

Animals were divided into four groups (see Materials and Methods), and each group contained 12 rats. After i.t. instillation for 24 h, the survival rate of animals was 67% without RSV pretreatment but was 83% for animals pretreated with RSV (Fig. 1). In addition, the mean survival time was significantly longer in rats pretreated with RSV than without (12.96⫾1.23 h vs. 5.37⫾0.72 h, P⬍0.01). All saline-instillated, control animals were healthy and survived through the entire experimental period. The bacterial burden during infection is dependent on a dynamic balance between the rates of bacterial proliferation and the host clearance efficacy, mainly through phagocytosis by the recruited macrophages [19]. The effect of RSV pretreatment on bacterial burden in the lungs was next investigated 24 h after bacterial challenge. As shown in Figure 2, no bacterial CFU was detected in the uninfected group. A log CFU of 7.52 ⫾ 1.13 was observed in the infected group, and the log CFU was reduced to 4.04 ⫾ 0.82 (P⬍0.001) in the RSVpretreated and infected group (Fig. 2A). A similar reduction in the bacterial CFU in BAL fluid was also observed (Fig. 2B). These results indicated that RSV pretreatment reduces the mortality rate and bacterial load during the process of S. marcescens-induced, acute pneumonia.

Immunofluorescent staining and confocal microscopy Two hours after LPS challenge, tissue sections (4 ␮m) were prepared and blocked with 5% normal donkey serum in PBS for 1 h and incubated with rabbit polyclonal anti-NF-␬B p65 antibodies (1:1000 dilution, Upstate Biotechnology, Lake Placid, NY, USA). Samples were washed once with PBS and incubated with 7-amino-4-methylcoumarin-3-acetic acid-conjugated donkey anti-rabbit IgG (1:1000 dilution, Chemicon International, El Segundo, CA, USA). Nuclei were counterstained with propidium iodide (1:2000 dilution, Molecular Probes, Eugene, OR, USA) and examined under a Zeiss confocal laser-scanning microscope (Model LSM 510, Zeiss, Germany).

Western blot analyses The membrane was blotted with antiphosphorylated I␬B␣, anti-I␬B␣ (Cell Signaling Technology, Beverly, MA, USA), or anti-NF-␬B (Upstate Biotechnology) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies and visualized by ECL (Amersham, Piscataway, NJ, USA).

NF-␬B DNA-binding assay NF-␬B activity was measured using a NF-␬B p65 ELISA-based assay kit (Chemicon International). Nuclear protein (45 ␮g; 50 ␮l) was incubated with immobilized oligonucleotides in a 96-well plate for 1 h containing a NF-␬B consensus sequence to allow the binding of the activated NF-␬B to the target nucleotides. Anti-NF-␬B p65 subunit antibody (1:1000 dilution) was then added and incubated for 1 h at room temperature, followed by HRP-conjugated secondary antibody (1:500 dilution). The binding activity was measured by absorbance at 450 nm (Bio-Rad).

Data analysis Statistical analysis was performed using ANOVA (SPSS12.0 software, SPSS, Chicago, IL, USA) with a correction for multiple comparisons. The difference between results of two assay conditions with a P value ⬍0.05 was considered to be significant.

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Fig. 2. Bacterial counts in lung tissue (A) and BAL fluid (B) isolated from rats, with or without RSV pretreatment and with or without S. marcescens infection. Data are expressed as mean ⫾ SEM from three independent experiments (n⫽6). ***, P ⬍ 0.001. ND, Not determined.

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Lung histopathology in S. marcescens-induced acute pneumonia Lung tissues were examined 24 h after bacterial challenge. In the uninfected lungs, no signs of pneumonia were observed (Fig. 3A, i and ii). In contrast, the infected lungs were diffusely reddened, wet, and swollen (congestion and edema; Fig. 3A, iii), and consolidations of lung tissue are evident. In contrast, in RSV-pretreated and infected lungs, tissue swelling and consolidations were reduced significantly (Fig. 3A, iv). Histological examinations of the infected lungs showed multiple parenchymal foci with alveolar inflammation, vascular congestion, alveolar epithelial cell hypertrophy, interstitial edema, hemorrhage, alveolar necrosis, and fibrin deposition. Infiltration of inflammatory cells including lymphocytes, neutrophils, and macrophages was also observed (Fig. 3B, iii). In RSV-pretreated and infected lungs, inflammation foci were clearly reduced and were mostly restricted to perivascular areas close to infected bronchioles, and the parenchymal injury

and neutrophil infiltration were greatly attenuated (Fig. 3B, iv). Histograms of the uninfected lungs are shown in Figure 3B, i and ii, for comparison.

RSV affects lung cellularity and inflammatory cell activities To investigate whether RSV pretreatment affects inflammatory cell recruitment, the cell composition of the BAL fluid was examined. BAL fluid was collected from killed animals. Twenty-four hours after postinfection, the number of lymphocytes, neutrophils, and macrophages was counted (Fig. 4). In the bacterial-challenged group, intensive leukocyte infiltration in lung tissues was evident, and differences were observed in total pulmonary leukocyte counts between rats with and without RSV pretreatment (2.60⫾0.34⫻106 vs. 2.23⫾0.27⫻106 total cells, respectively; P⫽0.014; Fig. 4A; n⫽6). Different types of cell populations of the BAL fluid, with and without RSV pretreatment, were next calculated. Dense consolidations

Fig. 3. Effect of RSV on gross and microscopic views of the lungs prepared from control and S. marcescensinfected rats. To prepare for histosections, lungs were excised 24 h after infection, fixed in 10%-buffered formalin, dehydrated in graded alcohol, and then embedded in paraffin. Sections (4 ␮m) were cut, mounted on poly-L-lysine-coated slides, and stained with H&E. At least 10 random fields were checked in each section at ⫻100 original magnification using a Zeiss microscope (Axioplan). The representative fields were photographed and presented. (A) Gross view. Arrows indicate lung consolidation areas. (B) Microscopic view. Arrows indicate alveoli filled with neutrophils. (Ai and Bi) –RSV, –S. marcescens infection (CH-1); (Aii and Bii) ⫹RSV, –S. marcescens infection (CH-1); (Aiii and Biii) –RSV, ⫹S. marcescens infection (CH-1); (Aiv and Biv) ⫹RSV, ⫹S. marcescens infection (CH-1).


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compared with that of the nonpretreated rats (P⬍0.01). This finding indicated that RSV pretreatment can modulate the response of NK cells in acute Serratia pneumonia.

RSV modulates BAL fluid cytokine profile and reduces cytokine gene transcription Complex interactions between S. marcescens and rat host are expected during acute lung infection. To further characterize the RSV effect on host innate immunity, LPS derived from S. marcescens was used to induce acute pneumonia, and the RSV effect was assessed. Animals were fed with RSV (0.1, 0.25, and 0.5 mg kg–1), respectively, or were unfed before LPS challenge. Six hours later, animals were killed, BAL was prepared, and the concentrations of TNF-␣, IL-1␤, and IL-6 were measured. In LPS-challenged rats, the BAL TNF-␣, IL-1␤, and IL-6 were increased markedly compared with those of the vehicle group. TNF-␣ was increased from 31.4 ⫾ 5.7 to 2265.3 ⫾ 198.5 pg ml–1 (Fig. 6A). Pretreatment with RSV resulted in a dosedependent decline of the BAL TNF-␣ (Fig. 6A); at a RSV dose

Fig. 4. Effect of RSV pretreatment on pulmonary leukocyte numbers and cell populations following i.t. infection with S. marcescens CH-1. (A) No significant effect of RSV pretreatment on leukocyte numbers was observed in rats instillated with saline. In contrast, RSV pretreatment reduced leukocyte infiltration in S. marcescens CH-1-infected lungs. (B) RSV pretreatment enhanced macrophage recruitment into S. marcescens CH-1-infected rat lungs and reduced the numbers of neutrophils recruited, as compared with saline-pretreated lungs. Data were presented as mean ⫾ SEM (n⫽6). **, P ⬍ 0.01; *, P ⬍ 0.05 (compared with the saline-pretreated and infected group).

comprising primarily neutrophils were observed in saline-pretreated, lung-infection rats (Fig. 4B). In contrast, macrophage infiltration was predominant in RSV-pretreated, bacterial-challenged lungs (Fig. 4B, P⬍0.05). Some macrophages had ingested bacteria in both of these groups (data not shown). Briefly, animals pretreated with RSV showed significantly less neutrophil infiltration but had increased macrophage infiltration in the lungs compared with animals pretreated with saline. We next examined the phagocytotic ability of the macrophages derived from lungs, with and without RSV pretreatment. At 4 h postinfection, the mean PI of the AMs was 63.7 ⫾ 11 and 37.9 ⫾ 8, respectively, between the nonpretreated and RSV-pretreated lungs (P⬍0.05; Fig. 5A). Thus, AMs from RSV-pretreated rats showed comparatively reduced phagocytic capacity after S. marcescens infection. NK cells have been reported to play pronounced roles in innate immunity against development of infections [20]. In this study, the effect of RSV pretreatment on the cytotoxicity of spleen NK cells against YAC-1 cells was investigated. As shown in Figure 5B, the cytotoxic activity of the NK cells derived from RSV-pretreated rats was enhanced significantly 1032


Fig. 5. Effect of RSV pretreatment on phagocytotic activity of the AMs and spleen NK cell activity, with and without S. marcescens CH-1 infection. (A) Phagocytotic activity of the AMs isolated from rat lungs, with or without RSV pretreatment and with or without S. marcescens infection. A total of 50,000 AMs was adhered to 96-well plates in serum-free medium for 1 h. Fluorescently labeled S. typhimurium were then added and further incubated for 2 h. Phagocytotic activity was evaluated by flow cytometry. *, P ⬍ 0.05, compared with AMs from saline-pretreated pneumonia rats. Data were representative of three independent experiments (n⫽6). (B) Cytotoxicity of spleen NK cells was determined as described in Materials and Methods. Data were presented as mean ⫾ SEM from three independent experiments (n⫽6). **, P ⬍ 0.01.


of 0.5 mg kg–1, the TNF-␣ concentration was reduced by 77%. The production of IL-1␤ and IL-6 was attenuated similarly by RSV pretreatment (Fig. 6, B and C). Dex, a conventional anti-inflammatory drug, also exerted a similar effect as that of RSV (Fig. 6). The effect of RSV on BAL cytokine profiles in LPS-challenged rats prompted us to examine whether the regulation is at the transcriptional level. The relative abundance of TNF-␣, IL-1␤, and IL-6 mRNA in the lung tissues was examined by RT followed by nested PCR. Total cellular RNA was isolated from lung homogenates 2 h after LPS challenge. As shown in

Figure D, although TNF-␣, IL-1␤, and IL-6 transcripts were not detected in unchallenged lung homogenates, a marked increase in these cytokine transcripts was observed in LPSchallenged animals. In RSV- and Dex-pretreated animals, the LPS-induced expression of TNF-␣, IL-1␤, and IL-6 transcripts was significantly attenuated.

RSV inhibits LPS-induced NF-␬B activation and I␬B␣ phosphorylation Expression of the proinflammatory cytokine genes is regulated by the NF-␬B signaling pathway. To see if the RSV effect is

Fig. 6. Effect of RSV pretreatment on the synthesis and gene expression of proinflammatory cytokines in lung tissues. Rats were pretreated, with or without RSV (0.1, 0.25, or 0.5 mg kg–1) or Dex (0.1, 0.25, or 0.5 mg kg–1) as indicated. Thirty minutes later, i.t. instillation of LPS (10 mg kg–1) was performed. Animals were killed at 6 h after LPS challenge. BAL fluid was prepared, and the amount of (A) TNF-␣, (B) IL-1␤, and (C) IL-6 was measured by ELISA. Results were expressed as mean ⫾ SEM (n⫽6). **, P ⬍ 0.01; *, P ⬍ 0.05, versus respective LPS-challenged group. For the measurement of gene expression, animals were pretreated with or without RSV or Dex, followed by challenging with or without LPS. Animals were killed 2 h later, and total RNA was prepared from lung tissue. Levels of cytokine gene (TNF-␣, IL-1␤, and IL-6) transcripts were measured by RT-nested PCR as described in the text. (D) The results are representative of three independent experiments.


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mediated through suppression of NF-␬B activation, we compared the nuclear translocation of the NF-␬B p65 subunit in LPS-challenged lungs with and without RSV pretreatment. Figure 7 shows that in vehicle-treated samples, most of the nuclei was stained negative with p65 (Fig. 7A). In LPS-challenged lungs, the p65-positive nuclei (purple) were markedly increased (Fig. 7B), indicating an increased NF-␬B activation by LPS administration. In RSV (0.5 mg kg–1)- or Dex (0.5 mg kg–1)-pretreated lungs, the number of p65-positive nuclei was reduced to a level comparable with that of the vehicle-treated lungs (Fig. 7, C and D). To confirm the results from confocal microscopy, Western blot analysis was performed to compare the relative abundance of NF-кB p65 in the nuclear fractions. Two hours after LPS challenge, lung tissues were harvested, and the nuclear extracts were prepared. As expected, LPS challenge significantly increased the nuclear NF-кB p65 content compared with that of the vehicle-treated sample. Again, similar to Dex, pretreatment with RSV suppressed the translocation (Fig. 8A). An increased nuclear translocation of NF-кB is expected to correlate with an increased DNA binding and vice versa. To see if such correlation does exist in LPSchallenged and LPS-challenged, RSV-pretreated animals, nuclear extracts were prepared and assayed for consensus nucleotide fragment binding by NF-кB p65. As shown in Figure 8B, the DNA-binding activity of NF-кB p65 in the nuclear extract was increased significantly upon LPS challenge compared with that of the vehicle group. Pretreatment with RSV or Dex before LPS challenge resulted in a significant decrease in NF-кB p65 DNA-binding activity (P⬍0.01). The results clearly indicated

that RSV attenuates LPS-induced lung inflammation via modulation of the NF-кB pathway. The translocation of NF-␬B to the nucleus is preceded by the phosphorylation, ubiquitination, and proteolytic degradation of I␬B␣ [21]. To determine whether inhibition of LPSinduced NF-␬B activation by RSV pretreatment was a result of inhibition of I␬B␣ phosphorylation, we compared the extent of I␬B␣ phosphorylation by Western blot analysis. Figure 8C showed that LPS challenge increases the level of I␬B␣ phosphorylation compared with vehicle-treated and RSV-pretreated, LPS-challenged rats. Furthermore, the amount of I␬B␣ was reduced by LPS treatment, and pretreatment of RSV increased the I␬B␣ level (Fig. 8D). Thus, the reduced nuclear translocation of NF-␬B by RSV pretreatment is closely related to a reduced I␬B␣ phosphorylation and increase of I␬B␣ concentration.

DISCUSSION In recent decades, S. marcescens has been consistently, closely related to hospital-acquired infections [4]. It causes a wide spectrum of infections in addition to pneumonia, such as meningitis, septicemia, urinary tract infection, endocarditis, conjunctivitis, and wound infection [5, 22]. S. marcescens readily adheres to invasive hospital instrumentation, such as catheters, endoscopes, and i.v. tubing [5], and is relatively resistant to standard disinfection protocols [22, 23]. A recent study about ventilated patients’ infection found that a relatively

Fig. 7. Localization of NF-␬B p65 in lung tissue by immunohistochemical analysis. Two hours after LPS administration, the rat lung tissues were collected and sectioned. Lung sections from the LPS-challenged rats with (C) or without (B) RSV (0.5 mg kg–1) pretreatment were immunohistochemically stained. Lung sections from saline-exposed rats (A) were used as negative controls. Blue fluorescence spots observed indicated localization of NF-␬B p65. For comparison, the nuclei of lung tissues were counterstained in red using propidium iodide. The number of NF-␬B p65-positive nuclei (purple) was markedly increased in the LPS-challenged rodents (B). In lungs pretreated with RSV (C) or Dex (D), the number of p65-positive nuclei was reduced to a level comparable with that of the vehicle-treated lungs (A). Each photograph is a representative of six separate animals, taken from at least three different fields (⫻400 original magnification).

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Fig. 8. RSV pretreatment inhibits I␬B␣ phosphorylation and NF-␬B activation. (A) Effect of RSV on LPS-induced NF-␬B p65 nuclear translocation. Pretreated animals were killed 2 h after LPS challenge, and lung tissues were collected. Nuclear fractions were prepared for quantification of NF-␬B p65 by Western blotting. Lamin B was blotted in parallel as the internal control. (B) Effect of RSV on NF-␬B p65 DNA binding. Animals were treated and killed 2 h after LPS challenge. Lung tissues were collected and nuclear fractions prepared. DNA-binding activity of the NF-␬B p65 in the nuclear fractions was assessed. Results were expressed as mean ⫾ SEM (n⫽6). **, P ⬍ 0.01, compared with that of the LPS-challenged group. (C) Effect of RSV pretreatment on I␬B␣ phosphorylation (p-I␬B␣) and amount of I␬B␣. Animals were killed 2 h after LPS challenge. Lung tissues were collected and cytosolic fractions prepared. LPS treatment increased I␬B␣ phosphorylation compared with that of the vehicle-treated group. RSV or Dex significantly suppressed the LPS-induced I␬B␣ phosphorylation. Total cellular I␬B␣ was reduced by LPS challenge and was increased by RSV or Dex pretreatment. ␤-Actin was used as the internal control. Results were expressed as mean ⫾ SEM (n⫽6). (D) The percentage of I␬B␣ band intensity relative to that of ␤-actin was measured by densitometry. §, Significant difference between LPS group and the vehicle control group (P⬍0.05); *, significant difference between RSV/Dex groups and the vehicle control group (P⬍0.05).

high rate of Gram-negative pneumonias was identified, with S. marcescens involved in 12.1% of cases [24]. As a result of the situation of prevalent multi-drug-resistant S. marcescens strains and the potentiality that use of RSV might not generate pressure for emergence of drug-resistant S. marcescens strains, the ability of RSV to modulate host immunity might make it particularly suitable for use in prevention of S. marcescens lung infections. RSV has been shown to have anti-oxidative and anti-inflammatory properties, and previous studies of RSV have largely been related to its effects on health benefits and chemoprevention of cancer and coronary heart disease [7]. Previous studies have shown that RSV is effective against some viral infections [25, 26], and in vitro studies also showed some antibacterial activities [27–30]; however, there has been no report shown that it can be used as an antibiotic agent. One of the main

reasons is probably because relatively high concentrations are needed to show bacteriostatic efficacy. In comparison, the in vivo prophylactic effect of RSV on bacterial infections had remained uncharacterized. In the present study, we showed that RSV pretreatment modulates host innate immunity during S. marcescens lung infection and significantly ameliorates the pathophysiology of resulting acute pneumonia. The pulmonary lesions in rats induced by S. marcescens CH-1 in this study were typical of those observed in acute pulmonary infection in humans with severe necrosis of bronchoalveolar structures, vascular congestion, and neutrophil infiltration. During the lung-infection process, cytokines have been shown to be important, soluble mediators responsible for coordinating the inflammation response [31]. Many cytokines are known to be involved in host inflammation and antibacterial defenses within the lungs. TNF-␣ is capable of recruiting Lu et al. Resveratrol attenuates acute pneumonia

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inflammatory cells to the site of infection, directly and via up-regulation of adhesion molecules [32]. After recruitment of phagocytic cells, TNF-␣ can also promote antimicrobial activity by activating respiratory burst activity [33] and increasing degranulation of macrophages [34]. These effects have been shown to be required for effective in vivo host defense against a range of microorganisms, including Klebsiella pneumoniae [35]. IL-1␤ shares several activities of TNF, including promotion of neutrophil recruitment and activation of macrophages at the site of infection [36]. In some situations, these two cytokines act synergistically to exert their effects [37]. IL-6 has been ascribed pro- and anti-inflammatory effects. IL-6 can activate monocytes [38] and synergize with TNF-␣ to increase the respiratory burst of neutrophils [39]. Our results indicated that RSV pretreatment reduced neutrophil influx into the alveolar compartment after S. marcescens infection, and this reduction in neutrophil influx was associated with decreased TNF-␣, IL-1, and IL-6 levels in BAL fluid during the early phase of infection (Fig. 6). These findings suggested that RSV pretreatment might have prevented greater lung damage through decreasing neutrophil aggregation and inflammatory responses. Our studies showed a pleiotropic, modulatory effect from RSV on host innate immunity during the process of S. marcescens lung infection. For example, RSV pretreatment decreased resulting lung inflammation, as evidenced by lesssevere lung pathology and reduced cytokine synthesis and neutrophil aggregation. Besides, macrophage infiltration was a predominant finding in lungs of rats pretreated with RSV in comparison with rats without RSV pretreatment (Fig. 4B). AMs are the resident mononuclear phagocytes of the lung and are vital in the initial host response to pulmonary infections [19]. They mediate the phagocytic response to infecting bacteria for bacterial clearance. An increase in aggregation of macrophages may indicate recruitment of a large number of macrophages in the encounter of heavy bacterial load during acute lung infection. This is beneficial for bacterial clearance. In response to S. marcescens infection of the distal airways, macrophages have the capacity to ingest bacteria and produce inflammatory mediators that are important for host defense. Interestingly, although outnumbered, AMs from RSV-pretreated rats exhibited a lower phagocytic capability (Fig. 5A). This was related to the observation that fewer macrophages took up bacteria in the lungs of RSV-pretreated rats (data not shown). As macrophage activity is closely related to respiratory burst responses, such as release of reactive oxygen species and reactive nitrogen species, which are generated endogenously in the rat host [40], it was not unexpected that a reduction in macrophage activity was observed with RSV pretreatment. Thus, for some reasons not totally understood, RSV pretreatment helped clear bacteria by recruitment of a large number of macrophages and at the same time, reduced the host respiratory burst inflammation by decreasing neutrophil aggregation, leading to better lung protection. A similar in vitro reduction of the RSV phagocytosis effect on Kluyveromyces lactis cells was also reported by Leiro et al. [40]. These results suggested RSV might involve balance of host innate immunity during S. marcescens lung infection. Another major effect of RSV pretreatment observed in this study was the increase in NK cell activity measured using 1036


harvested spleen cells (Fig. 5B). NK cells play important roles in modulation of host innate immunity against lung-infecting bacterial cells such as Mycoplasma spp. [41], Bordetella pertussis [42], and Mycobacterium tuberculosis [16]. Previous studies showed that NK cells, which are central components of innate immune response, do not directly attack invading bacterial cells but instead attack the infected host cells [20]. It is also reported that NK cells secrete IFN-␥, which recruits AMs to kill opsonized Mycoplasma pulmonis [43]. Our studies have detected a slight increase in IFN-␥ in BAL fluid of RSVpretreated rats after challenge with S. marcescens cells (data not shown). This finding indicated that RSV might increase the NK cell activity, leading to an increase of IFN-␥ production and recruitment of macrophages for protection of S. marcescens lung infection. After LPS administration, rats with RSV pretreatment showed lower levels of NF-␬B activation than those pretreated with vehicle (Fig. 7). The NF-␬B family of transcription factors is an important regulator of innate immunity [44]. Activation and nuclear translocation of NF-␬B have been associated with increased transcription of cytokines (TNF-␣, IL-1, and IL-6). In our experimental model, LPS challenge with vehicle pretreatment resulted in a significant increase in NF-␬B migration into the nuclear compartment 2 h after infection. By contrast, RSV pretreatment significantly inhibited translocation of NF-␬B p65 and p50 transcription factors into the nuclear compartment of lung resident cells (Fig. 8). We further observed that the anti-inflammatory effects occurred via suppression of the phosphorylation of I␬B␣ and the subsequent nuclear translocation of NF-␬B (Fig. 8). Thus, the underlying mechanism of amelioration of LPS or S. marcescens-induced acute pneumonia might be, at least in part, a result of negative regulation of NF-␬B signaling. The RSV effect observed is similar to those of previous studies about different research models, including skin cancer [45] and HSV-infected cells [46], further confirming that RSV protects against tissue damage by inhibition of NF-␬B activation. In summary, this study showed that pretreatment with RSV reduces the risk of S. marcescens lung infections and ameliorates lung tissue damage. Also, a clearer understanding of the cellular and molecular targets of RSV action within the innate immune system during lung infections initiated by S. marcescens cells or LPS was provided. Our findings also supported that the anti-inflammatory effects of RSV pretreatment occur through inhibition of the NF-␬B signaling pathway by suppressing the phosphorylation of I␬B␣ and the subsequent nuclear translocation of NF-␬B. These results implied that RSV has excellent potential to be developed as a pretreatment for patients at high risk of acute pneumonia as a result of S. marcescens infection. Further exploration of RSV as an agent for the treatment of existing antibacterial lung infection is also warranted.

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