ABSTRACT
The incidence of community-associated methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in previously healthy individuals has increased in the past 5 years. Such infections are associated with bronchiectasis and high mortality rates, making them a significant public health concern. The mechanisms of host defense against this pathogen are not well characterized. However, patients diagnosed with MRSA, as opposed to methicillin-susceptible S. aureus (MSSA), are more likely to have abused alcohol in the past, and these patients are more likely to die from sepsis. In the United States, USA300 is the predominant strain that causes necrotizing pneumonia. To investigate whether acute ethanol exacerbates MRSA pneumonia, mice were intraperitoneally (i.p.) administered 2 or 4 g/kg of ethanol 30 min prior to oropharyngeal inoculation of 2 × 107 CFU of USA300. An increased pulmonary bacterial burden was observed in alcohol-intoxicated mice at 16 and 24 h and was associated with decreased levels of interleukin 6 (IL-6). IL-6 activates signal transducer and activator of transcription 3 (STAT3) as part of an acute-phase response of infection. Reg3γ is an antimicrobial C-type lectin that is induced by STAT3 signaling in response to Gram-positive bacteria. Previously, in situ hybridization studies showed that Reg3g is highly expressed in lung epithelium. In the present study, we found that acute ethanol exacerbated USA300 in a murine model of USA300 pneumonia. This was associated with reduced IL-6 expression in vivo as well as inhibition of IL-6 induction of STAT3 signaling and Reg3g expression in mouse lung epithelial (MLE12) cells in vitro. Furthermore, recombinant Reg3γ administration 4 h after MRSA infection in alcohol-intoxicated mice rescued USA300 clearance in vivo. Therefore, acute alcohol intoxication leads to decreased MRSA clearance in part by inhibiting IL-6/STAT3 induction of the antimicrobial protein Reg3γ in the pulmonary epithelium.
INTRODUCTION
Globally, the economic burden of alcohol is estimated to be one to three percent of total health costs (1). Alcohol, as the most abused substance in the United States (2), causes many health problems, including liver disease and cancer, and increases the risk of several infectious diseases such as bacterial pneumonia. Historically, alcohol abuse has been associated with an increased susceptibility to infection with the pulmonary pathogens Klebsiella pneumoniae (3), Streptococcus pneumoniae (4), and Escherichia coli (5). Although 89 million Americans (30% of the population) are colonized with Staphylococcus aureus and 2.3 million people are colonized with methicillin-resistant S. aureus (MRSA) strains (6, 7), MRSA pneumonia has only recently been studied in the context of alcohol consumption. This opportunistic Gram-positive bacterium lies dormant in mucocutaneous epithelia such as the mouth or nose until a break in the skin or a decline in immune defenses lead to infections such as impetigo, folliculitis, or cellulitis (8). In an immunocompromised host, severe life-threatening infections such as meningitis, endocarditis, and pneumonia can occur, causing significant morbidity and mortality (8). A retrospective study of fatal outcomes from MRSA septicemia found a significant odds ratio (OR) with alcoholism (OR, 4.6; P < 0.001) (9). Furthermore, a prospective study of 190 patients with MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) found that MRSA patients are more likely to have abused alcohol than MSSA patients (10). The rise in community-acquired MRSA pneumonia cases among young healthy individuals (11) prompted this study on the impact of acute ethanol (EtOH) consumption on susceptibility to MRSA infection using a mouse model of MRSA pneumonia.
The lung epithelium secretes chemokines, cytokines, and antimicrobial proteins in response to bacterial pathogens (12) and provides the first line of immune defense against pulmonary pathogens. In addition, the epithelium plays a crucial role in serving as a structural barrier and orchestrating mucociliary clearance of pathogens. Although adaptive immunity is important to clear MRSA in the skin of mice (13), it is dispensable for clearance of MRSA in the lung because Rag2/Il2rg−/− mice, which are deficient in B, T, and NK cells, are resistant to infection (14, 15). However, patients with inherited (autosomal dominant) hyper-IgE syndrome (HIES) still present with recurrent S. aureus pulmonary infections due to loss-of-function mutations in signal transducer and activator of transcription 3 (STAT3) (16). The interleukin-6 (IL-6) family of cytokines binds to the Gp-130 receptor, phosphorylating STAT3 and allowing its dimerization. Activated STAT3 is a master transcription factor involved in the generation of Th17 cells, T cell-dependent IgG responses, and the induction of antimicrobial factors (e.g., β-defensins) in response to staphylococcal and Candida infections (16–18). In addition, STAT3 is important in epithelial homeostasis, wound healing, and regulation of antimicrobial production (19, 20). Although acute alcohol decreases the expression of STAT3-dependent genes in vitro (21), it is unclear whether this inhibition of STAT3-dependent genes plays a role in lung epithelial defense during MRSA challenge (22–24). This series of experiments tested the hypothesis that acute ethanol would suppress STAT3 phosphorylation in the lung and reduce clearance of MRSA.
Acute ethanol or vehicle control was administered prior to oropharyngeal aspiration (OA) of USA300. Acute alcohol markedly increased the pulmonary burden of USA300 at 16 and 24 h. This was also associated with decreased production of IL-6 and increased expression of the gene for suppressor of cytokine signaling 3 (Socs3), the main inhibitor of STAT3, and reduced Reg3g transcript levels in the lung; Reg3γ has previously been shown to be regulated by IL-6/Gp-130/STAT3 signaling and has both bacteriostatic activity and bactericidal activity against MRSA (14). Mouse lung epithelium (MLE12) cells exposed to 50 mM acute ethanol also displayed decreased IL-6-stimulated STAT3 activation and an increase in Socs3 expression. Consistent with inhibited STAT3 signaling, ethanol also reduced Reg3g expression in vitro. Finally, in vivo administration of recombinant Reg3γ was effective at augmenting clearance of USA300 infection in the presence of acute ethanol. Taking the results together, we determined that USA300 initiates a STAT3-dependent induction of Reg3γ and this signaling pathway is perturbed by acute ethanol. The use of recombinant Reg3γ may be a novel way to augment mucosal immunity to MRSA infection in at-risk hosts.
MATERIALS AND METHODS
Mice.C57BL/6 (wild type [WT]) mice obtained from Charles River Laboratories were housed in specific-pathogen-free rooms within animal facilities at the Louisiana State University Health Sciences Center or the Rangos Research Building at Children's Hospital of Pittsburgh. To model acute ethanol intoxication, WT mice received 0, 2, or 4 g/kg of 20% ethanol (EtOH) in phosphate-buffered saline (PBS) administered intraperitoneally. Mice were monitored for 30 min and then inoculated with 2 × 107 CFU of USA300 by oropharyngeal aspiration. The lung burden (CFU) was determined at 0, 4, 16, and 24 h after infection. All protocols were approved by the IACUCs at Louisiana State University Health Science Center and the University of Pittsburgh.
OA of MRSA in mice.A single colony of the MRSA USA300 strain (from Frank R. DeLeo's lab at NIAID) was grown for 18 h at 37°C in 5 ml of BBL Trypticase soy broth (TSB) (BD). After this period, fresh TSB was inoculated with the bacteria at a 1:100 dilution and incubated at 37°C for 2 h with shaking to obtain bacteria during the log phase of growth. Bacteria were washed with PBS, resuspended in 1 ml of PBS, and diluted five times to obtain approximately 4 × 108 CFU/ml of working stock. Fifty microliters of this working stock was administered with the tongue pull method at a dose of approximately 2 × 107 CFU of USA300 in the lung. The working stock was serially diluted and plated on LB plates to determine the exact concentration of the inoculum. For Reg3γ rescue experiments, recombinant Reg3γ-Fc versus IgG control or Reg3γ-cleaved versus PBS were administered at a dose of 60 mg per mouse by oropharyngeal aspiration (OA) to USA300-infected mice 4 h after infection.
Lung bacterial burden, mRNA, and protein analysis.Lungs were harvested in PBS, homogenized with glass homogenizer, and plated on LB in three serial dilutions. Colonies were counted after overnight incubation at 37°C. Gene expression was determined by RNA extraction with TRIzol (Invitrogen), reverse transcription by iScript (Bio-Rad), and quantitative PCR (qPCR) with TaqMan probes (Applied Biosystems) on an iCycler thermocycler (Bio-Rad). All threshold cycle (CT) values were normalized to Hprt. Lung homogenates were analyzed using bead-based Luminex assays (Millipore) for protein levels of IL-1β, IL-6, and Lif.
MLE12 cell culture and IL-6 stimulation.MLE12 (ATCC CRL-2110) cells were regularly passaged in Dulbecco modified Eagle medium (DMEM)–F-12 medium supplemented with 1.94 mM l-glutamine, 10 mM HEPES, 5 μg/ml insulin, 10 μg/ml transferrin, 10 nM hydrocortisone, 10 mM β-estradiol, penicillin, streptomycin, and 2% fetal bovine serum. Cells were seeded at 2 × 106 cells/ml in 24-well plates 1 day before stimulation. Cells were stimulated with IL-6 (10 ng/ml) (R&D) for 15 min to measure P-STAT3 by enzyme-linked immunosorbent assay (ELISA) and for 2 h to measure Socs3 and Reg3g mRNA levels. For the STAT3 reporter assay, MLE12 cells were stably transduced with a STAT3-luciferase reporter using a lentiviral construct (SA-Biosciences). Luminescence was read 24 h after stimulation with IL-6 (10 ng/ml).
Histologic analysis.Animals were euthanized after infection with USA300. The left lung was inflated with 10% neutral buffered formalin (Sigma Chemical Co, St. Louis, MO) at a pressure of 25 cm H2O for 10 min, removed from the animal, and placed in fresh 10% neutral buffered formalin for 24 h before processing and paraffin embedding. For histologic analysis, 5-μm sections were cut and stained with hematoxylin and eosin (H&E). Histologic scoring was performed by a pathologist reviewer (D.A.P.) on blinded lung sections. Lung injury induced by USA300 was assessed by a numerical scoring scale from 0 to 3 (increasing in severity): 1, mild peribronchiolar/perivascular inflammation; 2, peribronchiolar/perivascular and alveolar inflammation; and 3, peribronchiolar/perivascular and alveolar inflammation with debris in bronchiolar lumen.
Statistical analysis.For multiple comparisons and statistical analyses with three or more groups, data were analyzed using analysis of variance (ANOVA) followed by nonparametric Kruskal-Wallis testing. For two groups, nonparametric data were analyzed with the Mann-Whitney test and parametric data were analyzed with Student's t test. All analysis was performed with Prism software (GraphPad).
RESULTS
Acute alcohol intoxication increases pulmonary MRSA burden in a dose-dependent manner.Acute alcohol intoxication was modeled as previously described by i.p. administration of EtOH 30 min before OA aspiration of 2 × 107 CFU of MRSA USA300 (3). Acute alcohol at a dose of 2 g/kg increased the MRSA lung burden 5-fold at 16 h after infection. EtOH at 4 g/kg increased the MRSA lung burden 21-fold at 16 h and 8-fold at 24 h after infection (Fig. 1A). The higher dose of EtOH was also associated with increased peribronchial inflammation (Fig. 1B to E).
Acute alcohol intoxication increases the pulmonary MRSA burden (A). Quantification of the MRSA lung burden was done by CFU assay at 16 and 24 h after oropharyngeal aspiration (OA) administration of USA300 (2 × 107 CFU) in control mice and mice treated with 2 g/kg (lung burden increased 5-fold at 16 h after infection) or 4 g/kg (increased 21-fold at 16 h and 8-fold at 24 h) EtOH (n = 5 to 10 in each group). P values were calculated by ANOVA and the Kruskal-Wallis test/Dunn multiple-comparison test as indicated. Error bars represent the standard error of the mean (SEM). (B and C) Representative lung histology (magnification, ×100) at 16 h after USA3000 infection in control (B) or EtOH-treated (dose, 4 g/kg) (C) animals. (D) Representative histology demonstrating inflammatory exudate in the airway of EtOH-treated mice (magnification, ×400). (E) Histological scoring of lung inflammation in control and EtOH-treated (dose, 4 g/kg) mice (n = 5 per group, P value calculated by the Mann-Whitney test).
Acute alcohol intoxication attenuates IL-6 production during MRSA pneumonia.Since it was previously demonstrated that IL-1β and Gp-130/STAT3 signaling is necessary for MRSA clearance (14, 25), protein levels of IL-1β and Gp-130 ligands were analyzed by Luminex assay at 0, 4, 16, and 24 h after infection. IL-1β, Lif, and IL-6 were substantially induced at 4 h after infection and were decreased with MRSA clearance in control mice at 16 and 24 h (Fig. 2A to C). Although Lif was marginally increased with alcohol at 16 h after infection (P > 0.05), IL-6 induction was substantially decreased at 4 h after infection (P < 0.001) (Fig. 2B and C).
Acute alcohol intoxication attenuates IL-6 production during MRSA pneumonia. Lung homogenate cytokine levels at 0, 4, 16, and 24 h after MRSA administration were determined by Luminex assay in control mice versus mice treated with 2 g/kg EtOH (n = 5 in each group). P values were calculated by the unpaired t test with the Holm-Sidak multiple-comparison correction. Error bars represent the SEM.
Alcohol inhibits IL-6/Gp-130-stimulated STAT3 activation in lung epithelial cells.Activation of STAT3 as determined by immunohistochemistry for phosphorylated STAT3 is greatly enhanced in epithelial cells during MRSA pneumonia (14). STAT3 signaling in the epithelium leads to transcription of genes that regulate epithelial repair and barrier function, as well as antimicrobial factors (12). Since alcohol had varied effects on the induction of different Gp-130 ligands, it was hypothesized that ethanol may also inhibit activation of STAT3. To determine whether ethanol directly inhibits Gp-130-induced epithelial STAT3 activation, MLE12 cells were treated with IL-6 at 1 h after incubation with 50 mM EtOH. There were increased P-STAT3 levels seen with IL-6 stimulation, with a mild but not statistically significant attenuation with acute EtOH (Fig. 3A). However, IL-6 treatment resulted in a significant increase in STAT3-luciferase reporter-expressing MLE12 cells (Fig. 3B). The addition of 50 mM EtOH at 1 h prior to IL-6 treatment substantially reduced IL-6 induced STAT3 activation in the reporter assay (Fig. 3B).
Alcohol inhibits IL-6-stimulated STAT3 activation in MLE12 cells. (A) IL-6 (10 ng/ml) stimulation in MLE12 cells stimulated STAT3 activation as indicated by P-STAT3 ELISA (n = 4 per group) performed 10 min after stimulation, and the phosphor-ELISA signal trended lower with 50 mM ETOH (P = 0.09 by ANOVA with multiple-comparison correction). OD450, optical density at 450 nm. (B) STAT3 luminescence reporter activity at 24 h after stimulation (n = 11 in each group). P values were calculated using 2-way ANOVA and Dunn's multiple-comparison test. Error bars represent the SE.
Acute alcohol increases Socs3 expression in MLE12 cells in vitro and in lung tissue during MRSA pneumonia in vivo.It has been previously reported that in human monocytes acute ethanol ingestion can induce SOCS3, which in turn can inhibit Gp-130 activation of STAT3 (23). Thus, this mechanism may play a role in ethanol-induced exacerbation of MRSA pneumonia. Indeed during MRSA pneumonia, acute alcohol increased levels of Socs3 expression in vivo (Fig. 4A). Furthermore, IL-6-stimulated Socs3 expression was increased with 50 mM ethanol in MLE12 cells (Fig. 4B). Therefore, alcohol may act through Socs3 to inhibit Gp-130-induced STAT3 signaling during MRSA pneumonia.
Acute alcohol inhibits STAT3 activation by increasing Socs3 expression. (A) WT B6 mice administered 2 g/kg EtOH at 30 min before OA USA300 infection were euthanized at 0, 4, 16, and 24 h after infection. Socs3 expression was determined by real-time PCR and normalized to Hprt (n = 5 in each group). (B) MLE12 cells were exposed to 50 mM EtOH in the medium at 1 h before stimulation with 10 ng/ml IL-6. Socs3 expression was determined by real-time PCR of mRNA extracted 2 h after stimulation and normalized to Hprt. P values (determined with the Mann-Whitney test) are indicated. Error bars represent the SE.
Alcohol decreases IL-6 induction of Reg3g in MLE12 cells, and recombinant Reg3g rescues decreased MRSA clearance due to acute alcohol in vivo.Previous results have shown that STAT3 directly activates transcription of the antistaphylococcal protein Reg3γ from MLE12 cells (14). In the presence of alcohol, Reg3g expression in response to IL-6 stimulation was decreased (Fig. 5A). To determine whether Reg3γ can be used therapeutically, recombinant Reg3γ was administered by OA at 4 h after infection. Similar to the results shown in Fig. 1, there was a 20-fold increase in the lung burden in mice administered ethanol. Furthermore, the administration of Reg3γ reduced the lung burden by 4-fold and rescued the increase in lung burden due to 4 g/kg acute alcohol (Fig. 5B). Although Reg3γ treatment reduced CFU, the histologic scoring of peribronchial lung inflammation did not differ at this time point, with scores of 2.21 ± 0.32 and 1.86 ± 0.18 in the EtOH control IgG group and the EtOH Reg3γ group, respectively.
Alcohol decreases IL-6 induction of Reg3g, and recombinant Reg3γ rescues decreased MRSA clearance due to acute alcohol. (A) MLE12 cells were exposed to 50 mM EtOH in the medium at 1 h before stimulation with 10 ng/ml IL-6. Reg3g expression was determined by real-time PCR of mRNA extracted 2 h after stimulation and normalized to Hprt (n = 4 in each group). (B) WT B6 mice were i.p. administered 4 g/kg EtOH at 30 min before OA USA300 infection. Four hours after infection, recombinant Reg3γ-Fc (60 μg) or a mouse IgG2A control was OA administered. Lung burden was determined by CFU assay at 16 h after infection (n = 5 in each group). Data are representative of two independent experiments. P values were calculated using 2-way ANOVA and Tukey's multiple-comparison test. Error bars represent the SE.
DISCUSSION
Although alcoholism has been linked to increased mortality due to MRSA pneumonia as a part of a clinical study, this is the first scientific study to model the interactions between alcohol and MRSA pneumonia (10). Furthermore, no other study has examined the effects of acute alcohol in compromising host defense against MRSA pneumonia. One underlying mechanism is that acute alcohol inhibits STAT3 induction of the antistaphylococcal protein Reg3γ by increasing Socs3 activation in the pulmonary epithelium. Acute alcohol inhibits IL-6-stimulated P-STAT3 transcription and Reg3g expression in MLE12 cells and increases Socs3 expression in vivo during MRSA pneumonia. Furthermore, recombinant Reg3γ rescued the impaired MRSA clearance in acute alcohol-intoxicated mice.
Another consideration with the increased number of STAT3 signaling inhibitors proposed as cancer therapies is the potential for compromised immune host defense (26). STAT3 activates many genes involved in host defense, as shown by the clinical phenotypes of hyper-IgE syndrome patients with STAT3 mutations, who are predisposed to multiple skin and pulmonary infections (16). Acute alcohol compromises the host defense against Streptococcus pneumoniae by increasing STAT3-p27Kip1 signaling in response to colony-stimulating factor 3 (Csf3) in bone marrow cells and ultimately suppress granulopoiesis by inducing cell cycle arrest (24). The decreased STAT3 signaling due to acute alcohol in this current study might be an effect of acute EtOH limited to this specific cell type or localized to the IL-6/Gp-130 pathway. However, the inhibition of the IL-6/Gp-130/STAT3 pathway with acute alcohol has also been observed in human monocytes, and this suppression was mediated by increases in SOCS3 by alcohol (23).
The Reg3 family of soluble C-type lectins has been characterized in the gastrointestinal system. Epithelial production of Reg3γ is protective against intestinal Listeria monocytogenes (27). Recently, Reg3β and Reg3γ production in the intestine was shown to be suppressed by alcohol both in mice exposed to 3 weeks of alcohol and in chronically alcoholic human subjects (28). However, no other study has examined alcohol's effects on Reg3γ in the host defense against bacterial pneumonia. In conclusion, acute alcohol intoxication leads to an increased MRSA lung burden by inhibiting STAT3 activation of Reg3γ production by the pulmonary epithelium. Furthermore, recombinant Reg3γ was effective in rescuing the increased MRSA burden in mice administered alcohol and can potentially be developed as a therapeutic for MRSA pneumonia. Reg3γ did not reduce lung injury at the 16-h time point; however, this may be due to the fact that lung inflammation can be slower to resolve than bacterial clearance. Additionally there may be other STAT3-regulated genes or other antimicrobial factors such as the defensins or calgranulins that could also be affected by acute EtOH, which need to be considered as well.
A more comprehensive clinical study on chronic and acute alcohol intoxication should be performed to determine if specific types of alcohol ingestion predispose to MRSA pneumonias in humans. However, in this mouse model, acute alcohol exacerbates MRSA pneumonia. Thus, it may be important for clinicians to ascertain recent alcohol intake as a potential risk factor for complicated MRSA pneumonia.
ACKNOWLEDGMENTS
This work was supported by NIH NIAAA grants R01AA016688, 5P60AA009803, 5F31AA021061, and 5T32AA007577.
FOOTNOTES
- Received 7 August 2013.
- Returned for modification 8 September 2013.
- Accepted 31 December 2013.
- Accepted manuscript posted online 13 January 2014.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
