Published Ahead of Print 13 January 2014. 2014, 82(4):1402. DOI: 10.1128/IAI.00974-13. Infect. Immun. 

Jay K. KollsDerek A. Pociask, Rachel A. Armentrout, Kong Chen and Sun-Mi Choi, Jeremy P. McAleer, Giraldina Trevejo-Nunez, 

γEpithelial Production of Reg3aureus Clearance in the Lung by ImpedingMethicillin-Resistant Staphylococcus Acute Alcohol Intoxication Impairs

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Acute Alcohol Intoxication Impairs Methicillin-ResistantStaphylococcus aureus Clearance in the Lung by Impeding EpithelialProduction of Reg3�

Sun-Mi Choi,a Jeremy P. McAleer,b Giraldina Trevejo-Nunez,b Derek A. Pociask,b Rachel A. Armentrout,b Kong Chen,b Jay K. Kollsa,b

Department of Genetics, Louisiana State University Health Science Center, New Orleans, Louisiana, USAa; Richard King Mellon Foundation Institute for Pediatric Research,Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USAb

The incidence of community-associated methicillin-resistant Staphylococcus aureus (MRSA) pneumonia in previously healthyindividuals has increased in the past 5 years. Such infections are associated with bronchiectasis and high mortality rates, makingthem a significant public health concern. The mechanisms of host defense against this pathogen are not well characterized. How-ever, patients diagnosed with MRSA, as opposed to methicillin-susceptible S. aureus (MSSA), are more likely to have abused al-cohol in the past, and these patients are more likely to die from sepsis. In the United States, USA300 is the predominant strainthat causes necrotizing pneumonia. To investigate whether acute ethanol exacerbates MRSA pneumonia, mice were intraperito-neally (i.p.) administered 2 or 4 g/kg of ethanol 30 min prior to oropharyngeal inoculation of 2 � 107 CFU of USA300. An in-creased pulmonary bacterial burden was observed in alcohol-intoxicated mice at 16 and 24 h and was associated with decreasedlevels of interleukin 6 (IL-6). IL-6 activates signal transducer and activator of transcription 3 (STAT3) as part of an acute-phaseresponse of infection. Reg3� is an antimicrobial C-type lectin that is induced by STAT3 signaling in response to Gram-positivebacteria. 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 reducedIL-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 Reg3� administration 4 h after MRSA infection in alcohol-intoxicated micerescued USA300 clearance in vivo. Therefore, acute alcohol intoxication leads to decreased MRSA clearance in part by inhibitingIL-6/STAT3 induction of the antimicrobial protein Reg3� in the pulmonary epithelium.

Globally, the economic burden of alcohol is estimated to be oneto three percent of total health costs (1). Alcohol, as the most

abused substance in the United States (2), causes many healthproblems, including liver disease and cancer, and increases therisk of several infectious diseases such as bacterial pneumonia.Historically, alcohol abuse has been associated with an increasedsusceptibility to infection with the pulmonary pathogens Kleb-siella pneumoniae (3), Streptococcus pneumoniae (4), and Esche-richia coli (5). Although 89 million Americans (30% of the popu-lation) are colonized with Staphylococcus aureus and 2.3 millionpeople are colonized with methicillin-resistant S. aureus (MRSA)strains (6, 7), MRSA pneumonia has only recently been studied inthe context of alcohol consumption. This opportunistic Gram-positive bacterium lies dormant in mucocutaneous epithelia suchas the mouth or nose until a break in the skin or a decline inimmune defenses lead to infections such as impetigo, folliculitis,or cellulitis (8). In an immunocompromised host, severe life-threatening infections such as meningitis, endocarditis, and pneu-monia can occur, causing significant morbidity and mortality (8).A retrospective study of fatal outcomes from MRSA septicemiafound a significant odds ratio (OR) with alcoholism (OR, 4.6; P �0.001) (9). Furthermore, a prospective study of 190 patients withMRSA or methicillin-susceptible Staphylococcus aureus (MSSA)found that MRSA patients are more likely to have abused alcoholthan MSSA patients (10). The rise in community-acquired MRSApneumonia cases among young healthy individuals (11)prompted this study on the impact of acute ethanol (EtOH) con-sumption on susceptibility to MRSA infection using a mousemodel of MRSA pneumonia.

The lung epithelium secretes chemokines, cytokines, and anti-microbial proteins in response to bacterial pathogens (12) andprovides the first line of immune defense against pulmonarypathogens. In addition, the epithelium plays a crucial role in serv-ing as a structural barrier and orchestrating mucociliary clearanceof pathogens. Although adaptive immunity is important to clearMRSA in the skin of mice (13), it is dispensable for clearance ofMRSA in the lung because Rag2/Il2rg�/� mice, which are deficientin B, T, and NK cells, are resistant to infection (14, 15). However,patients with inherited (autosomal dominant) hyper-IgE syn-drome (HIES) still present with recurrent S. aureus pulmonaryinfections due to loss-of-function mutations in signal transducerand activator of transcription 3 (STAT3) (16). The interleukin-6(IL-6) family of cytokines binds to the Gp-130 receptor, phosphor-ylating STAT3 and allowing its dimerization. Activated STAT3 is amaster transcription factor involved in the generation of Th17cells, T cell-dependent IgG responses, and the induction of anti-microbial factors (e.g., �-defensins) in response to staphylococcaland Candida infections (16–18). In addition, STAT3 is important

Received 7 August 2013 Returned for modification 8 September 2013Accepted 31 December 2013

Published ahead of print 13 January 2014

Editor: A. J. Bäumler

Address correspondence to Jay K. Kolls, Jay.Kolls@chp.edu.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00974-13

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in epithelial homeostasis, wound healing, and regulation of anti-microbial production (19, 20). Although acute alcohol decreasesthe expression of STAT3-dependent genes in vitro (21), it is un-clear whether this inhibition of STAT3-dependent genes plays arole in lung epithelial defense during MRSA challenge (22–24).This series of experiments tested the hypothesis that acute ethanolwould suppress STAT3 phosphorylation in the lung and reduceclearance of MRSA.

Acute ethanol or vehicle control was administered prior tooropharyngeal aspiration (OA) of USA300. Acute alcohol mark-edly increased the pulmonary burden of USA300 at 16 and 24 h.This was also associated with decreased production of IL-6 andincreased expression of the gene for suppressor of cytokine signal-ing 3 (Socs3), the main inhibitor of STAT3, and reduced Reg3gtranscript levels in the lung; Reg3� has previously been shown tobe regulated by IL-6/Gp-130/STAT3 signaling and has both bac-teriostatic activity and bactericidal activity against MRSA (14).Mouse lung epithelium (MLE12) cells exposed to 50 mM acuteethanol also displayed decreased IL-6-stimulated STAT3 activa-tion and an increase in Socs3 expression. Consistent with inhibitedSTAT3 signaling, ethanol also reduced Reg3g expression in vitro.Finally, in vivo administration of recombinant Reg3� was effectiveat augmenting clearance of USA300 infection in the presence ofacute ethanol. Taking the results together, we determined thatUSA300 initiates a STAT3-dependent induction of Reg3� and thissignaling pathway is perturbed by acute ethanol. The use of re-combinant Reg3� may be a novel way to augment mucosal immu-nity to MRSA infection in at-risk hosts.

MATERIALS AND METHODSMice. C57BL/6 (wild type [WT]) mice obtained from Charles River Lab-oratories were housed in specific-pathogen-free rooms within animal fa-cilities at the Louisiana State University Health Sciences Center or theRangos Research Building at Children’s Hospital of Pittsburgh. To modelacute ethanol intoxication, WT mice received 0, 2, or 4 g/kg of 20% eth-anol (EtOH) in phosphate-buffered saline (PBS) administered intraperi-toneally. 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 wereapproved by the IACUCs at Louisiana State University Health ScienceCenter 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 mlof BBL Trypticase soy broth (TSB) (BD). After this period, fresh TSB wasinoculated with the bacteria at a 1:100 dilution and incubated at 37°C for2 h with shaking to obtain bacteria during the log phase of growth. Bac-teria were washed with PBS, resuspended in 1 ml of PBS, and diluted fivetimes to obtain approximately 4 � 108 CFU/ml of working stock. Fiftymicroliters of this working stock was administered with the tongue pullmethod 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 deter-mine the exact concentration of the inoculum. For Reg3� rescue experi-ments, recombinant Reg3�-Fc versus IgG control or Reg3�-cleaved ver-sus PBS were administered at a dose of 60 mg per mouse by oropharyngealaspiration (OA) to USA300-infected mice 4 h after infection.

Lung bacterial burden, mRNA, and protein analysis. Lungs wereharvested in PBS, homogenized with glass homogenizer, and plated on LBin three serial dilutions. Colonies were counted after overnight incubationat 37°C. Gene expression was determined by RNA extraction with TRIzol(Invitrogen), reverse transcription by iScript (Bio-Rad), and quantitativePCR (qPCR) with TaqMan probes (Applied Biosystems) on an iCyclerthermocycler (Bio-Rad). All threshold cycle (CT) values were normalized

to Hprt. Lung homogenates were analyzed using bead-based Luminexassays (Millipore) for protein levels of IL-1�, 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, 10mM HEPES, 5 �g/ml insulin, 10 �g/ml transferrin, 10 nM hydrocorti-sone, 10 mM �-estradiol, penicillin, streptomycin, and 2% fetal bovineserum. Cells were seeded at 2 � 106 cells/ml in 24-well plates 1 day beforestimulation. Cells were stimulated with IL-6 (10 ng/ml) (R&D) for 15 minto measure P-STAT3 by enzyme-linked immunosorbent assay (ELISA)and for 2 h to measure Socs3 and Reg3g mRNA levels. For the STAT3reporter assay, MLE12 cells were stably transduced with a STAT3-lucifer-ase reporter using a lentiviral construct (SA-Biosciences). Luminescencewas read 24 h after stimulation with IL-6 (10 ng/ml).

Histologic analysis. Animals were euthanized after infection withUSA300. The left lung was inflated with 10% neutral buffered formalin(Sigma Chemical Co, St. Louis, MO) at a pressure of 25 cm H2O for 10min, removed from the animal, and placed in fresh 10% neutral bufferedformalin for 24 h before processing and paraffin embedding. For histo-logic analysis, 5-�m sections were cut and stained with hematoxylin andeosin (H&E). Histologic scoring was performed by a pathologist reviewer(D.A.P.) on blinded lung sections. Lung injury induced by USA300 wasassessed 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/perivas-cular and alveolar inflammation with debris in bronchiolar lumen.

Statistical analysis. For multiple comparisons and statistical analyseswith three or more groups, data were analyzed using analysis of variance(ANOVA) followed by nonparametric Kruskal-Wallis testing. For twogroups, nonparametric data were analyzed with the Mann-Whitney testand parametric data were analyzed with Student’s t test. All analysis wasperformed with Prism software (GraphPad).

RESULTSAcute alcohol intoxication increases pulmonary MRSA burdenin a dose-dependent manner. Acute alcohol intoxication wasmodeled as previously described by i.p. administration of EtOH30 min before OA aspiration of 2 � 107 CFU of MRSA USA300(3). Acute alcohol at a dose of 2 g/kg increased the MRSA lungburden 5-fold at 16 h after infection. EtOH at 4 g/kg increased theMRSA lung burden 21-fold at 16 h and 8-fold at 24 h after infec-tion (Fig. 1A). The higher dose of EtOH was also associated withincreased peribronchial inflammation (Fig. 1B to E).

Acute alcohol intoxication attenuates IL-6 production dur-ing MRSA pneumonia. Since it was previously demonstrated thatIL-1� and Gp-130/STAT3 signaling is necessary for MRSA clear-ance (14, 25), protein levels of IL-1� and Gp-130 ligands wereanalyzed by Luminex assay at 0, 4, 16, and 24 h after infection.IL-1�, Lif, and IL-6 were substantially induced at 4 h after infec-tion and were decreased with MRSA clearance in control mice at16 and 24 h (Fig. 2A to C). Although Lif was marginally increasedwith alcohol at 16 h after infection (P � 0.05), IL-6 induction wassubstantially decreased at 4 h after infection (P � 0.001) (Fig. 2Band C).

Alcohol inhibits IL-6/Gp-130-stimulated STAT3 activationin lung epithelial cells. Activation of STAT3 as determined byimmunohistochemistry for phosphorylated STAT3 is greatly en-hanced in epithelial cells during MRSA pneumonia (14). STAT3signaling in the epithelium leads to transcription of genes thatregulate epithelial repair and barrier function, as well as antimi-crobial factors (12). Since alcohol had varied effects on the induc-tion of different Gp-130 ligands, it was hypothesized that ethanolmay also inhibit activation of STAT3. To determine whether eth-

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anol directly inhibits Gp-130-induced epithelial STAT3 activa-tion, MLE12 cells were treated with IL-6 at 1 h after incubationwith 50 mM EtOH. There were increased P-STAT3 levels seenwith IL-6 stimulation, with a mild but not statistically significantattenuation with acute EtOH (Fig. 3A). However, IL-6 treatmentresulted in a significant increase in STAT3-luciferase reporter-expressing MLE12 cells (Fig. 3B). The addition of 50 mM EtOH at1 h prior to IL-6 treatment substantially reduced IL-6 inducedSTAT3 activation in the reporter assay (Fig. 3B).

Acute alcohol increases Socs3 expression in MLE12 cells invitro and in lung tissue during MRSA pneumonia in vivo. It hasbeen previously reported that in human monocytes acute ethanolingestion can induce SOCS3, which in turn can inhibit Gp-130activation of STAT3 (23). Thus, this mechanism may play a role inethanol-induced exacerbation of MRSA pneumonia. Indeed dur-ing MRSA pneumonia, acute alcohol increased levels of Socs3 ex-pression in vivo (Fig. 4A). Furthermore, IL-6-stimulated Socs3 ex-pression was increased with 50 mM ethanol in MLE12 cells

FIG 1 Acute alcohol intoxication increases the pulmonary MRSA burden (A). Quantification of the MRSA lung burden was done by CFU assay at 16 and 24 hafter 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 16h 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 theKruskal-Wallis test/Dunn multiple-comparison test as indicated. Error bars represent the standard error of the mean (SEM). (B and C) Representative lunghistology (magnification, �100) at 16 h after USA3000 infection in control (B) or EtOH-treated (dose, 4 g/kg) (C) animals. (D) Representative histologydemonstrating inflammatory exudate in the airway of EtOH-treated mice (magnification, �400). (E) Histological scoring of lung inflammation in control andEtOH-treated (dose, 4 g/kg) mice (n 5 per group, P value calculated by the Mann-Whitney test).

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(Fig. 4B). Therefore, alcohol may act through Socs3 to inhibitGp-130-induced STAT3 signaling during MRSA pneumonia.

Alcohol decreases IL-6 induction of Reg3g in MLE12 cells,and recombinant Reg3g rescues decreased MRSA clearance dueto acute alcohol in vivo. Previous results have shown that STAT3directly activates transcription of the antistaphylococcal proteinReg3� from MLE12 cells (14). In the presence of alcohol, Reg3gexpression in response to IL-6 stimulation was decreased (Fig.5A). To determine whether Reg3� can be used therapeutically,recombinant Reg3� was administered by OA at 4 h after infection.Similar to the results shown in Fig. 1, there was a 20-fold increasein the lung burden in mice administered ethanol. Furthermore,the administration of Reg3� reduced the lung burden by 4-foldand rescued the increase in lung burden due to 4 g/kg acute alco-hol (Fig. 5B). Although Reg3� treatment reduced CFU, the histo-logic scoring of peribronchial lung inflammation did not differ atthis time point, with scores of 2.21 0.32 and 1.86 0.18 in theEtOH control IgG group and the EtOH Reg3� group, respectively.

DISCUSSION

Although alcoholism has been linked to increased mortality due toMRSA pneumonia as a part of a clinical study, this is the firstscientific study to model the interactions between alcohol andMRSA pneumonia (10). Furthermore, no other study has exam-ined the effects of acute alcohol in compromising host defenseagainst MRSA pneumonia. One underlying mechanism is thatacute alcohol inhibits STAT3 induction of the antistaphylococcalprotein Reg3� by increasing Socs3 activation in the pulmonaryepithelium. Acute alcohol inhibits IL-6-stimulated P-STAT3 tran-scription and Reg3g expression in MLE12 cells and increases Socs3expression in vivo during MRSA pneumonia. Furthermore, re-combinant Reg3� rescued the impaired MRSA clearance in acutealcohol-intoxicated mice.

Another consideration with the increased number of STAT3signaling inhibitors proposed as cancer therapies is the potentialfor compromised immune host defense (26). STAT3 activatesmany genes involved in host defense, as shown by the clinicalphenotypes of hyper-IgE syndrome patients with STAT3 muta-

FIG 2 Acute alcohol intoxication attenuates IL-6 production during MRSApneumonia. Lung homogenate cytokine levels at 0, 4, 16, and 24 h after MRSAadministration were determined by Luminex assay in control mice versus micetreated with 2 g/kg EtOH (n 5 in each group). P values were calculated by theunpaired t test with the Holm-Sidak multiple-comparison correction. Errorbars represent the SEM.

FIG 3 Alcohol inhibits IL-6-stimulated STAT3 activation in MLE12 cells. (A)IL-6 (10 ng/ml) stimulation in MLE12 cells stimulated STAT3 activation asindicated by P-STAT3 ELISA (n 4 per group) performed 10 min after stim-ulation, and the phosphor-ELISA signal trended lower with 50 mM ETOH(P 0.09 by ANOVA with multiple-comparison correction). OD450, opticaldensity at 450 nm. (B) STAT3 luminescence reporter activity at 24 h afterstimulation (n 11 in each group). P values were calculated using 2-wayANOVA and Dunn’s multiple-comparison test. Error bars represent the SE.

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tions, who are predisposed to multiple skin and pulmonary infec-tions (16). Acute alcohol compromises the host defense againstStreptococcus pneumoniae by increasing STAT3-p27Kip1 signalingin response to colony-stimulating factor 3 (Csf3) in bone marrowcells and ultimately suppress granulopoiesis by inducing cell cyclearrest (24). The decreased STAT3 signaling due to acute alcohol inthis current study might be an effect of acute EtOH limited to thisspecific cell type or localized to the IL-6/Gp-130 pathway. How-ever, the inhibition of the IL-6/Gp-130/STAT3 pathway withacute alcohol has also been observed in human monocytes, andthis suppression was mediated by increases in SOCS3 by alcohol(23).

The Reg3 family of soluble C-type lectins has been character-ized in the gastrointestinal system. Epithelial production of Reg3�is protective against intestinal Listeria monocytogenes (27). Re-cently, Reg3� and Reg3� production in the intestine was shown tobe suppressed by alcohol both in mice exposed to 3 weeks of alco-hol and in chronically alcoholic human subjects (28). However,no other study has examined alcohol’s effects on Reg3� in the hostdefense against bacterial pneumonia. In conclusion, acute alcoholintoxication leads to an increased MRSA lung burden by inhibit-ing STAT3 activation of Reg3� production by the pulmonary ep-ithelium. Furthermore, recombinant Reg3� was effective in res-cuing the increased MRSA burden in mice administered alcoholand can potentially be developed as a therapeutic for MRSA pneu-

monia. Reg3� did not reduce lung injury at the 16-h time point;however, this may be due to the fact that lung inflammation can beslower to resolve than bacterial clearance. Additionally there maybe other STAT3-regulated genes or other antimicrobial factorssuch as the defensins or calgranulins that could also be affected byacute EtOH, which need to be considered as well.

A more comprehensive clinical study on chronic and acutealcohol intoxication should be performed to determine if specific

FIG 4 Acute alcohol inhibits STAT3 activation by increasing Socs3 expression.(A) WT B6 mice administered 2 g/kg EtOH at 30 min before OA USA300infection were euthanized at 0, 4, 16, and 24 h after infection. Socs3 expressionwas determined by real-time PCR and normalized to Hprt (n 5 in eachgroup). (B) MLE12 cells were exposed to 50 mM EtOH in the medium at 1 hbefore stimulation with 10 ng/ml IL-6. Socs3 expression was determined byreal-time PCR of mRNA extracted 2 h after stimulation and normalized toHprt. P values (determined with the Mann-Whitney test) are indicated. Errorbars represent the SE.

FIG 5 Alcohol decreases IL-6 induction of Reg3g, and recombinant Reg3�rescues decreased MRSA clearance due to acute alcohol. (A) MLE12 cells wereexposed to 50 mM EtOH in the medium at 1 h before stimulation with 10ng/ml IL-6. Reg3g expression was determined by real-time PCR of mRNAextracted 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 OAUSA300 infection. Four hours after infection, recombinant Reg3�-Fc (60 �g)or a mouse IgG2A control was OA administered. Lung burden was determinedby CFU assay at 16 h after infection (n 5 in each group). Data are represen-tative of two independent experiments. P values were calculated using 2-wayANOVA and Tukey’s multiple-comparison test. Error bars represent the SE.

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types of alcohol ingestion predispose to MRSA pneumonias inhumans. However, in this mouse model, acute alcohol exacerbatesMRSA pneumonia. Thus, it may be important for clinicians toascertain recent alcohol intake as a potential risk factor for com-plicated MRSA pneumonia.

ACKNOWLEDGMENTS

This work was supported by NIH NIAAA grants R01AA016688,5P60AA009803, 5F31AA021061, and 5T32AA007577.

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