0. zhou et al 2013

of February 23, 2015.This information is current as

Induced Inflammation and RemodelingPattern/Influenza Virus and Cigarette Smoke

Pathogen-Associated Molecular Role of Ribonuclease L in Viral

Silverman, Chun Geun Lee and Jack A. EliasYang Zhou, Min-Jong Kang, Babal Kant Jha, Robert H.

http://www.jimmunol.org/content/191/5/2637doi: 10.4049/jimmunol.1300082August 2013;

2013; 191:2637-2646; Prepublished online 2J Immunol

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2.DC1.htmlhttp://www.jimmunol.org/content/suppl/2013/08/02/jimmunol.130008

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The Journal of Immunology

Role of Ribonuclease L in Viral Pathogen-AssociatedMolecular Pattern/Influenza Virus and CigaretteSmokeInduced Inflammation and Remodeling

Yang Zhou,*, Min-Jong Kang,*, Babal Kant Jha, Robert H. Silverman,

Chun Geun Lee,*, and Jack A. Elias*,

Interactions between cigarette smoke (CS) exposure and viral infection play an important role(s) in the pathogenesis of chronic

obstructive pulmonary disease and a variety of other disorders. A variety of lines of evidence suggest that this interaction induces

exaggerated inflammatory, cytokine, and tissue remodeling responses. We hypothesized that the 2-59 oligoadenylate synthetase

(OAS)/RNase L system, an innate immune antiviral pathway, plays an important role in the pathogenesis of these exaggerated

responses. To test this hypothesis, we characterize the activation of 2-59 OAS in lungs from mice exposed to CS and viral

pathogen-associated molecular patterns (PAMPs)/live virus, alone and in combination. We also evaluated the inflammatory

and remodeling responses induced by CS and virus/viral PAMPs in lungs from RNase L null and wild-type mice. These studies

demonstrate that CS and viral PAMPs/live virus interact in a synergistic manner to stimulate the production of select OAS

moieties. They also demonstrate that RNase L plays a critical role in the pathogenesis of the exaggerated inflammatory, fibrotic,

emphysematous, apoptotic, TGF-b1, and type I IFN responses induced by CS plus virus/viral PAMP in combination. These studies

demonstrate that CS is an important regulator of antiviral innate immunity, highlight novel roles of RNase L in CS plus virus

induced inflammation, tissue remodeling, apoptosis, and cytokine elaboration and highlight pathways that may be operative in

chronic obstructive pulmonary disease and mechanistically related disorders. The Journal of Immunology, 2013, 191: 26372646.

Chronic obstructive pulmonary disease (COPD) is a com-posite term that encompasses chronic bronchitis andemphysema (1). It is the fourth leading cause of death in

the world and, in Western societies, is strongly associated withcigarette smoke (CS) exposure (1). Chronic bronchitis is charac-terized by chronic inflammation and mucus metaplasia, whereasemphysema exhibits structural cell apoptosis and enlargement ofalveolar airspace (13). Recent studies have also highlighted theinteresting observation that the airways in COPD tissues are oftenremodeled and fibrotic, whereas the nearby alveoli manifest tissuerarification and septal rupture (1). The inflammation in COPDtissues is felt to be causally related to the emphysema and otherpathologic alterations in the lungs from these patients (1, 2, 4, 5)and worsens with disease progression (1, 4, 5). The mechanismsthat mediate these inflammatory and remodeling responses, how-ever, are not adequately understood.

Acute exacerbations are a leading cause of morbidity andmortality in COPD (6), and bacterial and respiratory virus infec-tions account for 5070% of these exacerbations (7, 8). Whencompared with other causes of COPD exacerbations, those causedby viral upper respiratory tract infections are associated with moresevere symptoms, more frequent hospitalizations, and longer re-covery times (9, 10). Influenza virus, rhinovirus, coronavirus,respiratory syncytial virus, parainfluenza, adenovirus, and met-apneumovirus are important causes of COPD exacerbations (4).The frequency of these exacerbations correlates with the rate ofdisease progression and loss of lung function as well as the overallhealth status of the patient (610). Thus, exacerbations are nowconsidered to be legitimate targets for disease therapy (8, 9).However, the mechanisms that mediate these exacerbations andtheir effects on tissue inflammation and remodeling have not beenadequately defined.A number of lines of evidence have demonstrated that innate

immune responses play important roles in the pathogenesis ofCOPD (1114). In keeping with these findings and their clinicalimport, studies from our laboratory and others have investigatedthe effects of CS, viruses, and viral pathogen-associated molecularpatterns (PAMPs), alone and in combination, on pulmonary innateimmunity. These studies demonstrated that CS and viruses/viralPAMPs interact in a synergistic manner to increase pulmonaryinflammation and fibrotic and emphysematous tissue remodeling(5). They also demonstrated that these exaggerated responses aremediated by a mitochondrial antiviral signaling moleculede-pendent cytokine cascade characterized by the early induction oftype I IFN and IL-18 and later induction of IL-12/IL-23p40 andIFN-g (5). However, the possibility that other innate/antiviral path-ways could also contribute to these responses has not been fullyaddressed.RNase L (also called RNase 4 or 2-5Adependent RNase) is an

IFN-stimulated, highly regulated endoribonuclease that is widely

*Section of Pulmonary and Critical Care Medicine, Yale University School of Med-icine, New Haven, CT 06520; Department of Internal Medicine, Yale UniversitySchool of Medicine, New Haven, CT 06520; and Department of Cancer Biology,Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195

Received for publication January 10, 2013. Accepted for publication June 25, 2013.

This work was supported in part by National Institutes of Health (NIH) Grant R01HL-079328 and Flight Attendant Medical Research Institute Grant 82571 (to J.A.E.)and NIH Grant R01 CA-044059 (to R.H.S.).

Address correspondence and reprint requests to Dr. Jack A. Elias, Section of Pulmo-nary and Critical Care Medicine and Department of Internal Medicine, Yale Univer-sity School of Medicine, New Haven, CT 06520. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: BAL, bronchoalveolar lavage; COPD, chronic obstruc-tive pulmonary disease; CS, cigarette smoke; OAS, oligoadenylate synthetase; PAMP,pathogen-associated molecular pattern; Poly(I:C), polyinosinic-polycytidylic acid;qRT-PCR, quantitative RT-PCR; RA, room air; SPC, surfactant apoprotein C; WT,wild-type.

Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300082

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expressed in mammalian tissues (15). It is produced as a latentenzyme, activated by 2-59 oligoadenylate synthetase (OAS) proteins,and destroys cellular and viral RNAs (16). It is a major effector ofIFN-induced antiviral responses that can inhibit the replication ofa variety of viruses including encephalomyocarditis virus (17, 18),mengovirus (19, 20), vaccinia virus (21), reovirus (22), West Nilevirus (23), and HSV type 1 (24). It also contributes to antibacterialinnate immunity and has antiproliferative and apoptotic effectorroles (15, 18, 2528). Its role in the pathogenesis of CS and virus-induced pulmonary responses like COPD, however, has not beeninvestigated.We hypothesized that RNase L contributes to the inflammatory

and remodeling responses induced by viral PAMPs or influenzavirus in CS-exposed mice. To test this hypothesis, we characterizedthe 2-59 OAS/RNase L system in mice exposed to CS and virus/viral PAMPs, alone and in combination, and defined the roles ofRNase L in the pathogenesis of the exaggerated inflammatory andremodeling responses in this modeling system. These studiesdemonstrate that CS interacts in a synergistic manner with viralPAMPs and live influenza virus to induce a number of OASmoieties. They also demonstrate that RNase L plays a critical rolein the exaggerated inflammation, fibrosis, emphysema, apoptosis,and TGF-b1 and type I IFN production induced by CS and viralPAMPs/virus in combination in the murine lung.

Materials and MethodsMice

RNase L2/2 mice were generated and genotyped as previously described(18). All mice were bred to be congenic on a C57BL/6 background. Allanimal experiments were approved by the Yale School of Medicine In-stitutional Animal Care and Use Committee in accordance with federalguidelines.

Treatment of mice

Eight-week-old wild-type (WT) or RNase L2/2mice were exposed to roomair (RA) or the smoke from nonfiltered research cigarettes (2R4; Univer-sity of Kentucky, Lexington, KY) as previously described by our labora-tory (5). During the first week, mice received a half cigarette twice a day toallow for acclimation. During the remainder of the exposure, they receivedthree cigarettes per day (one cigarette per session, three sessions per day).Polyinosinic-polycytidylic acid [Poly(I:C)] or influenza A virus were ad-ministrated after 2 wk of CS exposure. Poly(I:C) was used in theseexperiments to mimic viral dsRNA, which is produced during influenza (orvirus) replication. For Poly(I:C) treatment, the mice were anesthetized, and50 ng Poly(I:C) or PBS vehicle controls were administered twice per weekfor 2 wk via nasal aspiration. CS exposure was continued during this in-terval. The mice were sacrificed and evaluated 1 d after the last adminis-tration. For influenza A treatment, the mice were anesthetized, and 5.0 3103.375 50% tissue culture infective doses of A/PR8/34 influenza (equiv-alent to 0.05 LD50 in C57BL/6J mice) was administered via nasal aspi-ration in 70 ml PBS. CS exposure was continued during this interval. Themice were sacrificed and evaluated 7 and 14 d after the virus adminis-tration.

Bronchoalveolar lavage and histology

Mice were anesthetized, the thorax was opened, right heart perfusion wasaccomplished with calcium and magnesium-free PBS, and bronchoalveolarlavage (BAL) was undertaken as previously described (5). Total cell countswere determined using a hemocytometer, and aliquots were cytospun ontomicroscope slides and stained with Diff-Quick (Dade Behring) for cellulardifferential assessment. After the BAL, the lungs were removed from thethorax, inflated at 25 cm pressure with PBS containing 0.5% low meltingpoint agarose gel, fixed, embedded in paraffin, sectioned, and stained.H&E and Mallorys trichrome stains were performed in the ResearchHistology Laboratory of the Department of Pathology at the Yale Uni-versity School of Medicine.

Quantitative RT-PCR

Total RNA was isolated from whole-lung tissue using Trizol reagent(Invitrogen), and transcript levels were quantified using real-time quanti-

tative RT-PCR (qRT-PCR). The primer sequences that were employed havebeen previously described (5).

Immunofluorescence

Lung sections were rehydrated, blocked with 5% BSA, incubated withrabbit anti-mouse RNase L Ab generated using recombinant mouse RNaseL expressed in Escherichia coli, and purified by fast protein liquid chro-matography. The goat anti-mouse surfactant apoprotein C (SPC) Ab wasfrom Santa Cruz Biotechnology (Santa Cruz, CA). Slides were then in-cubated with anti-goat Alexa Fluor 594 and anti-rabbit Alexa Fluor 488(Invitrogen), and then coverslipped with Vectashield with DAPI (VectorLaboratories, Burlingame, CA).

Quantification of lung collagen

Animals were anesthetized, right heart perfusion was accomplished withcalcium and magnesium-free PBS, the heart and lungs were removed, andthe right lung was frozen in liquid nitrogen and stored at280C until used.Collagen content was determined by quantifying total soluble collagenusing the Sircol Collagen Assay kit (Biocolor) according to the manu-facturers instructions. The data are expressed as the collagen content ofthe entire right lung.

Assessment of alveolar size

Alveolar size was determined in lungs that had been fixed to pressure bymeasuring mean the chord length of H&E-stained lung sections. Briefly, 10random fields were evaluated by microscopic projection onto the NationalInstitutes of Health ImageJ program (version 1.63), and alveolar size wasestimated from the mean chord length of the air space as described pre-viously by our laboratory (5).

TUNEL analysis

End labeling of exposed 39-OH ends of DNA fragments in paraffin-embedded tissue was undertaken with the TUNEL in situ cell death de-tection kit AP (Roche Diagnostics). After staining, 810 random pictureswere obtained from each lung, and a minimum of 300 cells were visuallyevaluated in each section. The labeled cells were expressed as a percentageof total nuclei.

ELISA

TGF-b1 and IFN-b levels in the mouse BAL samples were quantified inusing ELISA kits (R&D Systems for TGF-b1; PBL Biomedical Labora-tories for IFN-b) following the manufacturers instructions.

Statistics

Values are expressed as the mean 6 SEM. As appropriate, groups werecompared by two-way ANOVA; follow-up comparisons between groupswere conducted using a two-tailed Student t test. A p value #0.05 wasconsidered to be significant.

ResultsCS interacts with Poly(I:C) or influenza A to enhance theexpression of multiple forms of OAS

In these studies, qRT-PCR was performed to define the levels ofmultiple forms of OAS in lungs from mice treated by nasal as-piration with CS and Poly(I:C) or influenza A, alone and in com-bination. These evaluations demonstrated that modest levels ofmRNA encoding OAS transcripts could be appreciated in lungsfrom mice in RA and mice exposed to CS only (Fig. 1). Poly(I:C)treatments stimulated the levels of a number of OAS transcripts(Fig. 1AE). Importantly, many of the OAS transcripts (OAS1D,OAS1G, OAS2, OASL1, and OASL2) were synergistically in-creased beyond the levels obtained with CS alone and Poly(I:C)alone, in mice exposed to CS and Poly(I:C) in combination (Fig.1AE). Other OAS genes, including OAS1A, OAS1B, OAS1C,OAS1E, OAS1H, and OAS3, were not synergistically induced byCS and Poly(I:C) in combination (data not shown). To begin tounderstand the effects of CS plus live virus, mice were exposed toRA or CS for 2 wk, infected with influenza A, and qRT-PCR wasused to quantitate the levels of multiple forms of OAS. Thesestudies demonstrated that CS and live virus, individually, did not

2638 RNase L IN VIRAL PAMP/VIRUS AND CS-INDUCED REMODELING

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alter levels of OAS2 and OAS3 transcripts and only modestlyincreased levels of OASL1 transcripts (Fig. 1FH). In contrast, CSexposure and virus infection interacted to synergistically increasethe expression of these OAS moieties (Fig. 1FH). Other forms ofOAS genes, including genes in the OAS1 family and OASL2,were not synergistically induced by CS and influenza A (data notshown). These studies demonstrate that CS and Poly(I:C) or in-fluenza A interact to synergistically stimulate multiple forms ofOAS in the murine lung.

RNase L is expressed in type II alveolar epithelial cells in thelungs from mice treated with CS plus Poly(I:C) or influenza A

Because OAS moieties are important activators of RNase L, im-munofluorescent studies were next undertaken to localize RNase Lin lungs from mice treated with CS and Poly(I:C) or influenza A,alone and in combination. These evaluations demonstrated thatweak RNase L staining could be appreciated in lungs from mice inRA and mice exposed to CS only (Fig. 2). Poly(I:C) or influenza Atreatments stimulated the expression of RNase L (Fig. 2). Im-portantly, the levels of RNase L expression in lungs from miceexposed to CS plus Poly(I:C) or influenza A in combination were

greater than the levels in mice treated with each moiety individ-ually (Fig. 2). This RNase L expression colocalized with the typeII alveolar epithelial cell marker SPC (Fig. 2, white arrows). Thus,these studies demonstrate that CS and Poly(I:C) or influenza Ainteract to synergistically stimulate RNase L expression in type IIalveolar epithelial cells in the murine lung.

The exaggerated inflammation induced by CS and Poly(I:C) orinfluenza A virus in combination is decreased in the absence ofRNase L

As previously reported by our laboratory (5), CS exposure causeda mild increase in BAL total cell recovery, Poly(I:C) significantlyincreased total cell, macrophage, lymphocyte, and neutrophil re-covery, and CS and Poly(I:C) interacted in a synergistic mannerto enhance these inflammatory responses (Fig. 3A). To evaluatethe importance of RNase L in these responses, we compared theeffects of these interventions in WT and RNase L2/2 animals.These studies demonstrated that the inflammatory effects of CS orPoly(I:C) individually were not significantly altered in the absenceof RNase L. In contrast, the synergistic interactions of Poly(I:C)treatment and CS exposure were significantly diminished in ani-

FIGURE 1. OAS gene expression in the lungs

from mice treated with CS plus Poly(I:C) or in-

fluenza A, alone or in combination. The levels of

mRNA encoding various forms of OAS were mea-

sured in whole-lung RNA extracts using qRT-PCR.

(A) OAS1D. (B) OAS1G. (C) OAS2. (D) OASL1.

(E) OASL2. (F) OAS2. (G) OAS3. (H) OASL-1.

The data are presented as the mean 6 SEM per-centage of concurrent b-actin evaluations. n = 4 for

each. *p # 0.05.

The Journal of Immunology 2639

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mals with null mutations of RNase L (Fig. 3A, 3B). This manifestsas a significant decrease in total inflammatory cell and macro-phage, lymphocyte, and neutrophil recovery in the BAL fluidsfrom RNase L2/2 mice compared with WT controls (Fig. 3A, 3B).

These data demonstrate that exaggerated pulmonary inflammatoryresponse induced by CS and Poly(I:C) in combination is signifi-cantly diminished in the lungs of RNase L2/2 mice comparedwith WT controls.

FIGURE 2. Localization of RNase L in the lungs from mice treated with CS plus Poly(I:C) or influenza A, alone or in combination. Type II epithelial cells

were stained with an anti-SPC Ab and then labeled with red fluorescence (Alexa Fluor 594). RNase L was labeled with green fluorescence (Alexa Fluor 488).

Nuclei are stained with DAPI (blue). Sites of colocalization of RNase L and SPC are highlighted with white arrows. Original magnification 340.

FIGURE 3. Role of RNase L in the pulmonary

inflammation in lungs from mice exposed to CS

plus Poly(I:C) or influenza A, alone and in com-

bination. WT or RNase L2/2 mice were exposed to

CS (+) or RA (2) and randomized to receive Poly(I:C), influenza A virus, or vehicle. In (A) and (C),

BAL fluid was collected, and total cell recovery

was determined. In (B) and (D), BAL cell differ-

ential was assessed. The noted values represent the

mean 6 SEM of evaluations in a minimum of fourmice. *p # 0.05.


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To define the role(s) of RNase L in the pulmonary inflammationinduced by CS plus influenza A, WT and RNase L2/2 mice wereexposed to RA or CS followed by influenza A infection. BALinflammation was evaluated 7 d after viral inoculation. Virus in-fection caused significant increases in total cell (Fig. 3C), mac-rophage, lymphocyte, and neutrophil recovery that peaked 7 dafter viral inoculation (Fig. 3D). Interestingly, CS and influenzaA also interacted in a synergistic manner to enhance this inflam-matory response (Fig. 3C, 3D). The stimulatory effects of influ-enza were not significantly altered in the absence of RNase L(Fig. 3C, 3D). In contrast, the augmented inflammation inducedby CS and virus in combination was markedly decreased in RNaseL null mice (Fig. 3C, 3D). There was a significant decrease in totalBAL cell, neutrophil, macrophage, and lymphocyte recovery inRNase L null mice versus WT controls (Fig. 3C, 3D). Together,these studies demonstrate that the heightened pulmonary inflam-matory response induced by CS plus virus is mediated by anRNase Ldependent mechanism(s).

RNase L plays a critical role in the airway fibrosis induced byCS plus Poly (I:C) or influenza A

As previously reported by our laboratory (5), CS and Poly(I:C)individually did not induce collagen deposition, whereas airwayfibrosis was a prominent feature in the lungs of mice that areexposed to CS and Poly(I:C) in combination (Fig. 4A, 4B). Todefine the role(s) of RNase L in these remodeling responses, wecompared the fibrosis in WT and RNase L null mice treated withCS and Poly(I:C), alone and in combination. As shown in Fig. 4,trichrome histologic evaluations and Sircol collagen assays dem-onstrated that the fibrosis and collagen accumulation in micetreated with CS or Poly(I:C) individually were not altered in theabsence of RNase L. In contrast, the fibrosis and collagen accu-mulation in mice treated with CS plus Poly(I:C) were significantlydecreased in lungs from RNase L null mice compared with sim-

ilarly treated WT controls. In accord with these findings, the levelsof BAL total TGF-b1 were significantly increased in WT micetreated with CS and Poly(I:C) in combination, and these inductiveeffects were significantly ameliorated in the absence of RNase L(Fig. 4C). Similarly, virus infection caused a modest increase incollagen accumulation that peaked 14 d after viral inoculation(Fig. 5A, 5B). This response was further augmented in micetreated with CS and influenza in combination (Fig. 5A, 5B). Thefibrotic response induced by influenza alone was not significantlyaltered in the absence of RNase L (Fig. 5A, 5B). In contrast,14 d after viral inoculation, the augmented response induced byCS and virus in combination was significantly decreased in theabsence of RNase L (Fig. 5A, 5B). In accord with these findings,the levels of BAL total TGF-b1 were modestly increased in miceinfected with influenza alone and synergistically increased in miceexposed to CS plus influenza (Fig. 5C). The levels of total TGF-b1 were decreased in mice infected with influenza A alone, butthese changes did not reach statistical significance (Fig. 5C). Incontrast, the induction in mice exposed to CS and virus was sig-nificantly decreased in the absence of RNase L (Fig. 5C). Whenviewed in combination, these data demonstrate that the fibrotictissue remodeling and TGF-b1 induced by CS plus Poly(I:C) orinfluenza A in combination are significantly diminished in thelungs from RNase L2/2 mice compared with WT controls.

RNase L plays a critical role in the airspace destructioninduced by CS plus Poly(I:C) or influenza A

As previously reported (5), CS and Poly(I:C) or virus individuallydid not induce significant alveolar remodeling, whereas alveolarremodeling and enlargement were readily appreciated in the lungsof mice that were exposed to CS plus Poly(I:C) or influenza A incombination (Figs. 6, 7). To define the role(s) of RNase L in thesealveolar remodeling responses, we compared the alveoli in WTand RNase L null mice treated with CS and Poly(I:C) or influenza

FIGURE 4. Role of RNase L in the airway fi-

brosis and TGF-b1 production in lungs from mice

exposed to CS plus Poly(I:C), alone and in com-

bination. WT or RNase L2/2 mice were exposed to

CS (+) or RA (2) and randomized to receive Poly(I:C) or vehicle. In (A), lungs were sectioned and

underwent trichrome staining. Original magnifica-

tion 320. In (B), lung collagen was measured usingSircol evaluations. In (C), total BAL TGF-b1 was

measured by ELISA. The sections in (A) are rep-

resentative of evaluations in a minimum of eight

mice each. The values in (B) and (C) represent the

mean 6 SEM of evaluations in a minimum of fourmice. *p # 0.05.


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A, alone and in combination. As shown in Fig. 6, treatment withCS or Poly(I:C) individually did not cause significant alveolar

enlargement in the presence or absence of RNase L. In contrast,alveolar chord length measurements were significantly increased

FIGURE 5. Role of RNase L in the airway fi-

brosis and TGF-b1 production in lungs from mice

exposed to CS plus flu virus, alone and in combi-

nation. WT or RNase L2/2 mice were exposed to

CS (+) or RA (2) and randomized to receive in-fluenza A virus or vehicle. In (A), lungs were sec-

tioned and underwent trichrome staining. Original

magnification 310. In (B), lung collagen contentwas measured using Sircol evaluations. In (C), total

BAL TGF-b1 was measured by ELISA. The sec-

tions in (A) are representative of evaluations in

a minimum of eight mice each. The values in (B)

and (C) represent the mean 6 SEM of evaluationsin a minimum of four mice. *p # 0.05.

FIGURE 6. Role of RNase L in the alveolar

destruction in lungs from mice exposed to CS plus

Poly(I:C), alone and in combination. WT and

RNase L2/2 mice were exposed to CS (+) or RA

(2) and were randomized to receive Poly(I:C) orvehicle. In (A), lungs were infused with fixative

under constant pressure (25 cm H2O), sectioned,

and underwent H&E staining. Each section is rep-

resentative of evaluations in eight mice with the

noted genotype. Original magnification 310. In(B), alveolar airspace size was calculated using

ImageJ analysis software. Data represent the mean 6SEM of chord length evaluations in a minimum

of four mice. In (C), lungs were processed for TUNEL

staining, and TUNEL-positive cells were counted.

The data are presented as the mean TUNEL scores 6SEM. *p # 0.05.


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in lungs from mice exposed to CS plus Poly(I:C) (Fig. 6A, 6B).Importantly, this alveolar remodeling was significantly amelio-rated in the absence of RNase L (Fig. 6A, 6B). Treatment with CSand Poly(I:C) in combination also caused significant alveolar in-jury with alveolar epithelial DNA injury/apoptosis on TUNELevaluations (Fig. 6C). This injury/cell death response was alsosignificantly decreased in mice that lacked RNase L (Fig. 6C).In the settings of live virus infection, alveolar remodeling peaked14 d after viral inoculation. Similar to Poly(I:C) treatment, the ef-fects of CS and virus individually were not significantly altered inthe absence of RNase L (Fig. 7A, 7B). In contrast, the heightenedalveolar remodeling in mice exposed to CS and virus, in combi-nation, was markedly decreased in RNase L null animals (Fig. 7A,7B). In accord with these findings, CS and virus individually didnot alter or only modestly increased the number of TUNEL-pos-itive alveolar epithelial cells, whereas CS exposure plus virussignificantly increase the number of TUNEL-positive cells inthese animals (Fig. 7C). Importantly, this augmented DNA injury/cell death response was markedly diminished in mice that lackRNase L (Fig. 7C). Together, these findings demonstrate that thealveolar remodeling and epithelial apoptosis in lungs from micetreated with CS plus Poly(I:C) or influenza A are mediated via anRNase Ldependent mechanism(s).

RNase L plays a critical role in the stimulation of type I IFN byCS plus Poly(I:C) or influenza A

Studies were next undertaken to define the role(s) of RNase L in thestimulation of type I IFNs in the lung. As previously described (5),CS did not cause major alterations; Poly(I:C) stimulated and CSplus Poly(I:C) interacted to further increase type I IFN mRNA andprotein accumulation (Fig. 8). The effects of CS and Poly(I:C)individually were not altered in the absence of RNase L (Fig. 8A

C). In contrast, the augmented stimulation of type I IFN by CSplus Poly(I:C) was markedly decreased in RNase L2/2 mice (Fig.8AC). In the settings of live virus infection, influenza A infectionresulted in type I IFN production that peaked on day 7 and di-minished thereafter. This type I IFN was significantly augmentedin mice exposed to CS (data not shown). Interestingly, after treat-ment with CS plus virus, null mutations of RNase L resulted indefective viral resistance and titers of virus that were 4-foldhigher in RNase L null versus WT mice at day 7 after viral in-oculation (Supplemental Fig. 1A). Despite this increase in virusaccumulation, the production of type I IFN was comparable oronly slightly increased in RNase L2/2 mice (Supplemental Fig.1BD). To obtain an index of the ability of the lungs from WT andRNase L null mice to produce type I IFN when infected, we lookedat the relationship between the levels of type I IFN and viral load.As can be seen in Fig. 8DF, when the levels of type I IFN werenormalized to viral titer, it is clear that lungs from RNase L nullmice have a decreased ability to produce type I IFNs comparedwith infected WT controls. In combination, these studies dem-onstrate that RNase L plays a critical role in type I IFN productioninduced by CS plus Poly(I:C) or influenza A.

DiscussionClinical observations suggest that interactions between CS expo-sure and viral infection play important roles in the pathogenesis ofa variety of diseases including COPD, the enhanced morbidity inrespiratory syncytial virusinfected, second hand smokeexposedchildren, and the enhanced mortality in virus-infected, otherwisenormal smokers (9, 10, 2934). In many of these settings, virusinfection/exposure is believed to result in inflammatory responsesthat overwhelm protective anti-inflammatory defenses (6, 35). Inkeeping with these clinical observations, studies from our labo-

FIGURE 7. Role of RNase L in the alveolar de-

struction in lungs from mice exposed to CS plus

virus, alone and in combination. WT or RNase L2/2

mice were exposed to CS (+) or RA (2) and ran-domized to receive influenza A virus or vehicle. In

(A), lungs were infused with fixative under constant

pressure (25 cm H2O), sectioned, and underwent

H&E staining. Each section is representative of

evaluations in eight mice with the noted genotype.

Original magnification 310. In (B), alveolar air-space size was calculated using ImageJ analysis

software. The data represent the mean 6 SEM ofchord length evaluations in a minimum of four

mice. In (C), lungs were processed for TUNEL

staining, and TUNEL-positive cells were counted.

The data are presented as the mean TUNEL scores 6SEM. *p # 0.05.


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ratory and others have demonstrated that CS selectively enhancesthe airway and parenchymal inflammatory, remodeling, and apo-ptotic responses induced by viral PAMPs and demonstrated theimportance of a type I IFNIL-18IL-12/IL-23 p40IFN-g cyto-kine cascade, the activation of dsRNA-dependent protein kinaseand eurkaryotic initiation factor 2a, and mitochondrial antiviralsignaling molecule in these events (5). The present studies add tothese observations by testing the hypothesis that the 2-59 OAS/RNase L pathway also contributes to these augmented responses.These studies demonstrate that CS and viral PAMPs/live virusinteract in a synergistic manner to stimulate OAS moieties. Theyalso demonstrate that RNase L plays a critical role in the patho-genesis of the exaggerated inflammatory, fibrotic, emphysematous,apoptotic, and cytokine responses induced by CS and virus/viralPAMPs in combination.The 2-59 OAS/RNase L system was one of the first antiviral

pathways discovered during investigations on how IFN inhibitsviral infection (15). IFNs induce OAS genes in higher vertebratesand OAS proteins are pathogen recognition receptors for viralPAMPs, which use ATP to synthesize 2-59 linked oligoadenylates

that activate RNase L (15). Regulated turnover and processing ofRNA by RNase L is essential for the complete IFN responseagainst some viral infections. Activated RNase L also has pro-apoptotic and growth inhibitory properties (15). Our studies add tothese observations a number of novel ways. First, they demon-strate that CS is an important regulator of viral/viral PAMP acti-vation of OAS moieties and RNase L. They also demonstrate thatthis pathway is impressively activated in lungs from mice exposedto CS and virus/viral PAMP in combination compared with miceexposed to either individually. In keeping with the importance ofIFNs in the pathogenesis of the inflammatory and remodelingresponses induced by CS and virus/PAMP in combination (5),they also implicate the 2-59 OAS/RNase L pathway in the path-ogenesis of COPD and emphysematous and fibrotic tissue re-modeling.Four classes of OAS genes (OAS1, OAS2, OAS3, and OAS-like)

have been identified in the human and mouse OAS gene family(3638). There are at least eight different isoforms of OAS1, inaddition to OAS2, OAS3, and two isoforms of OAS-like proteinsin the mouse (36). These proteins are expressed in a cell/tissue-

FIGURE 8. Role of RNase L in the production of

type I IFN in lungs from mice exposed to CS plus

Poly(I:C) or influenza A, alone or in combination.

WT or RNase L2/2 mice were exposed to CS (+) or

RA (2) and randomized to receive Poly(I:C) orinfluenza A or vehicle. Type I IFN mRNAs and

proteins were measured with RT-PCR and ELISA,

respectively. In (A), BAL IFN-b was assessed by

ELISA. In (B), IFN-b mRNA levels were measured

in whole-lung RNA extracts. In (C), IFN-a4 mRNA

levels were measured in whole-lung RNA extracts.

In (D), the levels of BAL IFN-b were assessed by

ELISA and normalized to viral titer. In (E), IFN-b

mRNA levels were measured in whole-lung RNA

extracts and were normalized to viral titer. In (F),

IFN-a4 mRNA levels were measured in whole-lung

RNA extracts and normalized to viral titer. The data

in (A) and (D) represent the mean 6 SEM of evalua-tions in a minimum of four mice. The data in (B),

(C), (E), and (F) are expressed as the mean 6 SEMpercentage of concurrently evaluated b-actin tran-

scripts. n = 4 for each. *p # 0.05, **p # 0.01.


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specific manner, display differences in levels of dsRNA requiredfor optimal activation, and have distinct roles in the responsesagainst specific viruses by synthesizing 2-5A of different lengths(19, 3941). Our studies demonstrate that all OAS subtypes areexpressed in the lung. They also demonstrate that these moietiesare differentially regulated by synthetic dsRNA and live virus.Specifically, only two isoforms of OAS1 were synergistically in-duced by CS and Poly(I:C) in combination, whereas only OAS2and OASL1 proteins were induced by live influenza virus. Themechanisms of the stimulus- and isoform-specific effects and theirimportance in CS plus viral PAMP/virus-induced inflammatory,apoptotic, destructive, fibrotic, and cytokine responses in the lungwill need additional investigation.The emphysematous alveolar destruction that is characteristic of

COPD has been attributed to a variety of abnormalities includingprotease excess and oxidant injury (42, 43). Recently, the apoptosisof endothelial and epithelial cells has been proposed to be a crit-ical event in the genesis of these abnormalities (44). In keepingwith the importance of this cell death response, we previouslynoted that the alveolar destruction that is induced by CS plus viralPAMPs/live virus are associated with exaggerated epithelial TUNELresponses (5). Our studies add to these findings by demonstratingthat RNase L is expressed in type II alveolar epithelial cells andplays a critical role in this cell death response. Although themechanism of this contribution is not clear, it is important to pointout that, in addition to viral RNA, RNase L can also target cellularRNAs. These targets include ribosome RNA and mitochondrialmRNA and can engender apoptosis of infected cells (25, 26, 28,4547). Thus, one can easily see how the sustained activationof RNase L in mice exposed to CS plus Poly(I:C)/flu virus cantrigger apoptotic alveolar destruction.Excessive extracellular matrix accumulation and fibrotic re-

modeling are common consequences of antipathogen immuneresponses (48, 49). In some cases, they are protective and serve towall the pathogen off or induce tissue repair. In others, the fibroticresponse itself causes tissue dysfunction and has adverse con-sequences. Airway fibrosis is well documented in COPD and hasbeen proposed to contribute to the airflow obstruction that thesepatients experience (50). Our studies provide interesting insightsinto the fibrosis in COPD and possibly other disorders. Specifi-cally, they demonstrate that RNase L plays a critical role(s) in thefibrosis that is induced by CS plus viral PAMPs in combination.They also provide a potential mechanism for this finding bydemonstrating that RNase L also plays an important role in thestimulation of TGF-b1. To our knowledge, this is the first dem-onstration that RNase L can play a role in the pathogenesis oftissue fibrosis or the regulation of TGF-b1. These observationsraise the interesting possibility that viruses, viral PAMPs, and/orthe 2-59 OAS/RNase L system may play similarly important rolesin the pathogenesis of other idiopathic fibrotic disorders such asidiopathic pulmonary fibrosis or scleroderma.As noted above, RNase L is indirectly activated by IFN in com-

bination with viral dsRNA. Interestingly, our studies also dem-onstrate that the induction of type I IFNs by CS plus viral PAMPsis decreased in the absence of RNase L. These findings may beexplained by the ability of RNase L to generate small RNAs, whichactivate the RIG-Iike helicase pathway and induce the transcriptionof type I IFN genes (51). Regardless, one can envision a positive-feedback loop in which viral infection/exposure induces type lIFN in CS-exposed tissues (via the RIG-Iike helicase or the 2-59OAS/RNase L pathways), which further activates RNase L andincreases IFN elaboration. One can envision how this sort ofa positive-feedback loop could contribute to the chronicity and/orintensity of CS plus virus-induced responses.

To thoroughly understand the interactions of CS exposure andviral infection, we used viral PAMPs and live virus. This com-parison was undertaken to avoid the confounding effects that couldbe seen with altered titers of live virus in RNase L null mice. Ourstudies with Poly(I:C) clearly demonstrate that the induction oftype l IFNs in CS-exposed mice was decreased in the absence ofRNase L. Similar results were not noted with live virus. In theseexperiments, the titers of influenza in RNase L null mice weresignificantly higher than in WT controls. Importantly, when theincreased titers of influenza in CS-exposed RNase L null mice weretaken into account, it is clear that CS-exposed RNase Ldeficientmice also have a defect in type l IFN elaboration. It is also im-portant to point out that, despite the increased titers of virus in theCS-exposed RNase L null mice, virus-induced phenotypes wereameliorated in lungs from these animals. These findings reinforcethe importance of RNase L in mediating the tissue effects of virusin smoke-exposed tissues.Influenza virus nonstructural protein 1 has the ability to bind

dsRNA and sequester the activation of OAS, thereby counteractingthe antiviral responses induced by the IFNOASRNase L system(52). This viral evasion mechanism is used by influenza A virusand numerous studies have demonstrated that RNase L has noeffect on viral replication in cells infected with influenza virus (52).In accord with these observations, we noted that when mice weretreated with Poly(I:C) or virus alone, null mutations of RNase Ldid not alter inflammatory or tissue-destructive responses, and sig-nificant differences in pulmonary pathology were not observedin comparisons of lungs from RNase L2/2 and WT mice. However,when mice were exposed to CS before inoculation with influenza,RNase L exhibited impressive antiviral effects and diminishedinflammatory and tissue-remodeling responses. These observationssuggest that CS alters the nonstructural protein 1based evasionmechanism that is operative in RA-exposed mice and enhances theimportance of RNase L in viral control and clearance.In conclusion, our studies demonstrate that the 2-59OAS/RNase

L system is activated in lungs from mice exposed to CS plus viralPAMPs/live virus. They also demonstrate that RNase L plays acritical role in the pathogenesis of the exaggerated inflammatory,emphysematous, fibrotic, apoptotic, and cytokine responses thatare induced by CS and virus/PAMP in combination. These find-ings suggest that this activation plays an important role in thepathogenesis of COPD and other diseases in which interactionsbetween CS exposure and viral infection play an important patho-genetic role. They also suggest that polymorphisms in the 2-59OAS/RNase L system might be able to contribute to the sus-ceptibility and heterogeneity of these disorders (53, 54). If theanti-inflammatory, remodeling, controlling, and antiviral proper-ties of RNase L can be appropriately balanced, RNase L may bean important therapeutic target in COPD and other disorders. Thisis an exciting prospect because naturally occurring RNase L in-hibitors have been described (55, 56). Additional investigation ofRNase L in health and disease is warranted.

DisclosuresThe authors have no financial conflicts of interest.

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