of April 8, 2018.This information is current as

Induced Immune Responses−Virus yncytialand Autophagy during Respiratory S

Sirtuin 1 Regulates Dendritic Cell Activation

LukacsAndrew J. Rasky, David B. Lombard and Nicholas W. Anna B. Owczarczyk, Matthew A. Schaller, Michelle Reed,

ol.1500326http://www.jimmunol.org/content/early/2015/07/08/jimmun

published online 8 July 2015J Immunol 

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The Journal of Immunology

Sirtuin 1 Regulates Dendritic Cell Activation and Autophagyduring Respiratory Syncytial Virus–Induced ImmuneResponses

Anna B. Owczarczyk,* Matthew A. Schaller,* Michelle Reed,* Andrew J. Rasky,*

David B. Lombard,*,† and Nicholas W. Lukacs*

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children worldwide. Sirtuin 1 (SIRT1),

an NAD+-dependent deacetylase, has been associated with the induction of autophagy and the regulation of inflammatory

mediators. We found that Sirt1 was upregulated in mouse lung after RSV infection. Infected animals that received EX-527,

a selective SIRT1 inhibitor, displayed exacerbated lung pathology, with increased mucus production, elevated viral load, and

enhanced Th2 cytokine production. Gene expression analysis of isolated cell populations revealed that Sirt1 was most highly

upregulated in RSV-treated dendritic cells (DCs). Upon RSV infection, EX-527–treated DCs, Sirt1 small interfering RNA–treated

DCs, or DCs from conditional knockout (Sirt1f/f-CD11c-Cre+) mice showed downregulated inflammatory cytokine gene expression

and attenuated autophagy. Finally, RSV infection of Sirt1f/f-CD11c-Cre+ mice resulted in altered lung and lymph node cytokine

responses, leading to exacerbated pathology. These data indicate that SIRT1 promotes DC activation associated with autophagy-

mediated processes during RSV infection, thereby directing efficient antiviral immune responses. The Journal of Immunology,

2015, 195: 000–000.

Respiratory syncytial virus (RSV), a single-stranded,negative-sense RNA virus of the Paramyxoviridae fam-ily, is a ubiquitous human pathogen. Although RSV

predominantly causes mild respiratory tract infection, it is theleading global cause of lower respiratory tract infection in children,and it is responsible for significant morbidity and mortality amonginfants, the elderly, and patients with chronic respiratory diseasesworldwide (1, 2). Unfortunately, no effective pharmacologictherapies against RSV infection exist, and attempts at developinga vaccine have failed despite years of effort (3). Infants hospi-talized with a severe RSV infection are at a greater risk for de-veloping allergic asthma and recurrent wheezing later in life (4,5), suggesting that a chronic alteration of the pulmonary immuneenvironment occurs after RSV infection.During RSV infection, pulmonary dendritic cells (DCs) drive

innate immune responses that direct the resultant adaptive immuneresponse. Activated DCs migrate to lung-draining lymph nodes

(LDLNs) and dictate T cell maturation via costimulatory markerpresentation, proinflammatory cytokine release, and Ag presen-tation. DCs detect viral Ags via pattern recognition receptors,including RIG-I, MyD88-dependent TLRs, and TRIF-dependentTLRs, which leads to the production of type I IFN and effectiveAPC function (6–8). Recent work in our laboratory (9, 10) and inothers (11) suggests that autophagy facilitates intracellular path-ogen recognition, DC maturation, and proinflammatory cytokineproduction. Because RSV enters the host cell cytosol directlythrough membrane fusion (12), DC activation relies on autophagicmachinery to mediate endosomal TLR-dependent cytokine pro-duction and proper innate immune responses.Autophagy is a conserved intracellular membrane trafficking

pathway whereby cytoplasmic material is sequestered withindouble-walled vesicles, which degrade upon fusion with lyso-somes. This process maintains cellular metabolic equilibrium andpromotes cell survival during physiological (aging, differentiation)and pathological (infection, degeneration, cancer) stress conditions(13). Autophagy plays critical roles in innate immunity, includingthe clearance of cytoplasmic pathogens (14), delivery of viral Agto endosomal TLRs (14), and the loading of Ag onto MHCmolecules for T cell presentation (15, 16). A family of autophagy-related (Atg) genes orchestrates the initiation, elongation/closure,and maturation of autophagosomes. It has been reported that threekey ATG proteins (ATG5, ATG7, ATG8/LC3) are deacetylatedand activated by Sirtuin 1 (SIRT1) (17).Sirtuins are a family of seven NAD+-dependent protein and

histone deacetylases/deacylases, also termed class III histone deace-tylases. SIRT1 impacts many areas of biology and pathophysiology,including cancer, metabolism, circadian rhythm, neurodegeneration,inflammation, and cardiovascular disease (18). Furthermore, Sirt1variants are associated with familial diabetes and childhood obe-sity (19, 20). Additionally, SIRT1 influences immune function indiverse ways by regulating processes such as lymphocyte activa-tion, T cell proliferation and differentiation, and macrophage se-cretion (21). However, the role of SIRT1 in DC biology and its

*Department of Pathology, University of Michigan Medical School, Ann Arbor, MI48109; and †Institute of Gerontology, University of Michigan Medical School, AnnArbor, MI 48109

Received for publication February 9, 2015. Accepted for publication June 16, 2015.

This work was supported by National Institutes of Health Grants HL-114858 (toN.W.L.), R01GM101171 (to D.B.L.), and R21CA177925 (to D.B.L.), as well as agrant from the Glenn Foundation for Medical Research (to D.B.L.).

Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, Departmentof Pathology, University of Michigan, 109 Zina Pitcher Place, 4059 BiomedicalScience Research Building, Ann Arbor, MI 48109-2200. E-mail address: nlukacs@med.umich.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AEC, alveolar epithelial cell; BMDC, bone mar-row–derived dendritic cell; COPD, chronic obstructive pulmonary disease; DC, den-dritic cell; dpi, days postinfection; ER, endoplasmic reticulum; KO, knockout;LDLN, lung-draining lymph node; 3-MA, 3-methyladenine; MOI, multiplicity ofinfection; PAS, periodic acid–Schiff; qPCR, quantitative real-time PCR; RSV, respi-ratory syncytial virus; siRNA, small interfering RNA; SIRT1, Sirtuin 1; TEM, trans-mission electron microscopy; WT, wild-type.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500326

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subsequent impact on adaptive immunity have not been wellelucidated.In this study, we demonstrate that SIRT1 promotes DC activation

and autophagy-mediated processes during RSV infection, and thatthe absence of SIRT1 activity alters the antiviral immune responsethrough the regulation of innate cytokine production. Collectively,these findings expand our understanding of the innate immuneresponse during RSV infection and may contribute to therapeuticstrategies, such as a viral vaccine, aimed at preventing severepathology.

Materials and MethodsReagents

EX-527 (SIRT1 inhibitor III, Calbiochem, Darmstadt, Germany) andSRT1720 (Calbiochem) were reconstituted in DMSO and diluted in culturemedium for in vitro work. Based on previous reports (22, 23), we verified1 mM as an appropriate dose in vitro, with no significant changes in DCcytokine production at greater concentrations. We observed comparableviability in control and EX-527–treated cells by flow cytometry (Live/Dead fixable yellow, Invitrogen, Waltham, MA) and exclusion dye stain(trypan blue). In the in vivo experiments, treated mice received daily i.p.injections of 100 ml (1 mg/kg) EX-527 reconstituted in DMSO and dilutedin normal saline; controls received DMSO-saline. Dose-response assaysrevealed that administering 10 mg/kg EX-527 to RSV-infected micecaused a rebound in Sirt1 and a reversal of the phenotype observed at the1 mg/kg EX-527 dose. 3-Methyladenine (3-MA, Sigma-Aldrich, St. Louis,MO) was reconstituted with PBS plus 0.1% BSA and used at 10 mM in celltreatments. Imiquimod (R837, InvivoGen, San Diego, CA) was recon-stituted in endotoxin-free water and used at 1 mg/ml. RPMI 1640 (Lonza)and HAM-F12 (Invitrogen) media were used for cell culturing. To induceamino acid starvation, the cell culture medium was exchanged with HBSS(Invitrogen).

Cell lines

MLE-12 and LA4 cells were purchased from the American Type CultureCollection (Manassas, VA). MLE-12 cells were maintained in HITESmedium, a supplemented RPMI 1640–based medium (13 insulin trans-ferrin selenium-X, 100 mg/ml streptomycin, 100 U/ml penicillin, 10 nMb-estradiol, 10 nM hydrocortisone, 2% FBS). LA4 cells were cultured inHAM-F12 medium supplemented with 1% penicillin/streptomycin plus10% FCS.

Mice

C57BL/6J (BL6), B6;129-Sirt1tm1Ygu/J (Sirt1f/f), and C57BL/6J-Tg(Itgax-cre,-EGFP)4097Ach/J (CD11c-Cre-GFP) mice were purchased at 6–7 wkof age from The Jackson Laboratory (Bar Harbor, ME). Sirt1f/f mice, inwhich two loxP sites flank Sirt1 exon 4, were crossed to CD11c-Cre-GFPtransgenic mice. As the Sirt1f/f mice were on a mixed C57BL/6J;129background, we backcrossed the Sirt1f/f-CD11c-Cre progeny to a C57BL/6Jbackground for six generations. Deletion of exon 4 produces a truncatedprotein that lacks catalytic activity, causing a Sirt1-null genotype (24).Thus, Cre+ mice lack a functional SIRT1 in CD11chigh cells. Sirt1f/f-CD11c-Cremouse breeding took place in-house at the University of Michigan (AnnArbor, MI). All work involving animals was reviewed and approved bythe University of Michigan University Committee on Care and Use ofAnimals.

RSV and plaque assays

The RSV strain 2-20, provided by Dr. Martin Moore (Emory University,Atlanta, GA), was originally isolated from a severely ill RSV-infected infant(25). Line 19 RSV (antigenic subgroup A), originally obtained from a sickinfant at the University of Michigan Hospital System, was shown in animalmodels to mimic human infection by eliciting airway mucus productionupon inoculation with 1 3 105 PFU RSV (26). RSV strains were propa-gated in our laboratory in HEp-2 cells (American Type Culture Collec-tion). Mice were infected intratracheally via tongue-pull with 1.5 3 105

PFU RSV. Plaque assays were performed on RSV-infected lungs. Wholelungs were harvested 4 d postinfection (dpi) and ground with sand usinga mortar and pestle. Supernatants were serially diluted and incubated withVero cells for 3 d. RSV plaques were detected using a specific polyclonalAb (Millipore, Temecula, CA).

Lung histology

Serial 6-mm sections were obtained from paraffin-embedded, 10%formalin-fixed left lungs stained with H&E. Intracytoplasmic and luminalmucin were assessed by periodic acid–Schiff (PAS) staining. Five sectionswere analyzed per mouse lung, with two lung slices per section per mouseto select representative slides. PAS-stained slides were blindly scored forgoblet cell hyperplasia by light microscopy. The following scoring systemwas used: 1, absent; 2, staining in multiple airways; 3, staining in multipleairways with mucus plugging; 4, severe mucus plugging in multiple air-ways.

DC and AEC cultures

Bone marrow–derived DCs (BMDCs) were isolated from whole bonemarrow of naive C57BL/6 mice, Sirt1f/f-CD11c-Cre+ mice, or littermatecontrols. Bone marrow cells were seeded into tissue culture flasks con-taining RPMI 1640–based complete medium supplemented with 15 ngGM-CSF/ml (R&D Systems, Minneapolis, MN). C57BL/6 mouse-derivedcells were fed on days 3 and 5 and harvested on day 7, a time point bywhich cells were $ 85% CD11b+ CD11c+ BMDCs by flow cytometricanalysis. Cells derived from the Sirt1f/f-CD11c-Cre mice were cultured for10 d (fed on days 3, 5, and 7) to achieve high Cre activity. PulmonaryCD11b+ DCs and CD103+ DCs were isolated from lungs and bronchi ofSirt1f/f-CD11c-Cre+ mice and littermate controls by enzymatic digestion aspreviously described (10). Alveolar epithelial cell (AEC) cultures wereprepared from whole lungs of naive mice as previously described (10). Allcells were infected with RSV at a 1:1 multiplicity of infection (MOI).

Lymph node restimulation and protein quantification

Lymph nodes were isolated by mechanical disruption, cultured, and thenrestimulated with RSV for 48 h before collecting supernatants for proteinanalysis on a Bio-Plex suspension array system (Bio-Rad, Hercules, CA),according to the manufacturer’s protocol. Custom kits containing Ab-coated beads for mouse IL-4, IL-5, IL-13, IL-17a, and IFN-g were usedto assay cytokine concentration (Bio-Rad). Results are reported as folddifference over concentrations in control or unstimulated cells.

Quantitative PCR

RNA was extracted as per TRIzol reagent protocol (Invitrogen) or perQiagen RNeasy mini kit protocol (Qiagen, Hilden, Germany), and 5–10 mgwas reverse transcribed into cDNA. mRNA was determined using pre-developed Applied Biosystems primer/probe sets and analyzed using anABI Prism 7500 sequence detection system (Applied Biosystems, FosterCity, CA). Transcription levels of Muc5ac, Gob5, Ifnb, RSV-G, RSV-F, andRSV-N were assessed using custom primers, as previously described (27).GAPDH served as the internal control to normalize gene expression, andfold change (Δ) values were calculated relative to an uninfected, untreated,or wild-type (WT) control group assigned an arbitrary value of 1.

Transfection and RNA interference

Small interfering RNAs (Dharmacon, Lafayette, CO: L-049440-00-0005,siSIRT1; D-001810-10-05, siControl) were introduced via electropora-tion at 20 mM per sample according to the manufacturer’s instructions withan Amaxa Nucleofector kit and Nucleofector II device (Lonza Cologne,Cologne, Germany). Transfection efficacy was verified by quantitativereal-time PCR (qPCR), demonstrating a 75–96% decrease in Sirt1 ex-pression. Viability of cells at 48 h posttransfection, while maintained inDC growth medium, was $80% and not different from transfected controlcells. Sirt1 knockdown was assessed by mRNA analysis. After 48 h oftransfection, the DCs were infected with RSV for 2 h.

Confocal microscopy

BMDCs were cultured as described and then plated in Lab-Tek chamberslides (Thermo Fisher Scientific, Waltham, MA). Cells were treated asindicated, fixed in 4% paraformaldehyde for 20 min, and then blocked for1 h at room temperature in PBS plus 5% normal goat serum plus 0.1%Tween 20. Cells were subsequently incubated with 1˚ Ab rabbit polyclonalanti-LC3B (NB600-1384, Novus Biologicals, Littleton, CO) for 2 h at37˚C and then incubated with 2˚ Ab goat anti-rabbit Alexa Fluor 568(A11011, Invitrogen). ProLong Gold antifade reagent plus DAPI (Invi-trogen) was added before the cells were imaged on a Nikon A1 confocallaser microscope system under oil immersion, using NIS-Elements ac-quisition software (Nikon Instruments). The number of LC3 puncta percell was counted in at least 15 cells per sample per condition per experi-ment. Images were converted to black and white in Adobe Photoshop tomore easily distinguish background staining from actual puncta.

2 SIRT1 REGULATES DC FUNCTION DURING RSV IMMUNE RESPONSE

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Transmission electron microscopy

Cells were spun down into a gelatin capsule with a clinical centrifuge. Cellpellets were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)at 4˚C for 1 h. The cell pellets were washed twice in 0.1 M cacodylatebuffer, postfixed with 2% osmium tetroxide for 1 h on ice, and then rinsedtwice with 0.1 M cacodylate buffer. The fixed cell pellets were dehydratedthrough an ethanol dilution series up to 100% ethanol and then immersedtwice in propylene oxide for 10 min. Pellets were then infiltrated in a 1:3(1 h), 1:1 (2 h), and finally 3:1 (overnight) eponate resin/propylene oxidemixture while rotating at room temperature, and subsequently embedded in100% Eponate resin in gelatin capsules and allowed to harden in a 65˚Coven overnight. After hardening, tissue blocks were ultrathin sectioned ata 70-nm thickness and placed on 200-mesh copper grids. Grids werecounterstained with saturated uranyl acetate and lead citrate and thenviewed on a Philips CM-100 electron microscope. Five grids (∼15–20cells/grid) were studied per treatment.

Statistical analysis

All data are presented as means 6 SEM. Data were evaluated and graphswere generated using GraphPad Prism software (version 6.0). Statisticalsignificance was assessed by one-way ANOVA, followed by a Newman–Keuls posttest. Significant differences were regarded as p # 0.05.

ResultsInhibition of SIRT1 augments RSV-induced lung pathology

SIRT1 has recently been associated with immune responses andlung diseases (21, 28–30). Sirt1 mRNA levels were measured in

RSV-infected C57BL/6J WT mice, and maximal Sirt1 expressionwas observed 4 dpi in lung tissue, coinciding with peak viral load

(31) (Fig. 1A). To test whether Sirt1 contributed to the outcome of

RSV infection, C57BL/6J WT mice were infected with RSV on

day 0 and received daily i.p. injections of EX-527 (1 mg/kg),

a SIRT1-selective chemical inhibitor (22). At 8 dpi, a time point

that corresponds to maximum lung pathology (32), the animals

were sacrificed to assess pathological parameters. There were sig-

nificant increases in the expression of Ifng, Il5, and Il10 in the

EX-527–treated, RSV-infected mice compared with control RSV-

infected mice (Fig. 1B). Histological examination of lung sections

from RSV-infected mice revealed greater peribronchial inflam-

mation (Fig. 1C) and goblet cell hyperplasia (Fig. 1D) in EX-527–

treated, RSV-infected lungs than in infected controls. The in-

creased mRNA levels of RSV proteins in the EX-527–treated,

RSV-infected animals suggested that viral clearance was reduced

in the absence of functional SIRT1 (Fig. 1E). RSV-restimulated

lymph nodes from the EX-527–treated, RSV-infected mice pro-

duced significantly higher levels of the Th2 cytokines IL-4, IL-5

and IL-13 than did RSV-infected controls (Fig. 1F), with no dif-

ference in IL-17a or IFN-g production (unpublished observations).

Therefore, this in vivo study proposes that systemic SIRT1 in-

hibition exacerbates RSV-induced lung pathology via alteration

of T cell cytokine production, and it suggests that SIRT1 pro-

FIGURE 1. Systemic SIRT1 inhibition augments RSV-induced lung pathology. (A) Lung mRNA expression of Sirt1 in RSV-infected WT BL6 mice was

obtained using qPCR and compared with naive controls. (B) Lung mRNA expression of cytokines 8 dpi were obtained using qPCR and compared with naive

controls. Note naive and EX-527 controls were not significantly different. (C) Representative lung histology from naive and infected (line 19 RSV) mice treated

with DMSO-saline control or EX-527 (8 dpi) stained with hematoxylin and PAS. Arrows point to goblet cells. Scale bar, 100 mm. (D) Histological mucus scores

as assessed from lung sections of control and experimental groups 8 dpi. Asterisks indicate significance compared with naive and EX-527 control mucus scores,

which were not statistically different from each other. (E) Viral protein mRNA transcript 8 dpi was obtained using qPCR and compared with naive controls. (F)

LDLNs of naive, or EX-527-treated, or RSV-infected, or EX-527 plus RSV–treated mice 8 dpi were dissociated into single-cell suspensions and restimulated in

culture with RSV. Cytokine concentrations in culture supernatants were assayed by Bio-Plex. Data are representative of three independent experiments. Values

represent mean 6 SEM (n = 5 mice/group). *p , 0.05, **p , 0.01, ***p , 0.001. ND, not determined.

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motes anti-RSV immune responses that result in efficient viralelimination.

Sirt1 upregulation in RSV-infected DCs is required for efficientDC activation and autophagy

Because RSV pathology was enhanced when SIRT1 function wasinhibited, we assessed whether there were cell-specific differencesin Sirt1 expression. Sirt1 mRNA levels were examined in mouseBMDCs, two primary pulmonary DC subsets, primary AECs, aswell as two immortalized pulmonary epithelial cell lines, MLEand LA4 (Fig. 2A, 2B). Whereas all cell populations showed in-creased Sirt1 expression over untreated controls, the DCs had the

highest increase (BMDCs ∼400-fold; pulmonary DCs ∼4-fold),and owing to their central role in directing immune responses,these cells became the focus of our studies. Sirt1 expression maynot be directly regulated by the extent of viral replication, as Sirt1continued to increase despite a decrease in RSV F expression(Supplemental Fig. 1A).Our laboratory has previously demonstrated that TLR-dependent

DC maturation and innate cytokine production in response to RSVrequire autophagy (9). Given the reported relationship betweenSIRT1 and autophagy proteins (17), DC activation upon RSVinfection was assessed in the presence of SIRT1 inhibition andcompared with autophagy inhibition. Cultured BMDCs infected

FIGURE 2. RSV-infected BMDCs upregulate Sirt1, whereas the inhibition of SIRT1 activity by EX-527 attenuates DC cytokine production and

autophagy. (A and B) mRNA expression of Sirt1 in WT BL6 mouse BMDCs, primary WT BL6 mouse pulmonary DCs, primary WT BL6 mouse AECs,

MLE, and LA4 cells as analyzed by qPCR and compared with untreated controls. (C) WT BMDCs cultured from BL6 mice were treated with DMSO, 1 mM

EX-527, or 10 mM 3-MA for 30 min before being infected with line 19 RSV (1:1 MOI) for 24 h. Cytokine gene expression was determined by qPCR. (D)

WT BL6 BMDCs were pretreated as in (C) and then stimulated for 4 h with 1 mg/ml imiquimod. (E) WT BL6 BMDCs were treated with 10 mM SRT1720

for 30 min before infection as in (C). Cytokine concentrations were measured by Bio-Plex assay. (F) Number of autophagosomes in WT BL6 BMDCs 2 h

after RSV 2-20 infection (R) with or without 1 mM EX-527 (E) or with or without 10 mM SRT1720 (S) as assessed by punctate LC3 staining and confocal

microscopy (scale bar, 10 mm); data quantified in (G) compared with respective controls. Data are representative of at least two independent experiments,

with at least three replicates per group. Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

4 SIRT1 REGULATES DC FUNCTION DURING RSV IMMUNE RESPONSE

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for 2 h in the presence of EX-527 or 3-MA, an autophagy in-hibitor, expressed lower levels of inflammatory cytokine genescompared with RSV-infected BMDCs (Fig. 2C), with no signifi-cant changes in viral gene expression (Supplemental Fig. 1B).These studies also demonstrated a significant reduction in cyto-kine production within EX-527– or 3-MA–treated DCs stimulatedwith imiquimod, a TLR7 agonist (Fig. 2D), with no observedchanges in Sirt1 expression (Supplemental Fig. 1C). Conversely,SRT1720, a SIRT1 activator, produced a significant increase inonly IL-12 and CCL5 production in RSV-infected BMDCs (Fig.2E). Flow cytometric studies were used to examine whetherSIRT1 was involved in the expression of maturation markers/costimulatory markers (MHC class II, CD40, CD80, CD86) as-sociated with APC function. We did not observe significantchanges in any of these cell surface markers in control, infectedcells versus EX-527–treated, infected cells (data not shown).Thus, although SIRT1 is not necessary for APC marker matura-tion, it is important for endosomal TLR stimulation, and thereforeappropriate DC cytokine production during RSV infection.The aforementioned results obtained using EX-527 and 3-MA

prompted experiments to determine whether SIRT1 was necessaryfor efficient autophagy in DCs. Cultured BMDCs were treated withEX-527, infected with RSV for 2 h, and immunostained for LC3(ATG8), a key marker for autophagosomes. Confocal staining forLC3 revealed an increase in autophagosomes during RSV infec-tion, supporting previous reports from our laboratory (9, 10) (Fig. 2F,2G). However, EX-527 treatment of RSV-infected BMDCs dra-matically reduced the number of autophagosomes formed com-pared with infected control cells (Fig. 2F, 2G). Additionally, theEX-527–treated, RSV-infected cells did not have an activatedmorphology, that is, the formation of dendrite-like projections.Moreover, SRT1720-treated, RSV-infected BMDCs had similarnumbers of autophagosomes as RSV-infected controls, suggestingthat SIRT1-induced autophagosome formation may reach a thresholdin the presence of a viral stimulus (Fig. 2F, 2G).To further explore SIRT1 involvement in autophagy responses,

DCs were depleted of SIRT1 using Sirt1-specific small interfer-ing RNA (siRNA). Examination by confocal microscopy dem-

onstrated that in presence of Sirt1 siRNA, fewer autophagosomeswere found in RSV-infected BMDCs (Fig. 3A, 3B). Additionally,Sirt1 knockdown significantly downregulated Ifnb expressionduring RSV infection and reduced the levels of other innatecytokines, Il1b and Ccl5 (Fig. 3C). Overall, these results illustratehow SIRT1 is crucial for proper DC activation associated withautophagy using both pharmacological and siRNA knockdownapproaches.

DCs from Sirt1f/f-CD11c-Cre+ mice have altered cytokineproduction and autophagy in response to RSV

To investigate the role of Sirt1 within DCs during RSV-inducedresponses in a more physiologic setting, we generated conditionalknockout (KO) mice (Sirt1f/f-CD11c-Cre), where the Cre+ prog-eny express catalytically inactive SIRT1 in CD11c+ myeloid cells.We verified Sirt1 excision in Cre+ BMDCs (Supplemental Fig.2A). We also observed no baseline differences in the number ofpulmonary immune cell subtypes from naive Sirt1f/f-CD11c-Cremice or in splenic cell subsets from RSV-infected Sirt1f/f-CD11c-Cre mice (Supplemental Fig. 2B–D). As shown in Fig. 4A,BMDCs from Sirt1f/f-CD11c-Cre+ mice did not upregulate in-flammatory cytokines in response to RSV infection compared withBMDCs from Cre2 littermate controls. Similar to EX-527 treat-ment, costimulatory marker expression was unaltered in naive orinfected Cre+ BMDCs compared with controls (data not shown).Likewise, RSV-infected CD11b+ pulmonary DCs isolated fromSirt1f/f-CD11c-Cre+ mice showed reduced cytokine gene expres-sion compared with Cre2 controls (Fig. 4B). Cre+ BMDCs orpulmonary DCs did not demonstrate significant differences in viralgene expression compared with Cre– BMDCs (Supplemental Fig.3A), confirming our EX-527 studies. Interestingly, Cre2 and Cre+

DCs showed a relatively modest, but significant, reduction in IL-12p40 and TNF production upon LPS activation (SupplementalFig. 3B), whereas TNF stimulation of Sirt1-deficient DCs showedno deficit in cytokine production (Supplemental Fig. 3C). Overall,these data suggest that SIRT1 dysfunction does not intrinsicallyimpair cytokine secretion, and thus further demonstrate cytokineproduction deficiency in RSV-infected Sirt1-deficient DCs.

FIGURE 3. Repressed autophagy

and autophagy-dependent innate cy-

tokine production in Sirt1 siRNA-

treated BMDCs. (A) WT BL6 BMDCs

were transfected by electroporation

with appropriate siRNA 48 h prior

to RSV 2-20 infection (MOI 1:1).

Two hours later, autophagosomes were

observed by confocal microscopy and

quantified in (B). Scale bar, 10 mm.

(C) Innate cytokine gene expression

assessed by qPCR inWT BL6 BMDCs

24 h after RSV infection. Cells were

transfected with control or Sirt1-

specific siRNA 48 h prior to infec-

tion. Data are representative of two

independent experiments, three rep-

licates per group. Values represent

mean 6 SEM. *p , 0.05, **p ,0.01, ***p , 0.001.

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With regard to autophagosome formation, Sirt1-deficientBMDCs had significantly attenuated punctate LC3 staining duringHBSS starvation-induced autophagy, compared with their litter-mate controls (Fig. 4C). Whereas the Cre+ BMDCs showed anincrease in LC3 puncta after RSV infection (Fig. 4C), the numberof autophagosomes was significantly reduced compared withinfected Cre2 BMDCs (Fig. 4D). Additionally, independent in-hibition of autophagy with 3-MA did not significantly alter RSVgene levels in either Cre2 or Cre+ BMDCs (data not shown).Overall, these results provide genetic evidence that Sirt1-deficientDCs harbor defects in their ability to mount effective anti-RSVresponses related to impaired autophagy.Recently it has been reported that LC3 can be recruited to other

membranes, including single-membrane vesicles, in a processknown as LC3-associated phagocytosis (33).Whereas LC3-associatedphagocytosis and autophagy both produce punctate LC3 immu-nostaining, a key distinguishing ultrastructural feature betweenthese processes is the formation of a single- versus double-walled

vesicle (34). To verify that the Sirt1-deficient DCs were notforming autophagosomes, starved or RSV-infected Cre2 and Cre+

BMDCs were examined by transmission electron microscopy(TEM). Double-walled and single-walled membranes were ob-served in Cre2 BMDCs at baseline, after starvation, and after RSVinfection, whereas double-walled autophagosomes or autolyso-somes were undetected in Sirt1-deficient BMDCs under the sameconditions (Fig. 4E). These TEM data verify the confocal mi-croscopy data that the formation of autophagosomes was severelyimpaired in Cre+ DCs.

Sirt1f/f-CD11c-Cre+ mice experience exacerbated lungpathology and delayed resolution of inflammation followingRSV infection

During the first 3 d of RSVinfection, the host response is dominatedby innate immunity. This response includes the activation of res-ident DCs, the secretion of early inflammatory mediators, and therecruitment of NK cells and neutrophils (32). Because DC influx

FIGURE 4. Altered cytokine production and autophagy after RSV infection in Sirt1f/f-CD11c-Cre+ DCs. (A and B) Innate cytokine gene expression in

BMDCs or pulmonary DCs from Sirt1f/f-CD11c-Cre mice, 24 h after RSV 2-20 (MOI 1:1) infection, obtained by qPCR and compared with noninfected

controls. (B) CD11b+ DCs were flow sorted from lungs of naive Cre2 or Cre+ mice prior to RSV infection. (C) Number of autophagosomes in Cre2 and

Cre+ BMDCs 2 h after no treatment, HBSS treatment (starvation medium), or RSV 2-20 infection (MOI 1:1), as assessed by LC3 immunostain and confocal

microscopy; data are quantified in (D). Scale bar, 10 mm. (E) Representative TEM images of Cre2 and Cre+ BMDCs, which were untreated, starved with

HBSS medium, or infected with RSV 2-20 for 2 h. Scale bar, 100 nm. Note the double membranes in the insets (Cre2 panel). Thick arrowheads indicate

autophagosomes. The asterisk indicates the initiation of an autophagosome, with an isolation membrane beginning to encompass cytosolic material. Thin

arrowheads indicate single-membrane vacuoles, some containing cellular material. Data are representative of at least three independent experiments. Values

represent mean 6 SEM (three replicates per group, five mice per sort). *p , 0.05, ***p , 0.001.

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begins as soon as 2 dpi (35), we used this time point to analyze earlyimmune responses to RSV in Sirt1f/f-CD11c-Cre mice. Whereasimmune cells began to infiltrate the lungs at 2 dpi (Fig. 5A), Cre+

mice had greater increases in lung mRNA levels of potentiallypathogenic cytokines Il13, Il17a, and Il10 compared with Cre2mice(Fig. 5B). Additionally, crucial innate cytokines, including Il6,Il12p40, and Tnfa, were downregulated in the lungs of Cre+ micepostinfection, recapitulating our in vitro observations in DC subsets.To specifically examine the impact of SIRT1 deletion in CD11c+

APCs during the height of RSV-induced lung pathology, we an-alyzed Sirt1f/f-CD11c-Cre+ mice and littermate controls 8 dpi.Histological examination revealed that Cre+ mice had increasedlevels of airway inflammation and goblet cell hyperplasia com-pared with Cre2 controls (Fig. 5A). RSV-infected Cre+ miceexpressed higher levels of pathogenic cytokine genes, includingIl4, Il13, and Il17a (Fig. 5B), and mucus-related genes (Fig. 5C)in their lungs than Cre2 mice. RSV protein genes were greaterin Cre+ lungs (Fig. 5D), suggesting diminished viral clearance.Therefore, we performed a plaque assay on 4 dpi and observedsignificantly more infectious virus in Cre+ mice compared withCre2 mice (Fig. 5E). Of interest, LDLN cultures prepared fromRSV-infected Cre+ mice secreted significantly greater amounts of

IL-17a and IFN-g upon restimulation with RSV than LDLN cul-tures from Cre2 mice (Fig. 5F).Because the Sirt1f/f-CD11c-Cre+ animals showed a proin-

flammatory lung environment by 2 dpi and an exacerbated pa-thology at 8 dpi, the 12 dpi time point was assessed to investigatethe resolution of the response. Cre+ mice had significantly ele-vated mRNA levels of Ifng, Il4, and Il5, coupled with 3-foldhigher Gob5 expression in their lungs (Fig. 5B, 5C). However,there were no significant differences in cytokines produced byLDLNs harvested at 12 dpi and restimulated with RSV (Fig. 5F).Despite viral elimination at 12 dpi, as indicated by the absence ofRSV protein mRNA (data not shown), histological examination ofthe lungs from Cre+ mice revealed persistent pathology and in-flammation (Fig. 5A, far right panels). Overall, these in vivoresults parallel the results of our SIRT1 inhibitor studies andsupport the concept that Sirt1 in DCs promotes effective antiviralimmunity and limits lung pathology.

DiscussionActivated APCs are instrumental in achieving immune responsesthat effectively clear an infection while limiting injury to sur-rounding tissue. Our results indicate that SIRT1 is necessary to

FIGURE 5. Sirt1f/f-CD11c-Cre+ mice suffer from exacerbated, prolonged RSV-induced lung pathology. (A) Representative lung histology from naive and

RSV 2-20–infected Sirt1f/f-CD11c-Cre mice 2, 8, and 12 dpi stained with hematoxylin and PAS. Insets highlight goblet cell hyperplasia. Note the mucus

plug occluding the Cre+ airway 12 dpi. Scale bar, 100 mm. mRNA expression of lung cytokines (B), mucus-associated gene Gob5 (C), and viral protein (D)

were obtained using qPCR and compared with naive controls. (E) RSV titers in lungs of Cre+ and Cre2 mice, determined by plaque assay on 4 dpi

(expressed as PFU/ml). (F) LDLNs from RSV-infected Cre+ and Cre2 mice 8 or 12 dpi were dissociated into a single-cell suspension and restimulated in

culture with RSV 2-20. Cytokine concentrations in culture supernatants were assayed by Bio-Plex. Data representative of two independent experiments,

three to five mice/group (values are means 6 SEM). *p , 0.05, **p , 0.01, ***p , 0.001.

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promote DC activation and autophagy during RSV infection, andthat in the absence of active SIRT1 within DCs, mice experiencea pathological antiviral immune response. The present studydemonstrates the importance of SIRT1 in DC biology by threeindependent approaches: 1) chemical inhibition of SIRT1 with EX-527, 2) siRNA knockdown of Sirt1, and 3) genetic ablation of Sirt1in DCs. Blocking Sirt1 by any of these methods resulted in at-tenuated cytokine production and inhibited autophagy withinRSV-infected DCs. We found that global inhibition of SIRT1(EX-527) and conditional (CD11c+ cell-specific) Sirt1 defi-ciency in an RSV infection model led to exacerbation of pul-monary pathology. These latter results suggest the adaptiveimmune response is skewed toward a more allergic (Th2) phe-notype in the setting of DC-specific Sirt1 deficiency. Further-more, the increased viral titer at 4 dpi and the decreased viralclearance at 8 dpi may have contributed to the lack of resolutionin pathology and inflammation at 12 dpi in Sirt1f/f-CD11c-Cre+

mice. Thus, to our knowledge this is the first study to charac-terize Sirt1 as having a beneficial impact in an antiviral re-sponse, and to link Sirt1 to autophagy and innate cytokinesecretion within virally infected DCs. As summarized in Fig. 6,these functions of SIRT1 appear to have a significant role indirecting the development of an effective antiviral, minimallypathologic immune environment.Previous work has revealed that SIRT1 can block immune and

inflammatory processes, including cytokine production in APCs(21, 36), likely due to its transcriptional repression of NF-kB(RelA/p65) via deacetylation (37). Furthermore, it has beendemonstrated that the ablation of Sirt1 in macrophages, usinga myeloid cell–specific Sirt1 KO (Mac-Sirt1 KO) mouse, renderedNF-kB hyperacetylated and resulted in increased transcriptionalactivation of proinflammatory target genes, including Il6, Il12,Tnfa, and Il1b (38). These studies concluded that by targeting NF-kB, SIRT1 acts as a brake on metabolically detrimental inflam-matory cytokine production by macrophages in a model of diet-induced diabetes. However, during RSV infection, DCs depend onautophagy for the trafficking of viral components to mediate TLR-induced innate cytokine upregulation. The lack of SIRT1 in DCsreduced critical cytokine production, including IFN-b and IL-12,which we suggest contributed to a pathological Th2/Th17 immuneresponse within the airways. Previous studies examining DC-specific KO mice reported no changes in DC maturation, differ-entiation, or development compared with WT DCs (39, 40), inagreement with our observations. Thus, our data indicate that

SIRT1 has a unique role in modulating DC cytokine production inthe context of a viral infection, such as RSV (12).Direct SIRT1 interaction with components of the autophago-

somal machinery has been shown in studies utilizing immuno-precipitation and confocal microscopy (17, 41). Interestingly,global Sirt12/2 mice resemble Atg52/2 (autophagy defective)mice in phenotype, including the accumulation of damagedorganelles in the cytoplasm, disruption of energy homeostasis, andearly perinatal mortality (17). Most recently, a study has elegantlyreported how SIRT1 selectively activates LC3 in the nucleus viadeacetylation during starvation, mediating LC3 export to the cy-toplasm to initiate autophagy. Importantly, SIRT1 has been shownto induce autophagy as a protective mechanism during stressconditions aside from nutrient deprivation, including hypoxia,oxidation, and the accumulation of toxic/unfolded proteins, ina wide range of cell types (42–44). Because virus-induced endo-plasmic reticulum (ER) stress is a known biological response (45),RSV-infected Sirt1f/f-CD11c-Cre+ DCs may experience alteredER stress due to Sirt1 deficiency. As various studies have linkedthe attenuation of ER stress with SIRT1 activation and function(46–48), future experiments will address the mechanistic details ofthe potential interactions of the autophagic pathway, ER stress,and SIRT1 during RSV infection.These present data allow further speculation toward specific

pulmonary disease states. Baseline respiratory dysfunction, as inthe case of chronic obstructive pulmonary disease (COPD) andallergic airway disease (e.g., asthma), can be exacerbated duringRSV infection (49). Of note, cigarette smoke reduced the levels ofSIRT1 in the lungs of patients with COPD and in rat models, aswell as in monocyte-macrophage cell lines (28). Perhaps mecha-nistically related, SIRT1 deacetylates target proteins such asFoxO3, p53, matrix metalloproteinase 9, and NF-kB, all of whichare implicated in the pathogenesis of COPD (28). Thus, reductionof SIRT1 may promote acetylation of these proteins, thereby en-hancing disease factors, including autophagy, cellular senescence,emphysema, fibrosis, and inflammation. Targeting SIRT1 in pre-clinical pulmonary disease models has yielded disparate results. Inasthma mouse models, administration of pharmacological sirtuininhibitors (sirtinol and cambinol) reduced allergic airway in-flammation and Th2 cytokine responses (29, 30). However, ina separate study using SRT1720, a SIRT1 activator, inhibition ofTh2 responses was observed during OVA-induced airway disease(50). Thus, compared with our studies of RSV-induced responses,SIRT1 may have a differential effect in a noninfectious setting.

FIGURE 6. SIRT1 promotes effective antiviral adaptive immune responses by driving DC activation and autophagy. Upon uptake of viral Ags, DCs

upregulate Sirt1 expression. SIRT1 contributes to the activation of autophagic processes within the DC, such as by deacetylating key ATG proteins, which

indirectly promote APC function. Likewise, SIRT1 may directly influence DC function (not elucidated), given its broad involvement in many cellular

pathways. Once activated, DCs produce crucial, instructive innate cytokines, skewing T cell differentiation toward an antiviral Th1 adaptive immune

response while suppressing pathologic Th2 and Th17 responses. As a result, SIRT1 within DCs dictates the development of an immune environment that

effectively clears the RSV and resolves the associated inflammation.

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These seemingly contradictory results may also be attributed tothe specificity of the inhibitors. Sirtinol antagonizes SIRT2 (IC50,58 mM) more potently than SIRT1 (IC50, 131 mM) (51). Cambinolinhibits SIRT1 and SIRT2 with similar IC50 values of 56 and59 mM, respectively (52). In contrast, EX-527 is a potent andselective SIRT1 inhibitor (IC50, 38 nM), with negligible potencyagainst SIRT2 (IC50, 19.6 mM) or SIRT3 (IC50, 48.7 mM) and itdoes not inhibit class I/II histone deacetylase activity (23). Thepresent study demonstrates the exacerbation of RSV infection inthe context of a highly selective SIRT1 inhibitor and in CD11c-specific Sirt1 KO mice, suggesting that during viral infection,SIRT1 promotes a protective immune environment linked toautophagy within DCs. These observations may be especiallyimportant, as most severe exacerbations in asthma and COPD areassociated with viral infections (49).Our studies cannot exclude the possibility that the heightened

pathology in our EX-527–treated mice was due to a synergisticeffect of SIRT1 inhibition on multiple relevant cell types, in-cluding AECs, alveolar macrophages, and T cells. Importantly,alveolar macrophages outnumber other pulmonary cell types interms of frequency and CD11c expression (53). Thus, it is plau-sible that our CD11c-specific Sirt1 KO mice harbor defects inSIRT1 function within all myeloid cells. Sorted alveolar macro-phages from CD11c-specific Sirt1 KO mice were able to effi-ciently upregulate inflammatory cytokine production during exvivo RSV stimulation (data not shown), whereas DCs from thesame animals had clear defects in cytokine production. Therefore,our in vivo experiments suggest an essential role for SIRT1-mediated DC cytokine production during RSV infection in fine-tuning the T cell–mediated adaptive immune response.Collectively, these data suggest crucial roles for the protein

deacetylase SIRT1 in the activation of DC cytokine secretion viaautophagy during RSV infection. Thus, SIRT1 pharmacologicalactivators, such as SRT1720 or resveratrol, may serve a part inpreventative therapies aimed at fortifying weak or insufficientimmunity in RSV-susceptible patients. Likewise, the creation ofvaccine adjuvants containing SIRT1-activating components may bebeneficial, as this could facilitate the development of a successfulRSV vaccine.

AcknowledgmentsWe thank J. Connett and C. Ptaschinski for editing assistance, L. Johnson for

histological slide preparation, R. Kunkel for designing Fig. 6, and members

of the Lukacs, Kunkel, and Hogaboam laboratories for helpful discussions.

Additionally, we acknowledge the technical assistance of the University of

Michigan Flow Cytometry and the Microscopy Imaging and Analysis Core

Facilities.

DisclosuresThe authors have no financial conflicts of interest.

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