The Journal of Immunology

Neonatal Rhinovirus Infection Induces Mucous Metaplasiaand Airways Hyperresponsiveness

Dina Schneider,* Jun Y. Hong,*,† Antonia P. Popova,* Emily R. Bowman,*

Marisa J. Linn,* Alan M. McLean,* Ying Zhao,* Joanne Sonstein,‡,x J. Kelley Bentley,*

Jason B. Weinberg,* Nicholas W. Lukacs,{ Jeffrey L. Curtis,‡,x Uma S. Sajjan,* and

Marc B. Hershenson*,†

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads

to an IL-13–driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA

persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-a, -b, and -g peaked 1 d postinfection, whereas

IFN-l levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls,

virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several

immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-g and of NKp46/CD335+, TCR-b+

cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC

expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC,

MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus

production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti–IL-13 neutralizing Ab attenuated

RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia.

Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that

neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are

mediated, at least in part, by IL-13. The Journal of Immunology, 2012, 188: 2894–2904.

Wheezing-associated acute respiratory viral infectionsin infancy, particularly those caused by respiratorysyncytial virus (RSV) (1, 2), have been long consid-

ered risk factors for asthma. However, recent studies suggest apossible role for human rhinovirus (RV) in the pathogenesis ofasthma. RV is the virus most commonly associated with acuterespiratory hospitalizations in infants and young children (3).Results from the Childhood Origins of Asthma study suggest that,in patients with a family history of asthma, wheezing-associatedillness with RV is the most important risk factor for subsequentasthma development (4, 5). In another study of infants hospital-ized for respiratory infection-associated wheezing, RV was asso-ciated with the development of asthma (6), in contrast to RSV,

which was negatively associated with later childhood asthma (7).In contrast, whereas it is conceivable that respiratory viral infec-tion might lead to the development of asthma by damaging thedeveloping airways or altering the immune response, viral infec-tions may simply reveal a pre-existing tendency for asthma.A number of rodent models aiming to elucidate the mechanisms

linking early life viral respiratory infections to asthma have beendeveloped. BALB/c mice infected with RSVat,1 wk of age showmore mucus-producing cells in the airways, mild airway tissueeosinophilia, elevated IL-13 production, and airway cholinergichyperresponsiveness (8). Mice infected with RSV at 1 wk of agealso show airways hyperresponsiveness and IL-13 production thatpersists 69 d postinfection (9). One- to 2-d-old BALB/c miceinfected with pneumonia virus of mice (PVM), a natural mousepathogen that is closely related to human RSV (10), show airwaylymphocytic inflammation, modest goblet cell metaplasia, andairways hyperresponsiveness at 7 wk of age, which is dependenton IL-4R signaling (10).Chronic airways hyperresponsiveness and goblet cell hyper-

plasia following viral infection have also been shown in matureC57BL6/J mice infected with Sendai virus (murine parainfluenzavirus type 1) (11). In these mice, IL-13 production by NK T cellsand alternatively activated macrophages is responsible for per-sistent mucous metaplasia (12).Despite the association of early-life RV infections with asthma

development in children (4–6), the effects of neonatal RV infectionhave not been tested in an animal model. We have recently de-veloped a mouse model of RV infection employing RV1B, a minorgroup virus that binds to low-density lipoprotein family receptors.In this model, RV1B replicates in the lungs of mature mice andinduces a robust IFN response (13). In mice with pre-existingallergic airways disease, alternatively polarized macrophages are

*Department of Pediatrics and Communicable Diseases, University of MichiganMedical School, Ann Arbor, MI 48109; †Department of Molecular and IntegrativePhysiology, University of Michigan Medical School, Ann Arbor, MI 48109;‡Department of Internal Medicine, University of Michigan Medical School, AnnArbor, MI 48109; xMedical Service, Veterans Affairs Ann Arbor Health System,Ann Arbor, MI 48109; and {Department of Pathology, University of Michigan Med-ical School, Ann Arbor, MI 48109

Received for publication May 16, 2011. Accepted for publication January 13, 2012.

This work was supported by National Institutes of Health Grants HL81420 (to M.B.H.)and HL056309 (to J.L.C.), the Research Enhancement Award Program from theBiomedical Laboratory Research and Development Service, Department of VeteransAffairs (to J.L.C.), and National Institutes of Health Grant T32HL07749-17 (to D.S.).

Address correspondence and reprint requests to Dr. Marc B. Hershenson, Univer-sity of Michigan Medical School, Medical Science Research Building 2, 1150 W.Medical Center Drive, Room 3570B, Ann Arbor, MI 48109-5688. E-mail address:mhershen@umich.edu

Abbreviations used in this article: PAS, periodic acid-Schiff; PVM, pneumonia virusof mice; RSV, respiratory syncytial virus; RV, rhinovirus; TCID50, 50% tissue cultureinfectivity dose.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101391

in part responsible for RV-induced airway inflammation andhyperresponsiveness (14). In the current study, we tested the earlyand late effects of RV1B infection in neonatal mice, focusing onthe role of IL-13. We also examined the effects of RV infection onNKT cell IL-13 expression and macrophage polarization.

Materials and MethodsGeneration of RV

RV1B (American Type Culture Collection, Manassas, VA) were grown inHeLa cells, concentrated, partially purified, and tittered, as described (15).Similarly concentrated and purified HeLa cell lysates were used for shaminfection. Fifty percent tissue culture infectivity doses (TCID50) weredetermined by the Spearman-Karber method.

RV exposure

Experiments were approved by the Institutional Animal Care and UseCommittee. Seven-day-old BALB/c or BALB/c-Il4ratm1Sz/J mice (TheJackson Laboratory, Bar Harbor, ME) were inoculated intranasally with15 ml 2 3 108 TCID50/ml RV1B or an equal volume of sham. For selectedexperiments, 8-wk-old BALB/c mice were inoculated with 45 ml 2 3 108

TCID50/ml RV1B or an equal volume of sham (13).

Lung inflammation

Toquantify inflammatorycells, lungdigestswereminced, lysed incollagenasetype IV (Life Technologies Invitrogen, Carlsbad, CA), and strained through70-mm nylon mesh (BD Falcon, San Jose, CA) (16). The pellet was treatedwith RBC lysis buffer (BD Pharmingen, San Diego, CA), and leukocyteswere enriched by spinning the cells through 20% Percoll (Sigma-Aldrich,St. Louis, MO). Cytospins were stained with Diff-Quick (Dade Behring,Newark, DE), and differential counts were determined from 200 cells.

Histology and immunohistochemistry

Lungs were fixed with 10% formaldehyde and paraffin embedded. Blockswere sectioned at 500-mm intervals at a thickness of 5 mm. Sections were

stained with periodic acid-Schiff (PAS; Sigma-Aldrich) to visualize mucusaccumulation. Additional sections were immunostained for goat anti-mouse muc5AC or its isotype control (Santa Cruz Biotechnology, SantaCruz, CA), labeled with biotinylated anti-goat IgG (Vector Laboratories,Burlingame, CA), and developed using the Vectastain Elite ABC kit(Vector Laboratories) and diaminobenzidine (Sigma-Aldrich) as a sub-strate. The number of PAS-positive cells per basement membrane lengthand alveolar chord length was determined using NIH ImageJ analysissoftware, as described (17).

Quantitative real-time PCR

Whole-lung RNA was extracted with RNeasy (Qiagen, Valencia, CA) andanalyzed by quantitative real-time PCR using specific primers and probes.Signals were normalized to GAPDH.

Measurement of lung cytokines

IL-4, IL-5, and IL-13 protein levels were measured by immune bioplexassay (Bio-Rad, Hercules, CA).

Flow cytometry

Staining and analysis by flow cytometry were performed, as described (18).Cells were analyzed on an LSR II flow cytometer (BD Biosciences, SanJose, CA) equipped with 488-nm blue, 405-nm violet, and 633-nm redlasers. Data were collected on an HP XW4300 Workstation (Hewlett-Packard, Palo Alto, CA) using FACSDiva software (BD Biosciences)with automatic compensation and were analyzed using FlowJo software(Tree Star, Ashland, OR). A range of cells (10,000–100,000) was analyzedper sample. Isotype-matched irrelevant control mAbs were tested simul-taneously in all experiments. We used mAbs against the following Ags(clones and fluorochromes shown in parentheses): Ly6G (1A8; FITC orPE), Siglec-F (E50-2440; PE), and CD19 (1D3; allophycocyanin-cyanine7) (each from BD Pharmingen, San Jose, CA); CD4 (GK1.5 PE-Cy7),CD11b (M1/70 Alexa Fluor 700), CD11c (N418 Pacific Blue), IFN-g(XMG1.2 Pacific Blue), IL-13 (eBio13A AF647), NKp46/CD335 (29A1.4eFluor 660), and TCR-b (H57-597 FITC or PE) (each from eBioscience,San Diego, CA); and CD206 (MR5D3 AF647) (AbD Serotec, Raleigh,NC). Appropriate isotype-matched controls were used in all experiments.

FIGURE 1. RV1B infection of neonatal mice elicits

a brisk IFN response. Seven-day-old BALB/c pups

were intranasally infected with RV1B or sham control,

and lungs were harvested for analysis 1–7 d postinfec-

tion. Positive-strand viral RNA (A) and mRNAs en-

coding IFN-a, -b, -g, and -l (B) were measured by

quantitative PCR. IFN expression was normalized by

expression of the housekeeping gene GAPDH. n = 3–5

animals per time point, *different from sham, p ,0.05, unpaired t test.

The Journal of Immunology 2895

For intracellular staining, freshly isolated aliquots of the lungmincewerestimulated for 5 h at 37˚C in 5% CO2 with PMA (50 ng/ml) and ionomycin(1 mg/ml) in the presence of brefeldin A. The lung minces were washed,and the cell pellets were resuspended in Dulbecco’s PBS. To eliminatepotential artifacts due to dead cell contamination, the cells were firststained with Aqua Live/Dead Fixable Dead Cell Stain Kit (Invitrogen,Carlsbad, CA) and then stained for surface Abs, as above. The lung mincecells were fixed, permeabilized, and then incubated with anti-mouse IFN-g,IL-13, and CD206, according to the eBioscience protocol for intracellularimmunofluorescent staining of cells for flow cytometry.

Airways responsiveness

Airway cholinergic responsiveness was assessed by measuring changes intotal respiratory system resistance in response to increasing doses ofnebulized methacholine. Mice were anesthetized with pentobarbital. Re-sistance was measured plethysmographically (Buxco, Wilmington, NC).

IL-13 neutralization

For IL-13 neutralization, 30 mg goat anti-mouse IL-13 or normal goat IgG(R&D Systems, Minneapolis, MN) was administered i.p. on days 0, 2, 4,

FIGURE 2. RV1B infection of neonatal

mice induces airway inflammation. Seven-

day-old BALB/c pups were intranasally

infected with RV1B or sham control, and

lung homogenates were analyzed for in-

flammatory cell counts (A–D). Cytospins

were stained with Diff-Quick. n = 3–4 ani-

mals per time point, *different from sham,

p , 0.05, unpaired t test. (E and F) Images

of lungs from sham (E)- and RV1B (F)-in-

fected mice. Lungs were stained with H&E.

Original magnification 3200. Arrows indi-

cate focal areas of inflammation. These

images are representative of three individual

experiments of three mice per group.

FIGURE 3. RV1B infection of 7-d-old BALB/c pups

increases lung cytokine mRNA levels of CXCL1 (A),

TNF-a (B), CXCL2 (C), and IL-13 (D). mRNA ex-

pression was measured by quantitative PCR. n = 3–10

animals per time point, *different from sham, p ,0.05, unpaired t test.

2896 EARLY LIFE RHINOVIRUS INFECTION IN MICE

and 6 postinfection. Mice were sacrificed for analysis 28 d after initial RVinfection.

OVA sensitization and challenge

For OVA sensitization, mice were injected i.p. on days of life 17 and 24 with100 ml PBS or a solution of alum and 50 mg endotoxin-free OVA (Sigma-Aldrich). Next, mice were challenged intranasally with 25 ml PBS or 50 mgOVA in PBS on days of life 32, 33, and 34. Mice were studied 1 d after thefinal challenge.

Data analysis

Data are represented as mean 6 SEM. Statistical significance was deter-mined by unpaired t test, rank sum test, or two-way ANOVA, as appro-priate. Differences were pinpointed by Newman–Keuls’ multiple range test.

ResultsRV1B infection elicits IFN responses and inflammation inneonatal mice

We initially characterized the response to viral inoculation in 7-d-old BALB/c pups. Pups were intranasally infected with RV1B orsham control, and lungs were harvested for analysis 1–7 d post-infection. Positive-strand viral RNA persisted in the mouse lungup to 7 d postinfection (Fig. 1A). The sustained level of viralcopies 3 d postinfection strongly suggests that RV causes a rep-licative infection in neonatal mice. Virus was not detected .7 dfollowing infection.mRNAs encoding IFN-a, -b, and g were each strongly induced

at 1 d after RV1B infection, but not after sham inoculation (Fig.1B). IFN-a, -b, and -g levels decreased thereafter. In contrast,IFN-l mRNA levels remained elevated through day 7. IFN-l re-ceptors are largely restricted to cells of epithelial origin (19), andIFN-l receptor knockout mice show attenuated antiviral responsesto dsRNA (20). It is therefore conceivable that dsRNA, releasedfrom RV-infected epithelial cells, stimulates further IFN-l pro-duction.Lung differential cell counts revealed modest, but statistically

significant increases in the numbers of neutrophils on days 3, 5, and7, and in the number of macrophages on days 5 and 7 postinfection(Fig. 2A–D). In addition, lymphocytes and eosinophils were in-creased 7 d postinfection. Nevertheless, histology showed minimalcellular infiltration of the lungs after RV infection in neonatalanimals (Fig. 2E, 2F).Lungs were examined for selected mRNAs encoding proin-

flammatory cytokines and chemokines. Based on the early neu-trophilic response to RV infection, we chose to examine threeneutrophil chemoattractants (CXCL1, CXCL2, and TNF-a), eachof which are induced by RV1B infection in mature mice (21).mRNAs encoding CXCL1, CXCL2, and TNF-a were each in-creased compared with sham-inoculated mice (Fig. 3). mRNAexpression of IL-13, which has previously been shown to be in-duced by neonatal RSV infection (8), was also increased.

Neonatal RV1B infection induces airwayshyperresponsiveness, inflammation, and mucus accumulation

We examined the airway cholinergic responsiveness of sham- andRV-exposedmice on day 35 of life (28 d postinfection).We detectedno viral RNA in either treatment group at 28 d postinfection (datanot shown). There was no difference in weight between sham- andRV-infected mice at this time point (sham, 29.56 1.9 g; RV, 29.462.9 g). However, there was a significant increase in methacholineresponsiveness in mice infected with RV as neonates (Fig. 4A). Incontrast, mature mice infected with RV1B showed no increase inairways responsiveness 28 d postinfection.To understand the change in responsiveness, we examined the

lungs of 35-d-old mice infected as neonates. Most airways werehistologically normal. Again, differences in inflammatory cells be-

tween sham- and RV-infected mice were slight. Selected airwaysfrom RV-infected mice showed a modest amount of peribronchialinflammation (Fig. 4B, 4C). In addition, RV-infected mice showedrare, localized collections of lymphocytes in the peribronchial,perivascular, and subpleural spaces (Fig. 4D, 4E). Compared withsham-infected mice, lung digests of RV-infected mice showeda small, but significant increase in the percentage of neutrophils

FIGURE 4. Neonatal RV infection induces persistent changes in airways

responsiveness and inflammation. (A) Effects of sham and RV1B infection

of neonatal and mature mice on airway cholinergic responsiveness, mea-

sured 4 wk after initial inoculation (day 35 of life for mice infected with

RV as neonates, and day 84 of life for mice infected with RV at maturity).

Airway cholinergic responsiveness was assessed by measuring changes in

total respiratory system resistance in response to increasing doses of

nebulized methacholine. n = 6–10 animals per group, *different from

sham-exposed mice of the same age, p , 0.05, two-way ANOVA. (B–E)

Representative images of lungs harvested 4 wk after neonatal sham (B) or

RV1B infection (C–E). Lungs were stained with H&E. Original magnifi-

cation 3200. These images are representative of three individual experi-

ments of three mice per group. (F) Lung homogenate inflammatory cells

from mice harvested 28 d after neonatal inoculation with sham or RV.

Cytospins were stained with Diff-Quick. n = 6 animals per group, *dif-

ferent from sham-exposed neonatal mice, p , 0.05, unpaired t test.

The Journal of Immunology 2897

(sham, 3.3 6 1.1% of total; RV, 12.5 6 1.9%; mean 6 SEM, p ,0.001, unpaired t test). Lungs of mice undergoing neonatal RV in-fection also demonstrated modest increases in total neutrophils,macrophages, lymphocytes, and eosinophils (Fig. 4F). There was nodifference in mean alveolar chord length between sham- and RV-infected mice (sham, 25.36 0.2 mm; RV, 24.96 0.9 mm; p = 0.71).The minimal cellular infiltration of the lungs after RV infection inneonatal animals, particularly at later time points when airwaysresponsiveness is increased, suggests that other mechanisms areresponsible for the observed airways hyperresponsiveness.To learn more about changes in leukocyte subsets following RV

infection, we conducted flow cytometry of cells from minced lungsobtained from mice 3 wk after neonatal infection, focusing onNKT cells and macrophage polarization. We found increases inGr1-positive neutrophils and Gr1-positive, SigF double-positiveeosinophils, CD4-positive T cells, CD19-positive B cells, and

CD11b-positive cells within monocyte/macrophage gates com-pared with sham controls (Fig. 5A–F). CD11b-positive cells fromRV-infected mice showed a significant increase in CD206 ex-pression (Fig. 5G), indicating alternative macrophage polarization.When cells were costained with Abs against IFN-g and IL-13,lung cells from RV-infected mice showed increases in the per-centage of CD4-positive T cells expressing IFN-g (Fig. 5H), andthe percentage of NKp46/CD335-positive TCR-b–positive cells(NKT cells) expressing IL-13 (Fig. 5I). Finally, CD11b-positivecells did not express IL-13 (data not shown).We then examined PAS staining. Lungs from 35-d-old mice

inoculated with sham on day 7 of life showed a range of PASstaining ranging from no signal to patches of dense staining in thelarge airways (Fig. 6A, 6B). In contrast, mice infected with RV atday 7 of life and studied at day 35 showed some large airways thatwere completely filled with PAS-positive epithelial cells, as well

FIGURE 5. Neonatal RV infection induces pro-

longed changes in airway inflammation. Cells from the

minced lungs of 35-d-old mice infected with sham or

RV at 7 d of age were analyzed by flow cytometry.

Compared with sham-exposed mice, RV-infected mice

showed significant increases in Gr1-positive neu-

trophils (A); Gr1-positive, SigF-positive eosinophils

(B); CD4-positive T cells (C); CD19-positive B cells

(E); and CD11b-positive cells (F). There was no

change in the number of CD8-positive cells (D). RV

infection increased the CD206 expression of CD11b-

positive cells (G; sham, gray line; RV, black line).

Lung digests from RV-infected mice showed increases

in the percentage of CD4-positive T cells expressing

IFN-g (H) and CD335-positive NK cells expressing IL-

13 (I). n = 5–6 animals per group, *different from

sham-exposed neonatal mice, p , 0.05, rank sum test.

2898 EARLY LIFE RHINOVIRUS INFECTION IN MICE

as PAS staining in the medium-sized and small airways (Fig. 6C,6D). Sham- and RV-infected mature mice showed only rare PAS-positive cells 28 d after treatment (data not shown). Mucus-secreting cells in the large airways were assessed 14, 21, and28 d after neonatal infection by counting PAS-positive cells perbasement membrane length. RV-infected mice showed signifi-cantly increased PAS staining (Fig. 6E). We also examined PASstaining 60 and 100 d after neonatal sham or RV treatment, ondays 67 and 107 of life. At these time points, both groups showedonly sparse PAS staining in the large airways, indicating that RV-induced mucous metaplasia had mostly resolved. However, somesmall airways of RV-infected 67-d-old mice continued to showPAS staining (Fig. 6F). Immunohistochemical staining for muc5AC

confirmed increased mucus expression in the large and smallairways of RV-treated mice (Fig. 6F–I).We examined the time course of the mucus-related genes gob5,

muc5AC, and muc5B. Given the sufficiency of pulmonary IL-13expression for mucus hypersecretion in transgenic mice (22), wealso examined the time course of this Th2 cytokine. The mRNAlevels of genes related to mucus production were increased inmice infected as neonates, but not mice infected as adults (Fig.7A–C). A similar pattern was observed for IL-13 mRNA, withsignificant increases on days 7, 14, and 21 (but not 28) postin-fection (Fig. 7D). Lung levels of the Th2 cytokines IL-4, IL-5, andIL-13 were each significantly increased 3 wk after RV inoculation(Fig. 7E).

FIGURE 6. Neonatal RV infection causes airway mucous metaplasia. Lungs from mice inoculated with sham or RV1B at 7 d of age were harvested on

day 35 and stained with PAS. PAS staining in the large airways of sham-infected mice ranged from no signal to patches of dense staining (A). Sham-

infected animals showed no staining in the small airways (B). In contrast, mice infected at day 7 of life showed some large airways that were completely

filled with PAS-positive epithelial cells (C), as well as PAS staining in the medium-sized and small airways (D). These images are representative of four

individual experiments of four to five mice per group. (E) PAS staining was quantified by using NIH ImageJ analysis software. RV-infected mice showed

significantly increased PAS staining 14, 21, and 28 d postinfection. n = 3 animals per group, *different from sham-exposed mice, p , 0.05, unpaired t test.

(F) We also examined PAS staining 60 and 100 d after neonatal sham or RV treatment, on days 67 and 107 of life. RV-infected 67-d-old mice continued to

show mucus (arrow, inset). These images are representative of two individual experiments of four to five mice per group. (G–J) Lung tissues from sham-

inoculated (G, H) and RV-infected mice (I, J) were also immunostained for MUC5AC. Signals were amplified and visualized using biotinylated anti-IgG and

diaminobenzidine as a substrate. These images are representative of four individual experiments of four to five mice per group. Original magnifications350

(A, C), 3100 (G, I), and 3200 (B, D, F, H, J).

The Journal of Immunology 2899

IL-13 neutralization attenuates RV-induced mucusaccumulation and airways responsiveness

We hypothesized that IL-13 is required for mucus hypersecretionand airways hyperresponsiveness. We therefore administered neu-tralizing Ab to IL-13 or nonimmune serum days 0–6 post-RV in-fection. As noted previously, airways responsiveness was increasedin BALB/c mice 28 d after neonatal RV infection. However, micetreated with anti–IL-13 showed a decreased level of methacholineresponsiveness (Fig. 8A). Anti–IL-13 decreased the number ofPAS-positive cells in the small airways, as defined by a circum-ference ,3 mm (Fig. 8B). Administration of anti–IL-13 had noeffect on lung digest differential cell counts (data not shown).

IL-4R null mice show reduced mucous metaplasia in responseto neonatal RV infection

BALB/c-Il4ratm1Sz/J mice (23) were inoculated with sham orRV1B, as described above. In contrast to wild-type mice, RV1B-treated IL-4R null mice showed no increase in PAS stainingcompared with sham-treated mice (Fig. 9A–C; compare with Fig.6C, 6D). In addition, there was no significant increase in gob5,muc5AC, muc5B, or IL-13 mRNA expression (Fig. 9D; comparewith Fig. 7).

Effect of early RV infection on the response to OVAsensitization and challenge

Micewere inoculated with sham or RVon day 7 of life, sensitized toPBS or OVA on days 17 and 24, and challenged with PBS or OVAon days 32 and 34. Mice were studied 1 d after the final challenge.Neonatal RV infection increased OVA-induced airway inflamma-

tion (Fig. 10A, 10B). Prior RV infection increased lung digestneutrophil and macrophage, but not lymphocyte or eosinophilcounts (Fig. 10C). Finally, prior neonatal RV infection increasedairways responsiveness following allergen sensitization and chal-lenge (Fig. 10D).

DiscussionWe demonstrate that, unlike mature mice, BALB/c mice inoculatedwith RV1B at 7 d of age display airways hyperresponsiveness,mucous metaplasia, and IL-13 production that last at least 1 mopostinfection. These data suggest that early life infection with RVcan induce a prolonged state of airway pathology that recapitulatesthree hallmark features of allergic asthma.It has previously been shown that infection of BALB/c mice

shortly after birth with RSV and the closely related PVM causespersistent modest goblet cell metaplasia, airways hyperrespon-siveness, and IL-13 production (9, 24). Infection of mature micewith RSV and PVM each result in the loss of cilia and sloughingof epithelial cells into the airway, airway wall edema, and packingof the airway lumen with polymorphonuclear leukocytes, fibrin,lymphocytes, and mucus (10, 25). Similar findings are present ininfants dying of RSV bronchiolitis (26). On this basis, perhaps it isnot surprising that RSV infection causes long-lasting effects onairway function. In contrast, RSV infection of 1- to 2-d-old miceinduced relatively modest airway changes (24), perhaps becauseof an attenuated immune response, which is required for full-blown RSV-induced clinical illness (27).In contrast to RSV, RV typically infects small clusters of cells in

the airway epithelium (28) and elicits minimal, if any, cytotoxicity

FIGURE 7. RV1B infection of 7-d-old

BALB/c pups induces persistent increases in

the expression of mucus-related genes (A–

C) and IL-13 (D). mRNA expression was

measured by quantitative PCR and normal-

ized for GAPDH. n = 4 animals per time

point, *different from sham-exposed mice

of the same age, p , 0.05, unpaired t test.

(E) IL-4, IL-5, and IL-13 protein levels from

the lungs of RV-infected mice harvested 3

wk after neonatal infection were signifi-

cantly elevated compared with sham-ex-

posed mice. Protein levels were measured

by immune bioplex assay (n = 4–5 animals

per group, *different from sham-exposed

mice, p , 0.05, unpaired t test).

2900 EARLY LIFE RHINOVIRUS INFECTION IN MICE

(29). In mature mice, RV infection causes modest neutrophilic andlymphocytic inflammation (13) and, as shown in this work, nomucous metaplasia. In the current study, lung digests from BALB/cmice infected on day 7 of life showed modest infiltration of theairways with neutrophils, lymphocytes, macrophages, and eosi-nophils. Similarly, at age 35 d, the lungs of mice undergoingneonatal RV infection showed only a minor amount of peribron-

chial inflammation, with small but significant increases in totalneutrophils, macrophages, and lymphocytes. Thus, the amount ofcellular infiltration of the airways is unlikely to be the sole causeof the observed airways hyperresponsiveness.We also observed peribronchial, perivascular, and subpleural

collections of lymphocytes in the lungs of RV- but not sham-infected mice. These collections, which resemble BALT, areonly occasionally present in normal mice, but may be induced bysensitization and challenge with heat-killed Pseudomonas aeru-ginosa (30) or by heterologous viral infections (31, 32). BALT isalso spontaneously increased in the peribronchial and perivascularlung tissue of mice deficient for the chemokine receptor CCR7(33, 34). In humans, BALT is commonly present in infants, but notin normal adult lungs (35). The presence of BALT-like infiltrates isevidence of an adaptive immune response against RV infection.RV infection of immature mice also induced significant mucous

metaplasia that was present 14 d postinfection and persisted until28 d postinfection. Consistent with this, RV-infected immaturemice showed significant increases in mRNAs encoding the gel-forming glycoproteins MUC5AC and MUC5B. MUC5AC pro-tein was also increased. Gob-5 (mCLCA3), a member of thecalcium-activated chloride channel family that drives mucin release(36, 37), was also significantly increased. Neither sham-treatedimmature mice nor mature mice showed such increases. LikeRV, RSV infection increases the number of mucus-producing cellsin immature, but not mature mice (8). Together, these data suggestthat the newborn airway is more susceptible to virus-inducedmucous metaplasia.Gob-5 expression and mucus secretion are positively regulated

by the Th2 cytokines IL-13, IL-10, and IL-9 (22, 38, 39), and IL-10–induced mucous metaplasia is IL-13 dependent (38). Persistentmucous metaplasia and airways hyperresponsiveness do not occurin PVM-infected IL-4R knockout mice, implying a requirementfor IL-13 in this process. We therefore examined the mRNA ex-pression of IL-13 in RV-infected mice. Quantitative PCR dem-onstrated a biphasic increase in IL-13 mRNA expression (Fig.3D), with an initial induction immediately after RV infection and

FIGURE 8. IL-13 neutralization partially attenuates RV-induced airways

hyperresponsiveness and mucous cell metaplasia. (A) Sham- and RV1B-

infected animals were treated with IgG or anti–IL-13. Airway cholinergic

responsiveness was measured 4 wk after initial inoculation. n = 4–6 ani-

mals per group, *different from sham-exposed mice, p , 0.05, two-way

ANOVA. (B) PAS staining in the large and small airways of RV-infected

mice treated with anti–IL-13 or IgG. n = 6–8 animals per group, *different

from sham-exposed mice, p , 0.05, unpaired t test.

FIGURE 9. Effect of neonatal RV infection on

IL-4R null mice. BALB/c-Il4ratm1Sz/J mice were

inoculated with sham or RV at day 7 of life, and

airway tissues were examined 28 d postinfection.

Unlike RV-infected wild-type BALB/c mice (see

Fig. 6), IL-4R null mice showed no PAS-positive

cells in large, medium, or small airways (A–C).

These images are representative of two individual

experiments of three mice per group. Original

magnifications 350 (A), 3100 (B), 3200 (C). (D)

Lung gob5, MUC5AC, MUC5B, and IL-13 mRNA

expression was measured by quantitative PCR.

Unlike wild-type mice (see Fig. 7), RV-infected IL-

4R null mice failed to show an increase in mucus or

IL-13 expression. n = 3–5 animals per group.

The Journal of Immunology 2901

a second peak 21 d postinfection (Fig. 7D). Administration ofanti–IL-13 neutralizing Ab significantly decreased PAS staining inthe small airways and partially decreased airways responsiveness.Finally, IL-4R null mice failed to show RV-induced mucousmetaplasia, implicating IL-13 in the pathogenesis of persistentairway changes in RV-infected neonatal mice.Chronic airways hyperresponsiveness and goblet cell hyper-

plasia following viral infection were first shown in C57BL6/J straininfected with Sendai virus (murine parainfluenza virus type 1) (11),a negative sense, ssRNA virus. In these mice, IL-13 production byNK T cells and alternatively activated macrophages is responsiblefor persistent mucous metaplasia (12). We have recently shownthat alternatively polarized macrophages are responsible for RV-induced airway inflammation and hyperresponsiveness in maturemice with allergic airways disease (14). In the current study, wefound increased expression of CD206, also known as macrophagemannose receptor 1 and C-type lectin domain family 13 memberD, in CD11b-positive macrophages, indicative of a switch inmacrophage activation state. Alternative macrophage activationhas been demonstrated to be IL-13 dependent in Cryptococcusneoformans-infected mice (40). We also found an increase in thepercentage of CD335-positive, TCR-b–positive cells expressingIL-13. Together, these results are consistent with the notion thatIL-13, produced by NKT cells, plays a significant role in chronicmucous metaplasia and hyperresponsiveness following viral in-fection. Other mechanisms are possible, however. We have notexcluded the possibility that IL-13–producing CD4-positiveTh2 lymphocytes play a role. It is therefore conceivable that RV

infection has direct effects on airway epithelial cell mucus pro-duction. RV infection of NCI-H292 airway epithelial cells inducesTLR3- and epidermal growth factor receptor-dependent MUC5ACexpression (41). We have also noticed that RV infection of primaryairway epithelial cells grown at air-liquid interface induces mu-cous metaplasia (U. Sajjan, M. Hershenson, unpublished obser-vations). In addition, the proinflammatory cytokine IL-17A hasbeen shown to regulate airway epithelial cell MUC5AC andMUC5B expression via an IL-6 paracrine loop (42). Finally,whereas it is conceivable that early RV infection increases airwayresponsiveness by inducing subtle alterations in airway and pa-renchymal development (43), importantly, neonatal RV infectionhad no effect on lung alveolarization.As noted above, infection of mature mice failed to inducemucous

metaplasia or long-lasting airways hyperresponsiveness as it did inneonatal mice. The mechanisms underlying this age-dependenteffect are unclear. First, it is conceivable that neonatal mice aremore susceptible to viral infection, leading to exaggerated airwayeffects. It has previously been shown that the activation and entry ofneonatal T cells into the lungs are delayed after influenza infection(44). In the current study, RV copy number was sustained for 3 d inneonatal mice, and lung lymphocyte infiltration did not increaseuntil 7 d postinfection. This contrasts with mature mice in whichviral copy number peaks 24 h postinfection and lymphocyte in-filtration increases 1 d postinfection. In addition, neutrophilic in-flammation and IFN-g expression by CD4+ T cells persisted forweeks after neonatal infection. Together, these results suggest adelayed and prolonged response to acute viral infection in neonatal

FIGURE 10. Effect of neonatal RV infection on the

response to allergen sensitization and challenge. Mice

were inoculated with sham or RV on day 7 of life,

sensitized with i.p. OVA on days 17 and 24, and

challenged with intranasal OVA on days 32, 33, and

34. Compared with sham- and OVA-treated mice (A),

RV-infected, OVA-treated mice showed greater peri-

bronchial inflammation (B). Lung sections were

stained with H&E. Original magnification 3100.

Images represent two individual experiments of two

mice per group. (C and D) RV-infected mice also show

greater OVA-induced bronchoalveolar neutrophils and

macrophages (C) and airways cholinergic respon-

siveness (D). n = 3–6 animals per group. *Different

from sham, p , 0.05, unpaired t test.

2902 EARLY LIFE RHINOVIRUS INFECTION IN MICE

animals. Second, the neonatal airway epithelium may be moresusceptible to rhinovirus infection than the mature epithelium.Mucous metaplasia has been shown to increase susceptibility ofthe airway epithelium to RV infection (45). In rhesus monkeys,goblet cells in the trachea decrease in abundance with increasingage (46). We noted that sham-infected 35-d-old mice show a rangeof PAS staining, extending from no signal to patches of densestaining in the large airways, whereas sham-infected 8-wk-oldmice show only rare PAS-positive cells. Third, it is possible thatearly life viral infection causes mucous metaplasia and airwayshyperresponsiveness by augmenting or maintaining the neonatalimmature response, which is skewed toward a Th2 phenotype (47–49). The relatively high IL-13 levels in both sham- and RV-treatedimmature mice are consistent with Th2 polarization.Neonatal RV infection also enhanced the inflammatory response

to allergen sensitization and challenge. Prior infection with RSVhas been shown to increase subsequent allergic airways responses inboth immature (9) and mature mice (50). Interestingly, in contrastto the previous study of neonatal RSV infection, prior infectionwith RV significantly increased OVA-induced neutrophilic in-flammation. The mechanisms by which allergen exposure inducesneutrophilic airways inflammation have not been well studied, butmay involve the activation of the IL-17/IL-23 axis (51, 52).Finally, we note that the effects of neonatal RV infection ap-

peared to fade with time, with mucous metaplasia substantiallyreduced by 60 d postinfection and absent by 100 d postinfection.Because babies are exposed to RV numerous times during infancy,it is conceivable that there could be additive or synergistic effects ofinfection over time.We conclude that RV1B infection of neonatal BALB/c mice

leads to airways inflammation, mucous metaplasia, and hyper-responsiveness, which are mediated, at least in part, by IL-13.These results are consistent with the notion that, under certaincircumstances, early infection with RVmay play a causal role in thedevelopment of asthma.

DisclosuresThe authors have no financial conflicts of interest.

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