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• W1981093593 abstract "We have followed throughout time the development of allergic airway disease (AAD) in both uninfected mice and mice infected intranasally with murine cytomegalovirus (MCMV). Histological evaluation of lung tissue from uninfected mice with AAD demonstrated mucus plugging after 14 and 21 days of ovalbumin-aerosol challenge, with resolution of mucus plugging occurring by 42 days. In MCMV/AAD mice, mucus plugging was observed after 7 days of ovalbumin-aerosol challenge and remained present at 42 days. The level of interleukin-13 in bronchoalveolar lavage fluid from MCMV/AAD mice was decreased compared with AAD mice and was accompanied by increased levels of interferon-γ. Levels of Muc5A/C, Muc5B, or Muc2 mucin mRNA in the lungs of MCMV/AAD mice were not elevated compared with AAD mice. MCMV was able to infect the airway epithelium, resulting in decreased expression of Foxj1, a transcription factor critical for ciliogenesis, and a loss of ciliated epithelial cells. In addition, an increase in the number of epithelial cells staining positive for periodic acid-Schiff was observed in MCMV/AAD airways. Together, these findings suggest that MCMV infection of the airway epithelium enhances goblet cell metaplasia and diminishes efficient mucociliary clearance in mice with AAD, resulting in increased mucus plugging. We have followed throughout time the development of allergic airway disease (AAD) in both uninfected mice and mice infected intranasally with murine cytomegalovirus (MCMV). Histological evaluation of lung tissue from uninfected mice with AAD demonstrated mucus plugging after 14 and 21 days of ovalbumin-aerosol challenge, with resolution of mucus plugging occurring by 42 days. In MCMV/AAD mice, mucus plugging was observed after 7 days of ovalbumin-aerosol challenge and remained present at 42 days. The level of interleukin-13 in bronchoalveolar lavage fluid from MCMV/AAD mice was decreased compared with AAD mice and was accompanied by increased levels of interferon-γ. Levels of Muc5A/C, Muc5B, or Muc2 mucin mRNA in the lungs of MCMV/AAD mice were not elevated compared with AAD mice. MCMV was able to infect the airway epithelium, resulting in decreased expression of Foxj1, a transcription factor critical for ciliogenesis, and a loss of ciliated epithelial cells. In addition, an increase in the number of epithelial cells staining positive for periodic acid-Schiff was observed in MCMV/AAD airways. Together, these findings suggest that MCMV infection of the airway epithelium enhances goblet cell metaplasia and diminishes efficient mucociliary clearance in mice with AAD, resulting in increased mucus plugging. Asthma is an inflammatory disease of the lower airways, characterized by airflow obstruction, airway hyperreactivity, and airway inflammation. Mucus hypersecretion can contribute to airway obstruction in asthmatic patients, particularly during periods of exacerbation of the disease.1Takizawa T Thurlbeck WM Muscle and mucous gland size in the major bronchi of patients with chronic bronchitis, asthma, and asthmatic bronchitis.Am Rev Respir Dis. 1971; 104: 331-336PubMed Google Scholar, 2Fahy JV Steiger DJ Liu J Basbaum CB Finkbeiner WE Boushey HA Markers of mucus secretion and DNA levels in induced sputum from asthmatic and from healthy subjects.Am Rev Respir Dis. 1993; 147: 1132-1137Crossref PubMed Scopus (87) Google Scholar In fact, several studies have reported that airway obstruction is a key feature in fatal asthma with occlusions being comprised of mucus, inflammatory cells, and shed epithelial cells.3Huber H Koessler K The pathology of bronchial asthma.Arch Intern Med. 1922; 30: 689-760Crossref Scopus (329) Google Scholar, 4Dunnill MS The pathology of asthma, with special reference to changes in the bronchial mucosa.J Clin Pathol. 1960; 13: 27-33Crossref PubMed Scopus (720) Google Scholar, 5Cardell P Death in asthmatics.Thorax. 1959; 14: 341-352Crossref Google Scholar, 6Kuyper LM Pare PD Hogg JC Lambert RK Ionescu D Woods R Bai TR Characterization of airway plugging in fatal asthma.Am J Med. 2003; 115: 6-11Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 7Jeffery PK Morphology of the airway wall in asthma and in chronic obstructive pulmonary disease.Am Rev Respir Dis. 1991; 143: 1152-1161Crossref PubMed Google Scholar The pattern of these elements within the plug suggests that they represent multiple or ongoing events rather than a single inflammatory response and emphasize the chronic nature of asthma.7Jeffery PK Morphology of the airway wall in asthma and in chronic obstructive pulmonary disease.Am Rev Respir Dis. 1991; 143: 1152-1161Crossref PubMed Google Scholar In humans, mucus is produced by the submucosal glands and goblet cells. Under healthy conditions, goblet cells comprise a small percentage of the airway epithelium, but in asthma can increase in number to become 20 to 25% of the epithelial population in both central and peripheral airways.8Shimura S Andoh Y Haraguchi M Shirato K Continuity of airway goblet cells and intraluminal mucus in the airways of patients with bronchial asthma.Eur Respir J. 1996; 9: 1395-1401Crossref PubMed Scopus (125) Google Scholar In contrast, submucosal glands in the mouse are few, surrounding only the trachae, and a limited number of goblet cells are present under normal conditions; however, this animal model has proven to be a useful tool in understanding certain mechanisms involved in mucus hypersecretion and goblet cell hyperplasia. In several mouse models of asthma, interleukin (IL)-13 has been shown to be the crucial driving factor for the production of mucus.9Wills-Karp M Luyimbazi J Xu X Schofield B Neben TY Karp CL Donaldson DD Interleukin-13: central mediator of allergic asthma.Science. 1998; 282: 2258-2261Crossref PubMed Scopus (2378) Google Scholar, 10Grünig G Warnock M Wakil AE Venkayya R Brombacher F Rennick DM Sheppard D Mohrs M Donaldson DD Locksley RM Corry DB Requirement for IL-13 independently of IL-4 in experimental asthma.Science. 1998; 282: 2261-2263Crossref PubMed Scopus (1719) Google Scholar, 11Cohn L Homer RJ Marinov A Rankin J Bottomly K Induction of airway mucus production by T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production.J Exp Med. 1997; 186: 1737-1747Crossref PubMed Scopus (401) Google Scholar, 12Cohn L Homer RJ MacLeod H Mohrs M Brombacher F Bottomly K Th2-induced airway mucus production is dependent on IL-4Ralpha, but not on eosinophils.J Immunol. 1999; 162: 6178-6183PubMed Google Scholar These findings have been supported by in vitro studies in human epithelial cells, where IL-13 has been shown to enhance a mucus hypersecretory phenotype by increasing goblet cell density.13Zuhdi Alimam M Piazza FM Selby DM Letwin N Huang L Rose MC Muc-5/5ac mucin messenger RNA and protein expression is a marker of goblet cell metaplasia in murine airways.Am J Respir Cell Mol Biol. 2000; 22: 253-260Crossref PubMed Scopus (214) Google ScholarViral respiratory infections have been closely associated with asthma, potentially resulting in three separate outcomes: causative, exacerbating, or preventative. Being prevalent during infancy, respiratory syncytial virus (RSV) infection is associated with the first episodes of wheezing in the majority of infants, causing airway inflammation and airway obstruction.14Henderson FW Clyde Jr, WA Collier AM Denny FW Senior RJ Sheaffer CI Conley III, WG Christian RM The etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice.J Pediatr. 1979; 95: 183-190Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 15Martinez FD Morgan WJ Wright AL Holberg CJ Taussig LM Diminished lung function as a predisposing factor for wheezing respiratory illness in infants.N Engl J Med. 1988; 319: 1112-1117Crossref PubMed Scopus (564) Google Scholar, 16Busse WW Respiratory infections: their role in airway responsiveness and the pathogenesis of asthma.J Allergy Clin Immunol. 1990; 85: 671-683Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 17Martinez FD Wright AL Taussig LM Holberg CJ Halonen M Morgan WJ Asthma and wheezing in the first six years of life. The Group Health Medical Associates.N Engl J Med. 1995; 332: 133-138Crossref PubMed Scopus (3252) Google Scholar However, by the age of 2 nearly all children have been infected with RSV, but not all children develop asthma. Thus, it is unclear whether RSV infection can initiate or cause asthma, placing some infants at risk for the development of the disease, or whether this is merely an association. In contrast, it is generally accepted that viral respiratory infections can lead to the exacerbation of asthma in individuals who have been diagnosed with the disease. Epidemiological studies have shown that viral respiratory infections, particularly rhinovirus, coincide with asthma attacks in 80 to 85% of school-age children and 44% of adults, resulting in decreased lung function and increased symptom scores.18Johnston SL Pattemore PK Sanderson G Smith S Lampe F Josephs L Symington P O'Toole S Myint SH Tyrrell DA Holgate ST Community study of role of viral infections in exacerbations of asthma in 9–11 year old children.BMJ. 1995; 310: 1225-1229Crossref PubMed Scopus (1661) Google Scholar, 19Nicholson KG Kent J Ireland DC Respiratory viruses and exacerbations of asthma in adults.BMJ. 1993; 307: 982-986Crossref PubMed Scopus (944) Google Scholar Finally, the idea that viral pathogens may protect individuals from the development of asthma is controversial and is based on the observation that larger families that experience more infections have a lower risk of developing asthma.20Strachan DP Family size, infection and atopy: the first decade of the “hygiene hypothesis.”.Thorax. 2000; 55: S2-S10Crossref PubMed Google Scholar, 21Shaheen SO Aaby P Hall AJ Barker DJ Heyes CB Shiell AW Goudiaby A Measles and atopy in Guinea-Bissau.Lancet. 1996; 347: 1792-1796Abstract PubMed Scopus (474) Google Scholar, 22Matricardi PM Rosmini F Ferrigno L Nisini R Rapicetta M Chionne P Stroffolini T Pasquini P D'Amelio R Cross sectional retrospective study of prevalence of atopy among Italian military students with antibodies against hepatitis A virus.BMJ. 1997; 314: 999-1003Crossref PubMed Scopus (451) Google Scholar, 23Oddy WH de Klerk NH Sly PD Holt PG The effects of respiratory infections, atopy, and breastfeeding on childhood asthma.Eur Respir J. 2002; 19: 899-905Crossref PubMed Scopus (194) Google ScholarThe mechanism by which viral respiratory pathogens influence asthma is unknown. It is clear, however, that respiratory viruses share a common ability to infect the airway epithelium. In this manner viruses can directly damage the epithelial barrier, allowing the passage of more irritants and allergens into the lung, as well as increase epithelial cell shedding.24Reisinger J Triendl A Kuchler E Bohle B Krauth MT Rauter I Valent P Koenig F Valenta R Niederberger V IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation.J Allergy Clin Immunol. 2005; 115: 973-981Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Repair and regeneration of damaged airway epithelium is complex and several transcription factors important in ciliogenesis and lung morphology have been shown to play a role.25Park KS Wells JM Zorn AM Wert SE Laubach VE Fernandez LG Whitsett JA Transdifferentiation of ciliated cells during repair of the respiratory epithelium.Am J Respir Cell Mol Biol. 2006; 34: 151-157Crossref PubMed Scopus (158) Google Scholar Furthermore, viral respiratory infections activate the host's immune system, triggering a nonspecific proinflammatory response that is necessary to control the viral infection and a virus-specific CD8+ T-cell response, resulting in the production of interferon (IFN)-γ and clearance of the virus. It is conceivable that such changes in the local cytokine environment, triggered by viral infection, may play an important role in influencing asthma.Although RSV and rhinovirus are the primary viral respiratory pathogens associated with changes in asthma, other viruses can also influence this disease. For example, human cytomegalovirus, a β-herpesvirus, has been associated with exacerbations of asthma in adults.26Atmar RL Guy E Guntupalli KK Zimmerman JL Bandi VD Baxter BD Greenberg SB Respiratory tract viral infections in inner-city asthmatic adults.Arch Intern Med. 1998; 158: 2453-2459Crossref PubMed Scopus (174) Google Scholar Therefore, we have examined the ability of murine cytomegalovirus (MCMV), which shares many biological properties with human cytomegalovirus, to influence the ovalbumin (OVA)-induced murine model of allergic airway disease (AAD). Our initial studies demonstrate that MCMV infection enhanced mucus plugging in small airways during acute AAD.27Wu CA Puddington L Whiteley HE Yiamouyiannis CA Schramm CM Mohammadu F Thrall RS Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease.J Immunol. 2001; 167: 2798-2807PubMed Google Scholar In the present studies we found that enhanced mucus plugging in MCMV/AAD mice was observed during both acute and chronic airway disease, but was not associated with increased mucin gene expression or increased IL-13 expression as compared to AAD mice. However, MCMV infection decreased Foxj1 expression, a transcription factor important in ciliogenesis, and resulted in changes in the ciliary phenotype of the airway epithelium that may hinder efficient mucociliary transport, thereby enhancing mucus plugging.Materials and MethodsAnimals and VirusesSeven-week-old female C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were provided sterile food and water, and housed in microisolators under specific pathogen-free conditions. Their welfare was in accordance with institutional and Office of Laboratory Animal Welfare guidelines. All animal handling and manipulations were preapproved by the Animal Care Committee at the University of Connecticut Health Center.MCMV strain K181, originally obtained from Dr. M.C. Jordan (Oregon Health Sciences University, Portland, OR), was commercially screened for the presence of other pathogens and scored negative. Recombinant MCMV, expressing the enhanced green fluorescent protein under the control of the immediate early 2 promoter, was a gift from Dr. John Hamilton (Duke University, Durham, NC).28Henry SC Schmader K Brown TT Miller SE Howell DN Daley GG Hamilton JD Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection.J Virol Methods. 2000; 89: 61-73Crossref PubMed Scopus (46) Google ScholarOVA Study ProtocolOVA sensitization and aerosol challenge of C57BL/6J mice has been previously described for our model of AAD.27Wu CA Puddington L Whiteley HE Yiamouyiannis CA Schramm CM Mohammadu F Thrall RS Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease.J Immunol. 2001; 167: 2798-2807PubMed Google Scholar Briefly, mice were given three weekly intraperitoneal injections of a suspension containing 25 μg of OVA (grade V; Sigma, St. Louis, MO) and 2 mg of aluminum hydroxide [Al(OH3)] in 0.5 ml of saline. One week after the last injection, mice were exposed to 0.2% aerosolized OVA generated by a BANG nebulizer (CH Technologies, Inc., Westwood, NJ) for 1 hour per day, delivered in a nose-only inhalation exposure chamber with space for exposing 48 mice simultaneously (In-Tox Production, Moriarty, NM). The concentration of OVA-aerosol used in these experiments differed from the 1% OVA solution used in our previous studies27Wu CA Puddington L Whiteley HE Yiamouyiannis CA Schramm CM Mohammadu F Thrall RS Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease.J Immunol. 2001; 167: 2798-2807PubMed Google Scholar and was chosen to maximize differences in parameters between AAD and MCMV/AAD mice. This procedure was repeated daily for 1, 3, 7, 10, 14, 21, or 42 days, as noted in the text, and mice were sacrificed 24 hours after the last OVA-aerosol challenge. Two control groups are routinely included in our studies: naïve mice that received no treatment and OVA-sensitized mice that received three weekly injections of OVA/Al(OH3), but are not exposed to OVA-aerosol. No differences in measured outcomes have been noted between these two controls.MCMV/AAD mice were sensitized and exposed to OVA-aerosol as described above with one addition. Viral infection was performed intranasally 1 week before the start of OVA-aerosol challenge, using 2 × 104 PFU of MCMV. Two control groups were included in these studies: MCMV/OVA-sensitized mice that received three weekly injections of OVA/Al(OH3) and were intranasally infected with MCMV, but were not exposed to OVA-aerosol and MCMV infected mice that were infected with MCMV, but not sensitized to OVA/Al(OH3) or exposed to OVA-aerosol. No differences in measured outcomes were noted between these two controls. At the time of sacrifice, confirmation of MCMV infection was determined by the presence of virus in the salivary gland using a standard plaque assay.Analysis of Bronchoalveolar Lavage Fluid (BALF)Twenty-four hours after the final OVA-aerosol challenge, the lungs from each animal were lavaged in situ with five 1-ml aliquots of sterile saline. The total number of viable leukocytes was determined by trypan blue exclusion using a hemocytometer. Leukocyte differentials were determined by May-Grunwald/Giemsa staining of cytocentrifuged BALF preparations.Measurement of Cytokines in BALF and SerumBALF, collected after 1, 3, 7, 10, 14, 21, or 42 days of OVA-aerosol challenge, was concentrated 10-fold using a Centriplus YM-10 filtration device (Amicon, Beverly, MA), aliquoted, and stored at −80°C until use. The presence of IFN-γ (Pierce-Endogen, Rockford, IL) and IL-13 (R&D Systems, Minneapolis, MN) were assayed by enzyme-linked immunosorbent assay, testing an undilute and a 1:2 dilution of concentrated BALF. The limit of detection was 10 and 1.5 pg/ml for IFN-γ and IL-13, respectively.RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)Fresh lung tissue was homogenized in 1 ml of Ultraspec RNA solution (Biotecx Laboratories, Houston, TX) and stored at −80°C until use. Total RNA was isolated by chloroform extraction and ethanol precipitation, according to the manufacturer's specifications, and used in RT-PCR assays. Five μg of total RNA was treated with 5 U of DNase I (Invitrogen, Carlsbad, CA) for 15 minutes at room temperature, and was then used to generate cDNA templates for PCR with the SuperScript First-Strand (Invitrogen) and random hexamers in 50-μl total volume. Reactions lacking the reverse transcriptase served as a control for the presence of genomic DNA. To determine the levels of mucin mRNA by PCR, 5 μl of cDNA was incubated with the following primers: Muc5A/C, 5′-TGGACCTCAGGTATTTCACAC-3′ (sense) and 5′-TAGTTCTTAGCCCTGCATTGC-3′ (antisense); Muc5B, 5′-GCACCTGCTGTCAGGAGAGCA-3′ (sense) and 5′-TGGTCAGTTGTGCAGGTTCTG-3′ (antisense); and Muc2, 5′-TTTGGGAACATGCAGAAGATCAAC-3′ (sense) and 5′-CGTGAGAGCACTGGCGCGAGA-3′ (antisense). GAPDH primers [5′-ATGTTCCAGTATGACTCCACT-3′ (sense) and 5′-CCACAATGCCAAAGTTGTCAT-3′ (antisense)] were added to each PCR reaction as an internal control. The following PCR conditions were implemented: 94°C for 3 minutes, 35 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, followed by a final extension at 72°C for 7 minutes. The lungs from three individual mice were examined at each time point and the resulting PCR products were resolved on a 2% agarose gel. The intensity of the mucin bands and GAPDH was determined using IPLab Gel software (BD Biosciences, Rockville, MD).Real-Time PCR for Mucin and Foxj1 mRNATotal RNA from lung tissue was treated with DNase I as described above. Real-time PCR was performed in duplicate for Muc5A/C, Muc5B, Gob-5, foxj1, and β-actin in a final volume of 25 μl containing 12.5 ng of total RNA and the iScript one-step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, CA) using an iCycler iQ real-time PCR detection system (Bio-Rad). The primers were: Muc5A/C, 5′-AAAGACACCAGTAGTCACTCAGCAA-3′ (sense) and 5′- CCACATGGGGTCACACTTC-3′ (antisense)29Kunert KS Keane-Myers AM Spurr-Michaud S Tisdale AS Gipson IK Alteration in goblet cell numbers and mucin gene expression in a mouse model of allergic conjunctivitis.Invest Ophthalmol Vis Sci. 2001; 42: 2483-2489PubMed Google Scholar; Muc5B, 5′-CATGCTCACAAAAGCAGATG-3′ (sense) and 5′-CCAGGGTTTATTGCAGCTTA-3′ (antisense); Gob-5, 5′-TCCAGGCTGTGGATAAGTCC-3′ (sense) and 5′-TATCTTCAGCACGTGGATGC-3′ (antisense)30Busse PJ Zhang TF Srivastava K Lin BP Schofield B Sealfon SC Li XM Chronic exposure to TNF-alpha increases airway mucus gene expression in vivo.J Allergy Clin Immunol. 2005; 116: 1256-1263Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar; Foxj1, 5′-GAGTGAGGGCAAGAGACTGG-3′ (sense) and 5′-TCAAGTCAGGCTGGAAGGTT-3′ (antisense)31Lin L Peng SL Coordination of NF-kappaB and NFAT antagonism by the forkhead transcription factor Foxd1.J Immunol. 2006; 176: 4793-4803PubMed Google Scholar; and β-actin, 5′-GATGCCACAGGATTCCATA-3′ (sense) and 5′-AGAGGGAAATCGTGCGTGAC-3′ (antisense) at concentrations of 325 nmol/L, 400 nmol/L, 300 nmol/L, 150 nmol/L, and 220 nmol/L, respectively. The PCR conditions were as follows: 50°C for 10 minutes for the RT step, 95°C for 3.5 minutes followed by 40 cycles of 95°C for 10 seconds, then 58°C for 20 seconds. The efficiency of all PCR reactions was greater than 90% and the relative fold-increase of mucin, Gob-5, or Foxj1 was calculated using the CT (threshold cycle) method as compared to the housekeeping gene β-actin.Lung Histology and ImmunohistochemistryUnmanipulated, noninflated lungs were removed, fixed with 10% buffered formalin, and embedded in paraffin by conventional methods. Tissue sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) or Alcian blue (data not shown). Histological examination of lung tissue infected with recombinant MCMV was processed in an identical manner. For analysis of Foxj1 expression, noninflated lungs were fixed with fresh 4% paraformaldehyde in phosphate-buffered saline for 24 to 48 hours at 4°C and embedded in paraffin. Five-μm tissue sections were deparaffinized in toluene, then rehydrated through graded alcohol to water. Exogenous peroxidase was quenched by incubating the slides in 3% H2O2 in water for 15 minutes at room temperature. Antigen retrieval was performed by incubating the slides in citrate buffer, pH 6.0, at 95°C for 20 minutes, followed by cooling at room temperature for 20 minutes. The samples were incubated with Foxj1 antibody (1:4000; Upstate, Lake Placid, NY) in phosphate-buffered saline (PBS) with 0.1% bovine serum albumin for 1 hour at room temperature, then washed in PBS. The secondary antibody, biotinylated anti-mouse IgG from the M.O.M kit (Vector Laboratories, Burlingame, CA), was used according to the manufacturer's instructions, then tissues were counterstained for 30 seconds with hematoxylin. In addition, the total number of cells, as well as the percentage of PAS-positive or Foxj1-positive cells, was determined as follows. Sequential lung tissue sections from the same AAD and MCMV/AAD mice, used for examination of Foxj1 expression above, were stained with PAS, followed by hematoxylin or Foxj1, followed by hematoxylin. The number of positive cells in airways that demonstrated PAS staining (<150 μm in diameter) was determined, examining at least four airways for each group and counting a total of six to eight sections with 50 μm of basement membrane.Statistical AnalysisA statistical comparison between two groups at a single time point was calculated using an unpaired t-test. Analysis of multiple time points or experiments with three or more groups was performed using one-way analysis of variance, followed by Fisher's PLSD. All tests were performed using the software StatView 4.5 (Abacus Concepts, Inc., Berkeley, CA). A probability of P < 0.05 was considered significant. Data are presented as a mean ± SEM.ResultsMCMV Infects the Bronchial EpitheliumAlthough it is known that MCMV can infect the lungs of mice, infection of the airway epithelium has not been demonstrated. Thus, C57BL/6 mice were infected intranasally with a recombinant MCMV that expresses the enhanced green fluorescent protein. The mice were sacrificed after 7 days, when peak viral loads of MCMV were present in the lung.27Wu CA Puddington L Whiteley HE Yiamouyiannis CA Schramm CM Mohammadu F Thrall RS Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease.J Immunol. 2001; 167: 2798-2807PubMed Google Scholar Unmanipulated lung tissue was fixed in formalin and sequential sections stained with H&E (Figure 1A) or examined by phase (Figure 1B) and fluorescent (Figure 1C) microscopy. Fluorescent green staining was evident in the bronchial epithelium of airways less than 200 μm in diameter. No staining of the surrounding lung tissue, including the endothelium of blood vessel, was noted. Although MCMV is a lytic virus, no gross structural damage was evident on examination. This histological analysis demonstrated that MCMV can infect the bronchial epithelium.Concomitant MCMV Infection Enhanced Mucus ProductionWe have previously shown that concomitant MCMV infection enhanced mucus plugging during the acute stage of AAD (after 7 days of OVA-aerosol challenge).27Wu CA Puddington L Whiteley HE Yiamouyiannis CA Schramm CM Mohammadu F Thrall RS Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease.J Immunol. 2001; 167: 2798-2807PubMed Google Scholar To determine the ability of MCMV to influence mucus plugging during the acute and chronic stages of disease, lung sections from uninflated, nonmanipulated lungs were isolated from AAD and MCMV/AAD mice after 1, 3, 7, 10, 14, 21, and 42 days of OVA-aerosol challenge and stained with PAS. As controls, lung sections from MCMV-infected and -uninfected naïve mice were also examined. Little to no PAS staining was observed in lung tissue from either AAD or MCMV/AAD mice on day 1 (not shown). Although the majority of airways from AAD and MCMV/AAD mice on day 3 did not stain with PAS, a limited number of airways displayed PAS-positive cells in both groups (Figure 2, A and B, respectively). By day 7, the number of PAS-stained airways increased in both AAD and MCMV/AAD mice (Figure 2, D and E, respectively), but mucus plugging of airways was evident only in MCMV/AAD mice. Extensive PAS staining of the airway epithelium, as well as mucus plugging, was present in both AAD and MCMV/AAD mice after 14 days (Figure 2, G and H) and 21 days (Figure 2, J and K) of OVA-aerosol challenge. By 42 days of OVA-aerosol challenge, only a few PAS-positive staining cells were observed in AAD mice and no mucus plugs were noted (Figure 2M). In contrast, mucus plugging was still evident in MCMV/AAD mice (Figure 2N). PAS staining was not present in lung tissue from naïve (Figure 2L) or MCMV mice corresponding to 3 (Figure 2C), 7 (Figure 2F), and 14 (Figure 2I) days of OVA-aerosol challenge (Figure 2, C, F, and I). Thus, concomitant MCMV infection in mice with AAD resulted in an earlier onset of mucus plugging (7 days of OVA-aerosol challenge) and prolonged mucus plugging (42 days of OVA-aerosol challenge).Figure 2Histological evidence of mucus plugging in airways from AAD and MCMV/AAD mice. After 3 (A, B), 7 (D, E), 14 (G, H), 21 (J, K), or 42 (M, N) days of OVA-aerosol challenge, mice were sacrificed and unmanipulated lung tissue was processed for histological evaluation. Lung tissue from MCMV-infected mice corresponding to 3 (C), 7 (F), and 14 days (I), along with lung tissue from naïve mice (L), served as controls. PAS-stained lung sections from AAD, MCMV/AAD, MCMV, and naive mice are shown here, with mucus plugs first observed in MCMV/AAD and AAD mice after 7 and 14 days of OVA-aerosol, respectively. Similar results were obtained with Alcian blue staining. Scale bar = 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IL-13 Production Was Decreased in MCMV/AAD MiceIL-13 is a pleiotropic cytokine, produced by activated CD4+ Th2 cells, that has been associated with many features of asthma, including increased mucus production.9Wills-Karp M Luyimbazi J Xu X Schofield B Neben TY Karp CL Donaldson DD Interleukin-13: central mediator of allergic asthma.Science. 1998; 282: 2258-2261Crossref PubMed Scopus (2378) Google Scholar, 10Grünig G Warnock M Wakil AE Venkayya R Brombacher F Rennick DM Sheppard D Mohrs M Donaldson DD Locksley RM Corry DB Requirement for IL-13 independently of IL-4 in experimental asthma.Science. 1998; 282: 2261-2263Crossref PubMed Scopus (1719) Google Scholar To determine whether differences in IL-13 were associated with enhanced mucus plugging in MCMV/AAD mice, BALF was collected from AAD and MCMV/AAD mice from the same experiment and examined after 1, 3, 7, 10, 14, 21, and 42 days of OVA-aerosol challenge. As shown in Figure 3A, elevated levels of IL-13 were observed in BALF from AAD mice after 3 and 7 days of OVA-aerosol challenge. In contrast, the levels of IL-13 were reduced in BALF from MCMV/AAD mice after 3 days of OVA-aerosol challenge (Figur" @default.
• W1981093593 created "2016-06-24" @default.
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• W1981093593 date "2008-03-01" @default.
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• W1981093593 title "Murine Cytomegalovirus Influences Foxj1 Expression, Ciliogenesis, and Mucus Plugging in Mice with Allergic Airway Disease" @default.
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