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Experimental and Toxicologic Pathology 65 (2013) 357– 364

Contents lists available at SciVerse ScienceDirect

Experimental and Toxicologic Pathology

jo u rn al h omepage: www.elsev ier .de /e tp

haracterization of the lung epithelium of wild-type and TLR9−/− mice afteringle and repeated exposures to chicken barn air

am Saran Sethia,1, David Schnebergerb,1, Baljit Singhb,∗

School of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, IndiaDepartment of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada

r t i c l e i n f o

rticle history:eceived 4 July 2011ccepted 29 November 2011

eywords:lara cellucus cell

LR9ype-II alveolar epithelial cell

a b s t r a c t

Exposure to chicken barn air causes lung injury resulting in lower and upper respiratory symptoms inthe poultry workers, and mechanisms of which are not fully understood. The lung injury can initiatemodifications such as proliferation of the airway epithelial cells such as Clara cells, type II alveolar (T2)cells and mucus producing goblet cells as part of the innate immune response. Toll-like receptors (TLR)have been suggested to play a role in cell division and proliferation. To understand the effect of TLR9 onClara cells, T2 and mucus-producing goblet cells, we quantified the numbers of these cells in the lungs ofwild-type (WT) and TLR9−/− mice exposed to chicken barn air. The mice were exposed for either one dayor five or 20 days for 8 h/day. Clara cells and T2 cells were labelled with antibodies, and the mucus cells

mmunohistochemistryhicken barn airung injury

were identified with Periodic-acid Schiff stain, and quantified in per unit tissue section area. The datashow decrease in the number of Clara cells and increase in mucus-producing goblet cells after exposureto chicken barn air in both WT and TLR9−/− mice. Numbers of T2 cells increased and decreased in WT andTLR9−/− mice, respectively, after exposure to poultry barn air. These data show that exposure to chickenbarn air can affect major lung epithelial cells, and allude to the role of TLR9 in regulation of some of theseresponses.

. Introduction

Workers in swine and poultry units are exposed to significantevels of organic dust and endotoxins (Iversen et al., 2000). Poultry

orkers suffer lower baseline lung function (Radon et al., 2001)rom work-related lower and upper respiratory symptoms suchs chronic cough, allergic and non-allergic rhinitis, organic dustoxic syndrome, chronic bronchitis, hypersensitivity pneumonitisFarmer’s Lung), toxin fever and occupational asthma or asthma-ike syndrome (Iversen et al., 2000; Radon et al., 2001; Redente and

assengale, 2006). The toxic components in the barn air engagehe respiratory epithelium, which is the first line of defense in theung (Puchelle et al., 2006; Redente and Massengale, 2006). Thepithelium in the lung provides protection against inhaled toxicolecules through formation of a physical barrier in the conducting

irways and the alveolar region, production of mucus, and expres-ion of various immune receptors.

∗ Corresponding author at: Department of Veterinary Biomedical Sciences, West-rn College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive,askatoon, SK S7N 5B4, Canada. Tel.: +1 306 966 7486; fax: +1 306 966 7405.

E-mail address: baljit.singh@usask.ca (B. Singh).1 These authors contributed equally to this work.

940-2993/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.etp.2011.11.002

© 2011 Elsevier GmbH. All rights reserved.

The respiratory epithelium is maintained through division ofcells such as basal cells, Clara cells and type II alveolar epithelial(T2) cells. Clara cells are non-ciliated and non-mucus-producingdome-shaped cells (Massaro et al., 1994; Plopper et al., 1980). Claracells secrete a 10 kD (CC10) protein immunomodulatory and anti-inflammatory protein, which is identical to human uteroglobulin(UG) (Miele et al., 1988). The expression of CC10 is altered in vari-ous lung diseases (Bernard et al., 1992; Lesur et al., 1995). T2 cellsperform many important functions including production of surfac-tant, ion transport and alveolar repair in response to lung injury(Gereke et al., 2007; Kalina et al., 1992). Clara cells synthesize sur-factant proteins SP-A, SP-B and SP-D (Fehrenbach, 2001; Kalinaet al., 1992; Voorhout et al., 1992). T2 cells produce all the surfactantproteins including SP-C, which appears to be exclusively producedby T2 cells (Beers et al., 1994; Fehrenbach, 2001). Both Clara cellsand T2 cells express enzymes such as P450 and GSH, which areinvolved in xenobiotic metabolism and may lead to the formationof toxic metabolites leading to injury and death to Clara cells andT2 (Plopper et al., 2001). There are few or no mucus-producing gob-let cells in the intrapulmonary bronchus and bronchiole of normalmice (Pack et al., 1981; Plopper et al., 1983). Increased mucus pro-

duction is part of the innate defense response in the conductingairways. There are data on the accumulation of mucus-producingcells in the mouse airways in asthma (Hayashi et al., 2004; Zhuet al., 1999) and following exposure to swine barn air and allergens

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Charavaryamath et al., 2005; Justice et al., 2002). It is believed thathe increase in goblet cells, which may contain mucus, is throughrans-differentiation of Clara cells (Tyner et al., 2006). There are noata on the impact of exposure to poultry barn air on Clara cells, T2ells and mucus cells, which are critical for the pulmonary defense.

Recent data on the biology of Toll-like receptors (TLR) havenhanced our understanding of innate host responses to pathogensstablishing that TLR9 is critical for the ligation of bacterial DNAuring endotoxemia (Beutler, 2002; Murad and Clay, 2009). TLR9

s expressed in the cytoplasm and the membrane of a variety ofells including macrophages, neutrophils and epithelial cells, whichre central to the recognition and phagocytosis of bacteria (Latzt al., 2004; Wagner, 2004). TLR9 also has been implicated in cancernd apoptosis suggesting its roles in cell division and proliferationDamiano et al., 2006; Tanaka et al., 2010). Many bacterial com-onents in the barn air may induce inflammation through theirinding to TLRs. Therefore, bacterial DNA in the barn air may induce

ung injury through its binding to TLR9 expressed on various lungells.

The mechanisms of lung injury following exposure to poultryarn air remain poorly understood. It is possible that the lungpithelium may be damaged upon exposure to poultry barn aireading to proliferation of Clara cells, T2 cells and goblet cells toeplace the dead cells. Clara, T2 and goblet cells may be directlyamaged following exposure to poultry barn air. Therefore, weuantified the numbers of Clara cells, T2 cells and goblet cells inhe lungs of WT and TLR9−/− mice exposed to poultry barn air.

. Material and methods

The animal experiment protocols were approved by Animalesearch Ethics Board, University of Saskatchewan, Saskatoon,anada, and were conducted according to the Canadian Council onnimal Care Guidelines. Specific pathogen-free, six-week-old, WT

TLR9+/+) and TLR9−/− mice were maintained in the animal carenit of Western College of Veterinary Medicine. All the personnel

nvolved in collection and analyses of samples were blinded to thereatment groups.

.1. Control and treatment groups

The WT and TLR9−/− (N = 40 each) mice were divided into controlN = 16 of each strain) and treatment groups (N = 24 of each strain).he mice were kept inside a chicken barn for 8 h per day for one,ve, and 20 days (N = 8 each group). The control animals of bothtrains were transported to barn in the same vehicle and were car-ied back to the animal care unit to normalize for any stress relatedo transport.

.2. Tissue collection and processing

Immediately following exposure to the barn air, mice were euth-nized (1 mg xylazine and 10 mg ketamine/100 g) to collect the lungissues for histology and immunohistochemistry. Three pieces fromach lung lobe (left and right) were fixed in 4% paraformaldehydeor 16 h and were processed to obtain 5 �m thick paraffin sectionsn poly l-lysine coated clean glass slides.

.3. Light microscopy

The lungs sections were stained with Periodic Acid Schiff (PAS)ethod to demonstrate mucus-producing cells. These PAS stained

ections were used for the quantification of positive cells asescribed previously (Charavaryamath et al., 2005).

ogic Pathology 65 (2013) 357– 364

2.4. Immunohistochemistry

Lung sections were processed for immunohistochemistry asdescribed previously (Charavaryamath et al., 2005). Briefly, thesections were deparaffinized, hydrated and incubated with 0.5%hydrogen peroxide for 20 min to quench endogenous peroxidase,treated with pepsin (2 mg/ml in 0.01 N HCl) for 45 min and 1%bovine serum albumin for 30 min. Following quenching of theendogenous peroxidase and blocking for non-specific binding ofthe antibodies, the sections were stained with primary antibodiesagainst human uteroglobulin (rabbit polyclonal, dilution 1:20,000;Ab40873) and pro-surfactant protein C (rabbit, dilution 1:5000,Ab3786) followed by appropriate biotinylated or horseradish per-oxidase (HRP)-conjugated secondary antibodies (1:100; DAKO A/S,Denmark) to identify Clara cells and T2 cells, respectively. The reac-tion was visualized using a color development kit (SK4600, Vectorlaboratories, USA). To eliminate the possibility of color reactiondue to non-specific binding of the secondary antibody, we omit-ted primary antibody from the staining or replaced it with anisotype-matched immunoglobulin. To test the integrity of the stain-ing methods, we stained lung sections with an antibody againstVon Willebrand factor (vWF) antibody (1:600) to delineate vascu-lar endothelium. The results for the controls are shown later alongwith other results.

2.5. Quantification

Clara cells stained with the antibody and PAS positive mucuscells were counted in only those airways that were fully cross-sectioned. These cells were counted in 10 fields/section from eachanimal manually in an area of 0.2 mm2 under 40× objective lens ofthe microscope so as to maintain the uniformity. Because of theneed for a higher magnification to resolve T2 cells, we countedthese cells manually per 0.08 mm2 area at 100× under oil immer-sion. Five animals from each group were randomly selected for thequantification of these cells. Because the data from one day and20 day normal WT and TLR9−/− mice were not different, the dataof one day control animals were used for further comparison. Theevaluator was blinded to the identity of the treatment groups.

2.6. Statistical analyses

The data was analyzed by single analyses of variance followedby group comparisons with post hoc tests. The significance wasaccepted at p < 0.05.

3. Results

Haematoxylin and eosin staining of mouse lungs yielded noapparent histological differences between control WT and TLR9−/−

(Fig. 1a and b) and WT and TLR9−/− (Fig. 1c and d) mice after singleand multiple exposures to chicken barn air.

3.1. Clara cells

The omission of primary antibody (Fig. 2a) or replacement withan isotype-matched immunoglobulin (data not shown) resulted inlack of color reaction in the lung tissues while vWF stained theendothelium (Fig. 2b). The uteroglobulin antibody stained Claracells (Fig. 2c) but not ciliated cells of the airways or the alveolarseptal cells such as T2 cells. Clara cells were evenly distributed

throughout the epithelium of small airways and few of these cellsshowed small dome shaped projection in lungs of WT mice (Fig. 2c).The TLR9−/− mice exposed for one day showed a reduction in cellsstained with uteroglobulin antibody (Fig. 2d).

R.S. Sethi et al. / Experimental and Toxicologic Pathology 65 (2013) 357– 364 359

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ig. 1. H&E staining: lung sections showed normal appearance of lung architectureve exposures did not reveal any significant difference in bronchioles (Br) or alveolair. Original magnification (a)–(d) 40×.

Numerical counts revealed a decrease in the number of Claraells in WT mice exposed multiple times to chicken barn air com-ared to the control mice (Fig. 2e). WT mice exposed 20 timeshowed a significant decrease in the number of Clara cells com-ared to those exposed once or five times. TLR9−/− mice showed aignificant decrease in the number of Clara cells following a singlexposure to chicken barn air (Fig. 2f). Further, there was a signifi-ant difference between the decrease in the number of Clara cellsn TLR9−/− mice exposed for 20 times compared to those exposedor one day or five days. However, the number of Clara cells did nothow any significant difference between TLR9−/− mice exposed forne day and five days. Number of cells stained with uteroglobu-in antibody was less in TLR9−/− mice compared to WT mice afteringle exposure to chicken barn air.

.2. Mucus producing cell

The mucus producing cells were stained pink with PAS methodFig. 3a–d). The data revealed a significant increase in the numberf PAS-positive cells in the lungs of all the exposed WT and TLR9−/−

ice compared to the respective controls (p < 0.05) (Fig. 3e). Thereas significant increase in PAS cells in WT and TLR9−/− mice

xposed for 20 days compared to those subjected to a single expo-ure (Fig. 3f).

.3. Type 2 alveolar cells

The type 2 alveolar (T2) cells, typically located in the corners of

he alveolar septal wall, were stained in the perinuclear areas withuman pro-surfactant protein C in both WT and

TLR9−/− mice (Fig. 4a and b). Type-1 alveolar epithelial cells andhe airway epithelial cells did not react with the antibody.

ntrol WT (a) and TLR9−/− mice (b). The lungs of WT (c) and TLR9−/− (d) mice afterons compared to control animals after single or multiple exposures to chicken barn

The number of T2 cells of the alveolar septa showed no dif-ference between the control groups of WT and TLR9−/− mice. Thesingle exposure to chicken barn air resulted in a significant increasein the number of T2 cells in WT mice (Fig. 4c) whereas TLR9−/− micedid not show any change in the number of these cells after singleexposure (Fig. 4d). Further, the number of T2 cells decreased sig-nificantly in TLR9−/− mice after five or 20 exposures compared toone day exposed group.

4. Discussion

Innate immune responses in the lung epithelium are regulatedthrough the secretions of various cells. Clara cells and T2 cells differ-entiate into bronchiolar and alveolar epithelial cells, respectively,under normal and pathological conditions. P450 and GSH expressedin Clara cells and T2 cells enable them to metabolize toxic materi-als (West et al., 2000; Yost et al., 1989), and the products of thesecells such as CC10 and surfactant proteins regulate inflammatoryresponses (Levin et al., 1986; Miele et al., 1988; Wright, 1997).Mucus produced by lung epithelial cells is an important part of theinnate immune system in the lungs (Hayashi et al., 2004). Because ofthe importance of Clara cells, T2 cells and mucus cells in pulmonarydefense and the potential of damage from various toxic moleculesin poultry barn air, we quantified their numbers in lungs of WT andTLR9−/− mice exposed to poultry barn air. The data showed effectsof exposures to poultry barn air on the numbers of Clara cells, T2cells and mucus-producing cells in the lungs of WT and TLR9−/−

mice.The present study demonstrates decrease in the number of Clara

cells in the airway epithelium of WT and TLR9−/− mice after expo-sure to poultry barn air compared to the control animals. WhileTLR9−/− mice showed a decrease after single exposure to poul-try barn air, the decrease was noticed in WT mice only after 5 or

360 R.S. Sethi et al. / Experimental and Toxicologic Pathology 65 (2013) 357– 364

Fig. 2. Immunohistochemistry and quantification of Clara cells in the airways. Lung section stained without primary antibody does not show any color development inairways or blood vessels (a). Antibody specific for vWF labels vascular endothelium (double arrowheads), but not the airways (double arrows) (b). Clara cell antibody reactswith Clara cells (double arrow heads) in the normal lungs of WT (c). The staining is observed in much reduced numbers of epithelial cells in one day exposed lungs of TLR9−/−

mice (d). Quantification of Clara cells in WT and TLR9−/− mice showed a significant decrease between control and 1-day, 5-day and 20-day exposed groups (e and f). Treatmentgroups have significant differences among them if different letters denote the groups. For example, WT control and WT 20d exposed groups. Original magnification (a) 20×;(b)–(d) 40×; inset 1000×.

R.S. Sethi et al. / Experimental and Toxicologic Pathology 65 (2013) 357– 364 361

Fig. 3. PAS staining and quantification of mucus producing cells in the airways: lung sections from normal WT and TLR9−/− mice showed no mucus producing cells in theairway epithelium (a and c). The exposed lung sections of WT and TLR9−/− mice showed a large number of pink stained mucus producing cells (double arrow heads) (b and d).Quantification of PAS-positive cells showed a significantly higher number of cells in 1-day, 5-day and 20-day exposed WT and TLR9−/− mice lungs compared to the controls(e and f). Treatment groups have significant differences among them if different letters denote the groups. For example, WT control and WT 1d exposed groups. Originalmagnification (a)–(d) 400×.

362 R.S. Sethi et al. / Experimental and Toxicologic Pathology 65 (2013) 357– 364

Fig. 4. Immunohistochemistry and Quantification of T2 cells in the alveolar septa. T2 cells (double arrow heads) were stained by using antibody, which reacts with alveolarcells in the normal, and the exposed lungs of WT mice (a) and (b). Quantification of T2 cells in WT and TLR9−/− mice showed a significant difference between control and1 nificac and (

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-day, 5-day and 20-day exposed groups (Fig. 3e and f). Treatment groups have sigontrol and WT 1 day, 5 days and 20 days exposed groups. Original magnification (a)

0 exposures. Similar decreases in Clara cells have been reportedn the airways of patients suffering from chronic diseases such assthma (Shijubo et al., 1999; Van Vyve et al., 1995), idiopathic pul-onary fibrosis (Lesur et al., 1995) and chronic obstructive disease

Bernard et al., 1992). Clara cells play major roles in metabolism oforeign chemicals through expression of GSH and P450 (West et al.,000), and the depletion of these enzymes is linked to damage andeath of Clara cells (Plopper et al., 2001). Clara cells also expressroteins that play a role in transport of gases such as ammonia,hich are present in poultry barn air and could be responsible for

he toxic effects seen in our study (Han et al., 2009). The declinen Clara cell numbers may be due to direct physical damage tohe cells or depletion of the protein used to identify them withmmunohistochemistry, and either of the reasons would indicateltered physiology of Clara cells following exposure to the barn air.lthough there was an early onset of decline in Clara cells in TLR9−/−

ice indicating probable role of TLR9, there were no differences in

he numbers between the two strains at any of the time points.

Mucus production by the epithelial cells in the conductingirways of the lung is a critical part of the innate immune responsend changes in the numbers of PAS-positive goblet cells are

nt differences among them if different letters denote the groups. For example, WTb) 1000×.

considered to be a reliable indicator of inflammatory response.We found an increase in PAS-positive cells in all the exposed miceirrespective of the number of exposures or the status of TLR9−/−.We have previously reported similar increase in PAS-positivecells in rats exposed to swine barn air (Charavaryamath et al.,2005). Others have reported similar changes in mucus cells in micechallenged intra-nasally with LPS (Vernooy et al., 2002), mousemodel of asthma (Pack et al., 1981; Plopper et al., 1983) and inacute and chronic diseases of the airways (Jeffery, 1994; Trevisaniet al., 1992). Endotoxin, which is present in chicken and swinebarn air, is a known agonist of mucus production (Charavaryamathet al., 2005; Harkema and Hotchkiss, 1993), and it may havecontributed to an increase in the number of cells actively engagedin production of mucus. Mucus traps inhaled particles and containsanti-microbial peptides. While increased production of mucus hasbeneficial effects, the impaired clearance of the mucus may leadto formation of plugs (Takeyama et al., 1999) and blockage of the

airway passages (Jackson, 2001). The data does not clarify whetherthere was an actual increase in the numbers of mucus-producingcells or an increase in the numbers of existing cells stimulated toproduce mucus. Nevertheless, the increase in PAS-positive cells

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ndicates protective inflammatory response in the lungs of micexposed to poultry barn air and seems to be independent of TLR9.

Alveolar T2 cells were stained with antibody against pro-urfactant protein C in their expected location in the corners oriches of the alveolar septa of normal WT and TLR9−/− mice. Previ-us immunohistochemical studies have localized SP-C and pro-SPCn T2 cells (Beers et al., 1994; Beytut, 2010). We observed immunos-aining in the perinuclear areas of T2 cells similar to the stainingattern reported earlier in sheep pulmonary adenomatosis (Beytutt al., 2009; Phelps and Floros, 1991) and lamb lungs infected withheep pox virus (Beytut, 2010) suggesting that the endoplasmiceticulum and Golgi apparatus located in the perinuclear area ofhe cells are involved in the synthesis of SP-C or its precursor.

hile WT mice exposed once or multiple times to poultry barnir showed increase in T2 cell numbers compared to the controls,he numbers of T2 cells were reduced following 5 or 20 exposuresn TLR9−/− mice. The reasons for these apparent differences wereot addressed in this study but could be due to the influence ofLR9−/− on the physiology of T2 cells.

The proliferation of cells such as T2 and Clara cells is required toeplace the damaged or dead epithelial cells in respective areas ofhe lung (Rennard et al., 1983). Therefore, an increase in the num-ers of T2 cells may indicate damage to Type-1 epithelial cells in WTice and the decrease in TLR9−/− mice may be due to direct dam-

ge to T2 cells or their rapid differentiation into type 1 alveolar cellss reported following exposure to NO2 (Evans et al., 1975) and oxy-en (Adamson and Bowden, 1974). Because we detected cells usingntibodies against one of their products, it is possible that deple-ion or increased production of that particular protein may resultn changes in cell numbers, and account for the difference in T2 cellumbers between WT and TLR9−/− mice. We do rule out that possi-ility because the morphological identification of T2 cells is easiernd we did not notice differences in their staining between the twoypes of mice used in our studies. There is obvious high variancen the values for the markers within the groups. We expected suchariance in this in vivo study because the mice were exposed to aighly complex environment in the barn. Because the environment

n the barn is altered by the daily cycle of work activity, we tookhe mice into the barn at the same time for each of the exposures,ept them in the barn in the same location and at the same heighto minimize the potential variation in responses. We have reportedimilar high variance in mucus-producing cells in previous studiesonducted in pig barns (Charavaryamath et al., 2005). Therefore,e remain cautious in interpretation of the data obtained in this

tudy and may require further investigation in a more controllednvironment.

We conclude there is a decrease in Clara cells and an increasen numbers of mucus producing goblet cells in the airway epithe-ium of WT and TLR9−/− mice after single and multiple exposureso chicken barn air. We found a possible TLR9-dependent changen T2 cell numbers after single exposure to chicken barn air,

hich definitely requires further investigation. Because of increas-ng intensity of chicken operations where large numbers of birdsre kept in confined facilities poses serious toxicology risks to theorkers. The risks such as those to the pulmonary system require

urther investigation to minimize occupational hazard to the work-rs.

cknowledgements

The study was supported through a grant from Natural Sciences

nd Engineering Research Council of Canada. David Schnebergers a recipient of a PHARE Graduate Scholarship from the Canadiannstitutes for Health Research and a Founding Chairs Fellowshiprom the Canadian Centre for Health and Safety in Agriculture,

ogic Pathology 65 (2013) 357– 364 363

University of Saskatchewan. Dr. R.S. Sethi’s visit was supportedthrough a collaborative program between Guru Angad Dev Vet-erinary and Animal Sciences University and the University ofSaskatchewan.

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