Secondhand smoke (SHS) exposure has been linked to the worsening of ongoing lung diseases. However, whether SHS exposure affects the manifestation and natural history of imminent pediatric muco-obstructive airway diseases such as cystic fibrosis remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice to SHS from postnatal day (PND) 3–21 and lung phenotypes were examined at PND22. Although a majority of filtered air (FA)-exposed Scnn1b-Tg+ (FA-Tg+) mice successfully cleared spontaneous bacterial infections by PND22, the SHS-exposed Scnn1b-Tg+ (SHS-Tg+) mice failed to resolve these infections. This defect was associated with suppressed antibacterial defenses, i.e., phagocyte recruitment, IgA secretion, and Muc5b expression. Whereas the FA-Tg+ mice exhibited marked mucus obstruction and Th2 responses, SHS-Tg+ mice displayed a dramatic suppression of these responses. Mechanistically, downregulated expression of IL-33, a stimulator of type II innate lymphoid cells, in lung epithelial cells was associated with suppression of neutrophil recruitment, IgA secretions, Th2 responses, and delayed bacterial clearance in SHS-Tg+ mice. Cessation of SHS exposure for 21 d restored previously suppressed responses, including phagocyte recruitment, IgA secretion, and mucous cell metaplasia. However, in contrast with FA-Tg+ mice, the SHS-Tg+ mice had pronounced epithelial necrosis, alveolar space consolidation, and lymphoid hyperplasia; indicating lagged unfavorable effects of early postnatal SHS exposure in later life. Collectively, our data show that early postnatal SHS exposure reversibly suppresses IL-33 levels in airspaces which, in turn, results in reduced neutrophil recruitment and diminished Th2 response. Our data indicate that household smoking may predispose neonates with muco-obstructive lung disease to bacterial exacerbations.

This article is featured in In This Issue, p.829

Poor indoor air quality is a serious health concern, especially for young children who spend 85–90% of their time indoors (1). Secondhand smoke (SHS) is a major contributor to indoor air pollution (2, 3). Early childhood SHS exposure accounts for nearly 300,000 annual cases of lower respiratory tract infections (4). The majority of these cases reflect SHS-induced exacerbations of existing muco-obstructive airway diseases (5). Superimposition of SHS exposure onto the muco-obstructive airways in early postnatal life may trigger complex interactions between various pathophysiological processes, including tobacco smoke–induced perturbations, muco-obstructive disease–associated inflammatory milieu, and early postnatal lung development (6). The existence and the nature of these interactions remain elusive. Therefore, in-depth understanding of the effect of SHS exposure on normal lung development and on imminent muco-obstructive lung disease is essential and warrants direct investigation.

The Scnn1b transgenic (Scnn1b-Tg+) mouse, an experimental model of muco-obstructive airway disease, was developed by Tg targeting of the epithelial sodium ion channel subunit β (βENaC) to airway epithelial cells (7). Overexpression of βENaC-encoding epithelial sodium channel, nonvoltage gated 1, β subunit (Scnn1b) transgene results in sodium ion hyper-absorption, airway surface liquid dehydration, mucus hyperconcentration, muco-obstruction, and airway inflammation (7, 8). As a result, Scnn1b-Tg+ pups exhibit a mucociliary clearance defect that leads to defective clearance of spontaneously aspirated bacteria from lung airspaces (9, 10). The Scnn1b-Tg+ airway inflammation is characterized by macrophage activation and increased immune-cell recruitment, including macrophages, neutrophils, lymphocytes, and eosinophils (7, 8, 10, 11). Early postnatal initiation of human cystic fibrosis (CF) or chronic bronchitis-like lung disease in this model provides a unique opportunity to investigate the initiation and progression of pediatric lung diseases.

Studies employing the Scnn1b-Tg+ adult mice have highlighted the effects of exposure to environmental toxicants, including nanoparticles, fungal spores, and mainstream tobacco smoke on airway-disease phenotypes in these mice (1214). A recent report investigated the effect of short-term mainstream tobacco smoke exposure (postnatal day [PND] 10–14) on the ongoing airway disease in Scnn1b-Tg+ neonates (15). The effects of early SHS exposure, however, on the initiation and progression of key airway disease–associated phenotypes, i.e., airspace inflammation, spontaneous bacterial infection, and mucus hypersecretion, remain unclear. Furthermore, a dearth of studies focused on the effects of SHS exposure in neonates necessitates further investigation.

To understand the complex interactions between early postnatal SHS exposure and muco-obstructive disease pathogenesis, two separate studies were conducted. The first time-point study, i.e., 22-d study, was conducted to test the hypothesis that SHS exposure from PND3–21 will perturb normal lung homeostasis in wild-type (WT) pups and exaggerate Scnn1b-Tg+ lung pathology. Because two major pathological events, i.e., spontaneous bacterial infection and Th2 response-mediated mucus hypersecretion, are initiated and progress during the first 21 d of postnatal lung development in Scnn1b-Tg+ mice; the effect of SHS exposure on bacterial clearance and modulation of Th2 responses was examined on PND22. To gain further insights into the long-term effects of early postnatal SHS exposure, a second time-point study, i.e., a 43-d study, was conducted. In this study, we tested the hypothesis that the cessation of SHS exposure will restore the Scnn1b-Tg+ pathological responses. A 21-d recovery period (PND22–42) was allowed following cessation of SHS exposure on PND21. To accomplish these investigations, bronchoalveolar lavage fluid (BALF) cell counting, BALF cytokine/IgA measurement, lung histopathological analyses, and mucous cell metaplasia/Th2 response–related gene expression analyses were performed on PND43. The results from these studies provide novel insights into the effects of early postnatal SHS exposure on the manifestation of responses associated with muco-obstructive lung diseases.

Cryopreserved embryos of Scnn1b-Tg+ mice [Tg(Scgb1a1-Scnn1b)6608Bouc/J] were recovered from a cryorepository at the Jackson Laboratory (Bar Harbor, ME). Offspring were shipped to and maintained at a pathogen-free facility at the Division of Laboratory Animal Medicine at Louisiana State University, Baton Rouge. The procured Scnn1b-Tg+ mice were on a C57BL/6-congenic background and were maintained on this background by controlled breeding between WT (Scnn1b-Tg) and Scnn1b-Tg+ breeders. All mice used in this study were genotyped by PCR for the presence of the Scnn1b transgene, as previously reported (7). Mice were maintained in individually ventilated cages on a 12-h dark/light cycle and were fed regular diet and water ad libitum except during the SHS-exposure period (5 h/d). All animal use and inhalation exposure procedures were approved by the Institutional Animal Care and Use Committee of Louisiana State University.

SHS is comprised of ∼85–90% side-stream smoke with the remainder being exhaled mainstream smoke. In this study, we used side-stream smoke as a surrogate for SHS as previously described (16). SHS exposures were conducted at the American Association for the Accreditation of Laboratory Animal Care–accredited Inhalation Facility at Louisiana State University. We used a TSE Cigarette Smoke Generator (TSE Systems, Chesterfield, MO) to generate SHS from 3R4F filtered research cigarettes (University of Kentucky, Lexington, KY). HEPA-filtered air (FA) or SHS in FA was supplied to 1.3-m3 Plexiglas exposure chambers fitted with stainless steel wire cages (17). Throughout the exposures, we recorded carbon monoxide (by infrared spectroscopy) and total suspended particulate (TSP) levels with a DustTrak and by gravimetric sampling. We selected a target TSP exposure level of 10 mg/m3, which has been previously used in in utero SHS-exposure studies (1618). Various ambient parameters, including particle concentration, carbon monoxide content, temperature, and relative humidity (RH) were monitored daily for FA and SHS chambers during the entire 19 consecutive days (PND3–21) of 5 h/d exposures (Figs. 1B, 5B).

Our investigation included two separate time-point studies, i.e., 22 d (Fig. 1A) and 43 d (Fig. 5A). The detail on total number of litters, litter sizes, and genders included for data analyses is provided in Supplemental Table I. The start of SHS exposure on PND3 circumvented the challenge of a pup-rejection response in dams and its resulting neonatal deaths (Y. Saini and B.W. Lewis, unpublished observations). BALF was harvested aseptically for total and differential cell counting, cytokine/IgA analyses, and microbiological analyses, as previously described (9, 10). Briefly, mice were anesthetized via i.p. injection of 2,2,2-tribromoethanol and were exsanguinated by incising the posterior vena cava. The thoracic cavity was exposed and the trachea was cannulated with appropriate stub adaptors (23 guage, 22 d; 20 guage, adults). The stub adaptor was secured in place with a silk suture (4.0). The left main-stem bronchus was ligated with a silk suture to prevent lavaging of the left lung lobe. The lavaging was performed with the calculated volume of calcium- and magnesium-free PBS following a “twice three-times” rule, as described previously (19). Left lung lobes were stored in 10% neutral buffered formalin and lavaged right lungs were snap frozen for further analyses.

Harvested BALF was centrifuged at 300 × g for 5 min and the supernatant was stored at −80°C for further analyses. The pellet was resuspended in 500 μl of PBS and total cell counts were determined with a hemocytometer (Brightline, Horsham, PA). Two hundred microliters of cell suspension was used for cytospin preparation (CytoSpin 3; Thermo Shandon, Pittsburgh, PA) followed by differential staining (Modified Giemsa kit; Newcomer Supply, Middleton, WI).

To estimate IgA concentration in BALF, cell-free BALF supernatant was analyzed with a Mouse IgA ELISA Kit (88-50450), according to the manufacturer’s instructions (eBioscience, San Diego, CA). Mouse IL-1α, IL-4, IL-5, IL-13, keratinocyte chemoattractant (KC), TNF-α, MCP-1, and Eotaxin levels were assayed in cell-free BALF supernatant using a Luminex-XMAP–based assay (MCYTOMAG-70K), according to the manufacturer’s instructions (EMD Millipore, Billerica, MA).

Lungs were fixed in 10% neutral buffered formalin for subsequent assessment of lung pathology and intracellular and extracellular muco-polysaccharides. We adopted a previously reported strategy for semiquantitative grading of histological changes in Scnn1b-Tg+ lungs, as explained in a previous report (19). Histopathological analyses were performed by a board-certified anatomic pathologist (I. Langohr) and anatomic pathology resident (S. Patial) in a blinded fashion.

Formalin-fixed, paraffin-embedded lung sections were used for immunohistochemical localization of IL-33. Five-micrometer sections were deparaffinized with Citrisolv (2 × 5 min each) and were rehydrated with graded ethanol (100, 100, 95, 70, 30%, distilled water; 3 min each). Ag retrieval was performed using a heat-induced, Ag-retrieval method (heating slides in 0.1 M sodium citrate solution [with 0.05% Tween 20] at 95–100°C for 30 min, followed by cooling to room temperature). The sections were then rinsed in deionized water (2 × 5 min each) and processed according to illustrated protocol (Immunocruz goat ABC Staining Kit, catalog number SC2023; Santa Cruz Biotechnology) with the following modifications. Polyclonal goat IL-33 primary Ab (AF3626; R&D Systems, Minneapolis, MN) and isotype control (goat IgG; Jackson ImmunoResearch) were used at a 1.25 μg/ml concentration with overnight incubation in the cold room. After primary incubation, quenching for endogenous peroxidases was performed with 3% hydrogen peroxide in deionized water for 10 min at room temperature. Coverslip-mounted slides were imaged by transmitted light microscopy (Nikon Ci-L microscope).

Frozen right lung lobes were processed for extraction of total RNA with a Quick-RNA MicroPrep kit (R1051), according to the manufacturer’s instructions (Zymo Research, Irvine, CA). Total RNA was analyzed spectrophotometrically for quantity and purity (NanoDrop, Wilmington, DE). All the samples used for further analyses had an A260/A280 ratio of 1.85 or greater. cDNA generation and RT-PCR were performed as described previously (20). Gene-specific primer sequences used in this study are listed in Supplemental Table II.

Aseptically harvested BALF from 22-d-old mice was processed for CFU enumeration as previously described (9, 10). The Columbia blood agar plates were incubated at 37°C in an anaerobic candle jar. The CFUs were counted and morphologically distinguished colonies were restreaked, expanded, and processed for 16S ribosomal DNA sequencing. Bacterial cultures derived from individual CFUs were processed for bacterial species identification using 16S rRNA gene sequencing. Briefly, frozen bacterial samples were heat inactivated at 100°C for 3 min. Bacterial genomic DNA was extracted using Maxwell 16 Cell DNA Extraction Kit (Promega, Madison, WI). Hypervariable regions of the 16S rRNA gene were amplified by using the primer pair 338F (5′-ACTYCTACGGRAGGCWGC-3′) and 1061R (5′-CRRCACGAGCTGACGAC-3′) using κ HiFi Master Mix (Kapa Biosystems, Wilmington, MA) at 55°C annealing temperature (21). Amplified PCR products were purified using RapidTips (Diffinity Genomics, West Chester, PA) and sequenced by both forward and reverse primers using ABI Prism 3130 Genetic Analyzer (Life Technologies, Carlsbad, CA). Obtained sequences were queried into the 16S rRNA sequence database at NCBI using the Basic Local Alignment Search Tool and genus or species of culture isolates were determined based on the sequence homology to the 16S rRNA gene sequence.

ANOVA followed by Tukey post hoc test for multiple comparisons was used to determine significant differences among groups. Measurements from two groups were compared using Student t test assuming unequal variance. All data were expressed as mean ± SEM. A p value <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). For selected endpoints, the summary of results is provided for individual genders in Supplemental Tables III and IV.

To determine whether SHS exposure can perturb airspace homeostasis, we exposed neonatal Scnn1b-Tg+ mice, a well-characterized mouse model of muco-obstructive airway disease, and littermate control WT mice, to SHS (TSP = 10 mg/m3) or HEPA-FA and assessed various BALF signatures of airspace homeostasis, including immune-cell counts and soluble mediators (Fig. 1), at PND22.

FIGURE 1.

SHS exposure alters immune cell composition in lung airspaces. (A) Experimental design for a 21-d study depicting exposure regimen and various outcomes examined. (B) Exposure parameters for 21-d study. (C) Total cell counts in the harvested BALF from 22-d-old FA- and SHS-exposed WT and Scnn1b-Tg+ mice (n = 7–14 per group, overall p value < 0.0001). (D) Differential cell counts (overall p value < 0.0001) and (E) their respective percentages (overall p value < 0.0001) are shown as a stacked bar graph [BALF macrophages (red), neutrophils (blue), eosinophils (green), lymphocytes (black)]. (F) Representative photomicrographs of BALF cytospins. (Fa) FA-WT, (Fb) SHS-WT, (Fc) FA-Tg+, and (Fd) SHS-Tg+ mice at PND22. Macrophages (red arrows), neutrophils (blue arrows), eosinophils (green arrows), and lymphocytes (black arrows) Cytospins were stained with Wright–Giemsa stain (modified Giemsa kit; Newcomer Supply, Middleton, WI). (G) BALF cytokine levels (picograms per milliliter) of (Ga) MCP-1 (overall p value = 0.0003), (Gb) Eotaxin (overall p value = 0.0089), (Gc) IL-5 (overall p value = 0.0002), (Gd) KC (overall p value < 0.0001), (Ge) TNF-α (overall p value = 0.2227), and (Gf) IL-1α (overall p value = 0.0026) in BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) mice (n = 6–8 per group). The dotted line represents lower limit of detection (LOD) for respective chemokine. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using ANOVA followed by Tukey multiple comparison post hoc test. RH, relative humidity.

FIGURE 1.

SHS exposure alters immune cell composition in lung airspaces. (A) Experimental design for a 21-d study depicting exposure regimen and various outcomes examined. (B) Exposure parameters for 21-d study. (C) Total cell counts in the harvested BALF from 22-d-old FA- and SHS-exposed WT and Scnn1b-Tg+ mice (n = 7–14 per group, overall p value < 0.0001). (D) Differential cell counts (overall p value < 0.0001) and (E) their respective percentages (overall p value < 0.0001) are shown as a stacked bar graph [BALF macrophages (red), neutrophils (blue), eosinophils (green), lymphocytes (black)]. (F) Representative photomicrographs of BALF cytospins. (Fa) FA-WT, (Fb) SHS-WT, (Fc) FA-Tg+, and (Fd) SHS-Tg+ mice at PND22. Macrophages (red arrows), neutrophils (blue arrows), eosinophils (green arrows), and lymphocytes (black arrows) Cytospins were stained with Wright–Giemsa stain (modified Giemsa kit; Newcomer Supply, Middleton, WI). (G) BALF cytokine levels (picograms per milliliter) of (Ga) MCP-1 (overall p value = 0.0003), (Gb) Eotaxin (overall p value = 0.0089), (Gc) IL-5 (overall p value = 0.0002), (Gd) KC (overall p value < 0.0001), (Ge) TNF-α (overall p value = 0.2227), and (Gf) IL-1α (overall p value = 0.0026) in BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) mice (n = 6–8 per group). The dotted line represents lower limit of detection (LOD) for respective chemokine. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using ANOVA followed by Tukey multiple comparison post hoc test. RH, relative humidity.

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SHS exposure alters immune-cell composition of BALF.

To examine the effect of SHS exposure on BALF immune-cell composition, we first compared FA-exposed WT (FA-WT) and SHS-exposed WT (SHS-WT) littermates. Although SHS exposure did not significantly alter total BALF cell counts in WT mice (Fig. 1C), differential cell counts revealed significantly increased numbers of neutrophils in SHS-WT mice as compared with FA-WT mice (SHS-WT, 3910 ± 510; FA-WT, not detected) (Fig. 1C–F). The total number of BALF cells was significantly increased in FA-exposed Scnn1b-Tg+ (FA-Tg+) mice (∼4.4-fold versus FA-WT mice, Fig. 1C). This was attributable to significantly increased numbers of macrophages (∼1.9-fold), neutrophils (∼36-fold), eosinophils (∼5-fold), and lymphocytes (∼3.5-fold) (Fig. 1D–F). A similar comparison between FA-WT or SHS-WT mice and SHS-exposed Scnn1b-Tg+ (SHS-Tg+) mice revealed a significant increase in the total number of cells in SHS-Tg+ BALF (∼2.2-fold versus FA-WT or SHS-WT mice, Fig. 1C), which was attributable to a significant increase only in macrophages (∼1.5-fold) and neutrophils (∼14-fold) (Fig. 1D–F). Further comparison of cell counts between FA-Tg+ and SHS-Tg+ mice revealed a significant reduction in total number of cells (∼0.7-fold lower in SHS-Tg+ mice versus FA-Tg+ mice) (Fig. 1C) which was accounted for by a significant reduction in neutrophils (∼1.4-fold), eosinophils (∼3.5-fold), and lymphocytes (∼1.2-fold) (Fig. 1D–F). These data suggest that early postnatal SHS exposure results in suppressed immune-cell recruitment to muco-obstructive airspaces.

Altered immune-cell recruitment is associated with altered BALF chemokine and IgA levels.

Because chemokines are responsible for recruitment of inflammatory cells into the inflammatory milieu, we next measured the levels of some BALF chemokines that have been demonstrated previously to be responsible for recruitment of inflammatory monocytes, eosinophils, and neutrophils. MCP-1, a monocyte and neutrophil chemoattractant, was below detection limits in both FA- and SHS-WT BALF, but was significantly elevated in FA-Tg+ BALF (∼26 pg/ml, Fig. 1Ga). Exposure of Scnn1b-Tg+ mice to SHS, however, resulted in significant reduction in BALF MCP-1 levels (∼7 pg/ml, i.e., ∼2.7-fold lower versus FA-Tg+ mice, Fig. 1Ga). Although macrophage counts were comparable between FA- and SHS-Tg+ BALF, a reduction in BALF neutrophils in SHS-Tg+ (Fig. 1D) was consistent with lower BALF MCP-1 levels.

Eotaxin (CCL11), a potent eosinophil chemoattractant, was significantly increased in SHS-WT (∼3.8-fold versus FA-WT mice, Fig. 1Gb) and, as expected from previous studies (8), in FA-Tg+ mice (∼7.6-fold versus FA-WT mice, Fig. 1Gb). Similarly, IL-5, a priming factor for eosinophil activation and recruitment, was significantly increased in FA-Tg+ mice (∼35.0-fold versus FA-WT mice, Fig. 1Gc). Consistent with the significant decrease in eosinophils, Eotaxin and IL-5 levels were significantly reduced in SHS-Tg+ mice (Eotaxin, ∼3.7-fold lower versus FA-Tg+ mice, Fig. 1Gb; and IL-5, ∼9.0-fold lower versus FA-Tg+ mice, Fig. 1Gc). As expected from previous studies (8), increased levels of the primary neutrophil chemokine, KC, in FA-Tg+ (∼106 pg/ml) mice mirrored a significant increase in neutrophils in this group compared with FA- or SHS-WT mice (Fig. 1Gd). In comparison with FA-Tg+ mice, however, the KC levels of SHS-Tg+ mice (∼90 pg/ml) were not decreased significantly (Fig. 1Gd).

To assess the effect of early postnatal SHS exposure on the release of soluble mediators of airway inflammation in WT and Scnn1b-Tg+ lungs, we measured selective proinflammatory mediators in the BALF. BALF concentrations for IL-1α, a damage- or danger-associated molecular pattern molecule, and TNF-α, a proinflammatory cytokine, were not increased in SHS-WT mice. As expected from previous studies (8, 9), the levels of IL-1α and TNF-α were significantly increased in FA-Tg+ BAL; SHS exposure, however, did not result in significant alteration in their release into the Scnn1b-Tg+ airspaces (Fig. 1Ge, Gf).

Because SHS-Tg+ mice have significantly reduced numbers of BALF lymphocytes (Fig. 1D–E), we speculated that the suppressive effects of early postnatal SHS exposure would also be reflected in the suppression of mucosal B-cell humoral immune responses. Therefore, we assessed BALF IgA levels as a readout of a B cell–specific adaptive immune response. BALF IgA levels were comparable between FA-WT and FA-Tg+ mice (Fig. 2A). SHS exposure, however, resulted in significant reduction in the levels of BALF IgA in WT as well as Scnn1b-Tg+ mice (Fig. 2A).

FIGURE 2.

SHS exposure reduces clearance of spontaneous bacterial infections. (A) IgA levels in cell-free BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 6–8 per group, overall p value = 0.0008). (B) CFUs in BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) mice (n = 10–16 per group, overall p value = 0.0130). The CFU values were log10-transformed with an offset of +1 (log10+1 transformation). Error bars represent SEM. (C) 16S ribosomal DNA sequencing–based species identification of cultured bacteria from BAL. ANOVA followed by Tukey multiple comparison post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

SHS exposure reduces clearance of spontaneous bacterial infections. (A) IgA levels in cell-free BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 6–8 per group, overall p value = 0.0008). (B) CFUs in BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) mice (n = 10–16 per group, overall p value = 0.0130). The CFU values were log10-transformed with an offset of +1 (log10+1 transformation). Error bars represent SEM. (C) 16S ribosomal DNA sequencing–based species identification of cultured bacteria from BAL. ANOVA followed by Tukey multiple comparison post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

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Early postnatal spontaneous bacterial infections, predominately due to the aspiration of normal microflora of oropharyngeal origin, are a consistent manifestation of defective mucociliary clearance in Scnn1b-Tg+ mice (10). These spontaneous bacterial infections of Scnn1b-Tg+ lungs clear almost completely by 3–4 wk of age (10), perhaps due to enhanced adaptive immune responses, e.g., formation of lymphoid nodules and IgA-mediated bacterial clearance. To determine the effect of early postnatal SHS exposure on spontaneous bacterial clearance, we harvested BALF aseptically and enumerated microaerophilic CFUs. Whereas only 1 of 10 FA-WT mice had CFUs (CFUs = ∼100 counts/ml), 5 of 16 SHS-WT mice had elevated CFU counts (average CFUs = ∼320 counts/ml) (Fig. 2B). As expected from previous studies (10), only 3 of 10 FA-Tg+ lungs had CFUs (average CFUs = ∼4100 counts/ml) (Fig. 2B). Following SHS exposure, however, 11 of 12 Scnn1b-Tg+ mice lungs had microaerophilic bacterial infections with significantly higher CFU counts (average CFUs = ∼6600 counts/ml), suggesting that SHS-Tg+ mice failed to clear infection.

Next, to determine the effect of SHS exposure on the diversity of microaerophilic microflora, we performed bacterial 16S DNA sequencing. These analyses on CFUs isolated from SHS-Tg+ mice revealed the presence of most of the previously reported bacterial species in Scnn1b-Tg+, including Pasteurella pneumotropica, Streptococcus sp., Staphylococcus sp., and Enterococcus sp. (Fig. 2C). Irrespective of mouse genotype, SHS exposure was associated with increased incidence of Micrococcus sp., suggesting that SHS exposure may have resulted in defective host defense specifically against this bacterium.

In agreement with the BALF lymphocyte cell counts (Fig. 1D, 1E), tissue lymphoid aggregates were not evident in either FA- or SHS-WT mice (Fig. 3A, 3C), although a significant increase in the number of lymphoid aggregates was observed in FA-Tg+ mice (Fig. 3Ae, 3C). This was consistent with significantly increased numbers of BALF lymphocytes in these mice (Fig. 1D, 1E). Further, as expected from BALF lymphocyte counts, SHS-Tg+ mice had significantly reduced numbers of lymphoid aggregates compared with FA-Tg+ mice (Fig. 3Af, 3C).

FIGURE 3.

SHS exposure alters Scnn1b-Tg+ lung pathology and suppresses Th2 responses associated with Scnn1b-Tg+ lung disease. (A) Representative photomicrographs from H&E-stained, left lung lobe sections from 22-d-old (Aa) FA-WT, (Ab) SHS-WT, (Ac and Ae) FA-Tg+, or (Ad and Af) SHS-Tg+ mice (n = 7–14 per group). (B) Representative photomicrographs from AB-PAS–stained left lung lobe sections from 22-d-old (Ba) FA-WT, (Bb) SHS-WT, (Bc) FA-Tg+, or (Bd) SHS-Tg+ mice (n = 7–14 per group). Mucus plugging and mucous cell metaplasia are apparent in FA-Tg+ (A) H&E- and (B) AB-PAS–stained sections. Lymphoid aggregates (black arrow), peri-bronchiolar neutrophilic inflammation (green arrow), lymphocytic infiltration (red arrow), and AB-PAS–stained mucous secretory cells (blue arrow). (C) Semiquantitative histopathology scores for mucus plugging, airway inflammation, epithelial necrosis, mucous secretory cells, parenchymal consolidation, and lymphoid hyperplasia. FA-WT (n = 7, white bar), SHS-WT (n = 9, orange bar), FA-Tg+ (n = 12, red bar), and SHS-Tg+ (n = 14, green bar). (D) BALF levels of IL-4 (picogram per milliliter) (n = 6–8 per group, overall p value = 0.0006); the dotted line represents lower limit of detection (LOD). (E) IL-13 (mean fluorescence intensity [MFI]) (n = 6–8 per group, overall p value = 0.0411) in FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar). (F) Absolute quantification of mRNA for selective genes related to mucous cell metaplasia and Th2 inflammation. Two values carrying identical designations (for example, or ) in the same row represent significant difference. Bold text indicates significantly higher values compared with SHS-Tg+ group. n = 5 mice per group. The data are presented as mean ± SEM. p < 0.05 using ANOVA followed by Tukey multiple comparison post hoc test. (G) Absolute quantification of Il13 mRNA expression; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 5 per group, overall p value < 0.0001). (H) Absolute quantification of Ifng mRNA expression; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 5 per group, overall p value = 0.1326). Absolute quantification (mRNA copies per 105 copies of Actb mRNA) of gene expression of selective genes in whole-lung homogenate from FA-WT or SHS-WT or Scnn1b-Tg+ mice. (I) Relative expression levels of Il13 as compared with Ifng. Expression values were normalized within gene group (by values from all samples with the lowest value). Normalized Il13 value for each sample was divided by normalized Ifng value. Obtained values indicating Il13/Ifng ratios were plotted (n = 5 per group, overall p value = 0.0039). (D, E, and G–I) The data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using ANOVA followed by Tukey multiple comparison post hoc test. Chi3l4, chitinase-like 4; Clca1, calcium-activated chloride channel regulator 1; Il13, IL-13; Ifng, IFN-γ; Foxa3, forkhead box A3; Gata3, GATA binding protein 3; Muc5ac, mucin 5, subtypes A and C; Muc5b, mucin 5, subtypes B; Retnla, resistin-like molecule α; Slc26a4, solute carrier family 26 member 4; Spdef, SAM pointed domain containing ETS transcription factor; Tff2, Trefoil factor 2.

FIGURE 3.

SHS exposure alters Scnn1b-Tg+ lung pathology and suppresses Th2 responses associated with Scnn1b-Tg+ lung disease. (A) Representative photomicrographs from H&E-stained, left lung lobe sections from 22-d-old (Aa) FA-WT, (Ab) SHS-WT, (Ac and Ae) FA-Tg+, or (Ad and Af) SHS-Tg+ mice (n = 7–14 per group). (B) Representative photomicrographs from AB-PAS–stained left lung lobe sections from 22-d-old (Ba) FA-WT, (Bb) SHS-WT, (Bc) FA-Tg+, or (Bd) SHS-Tg+ mice (n = 7–14 per group). Mucus plugging and mucous cell metaplasia are apparent in FA-Tg+ (A) H&E- and (B) AB-PAS–stained sections. Lymphoid aggregates (black arrow), peri-bronchiolar neutrophilic inflammation (green arrow), lymphocytic infiltration (red arrow), and AB-PAS–stained mucous secretory cells (blue arrow). (C) Semiquantitative histopathology scores for mucus plugging, airway inflammation, epithelial necrosis, mucous secretory cells, parenchymal consolidation, and lymphoid hyperplasia. FA-WT (n = 7, white bar), SHS-WT (n = 9, orange bar), FA-Tg+ (n = 12, red bar), and SHS-Tg+ (n = 14, green bar). (D) BALF levels of IL-4 (picogram per milliliter) (n = 6–8 per group, overall p value = 0.0006); the dotted line represents lower limit of detection (LOD). (E) IL-13 (mean fluorescence intensity [MFI]) (n = 6–8 per group, overall p value = 0.0411) in FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar). (F) Absolute quantification of mRNA for selective genes related to mucous cell metaplasia and Th2 inflammation. Two values carrying identical designations (for example, or ) in the same row represent significant difference. Bold text indicates significantly higher values compared with SHS-Tg+ group. n = 5 mice per group. The data are presented as mean ± SEM. p < 0.05 using ANOVA followed by Tukey multiple comparison post hoc test. (G) Absolute quantification of Il13 mRNA expression; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 5 per group, overall p value < 0.0001). (H) Absolute quantification of Ifng mRNA expression; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 5 per group, overall p value = 0.1326). Absolute quantification (mRNA copies per 105 copies of Actb mRNA) of gene expression of selective genes in whole-lung homogenate from FA-WT or SHS-WT or Scnn1b-Tg+ mice. (I) Relative expression levels of Il13 as compared with Ifng. Expression values were normalized within gene group (by values from all samples with the lowest value). Normalized Il13 value for each sample was divided by normalized Ifng value. Obtained values indicating Il13/Ifng ratios were plotted (n = 5 per group, overall p value = 0.0039). (D, E, and G–I) The data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using ANOVA followed by Tukey multiple comparison post hoc test. Chi3l4, chitinase-like 4; Clca1, calcium-activated chloride channel regulator 1; Il13, IL-13; Ifng, IFN-γ; Foxa3, forkhead box A3; Gata3, GATA binding protein 3; Muc5ac, mucin 5, subtypes A and C; Muc5b, mucin 5, subtypes B; Retnla, resistin-like molecule α; Slc26a4, solute carrier family 26 member 4; Spdef, SAM pointed domain containing ETS transcription factor; Tff2, Trefoil factor 2.

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Mucus obstruction or plugging, mucin hypersecretion, and mucous cell metaplasia are the hallmark pathologic features of Scnn1b-Tg+ lung disease (7, 8). To determine whether neonatal SHS exposure modulates these features, we compared airway histopathology in FA-Tg+ mice versus SHS-Tg+ mice. Mucus obstruction or plugging was not evident in FA- or SHS-WT mice (Fig. 3A–C), and the abundance of mucous secretory cells positive for Alcian blue–periodic acid–Schiff (AB-PAS) in the first and second generation of airways was comparable between these two groups (Fig. 3B, 3C). As expected from previous studies (7, 8), mucus obstruction or plugging and abundance of mucous secretory cells, particularly within the first and the second generation airways, was markedly increased in FA-Tg+ mice compared with WT mice; whereas both of these pathologic features were significantly reduced in SHS-Tg+ mice (Fig. 3A–C).

To test whether the reduction in the proportion of mucous secretory cells in SHS-Tg+ airways is a result of increased ciliated cell necrosis or of a diminished Th2 environment, further analyses of lung sections, cytokine analyses, and gene expression analyses were carried out. Detailed histopathological analysis of lung sections revealed 1) abundance of ciliated cells in SHS-Tg+ airways, and 2) a similar degree of necrosis between FA- and SHS-Tg+ airways (Fig. 3A–C). These results suggest that the reduction in the proportion of mucous secretory cells is not caused by SHS-induced loss of epithelial cells but may be attributable to the inhibition of trans-differentiation of ciliated or club cells to mucous cells, i.e., mucous cell metaplasia.

Because mucous cell metaplasia responses are primarily associated with a Th2 environment, we measured three major Th2 cytokines, IL-4 (Fig. 3D), IL-13 (Fig. 3E), and IL-5 (Fig. 1Gc), in the BALF. The levels of IL-4 and IL-5 were below the detection limits in both FA- and SHS-WT mice whereas basal levels of IL-13 were detected in both FA- and SHS-WT mice. In agreement with the increased number of mucous secretory cells (Fig. 3B, 3C), the BALF analysis revealed significantly elevated levels of IL-5 (Fig. 1Gc) and IL-4 in FA-Tg+ mice (Fig. 3D). As expected, SHS-Tg+ mice had significantly reduced levels of IL-5 (Fig. 1Gc), IL-4 (Fig. 3D), and IL-13 (baseline mean fluorescence intensity) (Fig. 3E).

To further determine the downstream effect of altered Th2 cytokine levels in BALF, we carried out RT-PCR–based gene expression analyses on whole-lung homogenates with a focus on selective Th2/mucous cell metaplasia–related genes, including Gata3, Spdef, Foxa3, Tff2, Agr2, Muc5ac, Muc5b, Clca1 (gob5/Clca3), Slc26a4 (Pds/pendrin), Retnla, and Chi3l4 (Fig. 3F). In comparison with FA-WT mice, SHS-WT mice did not exhibit any significant alterations in the expression levels of these genes (Fig. 3F). However, except for Gata3, Spdef, Muc5ac, and Chi3l4, all the analyzed genes were significantly upregulated in the lungs of FA-Tg+ mice. As expected from BALF cytokine and histopathological data, SHS-Tg+ mice exhibited significant downregulation of gene expression levels in most of the analyzed genes, i.e., Gata3, Foxa3, Tff2, Agr2, Muc5ac, Muc5b, Clca1 (gob5/Clca3), Slc26a4 (Pds/pendrin), Retnla, and Chi3l4 (Fig. 3F).

In addition to the involvement of Th2 cytokines, IFN-γ, a Th1 mediator, has been implicated in the mucous cell metaplasia (22, 23). To rule out the contribution of IFN-γ in suppression of mucous cell metaplasia in SHS-Tg+ mice, gene expression levels for Il13 (Fig. 3G) and Ifng (Fig. 3H) and the ratio of Il13/Ifng (Fig. 3I) were determined. Although the expression levels of Ifng were comparable between FA-WT, SHS-WT, and FA-Tg+ mice, significant suppression in its expression was observed in SHS-Tg+ mice (Fig. 3H). The relative levels of Il13 to Ifng expression suggest that the IL-13–dominant environment in FA-Tg+ is suppressed in SHS-Tg+ mice (Fig. 3I). These data suggest that the SHS exposure–induced suppression in mucous cell metaplasia is associated with suppression of the Th2 environment rather than induction of the Th1 environment. These data, along with suppressed IgA secretions (Fig. 2A), demonstrate that SHS exposure results in suppression of Th2 responses, as well as of B cell–mediated humoral responses in muco-obstructive airspaces.

The histopathological (Fig. 3A–C), BALF cytokine (Figs. 1G, 3D–E), and gene expression data (Fig. 3F–I) pointed to the possibility that SHS exposure directly affects the IL-4/-13–secreting cellular populations. IL-33 and two other cytokines, i.e., IL-25 and thymic stromal lymphopoietin (TSLP), are known to be released from stressed epithelial cells and stimulate IL-4/-5/-13–secreting type II innate lymphoid cells (ILC2s). SHS-induced suppression of the IL-33R, i.e., ST2, on ILC2 has been linked to the suppression of Th2 responses (24). Our preliminary screening (gene expression analyses on pooled cDNA samples [data not shown]) revealed significant suppression of Il33 expression, but not of Il25 and Tslp, in SHS-Tg+ mice. Therefore, we carried out RT-PCR on whole-lung homogenates to assess Il33 mRNA levels in all the experimental groups. Early postnatal SHS exposure of WT mice did not alter the expression levels of IL-33. In contrast, FA-Tg+ mice had an insignificant increase in Il33 mRNA levels, with a p value of 0.14, which was significantly reduced in SHS-Tg+ mice (Fig. 4A). To test whether these changes in gene expression are also reflected at the protein level, we measured both secreted as well as intracellular levels of IL-33 protein. To determine secreted levels of IL-33 into the airspaces, Western blot analysis was carried out on BALF (Fig. 4B). Similar to the mRNA levels, the secreted IL-33 protein levels were significantly upregulated in FA-Tg+ BALF but were significantly reduced to baseline levels in SHS-Tg+ BALF (Fig. 4B).

FIGURE 4.

SHS exposure causes downregulation of IL-33 expression in the Scnn1b-Tg+ mice. (A) Absolute quantification of Il33 mRNA expression; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 5 per group, overall p value = 0.0289) (B). Western blot analyses for secreted IL-33 in the BALF. (Top panel) Quantification (n = 4–6 per group, overall p value = 0.0006) (ImageJ analyses, normalized to the BALF volume yield normalized to the body weight) and (bottom panel) representative blot, a ∼33-KDa band was observed; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar). ANOVA followed by Tukey multiple comparison post hoc test. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative images from sections stained for immunolocalization of IL-33 protein in (a) FA-WT, (b) SHS-WT, (c, e, and g) FA-Tg+, or (d, f, and h) SHS-Tg+. Airway epithelium enclosed in red box in (c) (FA-Tg+) and green box in (d) (SHS-Tg+) are further enlarged as (e) and (f), respectively. Nuclear staining of alveolar epithelial cells (red arrow) in WT (FA-WT as well as SHS-WT) and FA-Tg+ lung sections. Cytoplasmic localization of IL-33 in airway epithelial cells (blue arrows) and macrophages (green arrow). Immunostaining was performed using 3′,3′-diaminobenzidine (DAB) substrate to detect immunolocalized HRP (brown chromogen). Tg+, Scnn1b-Tg+.

FIGURE 4.

SHS exposure causes downregulation of IL-33 expression in the Scnn1b-Tg+ mice. (A) Absolute quantification of Il33 mRNA expression; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 5 per group, overall p value = 0.0289) (B). Western blot analyses for secreted IL-33 in the BALF. (Top panel) Quantification (n = 4–6 per group, overall p value = 0.0006) (ImageJ analyses, normalized to the BALF volume yield normalized to the body weight) and (bottom panel) representative blot, a ∼33-KDa band was observed; FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar). ANOVA followed by Tukey multiple comparison post hoc test. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Representative images from sections stained for immunolocalization of IL-33 protein in (a) FA-WT, (b) SHS-WT, (c, e, and g) FA-Tg+, or (d, f, and h) SHS-Tg+. Airway epithelium enclosed in red box in (c) (FA-Tg+) and green box in (d) (SHS-Tg+) are further enlarged as (e) and (f), respectively. Nuclear staining of alveolar epithelial cells (red arrow) in WT (FA-WT as well as SHS-WT) and FA-Tg+ lung sections. Cytoplasmic localization of IL-33 in airway epithelial cells (blue arrows) and macrophages (green arrow). Immunostaining was performed using 3′,3′-diaminobenzidine (DAB) substrate to detect immunolocalized HRP (brown chromogen). Tg+, Scnn1b-Tg+.

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Finally, to identify the cellular source of secreted IL-33 protein, we carried out immunohistochemical staining of lung sections (Fig. 4C). Although both FA-WT and SHS-WT mice revealed intense nuclear staining in alveolar epithelial cells, the airway epithelial cells and macrophages remained unstained in these mice. The FA-Tg+ mice had comparable nuclear IL-33 staining in alveolar epithelial cells. In addition, airway epithelial cells and macrophages of these mice revealed cytoplasmic localization of IL-33. Because intense IL-33 nuclear staining of alveolar epithelial cells in WT groups was not reflected as secreted IL-33 in BALF of these mice, the increased level of cytoplasmic IL-33 in macrophages and airway epithelial cells of FA-Tg+ is the likely source of secreted IL-33 in BALF. In agreement with the gene expression and Western blot data, the IL-33 staining in SHS-Tg+ was not evident in the nuclei of alveolar epithelial cells. Further, the cytoplasmic staining for IL-33 in airway epithelial cells and macrophages of SHS-Tg+ lungs was remarkably reduced. These data revealed two novel findings: 1) IL-33 signaling is both upregulated in the airway epithelium and alveolar macrophages of Scnn1b-Tg+ lungs and is secreted into the airspaces, and 2) SHS exposure of neonates inhibits Il33 transcription in stressed Scnn1b-Tg+ lung macrophages and epithelial cells (Fig. 4).

To determine whether the suppressed immune responses observed in SHS-exposed mice were irreversible or reversible, and whether there are lagged responses or consequences of SHS exposure, we next performed a 42-d study whereby 3-d-old neonates were exposed to FA or SHS from PND3 onwards until the age of PND21, followed by maintenance of the weanlings in FA-supplied regular cages until PND43.

Cessation of early postnatal SHS exposure restores immune-cell recruitment into the airspaces.

To determine whether the cessation of SHS exposure will restore suppressed immune-cell recruitment into the lung airspaces, BALF was examined to measure cell counts and chemokines responsible for immune cell recruitment. Whereas total BALF cell counts were comparable between FA-WT and SHS-WT (Fig. 5C, 5D), FA-Tg+ mice had significantly higher total cells in BALF (Fig. 5C, 5D). In contrast with the 21-d study (Fig. 1C, 1D), SHS exposure to Scnn1b-Tg+ mice resulted in significantly increased total cells (SHS-Tg+, ∼408,000; FA-Tg+, ∼332,000) in BALF, which were largely attributed to increased numbers of macrophages, neutrophils, and lymphocytes (Fig. 5C, 5D). In comparison with the FA-Tg+ group, however, SHS-Tg+ mice had significantly reduced numbers of eosinophils in BALF (Fig. 5C, 5D).

FIGURE 5.

Cessation of SHS exposure restores immune-cell recruitment into the lung airspaces. (A) Experimental design for 42-d time point depicting exposure regimen and various endpoints examined. (B) Exposure parameters for 42-d study. (C) Total cell counts in the harvested BALF from 43-d-old FA- and SHS-exposed WT and Scnn1b-Tg+ mice (n = 11–17, overall p value < 0.0001). (D) Differential cell counts (overall p value < 0.0001) and (E) their respective percentages (overall p value < 0.0001) are shown as a stacked bar graph. BALF macrophage (red); neutrophil (blue), eosinophils (green), lymphocytes (black). (F) Representative photomicrographs of BALF cytospins. (Fa) FA-WT, (Fb) SHS-WT, (Fc) FA-Tg+, and (Fd) SHS-Tg+ mice at PND43. Macrophages (red arrows), neutrophils (blue arrows), eosinophils (green arrows), and lymphocytes (black arrows). (G) BALF cytokine levels (picograms per milliliter) of (Ga) MCP-1 (overall p value = 0.0104), (Gb) Eotaxin (overall p value = 0.3541), (Gc) IL-5 (overall p value = 0.0016), (Gd) KC (overall p value < 0.0001), (Ge) TNF-α (all values were below detection limits), and (Gf) IL-1α (overall p value < 0.0001) in BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) mice (n = 6–8 per group). The dotted line represents lower limit of detection (LOD) for respective chemokine. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using ANOVA followed by Tukey multiple comparison post hoc test. RH, relative humidity.

FIGURE 5.

Cessation of SHS exposure restores immune-cell recruitment into the lung airspaces. (A) Experimental design for 42-d time point depicting exposure regimen and various endpoints examined. (B) Exposure parameters for 42-d study. (C) Total cell counts in the harvested BALF from 43-d-old FA- and SHS-exposed WT and Scnn1b-Tg+ mice (n = 11–17, overall p value < 0.0001). (D) Differential cell counts (overall p value < 0.0001) and (E) their respective percentages (overall p value < 0.0001) are shown as a stacked bar graph. BALF macrophage (red); neutrophil (blue), eosinophils (green), lymphocytes (black). (F) Representative photomicrographs of BALF cytospins. (Fa) FA-WT, (Fb) SHS-WT, (Fc) FA-Tg+, and (Fd) SHS-Tg+ mice at PND43. Macrophages (red arrows), neutrophils (blue arrows), eosinophils (green arrows), and lymphocytes (black arrows). (G) BALF cytokine levels (picograms per milliliter) of (Ga) MCP-1 (overall p value = 0.0104), (Gb) Eotaxin (overall p value = 0.3541), (Gc) IL-5 (overall p value = 0.0016), (Gd) KC (overall p value < 0.0001), (Ge) TNF-α (all values were below detection limits), and (Gf) IL-1α (overall p value < 0.0001) in BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) mice (n = 6–8 per group). The dotted line represents lower limit of detection (LOD) for respective chemokine. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using ANOVA followed by Tukey multiple comparison post hoc test. RH, relative humidity.

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Next, we sought to determine whether the restored BALF cellularity was the result of reversal of chemokine suppression (Fig. 5Ga–Gf). BALF chemokine estimation for three major chemokines revealed a striking correlation between numbers of individual cell types and levels of respective chemokines that are known to recruit these cells. For example, MCP-1, a major monocyte chemoattractant, was restored in the SHS-Tg+ BALF; suggesting its contribution toward macrophage and neutrophil recruitment (Fig. 5Ga). A similar cell–chemokine correlation was clearly evident between eosinophils and neutrophils and their respective chemokines, i.e., Eotaxin (Fig. 5Gb) and KC (Fig. 5Gd), respectively. Similar to the PND21 age point (Fig. 1G), BALF levels for TNF-α and IL-1α were comparable between FA-Tg+ and SHS-Tg+ mice (Fig. 5Ge, 5Gf).

SHS-exposed neonates exhibit significantly enhanced adaptive immune responses in adulthood.

To determine whether the termination of SHS exposure for 3 wk will allow the reversal of adaptive immune suppression, we evaluated lung sections and BALF for the presence of lymphoid aggregates and secreted IgA, respectively. As seen at PND22 (Fig. 1D, 1E), the SHS exposure to WT mice did not increase BALF lymphocytes (Fig. 5D, 5E) and the number of lymphoid aggregates in lungs (Fig. 6A–C). FA-Tg+ mice, however, had significantly increased BALF lymphocytes (∼5-fold versus FA-WT mice, Fig. 5D, 5E) and lymphoid aggregates (Fig. 6A, 6C). However, unlike at PND22, adult SHS-Tg+ mice had significantly increased BALF lymphocytes (∼1-fold versus FA-Tg+ mice, Fig. 5D, 5E) and lymphoid aggregates (Fig. 6A–C). Furthermore, whereas only 3 out of 9 FA-Tg+ mice had well-defined lymphoid nodules (Fig. 6D), 8 out of 10 SHS-Tg+ mice had these nodules (Fig. 6D).

FIGURE 6.

Cessation of SHS exposure restores pathological features of Scnn1b-Tg+ lung and reinstates Th2 responses associated with Scnn1b-Tg+ lung disease. (A) Representative photomicrographs from H&E-stained left lung lobe sections from 43-d-old (Aa) FA-WT, (Ab) SHS-WT, (Ac and Ae) FA-Tg+, or (Ad and Af) SHS-Tg+ mice (n = 11–16). Lymphoid aggregates (black arrows); alveolar space consolidation (red arrows). (Aa–d) Scale bars, 400 μm. (Ae and Af) Scale bars, 50 μm. (B) Representative photomicrographs of AB-PAS–stained left lung lobe sections from 43-d-old (Ba) FA-WT, (Bb) SHS-WT, (Bc) FA-Tg+, or (Bd) SHS-Tg+ mice (n = 11–16). (Ba–d) Scale bars, 100 μm. (C) Semiquantitative histopathology scores for mucus plugging, airway inflammation, epithelial necrosis, mucous secretory cells, parenchymal consolidation, and lymphoid hyperplasia. FA-WT (n = 13, white bar), SHS-WT (n = 16, orange bar), FA-Tg+ (n = 11, red bar), and SHS-Tg+ (n = 11, green bar). ANOVA followed by Tukey multiple comparison post hoc test. Data are expressed as mean values (± SEM). (D) Number of BALT nodules in lung sections from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 11–16, overall p value = 0.0014). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) IgA levels in cell-free BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 6–8 per group, overall p value = 0.0001). (F) IL-4 cytokine levels in BALF (n = 6–8 per group, overall p value = 0.0342). (G) mRNA levels for selective genes related to mucous cell metaplasia and Th2 inflammation. Absolute quantification (mRNA copies per 105 copies of Actb mRNA) of gene expression of selective genes in whole-lung homogenate from FA-WT or SHS-WT or Scnn1b-Tg+ mice. The data are presented as mean ± SEM. Two values carrying identical designations (for example, or ) in same row represent significant difference. p < 0.05 using ANOVA followed by Tukey multiple comparison post hoc test. n = 5 mice per group. Bold text with p values in parenthesis indicates higher values compared with FA-WT group. Agr2, anterior gradient protein 2 homolog; Chi3l4, chitinase-like 4; Clca1, calcium-activated chloride channel regulator 1; Foxa3, forkhead box A3; Ifng, IFN-γ; Il4, IL-4; Il13, IL-13; Gata3, GATA binding protein 3; Muc5ac, mucin 5, subtypes A and C; Muc5b, mucin 5, subtypes B; Retnla, resistin-like molecule α; Slc26a4, solute carrier family 26 member 4; Spdef, SAM pointed domain containing ETS transcription factor; Tff2, Trefoil factor 2; Tg+, Scnn1b-Tg+.

FIGURE 6.

Cessation of SHS exposure restores pathological features of Scnn1b-Tg+ lung and reinstates Th2 responses associated with Scnn1b-Tg+ lung disease. (A) Representative photomicrographs from H&E-stained left lung lobe sections from 43-d-old (Aa) FA-WT, (Ab) SHS-WT, (Ac and Ae) FA-Tg+, or (Ad and Af) SHS-Tg+ mice (n = 11–16). Lymphoid aggregates (black arrows); alveolar space consolidation (red arrows). (Aa–d) Scale bars, 400 μm. (Ae and Af) Scale bars, 50 μm. (B) Representative photomicrographs of AB-PAS–stained left lung lobe sections from 43-d-old (Ba) FA-WT, (Bb) SHS-WT, (Bc) FA-Tg+, or (Bd) SHS-Tg+ mice (n = 11–16). (Ba–d) Scale bars, 100 μm. (C) Semiquantitative histopathology scores for mucus plugging, airway inflammation, epithelial necrosis, mucous secretory cells, parenchymal consolidation, and lymphoid hyperplasia. FA-WT (n = 13, white bar), SHS-WT (n = 16, orange bar), FA-Tg+ (n = 11, red bar), and SHS-Tg+ (n = 11, green bar). ANOVA followed by Tukey multiple comparison post hoc test. Data are expressed as mean values (± SEM). (D) Number of BALT nodules in lung sections from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 11–16, overall p value = 0.0014). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) IgA levels in cell-free BALF from FA-WT (white bar), SHS-WT (orange bar), FA-Tg+ (red bar), and SHS-Tg+ (green bar) (n = 6–8 per group, overall p value = 0.0001). (F) IL-4 cytokine levels in BALF (n = 6–8 per group, overall p value = 0.0342). (G) mRNA levels for selective genes related to mucous cell metaplasia and Th2 inflammation. Absolute quantification (mRNA copies per 105 copies of Actb mRNA) of gene expression of selective genes in whole-lung homogenate from FA-WT or SHS-WT or Scnn1b-Tg+ mice. The data are presented as mean ± SEM. Two values carrying identical designations (for example, or ) in same row represent significant difference. p < 0.05 using ANOVA followed by Tukey multiple comparison post hoc test. n = 5 mice per group. Bold text with p values in parenthesis indicates higher values compared with FA-WT group. Agr2, anterior gradient protein 2 homolog; Chi3l4, chitinase-like 4; Clca1, calcium-activated chloride channel regulator 1; Foxa3, forkhead box A3; Ifng, IFN-γ; Il4, IL-4; Il13, IL-13; Gata3, GATA binding protein 3; Muc5ac, mucin 5, subtypes A and C; Muc5b, mucin 5, subtypes B; Retnla, resistin-like molecule α; Slc26a4, solute carrier family 26 member 4; Spdef, SAM pointed domain containing ETS transcription factor; Tff2, Trefoil factor 2; Tg+, Scnn1b-Tg+.

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Further, to determine whether the increase in the incidence of lymphoid nodules was sufficient to restore the suppression of IgA levels that was detected at PND22, BALF IgA levels were determined at PND43 (Fig. 6E). Although both the WT groups, i.e., FA-WT and SHS-WT, had minimal levels of BALF IgA, significantly higher levels were detected in BALF from both FA-Tg+ mice (∼66.35 ± 17.70 ng/ml, Fig. 6E) as well as SHS-Tg+ mice (108.3 ± 14.01 ng/ml, Fig. 6E). These data indicate that the IgA levels in the BALF increase with increased incidence of lymphoid tissue, i.e., loose lymphoid aggregates and organized BALT nodules, and the cessation of SHS exposure restores lymphocyte homing to the lungs and BALF IgA secretion.

SHS-exposed neonates exhibit exaggerated lung pathology.

Although the SHS-WT mice appeared free of mucus plugging, airway inflammation, mucous cell metaplasia, parenchymal consolidation, and lymphoid hyperplasia responses, a significantly higher degree of epithelial necrosis was present in SHS-WT mice (Fig. 6C). As expected from previous studies (7, 8), the FA-Tg+ group had significantly elevated responses, including mucus plugging, airway inflammation, epithelial necrosis, mucous cell metaplasia, parenchymal consolidation, and lymphoid hyperplasia responses. In contrast, SHS-Tg+ mice exhibited all the features of Scnn1b-Tg+ lung pathology with exaggerated degree of airway inflammation, epithelial necrosis, parenchymal consolidation, and lymphoid hyperplasia (Fig. 6A, 6C, 6D).

Cessation of SHS exposure restores Th2 responses in Scnn1b-Tg+ adults.

Because the mucus plugging and mucous cell metaplasia features were restored in SHS-Tg+ adult mice, we sought to test whether the restoration of mucous cell metaplasia was a result of restored levels of Th2 cytokines in BALF. BALF cytokine estimation indicated that levels of IL-5 and IL-4 were below detection limits in WT mice, regardless of their exposure groups, whereas a significant increase in their levels was observed in Scnn1b-Tg+ mice (Figs. 5Gc, 6F). However, the levels of IL-5 and IL-4 were not significantly different between FA- and SHS-Tg+ mice (Figs. 5G, 6F), suggesting restoration of previously suppressed secretion of these cytokines.

Next, we examined whether the restoration of IL-4 levels was reflected in IL-4Rα signaling–mediated upregulation of Th2/mucous cell metaplasia–associated gene expression levels. Interestingly, although insignificantly, the mean expression levels of selective genes, i.e., Il33, Ifng, Agr2, and Muc5ac, were increased in SHS-WT mice; suggesting a long-term effect of early postnatal SHS exposure in WT mice. As speculated, the mRNA levels of all the genes (Il33, Il4, Il13, Ifng, Gata3, Spdef, Foxa3, Tff2, Agr2, Muc5ac, Muc5b, Clca1 (gob5/Clca3), Slc26a4 (Pds/pendrin), Retnla, and Chi3l4) that were suppressed in 22-d-old SHS-Tg+ lungs returned to the levels similar to FA-Tg+ lungs (Fig. 6G) at PND43. Of note, the restoration of expression level of Th2 responses–associated gene signatures showed strong correlation with restoration of morphological features of mucous cell metaplasia (Fig. 6B, 6C).

Mucosal host defense at respiratory surfaces is composed of multiple layers, e.g., physical epithelial barrier, mucociliary clearance, antimicrobial peptides and Igs, and immune cells, including macrophages and neutrophils (25). Direct or indirect interactions between mucosal host defense layers and inhaled airborne pollutants, such as SHS, may alter the normal physiology of the respiratory system, shift the natural history of an ongoing disease, or alter the manifestation of imminent diseases. Therefore, in vivo testing of interactions between mucosal host defense layers and airborne pollutants is critical to understanding of the initiation and progression of environmental lung diseases.

Because early childhood SHS exposure is considered as a risk factor for muco-obstructive lung diseases of pediatric origin, including asthma, CF, and chronic obstructive pulmonary disease (26, 27); detailed analyses of three-dimensional interactions between SHS exposure, normal lung homeostasis, and the natural history of disease pathogenesis due to intrinsic defects, e.g., ion channel defects in CF, are warranted. For this study, we focused on early postnatal exposures of SHS, a primary contributor to poor indoor air quality (2, 3), to the Scnn1b-Tg+ mouse model, in which muco-obstructive airway disease evolves during early postnatal lung development. Muco-obstructive airway diseases, as recapitulated in the Scnn1b-Tg+ mouse model, are characterized by airway surface liquid dehydration, airway inflammation, bacterial infection, mucus hypersecretion, and mucus obstruction.

Due to the early onset of lung disease, i.e., PND2–3, the Scnn1b-Tg+ model provides an excellent tool for studying muco-obstructive disease of pediatric origin along with other variables, e.g., airborne exposure to environmental pollutants. We therefore focused our investigation on the preadult phase, i.e., PND3–21, and investigated the effect of SHS exposure on 1) early postnatal airspace homeostasis and 2) the natural history of muco-obstructive airway disease. In particular, we examined the effects of early postnatal SHS exposure on inflammatory cell recruitment, mucous cell metaplasia, adaptive immune response, and bacterial clearance.

This study revealed that SHS exposure of Scnn1b-Tg+ mice results in significantly altered phenotypes, including suppressed immune cell recruitment, delayed bacterial clearance, and diminished adaptive immune responses. These data reveal that, rather than exaggerating immune-cell recruitment and Th2 responses or mucous cell metaplasia, early postnatal SHS exposure suppresses these responses. These observations highlight the important complex interaction between variables, including SHS exposure, ongoing muco-obstructive lung disease, and the developmental stage.

Immune-cell recruitment, a key component of host defense against inhaled insults, has been shown to be increased in acute smoke-exposed adults (28, 29) as well as in neonatal mice (30). In agreement with these studies, as compared with FA-WT mice, the SHS-WT mice in our study had significantly increased neutrophil counts. In contrast, however, perhaps due to a significantly low dose of smoke exposure levels (TSP = 10 mg/m3) adopted in our study as compared with previous reports (2830), macrophage counts were comparable between FA- and SHS-WT mice (Fig. 1C–E). As previously reported (8), the FA-Tg+ mice, in contrast to FA-WT mice, had significant increases in total, as well as differential, cell numbers and percentages. Contrary, however, to the expected exaggerated cellular recruitment (15), SHS-Tg+ mice exhibited significantly suppressed immune-cell recruitment responses. This reduction was not due to a decrease in macrophage counts but rather to reduced numbers of neutrophils, eosinophils, and lymphocytes (Fig. 1D). These data show that early postnatal SHS exposure results in suppressed cellular recruitment in Scnn1b-Tg+ mice.

Based on this suppressed cellular recruitment, we speculated that the reduced total cell counts in SHS-Tg+ lung airspaces might be a result of reduced chemokine levels in the airspaces of these mice. KC, a neutrophil chemoattractant (31), was found at comparable levels in FA-Tg+ and SHS-Tg+ BALF (Fig. 1G), suggesting the possibility that the cognate chemokine receptor for KC may either be suppressed due to systemic effects of SHS on blood cells, or is nonresponsive in neonates. MCP-1 (CCL2), a monocyte (32) and neutrophil chemoattractant (33), however, was significantly lower in SHS-Tg+ BALF; suggesting a direct effect of SHS exposure on the synthesis and release of this chemokine that resulted in inhibition of MCP-1-/CCR2-mediated neutrophil recruitment. These data indicate that SHS exposure can suppress resident cell functions and early postnatal innate immune responses. Whether the suppressed populations are epithelial cells or macrophages, or both, is still unclear and will require comprehensive analyses focused on the effect of SHS exposure on epithelium- and macrophage-derived chemokine mediators in healthy and diseased neonates.

Mucous cell metaplasia, an epithelial Th2 response, is a hallmark of Scnn1b-Tg+ airway disease (7, 8) as well as of human muco-obstructive airway diseases (34). Our previous report on gene expression analyses on whole lung tissues from 10- to 42-d-old WT and Scnn1b-Tg+ mice demonstrated significantly upregulated Th2 responses in the latter group (11), which were also confirmed in this study (Fig. 3F). In contrast, the SHS-Tg+ mice exhibited significantly suppressed mRNA levels for Gata3, a gene encoding a transcription factor controlling Th2 differentiation (35). These data clearly show a direct effect of diminished BALF Th2 cytokine (IL-4 and IL-13) levels on Th2 cell developmental machinery, i.e., Gata3-controlled Th2 responses. Along the same axis, the SHS-Tg+ mice exhibited downregulated expression of all Th2 response-related genes (Spdef, Foxa3, Tff2, Agr2, Muc5ac, Muc5b, Clca1 [gob5/Clca3], Slc26a4 [Pds/pendrin], Retnla, and Chi3l4; Fig. 3F). These data strongly suggest that SHS exposure at the onset of respiratory disease results in suppressed Th2 responses (Fig. 7).

FIGURE 7.

Hypothetical model for SHS exposure–induced suppression of Th2 immune responses. Airway surface liquid dehydration–induced stress in lung epithelium and macrophages of Scnn1b-Tg+ mice induces IL-33 secretion. Increased levels of IL-33 stimulate ILC2, which releases Th2 cytokines (IL-4, IL-13) into the airspaces. Th2 cytokines trigger GATA3-transcription factor–regulated development of Th2 cells and, eventually, IL4Rα-mediated gene expression changes (Spdef, Foxa3, Tff2, Agr2, Muc5ac, Muc5b, Clca1 [gob5/Clca3], Slc26a4 [Pds/pendrin], Retnla, and Chi3l4) leading to epithelial Th2 responses. SHS exposures superimpose additional stress onto the Scnn1b-Tg+ airspaces, resulting in suppression of IL-33 synthesis and release, thus leading to suppressed Th2 responses.

FIGURE 7.

Hypothetical model for SHS exposure–induced suppression of Th2 immune responses. Airway surface liquid dehydration–induced stress in lung epithelium and macrophages of Scnn1b-Tg+ mice induces IL-33 secretion. Increased levels of IL-33 stimulate ILC2, which releases Th2 cytokines (IL-4, IL-13) into the airspaces. Th2 cytokines trigger GATA3-transcription factor–regulated development of Th2 cells and, eventually, IL4Rα-mediated gene expression changes (Spdef, Foxa3, Tff2, Agr2, Muc5ac, Muc5b, Clca1 [gob5/Clca3], Slc26a4 [Pds/pendrin], Retnla, and Chi3l4) leading to epithelial Th2 responses. SHS exposures superimpose additional stress onto the Scnn1b-Tg+ airspaces, resulting in suppression of IL-33 synthesis and release, thus leading to suppressed Th2 responses.

Close modal

Based on histopathological (Fig. 3A–C), cytokine (Fig. 3D, 3E), and gene expression data (Fig. 3F–I), we speculated that the suppressed secretions of Th2-related cytokines, i.e., IL-4 and IL-13, in SHS-Tg+ BALF might have arisen due to SHS-induced suppression of resident cells, i.e., epithelial cells and macrophages. Epithelial cells, upon exposures to insults such as allergens (36, 37), nematodes (38), helminths (39), and viral infections (40), have been reported to release IL-33, an alarmin. IL-33, in association with IL-25 and TSLP, stimulates ILC2 to produce type 2 cytokines, including IL-4, IL-5, and IL-13 (41). Our data demonstrate that whereas FA-Tg+ mice had significantly increased levels of IL-33 expression (mRNA levels, intracellular IL-33 protein, and secreted BALF IL-33), the SHS-Tg+ mice had significant reductions in these endpoints (Fig. 4). This reduction in the expression levels of IL-33 suggests loss of SHS-induced ILC2 stimulation. Interestingly, a recent study on the role of IL-33/ST2 axis demonstrated that although mainstream smoke exposure of adult mice results in increased IL-33 release, these exposures simultaneously suppress the expression of the IL-33R, i.e., ST2, which mediates ILC2 stimulation (24). At present, we cannot determine whether the differences between these two studies stem from different exposure models (SHS versus mainstream smoke), age-group differences (neonates versus adults), or a combination of the two. Accordingly, our data suggest that the IL-33–ILC2–IL-4/-13–IL-4Rα axis mediates Th2 responses in Scnn1b-Tg+ neonates and that the SHS exposure directly suppresses IL-33 expression in epithelial and immune cells (Fig. 7). These alterations eventually result in suppression of downstream Th2 immune responses, most likely, due to disrupted IL-4/-13–IL-4Rα signaling.

Mucostasis creates a fertile anaerobic niche for the colonization of bacterial species (42). Scnn1b-Tg+ mice exhibit spontaneous infection with microflora of oropharyngeal origin (10), due to the muco-obstructive airways and resulting defective mucociliary clearance. These infections have been reported to clear in ∼70% of mice by the age of 4 wk (10). In agreement with the latter report, only 3 out of 10 FA-Tg+ mice in this study exhibited microaerophilic bacterial infection at PND22 (Fig. 2B). Despite significant reduction in mucus plugging and Th2-responses (Muc5ac and Muc5b), SHS-Tg+ mice exhibited defective bacterial clearance, i.e., 11 out of 12 SHS-Tg+ mice had persistent infections at PND22 (Fig. 2B). These data indicate that SHS exposure results in weakened host defense against spontaneously acquired microbial infections and that this defect is unrelated to mucostasis.

The roles of IL-33 in bacterial infections have also been explored in recent years in various infection models. Earlier, Lan et al. (43) and, recently, Robinson et al. (44) demonstrated that IL-33 facilitates neutrophil recruitment and improves bacterial clearance in different bacterial infection models. In line with these reports, reduced IL-33 levels in SHS-Tg+ mice was associated with reduced BALF neutrophil counts and delayed bacterial clearance. Further, in addition to neutrophils, secretory IgA (SIgA)–mediated neutralization of mucosal microorganisms (45) and microbial toxins (46) is a major component of mucosal host defense (47). In our study, SHS exposure resulted in a significant reduction in BALF SIgA levels both in WT and Scnn1b-Tg+ mice (Fig. 2A). Malik et al. (48) has recently established a link between IL-33 and SIgA levels in gut homeostasis and colitis. In line with this report, suppression in IL-33 levels in SHS-Tg+ mice may have resulted in suppressed SIgA levels, thus introducing an additional defect in antibacterial host defense (49). Accordingly, we speculate that the beneficial effect of IL-33 downregulation toward bacterial clearance, i.e., through reduction in mucostasis or mucus production, was counterbalanced or overpowered by the detrimental effects of IL-33 downregulation, i.e., through reduction in neutrophil counts (Fig. 1) and SIgA levels (Fig. 2A).

Although the effect of SHS exposure on WT mice was milder, as previously reported (7, 8), the presence of the Scnn1b transgene resulted in marked muco-obstructive lung disease. However, the simultaneous presence of these two challenges revealed significant alterations in the manifestation of imminent lung disease of Scnn1b-Tg+ neonates. Whether the presence of the SHS insult suppresses the immune responses associated with Scnn1b expression, or whether the presence of an Scnn1b expression–induced ion channel defect predisposes neonates to SHS-induced suppression, is still unclear and requires further investigation.

The gender-specific responses to SHS have been reported previously. In comparison with tobacco smoke–exposed adult males, the females exhibit exaggerated allergic responses (50), alveolar airspace enlargement (51), and increased airway remodeling (52, 53). Interestingly, our key findings, in particular neutrophil counts, cytokine levels, and mucous cell metaplasia, which form the basis of our study, followed similar trends for both genders (Supplemental Tables III, IV). This was not very surprising as the study was concluded at a juvenile age (PND22), well before the age of sexual maturity (PND42–56).

In the latter half of the study, we investigated whether the SHS-induced suppression of immune responses and Th2 inflammation in Scnn1b-Tg+ lungs would sustain or reverse upon cessation of SHS exposure. As expected, a majority of suppressed responses, including immune cell recruitment, gene expression signatures, chemokines, and mucous cell metaplasia, were restored at day 42 after cessation of SHS on day 21. In fact, some of these responses, including cell counts, mucous cell metaplasia, epithelial necrosis, and lymphoid hyperplasia, were exaggerated in SHS-Tg+ mice as compared with FA-Tg+ at day 42. These data suggest that the suppression of immune responses in SHS-Tg+ mice at PND22 were temporary and the cessation of SHS allows the restoration of immune responses. Early postnatal SHS exposure, however, results in lagged effects after cessation of SHS exposure, including structural alterations, particularly pulmonary consolidation, epithelial necrosis, and airway inflammation, which may have long-term consequences in adult life.

Although a number of studies have investigated the long-term effects of in utero SHS exposure (1618, 54), a limited number of studies have explored the short-term effects of neonatal SHS exposure. Given the highly sensitive stage of ongoing lung development, the superimposition of exposure to SHS or any other airborne environmental pollutant upon early postnatal life makes interpretation of these studies challenging. One limitation to this study was our inability to further explore the effect of SHS on individual cell types resident in the airspaces, i.e., macrophages and epithelial cells. For example, although FA-Tg+ macrophages revealed intense IL-33 staining (Fig. 4), we cannot determine whether increased IL-33 in macrophages indicates elevated endogenous IL-33 expression levels or phagocytosed IL-33 of epithelial origin. We therefore propose that further studies should be conducted to understand the effects of SHS exposures on cell-specific pathological responses of different neonatal age groups.

In conclusion, this study reveals previously underappreciated multidimensional interactions between SHS, early postnatal lung development, and muco-obstructive lung disease. Our data suggests that SHS exposure during early postnatal stage of muco-obstructive lung disease results in suppressed host responses, including: 1) suppressed cellular recruitment responses; 2) defective bacterial clearance; 3) suppressed mucous cell metaplasia; 4) diminished Th2 responses; and 5) suppressed adaptive immune response, i.e., IgA secretions. Whereas most of these responses are reversed upon cessation of SHS exposure, histologically, SHS-exposed lungs exhibit exaggerated pathological alterations indicating lagged effects of early postnatal SHS exposure that may have long-term consequences in adult life.

We thank Thaya Stoufflet for assistance with multiplex cytokine assays and 16S genomic sequencing, Christina Verret for technical assistance with inhalational exposures, and Sherry Ring for histological tissue processing.

This work was supported by the Flight Attendant Medical Research Institute grant (to Y.S.), the Louisiana State University–Tulane University Center of Biomedical Research Excellence (National Institute of General Medical Sciences Grant 5P30GM110760), Louisiana’s Governors Biotechnology Initiative, and the Louisiana State University School of Veterinary Medicine startup funds (to Y.S.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

AB-PAS

Alcian blue–periodic acid–Schiff

BAL

bronchoalveolar lavage

BALF

bronchoalveolar lavage fluid

CF

cystic fibrosis

βENaC

epithelial sodium ion channel subunit β

FA

filtered air

FA-Tg+

FA-exposed Scnn1b-Tg+

FA-WT

FA-exposed WT

ILC2

type II innate lymphoid cell

KC

keratinocyte chemoattractant

PND

postnatal day

RH

relative humidity

Scnn1b-Tg+

Scnn1b transgenic

SHS

secondhand smoke

SHS-Tg+

SHS-exposed Scnn1b-Tg+

SHS-WT

SHS-exposed WT

SIgA

secretory IgA

Tg

transgenic

TSLP

thymic stromal lymphopoietin

TSP

total suspended particulate

WT

wild-type.

1
U.S. Environmental Protection Agency. 2004. Air Quality Criteria for Particulate Matter. Washington, D.C: U.S. Environmental Protection Agency, Office of Research and Development, Final report. EPA 600/P-99/002aF-bF
.
2
Eisner
,
M. D.
2007
.
Indoor air, passive smoking, and COPD.
Am. J. Respir. Crit. Care Med.
176
:
426
427
.
3
Repace
,
J. L.
,
A. H.
Lowrey
.
1980
.
Indoor air pollution, tobacco smoke, and public health.
Science
208
:
464
472
.
4
U.S. Environmental Protection AgencyNational Cancer Institute (U.S.)Smoking and Tobacco Control Program
.
2004
.
Fact Sheet: Respiratory Health Effects of Passive Smoking.
U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, and U.S. Environmental Protection Agency
,
Washington, D.C
.
5
U.S. Department of Health and Human Services
.
2006
.
The Health Consequences of Involuntary Exposure to Tobacco Smoke: A Report of the Surgeon General.
Atlanda, GA
:
U.S. Dept. of Health and Human Services, Centers for Disease Control and Prevention, Coordinating Center for Health Promotion, National Center for Chronic Disease Prevention and Health Promotion, Office on Smoking and Health
.
6
Gibbs
,
K.
,
J. M.
Collaco
,
S. A.
McGrath-Morrow
.
2016
.
Impact of tobacco smoke and nicotine exposure on lung development.
Chest
149
:
552
561
.
7
Mall
,
M.
,
B. R.
Grubb
,
J. R.
Harkema
,
W. K.
O’Neal
,
R. C.
Boucher
.
2004
.
Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice.
Nat. Med.
10
:
487
493
.
8
Mall
,
M. A.
,
J. R.
Harkema
,
J. B.
Trojanek
,
D.
Treis
,
A.
Livraghi
,
S.
Schubert
,
Z.
Zhou
,
S. M.
Kreda
,
S. L.
Tilley
,
E. J.
Hudson
, et al
.
2008
.
Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.
Am. J. Respir. Crit. Care Med.
177
:
730
742
.
9
Saini
,
Y.
,
K. J.
Wilkinson
,
K. A.
Terrell
,
K. A.
Burns
,
A.
Livraghi-Butrico
,
C. M.
Doerschuk
,
W. K.
O’Neal
,
R. C.
Boucher
.
2016
.
Neonatal pulmonary macrophage depletion coupled to defective mucus clearance increases susceptibility to pneumonia and alters pulmonary immune responses.
Am. J. Respir. Cell Mol. Biol.
54
:
210
221
.
10
Livraghi-Butrico
,
A.
,
E. J.
Kelly
,
E. R.
Klem
,
H.
Dang
,
M. C.
Wolfgang
,
R. C.
Boucher
,
S. H.
Randell
,
W. K.
O’Neal
.
2012
.
Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation.
Mucosal Immunol.
5
:
397
408
.
11
Saini
,
Y.
,
H.
Dang
,
A.
Livraghi-Butrico
,
E. J.
Kelly
,
L. C.
Jones
,
W. K.
O’Neal
,
R. C.
Boucher
.
2014
.
Gene expression in whole lung and pulmonary macrophages reflects the dynamic pathology associated with airway surface dehydration.
BMC Genomics
15
:
726
.
12
Seys
,
L. J.
,
F. M.
Verhamme
,
L. L.
Dupont
,
E.
Desauter
,
J.
Duerr
,
A.
Seyhan Agircan
,
G.
Conickx
,
G. F.
Joos
,
G. G.
Brusselle
,
M. A.
Mall
,
K. R.
Bracke
.
2015
.
Airway surface dehydration aggravates cigarette smoke-induced hallmarks of COPD in mice.
PLoS One
10
:
e0129897
.
13
Geiser
,
M.
,
C.
Wigge
,
M. L.
Conrad
,
S.
Eigeldinger-Berthou
,
L.
Künzi
,
H.
Garn
,
H.
Renz
,
M. A.
Mall
.
2014
.
Nanoparticle uptake by airway phagocytes after fungal spore challenge in murine allergic asthma and chronic bronchitis.
BMC Pulm. Med.
14
:
116
.
14
Geiser
,
M.
,
O.
Quaile
,
A.
Wenk
,
C.
Wigge
,
S.
Eigeldinger-Berthou
,
S.
Hirn
,
M.
Schäffler
,
C.
Schleh
,
W.
Möller
,
M. A.
Mall
,
W. G.
Kreyling
.
2013
.
Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease.
Part. Fibre Toxicol.
10
:
19
.
15
Jia
,
J.
,
T. M.
Conlon
,
C.
Ballester-Lopez
,
M.
Seimetz
,
M.
Bednorz
,
Z.
Zhou-Suckow
,
N.
Weissmann
,
O.
Eickelberg
,
M. A.
Mall
,
A. O.
Yildirim
.
2016
.
Cigarette smoke causes acute airways disease and exacerbates chronic obstructive lung disease in neonatal mice
.
Am. J. Physiol. Lung Cell. Mol. Physiol.
311
:
L602
L610
.
16
Xiao
,
R.
,
Z.
Perveen
,
D.
Paulsen
,
R.
Rouse
,
N.
Ambalavanan
,
M.
Kearney
,
A. L.
Penn
.
2012
.
In utero exposure to second-hand smoke aggravates adult responses to irritants: adult second-hand smoke.
Am. J. Respir. Cell Mol. Biol.
47
:
843
851
.
17
Penn
,
A. L.
,
R. L.
Rouse
,
D. W.
Horohov
,
M. T.
Kearney
,
D. B.
Paulsen
,
L.
Lomax
.
2007
.
In utero exposure to environmental tobacco smoke potentiates adult responses to allergen in BALB/c mice.
Environ. Health Perspect.
115
:
548
555
.
18
Xiao
,
R.
,
Z.
Perveen
,
R. L.
Rouse
,
V.
Le Donne
,
D. B.
Paulsen
,
N.
Ambalavanan
,
A. L.
Penn
.
2013
.
In utero exposure to second-hand smoke aggravates the response to ovalbumin in adult mice.
Am. J. Respir. Cell Mol. Biol.
49
:
1102
1109
.
19
Livraghi
,
A.
,
B. R.
Grubb
,
E. J.
Hudson
,
K. J.
Wilkinson
,
J. K.
Sheehan
,
M. A.
Mall
,
W. K.
O’Neal
,
R. C.
Boucher
,
S. H.
Randell
.
2009
.
Airway and lung pathology due to mucosal surface dehydration in beta-epithelial Na+ channel-overexpressing mice: role of TNF-alpha and IL-4Ralpha signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.
J. Immunol.
182
:
4357
4367
.
20
Saini
,
Y.
,
S. P.
Proper
,
P.
Dornbos
,
K. K.
Greenwood
,
A. K.
Kopec
,
S. G.
Lynn
,
E.
Grier
,
L. D.
Burgoon
,
T. R.
Zacharewski
,
R. S.
Thomas
, et al
.
2015
.
Loss of Hif-2α rescues the Hif-1α deletion phenotype of neonatal respiratory distress in mice.
PLoS One
10
:
e0139270
.
21
Ong
,
S. H.
,
V. U.
Kukkillaya
,
A.
Wilm
,
C.
Lay
,
E. X.
Ho
,
L.
Low
,
M. L.
Hibberd
,
N.
Nagarajan
.
2013
.
Species identification and profiling of complex microbial communities using shotgun Illumina sequencing of 16S rRNA amplicon sequences.
PLoS One
8
:
e60811
.
22
Eichinger
,
K. M.
,
L.
Egaña
,
J. G.
Orend
,
E.
Resetar
,
K. B.
Anderson
,
R.
Patel
,
K. M.
Empey
.
2015
.
Alveolar macrophages support interferon gamma-mediated viral clearance in RSV-infected neonatal mice.
Respir. Res.
16
:
122
.
23
Cohn
,
L.
,
R. J.
Homer
,
N.
Niu
,
K.
Bottomly
.
1999
.
T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production.
J. Exp. Med.
190
:
1309
1318
.
24
Kearley
,
J.
,
J. S.
Silver
,
C.
Sanden
,
Z.
Liu
,
A. A.
Berlin
,
N.
White
,
M.
Mori
,
T. H.
Pham
,
C. K.
Ward
,
G. J.
Criner
, et al
.
2015
.
Cigarette smoke silences innate lymphoid cell function and facilitates an exacerbated type I interleukin-33-dependent response to infection.
Immunity
42
:
566
579
.
25
Whitsett
,
J. A.
,
T.
Alenghat
.
2015
.
Respiratory epithelial cells orchestrate pulmonary innate immunity.
Nat. Immunol.
16
:
27
35
.
26
Pattenden
,
S.
,
T.
Antova
,
M.
Neuberger
,
B.
Nikiforov
,
M.
De Sario
,
L.
Grize
,
J.
Heinrich
,
F.
Hruba
,
N.
Janssen
,
H.
Luttmann-Gibson
, et al
.
2006
.
Parental smoking and children’s respiratory health: independent effects of prenatal and postnatal exposure.
Tob. Control
15
:
294
301
.
27
Eisner
,
M. D.
,
C.
Iribarren
,
E. H.
Yelin
,
S.
Sidney
,
P. P.
Katz
,
G.
Sanchez
,
P. D.
Blanc
.
2009
.
The impact of SHS exposure on health status and exacerbations among patients with COPD.
Int. J. Chron. Obstruct. Pulmon. Dis.
4
:
169
176
.
28
John
,
G.
,
K.
Kohse
,
J.
Orasche
,
A.
Reda
,
J.
Schnelle-Kreis
,
R.
Zimmermann
,
O.
Schmid
,
O.
Eickelberg
,
A. O.
Yildirim
.
2014
.
The composition of cigarette smoke determines inflammatory cell recruitment to the lung in COPD mouse models.
Clin. Sci.
126
:
207
221
.
29
D’hulst
,
A. I.
,
K. Y.
Vermaelen
,
G. G.
Brusselle
,
G. F.
Joos
,
R. A.
Pauwels
.
2005
.
Time course of cigarette smoke-induced pulmonary inflammation in mice.
Eur. Respir. J.
26
:
204
213
.
30
McGrath-Morrow
,
S.
,
D.
Malhotra
,
T.
Lauer
,
J. M.
Collaco
,
W.
Mitzner
,
E.
Neptune
,
R.
Wise
,
S.
Biswal
.
2011
.
Exposure to neonatal cigarette smoke causes durable lung changes but does not potentiate cigarette smoke-induced chronic obstructive pulmonary disease in adult mice.
Exp. Lung Res.
37
:
354
363
.
31
Craig
,
A.
,
J.
Mai
,
S.
Cai
,
S.
Jeyaseelan
.
2009
.
Neutrophil recruitment to the lungs during bacterial pneumonia.
Infect. Immun.
77
:
568
575
.
32
Deshmane
,
S. L.
,
S.
Kremlev
,
S.
Amini
,
B. E.
Sawaya
.
2009
.
Monocyte chemoattractant protein-1 (MCP-1): an overview.
J. Interferon Cytokine Res.
29
:
313
326
.
33
Balamayooran
,
G.
,
S.
Batra
,
T.
Balamayooran
,
S.
Cai
,
S.
Jeyaseelan
.
2011
.
Monocyte chemoattractant protein 1 regulates pulmonary host defense via neutrophil recruitment during Escherichia coli infection.
Infect. Immun.
79
:
2567
2577
.
34
Saetta
,
M.
,
G.
Turato
,
S.
Baraldo
,
A.
Zanin
,
F.
Braccioni
,
C. E.
Mapp
,
P.
Maestrelli
,
G.
Cavallesco
,
A.
Papi
,
L. M.
Fabbri
.
2000
.
Goblet cell hyperplasia and epithelial inflammation in peripheral airways of smokers with both symptoms of chronic bronchitis and chronic airflow limitation.
Am. J. Respir. Crit. Care Med.
161
:
1016
1021
.
35
Zheng
,
W.
,
R. A.
Flavell
.
1997
.
The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.
Cell
89
:
587
596
.
36
Divekar
,
R.
,
H.
Kita
.
2015
.
Recent advances in epithelium-derived cytokines (IL-33, IL-25, and thymic stromal lymphopoietin) and allergic inflammation.
Curr. Opin. Allergy Clin. Immunol.
15
:
98
103
.
37
Bartemes
,
K. R.
,
K.
Iijima
,
T.
Kobayashi
,
G. M.
Kephart
,
A. N.
McKenzie
,
H.
Kita
.
2012
.
IL-33-responsive lineage- CD25+ CD44(hi) lymphoid cells mediate innate type 2 immunity and allergic inflammation in the lungs.
J. Immunol.
188
:
1503
1513
.
38
Yasuda
,
K.
,
T.
Muto
,
T.
Kawagoe
,
M.
Matsumoto
,
Y.
Sasaki
,
K.
Matsushita
,
Y.
Taki
,
S.
Futatsugi-Yumikura
,
H.
Tsutsui
,
K. J.
Ishii
, et al
.
2012
.
Contribution of IL-33-activated type II innate lymphoid cells to pulmonary eosinophilia in intestinal nematode-infected mice.
Proc. Natl. Acad. Sci. USA
109
:
3451
3456
.
39
Hung
,
L. Y.
,
I. P.
Lewkowich
,
L. A.
Dawson
,
J.
Downey
,
Y.
Yang
,
D. E.
Smith
,
D. R.
Herbert
.
2013
.
IL-33 drives biphasic IL-13 production for noncanonical type 2 immunity against hookworms.
Proc. Natl. Acad. Sci. USA
110
:
282
287
.
40
Monticelli
,
L. A.
,
G. F.
Sonnenberg
,
M. C.
Abt
,
T.
Alenghat
,
C. G.
Ziegler
,
T. A.
Doering
,
J. M.
Angelosanto
,
B. J.
Laidlaw
,
C. Y.
Yang
,
T.
Sathaliyawala
, et al
.
2011
.
Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus.
Nat. Immunol.
12
:
1045
1054
.
41
Kim
,
B. S.
,
E. D.
Wojno
,
D.
Artis
.
2013
.
Innate lymphoid cells and allergic inflammation.
Curr. Opin. Immunol.
25
:
738
744
.
42
Boucher
,
R. C.
2004
.
New concepts of the pathogenesis of cystic fibrosis lung disease.
Eur. Respir. J.
23
:
146
158
.
43
Lan
,
F.
,
B.
Yuan
,
T.
Liu
,
X.
Luo
,
P.
Huang
,
Y.
Liu
,
L.
Dai
,
H.
Yin
.
2016
.
Interleukin-33 facilitates neutrophil recruitment and bacterial clearance in S. aureus-caused peritonitis.
Mol. Immunol.
72
:
74
80
.
44
Robinson
,
K. M.
,
K.
Ramanan
,
M. E.
Clay
,
K. J.
McHugh
,
H. E.
Rich
,
J. F.
Alcorn
.
2017
.
Novel protective mechanism for interleukin-33 at the mucosal barrier during influenza-associated bacterial superinfection.
Mucosal Immunol.
In press.
45
Phalipon
,
A.
,
A.
Cardona
,
J. P.
Kraehenbuhl
,
L.
Edelman
,
P. J.
Sansonetti
,
B.
Corthésy
.
2002
.
Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo.
Immunity
17
:
107
115
.
46
Fernandez
,
M. I.
,
T.
Pedron
,
R.
Tournebize
,
J. C.
Olivo-Marin
,
P. J.
Sansonetti
,
A.
Phalipon
.
2003
.
Anti-inflammatory role for intracellular dimeric immunoglobulin a by neutralization of lipopolysaccharide in epithelial cells.
Immunity
18
:
739
749
.
47
Corthésy
,
B.
2013
.
Role of secretory IgA in infection and maintenance of homeostasis.
Autoimmun. Rev.
12
:
661
665
.
48
Malik
,
A.
,
D.
Sharma
,
Q.
Zhu
,
R.
Karki
,
C. S.
Guy
,
P.
Vogel
,
T. D.
Kanneganti
.
2016
.
IL-33 regulates the IgA-microbiota axis to restrain IL-1α-dependent colitis and tumorigenesis.
J. Clin. Invest.
126
:
4469
4481
.
49
Lugade
,
A. A.
,
P. N.
Bogner
,
T. H.
Thatcher
,
P. J.
Sime
,
R. P.
Phipps
,
Y.
Thanavala
.
2014
.
Cigarette smoke exposure exacerbates lung inflammation and compromises immunity to bacterial infection.
J. Immunol.
192
:
5226
5235
.
50
Seymour
,
B. W.
,
K. E.
Friebertshauser
,
J. L.
Peake
,
K. E.
Pinkerton
,
R. L.
Coffman
,
L. J.
Gershwin
.
2002
.
Gender differences in the allergic response of mice neonatally exposed to environmental tobacco smoke.
Dev. Immunol.
9
:
47
54
.
51
Awji
,
E. G.
,
J. C.
Seagrave
,
Y.
Tesfaigzi
.
2015
.
Correlation of cigarette smoke-induced pulmonary inflammation and Emphysema in C3H and C57Bl/6 mice.
Toxicol. Sci.
147
:
75
83
.
52
Tam
,
A.
,
A.
Churg
,
J. L.
Wright
,
S.
Zhou
,
M.
Kirby
,
H. O.
Coxson
,
S.
Lam
,
S. F.
Man
,
D. D.
Sin
.
2016
.
Sex differences in airway remodeling in a mouse model of chronic obstructive pulmonary disease.
Am. J. Respir. Crit. Care Med.
193
:
825
834
.
53
Tam
,
A.
,
J. H.
Bates
,
A.
Churg
,
J. L.
Wright
,
S. F.
Man
,
D. D.
Sin
.
2016
.
Sex-related differences in pulmonary function following 6 months of cigarette exposure: implications for sexual dimorphism in mild COPD.
PLoS One
11
:
e0164835
.
54
Xiao
,
R.
,
A.
Noël
,
Z.
Perveen
,
A. L.
Penn
.
2016
.
In utero exposure to second-hand smoke activates pro-asthmatic and oncogenic miRNAs in adult asthmatic mice.
Environ. Mol. Mutagen.
57
:
190
199
.

The authors have no financial conflicts of interest.

Supplementary data