Scnn1b-Tg+ (Tg+) mice [B6N.Cg-Tg(Scgb1a1-Scnn1b)6608Bouc/J] were procured from the Jackson Laboratory (Bar Harbor, ME) and maintained at the Division of Laboratory Animal Medicine (DLAM) vivarium of Louisiana State University (LSU). Club cell-specific Cre recombinase (CCSP-Cre⁺) mice were provided by Dr. Francesco J. DeMayo (NIEHS, North Carolina (NC)), and Egfr floxed (Egfr ^fl/fl) mice were provided by Dr. David Threadgill (Texas A&M University, Texas). All three mice strains were interbred to generate various parental genotypes. EGFR-sufficient Tg+ (CCSP-Cre^-/Egfr ^fl/fl/Tg+) and airway epithelial cell-specific EGFR-deficient Tg+ (CCSP-Cre⁺/Egfr ^fl/fl/Tg+) and their wild-type (WT) counterparts, i.e., CCSP-Cre^-/Egfr ^fl/fl/WT and CCSP-Cre⁺/Egfr ^fl/fl/WT, were generated by reciprocal crosses between CCSP-Cre⁺/Egfr ^fl/fl/WT and CCSP-Cre^-/Egfr ^fl/fl/Tg+ (or CCSP-Cre^-/Egfr ^fl/fl/WT and CCSP-Cre⁺/Egfr ^fl/fl/Tg+) parental genotypes. The CCSP-Cre⁺/Egfr ^fl/fl/Tg+ mice are expected to have EGFR deficiency only in the CCSP+ cells. However, our previous study employing CCSP-Cre mice (31) have shown that the CCSP-Cre⁺ causes recombination in almost all the airway epithelial cells. Therefore, we will be using the term “airway epithelial cell-specific EGFR-deficient mice” and not “Club cell-specific EGFR deficient mice”. Genotypes of all four experimental groups were confirmed with polymerase chain reaction (PCR). Nucleotide sequences of primers used for genotyping are included in Supplementary Table 1 . Mice were housed in hot-washed and individually ventilated cages on 12h day/night cycle at DLAM vivarium of LSU. Mice were provided with a regular diet and water ad libitum. All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Louisiana State University.

Juvenile mice (Postnatal day 21, PND21) were anesthetized via intraperitoneal (IP) injection of 2,2,2-tribromoethanol (Millipore Sigma, Burlington, MA). The left main stem bronchus was ligated, and the right lung lobes were aseptically lavaged with a body weight-adjusted volume of Dulbecco’s Phosphate Buffered Saline (DPBS) (Corning, Manassas, VA). A fraction of the harvested bronchoalveolar lavage fluid (BALF) was used for the estimation of colony forming units (CFUs), and the remaining BALF was centrifuged at 4°C for 500 x g for 5 min. Cell-free BALF supernatant was collected and stored at -80°C for the total protein, dsDNA, and cytokine analyses. Cell pellets were resuspended in 250 µl of DPBS and used for total and differential cell counts estimation as described previously (32). Lavaged right lung lobes were snap-frozen and stored at -80°C for gene expression analyses. Unlavaged left lung lobes were fixed in 10% neutral buffered formalin and processed for histological analyses, as described previously (33).

Total protein and dsDNA contents in the BALF were determined by Bradford assay (Bio-Rad, Hercules, CA) and spectrophotometric assay using NanoDrop 8000 (Thermo Fisher Scientific, Waltham, MA), respectively.

Cell-free BALF samples were assayed for various soluble mediators as previously described (34). Briefly, the BALF levels of various cytokines and chemokines were determined using Luminex XMAP-based assay (MCYTOMAG-70K), according to the manufacturer’s instructions (EMD Millipore, Billerica, MA). The list of cytokines and chemokines is included in Supplementary Table 2 .

IL-13 levels were analyzed in the cell-free BALF samples using Mouse IL-13 ELISA Kit-Quantikine (Cat # M1300CB, R&D systems, Minneapolis, MN), according to the manufacturer’s instructions.

The aseptically harvested BALF was serially diluted and plated onto Columbia blood agar (CBA) plates (Hardy Diagnostics, Santa Maria, CA). The plates were incubated in anaerobic candle jars at 37°C for 48h. The CFUs were counted, and the morphological characteristics of the colonies were recorded, as previously described (32).

The formalin-fixed left lung lobes were embedded in paraffin and sectioned at 5µm thickness. Alcian blue-periodic acid-Schiff (AB-PAS) staining was performed to stain mucopolysaccharide materials in the airway lumen and mucins content within the airway epithelial cells. Mucus obstruction was graded using the histological semiquantitative grading strategy as previously described (32). For MCM analyses, the photographs of AB-PAS-stained large airways were captured under the 40X objective of the ECLIPSE Ci-L microscope with DS-Fi2 camera attachment (Nikon, Melville, NY). Thereafter, the analyses were performed by quantifying the number of mucous cells per millimeter (mm) of basement membrane using the Fiji software (35). All the slides were graded by a board-certified anatomic pathologist in a blinded manner.

Formalin-fixed paraffin-embedded left lung sections were used for the immunohistochemical localization of MUC5AC, MUC5B, and E-cadherin. The sections were stained with the corresponding primary antibodies: rabbit polyclonal MUC5AC antibody (UNC 294, a kind gift by Dr. Camille Ehre, University of North Carolina, Chapel Hill, NC), rabbit polyclonal MUC5B antibody (UNC223, a kind gift by Dr. Camille Ehre, University of North Carolina, Chapel Hill, NC), and Rabbit monoclonal E-cadherin primary antibody (3195, Cell Signaling Technology, Danvers, MA) using previously published procedure (36–38). The immunostained slides were analyzed by a board-certified anatomic pathologist without prior knowledge of genotypes. The photographs were captured under the 40X or 4X objective of the ECLIPSE Ci-L microscope with DS-Fi2 camera attachment (Nikon, Melville, NY). Thereafter, captured images were processed using the Fiji software (35) to determine the percentage of stained area (MUC5B and MUC5AC).

Total RNA isolation from right lungs, analysis of quantity and purity of isolated total RNA, cDNA generation, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were done as described previously (39). The nucleotide sequences of primers used in RT-qPCR are included in Supplementary Table 1 .

Left lungs were fixed in 10% neutral buffered formalin for 24 hrs. and embedded in paraffin. These formalin-fixed, paraffin-embedded left lung sections were used to perform RNA in situ localization of Egfr mRNA using BaseScope technology (ACD, Newark, CA). Briefly, the lung sections were baked at 60°C in a hybridization oven, deparaffinized in xylene and dehydrated in 100% ethanol followed by air-drying the slides for 5 min at 60°C. The slides were then incubated for 10 min at room temperature (RT) with 3% hydrogen peroxide (322335; ACD, Newark, CA) to quench the endogenous peroxidase activity. Target retrieval was performed using a 1X RNAscope Target Retrieval Reagent (322000; ACD, Newark, CA) at 98-102°C for 15 minutes, followed by three washes in distilled water. RNAscope Protease IV (322336; ACD, Newark, CA) was added to the lung sections and incubated at 40°C in a hybridization oven for 30 minutes followed by two washes of distilled water. Custom designed Egfr probe (1307511-C1; ACD, Newark, CA) equilibrated to room temperature was subsequently added to the lung sections and incubated in a hybridization oven for 2 hours at 40°C. After two washes with the wash buffer (310091, ACD, Newark, CA), the sections underwent eight amplification steps to enhance the signals. BaseScopeTM Detection Reagents v2– RED (Cat. No. 323910; ACD, Newark, CA) were used to detect the red signal. Finally, the sections were counterstained with 50% Gill’s Hematoxylin I for 2 min at RT followed by 3–5 times rinse with tap water. The slides were further washed 2–3 times in 0.02% Ammonia water, followed by 3-time wash in tap water. The slides were dehydrated at 60°C in a hybridization oven until the slides were completely dry, and coverslipped using VectaMount mounting media (H-5000; Vector Laboratories, Burlingame, CA). The stained slides were analyzed by a board-certified anatomic pathologist without prior knowledge of genotypes. The stained slides were captured under the 100X objective of the ECLIPSE Ci-L microscope with DS-Fi2 camera attachment (Nikon, Melville, NY).

One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine statistically significant differences among the groups. All data were presented as mean ± standard error of the mean (SEM). Grubbs’ test was used to remove the outliers. To reduce the number of horizontal bars indicating statistically significant differences between experimental groups, we used a single bar when one group showed significant differences compared to multiple other groups (with vertical ticks). A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software Inc., La Jolla, CA).

The airway epithelial cell-specific EGFR-deficient Tg+ (Cre⁺/Tg+) and control (Cre^-/Tg+, Cre⁺/WT, and Cre^-/WT) mice were generated ( Figure 1A ), and three-week-old juveniles (Postnatal day 21; PND21) were assessed for endpoints including postnatal survival, RT-qPCR for genes relevant to mucoinflammatory responses, mucus obstruction, MCM, immune cell recruitment, cytokine levels, and bacterial load. The deletion of EGFR in the airway epithelial cells was confirmed by BaseScope assay ( Figure 1B ).

The germline deletion of EGFR in mice results in mortality between midgestation and PND 20, depending on the genetic background of the mice (14). Therefore, to determine whether the airway-epithelial cell-specific EGFR deficiency contributes to the embryonic mortality, we analyzed the possibility of embryonic mortality by calculating the mendelian ratio of the expected genotypes of neonates, i.e., Cre^-/WT (n=80), Cre⁺/WT (n=72), Cre^-/Tg+ (n=83), and Cre⁺/Tg+ (n=66). The mendelian ratio for the Cre^-/WT, Cre⁺/WT, Cre^-/Tg+, and Cre⁺/Tg+ progeny was 1.06:0.96:1.10:0.88, respectively. The calculated χ2 value of 0.498 suggested that the observed mendelian ratio was not significantly deviated from the expected mendelian ratio of 1:1:1:1. These data suggest that airway epithelial cell-specific deletion of EGFR doesn’t compromise embryonic viability.

The Tg+ mice exhibit significant postnatal mortality within the first 3 weeks of life (11, 26, 27). To determine the effect of airway epithelial cell-specific EGFR deletion on postnatal survivability of the Tg+ mice, we observed pups until PND20. The survivability was comparable between Cre^-/WT and Cre⁺/WT pups ( Figure 1C ), suggesting that airway epithelial cell-specific EGFR deletion does not compromise postnatal survival in WT pups. Consistent with the previous studies (11, 26, 27, 39), Cre^-/Tg+ exhibited ~41.5% mortality between PND0-PND20, with most of the deaths observed between PND7-PND17 ( Figure 1C ). As compared with the Cre^-/Tg+ group, the Cre⁺/Tg+ mice showed significantly higher mortality rate of ~56.1% ( Figure 1C ). However, similar to the Cre^-/Tg+ group, most of the deaths in Cre⁺/Tg+ mice occurred between PND7-PND17. We also investigated the effect of airway epithelial cell-specific deletion of EGFR on postnatal distress in mice by recording their body weights at PND21. Although the Tg+ pups of both genotypes displayed lower body weight at PND21, the airway epithelial cell-specific deletion of EGFR did not alter the postnatal body weight in WT and Tg+ juveniles ( Figure 1D ). These data suggest that the EGFR deletion promotes mucus obstruction, a pathogenic pathway that contributes to the neonatal mortality in Tg+ mice (11, 26, 27).

The Tg+ mice predominantly exhibit type 2 inflammation characterized by increased levels of Th2 cytokines, i.e., IL-4 and IL-13, and elevated type 2 inflammation-associated gene signatures (27, 30, 32, 39). Indeed, type 2 inflammation-associated mucus obstruction is known to be the primary cause of postnatal mortality in Tg+ mice (11, 26, 27). Here, we hypothesized that the exaggerated type 2 inflammation in the Cre⁺/Tg+ mice contributes to excessive mucus obstruction, which results in their increased mortality.

First, we analyzed the expression levels of type 2 inflammation-associated gene signatures, including Slc26a4, Retnla, Chi3l4, and Clca1. The mRNA levels for Slc26a4, Retnla, Chi3l4, and Clca1 were comparable in Cre^-/WT and Cre⁺/WT mice ( Figures 2A–D ), which suggest that the EGFR deletion in the healthy airways does not induce type 2 inflammation. As reported previously (30, 32), the Slc26a4 mRNA levels trended higher in Cre^-/Tg+ mice as compared with Cre^-/WT and Cre⁺/WT mice ( Figure 2A ). The Slc26a4 mRNA levels were significantly higher in Cre⁺/Tg+ mice as compared with all the other three experimental groups ( Figure 2A ). The mRNA levels of Retnla and Chi3l4 were higher in Cre^-/Tg+ versus Cre^-/WT mice ( Figures 2B, C ). As compared with the Cre^-/Tg+ mice, the mRNA levels of Retnla, Chi3l4, and Clca1 were higher in Cre⁺/Tg+ mice, with only Retnla showing a significant increase ( Figures 2B–D ). Because IL4Rα, a common receptor subunit for IL-4 and IL-13, is known to cause mucous cell metaplasia in Tg+ mice (11), we assessed the levels of IL-4 and IL-13. The IL-4 protein levels were comparable in BALF from Cre^-/WT and Cre⁺/WT mice ( Figure 2E ). As reported previously (32, 40), Cre^-/Tg+ mice had elevated IL-4 levels as compared with their WT counterparts ( Figure 2E ). As compared with the Cre^-/Tg+ mice, the Cre⁺/Tg+ mice had insignificantly elevated (p =0.77) IL-4 levels ( Figure 2E ). The Il13 mRNA levels in the lung homogenates were comparable among Cre^-/WT and Cre⁺/WT mice and significantly higher in the Cre⁺/Tg+ group versus the other three experimental groups ( Figure 2F ). Consistent with the mRNA levels, the IL-13 protein levels showed higher trend in Cre⁺/Tg+ mice compared to Cre^-/Tg+ (p =0.06) ( Figure 2G ).

Airway mucous cell metaplasia (MCM) and mucus obstruction are consistent features of lung disease in Tg+ mice, where two gel-forming mucins, i.e., MUC5B and MUC5AC, primarily contribute to airway mucus obstruction (27). To investigate the effect of airway epithelial cell-specific EGFR deletion on MCM and mucus obstruction, we performed AB-PAS, MUC5B, and MUC5AC staining on the lung sections. While only a very small proportion of Cre^-/WT and Cre⁺/WT mice showed minimal airway mucus obstruction, Cre^-/Tg+ mice had a marked increase in the proportion of mucous cells (AB-PAS+, MUC5B+, and MUC5AC+) and the degree of airway mucus obstruction ( Figures 3A–G , Supplementary Figure 1 ). The extent of MCM, as indicated by the proportion of AB-PAS+ and MUC5B+ airway epithelial cells, and the degree of airway mucus obstruction, as indicated by AB-PAS- and MUC5B-stained airway luminal contents, were significantly higher in Cre⁺/Tg+ mice compared with Cre^-/Tg+ mice ( Figures 3A, B, D–F ). The proportion of MUC5AC+ airway epithelial cells was comparable between Cre⁺/Tg+ mice versus Cre^-/Tg+ mice ( Figures 3C, G ). These data suggest that airway epithelial cell-specific EGFR deficiency results in increased MUC5B production and mucus obstruction in Tg+ juveniles.

Increased total protein and dsDNA contents in the BALF are indicative of lung inflammation (32, 41, 42). Therefore, to determine the effect of airway epithelial cell-specific EGFR deletion on lung inflammation, we assessed total protein and dsDNA contents in the BALF. BALF total protein and dsDNA contents were comparable among Cre^-/WT and Cre⁺/WT mice ( Figures 4A, B ). Cre^-/Tg+ mice, on the other hand, had significantly elevated BALF total protein and an increasing trend of dsDNA contents, as compared with Cre^-/WT and Cre⁺/WT mice ( Figures 4A, B ). Deletion of EGFR in the airway epithelial cells significantly increased the BALF total protein and dsDNA contents in Cre⁺/Tg+ mice compared with all other experimental groups ( Figures 4A, B ), suggesting that airway epithelial cell-specific deletion of EGFR contributes to exaggerated lung inflammation in Tg+ juveniles.

To determine whether the increase in the BALF protein levels in Cre⁺/Tg+ mice is caused by epithelial barrier dysfunction, we assessed the mRNA levels of key apical junction complex proteins that are critical for epithelial barrier function. The mRNA levels of genes encoding adherens junction (AJ) proteins, i.e., E-Cadherin (Cdh1), were significantly reduced in Cre⁺/Tg+ mice as compared with Cre^-/Tg+ mice ( Supplementary Figure 2A ). The mRNA levels of genes encoding tight junction (TJ) proteins, including Claudin 5 (Cldn5), Occludin (Ocln), and ZO1 (Tjp1), adherens junction (AJ) proteins, i.e., Beta-catenin (Ctnnb1), and gap junction protein, i.e., Connexin 43 (Cx43) trended lower in Cre⁺/Tg+ mice as compared with Cre^-/Tg+ mice ( Supplementary Figures 2B–F ). Consistent with the reduced Cdh1 mRNA levels, the immunohistochemical staining of E -Cadherin showed reduced staining intensity in Cre⁺/Tg+ mice as compared with Cre^-/Tg+ mice ( Supplementary Figure 2G ).

Next, we determined the effect of airway epithelial cell-specific EGFR deletion on immune cell recruitment into the airspaces by performing immune cell analyses in the BALF from all four experimental groups. Cre^-/WT and Cre⁺/WT mice had comparable total immune cells in the BALF ( Figures 5A, D ). The proportion of four types of immune cells, i.e., macrophages, neutrophils, eosinophils, and lymphocytes, were also comparable among Cre^-/WT and Cre⁺/WT mice ( Figures 5B–D ). These data suggest that the airway epithelial cell-specific deletion of EGFR doesn’t alter immune cell composition in the airspaces of WT mice. Consistent with previous reports (26, 27, 32, 40), as compared with the Cre^-/WT and Cre⁺/WT mice, the total cell counts were significantly increased in Cre^-/Tg+ mice, which was mainly attributable to increase in macrophages, neutrophils, eosinophils, and lymphocytes ( Figures 5A–D ). The total cell counts were significantly increased in Cre⁺/Tg+ as compared with Cre^-/Tg+ mice ( Figures 5A–D ), which was attributable to the increased numbers of macrophages and neutrophils ( Figures 5B–D ). These data suggest that airway epithelial cell-specific deficiency of EGFR enhances neutrophil and macrophage recruitment into the airspaces of Tg+ mice.

Next, we assessed the effect of airway epithelial cell-specific EGFR deletion on inflammatory mediators released into the lung airspaces by determining BALF cytokines and chemokines levels. The levels of primary neutrophil-specific chemokines, including KC/CXCL1, G-CSF, and MIP-2/CXCL2, were comparable among Cre^-/WT and Cre⁺/WT mice ( Figures 6A–C ). Consistent with the increased neutrophil counts in Cre^-/Tg+ mice, KC/CXCL1, G-CSF, and MIP-2/CXCL2 levels trended higher in Cre^-/Tg+ compared with Cre^-/WT and Cre⁺/WT mice ( Figures 6A–C ). Mirroring the additional increase in neutrophil counts in Cre⁺/Tg+ mice compared with Cre^-/Tg+ mice, KC/CXCL1, G-CSF, and MIP-2/CXCL2 levels were significantly increased in Cre⁺/Tg+ mice compared with Cre^-/Tg+ mice ( Figures 6A–C ). Consistent with the increased macrophage counts, the levels of MIP-1α/CCL3 and MIP-1β/CCL4, the chemokines reported to drive macrophage/monocyte recruitment (43–45), were significantly increased in Cre⁺/Tg+ mice compared with all other three experimental groups ( Figures 6D, E ). Other inflammatory mediators, such as TNF-α, showed significantly higher expression levels in Cre⁺/Tg+ mice compared with all other experimental groups ( Figure 6F ), and IL-6 showed significantly higher expression levels in Cre⁺/Tg+ mice compared with WT groups and a higher trend in Cre⁺/Tg+ mice than Cre^-/Tg+ mice ( Figure 6G ). We also assessed the levels of IL-5, a type 2 cytokine that plays a key role in the proliferation, maturation, and differentiation of eosinophils (46, 47). IL-5 levels were significantly higher in Cre^-/Tg+ mice and trended higher in Cre⁺/Tg+ mice versus their WT counterparts ( Figure 6H ). Consistent with the comparable eosinophil counts, IL-5 levels did not differ significantly between Cre^-/Tg+ and Cre⁺/Tg+ mice ( Figure 6H ). These data suggest that the airway epithelial cell-specific deletion of EGFR promotes a pro-inflammatory microenvironment in Tg+ airways that dictates the recruitment of inflammatory immune cells into the airspaces.

Spontaneous bacterial infections due to a defect in the mucociliary clearance resulting from mucostasis are a consistent feature of Tg+ lung disease (29, 31, 32, 39, 40, 48). These bacterial infections are, however, reported to be cleared in the early adulthood (29). To determine the effect of airway epithelial cell-specific EGFR deletion on the clearance of spontaneous bacterial infections, we estimated airspace bacterial burden by determining the colony-forming unit (CFU) counts in BALF at PND21. The BALF collected from Cre^-/WT and Cre⁺/WT mice were devoid of bacterial colonies ( Figure 7 ). Approximately 50% (6 out of 12) of Cre^-/Tg+ mice still had lower bacterial counts (mean CFU= ~108/ml) ( Figure 7 ). In contrast, 91.6% (11 out of 12 mice) of Cre⁺/Tg+ mice showed bacterial burden (mean CFU= ~3907/ml), which was significantly higher as compared with Cre^-/Tg+ mice ( Figure 7 ). These data suggest that the airway epithelial cell-specific EGFR deletion delays bacterial clearance in Tg+ mice.