Prenatal smoke exposure dysregulates lung epithelial cell differentiation in mouse offspring: role for AREG-induced EGFR signaling.

One-day-old C57Bl/6 mouse offspring of smoke- or air-exposed dams

C57Bl/6 neonates euthanized one day after birth. n=16 offspring from nonsmoking mothers (n=3 dams), n=24 from smoking mothers (n=4 dams). Dams were 8-10 wk old at the start of exposure. Offspring themselves were never directly exposed. The left lung was used for RNA isolation (RT-qPCR) and the right lung was formalin-fixed and paraffin-embedded for histology.

Three-dimensional mouse bronchiolar lung organoid

Three-dimensional mouse alveolar lung organoid

Ciliated cell number in neonatal lung - prenatal air vs prenatal smoke

Tubulin-positive (ciliated) cells counted manually in all airways of 1-day-old offspring and corrected per millimetre of airway (IHC, DAB). Prenatally smoke-exposed neonates had lower numbers of ciliated cells than controls (P<0.001), seen in both male and female offspring (P<0.0001). The paper reports these counts only as individual data points in Fig. 1B/1b, with no numeric mean, so no value is recorded here.

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Evidence

Ciliated cell number in bronchiolar lung organoids - non-treated vs AREG

Tubulin-positive cells counted in whole bronchiolar mouse lung organoids after 14 days (triple IF for tubulin, CC10 and KRT5), corrected for the total number of bronchial epithelial cells. AREG-treated organoids had lower numbers of tubulin-positive cells than non-treated organoids (P<0.001, Fig. 5D). This arm uses organoids derived from unexposed adult mice and tests AREG directly, not smoke. Counts are reported only as individual data points, so no value is recorded.

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Evidence

Foxj1 mRNA in neonatal lung - prenatal air vs prenatal smoke

Foxj1 mRNA measured by TaqMan RT-qPCR in RNA isolated from the left lung of 1-day-old offspring, normalized to Gapdh (2^-dCp). Lower Foxj1 expression in prenatally smoke-exposed offspring, especially in males (P=0.03 overall; male P=0.04; Fig. 1E/1e). Relative expression is plotted as individual values only, so no numeric level is recorded.

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Evidence

Spdef mRNA in neonatal lung - prenatal air vs prenatal smoke

Spdef mRNA by TaqMan RT-qPCR on left lung RNA, normalized to Gapdh. Higher in prenatally smoke-exposed offspring (P<0.001 overall; male P<0.001; Fig. 1G/1g). SPDEF is the master transcription factor for goblet cell differentiation in the airway epithelium. Individual values only; no numeric level recorded.

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Evidence

Foxm1 mRNA in neonatal lung - prenatal air vs prenatal smoke

Foxm1 mRNA by TaqMan RT-qPCR on left lung RNA, normalized to Gapdh. Higher in prenatally smoke-exposed offspring (P=0.014 overall; male P=0.01, female P=0.04; Fig. 1F/1f). FOXM1 promotes SPDEF activity. Individual values only; no numeric level recorded.

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Evidence

Pdpn mRNA in neonatal lung - prenatal air vs prenatal smoke

Pdpn (AECI marker) mRNA by TaqMan RT-qPCR on left lung RNA, normalized to Gapdh. Lower in prenatally smoke-exposed offspring (P<0.0001 overall; male P=0.04, female P=0.006; Fig. 3C/3c). Notably, the number of PDPN-positive AECI cells itself was NOT changed (Fig. 3B/3b), so the deficit is at the transcript level. Individual values only; no numeric level recorded.

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Evidence

Gramd2 mRNA in neonatal lung - prenatal air vs prenatal smoke

Gramd2 (AECI marker) mRNA by TaqMan RT-qPCR on left lung RNA, normalized to Gapdh. Lower in prenatally smoke-exposed offspring (P<0.0001 overall; both sexes P<0.001; Fig. 3E/3e). Aqp5 mRNA, another alveolar marker measured in the same panel, was not affected. Individual values only; no numeric level recorded.

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Evidence

pro-SPC-positive AECII cell number in neonatal lung - prenatal air vs prenatal smoke

pro-SPC-positive (AECII) cells quantified morphometrically by IHC and corrected for the total lung tissue area per section. Higher in prenatally smoke-exposed offspring in both sexes (P=0.001 overall; both sexes P<0.05; Fig. 2B/2b). Double staining with Ki67 showed more proliferating (Ki67/SPC-positive) AECII in smoke-exposed neonates (P=0.045), read from the local full text only. Individual values only; no numeric count recorded.

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Evidence

NKX2.1-positive AECII cell number in neonatal lung - prenatal air vs prenatal smoke

NKX2.1-positive (AECII) cells quantified morphometrically by IHC and corrected for the total lung tissue area per section. Higher in prenatally smoke-exposed offspring (P=0.0005 overall; male P=0.05, female P=0.006; Fig. 2D/2d). This is the second, independent AECII marker supporting the same finding. Individual values only; no numeric count recorded.

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Evidence

pro-SPC-positive AECII cell number in alveolar lung organoids - non-treated vs AREG

pro-SPC-positive (AECII) cells counted manually in alveolar mouse lung organoids after 14 days (double IF with PDPN) and corrected for the total number of alveolar epithelial cells. More pro-SPC-positive cells in AREG-treated than in non-treated alveolar organoids (P=0.03, Fig. 5G); PDPN-positive AECI numbers were unaffected (Fig. 5H). Individual values only; no numeric count recorded.

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Evidence

KRT5-positive basal cell number in bronchiolar lung organoids - non-treated vs AREG

KRT5-positive (basal) cells counted in whole bronchiolar mouse lung organoids after 14 days (triple IF with tubulin and CC10) and corrected for the total number of bronchial epithelial cells. Higher numbers of KRT5-positive cells in AREG-treated than in non-treated organoids (P=0.02, Fig. 5C); CC10-positive club cell numbers were unaffected (Fig. 5E). Individual values only; no numeric count recorded.

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Evidence

AREG expression in neonatal lung - prenatal air vs prenatal smoke

Two readouts of the EGFR ligand amphiregulin in 1-day-old offspring: (1) AREG-expressing cells by IHC, found mainly in the lung parenchyma, higher in prenatally smoke-exposed offspring (P=0.04 overall, most pronounced in females P=0.012; Fig. 4B/4b); (2) Areg mRNA by TaqMan RT-qPCR on left lung RNA, higher in smoke-exposed offspring in both sexes (P=0.0014 overall; both sexes P<0.05; Fig. 4C/4c). Egfr mRNA itself was NOT changed by prenatal smoke exposure (Fig. 4E/4e), so the paper's inference of increased EGFR signalling rests on ligand availability (more AREG, less EGF) rather than on any direct measure of EGFR phosphorylation, which was not assayed. Individual values only; no numeric level recorded.

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Evidence

Egf mRNA in neonatal lung - prenatal air vs prenatal smoke

Egf mRNA by TaqMan RT-qPCR on left lung RNA of 1-day-old offspring, normalized to Gapdh. Lower in prenatally smoke-exposed offspring (P=0.0018 overall; male P=0.02; Fig. 4D/4d). EGF has a higher affinity for EGFR than AREG and is associated with epithelial cell differentiation, so the paper reads lower Egf alongside higher Areg as a shift in which ligand drives EGFR. Individual values only; no numeric level recorded.

Exposure conditions

Outputs

Evidence

Ciliated cell number, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P<0.001 for the pooled comparison (and P<0.0001 in each sex separately) without an exact value; the tabulated p_value 0.001 is therefore the stated upper bound, not an exact figure. No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Foxj1 mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P=0.03 for the pooled comparison (male P=0.04). No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Spdef mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P<0.001 without an exact value; the tabulated p_value 0.001 is the stated upper bound. No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Foxm1 mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P=0.014 for the pooled comparison (male P=0.01, female P=0.04). No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Pdpn mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P<0.0001 without an exact value; the tabulated p_value 0.0001 is the stated upper bound. No magnitude of change is reported. The number of PDPN+ AECI cells was unchanged.

Statistical test: two-tailed Mann-Whitney U test

Gramd2 mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P<0.0001 without an exact value; the tabulated p_value 0.0001 is the stated upper bound. No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

pro-SPC+ AECII number, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P=0.001 for the pooled comparison (both sexes P<0.05). No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

NKX2.1+ AECII number, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P=0.0005 for the pooled comparison (male P=0.05, female P=0.006). No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Areg mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P=0.0014 for Areg mRNA in the pooled comparison (both sexes P<0.05); the number of AREG-expressing cells by IHC was higher at P=0.04 (female P=0.012). No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Egf mRNA, prenatal smoke vs prenatal air (neonatal lung)

Paper reports P=0.0018 for the pooled comparison (male P=0.02). No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

Tubulin+ ciliated cell number, AREG vs non-treated (bronchiolar organoids)

Paper reports P<0.001 without an exact value; the tabulated p_value 0.001 is the stated upper bound. No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

KRT5+ basal cell number, AREG vs non-treated (bronchiolar organoids)

Paper reports P=0.02. No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

pro-SPC+ AECII number, AREG vs non-treated (alveolar organoids)

Paper reports P=0.03. No magnitude of change is reported.

Statistical test: two-tailed Mann-Whitney U test

EGFR activation leads to altered ciliogenesis

AREG treatment of bronchiolar lung organoids reduced tubulin-positive ciliated cell numbers, reproducing the ciliated cell loss and Foxj1 downregulation seen in prenatally smoke-exposed neonates that had higher Areg and lower Egf expression.

EGFR activation leads to basal cell hyperplasia

AREG treatment of bronchiolar lung organoids increased KRT5-positive basal cell numbers (P=0.02), applying the EGFR ligand directly and so establishing the link on this paper's own evidence.

EGFR activation leads to alveolar epithelial type II cell hyperplasia

AREG treatment of alveolar lung organoids increased pro-SPC-positive AECII numbers (P=0.03), reproducing the higher AECII numbers found in prenatally smoke-exposed neonates.

EGFR activation leads to mucus secretory program activation

Prenatally smoke-exposed neonates with higher Areg and lower Egf expression showed higher expression of the mucus-related transcription factors Spdef and Foxm1. The organoid arm did not test the secretory program directly, so this link rests on the neonatal co-occurrence rather than on an AREG intervention.

EGFR activation leads to impaired alveolar epithelial type I cell differentiation

Prenatally smoke-exposed neonates with higher Areg and lower Egf expression showed lower Pdpn and Gramd2 mRNA, although PDPN-positive AECI cell numbers were unchanged and AREG treatment of alveolar organoids did not change PDPN-positive cell numbers either. The link is therefore transcript-level only and untested by intervention.

Maternal cigarette smoke exposure (prenatal window)

Mainstream cigarette smoke generated with a TE-10 smoke exposure system (Teague Enterprises, Woodland, CA) using Kentucky 2R4F research-reference filtered cigarettes. Two sessions of 50 min per day with a 3-h interval between exposures; smoke of 10 cigarettes generated per session. Adaptation: 3 cigarettes/session on day 1, 5 on day 2, 7 on day 3, 10 from day 4 onward. Per session this generated total particulate matter of 211 +/- 30 mg/m3 and a CO level of 205 +/- 35 parts per million (max 241 ppm). Dams were exposed from 7 days before mating until the day of euthanization, constant throughout pregnancy; mothers and offspring were not exposed after birth. Simultaneous cycling induced with 1.25 IU pregnant mare's serum gonadotropin and 1.25 IU human chorionic gonadotrophin; mating confirmed by vaginal plug detection. Approved by the IACUC of the University of Groningen (Permit No. 6589a).

Equipment: TE-10 smoke exposure system (Teague Enterprises, Woodland, CA); Kentucky 2R4F research-reference filtered cigarettes (The Tobacco Research Institute, University of Kentucky)

Immunohistochemistry on neonatal lung tissue

3 um sections of formalin-fixed, paraffin-embedded right lung. Deparaffinized, antigen retrieval in citrate (pH 6.0) and Tris-HCl (pH 7.4) buffer in a microwave oven for 15 min, then 0.3% H2O2 in PBS for 30 min. Primary antibodies against tubulin (ciliated cells), CC10 (club cells), pro-SPC and NKX2.1 (AECII), PDPN (AECI), Ki67 (proliferation) and amphiregulin. Detection with HRP enzyme and biotinylated secondary antibodies (30 min), colour reaction with 0.05% diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC). Slides were coded and examiners blinded to groups. Tubulin+ and CC10+ cells counted manually in all airways and corrected per millimetre of airway; pro-SPC+, NKX2.1+, AREG+, PDPN+ and pro-SPC/Ki67+ cells quantified morphometrically and corrected for the total lung tissue area per section.

Equipment: Aperio-ImageScope viewing software (Aperio 11.2.0.780, Vista, CA); TissueFAXS imaging system (TissueGnostics, Austria); ImageJ

TaqMan RT-qPCR on neonatal lung tissue

Total RNA isolated from left lung with an RNA isolation TRIzol Kit (Invitrogen); cDNA reverse transcribed with SuperScript-II Reverse Transcriptase (Invitrogen). TaqMan on-demand gene expression assays (Applied Biosystems) for Gapdh (Mm99999915_g1), Foxj1 (Mm01267279_m1), Scgb1a1 (Mm00442046_m1), Foxm1 (Mm00514924_m1), Spdef (Mm00600221_m1), Pdpn (Mm00447558_m1), Aqp5 (Mm00437578_m1), Gramd2 (Mm01174485_m1), Nkx2.1, Egf (Mm00438696_m1), Areg (Mm01354339_m1) and Egfr (Mm01187858_m1). Reactions in triplicate, 10 uL total (2 uL MilliQ water, 5 uL PCR MasterMix (Eurogentec), 0.5 uL assay mix, 2.5 uL cDNA), run on a LightCycler 480 Real-Time PCR System (Roche) and analysed with LightCycler 480 SW 1.5 software using the fit points method. Normalized to Gapdh by 2^-dCp; undetectable Cp values (>40) were interpreted as the maximum Cp value.

Equipment: LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland); TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA); TRIzol RNA isolation kit (Invitrogen)

Three-dimensional mouse lung organoid culture with AREG treatment

EpCAM+ cells isolated from lungs of pathogen-free male (n=3) and female (n=2) mice aged 8-12 wk. Approximately 10,000 EpCAM+ lung cells seeded with an equal number of fibroblasts per insert in 100 uL growth factor-reduced Matrigel diluted 1:1 with DMEM/F12 containing 5% FBS, into transwell inserts for 24 wells. In the treated groups AREG (10 ng/mL; R&D Systems, Minneapolis) was freshly added every 2 days. Cultures maintained at 37C in a humidified incubator with 5% CO2. After 14 days organoids were fixed in acetone diluted 1:1 (vol/vol) with methanol for 15 min at -20C. For bronchial analyses organoids were stabilized in 2% agarose, dehydrated and paraffin-embedded for cross-sectional histology.

Equipment: Growth factor-reduced Matrigel; Transwell inserts for 24 wells; Recombinant AREG (R&D Systems, Minneapolis)

Immunofluorescence on lung organoids

Paraffin-embedded bronchiolar organoids double- or triple-stained with primary antibodies against KRT5 (basal cell), CC10 (club cell) and tubulin (ciliated cell), visualized with fluorescent secondary antibodies and DAPI-containing mounting medium (Abcam). Alveolar organoid cultures washed with PBS, blocked in PBS with 5% (wt/vol) BSA, incubated overnight at 4C with primary antibodies against pro-SPC (AECII) and PDPN (AECI) diluted in PBS with 0.1% BSA and 0.1% Triton, washed 30 min and detected with fluorescent secondary antibodies and DAPI. Slides scanned on the TissueFAXS imaging system; images acquired on a Leica SP8 confocal microscope and a Leica 4000b microscope at x40 magnification. Positive cells counted manually and corrected for the total number of bronchial and alveolar epithelial cells in the organoids.

Equipment: Leica SP8 confocal microscope; Leica 4000b microscope; TissueFAXS imaging system (TissueGnostics, Austria); ImageJ