Maternal and perinatal obesity induce bronchial obstruction and pulmonary hypertension via IL-6-FoxO1-axis in later life

Abstract

Obesity is a pre-disposing condition for chronic obstructive pulmonary disease, asthma, and pulmonary arterial hypertension. Accumulating evidence suggests that metabolic influences during development can determine chronic lung diseases (CLD). We demonstrate that maternal obesity causes early metabolic disorder in the offspring. Here, interleukin-6 induced bronchial and microvascular smooth muscle cell (SMC) hyperproliferation and increased airway and pulmonary vascular resistance. The key anti-proliferative transcription factor FoxO1 was inactivated via nuclear exclusion. These findings were confirmed using primary SMC treated with interleukin-6 and pharmacological FoxO1 inhibition as well as genetic FoxO1 ablation and constitutive activation. In vivo, we reproduced the structural and functional alterations in offspring of obese dams via the SMC-specific ablation of FoxO1. The reconstitution of FoxO1 using IL-6-deficient mice and pharmacological treatment did not protect against metabolic disorder but prevented SMC hyperproliferation. In human observational studies, childhood obesity was associated with reduced forced expiratory volume in 1 s/forced vital capacity ratio Z-score (used as proxy for lung function) and asthma. We conclude that the interleukin-6-FoxO1 pathway in SMC is a molecular mechanism by which perinatal obesity programs the bronchial and vascular structure and function, thereby driving CLD development. Thus, FoxO1 reconstitution provides a potential therapeutic option for preventing this metabolic programming of CLD.

Fig. 1. A maternal prenatal and perinatal high-fat diet (HFD) and metabolic disorder induce transient early-onset obesity, elevated adipocytokine levels, and impaired glucose tolerance in offspring.

A Animal model of metabolic programming. Female mice were fed a HFD for 7 weeks prior to mating; control mice received a standard diet (SD). The dams remained on their respective diets throughout gestation and lactation. The offspring of both groups were weaned at postnatal day 21 (P21) and fed SD until P70. B–D Body weight (B), body weight gain (C), and intraperitoneal glucose tolerance test (ipGTT) results (D) of dams prior to mating. E The body weight, ratio of epigonadal white adipose tissue (WAT) relative to body weight and mRNA expression of Il6 (interleukin-6) and Lep (leptin) in the WAT of offspring at P21 using qRT-PCR; HFD relative to SD. F Serum IL-6 and leptin concentrations in the offspring were assessed by ELISA at P21; offspring were subjected to the ipGTT. G The body weight, ratio of WAT relative to the body weight and expression of Il6 and Lep in the WAT of offspring at P70 using qRT-PCR; HFD relative to SD. H Serum IL-6 and leptin concentrations in the offspring at P70 using ELISA. Offspring were subjected to the ipGTT at P70. Data are shown as mean ± standard error of the mean B: SD n = 32, HFD n = 40; C SD n = 32, HFD n = 40; D SD n = 36, HFD n = 49; E SD n = 9–25, HFD n = 7–27; F SD n = 5–30, HFD n = 5–7; G SD n = 10–68, HFD n = 7–31; H SD n = 6–21, HFD n = 3–7. Statistical analyses were performed using the two-sided Mann–Whitney test, two-sided Student’s t test or two-way ANOVA followed by the Bonferroni post-test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray = standard diet; white = high-fat diet. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 2. A maternal high-fat diet (HFD) is associated with thickening of the bronchial and vascular smooth muscle cell (SMC) layer as well as bronchial obstruction and pulmonary arterial hypertension beyond infancy.

A Representative images of the lungs of offspring at postnatal day 70 (P70). Immunohistochemistry for α-smooth muscle actin (αSMA) was performed to assess the bronchial SMC layer in the offspring of dams fed a HFD or standard diet (SD). B Quantitative measurement of the bronchial SMC layer in bronchi with diameters <150 µm and 150–200 µm. C Assessment of respiratory airway resistance using body plethysmography at P70. D–F Representative images of the lungs of offspring at P70. Dual staining for αSMA and von Willebrand factor (vWF) (D) was used to assess the muscularization of microvessels (20–100 µm) (E) and the medial wall thickness of microvessels (diameters: 20–70 µm and 70–150 µm) (F). G–K Echocardiographic assessment of heart rate (G), cardiac output (CO, H), stroke volume (SV, I), right ventricular internal diameter (RVID, J) and tricuspid annular plane systolic excursion (TAPSE, K). L, M Hemodynamic measurement of right ventricular systolic pressure (RVSP, L), and total pulmonary vascular resistance index (TPVRI, M). N Echocardiographic assessment of the left ventricular (LV) ejection fraction (EF). O, P Measurement of the systolic blood pressure (SBP) and diastolic blood pressure using invasive catheterization. Q qRT-PCR assessment of Nppa expression in the heart at P70. R Immunoblot analysis of αSMA protein in total lung homogenates at P21; β-actin was used as a loading control. A summary of the αSMA abundance relative to β-actin is displayed next to the immunoblot. S qRT-PCR assessment of Acta2 expression in laser-microdissected bronchi and vessels at P21; 18 s rRNA served as a housekeeping gene. Data are shown as mean ± standard error of the mean. B, C, E–S SD n = 4–17, HFD n = 6–9. Statistical analyses were performed using the two-sided Mann–Whitney test or two-sided Student’s t test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray = standard diet; white = high-fat diet. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 3. Identification of FoxO1-STAT3 signaling in bronchi, microvessels, and adjacent lung tissue from offspring at postnatal day 21 (P21).

A The heatmap from a transcriptomic analysis includes only genes with an absolute log-fold change (FC) > 2 (adjusted P value <0.05); standard diet (SD) and high-fat diet (HFD) represent the offspring of standard diet (SD)-fed dams (n = 2) and HFD-fed dams (n = 3), respectively. Z-Score is indicated in a color score. B Volcano plot analysis; genes with an absolute FC > 2 and adjusted P value <0.05 are indicated in red; genes with an FC > 2 but adjusted P value >0.05 are green (DEGs in SD compared to HFD). Labels in the volcano plot indicate cell cycle and FoxO1 target genes. P values are calculated using Wald test and P-adjusted values using the FDR/Benjamini–Hochberg approach. C, D Venn diagrams depicting the putative binding sites for the transcription factors Foxo1, STAT3, Jun, Nfe2l2, and Pparγ in the promoters of the top 100 upregulated and downregulated genes with adjusted P values <0.01. E Gene expression of Stat3, Socs3, and Jun in bronchial smooth muscle cells (bSMCs) isolated from offspring of SD- and HFD-fed dams at P21 (n = 4/group); Gapdh served as the housekeeping gene. F immunoblot for c-Jun and total STAT3 in total lung homogenates from HFD and SD at P21 (n = 6/group); a densitometric summary of the STAT3 and c-Jun data relative to β-actin is displayed next to the respective immunoblot. G A functional enrichment analysis to identify the upregulated biological processes (SD vs HFD) associated with differentially expressed genes (DEGs) with adjusted P values <0.05. A KEGG pathway analysis was used to identify the top ten upregulated pathways in the bronchi (SD vs HFD), microvessels, and adjacent lung tissue at P21. E, F Data are shown as mean ± standard error of the mean. The statistical analysis was conducted using the two-sided Mann–Whitney test and two-sided Student’s t test; *P < 0.05; ***P < 0.001. Gray = standard diet; white = high-fat diet. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 4. A maternal high-fat diet (HFD) induces proliferative STAT3 and AKT signaling and inactivates FoxO1 in bronchial and vascular smooth muscle cells (SMC).

A–D Representative dual immunofluorescent stainings for phosphorylated STAT3 (pSTAT3) or phosphorylated AKT (pAKT) and α-smooth muscle actin (αSMA) of lung sections (bronchi and microvessels) from offspring of HFD- or standard diet (SD)-fed dams at postnatal day 21 (P21) (A, C). The amount of pSTAT3 and pAKT was quantified and is displayed as the intensity of pSTAT3 or pAKT per αSMA-positive cell (B, D). E, F Representative images showing a dual-staining for αSMA and Ki67 (proliferation marker) of the bronchi (E) and microvessels (F) of offspring at P21; quantification of Ki67-positive nuclei relative to total nuclei among αSMA-positive cells in the bronchi (diameter: 150–250 µm) and microvessels. G–I Representative co-immunofluorescence staining to detect αSMA (red), FoxO1 (green), and DAPI (blue, nuclear staining) (G). Quantitative assessment of the nuclear and cytoplasmatic FoxO1 intensities (H) and the nuclear and cytoplasmic FoxO1 levels relative to total FoxO1 in αSMA-positive bronchial cells (I). J–L Representative co-immunofluorescence staining of αSMA (red), FoxO1 (green), and DAPI (blue) (J). Quantification of the nuclear and cytoplasmatic FoxO1 intensities (K), and nuclear and cytoplasmatic FoxO1 relative to total FoxO1 in αSMA-positive microvascular SMCs (L). M, N qRT-PCR for gene expression in laser-microdissected bronchi and vessels: Foxo1, Foxo3, Foxo4, and Fox6 mRNA (M) as well as genes regulated by FoxO1: Ccnb1, Cdkn1b, Bcl6, and Faslg mRNA (N). O, P qRT-PCR assessment of Foxo1, Foxo3, Foxo4, Fox6, Cdkn1b, Bcl6, and Gadd45a mRNA expression in primary bronchial SMCs from HFD and SD offspring (bSMC^HFD and bSMC^SD); SD (control) is set to 1. Data are shown as mean ± standard error of the mean; B, D, E, F SD n = 4–6, HFD n = 5–6; H, I, K–P SD n = 4–5 HFD n = 4–7. The statistical analysis was conducted using the two-sided Mann–Whitney test or two-sided Student’s t test; *P < 0.05; **P < 0.01. Gray = standard diet; white = high-fat diet. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 5. Interleukin (IL)−6-STAT3-AKT induces the proliferation of bronchial smooth muscle cells (SMC) via FoxO1.

A Immunoblot showing phosphorylated STAT3 (pSTAT3), and total STAT3 in primary murine bronchial smooth muscle cells (bSMC) from wild-type mice (WT). B Nuclear and cytoplasmatic phosphorylated FoxO1 (pFoxO1) fraction in % of total cell pFoxO1 per bSMC; pFoxO1 (green) and DAPI (blue, nucleus). C Nuclear and cytoplasmatic total FoxO1 fraction in % of total cell FoxO1 per bSMC. D Total FoxO1 in WT-bSMC. E Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 and total nuclear FoxO1 per cell in WT-bSMC. F Proliferation (cell count) or viability (MTT assay) in WT-bSMC. G Viability (MTT assay), proliferation (BrdU), and apoptosis (active caspase3/7 assay) in WT-bSMC. H Immunoblot showing phosphorylated AKT (pAKT), and total AKT in WT-bSMC. I Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per WT-bSMC. J Immunoblots showing pSTAT3, total STAT3, pAKT, and total AKT in WT-bSMC. K Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per WT-bSMC. L, M Nuclear and cytoplasmatic total FoxO1 fraction in % per bSMC from mice with inducible activation of FoxO1 (FoxO1^ADA mice). N Immunoblot of WT-bSMC and IL-6⁻/⁻bSMC showing pSTAT3 and total STAT3. O Nuclear and cytoplasmatic total FoxO1 fraction in % of total cell FoxO1 per bSMC. P Viability (MTT assay) and proliferation (BrdU). Q–S immunoblot for pSTAT3 and STAT3 in WT-bSMC (Q). Viability (MTT assay), proliferation (cell count), and pSTAT3 per cell using immunocytochemistry in bSMC from mice with a specific ablation of FoxO1 in SMCs (SMC^{FoxO1 KO}) and WT mice (R, S). For immunoblots, β-actin was used as a loading control; densitometric summary is displayed. Interleukin-6 and soluble IL-6 receptor (IL-6 + R), Stattic (STAT3 inhibitor), insulin, AKT Inhibitor (AKT-Inh.), Paclitaxel, and FoxO1 Inhibitor (FoxO1^Inh). All bSMC are isolated from 21-day-old mice. Data are shown as mean ± standard error of the mean. A–S n = 3–9 biological independent samples or experiments per group; (repeated measure) one-way ANOVA followed by the Bonferroni post-test, two-sided Mann–Whitney test and two-sided Student’s; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray = vehicle; white = stimulation; orange=Foxo1;^ADA blue=IL-6^−/−. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 6. Interleukin (IL)−6 regulates the proliferation of human bronchial smooth muscle cells (hbSMC) via FoxO1.

For the experiments with STAT3-inhibitor (Stattic) and AKT inhibitor (AKT-Inh.), hbSMC were pre-exposed to either Stattic, AKT-Inh. or vehicle for 2 h. Subsequently, the hbSMC were stimulated as indicated. A Immunoblot showing phosphorylated STAT3 (pSTAT3) in hbSMC after exposure to IL-6 with soluble IL-6 receptor (IL-6 + R) or vehicle for 30 min. B Immunoblot showing pAKT and total AKT in hbSMC after exposure to vehicle, IL-6 + R, or Stattic for 30 min. β-actin was used as a loading control. A densitometric summary is displayed. C Nuclear and cytoplasmatic phosphorylated FoxO1 (pFoxO1) fraction in % of total cell pFoxO1 per hbSMC using immunofluorescent staining; hbSMC were treated with IL-6 + R alone or combined with AKT-Inh. or vehicle for 30 min. D Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per hbSMC using immunofluorescent staining; hbSMC were treated with insulin alone or combined with AKT-Inh. or Stattic. E Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per hbSMC using immunofluorescent staining; hbSMC were treated with IL-6 + R or insulin alone or both in combination with AKT-Inh. or Stattic for 30 min. F Viability (MTT assay) of hbSMC after exposure to vehicle, IL-6 + R, Paclitaxel and/or Stattic. G–I hbSMCs were exposed to IL-6 + R or vehicle with or without Paclitaxel for 30 min (G) or 24 h (H, I). Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per bSMC (G); total FoxO1 per hbSMC (H), and proliferation (BrdU) of hbSMC (I). Data are shown as mean ± standard error of the mean. A–I n = 3–5 biological independent samples or experiments per group; (repeated measure) one-way ANOVA followed by the Bonferroni post-test or the two-sided Mann–Whitney test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Gray = vehicle; white = stimulation; orange = Paclitaxel; blue = IL-6. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 7. Offspring of Interleukin (IL)−6-deficient dams fed a perinatal high-fat diet (HFD) are protected from structural and functional lung changes at postnatal day 70 (P70) despite early-onset obesity and impaired glucose tolerance.

A Body weight and ratio of white adipose tissue (WAT) relative to body weight of the offspring of HFD- and standard diet (SD)-fed dams at P21 and P70. B Serum insulin and leptin concentrations at P21 using ELISA. C Offspring of HFD- and SD-fed dams were subjected to an intraperitoneal glucose tolerance test (ipGTT) at P21. D–F Representative images of lungs from offspring at P70. Tissues were stained for α-smooth muscle actin (αSMA) to assess the bronchial SMC layer (D). Quantitative measurement of the bronchial SMC (bSMC) layer in bronchi with a diameter of <150 µm and 150–200 µm (E). Assessment of respiratory airway resistance using body plethysmography at P70 (F). G–I Representative images of lungs from offspring at P70. Tissues were co-stained for αSMA and von Willebrand factor (vWF) (G) to assess the muscularization (H) and medial wall thickness of the microvessels (I). J assessment of Nppa mRNA in the right ventricle at P70 using qRT-PCR. K, L Representative immunofluorescent images of lungs from offspring at P21. Tissues were stained for αSMA and Ki67. The Ki67-positive nuclei/total nuclei were calculated for αSMA-positive cells in bronchi (diameter: 150–250 µm diameter) (K) and in microvessels (20–100 µm) (L). Values are shown next to the respective images. M, N Representative co-immunofluorescence staining to detect αSMA (red) and FoxO1 (green) in bronchi (M) and microvessels (N); DAPI was used for nuclear staining. Quantitative assessment of the nuclear and cytoplasmatic FoxO1 fraction in % of total cell FoxO1 in αSMA-positive bronchial (M) and vascular cells (N). Data are shown as mean ± standard error of the mean. A–N SD n = 3–19, HFD n = 3–17. Data were analyzed using the two-sided Mann–Whitney test and two-way ANOVA followed by the Bonferroni post-test; *P < 0.05; ***P < 0.001; ****P < 0.0001. Gray = standard diet; white = high-fat diet; blue=IL-6^−/−. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 8. Smooth muscle cell (SMC)-specific ablation of FoxO1 induces airway resistance and vascular muscularization in mice.

Genetically modified mice exhibiting a specific ablation of FoxO1 in SMC (SMC^FoxO1-KO) were compared to the respective wild-type controls (WT). A Representative images for α-smooth muscle actin (αSMA), an indicator of SMC, to assess the bronchial SMC layer. B Quantitative measurement of the SMC layer in the bronchi (diameter: 150–200 µm) of adult mice. C Assessment of respiratory airway resistance in adult mice using body plethysmography. D Measurement of the medial wall thickness in pulmonary microvessels with diameters of 20–70 µm, 70–150 µm and >150 µm. E Measurement of gene expression of Foxo3, Foxo4, and Foxo6 in total lung homogenates using qRT-PCR at P70; β-actin served as housekeeping gene; the expression is shown relative to WT and WT is set 1. F–H Immunoblots for pSTAT3 and total STAT3 (F), p c-Jun and total c-Jun (G), and PPARγ (H) in total lung homogenates of WT and SMC^{FoxO1 KO}; β-actin served as a loading control. The densitometric summaries of pSTAT3 relative to total STAT3, p c-Jun relative to total c-Jun, and PPARγ relative to β-actin are displayed next to the respective immunoblot. Data are shown as mean ± standard error of the mean. B–H WT n = 4–6, SMC^FoxO1-KO n = 4–7. Data were analyzed using the two-sided Mann–Whitney test; *P < 0.05; **P < 0.01. Gray = wild-type; orange = Foxo1^ADA. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Fig. 9. Paclitaxel (Pax) mitigates perinatal high-fat diet (HFD)-associated bronchial and vascular remodeling.

A The offspring of HFD- or standard diet (SD)-fed dams received an intravenous (i.v.) administration of vehicle or Pax, a FoxO1 activator, at postnatal day 50 (P50). All endpoints were assessed at P70. B–D Body weight (B), white adipose tissue (WAT) (C), and WAT relative to body weight (D). E Representative immunofluorescent lung images for α-smooth muscle actin (αSMA, red) and FoxO1 (green); the quantitative FoxO1 intensities per defined area (10 µm²) are displayed. F Respiratory airway resistance. G Analysis of the bronchial SMC (bSMC) layer in bronchi (diameter: 150–250 µm). H, I Complete microvascular muscularization (20–100 µm) (H). Right ventricular weight relative to body weight (I). J, K Representative immunofluorescent lung images for αSMA (red) and Ki67 (green, proliferation); the number of Ki67⁺ nuclei relative to total nuclei of αSMA-positive cells in bronchi (diameter: 150–250 µm) (J) and microvessels (20–100 µm) (K). Values are shown under the respective images. Data are shown as mean ± standard error of the mean. B–K SD n = 3–7, HFD n = 3–5; two-sided Mann–Whitney test and two-sided Student’s t test; *P < 0.05; ***P < 0.001; ****P < 0.0001. Black = SD + vehicle; white = HFD + vehicle; beige = SD + Paclitaxel; red = HFD + Paclitaxel. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file. L Proposed working model: effects of HFD-induced maternal and perinatal obesity with early-onset offspring obesity and elevated circulating levels of the adipocytokine Interleukin (IL)-6 on the developing lung. During lung development, this chronic subacute inflammatory state promotes the activation of STAT3 and AKT signaling in the lung and the proliferation of smooth muscle cells (SMC) via the cytoplasmatic sequestration of the transcription factor FoxO1. Bronchial and vascular SMC hyperproliferation is ultimately related to bronchial obstruction and pulmonary hypertension, respectively. Both IL-6 deficiency and FoxO1 activation prevent these structural and functional impairments of the bronchi and vessels after perinatal obesity, suggesting an IL-6-STAT3-AKT-FoxO1 axis and a new therapeutic approach to mitigate the early metabolic origins of chronic lung diseases (CLDs). The illustration has been created by the authors.