beta6 Integrin subunit deficiency alleviates lung injury in a mouse model of bronchopulmonary dysplasia

Abstract

Pulmonary inflammation is associated with the development of bronchopulmonary dysplasia in premature infants. We have previously shown that perinatal pulmonary expression of human IL-1beta is sufficient to cause a lung disease similar to bronchopulmonary dysplasia, characterized by inflammation, impaired alveolarization, poor postnatal growth, and increased mortality in infant mice. The alphavbeta6 integrin plays a critical role in regulating inflammation in the adult lung. To study the role of the beta6 integrin subunit in neonatal inflammatory lung disease, we compared the pulmonary development in IL-1beta-expressing infant mice with wild-type or null beta6 integrin loci. Absence of the beta6 integrin subunit decreased the mortality and improved the postnatal growth of IL-1beta-expressing pups. The disrupted alveolar development of IL-1beta-expressing mice was improved by beta6 integrin deficiency. IL-1beta-expressing beta6(-/-) pups had shorter alveolar chord length and thinner alveolar walls than IL-1beta-expressing beta6(+/+) pups. In addition, the absence of the beta6 integrin subunit reduced IL-1beta-induced neutrophil and macrophage infiltration into the alveolar spaces. beta6 integrin subunit deficiency suppressed inflammation and goblet cell hyperplasia in the airways and alleviated airway remodeling in IL-1beta-expressing mice. The expression of the chemoattractant proteins, keratinocyte-derived chemokine, macrophage-inflammatory protein-2, calgranulin A, and calgranulin B, of osteopontin, and of the chitinase-like lectins, Ym1 and Ym2, was lower in IL-1beta-expressing beta6(-/-) than in IL-1beta-expressing beta6(+/+) mice. We conclude that absence of the beta6 integrin subunit protects the infant murine lung against IL-1beta-induced inflammation and injury.

Figure 1.

The absence of the β6 integrin subunit improved the survival and postnatal growth of IL-1β–expressing mice. Doxycycline was administered to the dams from the beginning of pregnancy until the pups were killed at Postnatal Day (PN) 7. (A) Postnatal survival until PN7. IL-1β decreased the survival rate of β6^+/+ pups (log-rank test, P < 0.0001 IL-1β/β6^+/+ versus all other groups), but not of β6^−/− pups (P = 0.3 IL-1β/β6^−/− versus control/β6^−/−). All control/β6^−/− pups survived until PN7, whereas two control/β6^+/+ pups died at or soon after birth (P = 0.2 control/β6^+/+ versus control/β6^−/−). The total numbers of animals studied were: control/β6^+/+, 47; control/β6^−/−, 41; IL-1β/β6^+/+, 48; IL-1β/β6^−/−, 42. (B) Body weights at PN7. IL-1β/β6^−/− pups weighed more than IL-1β/β6^+/+ pups, although control/β6^−/− pups weighed less than control/β6^+/+ pups. Data are presented as means (±SEM) (n = 20–34 per group). ***P < 0.001.

Figure 2.

Alveolar septation and alveolar wall thinning in IL-1β–expressing mice was improved in mice deficient in β6 integrin subunit. Doxycycline was administered to the dams from the beginning of pregnancy until the pups were killed. (A–D) Lung histology at PN7. Normal alveolar septation is seen in control/β6^+/+ and control/β6^−/− mice (A and C). Lack of septation in IL-1β/β6^+/+ appears less severe in IL-1β/β6^−/− mice (B and D). Scale bar, 200 μm. (E) Mean alveolar chord length. IL-1β increased the chord length in both β6^+/+ and β6^−/− mice compared with control animals. The chord length was shorter in IL-1β/β6^−/− mice than in IL-1β/β6^+/+ mice. Data are presented as means (±SEM) (n = 6 per group). (F) Alveolar wall thickness. IL-1β–expressing mice had thicker alveolar walls than control mice at PN7. The alveolar walls were thinner in IL-1β/β6^−/− mice than in IL-1β/β6^+/+ mice. Data are presented as means (±SEM) (n = 6 per group). **P < 0.01, ***P < 0.001. NS, not significant.

Figure 3.

Neutrophils and macrophages in the lungs of infant mice. Dams received doxycycline from the beginning of pregnancy until the pups were killed at PN7. Neutrophils and macrophages were detected by immunohistochemistry and counted in the alveolar airspaces and alveolar septal walls in five to six animals per group. (A) The number of neutrophils in the alveolar airspaces was higher in IL-1β/β6^+/+ mice than in IL-1β/β6^−/− mice. The number of cells is expressed per square millimeter of airspace area. (B) IL-1β increased the number of neutrophils in the alveolar septa in both β6^+/+ and β6^−/− mice. The number of cells is expressed per square millimeter of alveolar septal area. (C) The distribution of neutrophils between the alveolar lumen and the alveolar walls was different in IL-1β/β6^+/+ and IL-1β/β6^−/− mice. Most of the neutrophils in IL-1β/β6^+/+ mice and IL-1β/β6^−/− mice were in the alveolar lumen and alveolar walls, respectively. The IL-1β–induced infiltration of macrophages to the alveolar spaces (D) and alveolar walls (E) was decreased in β6^−/− mice. The number of macrophages is expressed per square millimeter of distal airspace (D) and septal area (E). (F) The majority of macrophages was found in the alveolar spaces in all groups of mice. Data are presented as means (±SEM). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

mRNA expression and protein levels of CC and CXC chemokines. Dams were given doxycycline throughout pregnancy and postnatally until the pups were killed at PN7. The IL-1β–induced mRNA expression and protein production of the CXC chemokines, keratinocyte-derived chemokine (KC) (A and B) and macrophage inflammatory protein (MIP)–2 (C and D), were decreased in β6^−/− mice. IL-1β increased CXCR2 mRNA expression (E) similarly in β6^+/+ and β6^−/− mice. Absence of the β6 integrin subunit increased the mRNA expression and protein production of monocyte chemoattractant protein (MCP)–1 (F and G), and the mRNA expression of MCP-3 (H), in both control mice and IL-1β–expressing mice. The mRNA expression levels are shown relative to the mRNA expression level of control/β6^+/+ mice, which was set to 1.0. Data are presented as means (±SEM) (n = 7–12 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

mRNA expression and protein production of inflammatory mediators in the lungs of infant mice. Dams were given doxycycline from beginning of pregnancy until the pups were killed at PN7. The IL-1β–induced expression of calgranulin A (S100A8) (A), calgranulin B (S100A9) (B), and serum amyloid A3 (SAA3) (C) was decreased in β6^−/− mice. Absence of the β6 integrin subunit decreased the expression and production of osteopontin (D and E) in both control and IL-1β–expressing mice. The mRNA expression levels are shown relative to the mRNA expression level of control/β6^+/+ mice, which was set to 1.0. Data are presented as means (±SEM) (n = 8–11 per group). **P < 0.01, ***P < 0.001.

Figure 6.

Airway structure and expression of the chitinases, Ym1 and Ym2. Dams were given doxycycline from the beginning of pregnancy until the pups were killed at PN7. (A–D) Airway histology, periodic acid Schiff (PAS) staining. The airways of control/β6^+/+ and control/β6^−/− mice appear normal (A and C). Goblet cell hyperplasia, thickening of the airway epithelium, and accumulation of inflammatory cells are seen in the airways of IL-1β/β6^+/+ mice (B). In IL-1β/β6^−/− mice, the airways were thinner, mucus production was sparse, and airway inflammation was suppressed (D). Scale bars, 100 μm. (E) Distribution (%) of airways with different percentages of PAS-positive cells. Open bars, airways having less than 20% goblet cells; shaded bars, airways having 20–80% goblet cells; filled bars, airways having more than 80% goblet cells. Absence of the β6 integrin decreased the IL-1β–induced goblet cell hyperplasia. β6 integrin deficiency reduced the expression of Ym1 (F) and Ym2 (G) in control and IL-1β–expressing mice. The mRNA expression levels are shown relative to the mRNA expression level of control/β6^+/+ mice, which was set to 1.0. Data are presented as means (±SEM) (n = 9–10 per group). **P < 0.01, ***P < 0.001.

Figure 7.

Apoptosis and proliferation in the distal lung. Dams were given doxycycline from the beginning of pregnancy until the pups were killed at PN0 or PN7. (A) Total number of distal terminal transferase dUTP nick-end labeling–positive cells (apoptotic cells) per high-power field (HPF). Apoptotic cells in the distal septa (B) and distal airspace (C) per square millimeter septal and airspace area, respectively. (D) Total number of Ki-67 (proliferation marker)–positive cells in the distal lung per HPF. Open bars, control mice; shaded bars, IL-1β–expressing mice. Data are presented as means (±SEM) (n = 4–6 per group). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

Active and total transforming growth factor (TGF)–β1 in the lungs of infant mice. Dams were given doxycycline from the beginning of pregnancy until the pups were killed at PN7. (A) The level of active TGF-β1 was increased in the lungs of IL-1β/β6^+/+ and IL-1β/β6^−/− mice compared with control/β6^+/+ and control/β6^−/− mice, respectively. (B) The level of total TGF-β1 did not significantly differ between the genotypes. The level of TGF-β1 was determined by ELISA in lung homogenates from 5–10 animals per group. Data are presented as means (±SEM). *P < 0.05, ***P < 0.001.