Hedgehog signaling in neonatal and adult lung

Abstract

Sonic Hedgehog (Shh) signaling is essential during embryonic lung development, but its role in postnatal lung development and adult lung are not known. Using Gli1(nlacZ) reporter mice to identify cells with active Hh signaling, we found that Gli1(nlacZ)-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1(nlacZ)-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1(nlacZ)-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. To assess its functional relevance, we influenced Hh signaling in the developing postnatal lung and adult injured lung. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. After bleomycin injury in the adult lung, there are abundant Gli1(nlacZ)-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1(nlacZ)-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated overexpression of Shh increases collagen production in this model. Our data provide strong evidence that Hh signaling can regulate lung stromal cell function in two critical scenarios: normal development in postnatal lung and lung fibrosis in adult lung.

Figure 1.

Hedgehog (Hh) signaling occurs during postnatal development. Gli1^nlacZ/+ lung sections from postnatal day (P)0, P2, P7, P14, and P21 were stained for β-gal activity (blue). (A–E) Low power images show Gli1⁺ cells are initially present in alveolar walls and around bronchi but decrease significantly in the alveolar compartment by P14. At P21, Gli1⁺ cells are mostly seen around airways and vessels. (F–H) High-power images reveal that Gli1⁺ cells, present in alveolar walls and septa (arrowheads), decrease over time.

Figure 2.

Gli1^nlacZ and Gli2^nlacZ reporter expression patterns reveal Hh pathway activation in adult lung fibroblasts. (A–C) Gli1^nlacZ/+ lung sections and (D–F) Gli2^nlacZ/+ lung sections were stained for β-gal activity (blue). (A, F) Low-power images demonstrate prominent distribution of Gli1⁺ (A) and Gli2⁺ (D) cells around large airways and accompanying vessels. (B, C) High-power images show the absence of Gli1⁺ cells (B) and Gli2⁺ cells (E) in airway epithelia and large vessel endothelia. (C, F) High-power images of lung periphery show Gli1⁺ (C) and Gli2⁺ (F) cells adjacent to the pleural boundary and in more proximal alveolar parenchyma. In the alveolar compartment, Gli2⁺ cells are more numerous than are Gli1⁺ cells. (G–J) The majority of Gli1⁺ adult lung cells are collagen-producing fibroblasts. Merged immunofluorescent images of immunostained Gli1^nlacZ/+ lung sections are shown (β-gal: green, other markers: red; DAPI-stained DNA: blue). (G, H) Most β-gal⁺ cells are embedded in a Col1⁺ matrix (red). (I, J) β-gal⁺ cells closely surround the smooth muscle layers of vessels and large airways, but only a few coexpress α smooth muscle actin. b = bronchus; p = pleura; v = vessel.

Figure 3.

Bleomycin-induced fibrosis is characterized by increased numbers of Gli1⁺ fibroblasts and myofibroblasts. (A–D) Gli1^nlacZ/+ lung sections, 4 weeks after bleomycin exposure, were stained for β-gal activity (blue) and counter-stained with H&E (A, B, D) or picrosirius red (C). (A) Low-power image illustrates high number and heterogeneous distribution of Gli1⁺ cells in fibrotic zones of bleomycin-treated lungs. (B) High-power image shows the clustered, linear arrangement of β-gal⁺ nuclei (arrows), characteristic of fibrotic regions. β-gal⁺ cells extend along eosin-stained fiber tracks. (C) β-gal⁺ cells are largely located within areas of yellow birefringence (arrows) produced by picrosirius-stained collagen fibrils. (D) High-power image shows Gli1⁺ cells (arrowheads) in the alveolar septa of histologically normal distal lung parenchyma in lungs exposed to bleomycin 4 weeks earlier. (E) Graph shows comparison of the number of Gli1⁺ cells and Gli2⁺ cells in alveolar regions of normal lungs with the number of Gli1⁺ cells in areas of preserved architecture 4 weeks after bleomycin exposure (n = 3/group). Data are shown as mean and SD of each group. BLM = bleomycin. (F–I) Gli1⁺ cells in bleomycin-induced fibrotic lesions are fibroblasts and myofibroblasts. (F–I) Merged immunofluorescent images of Gli1^nlacZ/+ lung sections, 4 weeks after bleomycin exposure, were costained with antibodies against β-gal protein (green) and either Col1 (F, G) or α smooth muscle actin (α-SMA) (H, I) (both red). Nuclei are stained with DAPI (blue); β-gal⁺ nuclei appear blue-green in the merged photographs. Col1⁺/β-gal⁺ (G, arrow) and Col1−/β-gal⁺ cells (G, arrowhead) can be identified. Many β-gal⁺ cells in fibrotic zones also express α-SMA (I, arrow); β-gal+/α-SMA− cells are also present (I, arrowhead).

Figure 4.

Epithelial Sonic Hedgehog (Shh) overexpression increases bleomycin-induced lung fibrosis, whereas inhibition of Shh does not affect lung fibrosis. (A) C57BL/6J mice, exposed to bleomycin (1.3 U/kg), received treatment with Shh neutralizing antibody 5E1 or IgG isotype at Day −1 (60 mg/kg), Day 7 (30 mg/kg), Day 14 (30 mg/kg) or treatment with Shh-expressing adenovirus (Ad-Shh, 10⁹ plaque-forming units per mouse) or control adenovirus (Ad-ctl) at Day 21. Lungs harvested on Day 21 or 28, respectively, were measured for collagen content (μg/lung) using hydroxyproline assay (n = 13 per group for 5E1 experiment; n = 8 per group for Ad-Shh experiment; n = 2 for normal saline [NS]-treated control lungs). (B) HEK293 cells (HEK), infected with Ad-Shh or Ad-ctl, were placed in nonadherent coculture with primary Gli1^nlacZ/+ lung fibroblasts (Gli1-lacZ fb). X-gal staining after 72 hours shows β-gal⁺ cells only in coculture sphere containing Ad-Shh infected HEK cells (lower panel). Recombinant Shh (rShh) was used to show Shh responsiveness of Gli1^nlacZ/+ fibroblasts (upper panel). (C) Primary lung fibroblasts were cultured as nonadherent spheres (35,000 cells/sphere) and stimulated with rShh for a maximum of 6 days. Trypsinized cells at later time points show significantly more viable cells in rShh-treated spheres (red line) than in the control spheres (blue line). Data are shown as mean and SD of each group. *P < 0.05.

Figure 5.

Inhibition of Hh signaling affects postnatal lung development. C57BL/6J mice or Gli1^nlacZ/+ mice were treated with 5E1 antibody (30 mg/kg) or isotype IgG at P3. Lungs were inflated at P9 with 4% paraformaldehyde at 25 cm H₂O pressure for 10 minutes (A, B) or with OCT/sucrose at the same volumes (C, D) then stained with H&E for morphometric analysis (A, B) or X-gal and Nuclear Acid Fast stain (C, D). (A, B) Low-power images of 5E1-treated lungs show enlarged alveolar airspaces when compared with IgG controls. High-power images (insets) show the presence of septa in 5E1 and IgG. (C, D) Low-power images show Gli1⁺ cells are almost absent in the alveolar compartment in 5E1-treated mice when compared with IgG control. Some Gli1⁺ cells remain present around bronchi and vessels. Higher-power images (inserts) show the absence of Gli1⁺ cells at septal tips. (E) Mean linear intercept (MLI) of mice treated with 5E1 or IgG at P3 and analyzed at P9 (n = 8 per group). MLI of control mice inflated at different inflation pressures show increasing MLI with higher pressures. (F) Weights of 5E1- and IgG-treated mice are not different from P3 to P9 (n = 8 per group). Data are shown as mean and SD of each group. b = bronchus; v = vessel.