AMPK-driven Macrophage Responses Are Autophagy Dependent in Experimental Bronchopulmonary Dysplasia

Abstract

The pathogenesis of bronchopulmonary dysplasia (BPD) remains incompletely understood. Recent studies suggest insufficient AMP-activated protein kinase (AMPK) activation as a potential cause of impaired autophagy in rodent and nonhuman primate models of BPD. Impaired autophagy is associated with enhanced inflammatory signaling in alveolar macrophages (AMs) and increased severity of murine BPD induced by neonatal hyperoxia exposure. The goal of this study was to determine the role of autophagy and AMPK activation in macrophage responses in murine BPD. C57BL/6J mice were exposed to neonatal hyperoxia starting on postnatal day (P)1 and treated with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) between P3 and P6. Mice were euthanized on P7, and markers of AMPK activation and autophagy were assessed by immunoblotting. Alveolarization was assessed using radial alveolar counts, mean linear intercept measurements, and quantification of alveolar septal myofibroblasts. Relative mRNA expression of M1-like and M2-like genes was assessed in AMs isolated from BAL fluid from wild-type, LysMCre^--Becn1^fl/fl, and LysMCre⁺-Becn1^fl/fl mice after neonatal hyperoxia exposure. AICAR treatment resulted in AMPK activation and induction of autophagic activity in whole-lung and BAL cell lysates and attenuated hyperoxia-induced alveolar simplification in neonatal lungs. AICAR-treated control but not Beclin1-deficient AMs demonstrated significantly decreased expression of M1-like markers and significantly increased expression of M2-like markers. In conclusion, pharmacologic activation of AMPK by AICAR resulted in induction of autophagy and played a protective role, at least in part, through attenuation of proinflammatory signaling in AMs via autophagy-dependent mechanisms in a murine model of BPD.

Keywords: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK; alveolar macrophages; autophagy; bronchopulmonary dysplasia.

Figure 1.

5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) treatment induced AMP-activated protein kinase (AMPK) activation and autophagy in neonatal hyperoxia–exposed lungs. (A) Representative immunoblot analysis for markers of AMPK activation and autophagy using whole-lung homogenates harvested at P7 from vehicle control (VC) and AICAR-treated hyperoxia-exposed C57BL/6J mice. (B) Relative protein concentrations normalized to β-actin (ACTB) by densitometry are indicated for all blots except for the AMPK blot, for which the phospho-AMPK (p-AMPK)/AMPK ratio is shown. (C) Representative immunoblot analysis for p-AMPK, p62, LC3-I, and LC3-II using pooled BAL cell lysates (n = 3/lane) harvested from VC and AICAR-treated hyperoxia-exposed mice at P7. (D) Relative protein concentrations normalized to ACTB are indicated for p-AMPK and p62 blots, and LC3-II/LC3-I ratio is indicated for the LC3 blot. Data are shown as mean ± SEM; n = 5 per group. *P < 0.05 and **P < 0.01. P = Postnatal Day.

Figure 2.

AICAR treatment attenuated alveolar simplification and enhanced vascular development in hyperoxia-exposed neonatal murine lungs. (A) C57BL/6J mice were exposed to normoxia (21% O₂) or hyperoxia (75% O₂) between P1 and P7 and treated with VC or AICAR from P3 to P6. After euthanasia at P7, lungs were inflated with 10% neutral buffered formalin and embedded in paraffin. Hematoxylin and eosin–stained sections are shown. Scale bars, 50 μm. (B) Radial alveolar counts were assessed as a measure of alveolarization. n = 6–8 per group. (C) Mean linear intercepts were measured as a surrogate for alveolar diameter. n = 5 per group. (D and E) ACTA2-positive myofibroblasts localized at septal tips (indicated by circles) as a surrogate marker of secondary septation were quantified using ImageJ and NIS-Elements Basic Research software. n = 5 per group. Scale bars, 50 μm. (F) Representative immunoblot analysis for the expression of CD31, a pan–endothelial cell marker, in whole-lung homogenates of VC or AICAR-treated C57BL/6J mice. (G) Densitometric analysis of relative protein concentration of CD31 normalized to ACTB is shown. n = 5 per group. Data were obtained from a minimum of three separate litters and are shown as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. ns = not significant.

Figure 3.

AICAR-induced AMPK activation ameliorates inflammatory responses in hyperoxia-exposed neonatal mice. Immunofluorescence staining was performed on paraffin-embedded sections at P7 from C57BL/6J neonatal murine lungs treated with VC or AICAR with primary antibodies against myeloperoxidase (MPO) and FABP4 to assess neutrophil (A) and macrophage (B) accumulation in lung tissues, respectively. The secondary antibody was Alexa Fluor 594 goat antirabbit IgG (red). White arrowheads indicate MPO-expressing neutrophils. Quantitative assessment of neutrophil (C) and macrophage (D) counts per HPF was performed using ImageJ and NIS-Elements Basic Research software. Representative images are shown. n = 3–5 per group. Scale bars, 50 μm. (E) BAL fluid was obtained from VC and AICAR-treated hyperoxia-exposed C57BL/6J neonatal mice at P7, and alveolar macrophages were isolated by adhesion purification. Total RNA was isolated, and quantitative real-time PCR was performed for a panel of M1-like (TNF-α, IL-1β, and CXCL1) and M2-like (CD206, Arg1, and Fizz1) genes as indicated on the figure. n = 5–8 per group. Representative immunoblot and densitometric analysis for CD206 protein expression in pooled BAL cell lysates (F) (n = 3/lane) and whole-lung homogenates (G) harvested from VC and AICAR-treated hyperoxia-exposed mice at P7. Relative protein concentrations were normalized to ACTB. n = 3–5 per group. Data were obtained from a minimum of three separate litters and are shown as mean ± SEM. *P < 0.05, ***P < 0.001, and ****P < 0.0001. HPF = high-power field. Arg1 = arginase1; Fizz1 = found in inflammatory zone 1.

Figure 4.

Antiinflammatory effects of AICAR on alveolar macrophage responses in neonatal hyperoxia are autophagy dependent. (A) Representative immunoblot analysis for the expression of Beclin1 and NLRP3 in pooled BAL cell lysates harvested from 6-week-old Becn1^F/F;LysM-cre mice and Becn1^F/F littermates. n = 3/group. (B) Relative protein concentrations normalized to ACTB. (C) BAL fluid was obtained at P7 from Becn1^F/F;LysM-cre mice and Becn1^F/F littermates and (D) AICAR-treated Becn1^F/F;LysM-cre mice and Becn1^F/F littermates after neonatal hyperoxia exposure, and alveolar macrophages were isolated by adhesion purification. Total RNA was isolated, and quantitative real-time PCR was performed for a panel of M1-like (TNF-α, IL-1β, and CXCL1) and M2-like (CD206, Arg1, and Fizz1) genes as indicated on the figure. n = 7–17 per group. Data were obtained from a minimum of three separate litters and are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.