microRNA-33 deficiency in macrophages enhances autophagy, improves mitochondrial homeostasis, and protects against lung fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of sterol regulatory element binding protein (SREBP) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immunometabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in bronchoalveolar lavage (BAL) cells isolated from patients with IPF compared with healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti-miR-33 peptide nucleic acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. These studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a potentially novel therapeutic approach to treat this disease.

Keywords: Autophagy; Fatty acid oxidation; Fibrosis; Metabolism; Pulmonology.

Figure 1. miR-33 levels increase in BAL and lung CD45⁺ cells in patients with IPF, and its target gene expressions decrease in IPF BAL and lung.

(A) miR-33 relative expression in cells isolated from BAL of patients with IPF (n = 62) compared with healthy controls (n = 10) by qPCR analysis. (B) miR-33 relative expression in hematopoietic cells (CD45⁺) isolated from IPF (n = 9) compared with healthy controls (n = 4). (C) GSVA of miR-33-5p targets in BAL data set (GSE70866), 212 patients with IPF, and 20 healthy donors. (D) GSVA of miR-33-5p targets in the Lung LTRC data set (GSE47460), 254 patients with IPF, and 108 healthy donors. All PCR data were analyzed by nonparametric tests (Mann–Whitney U test or Kruskal-Wallis test where appropriate) and are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. Loss of miR-33 in myeloid cells is protective against bleomycin-induced lung fibrosis and increases mitochondrial-related target gene expression.

Evaluation of bleomycin-induced lung fibrosis in myeloid-specific miR-33–KO mice (miR33^M/M–/–) versus controls (miR33^M/M+/+) in bleomycin (red) compared with saline (blue) n = 8 for saline and n = 16 for bleomycin groups. (A) miR-33 relative expression by qPCR analysis in AM isolated from miR33^M/M–/– versus controls miR33^M/M+/+. (B) Quantitative analysis of hydroxyproline in lung homogenates from indicated groups of mice. (C and D) Acta2 and Col1a1 relative gene expression by qPCR analysis in mice lungs from indicated groups. (E and F) Representative images and quantitative measurements of Masson’s trichrome staining of lung sections in miR33^M/M–/– versus controls miR33^M/M+/+ with saline and bleomycin. (G) Differential cell counts in BAL were harvested from indicated groups. (H–O) BAL cytokines inflammatory panel in indicated groups: (H) TNF-α, (I) IL-12p70, (J) IL-2, (K) INF-γ, (L) KC-GRO, (M) IL-1β, (N) IL-13, and (O) IL-4. (P–T) qPCR analysis of mitochondrial-related miR-33 target genes: (P) Pgc-1α, (Q) Crot, (R) Abca1, (S) Cpt1α, and (T) Sirt3 in miR33^M/M–/– versus controls miR33^M/M+/+ with saline and bleomycin. All data were analyzed by ANOVA or Kruskal-Wallis tests, followed by post hoc analysis, and are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001. Total original magnification, 4×.

Figure 3. Pharmacological inhibition of miR-33 using PNA-33 in lung macrophages protects against bleomycin-induced pulmonary fibrosis in in vivo mouse model of PF.

(A) Binding efficiency of PNA-33 to the known miR-33 targets by gel shift assay on 10% nondenaturing polyacrylamide gel. Bound and the unbound fraction of target miR-33 were visualized by staining the gel in SYBR Gold. (B) Two-photon microscopy imaging of PNA-33 TAMRA conjugated in WT mice 24 hours after i.v. administration (red arrows indicate orange accumulation of TAMARA dye in macrophages). Total original magnification, (top panel) 4×, (Lower panels) 20×. (C) miR-33 relative expression in AM of WT mice after i.n. administration of PNA-33 and scrambled control in different time points (days 0, 1, 3, and 5). (D) Quantitative analysis of hydroxyproline in lung homogenates from indicated groups of mice in bleomycin-induced lung fibrosis model. (E and F) Acta2 and Col1a1 relative gene expression by qPCR analysis in mice lungs from indicated groups. (G and H) Representative images and quantitative measurements of Masson’s trichrome staining of lung sections after administration of PNA-33/scrambled control in saline and bleomycin. Total original magnification, 4×.(I and J) qPCR analysis of mitochondrial related miR-33 target genes: Pgc-1α and Abca1 after PNA-33/scrambled control in saline and bleomycin groups. Bleomycin; red, Saline; blue. n = 6 for saline, n = 8 for bleomycin groups. All data were analyzed by ANOVA or Kruskal-Wallis tests, followed by post hoc analysis, and are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001.

Figure 4. The absence of miR-33 in macrophages improves mitochondrial homeostasis (function and structure) at baseline and after bleomycin injury.

(A–D) qPCR analysis of miR-33 and target gene expression in response to inhibition by miR-33 antagomir in AM in vitro with and without bleomycin (n = 10). miR-33, Pgc-1α, Abca1, and Sirt3 relative expressions. (E–H) Seahorse analysis of AM isolated from miR33^M/M–/– versus miR33^M/M+/+ in response to bleomycin and saline. The analysis was measured under basal conditions followed by the addition of oligomycin, FCCP, rotenone, and antimycin (n = 16 in all groups). (E and F) Oxygen consumption rate (OCR, pmol/min). (G and H) Extracellular acidification rate (ECAR, pmpH/min). (I) Measurement of free circulating mtDNA by qPCR in the BAL isolated from miR33^M/M–/– versus miR33^M/M+/+ mice in response to bleomycin and saline (n = 6 for saline, n = 10 for bleomycin groups). (J–M) Representative images of transmission electron microscopy (TEM) imaging on lung tissues isolated from miR33^M/M–/– versus miR33^M/M+/+ mice in response to bleomycin and saline (n = 8). Red arrows indicate mitochondria. (N) Blinded measurements of mitochondria in TEM images from mice AM in miR33^M/M–/– versus miR33^M/M+/+ mice in bleomycin-treated mice by counting the dysmorphic versus normal-looking mitochondria in different groups (n = 8). (O) Ultrastructural qualitative and quantitative analysis of mitochondria in mice lung TEM images represented as mitochondrial area (au) in miR33^M/M–/– versus miR33^M/M+/+ mice in bleomycin- and saline-treated mice. The statistical test used were ANOVA or Kruskal-Wallis tests, followed by post hoc analysis. All data are presented as mean ± SEM. *P ≤ 0.05, **P <0.01, ***P < 0.001. Total original magnification, 4× (J and K, top panels in L and M) and 10× (lower panels in L and M).

Figure 5. Genetic ablation of miR-33 in lung macrophages induces autophagy after bleomycin injury.

(A) Representative images of transmission electron microscopy (TEM) imaging on lung tissues isolated from miR33^M/M–/– after bleomycin. There is a dramatic increase in autophagosome contents, only in the miR33^M/M–/– macrophages after bleomycin along with the improvement in mitochondrial structure in these cells (n = 6). Red arrows indicate mitochondria, and purple arrows indicate autophagosome. Total original magnification, 4×, 10×, and 20×. (B) Western blot analysis of phospho–AMPK-α (Ph.–AMPK-α) and PGC-1α in the lung homogenates isolated from miR33^M/M–/– and controls after bleomycin and saline. (C) Western blot analysis of Ph.–AMPK-α, LC3A/B, and P62 in AM after inhibiting miR-33 by miR-33 antagomir. (D and E) Representative images and quantification of IHC staining of LC3A/B in lung tissues isolated from miR33^M/M–/– and controls after bleomycin and saline treatment. (F and G) Representative images and quantification of IHC staining of P62 in lung tissues from miR33^M/M–/– and controls after bleomycin and saline treatment. All data were analyzed by ANOVA or Kruskal-Wallis tests, followed by post hoc analysis, and are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01. Total original magnification, 4× (D and F).

Figure 6. The absence of miR-33 in macrophages induces mitophagy in response to injury and alters cytokine-induced gene expressions in AM.

(A and B) Mitophagy assay in primary AM isolated from WT mice treated with PNA-33 (2 nM) or scrambled control after bleomycin (15 nM) or saline. (A) Representative images in indicated groups. (B) Quantitation of mitophagy staining. (C and D) Pink1 and Parkin expression in PNA-33/scramble-treated primary mice AM after bleomycin/saline. (E) Mitophagy measurement (RFU) in CD45⁺ cells isolated from human IPF lungs in response to PNA-33 versus scrambled control after 24 hours in culture (n = 4). (F–L) Evaluation of the effects of PNA-33/scramble on primary mice AM after cytokines stimulation. Primary mice AM were treated with PNA-33 (2 nM) or scrambled control for 24 hours before exposing them to IL-13 or INF-γ + LPS for another 24 hours. (F–I) Expression of Arg1, Chi3l1, IL-12, and Sirt1 in INF-γ–treated cells PNA-33/scramble treatments. (J–L) Expressions of Ym1, Abca1, and Pparg in IL-13–treated cells after PNA-33/scramble treatments (n = 7). All data were analyzed by ANOVA or Kruskal-Wallis tests, followed by post hoc analysis, and data are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001. Total original magnification, 4× and 10×.

Figure 7. The absence of miR-33 in lung macrophages improves mitochondrial homeostasis and decreases cell death in AT2 after bleomycin injury.

(A) Representative images of transmission electron microscopy (TEM) on lung tissues isolated from miR33^M/M–/– and miR33^M/M+/+ after bleomycin and saline treatment. Red arrows indicate mitochondria in AT2 (n = 8 per group). Total original magnification, 4× and 10×. (B) Blinded measurements of mitochondria in mice AT2 after bleomycin in TEM images by counting the dysmorphic versus normal-looking mitochondria in miR33^M/M–/– and control groups (n = 8). (C) Ultrastructural qualitative and quantitative analysis of mitochondria in mice lung AT2s in TEM images represented as mitochondrial area (au). (D and E) Representative images and quantification analysis of TUNEL IF staining on lung sections from miR33^M/M–/– and miR33^M/M+/+ after bleomycin and saline treatment (n = 6). Green, TUNEL⁺ cells. Total original magnification, 4×. (F) Representative images of TUNEL IF staining with Pro-SPC on lung sections from miR33^M/M–/– and miR33^M/M+/+ after bleomycin treatment (n = 6). Green, TUNEL; red, SPC. Total original magnification, 4× and 20×. (G) Quantification analysis of TUNEL⁺SPC⁺ cells on lung sections from miR33^M/M–/– and miR33^M/M+/+ after bleomycin (n = 6). (H and I) Schematic experimental planning and quantification of Caspase 3/7 activity measured in small airway epithelial cells (SAEC) with and without bleomycin after exposure to the supernatants harvested from ablated miR-33 AM (using PNA-33 or scrambled control) from WT mice. All data were analyzed by ANOVA or Kruskal-Wallis tests, followed by post hoc analysis, and are presented as mean ± SEM. *P ≤ 0.05, **P <0.01, ***P < 0.001.

Figure 8. Pharmacologic inhibition of miR-33 using PNA-33 ameliorates fibrosis in mice and human ex vivo models of PF.

(A–E) Evaluation of antifibrotic effects of PNA-33 in murine ex vivo model. Mouse PCLS isolated at day 14 after bleomycin or saline treatment. Bleomycin, red; saline, blue. (A) Two-photon microscopy imaging of mice PCLS from the bleomycin-treated group at the end of 5 days of stimulation with PNA-33 TAMRA conjugated or scrambled control. Blue, collagen; orange, TAMRA accumulation in macrophages. Total original magnification, 4× and 10×. (B–D) qPCR analysis of Acta2, Col1a1, Pgc-1α, and Abca1 in mouse PCLS after bleomycin following 5 days of stimulation with PNA-33 or scramble (n = 6 per group). All data were analyzed by ANOVA or Kruskal-Wallis tests, followed by post hoc analysis, and are presented as mean ± SEM. *P ≤ 0.05, **P < 0.01, ***P < 0.001. (F–H) Evaluation of antifibrotic effects of PNA-33 in human ex vivo model. hPCLS prepared from human IPF lungs isolated and treated with PNA-33 or scrambled control for 5 days before performing RNA-Seq. (F) Heatmap showing the fibrotic gene expression in IPF PCLS treated with PNA-33 or scramble. (G) Heatmap showing the profibrotic macrophage gene expression alterations by PNA-33 in IPF PCLS. (H) Scatterplot of genes found significantly differentially expressed in IPF lung macrophage versus controls (in single-cell RNA-Seq analysis) compared with IPF PCLS treated or untreated with miR-33 inhibitor (PNA-33). The x axis corresponds to the log fold change differences in IPF versus control lung macrophage reported in single-cell RNA-Seq analysis, the y axis corresponds to fold change differences in IPF PCLS following treatment with PNA-33. The size of each dot corresponds to the negative log_–10 transformed P values of a comparison of IPF PCLS with or without miR-33 treatment.