Itaconic acid regulation of TFEB-mediated autophagy flux alleviates hyperoxia-induced bronchopulmonary dysplasia

Abstract

Background

Premature infants often require oxygen supplementation, which can elicit bronchopulmonary dysplasia (BPD) and lead to mitochondrial dysfunction. Mitochondria play important roles in lung development, in both normal metabolism and apoptosis. Enhancing our comprehension of the underlying mechanisms in BPD development can facilitate the effective treatments.

Methods

Plasma samples from BPD and non-BPD infants were collected at 36 weeks post-menstrual age and used for metabolomic analysis. Based on hyperoxia-induced animal and cell models, changes in mitophagy and apoptosis were evaluated following treatment with itaconic acid (ITA). Finally, the mechanism of action of ITA in lung development was comprehensively demonstrated through rescue strategies and administration of corresponding inhibitors.

Results

An imbalance in the tricarboxylic acid (TCA) cycle significantly affected lung development, with ITA serving as a significant metabolic marker for the outcomes of lung development. ITA improved the morphological changes in BPD rats, promoted SP-C expression, and inhibited the degree of alveolar type II epithelial cells (AEC II) apoptosis. Mechanistically, ITA mainly promotes the nuclear translocation of transcription factor EB (TFEB) to facilitate dysfunctional mitochondrial clearance and reduces apoptosis in AEC II cells by regulating autophagic flux.

Conclusion

The metabolic imbalance in the TCA cycle is closely related to lung development. ITA can improve lung development by regulating autophagic flux and promote the nuclear translocation of TFEB, implying its potential therapeutic utility in the treatment of BPD.

Graphical abstract

Highlights

• •Itaconic acid (ITA) is a significant metabolic biomarker for human lung development.
• •ITA suppresses apoptosis in alveolar type II epithelial cells by regulating mitophagy.
• •ITA promotes TFEB-mediated mitochondrial autophagy flux.

Abbreviations

BPD

bronchopulmonary dysplasia

ITA

itaconic acid

TCA

tricarboxylic acid

AEC II

alveolar type II epithelial cells

PMA

post-menstrual age

IVH

Intraventricular hemorrhage

hsPDA

hemodynamically significant patent ductus arteriosus

NEC

Necrotizing enterocolitis

SGA

small for gestational age

EUGR

Extrauterine growth retardation

TFEB

transcription factor EB

H&E

hematoxylin and eosin

MLI

mean linear intercept

LC-MS

liquid chromatography–mass spectrometry

SD

Sprague-Dawley

TEM

Transmission electron microscopy

CQ

chloroquine

CCK-8

Cell counting kit-8

FITC

fluorescein isothiocyanate

KEGG

Kyoto Encyclopedia of Genes and Genomes

1. Introduction

Bronchopulmonary dysplasia (BPD) severs as the predominant morbidity associated with premature birth, affecting about 10,000 to 15,000 preterm infants annually in the USA [1]. Oxygen treatment can paradoxically worsen the condition, necessitate increased oxygen supplementation and exacerbate lung injury, and perpetuate a harmful cycle of injury consequently [2]. While BPD patients are gradually weaned off oxygen support in the weeks or months following birth, they may still experience respiratory complications, abnormal blood vessel formation, and delayed neurological development due to hyperoxia-induced [3,4]. Currently, the comprehensive treatment plan aiming at reducing the risk of BPD includes the administration of caffeine, antenatal steroids, pulmonary surfactants, and other medications in clinical practice [5], which has only minimally reduced the prevalence of BPD and lung injury [6]. The absence of effective treatment for BPD persists resulting from our insufficient understanding of the intricate molecular mechanisms underlying its development.

Infants with severe BPD often exhibit symptoms of growth retardation and metabolic dysregulation, indicating abnormalities in metabolic processes. Accumulating evidence suggests that metabolic imbalance can disrupt lung development homeostasis, ultimately resulting in the physiological and pathological changes associated with BPD [7,8]. While much of this evidence is derived from small-sample, single-center clinical studies or animal experiments, it prompts us to delve deeper into the metabolic alterations occurring during the development of BPD. Extensive research has shown that infants with BPD exhibit oxidative stress, mitochondrial dysfunction, and abnormalities in glucose metabolism, amino acid metabolism and lipid metabolism, which can lead to cellular apoptosis, differentiation abnormalities, and other related issues [4,9].

Mitochondria are cellular organelles responsible for energy metabolism and signal transduction, serving as high-speed processing centers. Hyperoxia can induce mitochondrial dysfunction. Strategies to optimize mitochondrial function not only involve reducing the production of dysfunctional mitochondria but also rapidly clearing dysfunctional mitochondria [10]. In this study, we metabolically profiled peripheral blood from BPD patients and no BPD patients and identified a crucial and easily overlooked role of itaconic acid (ITA) in the development of BPD.

ITA is a metabolic product related to cis-aconitic acid, biosynthesis typically occurs in the tricarboxylic acid (TCA) cycle, a decarboxylation reaction catalyzed by the enzyme immune responsive gene-1 [11]. ITA acts as a conditional factor with anti-inflammatory properties during the processes of tumor development, severe infections, and tissue injury [12,13]. Mechanistically, we found that ITA reduced apoptosis by regulating mitochondrial autophagic flux through TFEB nuclear translocation in vitro and in vivo experiments. Our findings reveal the underappreciated pathway whereby ITA regulates hyperoxia-induced apoptosis and may help to highlight ITA as a potential therapeutic agent in BPD.

2. Material and methods

2.1. Participant population and study design

The subjects of this study were premature infants born before 32 weeks of gestation, who were admitted to the neonatal intensive care unit of Xinhua Hospital (Shanghai, China) between July 2016 and January 2017. The study design received the approval from the hospital's ethics committee (XHEC-C-2017-015). The parents or legal guardians have signed the informed consent forms to agree to the participation of the preterm infants in this study. Divide these premature infants into two groups: non-BPD (n = 12) and BPD (n = 12). The infants of BPD group were diagnosed with a moderate or severe medical condition, characterized by the necessitation of >21% oxygen therapy for a minimum of 28 days, as well as the requirement for supplemental oxygen or positive pressure therapy at 36 weeks of post-menstrual age (PMA) [14]. The non- BPD group comprised infants who required oxygen therapy for fewer than 28 days.

2.2. Data collection

Clinical data were extracted from electronic medical records. Following enrollment, maternal information, demographic details, and clinical data were gathered for each infant. Antenatal data, encompassing maternal age, duration of pregnancy and labor, pregnancy-related conditions, and antenatal treatment, were compared. Follow-up information was obtained from outpatient follow-up reviews or telephone follow-ups up to 2 years old in premature infants. We paid attention on respiratory outcomes related to premature infants at the corrected gestational age of 2 years.

2.3. Definitions

Late-onset sepsis was diagnosed either clinically or confirmed by blood culture. According to the Papile classification system, severe intraventricular hemorrhage (IVH) referred to greater than or equal to grade 3. The diagnostic criteria for hemodynamically significant patent ductus arteriosus (hsPDA) typically included a ductus diameter＞1.5 mm, left atrial inner diameter/aortic root ratio ＞ 1.4, and the presence of a combined left-to-right shunt as determined by echocardiography [15]. Necrotizing enterocolitis (NEC) was diagnosed by evaluating clinical and radiographic gastrointestinal signs, defined as stage 2 or higher according to the modified Bell's staging criteria [16]. Weight, length, and head circumference were recorded on the growth charts. Infants classified as Small for gestational age (SGA) had birth weights below the 10th percentile for their respective gestational age. The definition of EUGR was still below the 10th percentile of average growth parameters for their gestational age when premature infants were discharged with growth parameters. Weight and length at two years of corrected age were calculated following the WHO multicenter growth study in 2006 (https://apps.who.int/iris/bitstream/handle/10665/43413/9,241,546 93X_eng.pdf). Underweight was defined as being more than 2 standard deviations (SD) below the mean weight for age, while short stature was defined as falling more than 2 SD below the mean length for age. Diagnosis of airway hypersensitivity disorder was based on the presence of symptoms such as recurrent coughing, or wheezing, which were partially relieved with anti-asthma medications. Adverse respiratory outcomes were defined as above two times of the readmission for respiratory diseases at the corrected gestational age of 2 years.

2.4. Plasma sample collection and preparation for metabolomics analysis

The peripheral blood samples of participants at 36 ± 3 weeks PMA were collected and stored at 4 °C for no more than 24 h. Following centrifugation at 1250 g for 5 min, plasma samples were collected and promptly stored at −80 °C.

2.5. Liquid chromatography–mass spectrometry (LC-MS) analysis

Subsequently, mixed 100 μL of each plasma sample with methanol, following centrifugation at 12,000 g for 15 min at 4 °C. After removing the supernatant, dried it by a centrifugal concentrator. Finally, each sample was transferred for subsequent LC-MS/MS analysis.

2.6. Animal procedures

Sprague-Dawley rats were purchased from GemPharmatech (Jiangsu, China). The Animal Care and Use Committee of Xinhua Hospital approved the experimental protocol. The rats were accommodated in environmentally controlled chambers, where humidity and temperature were regulated, maintaining a 12-h light-dark cycle. And they were provided ample access to both food and water.

2.7. Neonatal hyperoxia-induced lung injury model and study design

Experiments were performed in newborn rats exposed to a hyperoxic condition after birth [17]. Briefly, pups were pooled into dams and assigned to the control, BPD, or BPD with ITA groups randomly on the day of birth, counted as the postnatal day 0 (P0). The newborn rats were exposed to 85% O₂ as BPD group or room air as control group, whereas the BPD with ITA group was treated intraperitoneally with 100 mg/kg ITA (4-octyl itaconate, 4-OI, MCE, Cat No.: HY-112675) in 40% cyclodextrin/PBS (or 40% cyclodextrin/PBS as a vehicle was applied to the control and BPD groups) from P0 until P14. Nursing dams were rotated daily between hyperoxic and normoxic conditions.

2.8. Tissue preparation

The rats were sacrificed, and the lungs were excised at P7 and P14, as previously described [18]. The left lung was fixed under pressure using 4% paraformaldehyde in PBS, while the right lung was promptly frozen, and stored at −80 °C for subsequent detection assays.

2.9. Quantitative histomorphometric analysis

The left lung was fixed in formaldehyde and embedded in paraffin according to established protocols outlined in prior studies conducted by our group [19]. Serial 5-μm-thick sections were stained with hematoxylin and eosin (H&E) sequentially. The images from randomly selected non-overlapping fields, avoiding blood vessels, large airways, and small airways were captured using an inverted microscope (Houston, USA). Each experimental group comprised three pups, with morphometric analysis conducted on three random fields per pup. Onto the image, the mean linear intercept (MLI) was calculated by overlaying a pre-determined grid, consisted of randomly positioned lines totaling 1 mm in actual length. The number of times these lines intersected the air-tissue interface, terminal airspaces and secondary septa were manually counted.

2.10. Transmission electron microscopy (TEM)

The lungs were excised at P7 which fixed with 2.5% glutaraldehyde solution, dehydrated. The ultrathin sections were first mounted onto copper grids, then stained with uranyl acetate and lead citrate prior to imaging with a HT7800 TEM (HITACHI, Tokyo, Japan).

2.11. Cell culture

For the in vitro experiments, murine lung epithelial cells (MLE12) were cultured in DMEM medium containing 10% (v/v) FBS, for exposed to 21% as the control group and 85% oxygen as HYX group.

The concentration of chloroquine (CQ, MCE, Cat No.: HY-17589 A) used was 50 μM to inhibit autophagy [20]. Eltrombopag, a potent TFEB inhibitor, was employed at a concentration of 10 μM to suppress TFEB transcriptional activity by hindering the autophagy response induced [21].

2.12. Western blotting analysis

By lysing with RIPA lysis buffer and centrifugating at 13,000 g for 25 min for the supernatant, the concentrations of proteins from lung tissues and cells were detected by the bicinchoninic acid assay (BCA Protein Assay Kit, Byeotime, Cat No.: P0010). Nuclear and cytoplasmic proteins could be easily extracted from cultured cells by the Nuclear Protein and Cytoplasmic Protein Extraction Kit (Beyotime, Cat No.: P0027). The proteins mixed with SDS loading buffer were separated on 12.5% SDS-polyacrylamide gels after incubation, transferred to polyvinylidene difluoride membranes with a pore size of 0.45 μm, and then blocked with 5% bovine serum albumin. Sequentially, the membranes were incubated with primary antibodies against Bax (CST, 2772, 1:2000), Bcl-2 (CST, 3498, 1:2000), cleaved caspase-3 (CST, 9661, 1:2000), LC3 (CST, 12741, 1:2000), p62 (CST, 5114, 1:2000), β-actin (Servicebio, GB15001-100, 1:10,000), TFEB (Abcam, ab245350, 1:1000) and Lamin B (Servicebio, GB111802-100, 1:10,000) at 4 °C overnight respectively, and with corresponding IgG-HRP secondary antibodies (Proteintech, 1:20,000) at room temperature for 2 h. The images of protein bands were captured by a Tanon 5200 imaging system. Densitometric analysis was conducted using the ImageJ software (Media Cybernetics, Silver Spring, USA).

2.13. Cell counting kit-8 (CCK-8) assay

The MLE12 cells, exhibiting robust growth, were plated at a density of 5 × 10^3 cells per well in 96-well plates. Subsequently, ITA treatments were performed at concentrations of 10, 20, 40, 60, 80, and 100 μM for 24 h. Then, 10 μL of CCK-8 assay solution (Beyotime, C0038) was added to each well and incubated for 4 h. Subsequently, the absorbance of each well at 450 nm was measured using Thermo FC microplate reader (CA, USA).

$$Cellproliferationrate(\%)=\frac{ncellsat{t}_n–ncellsat{t}_0}{ncellsat{t}_0}\times 100\%\text{.}$$

2.14. Immunofluorescence staining

Sections were incubated with primary antibodies against AQP-5 (Affinity, AF5169, 1:200), SP-C (Affinity, DF6647, 1:200), LC3 (CST, 12741, 1:200) or cleaved caspase-3 (CST, 9661, 1:200) for 60 min, and therewith incubated with secondary coralite488-conjugated anti-rabbit IgG (Proteintech, SA00013-2, 1:10000) or coralite594-conjugated anti-mouse IgG antibodies (Proteintech, SA00013-3, 1:10000) for 60 min. The cells were counterstained with DAPI (Servicrbio, G1012, 1 μg/mL), and observed under a fluorescence microscope (Leica Microsystems).

2.15. Statistical analysis

Statistical analyses were conducted by IBM SPSS Statistics 26 (Chicago, IL, USA) and GraphPad Prism 7 (GraphPad Software, Boston, USA). Data were expressed as mean ± SD. In the analysis of the relationship between population data and metabolites, the χ² or Fisher's exact test was used for categorical variables, while the Kruskal-Wallis test was used for continuous variables. Spearman's rank correlation coefficient was utilized to determine the correlations between metabolites and the outcomes of premature infants. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was utilized for pathway evaluations. In experiments involving animals and cells, For paired variables, group differences were analyzed using a two-tailed Student's t-test, while comparison of multiple variables was conducted using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3. Results

3.1. ITA is an important biomarker for the development of BPD

To discern disparities in infant metabolites between the non-BPD and BPD groups (Fig. 1A), a series of multivariate variable pattern recognition analyses were performed. Table 1 displays a comparison of clinical data between the two groups of the premature infants. The levels of more than 300 metabolites in the peripheral blood were measured using targeted metabolomics, and their correlations were evaluated in combination with clinical respiratory system-related outcomes. Distinct metabolic profiles were observed in the OPLS-DA score plots between the non-BPD and BPD groups (R2Y = 1, Q2Y = 0.417, R2X = 0.509; Fig. S1A). Exploration of the function of the differential metabolites revealed enrichment in the TCA cycle, as indicated by KEGG pathway analysis (Fig. S1B). There were significant differences in some TCA cycle-related metabolites in patients with BPD, including cis-aconitic acid, ITA, and isocitric acid (Fig. 1B and C). Hierarchical cluster analysis was utilized to visualize the correlation between metabolites and clinical characteristics, employing Spearman's correlation coefficients (Fig. 1D–F). ITA levels were significantly correlated with BPD and high respiratory readmission rates (Fig. 1E–G). Collectively, metabolic dysregulation in the TCA cycle significantly affects lung development, prompting us to investigate the role of ITA in BPD development.

Fig. 1.

The differential metabolites and pathways significantly varied between the Non-BPD and BPD groups. A Flow chart of two groups (Created with BioRender.com). B The heatmap represents upregulated and downregulated metabolites in the TCA cycle. Elevated the levels of metabolites are represented by red and blue colors, respectively. C The concentration (μmol/L) of differential metabolites in the TCA cycle. *p < 0.05 (Created with BioRender.com). D Clustering heatmap based on Spearman's correlation analysis. The horizontal direction shows the differential metabolites, and the longitudinal direction shows the clinical characteristics and short-term outcomes. Red indicates a positive correlation, and blue shows a negative correlation. *p < 0.05; **p < 0.01. E ROC of plasma metabolic biomarkers and clinical dates to predict the BPD at 36 w PMA. F Clustering heatmap based on Spearman correlation analysis. The horizontal direction shows the differential metabolites, and the longitudinal direction shows the clinical characteristics and long-term outcomes in the first 2 years of life. Red indicates a positive correlation, and blue shows a negative correlation. *p < 0.05; **p < 0.01. G ROC of plasma metabolic biomarkers and clinical dates to predict the high respiratory readmission rate in the first 2 years of life. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 1.

Clinical characteristics of the infants with and without BPD.

Characteristics	BPD (n = 12)	No BPD (n = 12)	p-value
GA weeks, median (IQR)	27.1 (26.7, 28.5)	26.6 (25.7, 28.4)	0.33
BW g, mean ± SD	1050.0 ± 155.3	1033.3 ± 202.1	0.82
SGA, n (%)	1 (8.3%)	1 (8.3%)	1.00
Males, n (%)	6 (50.0%)	5 (41.7%)	1.00
Singleton, n (%)	8 (66.7%)	9 (75.0%)	1.00
Cesarean delivery, n (%)	2 (16.7%)	3 (25.0%)	1.00
Gestational hypertension, n (%)	0	1 (8.3%)	1.00
Maternal GDM, n (%)	1 (8.3%)	1 (8.3%)	1.00
Apgar at 5 min ≤7, n (%)	5 (41.7%)	8 (66.7%)	0.41
Surfactant treatment, n (%)	7 (58.3%)	9 (75.0%)	0.67
hsPDA, n (%)	1 (8.3%)	2 (16.7%)	1.00
Days on oxygen, days, mean ± SD	53.6 ± 16.7	83.3 ± 28.2	＜0.01**
Late-set sepsis, n (%)	4 (33.3%)	4 (33.3%)	0.15
NEC (≥stage Ⅱ), n (%)	2 (16.7%)	1 (8.3%)	1.00
IVH (III-IV)/PVL, n (%)	2 (16.7%)	2 (16.7%)	1.00
NICU days, mean ± SD	73.4 ± 20.0	88.8 ± 25.9	0.12
Weight at discharge, g, mean ± SD	2429.7 ± 444.8	2527.3 ± 304.5	0.54
EUGR at discharge, n (%)	9 (75.0%)	8 (66.7%)	1.00
Home oxygen, n (%)	0	3 (25.0%)	0.22

GA, gestational age; BW, birth weight; SGA, small for gestational age; GDM, gestational diabetes mellitus; hsPDA, hemodynamically significant PDA; NEC, necrotizing enterocolitis; IVH, intraventricular hemorrhage; EUGR, Extrauterine growth retardation. **p < 0.01.

3.2. ITA improves lung development in hyperoxia-induced BPD rat

Based on the hyperoxia-induced BPD animal model (Fig. 2A), no statistically significant difference in body weight and mortality between the BPD and control group was observed (Fig. 2B). The lung tissue of BPD rat exhibited characteristics of alveolar simplification, including enlarged alveoli with reduced the numbers of terminal airspace and secondary septa. We investigated the impact of ITA intervention on hyperoxia-induced alveolar simplification (Fig. 2C). ITA treatment group showed significantly increased the numbers of terminal airspace and secondary septa and significantly reduced the MLI, compared to that in BPD group. In addition, ITA increased the expression level of SP-C, as a specific marker representing alveolar type II epithelial cells (AEC II), whereas there was no noticeable change in the expression level of AQP-5, as a specific marker representing alveolar type I epithelial cells (Fig. 2D). These findings imply that ITA effectively alleviates hyperoxia-induced lung alveolar structure simplification in BPD. Furthermore, ITA is associated with AEC II in the development of BPD.

Fig. 2.

Analysis of lung development; itaconic acid (ITA) improves the alveolar simplification in BPD rats. A Flow chart of hyperoxia-induced BPD rat model. Control group pups are housed in room air (21%), BPD group pups are housed in O₂-enriched air (85%) and injected daily with solvent, and age-matched O₂-exposed (85%) pups were injected daily with itaconic acid (ITA; 100 mg/kg) until 14 days of age (Created with BioRender.com). B The body weights and mortality in BPD VS control group. C Newborn rats are sacrificed on the 7th and 14th day post birth. Lung morphometry, including the quantification of terminal air spaces, secondary septa counts, and mean linear intercept, is analyzed using hematoxylin and eosin staining of representative lung tissue sections. N = 3 animals/group. Bar = 100 μm. Values are expressed as mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. D Immunofluorescence staining of SP-C (red) and AQP-5 (green) in lung tissues at P7. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. ITA inhibits hyperoxia-induced cell apoptosis

Evaluating the expression of cleaved caspase-3 in the lung by immunofluorescence staining, ITA could inhibit the apoptosis was observed (Fig. 3A). The expression of cleaved-capase-3 in the lungs of BPD rats was increased in hyperoxia, which could be reduced after ITA treatment, as seen in Fig. 3A. In the hyperoxia-induced cellular model of MLE 12, the optimal therapeutic concentration of ITA selected by CCK-8 was 10 μmol/L (Fig. 3B). ITA also suppressed cleaved caspase-3 levels in MLE12 cells (Fig. 3C). In addition, the levels of Bcl-2, Bax and cleaved caspase-3 in lung tissues (P7) and MLE12 cells were also detected by western blotting for assessing the extent of apoptosis (Fig. 3D and E). Consistently, it was confirmed that ITA upregulated Bcl-2 expression while downregulating the expression of Bax and cleaved-caspase-3. The phenomemon indicates that the exogenous administration of ITA can successfully reverse severe apoptosis in vitro and in vivo.

Fig. 3.

ITA inhibits hyperoxia-induced cell apoptosis. A Representative histological images of lung tissues obtained through immunofluorescence staining, depicting cleaved caspase-3 (green), SP-C (red), and DAPI (blue). Bar = 0.05 mm. B The viability of MLE12 cells incubated with varying concentrations of ITA, was evaluated using the CCK-8 kit following the manufacturer's protocol. C Immunofluorescence staining of MLE12 cells with cleaved-capase 3 (red) and DAPI (blue) 24 h after ITA treatment (10 μmol/L). Bar = 100 μm. D Western blotting analysis of cleaved caspase-3, Bax, Bcl-2 in BPD rat lungs at P7 and MLE12 cells. E Densitometric analysis of Western blot bands, normalized to β-actin (n = 3). Data are shown as the mean ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. ITA promotes the mitophagy to maintain the mitochondrial balance

To gain further insight into how ITA regulates apoptosis, mitochondrial changes in AEC II cells were identified using TEM. As depicted in Fig. 4A, the primary changes of AEC II ultrastructure in the BPD group were nuclear chromatin condensation, swollen mitochondria, and reduction in the number of lamellar bodies. ITA treatment attenuates the mitochondrial change dramatically, given its important homeostatic role in the mitochondria. The replacement or repair of dysfunctional mitochondria may contribute to the recovery of organ function during hyperoxia. LC3, a key participant in mitophagy, undergoes degradation from the protein complex LC3 I to LC3 II upon initiation, subsequently recruited to autophagosomes and interacting with p62. The formation of the lysosomal complex ultimately results in total proteolysis [22]. Immunofluorescence staining experiments showed that the accumulation of LC3 was increased by ITA treatment in lung tissues (Fig. 4B) and MLE12 (Fig. 4C) with hyperoxia. Activation of the LC3 protein and expression of P62 were markers of mitophagy, and ITA treatment increased the level of LC3 II/I and reduced the level of p62 (Fig. 4D and E). The mitophagy pathway plays a significant role in cell death, and these results suggest that ITA induces mitophagy in hyperoxia-induced dysfunctional mitochondria to maintain mitochondrial mass balance.

Fig. 4.

ITA promotes the mitophagy through LC3/P62. A Samples were subsequently examined using electron microscopy. Red arrows indicate laminar bodies. Black arrows indicate mitochondria. B Representative histological images of immunofluorescence staining of lung tissues for LC3 (green), SP-C (red), and DAPI (blue). Bar = 0.05 mm. C Fluorescence staining of MLE12 cells with LC3 (green), SP-C (red), and DAPI (blue) 24 h after ITA treatment (10 μmol/L). Bar = 100 μm. D Western blotting analysis of P62 and LC3II/I in BPD rat lungs and MLE12 cells. E Densitometric analysis of Western blot bands, normalized to β-actin (n = 3). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01, and ****p < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.5. The regulation of mitophagy by ITA can be blocked by CQ

Immunofluorescence staining revealed markers of mitophagy and apoptosis. Currently, the antimalarial drug, CQ, is the only clinically available drug that inhibits autophagy [23]. Mitophagy was blocked by CQ, and the effects of ITA on suppressing apoptosis were blocked in vitro and in vivo. The level of LC3 and cleaved caspase-3 in the BPD lung increased, while the level of LC3 was increased and cleaved caspase-3 was inhibited after ITA treatment. This finding indirectly indicates that ITA regulates apoptosis by critically impacting mitophagy. Then, CQ inhibits ITA-induced mitophagy and increases the degree of apoptosis (Fig. 5A). Similar phenomena were also observed in MLE12 cells (Fig. 5B).

Fig. 5.

The regulation of mitophagy by ITA can be blocked by chloroquine. A Representative histological images from immunofluorescence staining of lung tissues for cleaved LC3 (green), Cleaved caspase-3 (red), and DAPI (blue). Bar = 0.05 mm. B Immunofluorescence staining of MLE12 cells with LC3 (green), Cleaved caspase-3 (red), and DAPI (blue). Bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.6. ITA promotes nuclear translocation of TFEB to facilitate autophagy flux for dysfunctional mitochondria clearance

Furthermore, to investigate autophagosome maturation and disintegration, a robust autophagic flux reporter system was employed. This system is based on tandem fusions of LC3 with acid-sensitive GFP and acid-insensitive mRFP. Initially, the GFP and mRFP fluorescence are visible at the same time as yellow dots in the autophagosome. For the degradation of GFP by acidic lysosomal proteases during the fusion of autophagosomes with lysosomes, the green fluorescence is extinguished. This results in LC3 emitting only red fluorescence [24]. On this basis, MLE12 cells stably expressing mRFP-GFP-LC3 were generated. Compared to the hyperoxia group, the fluorescence observed following ITA intervention, exhibited an increase in red and yellow dots, respectively representing autophagolysosomes and autophagosomes (Fig. 6A). ITA induces the nuclear translocation of TFEB by modifying it at the Cys212 site, and nuclear TFEB subsequently coordinates lysosomal biogenesis to aid in bacterial clearance [25]. As shown in Fig. 6A, eltrombopag, the TFEB inhibitor suppressing the transcriptional activity, led to a decrease in yellow and red dots, which suggests that the autophagy flux was blocked. Moreover, the levels of TFEB in cytoplasm and nucleus were detected by western blotting. ITA increased the level of TFEB in the nucleus (Fig. 6B and C). Eltrombopag reduced the degree of mitophagy (Fig. 6D and E), which reveals the mechanism that ITA promotes autophagy flux and dysfunction of mitochondria.

Fig. 6.

ITA promotes nuclear translocation of TEFB to facilitate autophagy flux for dysfunctional mitochondria clearance. An Immunofluorescence staining of MLE12 cells with LC3 of mitochondrial membrane (green), LC3 of lysosomes (red) and DAPI (blue). Eltrombopag, Elt. Bar = 10 μm. B Western blot analysis of the levels TFEB of cytoplasm and nucleus in MLE12 cells. Densitometric analysis of Western blot bands, normalized to β-actin and Lamin B (n = 3). C Densitometric analysis of Western blot bands, normalized to β-actin or Lamin B (n = 3). Data are shown as the mean ± SD. D Western blotting analysis for the levels LC3 II/LC3 I and P62 in MLE12 cells. E Densitometric analysis of Western blot bands, normalized to β-actin (n = 3). Data are shown as the mean ± SD. *p < 0.05, and **p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

BPD, a prevalent chronic lung condition, significantly impacts the long-term quality of life of premature infants. Emerging evidence indicates that premature infants often undergo dysregulated metabolism involving glucose, lipids, and amino acids. Animal studies and some cell-based research have highlighted that such metabolic imbalances often lead to apoptosis, inflammation, abnormal proliferation, or senescence in response to oxygen stress. Moreover, correcting these metabolic dysfunctions has been demonstrated to alleviate hyperoxia-induced lung injury. In our study, we present firstly the regulatory mechanism of TCA-cycle-related metabolite ITA through TFEB-mediated autophagy flux to alleviate hyperoxia-induced BPD. Following a combination of clinical study and the hyperoxia-induced BPD animal model, we observed that the accumulation of ITA in AEC II improved lung development.

Oxygen therapy is routinely administered to enhance the survival of premature infants; nevertheless, it also elevates the potential for oxygen toxicity across multiple organs. Previous research has underscored the impact of hyperoxia in prompting alterations in metabolic pathways via a phenomenon known as metabolic reprogramming [26,27]. Dysregulated metabolic processes, including abnormalities in lipid, amino acid, and glucose metabolism, have been shown in recent studies to influence the onset and progression of hyperoxia-induced neonatal diseases [1]. After exposure to hyperoxia, human umbilical vein endothelial cells from infants with BPD exhibited lower basal and maximal rates of oxygen consumption compared to healthy infants. This suggests an increased reliance on glycolysis [28]. Herein our aim was to evaluate the significance of metabolism and the outcomes of premature infants. Our findings indicate that dysregulated activation of the TCA cycle serves as a negative regulatory mechanism for human lung development. Intriguingly, along with functioning as a metabolic marker for the diagnosis of BPD, ITA also plays a crucial role in predicting outcomes of lung development.

Increasing evidence suggests that dysregulated metabolism acts as a disruptor of lung homeostasis, precipitating a cascade of complex physiological and pathological alterations. These include abnormal polarization and migration of alveolar macrophages, impairment of ciliary function, and induction of cellular senescence in alveolar epithelial cells. Such dysregulation contributes to the development of BPD [8]. The observation that ITA alleviates hyperoxia-induced alveolar structural simplification both in vivo and in vitro models is intriguing, and prompts its potential application in future prevention and treatment of BPD. Recently, metabolites such as ITA have garnered research interest, particularly for their anti-inflammatory roles in immune processes [29,30]. The mechanisms underlying the regulation of inflammatory response and oxidative stress by ITA, as discussed earlier [31], include regulating the transcription of the ATF3/IκBζ axis, the protein modification of JAK1/STAT6 pathway, and glycolytic enzyme metabolic action. In the context of cancer, ITA have been shown to have tumor-promoting properties [32]. Recently, investigators have developed an injectable hydrogel using ITA to create optimized microenvironments for cardiac stromal cells, with the goal of promoting heart repair after myocardial infarction [33]. In this study, we explored whether ITA can not only be used as a metabolic marker of BPD and whether it can regulate apoptosis in AEC II.

Our results are important when considering how extracellular ITA may target AEC II. Mitochondria, serving as the sites of the TCA cycle, play a crucial role in energy metabolism. Mitochondrial fission is critically important for controlling the generation of reactive oxygen species (ROS), cell proliferation, and mitochondrial quality control through mechanisms such as mitophagy and apoptosis [28]. This process aids in preventing excessive ROS generation, which occurs due to the accumulation of dysfunctional mitochondria under various adverse conditions [34]. Alveolar epithelial cell apoptosis is closely related to mitochondrial dysfunction [35]. Our data demonstrated that the inhibition of apoptosis in AEC II by ITA improved the simplified alveolar structure of BPD through efficient and prompt clearance of dysfunctional mitochondria. Mechanistically, ITA mainly promotes the unclear translocation of TFEB to facilitate dysfunctional mitochondrial clearance and reduces apoptosis in AEC II by regulating autophagy flux. Researchers have indirectly demonstrated that ITA induces the nuclear translocation of TFEB by modifying it at the Cys212 site, and nuclear TFEB subsequently coordinates lysosomal biogenesis to aid in bacterial clearance [25]. However, this study did not directly knock out the site interacting with ITA. We attempted to block the nuclear translocation of TFEB to see if the ITA effect was affected by eltrombopag, the TFEB inhibitor suppressing the transcriptional activity. This part of the experiment also provided a supplementary evidence for proving the mechanism of action of ITA in previous experiments.

ITA, as a carboxylic acid, is highly polar and is not easily permeable to cell membranes [30], making it not suitable for the intervention in both in vivo and in vitro models. To address this limitation, 4-OI, a cell-permeable itaconate derivative, was synthesized as a suitable substitute for ITA. However, we did not measure the concentration of ITA in the blood and lung tissues of the BPD rats following ITA supplementation. This aspect could have provided a deeper understanding of the relationship between exogenous ITA supplementation and endogenous ITA conversion. We recognize this as a limitation of our study and suggest that further exploration is warranted to investigate potential correlations. Moreover, precise verification of appropriate concentrations and intervention methods of ITA is still needed, particularly considering the potential toxicity under specific metabolic conditions during the neonatal period, as no relevant studies are currently available. This necessitates further in-depth investigation, which remains a focal point of our group. A more comprehensive understanding of the role of ITA in lung development will offer greater theoretical support for future clinical applications in the prevention and treatment of BPD.

5. Conclusion

Our study revealed a new function of ITA, as a regulator of apoptosis by promoting TFEB-mediated autophagy flux in AEC II. Apart from its anti-inflammatory effect, we identified an expanded role of ITA as a predictive and therapeutic agent for the lung development, underscoring the use of ITA as a therapeutic administration to improve lung development in BPD patients.

Disclosure of interest

The authors have indicated they have no potential conflicts of interest to disclose.

Funding

This work was supported by the National Natural Science Foundation of China [Grant No. 82371721 to Xingyun Wang, No.82271745 to Yongjun Zhang]; Shanghai Rising-Star Program [Grant No. 22QB1401000 to Xingyun Wang, No. 20YF1429200 to Chengbo Liu].

Data statement

Data will be made available on request.

CRediT authorship contribution statement

Chengbo Liu: Writing – review & editing, Writing – original draft, Validation, Methodology, Funding acquisition, Data curation, Conceptualization. Changchang Fu: Visualization, Validation, Project administration, Conceptualization. Yazhou Sun: Visualization, Validation, Investigation, Data curation, Conceptualization. You You: Software, Data curation. Tengfei Wang: Validation, Project administration. Yongjun Zhang: Supervision, Project administration, Funding acquisition. Hongping Xia: Supervision, Project administration, Conceptualization. Xingyun Wang: Writing – review & editing, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors have indicated they have no potential conflicts of interest to disclose.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

figs1.

Data availability

Data will be made available on request.