Introduction
Chest radiotherapy is critical in patients with thoracic and breast malignancies. However, radiation-induced damage to the lungs remains an important barrier to better implementation of radiotherapy. Radiation-induced lung injury (RILI) can be divided into early radiation pneumonitis (RP) and late radiation-induced pulmonary fibrosis (RPF) [
1]. RILI is associated with an increased risk of death, disability, and a decline in quality of life [
2]. RPF is characterized by fibroblast proliferation, collagen deposition, and destruction of normal lung architecture, resulting in dyspnea and respiratory failure [
3]. Given the increasing number of long-term cancer survivors, it’s of vital importance to mitigate or prevent late effects of radiotherapy. However, the biology and molecular mechanisms of RPF have not been fully elucidated.
Inflammation is a key element of RILI. Inflammasomes are essential for innate immunity and responses to cellular damage. The activation of inflammasomes leads to inflammatory death called pyroptosis [
4]. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP)3 inflammasome is the most widely studied inflammasome and can be activated by an array of stimuli, and has been linked to the pathogenesis of several inflammatory disorders, including cryopyrin-associated periodic syndromes, Alzheimer’s disease, gout, autoinflammatory diseases, and atherosclerosis [
5]. Previous studies have also shown that NLRP3 inflammasome is involved in fibrotic diseases, including idiopathic pulmonary fibrosis (IPF) [
6]. NLRP3 interacts with a bipartite adaptor protein, known as an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), and promotes the recruitment of pro-caspase-1 (CASP1) to the inflammasome complex. Active CASP1 then cleaves the cytokines pro-interleukin (pro-IL)-1β and pro-IL-18 into mature and biologically active forms [
7]. Cytokine interleukin (IL)-1β is a potent pro-inflammatory cytokine that has been proven to promote collagen synthesis [
8]. IL-1β has been shown to play a role in the occurrence, invasion, and metastasis of multiple kinds of tumors [
9]. Whether inhibiting the NLRP3 inflammasome has the potential to mitigate radiation damage to normal lung tissue and to inhibit further progression of tumors remains to be explored.
Some studies have suggested the involvement of inflammasomes in RILI, but they have mainly focused on macrophages [
10‐
12]. The role of lung epithelial cells in RILI remains to be elucidated. Airway epithelial cells are considered the host’s first line of defense against harmful invasion and participate in the initiation and progression of inflammation. Airway epithelial cells express a large number of pattern recognition receptors, which can quickly perceive pathogen-associated molecular patterns as well as damage-associated molecular patterns released by damaged tissue, and then make a series of responses, such as the release of cytokines and chemokines [
13]. In asthma, airway epithelial cells sense allergens and recruit immune cells. Recruited immune cells secrete a large number of chemokines and cytokines that cause further damage to epithelial cells, resulting in the enhancement and persistence of the inflammatory response [
14]. Therefore, studying the role of inflammasomes in the airway epithelium in RILI may provide new insights for the prevention of epithelial dysfunction.
Metabolic alterations are increasingly being recognized as important pathogenic processes that underlie fibrotic diseases [
15]. Glucose metabolism begins with its conversion into pyruvate and ends with lactic acid production in the cytoplasm, termed glycolysis [
16]. The metabolic shift that cancer cells undergo towards aerobic glycolysis, rather than being fully metabolized to carbon dioxide via mitochondrial oxidative phosphorylation (OXPHOS), was identified as the Warburg effect [
17]. The Warburg effect results in quicker production of biosynthetic intermediates, as well as adenosine 5′-triphosphate (ATP), which can then be used for protein synthesis and cell proliferation. Similarly, enhanced protein synthesis and production of the same biosynthetic intermediates are hallmark of fibrosis [
18]. In addition, the Warburg effect leads to IPF by enhancing myofibroblast activation [
19]. Therefore, targeting the Warburg effect may be a potential therapeutic strategy for fibrosis. However, its role in RPF remains unclear.
Here, we aimed to differentiate the radiation response between lung tumor cells and normal lung epithelial cells, and investigated the function of NLRP3 inflammasomes in lung epithelial cells in RILI. Notably, we preliminarily revealed the relationship between glucose metabolism and inflammasome activation in RILI. These data provide evidence that NLRP3 may serve as a promising therapeutic target in RILI.
Methods and materials
Cell isolation and culture
The human bronchial epithelial cell line BEAS-2B and HBE, non-small cell lung cancer cell line A549, and small cell lung cancer cell line H446 were purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China) and they were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% carbon dioxide.
. Primary human bronchial epithelial cell (PHBE) cells and primary lung fibroblasts (HLF) were isolated and cultured from freshly surgically removed human lung tissue as previously described [
20]. Primary mouse embryo fibroblasts (MEF) were isolated from embryos of pregnant C57/BL6 mouse. The uterus was removed and placed in balanced phosphate solution. After removing the head and other internal organs, the embryos were cut into small pieces. The tissue was centrifugated at 1000 rpm/5 min and resuspended the in trypsin, and then was digested in a 37 °C incubator for approximately 30 min. The cells were centrifuged at 1000 rpm for 5 min and cultured in RPMI-DMEM containing 10% FBS.
Human lung specimens
Fibrotic lung specimens were obtained from 8 patients with organizing pneumonia who underwent partial lung resection. Control lung tissue was obtained from 11 para-cancerous tissues of patients with lung cancer. The use of these samples was approved by the ethics review board.
Irradiation of cells
The cells were irradiated with X-ray radiation by using the Varian Vital Beam Linear Accelerator (Varian Medical Systems) (dose rate of 4 Gy/min, source–surface distance of 100 cm). The selection of radiation dose was based on our previous studies and other publications [
21‐
23].
Quantification of cytokines
Bronchoalveolar lavage fluid (BALF) and serum samples of mice were collected and centrifuged. The supernatants were used to quantify IL-1β and IL-18 levels. IL-1β and IL-18 levels in the culture supernatants of cells, BALF, or serum of mice were measured by enzyme-linked immunosorbent assay (ELISA) (Proteintech) according to the manufacturer’s instructions.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total ribonucleic acid (RNA) was extracted from the cells using TRIzol reagent (Invitrogen) according to standard procedures. qRT-PCR was performed using SYBR Premix Ex Taq reagents (TaKaRa) in two steps according to the manufacturer’s instructions. Primer sequences used are listed in Additional file
2: Table S1.
Intracellular reactive oxygen species (ROS), ATP and nicotinamide adenine dinucleotide phosphate (NADP)+/NADP(H) measurement
Intracellular ROS were detected using the fluorescent probe DCFH-DA (Beyotime). Briefly, lung epithelial cells were stained with DCFH-DA for 30 min, harvested, centrifuged, and washed with phosphate-buffered saline (PBS). The stained cells (approximately 105 cells/sample) were analyzed by flow cytometry using a FACSCalibur system. Intracellular ATP and NADP+/NADPH were detected using ATP Assay Kit (Beyotime) and NADP+/NADPH Assay Kit with WST-8 (Beyotime), respectively, according to the manufacturer’s instructions.
Measurement of lactic acid secretion and glucose uptake
Culture supernatants of irradiated lung epithelial cells were collected at different time points, and lactic acid and glucose levels were measured using a lactic acid assay kit (Nanjing Jiancheng) and Glucose Assay Kit (Nanjing Jiancheng), respectively, according to the manufacturer’s instructions.
EdU essay
The EdU Cell Proliferation Kit (Beyotime) was used to detect the proliferation ability of fibroblasts, following the manufacturer’s instructions. Briefly, 24 h after IL-1β treatment, EdU was added to the cells and incubated for 2 h, after which the cells were fixed and washed again. Following permeabilization, the click additive solution was added to the cells and incubated for 30 min. Finally, cells were stained with Hoechst 33,342 for 10 min, observed, and photographed under a microscope.
Western blotting
Western blot analysis was performed according to standard procedures, as previously described [
21]. The following primary antibodies were used: anti-NLRP3 (1:1000, Abcam), anti-ASC (1:1000; Proteintech), anti-GSDMD (1:1000; Abcam), anti-CASP1 (1:1000, Proteintech), anti-DPYSL4 (1:1000; Abcam), anti-p53 (1:1000; Santa Cruz), and anti-β-actin (1:5000; Proteintech). Following incubation with the primary antibody, the membranes were washed and probed with secondary antibodies (1:5000; Proteintech) for 1 h. Proteins were visualized by chemiluminescence.
Silencing of NLRP3, DPYSL4 and IL-1β by small interfering RNA (siRNA)
Transfection of NLRP3, DPYSL4, or IL-1β siRNA and negative control siRNA was performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Transfection efficacy was detected by qRT-PCR and western blotting 48 h post transfection. The siRNA sequences were as follows:
-
Scramble siRNA, 5′-UUCUCCGAACGUGUCACGU-3′
-
SiNLRP3, 5′-GCUUCAGGUGUUGGAAUUA-3′
-
SiIL-1β, 5′-CGAUUUGUCUUCAACAAGA-3′
-
SiDPYSL4, 5′-CGGUUCACACUACUGGAGCAA-3′
Histopathology and histochemistry
Serial paraffin sections of lung tissue were stained with Masson trichrome stain for analysis of pathological changes in the airway. Immunohistochemical (IHC) staining was performed as previously described [
24]. NLRP3 primary antibody (1:50; Novus) and DPYSL4 primary antibody (1:200; Abcam) was used. The IHC score was categorized as negative (1), weakly positive (2), moderately positive (3), and strongly positive (4).
Immunofluorescence
Approximately 2 × 104 BEAS-2B cells were seeded in 24 well plates coated with sterile glass coverslips. The next day the cells were given 10 Gy X-ray radiation, then the cells were fixed with 4% paraformaldehyde 24 h later. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 15 min. Next, the cells were blocked with 5% bovine serum albumin and incubated with primary antibodies (NLRP3: NOVUS, 1:100; ASC: Proteintech, 1:100) at 4 °C overnight. The next day, the cells were washed and incubated with secondary antibodies. The cells were then stained with DAPI, observed, and photographed under a microscope.
Lung cancer cells treated with MCC950 or PBS for 24 h were seeded in six-well plates (200 cells per well). After 2 weeks, the cells were fixed and stained with crystal violet, and colonies that consisted of at least 50 cells were counted.
In vivo experiments
C57BL/6 mice (6 weeks old, female) were raised at the standard specific pathogen free animal housing. IL-1 receptor (IL-1R) knockout mice were kindly provided by professor Feng Shao (National Institute of Biological Sciences, Beijing, China). All animal procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
To establish the RILI mouse model, mice were subjected to a single 16 Gy thoracic X-ray irradiation. Mice were sacrificed at different time points. Lung tissue, BALF, and serum were collected for further examination.
To deplete the lung macrophages of the mice, clodronate liposomes (FomuMax) were administered to C57BL/6 mice. Briefly, the mice received both intranasal and tail vein administration of the first dose, and then clodronate liposomes were administered through the tail vein every 2 days. The depletion efficiency was confirmed by flow cytometry.
For NLRP3 inhibition, the mice were given intraperitoneal injection of MCC950 (10 mg/kg) 24 h after 16 Gy thoracic radiation and continued for 2 months with injections every other day. Mice were sacrificed 5 months after radiation.
Genotyping of mice
Mouse tail deoxyribonucleic acid (DNA) was extracted using Quick Genotyping Assay Kit for Mouse Tails (Beyotime). Polymerase chain reaction (PCR) and gel electrophoresis were performed to detect the genotypes of the mice. The primer sequences used are listed in Additional file
2: Table S2.
ROS measurement of mouse lung tissue
The levels of ROS in lung tissues of mouse were measured with Reactive Oxygen Species Assay Kit (Applygen) according to the manufacture’s instruction. The single lung cell suspensions were incubated with the DHE fluorescent probe for 30 min at the final concentration of 10 µM. Then the cells were washed with PBS and the fluorescence intensity was measured.
RNA sequencing
Cells were lysed with TRIzol reagent and sent for transcriptome sequencing in liquid nitrogen (BGI, Shenzhen, China). Total RNA was extracted from cells. Oligo(dT)-attached magnetic beads were used to purify the mRNA. Single-stranded circular DNA was used as the final library. The final library was amplified with phi29 to form a DNA nanoball (DNB), which had more than 300 copies of one molecule. DNBs were loaded into the patterned nanoarray and single end 50 base reads were generated on the BGIseq500 platform (BGI; Shenzhen, China). The original RNA-seq data generated in the study has been uploaded to the Gene Expression Omnibus under registration number GSE211118.
Differential expression analysis was performed with a Q-value of < 0.05. In addition, functional enrichment analysis, including KEGG pathways, Gene Ontology [
25] enrichment, and gene set enrichment analysis (GSEA), was performed.
Statistical analysis
Experimental data are expressed as the mean ± standard error of the mean. Statistical differences among groups were assessed using SPSS 26.0 (IBM Corp; Armonk, NY). In all experiments, categorical variables between groups were compared using the χ2 test, and continuous variables were analyzed using Student’s t-test (two-tailed) or analysis of variance, as appropriate. All data met the assumptions of the tests, and statistical tests were justified, as appropriate. Differences were considered statistically significant at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). All experiments were repeated at least three times.
Discussion
RILI is one of the most common fatal complications of thoracic radiotherapy [
31]. Previous studies have mainly focused on how to increase the radiosensitivity of tumor cells, thus decreasing the total radiation dose to decrease radiation injury [
32]. However, most molecular targets that sensitize tumor cells to radiotherapy also increase damage to the normal lung epithelium.
This study investigated the different response patterns of normal lung cells and tumor cells to radiation, thus discovering potential radioprotective drugs. We reported tremendous differences in molecular pathway changes between normal and malignant lung cells after radiation damage using RNA-seq. Despite the different activation of p53 and cell cycle pathways. Surprisingly, the inflammatory response involving NLRP3 was only activated in lung epithelial cells but not in lung cancer cells after radiation. We found that NLRP3 is the highest-ranked inflammasome and its canonical pathway activation is associated with altered glucose metabolism. IL-1β, as the direct downstream protein of NLRP3, accelerates RILI by promoting fibroblast proliferation, migration, and activation as well as lung tissue remodeling. The repression of NLRP3 or IL-1β significantly reversed RILI in vitro and in vivo. All phenotypes were also observed in human-derived primary cells.
The tumor-killing effect of radiation can also damage the normal epithelium. The tolerable dose to normal tissues is a key issue limiting the implementation of radiotherapy. The concurrent combination of chemotherapy or immunotherapy and radiotherapy improves efficacy but also promotes the occurrence of RILI [
33]. Studies have revealed that the development of RILI involves multiple processes, including DNA damage and ROS generation, inflammatory cell infiltration, hypoxia, and lung tissue remodeling [
10,
31,
34]. To identify a potential protective agent against RILI, we analyzed the different response patterns of lung cancer and lung epithelial cells to radiation injury using RNA-seq. Consistent with previous studies, radiation triggers a series of DNA damage response pathways in cancer cells [
35,
36], including those helping cells recover from radiation injuries, such as activation of DNA damage sensing and early transduction pathways, cell cycle arrest, and DNA repair. The epithelium tends to undergo DNA damage-induced senescence and death, and triggers an inflammatory response. These differences provide a direction for us to seek potential agents for the prevention of RILI.
The NLRP3 inflammasome is widely expressed in epithelial and immune cells [
37]. It has been reported to play an important role in autoimmune and metabolic diseases by activation of CASP1 and production of IL-1β and IL-18. It is involved in the development of IPF and infective lung injury [
38]. However, their role in cancer remains controversial [
39]. Canonical activation of the NLRP3 inflammasome enhances the proliferation and metastasis of lung adenocarcinoma cells [
40]. It has also been shown to have an anti-tumorigenic role by promoting dendritic cell-mediated priming of IFN-γ-producing T lymphocytes against tumor cells [
41]. Activation of the NLRP3 inflammasome pathway has also been revealed in cancer-associated fibroblasts to functionally promote tumor progression and metastasis by modulating the tumor microenvironment towards an immunosuppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells [
42]. Consistent with previous findings, we found that MCC950, a specific small-molecule inhibitor of NLRP3, could significantly inhibit the proliferation of both small cell lung cancer (SCLC) and non-SCLC cells. Furthermore, it significantly alleviated lung fibrosis induced by radiation. Although the NLRP3 inflammasome was first identified and well-studied in immune cells, it can be activated by low-dose irradiation both in vitro and in vivo in macrophage [
43], and inhibition or deletion of NLRP3 can specifically alleviate radiation-induced lung inflammation in radiation [
10]. We reported that the NLRP3 inflammasome canonical pathway is activated in lung epithelial cells in response to radiation-induced damage, contributing to pulmonary fibrosis by activating fibroblasts. We used a macrophage scavenger to eliminate the effect of macrophages in the lungs in an in vivo mouse model of RILI. Mice eliminated with macrophages showed a higher level of IL-1β, suggesting that the role of macrophages may be protective in the early phase of RILI. The limitation here is that, since the differentiation of macrophages is heterogeneous, we did not distinguish the roles of different subtypes of macrophages. In addition, we only observed the early effects of macrophage depletion, and its role in the late phase of RILI remains unclear.
IL-1β is a classical downstream effector molecule of NLRP3, and previous studies have demonstrated the role of IL-1β in pulmonary fibrosis [
8]. Here, we demonstrated that it is the actual functional protein of NLRP3 that induces fibroblast proliferation and migration. We confirmed that the activation and accumulation of fibroblasts significantly induced the synthesis of type I collagen, which is a key element in pulmonary fibrosis. Another important feature of pulmonary fibrosis is pulmonary remodeling [
44]. We found that IL-1β induced TIMP-1 and MMP-3 expression, indicating the role of IL-1β in pulmonary remodeling. These data support the central role of IL-1β in RPF, making it an attractive novel therapeutic target.
We observed that early glycolysis products may be diverted to lactic acid instead of the citric acid cycle when in need of faster ATP production. Previous evidence suggests that alterations in glycolysis and glucose metabolism contribute to IPF progression [
45]. The Warburg effect induces myofibroblast differentiation and contributes to fibrosis in IPF [
15]. Lactic acid in the tumor microenvironment has been reported to suppress anticancer immunity [
46]. Therefore, targeting glucose metabolic dysregulation and suppressing the Warburg effect may be a promising future direction for RILI. We found that DPYSL4, a p53-inducible gene, was upregulated in irradiated epithelial cells. DPYSL4 is associated with mitochondrial supercomplexes, stimulates ATP production, and suppresses cancer cell invasion [
28]. Our study revealed that DPYSL4 has a protective role in irradiated epithelial cells by increasing ATP and NADPH levels and decreasing ROS levels, thus suppressing the occurrence of pyroptosis. Future research should focus on unveiling the genes and metabolites involved in glucose metabolism and their association with RILI.
Given the important role of NLRP3 and IL-1β in RILI development and progression, it is likely that repressing them using inhibitors may contribute to protecting lung function from radiation. IL-1β inhibitors, such as canakinumab, have been proven safe in lung cancer patients and have a survival benefit for certain groups [
47]. The relationship between metabolic alterations and inflammasome activation provides insight into the treatment and prediction model of RILI. This may be a new direction for research on the mechanisms of radiation damage and a novel target for developing new drugs in the future.
In conclusion, our data showed that glucose metabolism-related NLRP3 inflammasome activation plays a crucial role in RILI via IL-1β. This suggests the potential clinical application of IL-1β and NLRP3 inhibitors as radioprotective agents.
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