Monophosphoryl lipid A ameliorates radiation-induced lung injury by promoting the polarization of macrophages to the M1 phenotype

Adult male C57BL/6N mice (6–8-week-old, 18‐20 g) were purchased from Vital River Experimental Animal Company (Beijing, China). The protocols for animal experiments were approved by the Animal Care and Use Committee of the Academy of Military Medical Sciences (Beijing, China). The mice were maintained in rooms under a 12-h light/dark cycle, 20–26 °C, and at a relative humidity of 40–70%. The mice were acclimatized for one week and were fed enough food and water before the experiments.

Before irradiation, the mice were randomly divided into four groups as follows: normal control (CON group); MPLA without irradiation group (MPLA group); irradiation only group (IR group), and irradiation plus MPLA group (MPLA + IR group). MPLA was purchased from InvivoGen (Lot: 5978–42-01). Based on our previous research results[12], mice in the MPLA + IR group and MPLA group were injected intragastrically with 50 μg/kg MPLA dissolved in 0.1 mL phosphate buffer saline 12 h before irradiation. Mice in the IR group were injected with 0.1 mL phosphate buffer saline. In the previous research, the RILI model has been successfully constructed and verified many times [15]. Mice were then anesthetized by intraperitoneal injection with 1% pentobarbital sodium (50 mg/kg body weight) before thoracic irradiation (20 Gy) with a ⁶⁰Co γ-ray (Beijing Institute of Radiation) at a rate of 60.87 cGy/min. Except for the thorax, the other parts of the mice were covered with 10-cm-thick lead bricks.Lung tissue samples were collected at weeks 1, 4, 8, and 16 after radiation.

The mice alveolar epithelial cell (MLE‐12), the mice monocyte cell line (RAW 264.7) and the human lung epithelial cell (Beas-2B) were obtained from Otwo Biotech (Shenzhen, China). The cells were cultured in DMEM medium (10% fetal calf serum) at 37 °C in a 5% CO₂ humidified chamber. MLE-12 cells were radiated at dose of 8 Gy (clonogenic assay with 0 Gy, 2 Gy, 4 Gy, and 8 Gy) [16].

We used different culture supernatants as a conditioned medium to observe their effects on MLE-12 cells. We cultured the RAW 264.7 cells when the confluence was approximately 50–60%, replaced the original medium with DMEM medium, and continued to cultivate for 12 h. After this, we collected the supernatants and centrifuged them for 10 min at 2000 rpm, collected the supernatant as RAWsup; We added 1 µg/mL MPLA to the medium DMEM medium, then maintained cultured RAW264.7 cells for an additional 12 h. Next, collected the supernatants and centrifuged them for 10 min at 2000 rpm, the supernatant was MPLAsup; Unlike MPLAsup, we cultured RAW264.7 for 2 h with a medium containing GW4689 of 20 μM concentration before using MPLA [17, 18], the following steps were the same as the MPLAsup, we collected the supernatant as (GW4869 + MPLA)sup.

The cell proliferation potential was assessed based on the survival rate of the clones. Specific number of cells was seeded in 6-well plates and irradiated with 0 Gy, 2 Gy, 4 Gy, and 8 Gy. After 7–10 days of incubation, the plates were fixed with paraformaldehyde, stained with 1% crystal violet for 30 min, washed with PBS, and colonies (≥ 50 cells as a colony) counted under a dissecting microscope. The surviving fraction (SF) curve was based on the multi-target single-hit model.

FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) was used for analysising cell appotosis. After different experimental conditions, the MLE-12 cells were washed twice with cold PBS and resuspended in 1X Binding Buffer at 1 × 10⁶ cells/mL. Thereafter, 100 μL of the solution (1 × 10⁵ cells) was transferred into a 5 mL culture tube before adding 5 μL of FITC Annexin V and 5 μL of PI. The cells were gently vortexed and incubated for 15 min at 25 °C in the dark. Then, 400 μL of 1X Binding Buffer was added to each tube. Apoptosis rate analysis was performed within 1 h using flow cytometry.

Protein extracts were prepared from a different group of MLE-12 cells. The samples were incubated for 2 h with primary antibodies against TLR4 (proteintech, 1:5000), γ-H2AX (Cell Signaling Technology; 1:1000), E-cadherin (Cell Signaling Technology, 1:1000), Vimentin (Cell Signaling Technology, 1:1000), and α-SMA (Cell Signaling Technology, 1:1000) in 5% nonfat milk. The samples were then incubated with HRP-conjugated IgG (proteintech, 1:5000) for 1 h.

Immunofluorescence assay was used to detect the number of γ-H2AX foci. Cells were seeded in 24-well plates. After washing with PBS, cells were fixed in 3% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS. The cells were then stained with γ-H2AX primary antibody (Cell Signaling Technology; 1:200) and thereafter the secondary antibody (fluorescein goat anti-rabbit IgG, Invitrogen; 1:1000). Cells were stained with a DAPI (Antifade Mounting Medium with DAPI, Beyotime). The images were captured using an Olympus BX60 fluorescent microscope (Olympus America Inc).

The serum levels of tumor necrosis factor-alpha (TNF-α), transforming growth factor-β (TGF-β), IL-1β, and IL-10 were measured using ELISA, following the manufacturer’s instructions (BOSTER).

Data for TLR4 expression were downloaded from the TCGA and GTEx databases (http://​gepia2.​cancer-pku.​cn/​#index). GO analysis was analyzed with the R software.

All quantitative data were presented as the mean ± standard error of the mean (SEM). All experiments were performed in 3 independent replicates. Differences between two groups were analyzed using the t-test, while differences among multiple groups were analyzed using the one-way ANOVA and Tukey’s test. Statistical significance was set at 5%. All statistical analyses were two-sided, and data were analyzed using the Prism 9.0 software (GraphPad).

MPLA plays a critical radioprotective role in many tissues except the lung. To investigate the radioprotective effect of MPLA on mice lung tissue, the thoracic regions of mice were irradiated and analyzed. After 20 Gy local irradiation, the infiltration of cells was observed at weeks 1 and 4, and alveolar septal thickening was observed primarily at weeks 8 and 16 by Hematoxylin staining. Compared with the single irradiation group, radiation-induced inflammation and alveolar wall thickening were significantly lower in the MPLA + IR group (Fig. 1A–D). Quantitative analysis showed that MPLA modulated radiation-induced pneumonitis (Fig. 1E).

Advanced pulmonary RILI is mainly characterized by excess fibrosis [19]. Masson’s trichrome staining revealed that collagen deposition increased from 1-week to 16-week post-irradiation. Compared with the single irradiation group, collagen deposition induced by radiation in the MPLA + IR group was significantly low (Fig. 1A–D). Quantitative analysis showed that MPLA inhibited radiation-induced collagen deposition and pulmonary fibrosis (Fig. 1F).

TUNEL assay is a critical method for quantifying IR-Induced apoptosis in mice lungs. The apoptosis rate was analyzed using respective markers. The results showed that the MPLA significantly reduced the apoptosis of alveolar epithelial cells after radiation (Fig. 1A–D). The quantitative analysis presented as Fig. 1G.

Lung coefficient (lung weight/body weight) indicates the degree of pulmonary edema and high vascular permeability. We found that MPLA treatment restored normal lung weight after irradiation (Fig. 1H), suggesting that MPLA prevents RILI in vivo.

There is a close relationship between radiation-induced EMT and pulmonary fibrosis. The expression of EMT markers reflects the EMT severity [20, 21]. In the present study, the expression of EMT markers (E-cadherin and vimentin and α-SMA) in the epithelial lung cells were analyzed by immunofluorescence staining. The results showed that E-cadherin expression was downregulated in the IR group. In contrast, vimentin and α-SMA expression were upregulated in the alveolar epithelium after irradiation. However, MPLA inhibited the downregulated E-cadherin expression induced by radiation and inhibited the overexpression of α-SMA and vimentin in (Fig. 2A–D). Quantitative analysis revealed that irradiation reduced the expression of E-cadherin, but MPLA treatment reversed this phenomenon (Fig. 2E). MPLA inhibited the expression of vimentin and α-SMA in irradiated lung tissues (Fig. 2F–G). This result indicated that MPLA ameliorated radiation-induced EMT in mice lung tissue.

Our experiments demonstrated the radioprotective effect of MPLA on mice lung tissue in vivo. In vitro experiments were performed to explore whether MPLA also has a radioprotective effect on mice lung epithelial cells. We tested the toxicity of MPLA on MLE-12 cells from a concentration range from 0 to 50 μg/mL. Cytotoxicity test showed that a concentration of 0 to 10 μg/mL of MPLA was non-toxic to MLE-12 cells (Fig. 3A). In previous studies, we demonstrated that MPLA at a concentration of 1 μg/mL played an effective role in radiation protection [12]. Therefore, 1 μg/mL of MPLA was used in the subsequent experiments. MPLA is a TLR4-specific agonist. Previous studies using mice models have shown that MPLA protects against ionizing radiation damage to intestines by activating the TLR4 signaling pathway [11]. Interestingly, ionizing radiation modulates the expression of TLR4 in the lung tissue [12]. The data from TCGA and GTEx databases showed that the expression of TLR4 was higher in normal lung tissue than in tumor tissue (Fig. 3B). In the present study, the expression of TLR4 in MLE-12 cell, Beas-2B cell, and macrophage RAW264.7 was analyzed using the Western blot assay. We found that TLR4 expression was very low in MLE-12 and Beas-2B cells with or without MPLA treatment and radiation treatment. While TLR4 was highly expressed in RAW264.7 cells particularly after radiation, MPLA treatment did not upregulate TLR4 expression in RAW264.7 cells (Fig. 3C, D). Considering the differential expression of TLR4 in different cell types of the lung tissue, we assessed whether MPLA, a TLR4 agonist, directly enhanced the survival and alleviated irradiation-induced damage on MLE-12 cells or by activating macrophages (highly expressed TLR4). Then we subjected a clonal formation assay, as we could see, the supernatant of RAW264.7(RAWsup group) had no radiation protection effect against MLE-12, but it significantly reduces cell death after irradiation in the MPLAsup group(Fig. 3E). The “multi-target-single-hit model” showed that MPLA has no direct radioprotective effect on MLE-12 cells (Fig. 3F). Previous studies have shown that macrophage-derived exosomes in the testis are radioprotective [12]. The biological functions and pathways regulated by TLR4 were explored by functional enrichment analysis using data from the TCGA database. The GO results showed that TLR4 activity was enriched in exosome-related terms, such as secretory granule membrane (Fig. 3G). Therefore, RAW264.7 was treated with GW4869 (an inhibitor of exosome biogenesis/release) for 2 h before MPLA. Surprisingly, clonal formation assay revealed no significant radio-protection effect between the GW4869 + MPLA and the MPLAsup group (Fig. 3H, I). These results implied that MPLA protected lung tissues against irradiation damage by stimulating macrophages to produce exosomes.

A previous study demonstrated that thoracic irradiation stimulated the apoptosis of Type II Alveolar Epithelial Cells (AECIIs) [22]. In the present study, we explored the effect of MPLA on the apoptosis of MLE-12 cells after irradiation using flow cytometry. The experimental result proves that the total number of apoptosis detected in MPLAsup group was significantly less than that in groups NC and MPLA (Fig. 4A, B). Next, we inhibited exosomes with GW4869, surprisingly, in (GW4869 + MPLA)sup group, the inhibition of apoptosis was barely observed (Fig. 4C, D). This result suggests that macrophage-derived exosomes play a key role in MPLA-mediated radioprotection.

IR induces breakage of double-stranded DNA breaks and the subsequent apoptosis of corresponding cells. In the present study, we used γ-H2AX expression as an indicator of double-stranded DNA breaks at different time points after irradiation. The result showed that γ-H2AX overexpressed in CON group at 1 h after irradiation and partially recovered at 8 h after irradiation. γ-H2AX expression in MPLAsup group at 1 h and 8 h after radiation was significantly lower than that in the CON group (Fig. 5A, C). This experiment illuminated that MPLAsup alleviated radiation-induced DNA damage. Identically, we then used GW4869 to inhibit the exosomes from macrophage stimulated by MPLA, there was no significant difference in γ-H2AX expression between group (GW4869 + MPLA)sup and group CON, whether it was 1 h or 8 h (Fig. 5B, D). Then, we detected the γ-H2AX foci by immunofluorescence method, numbers of γ-H2AX foci were much higher in IR group compared to MPLAsup group, the (GW + MPLA)sup group had no significant difference to the IR group (Fig. 5E, F). Overall, the results showed that macrophage-derived exosomes alleviated DNA damage in MLE-12 cells.

Classically activated M1 macrophages promote inflammation and inhibit fibrosis, while alternatively activated M2 macrophages modulate inflammation and promote fibrosis [23, 24]. Lipopolysaccharide (LPS) induces M1 polarization of macrophages [25, 26]. To clarify the effect of MPLA on macrophage polarization, RAW264.7 cells were cultured in media supplemented with MPLA. The supernatant was collected after 12 h of culture to analyze the expression levels of TNF-α, TGF-β, IL-1β, and IL-10. We found that TNF-α and IL-1β were over secreted, suggesting that MPLA stimulated M2 polarization of RAW264.7 cells (Fig. 6A–D).

Irradiation induces EMT of epithelial cells following pulmonary fibrosis [27]. Our results showed that the expression of the epithelial marker E-cadherin was decreased and the expression of the mesenchymal markers Vimentin and α-SMA was upregulated after irradiation. However, the overexpression of α-SMA and Vimentin was inhibited in the MPLAsup group. Meantime, the downregulation of epithelial markers E-cadherin was inhibited in the MPLAsup group. There were no differences in the GW + MPLAsup group compared with the IR group(Fig. 6E–H). In conclusion, our experiments demonstrated that macrophage-derived exosomes can attenuate RILI. The radiation protection effect might be related to inhibition of EMT process.