Contributed equally
The present study evaluated the role of annexin A1 (ANXA1) in the treatment of acute radiation-induced lung injury (RILI) and investigated the mechanism of its action. The expression of ANXA1, interleukin-6 (IL-6) and myeloperoxidase (MPO) in the plasma of patients with RILI prior to and following hormonotherapy was assessed by enzyme-linked immunosorbent assay. The association of plasma ANXA1 concentration with clinical effect, and the correlation between the expression of ANXA1 and that of IL-6 and MPO were evaluated. ANXA1 was overexpressed or knocked down in a macrophage cell line, and its impact on IL-6 and MPO expression was measured. Following glucocorticoid hormonotherapy, patients with RILI exhibited a higher plasma concentration of ANXA1 compared with that prior to treatment, while IL-6 and MPO levels were lower. The concentration of ANXA1 in plasma was negatively correlated with IL-6 and MPO levels, with a correlation coefficient of −0.492 and −0.437, respectively (P<0.001). The increasing concentration of ANXA1 in plasma following treatment was associated with the clinical effect in patients with RILI (P=0.007). The expression levels of of IL-6 and MPO were inhibited both in the cytoplasm and in the culture solution, when ANXA1 expression was upregulated in a macrophage cell line. In conclusion, ANXA1 inhibited the synthesis and secretion of IL-6 and MPO inflammatory cytokines, indicating that ANXA1 may have therapeutic potential as a treatment target for RILI.
Radiation-induced lung injury (RILI) is the most common dose-limiting side effect in patients with thoracic tumors following radiotherapy (
The pathogenesis of RILI is complicated. Alveolar type II epithelial cells and lung capillary endothelial cells are the main targets of RILI (
Annexin A1 (ANXA1) is a protein regulated by glucocorticoids. ANXA1 mimics the anti-inflammatory effect of glucocorticoids (
The role of ANXA1 in the pathogenesis of acute RILI and the mechanism of its clinical benefit on RILI are unclear. The present study evaluated the therapeutic potential of ANXA1 against RILI by assessing ANXA1 expression in the plasma of patients with RILI prior to and following 4 weeks of glucocorticoid treatment. The anti-inflammatory mechanism of ANXA1 was investigated by upregulating and downregulating ANXA1 expression in a macrophage cell line to assess the impact on IL-6 and MPO expression. Further understanding of the anti-inflammatory effect of ANXA1 on RILI may provide pre-clinical evidence to support targeting ANXA1 in the prevention and treatment of RILI.
A total of 50 patients with thoracic tumors suffering with RILI following radiotherapy between January 2014 and October 2016 at Taizhou People's Hospital (Taizhou, China) were included in the present study. The patients had experienced RILI with a severity of grade 2 or more after therapy, according to the Common Terminology Criteria for Adverse Events from the National Cancer Institute (version 3.0; National Institutes of Health, Rockville, MD, USA) (
Glucocorticoid (methylprednisolone or dexamethasone) was administered to patients with RILI as the basic comprehensive treatment immediately after diagnosis. Methylprednisolone was administrated intravenously (iv), with a starting dose 1–2 mg/kg/day, and the dose would reduce by half every 4 days. Dexamethasone was administrated with a starting dose of 10 mg/day iv, and reduced as described previously. Routine blood examination was performed weekly during treatment, and a thoracic computed tomography scan was performed after 4 weeks of treatment. Response to treatment was assessed by alleviation of clinical symptoms (e.g. cough, fever, chest distress, dyspnea) and imaging (X-ray, CT scan) and laboratory findings (white blood cell count). The treatment outcome was evaluated comprehensively based on the patients' clinical symptoms and the aforementioned indicators, including CT scan and white blood cell count and neutrophils count.
Peripheral venous blood was drawn from each patient weekly from the time of diagnosis until 1 month post-treatment. Then, the neutrophil count was measured through routine blood tests, and the expression of ANXA1, IL-6 and MPO was assessed using ELISA in the plasma extracted by centrifugation from the blood samples. The ANXA1 (cat. no. ab222868; antibody name: AF3770) and IL-6 (cat. no. PD6050; antibody name: MAB206) kits, and the primary antibody were purchased from R&D Systems China Co., Ltd. The MPO kit was purchased from Abcam Shanghai Co., Ltd. (cat. no. ab119605; antibody name: ab45977). Measurements were performed in triplicate for each sample. The absorbance was read at 450 nm and the protein levels were calculated based on the standard curve. Blood samples from 20 healthy donors were also assessed as the negative control.
Eca109 human esophageal cancer cells, purchased from Shanghai Institute of Biochemistry and Cell Biology Chinese Academy of Sciences were cultured in DMEM with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and antibiotics at 37°C with 5% CO2. RNA from Eca109 human esophageal cancer cells was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc., Shanghai, China). The AMV reverse transcription kit (Promega Corporation) was subsequently used for reverse transcription to obtain the cDNA which was used as a template, together with ANXA1 primers (synthesized by Invitrogen; Thermo Fisher Scientific, Inc. Shanghai, China) for PCR amplification: The primer sequences were as follows: Sense, 5′-ATGGCAATGGTATCAGAATTCCTC-3′ and antisense, 5′-TTAGTTTCCTCCACAAAGAGCCACC-3′. PrimerSTAR HS DNA polymerase (Takara Biotechnology Co., Ltd., Dalian, China) was used for amplification. The reaction parameters were as follows: 95°C ×3 min, 94°C ×30 sec, 58°C ×30 sec, 72°C ×5 min, 72°C ×5 min, for 27 cycles. The PCR product was purified using the QIAquick PCR Purification kit (Qiagen China Co., Ltd., Shanghai, China). ANXA1 cDNA was cleaved with the restriction endonucleases
Human peripheral-blood monocytes are used as an established
THP-1 cells were induced into macrophage 48 h after transfection. The pIRES2-ANXA1 expression plasmid was transfected into THP-1 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The pIRES2-EGFP empty plasmid was transfected as the control. Prior to transfection, the plasmid (1 µg/ul) was mixed with liposomes and allowed to stand for 20 min. The mixture was dispensed into culture wells (2 µg DNA and 5 ul liposomes per well) following the manufacturer's protocol. The transfection efficiency was assessed by fluorescence microscopy and western blot analysis. The transfected THP-1 cells were then induced to generate ANXA1-overexpressing macrophages. Macrophages harboring the ANXA1 plasmid comprised the pIRES2-ANXA1 group and those with the empty plasmid comprised the no-load group. Non-transfected macrophages comprised the blank control. Each group of cells was partitioned into 3 wells and the experiments were repeated 3 times.
Small interfering RNAs (siRNAs) against ANXA1 were transfected into THP-1 cells to inhibit the expression of ANXA1. The siRNAs included 2 sequences targeting ANXA1 and a scrambled control sequence as follows: siRNA (a), 5′-CAGCGUCAACAGAUCAAAG-3′; siRNA (b), 5′-CCGAUCUGAGGACUUUGGU-3′; and control siRNA, 5′-CAGUCGCGUUUGCGACUGG-3.′ These siRNA sequences were designed as previously described (
Macrophages in each well were trypsinized and total cell protein was extracted using cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology). Sample protein concentrations were determined for each group using the DC Protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). SDS-PAGE (15%) was performed for each group using 50 µg protein sample per lane, and proteins were then transferred from the gel to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following a 1-h incubation in blocking buffer (cat. no. C-0042; 5% bovine serum albumin; Xiamen, BOYAO Biotechnology Co., Ltd.) at 4°C, membranes were incubated with the following primary antibodies at 4°C overnight with agitation: Anti-IL-6 (1:1,000; cat. no. RAB0307; Sigma-Aldrich; Merck KGaA), anti-ANXA1 (1:1,000; cat. no. SAB1405457; Sigma-Aldrich; Merck KGaA), anti-MPO (1:1,000; cat.no. 76923; Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-GAPDH (1:3,000; cat.no. sc-47724; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following washing with stripping buffer [β-mercaptoethanol 342 µl; 20% SDS 5 ml; Tris-Cl (pH 6.7) 3.125 ml; ddH2O 41.533 ml to total volume 50 ml], the membranes were incubated with agitation at room temperature for 1 h with a secondary antibody (m-IgGκ BP-HRP: sc-516102;Santa Cruz Biotechnology, Inc.) labeled with horseradish peroxidase in the presence of 2% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc), then HRP was visualized by Chemiluminescent Western Blot Detection kit (SuperSignal West Femto; Thermo Fisher Scientific, Inc.). The membranes were washed and imaged, and the bands were analyzed semi-quantitatively with ImageJ 1.8 software (
M2 macrophages in which ANXA1 was overexpressed or inhibited were resuspended for cell counting following transfection to ensure that the cell density in each well for further cultivation was the same. Following 72 h in culture, ELISA was performed on the medium to evaluate the level of IL-6 and MPO secreted by the macrophages, and western blot analysis was performed to evaluate the levels of IL-6 and MPO protein in the macrophages. ELISA was performed as aforementioned for peripheral venous blood samples.
Measurement data are presented as the mean ± standard deviation. SPSS (version 19.0; IBM Corp., Armonk, NY, USA) was utilized to create the database. Differences between means of two groups were compared using independent-samples t-test and those among multiple groups were compared using one-way analysis of variance (ANOVA). ANOVA was used to compare the means among baseline group and 1, 2, 3 and 4 weeks after treatment groups. Then, the Least Significant Difference post hoc test was used to compare between every two groups. Associations between treatment outcome and ANXA1, IL-6 and MPO levels were compared using a χ2 test. Correlation analysis was performed using Spearman's rank correlation method. P<0.05 was considered to indicate that a difference was statistically significant.
Among the 45 cases with RILI, the baseline level of ANXA1, IL-6 and MPO in the plasma prior to glucocorticoid treatment was 317.35±191.06, 258.45±172.32 pg/ml and 139.24±57.94 ng/ml, respectively. The corresponding level of healthy control group was 22.75±9.56, 41.05±12.33 pg/ml and 62.24±27.39 ng/ml, respectively. There was significant difference of ANXA1, IL-6 and MPO between the baseline of RILI patients and the healthy control (P<0.001) Following glucocorticoid administration, the expression of ANXA1 increased gradually (ANOVA test, F=31.503; P<0.001). The post hoc test showed that ANXA1 of 1, 2, 3 and 4 weeks after treatment was significantly higher compared with that of baseline, (P=0.003, <0.001, <0.001, <0.001, respectively), while that of IL-6 and MPO decreased, and the number of neutrophils remained unchanged (
Among the 45 cases, recovery occurred in 20 patients 2 weeks after glucocorticoid treatment, and a further 11 patients with repeat symptoms were cured following 4 weeks of treatment. These 31 cases were considered to have had an efficient treatment outcome. Interstitial lung diseases combined with chronic lung infection occurred in the remaining 14 cases, which were considered to have progressive disease. Cases were grouped by these two outcomes-efficient or progressive group. According to the increase of ANXA1 after hormone treatment, patients were divided into two groups: Patients with more than twice the baseline level and patients with less than two times the baseline level. Similarly, patients were divided into two groups according to the decrease of IL-6 and MPO, which were less than half of the baseline level and more than half of the baseline level group. Then, χ2 testing was performed to compare treatment outcome to expression levels of ANXA1, IL-6 and MPO in plasma following glucocorticoid treatment. The results indicated that the expression levels of ANXA1, IL-6 and MPO were associated with the treatment outcome for RILI. The increased expression of ANXA1 following 4 weeks of treatment was associated with improved treatment outcome for RILI (P=0.007), while decreased expression of IL-6 and MPO expression following 4 weeks of treatment was associated with improved treatment outcome (P=0.042 and P=0.003, respectively) (
PMA and IL-4 were added to the culture medium of THP-1 cells to induce differentiation to an M2 macrophage phenotype. THP-1 cells were round or quasi-circular, uniform in size and suspended in culture medium (
Following transfection of M2 macrophages with the p-IRES2-ANXA1 plasmid or siRNA against ANXA1, western blot analysis was used to determine ANXA1 protein expression. The cells transfected with pIRES2-ANXA1 exhibited a stronger ANXA1 signal compared with the cells in the blank control and no-load group. In macrophages transfected with siRNA (a) and siRNA (b), the ANXA1 band was weaker compared with that of the blank control and siRNA (control) groups (
Western blot results revealed decreased IL-6 protein expression in macrophages transfected with p-IRES2-ANXA1, while ANXA1 inhibition caused by siRNA led to enhanced IL-6 expression (
MPO protein expression decreased in macrophages transfected with p-IRES2-ANXA1, while ANXA1 inhibition enhanced MPO protein expression (
To date, efforts to treat acute RILI have focused on neutralizing pro-inflammatory cytokines or attenuating the infiltration of inflammatory cells (
The present study observed that glucocorticoid treatment is associated with increased ANXA1 in plasma. An increased concentration of ANXA1 in plasma following glucocorticoid treatment was associated with a more positive treatment outcome for RILI, indicating that ANXA1 may serve a beneficial role in the treatment of acute RILI.
ANXA1 in the plasma of patients with RILI was negatively correlated with the plasma levels of IL-6 and MPO, implying that ANXA1 may impact RILI treatment outcome by influencing the expression of these pro-inflammatory cytokines. IL-6 is an important pro-inflammatory factor, which is synthesized by activated alveolar macrophages, T helper 2 cells, lung fibroblasts and alveolar type II epithelial cells. IL-6 is involved in the responses to lung injury and early-stage inflammation (
Extracellular MPO is an alkaline protein with positive charge, which is easily adsorbed on the surface of negatively charged cell membrane (
In the present study, ANXA1 was increased in the plasma of patients with acute RILI following glucocorticoid therapy. These results are consistent with several reports (
In the present study, the overexpression of ANXA1 in macrophages inhibited the synthesis and secretion of IL-6 and MPO, while the inhibition of ANXA1 promoted these events, indicating that ANXA1 could serve an anti-inflammatory role, as previously reported (
In conclusion, ANXA1 reduces the expression of IL-6 and inhibits the release of MPO from monocytes and macrophages. This anti-inflammatory effect may underscore the mechanism by which ANXA1 in the plasma of patients with RILI is associated with treatment outcome. Although glucocorticoids are common drugs in RILI treatment, the present study indicates the potential of ANXA1 to mimic the anti-inflammatory effect of glucocorticoids, which would potentially avoid certain adverse effects of this therapy.
Not applicable.
This study was supported by the Scientific Program of the Health and Family Planning Commission of Jiangsu Province (grant No. H2017076) and the Foundation of Jiangsu Provincial Medical Innovation Team (grant No. CXTDA2017042).
All data generated or analyzed during the present study are included in this published article.
GH and JH conceived and were responsible for the design of the present study. GS, WX and KL analyzed and interpreted the patients' data. CD was responsible for the diagnosis of RILI and assessment of the curative effect based on imaging examinations. SZ and JY analyzed and interpreted the laboratory data. GH and SZ were the major contributors in writing the manuscript. All authors read and approved the present manuscript.
This research was approved by the Ethics Committee of Taizhou People's Hospital, and written informed consent was provided by each patient prior to the study.
All patients signed an informed consent for publication approved by the institutional Review Board.
The authors declare that they have no competing interests.
ANXA1, IL-6, MPO and neutrophils in the plasma of patients with acute radiation-induced lung injury following glucocorticoid administration for 4 weeks. ANXA1 at 4 weeks was significantly higher compared with the baseline level (317.35 vs. 586.75 pg/ml; P<0.001). IL-6 decreased gradually, and was significantly lower at 4 weeks compared with the baseline (258.45 vs. 139.35 pg/ml; P=0.003). The neutrophil count did not change with glucocorticoid administration (33.5×109 vs. 31.78×109 cells/dl; P=0.247). MPO decreased and was notably lower compared with the baseline level following 4 weeks of glucocorticoid treatment (139.24 vs. 51.35 ng/ml; P=0.018). ANXA1, annexin A1; IL-6, interleukin 6; MPO, myeloperoxidase; GRA, granulocyte count, absolute.
The correlation between the expression level of ANXA1 and IL-6 or MPO in plasma of patients with RILI treated with glucocorticoid. (A) Negative correlation between ANXA1 and IL-6; r=−0.974. (B) Negative correlation between ANXA1 and MPO, r=−0.956. ANXA1, annexin A1; IL-6, interleukin 6; MPO, myeloperoxidase; RILI, radiation-induced lung injury.
Differentiation of THP-1 cells to M2 macrophages. (A) THP-1 cells were circular or quasi-circular, growing in a state of suspension (magnification, ×200). (B) THP-1 cells induced with phorbol 12-myristate 13-acetate and interleukin-4 became polygonal shape with outgrowth, and grew adherent to the plate wall (magnification, ×200). (C) CD206 expression was observed by flow cytometry 48 h after induction. (D) Green fluorescent protein in macrophages transfected with pIRES2-ANXA1: The cells were spindle-like or pleomorphic (magnification, ×200). ANXA1, annexin A1; CD206, macrophage mannose receptor 1; PE, phycoerythrin.
Expression of ANXA1 and GAPDH in untransfected macrophages (blank control) or macrophages transfected with pIRES2-GFP (No-load), pIRES2-ANXA1, siRNA (a), siRNA (b) or siRNA (control). ANXA1, annexin A1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; siRNA, short interfering RNA.
IL-6 and MPO expression in M2 macrophages transfected with pIRES2-ANXA1 or siRNA against ANXA1, siRNA (a) and siRNA (b). (A) IL-6 protein expression was decreased in macrophages with overexpression of ANXA1, while ANXA1 inhibition induced by siRNA led to increased IL-6 expression. (B) IL-6 in culture medium was decreased with overexpression of ANXA1 (*P<0.05 compared with blank control) and increased with siRNA-mediated ANXA1 inhibition (**P<0.05 and ***P<0.05, compared with blank control). (C) MPO protein expression was decreased in macrophages with overexpression of ANXA1, while ANXA1 inhibition induced by siRNA led to increased IL-6 expression. (D) MPO in cell culture medium was decreased with overexpression of ANXA1 («P<0.01, compared with blank control), and increased with siRNA-mediated ANXA1 inhibition (««P<0.05 and «««P<0.05 compared with blank control). ANXA1, annexin A1; IL-6, interleukin 6; MPO, myeloperoxidase; siRNA, short interfering RNA.
Association between the clinical effect against RILI and the plasma levels of ANXA1, IL-6 and MPO at 4 weeks of glucocorticoid treatment.
ANXA1 in plasma | IL-6 in plasma | MPO in plasma | ||||
---|---|---|---|---|---|---|
Clinical effect against RILI | >2× baseline level | <2× baseline level | <50% of baseline level | >50% of baseline level | <50% of baseline level | >50% of baseline level |
Efficient, n | 20 | 11 | 19 | 12 | 19 | 12 |
Progressive, n | 3 | 11 | 4 | 10 | 2 | 12 |
χ2 | 7.166 | 4.132 | 8.562 | |||
P-value | 0.007 | 0.042 | 0.003 |
RILI, radiation-induced lung injury; ANXA1, annexin A1; IL-6, interleukin 6; MPO, myeloperoxidase.