Soy Isoflavones Promote Radioprotection of Normal Lung Tissue by Inhibition of Radiation-Induced Activation of Macrophages and Neutrophils

Abstract

Introduction:

Radiation therapy for lung cancer is limited by toxicity to normal lung tissue that results from an inflammatory process, leading to pneumonitis and fibrosis. Soy isoflavones mitigate inflammatory infiltrates and radiation-induced lung injury, but the cellular immune mediators involved in the radioprotective effect are unknown.

Methods:

Mice received a single dose of 10 Gy radiation delivered to the lungs and daily oral treatment of soy isoflavones. At different time points, mice were either processed to harvest bronchoalveolar lavage fluid for differential cell counting and lungs for flow cytometry or immunohistochemistry studies.

Results:

Combined soy and radiation led to a reduction in infiltration and activation of alveolar macrophages and neutrophils in both the bronchoalveolar and lung parenchyma compartments. Soy treatment protected F4/80⁺CD11c⁻ interstitial macrophages which are known to play an immunoregulatory role and are decreased by radiation. Furthermore, soy isoflavones reduced the levels of nitric oxide synthase 2 expression while increasing arginase-1 expression after radiation, suggesting a switch from pro-inflammatory M1 macrophage to an anti-inflammatory M2 macrophage phenotype. Soy also prevented the influx of activated neutrophils in lung caused by radiation.

Conclusions:

Soy isoflavones inhibit the infiltration and activation of macrophages and neutrophils induced by radiation in lungs. Soy isoflavones-mediated modulation of macrophage and neutrophil responses to radiation may contribute to a mechanism of resolution of radiation-induced chronic inflammation leading to radioprotection of lung tissue.

INTRODUCTION

The primary goal of combining a drug modality with radiation therapy is to maximize therapeutic benefit while mitigating severe off-target side effects in the treatment of cancer patients. Radiation injury to normal lung parenchyma is a major concern in non-small cell lung cancer (NSCLC). Radiotherapy given concurrently with chemotherapy is the conventional treatment for locally advanced NSCLC presenting as unresectable, stage III disease in approximately 50,000 Americans per year. There is an associated overall 5-year survival rate of 20%, emphasizing the need to improve the therapeutic ratio of concurrent chemo-radiotherapy.^{1, 2} High intensity radiotherapy could be more effective but is limited by lung tissue toxicity presenting as radiation pneumonitis that develops in up to 30% of patients after thoracic radiation.^{3, 4} Radiation pneumonitis is caused by an early inflammatory process triggered by damage to lung parenchyma, epithelial cells, vascular endothelial cells and stroma. This process involves induction of pro-inflammatory cytokines and chemokines which recruit inflammatory immune cells to the lung tissue resulting in pneumonitis and late fibrosis.^5–7 Early acute pneumonitis occurs by 2–4 months after radiotherapy, while late chronic pneumonitis manifests clinically over 6–24 months.^{3, 4} At late stages, radiation-induced pulmonary fibrosis results from aberrant resolution of inflammation in contrast to classical wound healing processes.⁶ These adverse events after radiotherapy affect patients’ breathing and their quality of life. Various strategies to decrease the extent of pneumonitis have been investigated but need further research efforts.⁸

We previously explored a complementary approach to alleviate lung radiation toxicity using soy isoflavones, consisting of genistein, daidzein and glycitein phytoestrogens extracted from soy beans. Although these isoflavones are similar in their chemical structure to estrogens, they have weak estrogenic activity and act as chemopreventive agents.^9–11 Our studies demonstrated that soy isoflavones have the dual capability of protecting normal lung from radiation injury and simultaneously enhancing radiation damage in the malignancy^{12, 13}. Soy mitigated the vascular damage, inflammation and fibrosis caused by radiation injury to lung tissue in a lung cancer model suggesting that soy can alter the radiation-induced inflammatory response.^{12, 13} In naïve mice, soy isoflavones supplementation given pre- and post-thoracic radiation protected the lungs against adverse effects of radiation including skin injury, hair loss, increased breathing rates, inflammation, pneumonitis and fibrosis.¹⁴ These findings in naïve mice corroborated our findings in lung tumor models and provided evidence for a radioprotective effect of soy isoflavones. Importantly, soy isoflavones also sensitized cancer cells to radiation both in vivo and in vitro in pre-clinical tumor models of lung cancer, demonstrating a differential effect of radioenhancement on lung tumors with simultaneous radioprotection of normal lung tissue.^{12, 13, 15}

We have reported that supplementation of soy isoflavones with thoracic irradiation mitigates radiation-induced inflammatory cytokines, infiltration of inflammatory cells and fibrosis,^12–14 but the cellular mediators of radioprotection remain unclear. In the current study, we investigated the role of macrophages and neutrophils in the mitigation of radiation-induced inflammatory events by soy isoflavones in lung tissue. Macrophages are recruited as a first response to radiation-induced damage in the tumor microenvironment or in normal tissues.¹⁶ Macrophages play distinct roles in the early versus late stages of inflammatory response.^17–19 Monocytes can differentiate into functionally different macrophage subsets. Inflammatory cytokines (TNF-α, GM-CSF, IFN-γ) generate classically activated M1 macrophages that mediate acute inflammation and participate in Th1 reactions.²⁰ M2 macrophages can be activated by IL-4, IL-13, IL-10, TGF-β, or immune complexes, participate in Th2 and Treg reactions, and promote tumor growth and fibrosis.^{21, 22} M1 predominates during acute inflammation, and then switches to M2 during the wound-healing phase at later stages.²¹ We tested whether soy influences macrophage skewing to M1 or M2 subsets, and if this altering of macrophage phenotypes could dictate normal lung response to radiation-induced damage.

Activation and infiltration of neutrophils is a hallmark event in the progression of acute lung injury,²³ and have been shown to be involved in radiation-induced alveolitis.²⁴ Therefore, the effect of soy isoflavones on infiltration and activation status of neutrophils was studied after radiation to the lungs. Our findings suggest that soy can inhibit the infiltration and activation of macrophages and neutrophils induced by radiation in lung parenchyma. Radiation induced a pro-inflammatory M1 macrophage phenotype in lungs, while mice receiving soy isoflavones and radiation switched to an anti-inflammatory M2 macrophage subtype. These data indicate that soy isoflavones modulate the cellular mediators of the inflammatory response induced by radiation.

MATERIALS AND METHODS

Mice

Female BALB/c mice (Harlan, Indianapolis, IN) 5–6 weeks old, were housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The animal protocol was approved by Wayne State University Institutional Animal Care and Use Committee.

Soy isoflavones

The soy isoflavone mixture G-4660 used is a pure extract of 98.16% isoflavones from soybeans consisting of 83.3% genistein, 14.6% daidzein and 0.26% glycitein (manufactured by Organic Technologies and obtained from the National Institutes of Health [NIH], Bethesda, MD). The soy isoflavone mixture was dissolved in DMSO and mixed with sesame seed oil at a 1:20 ratio just prior to treatment to facilitate gavage and avoid irritation of the esophagus by DMSO.^12–14

Lung irradiation

Radiation was delivered to the whole lung. Three anesthetized mice, in jigs, were positioned under a 6.4 mm lead shield with 3 cut-outs in an aluminum frame mounted on the X-ray machine for selective irradiation of the lung, as previously described.¹³ The dose rate was 101 cGy/min and half value layer was 2 mm Cu. Photon irradiation was performed at a dose of 10 Gy with a Siemens Stabilipan X-ray set operated at 250 kV, 15 mA with 1 mm copper filtration at a distance of 47.5 cm from the target.

Experimental Design

Mice were pre-treated with oral soy isoflavones for 3 days at a dose of 5mg/day (250mg/kg). Then, the lung was selectively irradiated with 10 Gy. Soy treatment was continued at 5mg/day for 10 days and then switched to a lower dose of 1mg/day (50mg/kg), given 5 days a week for up to 18 weeks, based on previous studies.^{12, 25} We have reported that these doses of soy isoflavones result in plasma levels comparable to those measured in Asian populations consuming foods rich in soy isoflavones (1–4 μM).¹⁴ At different time points, mice were either processed to harvest bronchoalveolar lavage (BAL) fluid for differential cell counting and lungs for flow cytometry or immunohistochemistry studies.

Analysis of immune cells by differential cell counting in BAL fluid and flow cytometry on single-cell suspension from lungs

BAL was performed prior to lung resection at 1, 8, 12, and 18 weeks after irradiation. Cells were loaded onto slides using a cytospin centrifuge and stained using a DiffQuik staining kit (IMEB Inc., San Marcos, CA,). Differential cell counts of leukocyte subsets were performed by counting at least 300 nucleated cells.²⁶

Following collection of BAL fluids, the same mice provided the lungs for flow cytometry studies. Lungs were digested with 0.4 mg/mL collagenase IV and red blood cells were lysed. Lung single-cell suspensions were incubated with Fc receptor-blocking antibody (eBioscience, San Diego, CA) prior to staining. For morphological characterization of leukocytes, CD45⁺ cells were sorted by fluorescence-activated cell sorting (FACS) using a BD FACSVantage SE. CD45⁺ cell subsets were gated according to cell size and granularity. Cell subsets obtained from each gate were spun onto slides using a cytospin, and stained using a DiffQuik staining kit. To determine immunophenotype, cells were immunostained using a 5-color fluorophore combination of antibodies consisting of CD45-APC, CD11b-FITC, F4/80-PE, CD11c-APC-eFluor780, and Ly6G-PerCp-Cy5.5 (eBioscience). Fixable viability dye eFluor450 was used to exclude dead cells. Cells were analyzed by flow cytometry using a BD LSR II flow cytometer followed by analysis on FlowJo v10 software.

Immunohistochemistry (IHC)

Lungs were intratracheally instilled with 10% formalin, resected, embedded in paraffin, and sectioned. Sections were incubated with primary antibodies directed against F4/80, nitric oxide synthase 2 (NOS2), arginase-1 (Arg-1), Gr-1 (Ly6C/Ly6G), and myeloperoxidase (MPO) (Abcam, Cambridge, UK) followed by biotinylated secondary antibodies (Vector Labs, Burlingame, CA). Staining was amplified using the avidin-biotin immunoperoxidase technique (Vector Labs). All slides were examined on a Nikon E800 microscope. Quantitation of the number of macrophages and measurement of cell areas were performed using ImageJ software in 10 fields of 40× per slide. For quantitation of overall staining, whole slide imaging was performed using a slide scanner and DensitoQuant analysis (3D Histech). The percentage of positive area was calculated as the number of positive pixels divided by total number of pixels.

Preparation of Lung Tissue Protein Lysates and Western Blot Analysis

Protein lysates were prepared from frozen lungs using a gentleMACS tissue dissociator. Lung protein extracts (50 μg) were loaded and separated on 10% SDS-PAGE and transferred to Whatman membranes. Membranes were incubated with anti-MPO followed by IgG-HRP secondary antibody. Membranes were re-probed with anti-β-actin Ab as a loading control, visualized by SuperSignal West Pico Chemiluminescent Substrate, and captured on a Fotodyne imaging system.¹⁵

Statistical Analysis

Comparisons between means of two treatment groups were analyzed by two-tailed unpaired Student’s t test. A value of p<0.05 was considered statistically significant.

RESULTS

Effect of radiation and soy isoflavones on immune cell subsets recovered from bronchoalveolar space

To identify the nature of inflammatory infiltrates in lungs induced by radiation and the effect of soy isoflavones on these cells, we first analyzed immune cell subsets recovered from BAL.²⁶ Alveolar macrophages constituted 80% to 90% of cells recovered from BAL fluids, and the majority presented as small macrophages in both control and soy-treated BAL (Figure 1). At 1 week after radiation, there was a significant decrease from 90.7±0.8% to 24.1±2.9% in small macrophages compared to control (p<0.0001). A concomitant significant increase from 9.1±0.7% to 75.8±3.0% in enlarged foamy macrophages (p<0.0001) with a morphology typical of activated macrophages was observed (Figure 1A, inset). This trend was consistent at 8, 12 and 18 weeks after radiation (Figure 1B, C, D). At 8, 12 and 18 weeks following radiation and soy, there was a significant increase in small macrophages associated with a concomitant decrease in enlarged foamy macrophages (p<0.01) with ratios comparable to those of control mice.

FIGURE 1. Effect of soy isoflavones on immune cells obtained from BAL fluid at different time points after radiation.

BAL fluids were harvested at early and late time points post-radiation. At 1 week (A), 8 weeks (B), 12 weeks (C), and 18 weeks (D) post-radiation, differential cell counts on BAL fluid cytospins were performed and the percentages of macrophages, neutrophils, and lymphocytes were calculated. The ratios of non-activated macrophages and enlarged, foamy activated macrophages (see inset 1A), as well as those of neutrophils and lymphocytes, are shown from BAL fluid obtained from treated and control mice. The data are presented as mean ± SEM (n = 3–5 mice/group/time point) and p-values shown represent significant differences between radiation + soy compared to radiation alone. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, radiation compared to control or radiation + soy compared to radiation alone.

Whereas BAL fluids from control mice have undetectable numbers of neutrophils and lymphocytes, neutrophil counts showed a significant increase induced by radiation at 12 weeks (19.0±2.1%, p<0.0001) which was decreased in radiation and soy isoflavones-treated mice (8.4±3.2%, p<0.05) (Figure 1C). A measurable increase in BAL lymphocytes was also observed after radiation; however, this increase was not seen in radiation and soy-treated mice.

Flow cytometry phenotypic analysis of immune myeloid cells infiltrating lung parenchyma following treatment with radiation and soy isoflavones

Analysis of the bronchoalveolar compartment revealed that radiation caused a switch from non-activated to activated macrophages at 2–4 months after radiation, which was prevented by soy isoflavones. To further determine the immunophenotype of the cells involved in the soy radioprotection, we have analyzed immune cell subsets in the lung parenchyma compartment by flow cytometry.

In a first step, lung cell suspensions from control mice were stained with anti-CD45 to separate and sort leukocytes by FACS. Gate G1 contained lymphocytes, gate G2 consisted of a majority of neutrophils/granulocytes and a few macrophages, and gate G3 was primarily composed of alveolar macrophages and some granulocytes (Figure 2A). Consistent with findings in BAL, lymphocytes in gate G1 did not show major alterations by treatment (personal communications); therefore we focused on myeloid cells.

FIGURE 2. Flow cytometry analysis of macrophages and neutrophils isolated from lung tissues.

(A). Morphology of FACS-sorted CD45⁺ immune cell subsets in normal lung tissue. Normal lungs from untreated mice were dissociated into single cell suspensions. Cells were immunostained with fluorescent anti-CD45 and sorted by FACS. CD45⁺ lung leukocyte subsets were gated in gates G1, G2, and G3 based on cell size [forward scatter (FSC), x-axis] and granularity [side scatter (SSC), y-axis]. Cells in each gate were sorted by FACS, cytospun onto slides and stained with DiffQuik, revealing that gate G1 contained lymphocytes, gate G2 consisted of a majority of neutrophils/granulocytes and fewer macrophages, and gate G3 was primarily composed of alveolar macrophages and some granulocytes. All magnifications are at 40×. (B). Interstitial and alveolar macrophages analysis. At 12 weeks after radiation, lungs from control (Con) mice, and mice treated with soy (Soy), radiation (Rad) or radiation + soy (R+S) were dissociated into single cell suspensions. Cells were stained with anti-CD45, anti-F4/80, and anti-CD11c fluorescent antibodies to analyze interstitial (F4/80⁺CD11c⁻) and alveolar (F4/80⁺CD11c⁺) tissue macrophages within CD45⁺ leukocyte populations by flow cytometry. Representative flow cytometry plots are presented, showing the gating strategy of CD45⁺ myeloid lung cells for analysis of F4/80⁺CD11c⁻ interstitial macrophages (IM in pink rectangle gate) and F4/80⁺CD11c⁺ alveolar macrophage (AM in pink circle gate). Percentages of F4/80⁺CD11c⁻ IM subsets and F4/80⁺CD11c⁺ AM subsets within CD45⁺ cells are shown for lungs from control and treated mice. The data are presented as mean ± SEM (n= 4–5 mice/group) and are representative of three separate experiments. (C). Analysis of CD45⁺CD11b⁺Ly6G⁺ neutrophils. Cells were stained with fluorescent anti-CD45, anti-CD11b, and anti-Ly6G to analyze neutrophil subsets by flow cytometry. Representative flow cytometry plots are presented, showing the gating strategy for analysis of CD11b⁺Ly6G⁺ neutrophils within CD45⁺ leukocyte populations. Percentages of CD11b⁺Ly6G⁺ neutrophils within CD45⁺ cells are shown for lungs from control and treated mice. The data are presented as mean ± SEM (n = 5 mice/group) and are representative of two separate experiments. *p<0.05, radiation compared to control or radiation + soy compared to radiation alone.

To analyze the phenotype of myeloid cells obtained from lungs at 12 weeks after radiation, cells were immunostained for CD45, CD11b, F4/80, CD11c, and Ly6G. Gates for CD45⁺ leukocytes were set as shown in Figure 4A and myeloid cells from gates G2 and G3 were further analyzed for specific macrophages and neutrophil markers. Macrophages were gated based on differential expression of F4/80 and CD11c to analyze interstitial macrophages (IM, F4/80⁺CD11c⁻) and alveolar macrophages (AM F4/80⁺CD11c⁺) (Figure 2B).²⁷ Percentages of F4/80⁺CD11c⁻ IM subsets showed a significant decrease in interstitial macrophages induced by radiation compared to control (p<0.05), whereas this treatment with soy isoflavones protected this population in irradiated lung tissue (p<0.05) (Figure 2B). F4/80⁺CD11c⁺ AM subsets did not show difference between treatments (Figure 2B), in contrast to BAL findings that showed an increase in AM by radiation and decrease by radiation + soy. The discrepancies between alveolar macrophages in BAL fluid versus lung tissue compartments could be due to lavage of loose alveolar macrophages and subsequent processing of lungs for FACS. Neutrophils were gated by expression of CD11b, and Ly6G/Gr-1 markers within the CD45⁺ population (Figure 2C).²⁸ CD11b⁺Ly6G⁺ neutrophils in lungs are significantly increased after radiation compared to control (p=0.01), however supplementation of soy to radiation did not significantly change the percent of neutrophils (Figure 2C).

FIGURE 4. NOS2 and Arg-1 functional macrophage markers in lungs treated with radiation and soy isoflavones.

Lung tissue sections were obtained from control (Con) mice and mice treated with radiation (Rad) or radiation + soy (Rad+Soy) at 18 weeks after radiation. Sections were stained by IHC for the markers NOS2 and Arg-1 to determine macrophage activation status in the lungs. (A). NOS2 staining revealed an abundance of activated macrophages caused by radiation presenting as clusters of enlarged NOS2⁺ cells in areas of pneumonitis (see insets). Lower levels of NOS2⁺ macrophages were seen in lungs of mice treated with radiation + soy treated lungs or control which presented as smaller cells (see inset). (B). Whole slide scanning for quantitation of NOS2 positive staining confirmed these findings. (C). Compared to radiation, Arg1 expression was greater with radiation + soy. (D). Whole slide scanning for quantitation of revealed higher levels of Arg-1 in radiation + soy compared to low levels after radiation alone. For analysis of whole slide scanning, the percentage of positive area was calculated as the total number of positive pixels divided by total number of pixels. All magnifications are at 40× and insets at 100× to reveal the extent of positive staining and morphology of immune cell subsets.

Skewing toward anti-inflammatory M2 macrophage phenotype in lung tissue treated with radiation and soy

To further study whether soy inhibits the activation of macrophages induced by radiation, as suggested in the BAL studies, we investigated macrophage subsets and their functional status in lungs treated with radiation only and radiation + soy. Radiation caused a striking infiltration of F4/80⁺ macrophages that was prominent at 18 weeks after radiation and observed in areas of thickened alveolar septa, reflective of pneumonitis,^12–14 (Figure 3A, arrowheads). Numerous alveolar macrophages were particularly enlarged with abundant cytoplasm showing the morphology of activated macrophages compared to small macrophages in control lungs (Figure 3A, see arrows and inset). Lungs from mice treated with radiation + soy had a lower density of F4/80⁺ macrophages and thinner alveolar septa (Figure 3A), compared to radiation–treated lungs, showing decreased pneumonitis.^{13, 14} Moreover, the morphology of the alveolar macrophages was much smaller than those of radiation-treated lungs (see insets, Figure 3A). Quantitation of F4/80⁺ cells showed that there was a significant increase in the number of alveolar macrophages in lungs treated with radiation compared to control lungs (p<0.001, Figure 3B). Irradiated mice supplemented with soy isoflavones showed a significantly reduced number of alveolar macrophages compared to radiation alone (p<0.001, Figure 3B), to levels similar to those of control mice. Measurements of the average size of the alveolar macrophages showed that radiation increased the size of these cells significantly compared to either radiation + soy (p<0.01, Figure 3C) or control (p<0.0001, Figure 3C).

FIGURE 3. Alveolar macrophages in lungs treated with radiation and soy isoflavones at 18 weeks post-radiation.

Lung tissue sections were obtained from control (Con) mice and mice treated with radiation (Rad) or radiation + soy (Rad+Soy) at 18 weeks after radiation. Sections were stained by IHC for the marker F4/80 to detect alveolar macrophages in the lungs. Arrows indicate positive staining of F4/80⁺ alveolar macrophages. (A). Radiation caused a marked increase in macrophages in thickened alveolar septa areas (arrowheads). Numerous alveolar macrophages were particularly enlarged with abundant cytoplasm showing the morphology of activated macrophages compared to small macrophages in control lungs (see inset). The density of F4/80⁺ macrophages was much lower in radiation + soy treated lungs at18 weeks after radiation compared to radiation–treated lungs. Alveolar macrophages were smaller, resembling those of control lungs (see inset) and the architecture of the alveolar septa was thinner akin to control lung tissue showing decreased pneumonitis compared to radiation–treated lungs. (B). Using ImageJ analysis of IHC slides, the numbers of F4/80⁺ alveolar macrophages were counted in 10 fields of 40× per slide and the average number of F4/80⁺ cells per field ± SE is reported for each treatment group. (C). Measurement of the F4/80⁺ alveolar macrophage cell areas were performed using ImageJ software in 10 fields of 40× per slide and the average cell area of macrophages per field ± SE in each treatment group is reported. The total number of macrophages counted and measured in 10 fields were 125 for control, 285 for radiation, and 101 for radiation + soy-treated mice. The means are reported in (B) and (C). All magnifications are at 40× and insets at 100× to reveal the extent of positive staining and morphology of immune cell subsets. **p<0.01, ***p<0.001, ****p<0.0001, radiation compared to control or radiation + soy compared to radiation alone.

To further clarify the functional phenotype of infiltrating macrophages, the NOS2 activation marker for M1 macrophages and Arg-1 activation marker for M2 macrophages were used to differentiate between pro-inflammatory M1 macrophage phenotype and anti-inflammatory M2 macrophage phenotype. Radiation caused a prominent increase in NOS2 staining of lung tissue by 18 weeks after radiation (Figure 4A), that was apparent in enlarged alveolar macrophages (Figure 4A, inset). These findings were confirmed by slide scanning quantitation for the level of staining (Figure 4B). In contrast, a lower level of NOS2 staining was observed following radiation + soy (Figure 4A, B). Staining of lungs with Arg-1 showed a prominent increase in radiation + soy treated lungs compared to radiation alone (Figure 4C, D).

Radiation caused an increase in pro-inflammatory M1 macrophage phenotype defined by high NOS2 and low Arg-1 levels at 18 weeks after radiation (Figure 4A, B). This is in contrast to relatively low NOS2 levels and high Arg-1 levels observed in lungs treated with radiation + soy or in controls (Figure 4B, C).

Radiation-induced infiltration and activation of granulocytes/neutrophils in lung tissue is decreased by soy isoflavones.

Analysis of BAL immune cells showed an increase in neutrophils by 12 weeks after radiation. Granulocyte/neutrophil phenotype and function were determined by IHC staining of lung tissue sections for Gr-1 (Ly6C/Ly6G) and the neutrophil marker NIMP at 12 weeks after radiation. Radiation caused a pronounced increase in clusters of Gr-1⁺ granulocytes in areas of thickened septa at 12 weeks after radiation (Figure 5, see inset). In contrast, following radiation + soy the alveolar septa were not as thickened and contained much lower levels of Gr-1⁺ cells (Figure 5). NIMP staining for neutrophils followed the same patterns with increased neutrophil infiltrates induced by radiation and decreased by radiation + soy (Figure 5).

FIGURE 5. Effect of soy isoflavones on radiation-induced infiltration of granulocytes/neutrophils in lung tissue.

Lungs tissue sections were obtained from control (Con) mice and mice treated with radiation (Rad) or radiation + soy (Rad+Soy) at 12 weeks after radiation. Sections were stained by IHC for Gr-1 (Ly6C/Ly6G) and NIMP to detect granulocytes/neutrophils. Staining of Gr-1⁺ granulocytes showed that radiation caused a pronounced increase in clusters of granulocytes in areas of thickened septa at 12 weeks after radiation (see inset). In contrast, following radiation + soy treatment the alveolar septa were not as thickened and much lower levels of positive cells for Gr-1 were observed. All magnifications are at 40× and insets at 100× to reveal the extent of positive staining and morphology of immune cell subsets.

Concurrent with increased infiltration of Gr-1⁺ and NIMP⁺ cells, lungs treated with radiation also showed multiple cells with intense MPO staining (Figure 6). MPO⁺ cells formed clusters in areas of thickened alveolar septa, which are indicative of a massive infiltration of activated neutrophils, as confirmed by quantitative analysis of the level of positive staining (Figure 6A, B). However, MPO⁺ infiltrates were reduced in lungs treated with radiation + soy (Figure 6A, B). These data indicate that radiation-induced neutrophil activation in lung tissue is inhibited by soy isoflavones. These findings were confirmed by western blot analysis of MPO expression in lung tissue lysates showing an increase induced by radiation, which was inhibited by the addition of soy isoflavones (Figure 6C).

FIGURE 6. Inhibition of radiation-induced activation of neutrophils in lung tissue.

Lungs tissue sections were obtained from control (Con) mice and mice treated with radiation (Rad) or radiation + soy (Rad+Soy) at 12 weeks after radiation. (A, B). Sections were stained by IHC for the neutrophil activation marker myeloperoxidase (MPO). Radiation caused extensive MPO staining in the lung tissue that is indicative of activated neutrophil infiltration. MPO⁺ activated neutrophils were present in clusters in areas of thickened alveolar septa (see inset) at 12 weeks after radiation. The levels of MPO were greatly reduced in radiation + soy treated lungs. (B). Whole slide scanning for quantitation of MPO positive staining confirmed these findings. For analysis of whole slide scanning, the percentage of positive area was calculated as the total number of positive pixels divided by total number of pixels. NIMP⁺ neutrophils were also increased in lung tissue by radiation, but not by radiation + soy. All magnifications are at 40× and insets at 100× to reveal the extent of positive staining and morphology of immune cell subsets. (C). Western blot analysis of MPO on whole tissue lysates obtained from lungs showed an increase induced by radiation, which was inhibited by the addition of soy isoflavones. Band intensities were quantified using ImageJ (NIH) densitometry analysis.

DISCUSSION

Conventional fractionated radiotherapy concurrent with chemotherapy is currently the standard of care for unresectable locally advanced NSCLC. Treatment success for this patient population has been severely constrained by pneumonitis and later fibrosis.^{29, 30} Hypofractionated radiotherapy is an emerging modality for early-stage lung cancer using high dose per fraction over a short time period to improve effectiveness of tumor destruction and reduce the number of visits for therapy.^31–33 However, high intensity radiotherapy can also be associated with greater damage to lung tissue, emphasizing the need to develop complementary approaches to alleviate radiation-induced injury to normal lung structures and function³⁴. Our pre-clinical murine studies^12–14 demonstrate that soy isoflavones can reduce the extent of inflammatory infiltrates and vascular damage caused by radiation in the lungs, suggesting that soy modulates immune responses triggered by injury. Studies on the effect of soy and the immune system in other diseases besides cancer also support this hypothesis. Genistein down-regulated cytokine-induced pro-inflammatory pathways in human brain microvascular endothelial cells.³⁵ Soy isoflavones had anti-inflammatory mechanisms via modulation of leukocyte-endothelial cell interactions in the study of atherosclerosis.³⁶ The goals of our current study were to determine whether soy isoflavones modulate innate immune cells involved in radiation-induced inflammation in normal lungs by examining the bronchoalveolar space and lung parenchyma compartments.

Macrophages are recruited as a first response to radiation-induced damage in the tumor microenvironment.^{16, 37} Alterations in lung macrophages after radiation have been observed during early and late phases of tissue injury^{38, 39} supporting the idea that macrophage activation contributes, at least partially, to the pathogenesis of radiation-induced lung injury. Therefore, modulation of the response of macrophages to radiation could be a mechanism of radioprotection by soy isoflavones. By 2–4 months after radiation, an increase in the number and size of macrophages was observed both in the bronchoalveolar space and lung parenchyma compartments, indicative of macrophage activation, confirming previous reports.^{38, 39} Soy isoflavones durably decreased the frequency and size of macrophages found in the lung after radiation.

Our flow cytometry analysis of lung parenchyma after lavage identified subsets of residual F4/80⁺CD11c⁺ alveolar macrophages and F4/80⁺CD11c⁻ interstitial macrophages, as reported by others.⁴⁰ A decrease in interstitial macrophages was induced by radiation, whereas this subset was protected by the addition of soy isoflavones. Interstitial macrophages played immunoregulatory roles in the maintenance of lung homeostasis and in pathologic conditions.⁴¹ Soy could potentially inhibit inflammatory responses by protecting interstitial macrophages. In contrast, alveolar macrophages exhibit a greater capacity to functionally contribute to pulmonary inflammation and anti-microbial defense.⁴² We found that soy isoflavones inhibited alveolar macrophage infiltration and activation induced by radiation, a possible mechanism controlling inflammatory processes. These findings suggest that soy modulation of macrophage subset functions in response to radiation may play a critical role in soy-mediated radioprotective effects in lungs.

In radiation-treated lungs, our analysis of myeloid cells showed extensive infiltration of inflammatory cells at sites of pneumonitis, consisting of macrophages and neutrophils. Both types of immune cells were morphologically and molecularly in a status of activation. In contrast, soy supplementation to radiation decreased both the infiltration and activation of myeloid cells. The influence of soy isoflavones on M1 and M2 macrophage polarization in irradiated lungs could be a mechanism of radioprotection. Macrophages possess the plasticity to respond to environmental stressors in tissues that functionally range from M1 pro-inflammatory to M2 immunosuppressive, anti-inflammatory phenotypes.^{43, 44} These two phenotypes can be distinguished by expression of NOS2 and Arg-1.⁴⁵ Normal tissue exposed to ionizing radiation generates “damage” signals and type 1 cytokines, such as IL-1β, IL-6, and TNF-α, that classically activate macrophages (M1) and drive the acute/chronic pulmonary inflammation induced by radiation.^{46, 47} M1 macrophages produce NOS2, which generates reactive NO species, thus promoting inflammation. Alternatively activated macrophages (M2) are important for the resolution of inflammation.⁴⁸ M2 macrophages produce Arg-1, which generates L-orthonine from arginine which is a precursor of proline, known to enhance collagen synthesis, thus promoting tissue repair and resolution of inflammation.^{43, 49} Our studies now demonstrate that radiation induced a pro-inflammatory M1 phenotype in lungs at late time points, while mice receiving soy isoflavones and radiation switched to an anti-inflammatory M2 subtype with increased levels of Arg-1 and decreased NOS2. These data indicate that soy isoflavones supplementation to radiation could result in skewing of alveolar macrophages from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype. These data are in agreement with soy isoflavones inhibition of the release of TNF-α, IL-1β, IL-6, and IFN-γ pro-inflammatory cytokines induced by radiation in lung tissues that promote an M1 macrophage phenotype.¹³

Infiltration and activation of neutrophils into the lung are key factors that occur after damage to lung tissue.^{23, 38} Therefore, inhibition of the inflammatory neutrophil response induced by radiation in the pulmonary environment may result in reduced host tissue damage. Our flow cytometry studies of lung single cell suspensions showed that CD11b⁺Ly6G⁺ neutrophils were increased after radiation. Immunostaining confirmed clusters of activated neutrophils, as confirmed by MPO staining, in sites of pneumonitis caused by radiation. Treatment with soy isoflavones inhibited radiation-induced neutrophil infiltration and activation, suggesting a mechanism of protection from tissue damage by soy.

In summary, our pre-clinical study in lung suggests that a radioprotective mechanism of soy isoflavones could involve inhibition of infiltration and activation of macrophages and neutrophils in irradiated lungs. These findings indicate that soy isoflavones used as a complementary intervention to radiotherapy for lung cancer could potentially reduce lung toxicity. This approach has been translated into an ongoing Phase I clinical trial of chemoradiotherapy for advanced stage III NSCLC to evaluate the safety and efficacy of soy isoflavone supplementation.