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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Health Phys. 2021 Oct 1;121(4):331–344. doi: 10.1097/HP.0000000000001443

AEOL 10150 alleviates radiation-induced innate immune responses in nonhuman primate lung tissue

Wanchang Cui 1,2,3,†, Pei Zhang 2, Kim G Hankey 2, Mang Xiao 1, Ann M Farese 2, Thomas J MacVittie 2,†
PMCID: PMC8601036  NIHMSID: NIHMS1749432  PMID: 34546215

Abstract

Purpose:

To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model.

Methods and Materials:

Rhesus macaques were irradiated with lethal doses of radiation to the whole thorax. A subset of the irradiated animals was treated with AEOL 10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Lung tissues were collected at necropsy for molecular and immunohistochemical (IHC) studies.

Results:

Microarray expression profiling in the irradiated lung tissues identified differentially expressed genes (DEGs) and pathways important in innate immunity. The elevated expression of cytokines (CCL2, CCL11, IL-8), complement factors (CFB, C3), apoptosis-related molecules (p53, PTEN, Bax, p21, MDM2, c-Caspase 3) and adhesion molecules (fibronectin, integrin β6, ICAM-1) were further studied using realtime PCR, Western blot, or IHC. Oxidative stress and pulmonary inflammatory cell infiltration were increased in the irradiated lungs. Treatment with AEOL 10150 significantly decreased oxidative stress and monocyte/macrophage infiltration.

Conclusions:

Cytokine/chemokine induced excessive innate immune response after thoracic irradiation plays important roles in RILI. To our knowledge, this is the first study to highlight the role of cytokine/chemokine induced innate immune responses in radiation-induced pulmonary toxicity in a NHP model.

Keywords: Radiation damage, lung, microarray, innate immunity, non-human primate (NHP)

Introduction

The acute radiation syndrome (ARS) is a serious health threat after accidental or deliberate radiation exposure. Hematopoietic and gastrointestinal sub-syndromes are the two main acute syndromes that patients may experience after exposure. For victims that have survived the ARS, the delayed effect of acute radiation exposure (DEARE) on multiple organs may contribute to significant co-morbidities and mortality (MacVittie et al. 2019). The pulmonary system is one of the organs affected by DEARE. Lung is a radio-sensitive organ with an estimated threshold of pneumonitis at 8 Gy in humans and the threshold for potential lethality at 10 Gy (Van Dyk et al. 1981, Hill 2005). Radiation-induced lung injury (RILI) is reported in over 50% of victims after radiation accidents often in conjunction with multi-organ failure (Medhora et al. 2012). In the 134 patients with confirmed diagnosis of ARS after the Chernobyl accident, radiation pneumonitis was detected in 8 patients (Mettler et al. 2007) . In the clinical setting, lung is also a major organ to receive high dose radiation in patients receiving thoracic radiation for treatment of lung and breast cancers. RILI may take months or even years to present clinical manifestations. Classically, RILI has been described into two phases, acute pneumonitis and late fibrosis (Hill 2005). The radiation-induced pneumonitis manifests after approximately 2 months, while the fibrosis presents months to years later (Marks et al. 2003, Hill 2005). In a meta-analysis of patients who underwent radiotherapy, it was found that 29.8% of patients developed pneumonitis and 1.9% of patients developed fatal pneumonitis (Palma et al. 2013). There are no controlled, randomized trials on the treatment of radiation-induced pneumonitis, however, corticosteroids are generally prescribed for patients with symptomatic radiation pneumonitis (Sekine et al. 2006). There is no proven effective treatment for radiation-induced lung fibrosis.

It is important to understand and elucidate the mechanisms that underlie RILI. In a nonhuman primate (NHP) model of whole thorax lung irradiation (WTLI), the dose response relationship and time course of the delayed pulmonary sequela and consequent morbidity and mortality was established (Garofalo et al., 2014). The WTLI model can be used to test the efficacy of medical countermeasures (MCM) for RILI. In a pilot study, it was shown that treatment with a small molecular weight catalytic metalloporphyrin antioxidant (AEOL 10150) from day 1-28 led to reduced clinical and radiographic RILI (Garofalo et al. 2014). A larger study using the same protocol showed that administration of AEOL 10150 from day 1-60 significantly increased survival after 10.74 Gy WTLI in NHP (MacVittie et al. 2017).

In the current study, global mRNA expression profiling was performed on the lung tissues from non-irradiated and the irradiated NHP used to establish the WTLI model. Differentially expressed genes (DEGs) between non-irradiated and irradiated animals were identified. KEGG pathway analysis was performed to identify the potentially affected pathways after thoracic irradiation. The expression of selected DEGs was further confirmed using qRT-PCR, Western blot and/or immunohistochemical (IHC) staining. Many DEGs that were significantly changed upon irradiation were related to the innate immune responses, such as chemokines and complement systems. Inflammatory cell infiltration into the irradiated lung tissues was studied using IHC staining. Treatment with AEOL 10150 significantly decreased the oxidized phospholipid (OxPL) (suggested as triggers of inflammation) level and monocyte/macrophage infiltration in the irradiated lungs. AEOL 10150 is not approved by FDA as a treatment for radiation injury and it is an experimental drug. To our knowledge, this is the first large-scale analysis of gene expression in a large animal WTLI model and suggests that innate immunity plays important roles in RILI.

Methods

Study animals, radiation exposure and dosimetry

Study animals were from two previously published studies (Garofalo et al. 2014, Garofalo et al. 2014). Briefly, Rhesus macaques (Macaca mulatta) were irradiated using a 6 MV photon source LINAC. NHPs were irradiated to the whole thorax at a dose rate of 0.80 Gy min−1. The dose was prescribed to midplane in the thorax. NHP were exposed in an antero-posterior (AP)/postero-anterior (PA) technique with approximately 50% dose contribution from the AP beam and 50% contribution for the PA beam. Real-time exposure doses were confirmed with dosimeters (Landauer® nanoDot™ system, Glenwood, IL 60425) placed on each animal at the time of irradiation. In the dose response study, NHP were exposed to doses from 9.0 to 12.5 Gy. In the MCM pilot study, NHP were exposed to 11.5 Gy WTLI, and then treated with vehicle or AEOL 10150 (5 mg kg−1 daily for a total of 28 days, with the first injection given at 24 hours after radiation exposure). Cageside and clinical observations were performed twice daily. Medical management (supportive care) was provided to all study animals per clinical signs to initiate and stop treatment. The main medical management was the corticosteroids (Dexamethasone) treatment for radiation pneumonitis (defined as non-sedated respiratory rate ≥ 80 breaths per minute). All animals that met euthanasia criteria prior to the end of study were euthanized. Five to seven non-irradiated, control NHP was included in this study. Numbers of the control samples in each experiment may vary due to tissue availability. All experiments were performed with prior approval from the University of Maryland Institutional Animal Care and Use Committee (IACUC).

Microarray and Statistical Methods

Lung tissues were collected and preserved in RNAlater® solution (Life Technologies, Carlsbad, CA), snap frozen or formalin fixed at necropsy. Effort was made to collect morphologically damaged lung tissue from each lobe of the irradiated NHP. There was generally no damaged lung tissue from the non-irradiated NHP. For molecular/histo studies, lung tissue from different animals on the same lobe was compared. Total RNA was extracted using miRNeasy mini Kit (Qiagen, Vlencia, CA). 100 ng total RNA was used for generating sense-strand cDNA using Ambion® WT Expression Kit (Life Technologies, Carlsbad, CA) and subsequent fragmentation and labeling using the GeneChip® WT Terminal Labeling kit (Affymetrix, Santa Clara, CA). Fragmented and labeled DNA samples were hybridized to Rhesus Gene 1.1 ST Array strips (catalog # 901799, Affymetrix Inc., Santa Clara, CA) at 48°C for 16 hours. Hybridized strips were then washed and stained using an Affymetrix Fluidics Workstation. Array images were scanned using a GeneAtlas® Imaging Station (Affymetrix Inc.). Probe set summarization and initial data quality examination was performed using the Expression Console™ software (Affymetrix Inc.). DEGs were identified using the Transcriptome Analysis Console (TAC) software (Affymetrix Inc.). DEGs were defined with at least 2-fold change and with ANOVA p < 0.05 and FDR < 0.05.

Bioinformatics analysis

To elucidate the biological processes and pathways related to the changed genes, DEGs were functionally annotated and classified using the WebGestalt (Wang et al. 2013) and the DAVID Bioinformatics Resources (Huang da et al. 2009). Because of the lack of bioinformatics information for rhesus macaque (Macaca mulatta), human orthologs for the significantly changed rhesus genes were used for the analysis.

Real-time quantitative reverse transcription PCR (qRT-PCR) validation

The reverse transcription reaction was performed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). An aliquot of the diluted cDNA was used to measure rhesus CCL2 (Rh02621753_m1), CCL11 (Rh02913931_m1), IL-8 (Rh02789706_m1) gene expression with the TaqMan primer and probe mixes from Life Technologies (Grand Island, NY). Actin (Rh03043379_gH) was used as an endogenous control for normalization. Real-time PCR was performed using a CFX96 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA).

Western blot

Snap frozen lung tissues were lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) using a TissueLyser (Qiagen, Hilden, Germany) with stainless steel beads. Lysates were centrifuged at 13,000 g for 10 minutes at 4°C. Supernatants were collected and centrifuged again. Protein concentration in the supernatant was quantified using QuickStart Bradford Protein Assay (Bio-Rad Laboratories). Lysates containing 20-40 μg total protein were resolved by tris-glycine SDS-PAGE gel electrophoresis. Proteins were transferred to PVDF membranes using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories). Membranes were blocked with 5% dry milk and then incubated with diluted primary antibodies at 4°C overnight. Bound primary antibodies were detected using horseradish-peroxidase-conjugated secondary antibodies. Chemiluminescent signals were developed using the SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL) and acquired using a GE ImageQuant LAS4000 Luminescent Image Analyzer (GE Healthcare Bio-Sciences, Piscataway, NJ). Densitometry values were quantified using NIH ImageJ (version 1.45S) analysis software (National Institutes of Health, Bethesda, MD). SDS-PAGE gels and Western blot reagents/supplies were from Bio-Rad Laboratories. The antibodies used in this study were listed in Table 1. PVDF membranes were stripped using the Restore (TM) Western Blot Stripping Buffer (Fisher Scientific, Pittsburgh, PA) to remove the bounded antibodies and re-probed for additional targets. For the Western blot of the complement system, the gels were run under non-reducing conditions. All Western blot experiments were performed using samples from at least two lobes from each animal, with at least two technical repeats.

Table 1.

Antibodies used in the current study.

Antibody name Antibody abbreviations Vendor Catalog number Applications MW kDa
CCL2/MCP-1 CCL2 Pierce Antibody Products 710002 Western Blot, IHC 11
CCL 11/Eotaxin-1 CCL11 Fitzgerald Industries 70R-7852 Western Blot, IHC 8
Interleukin 8 IL-8 BD Biosciences 550419 Western Blot 6-8
Complement factor B CFB Complement Technology, Inc. A235 Western Blot, IHC 97
Complement component 3 C3 Complement Technology, Inc. A213 Western Blot 187
C3c complement C3 Dako F0201 IHC
p53(DO-1) p53 Santa Cruz Biotechnology, Inc. Sc-126 Western Blot 53
PTEN PTEN Cell Signaling Technology, Inc. 9552 Western Blot 54
BAX BAX Cell Signaling Technology, Inc. 2772 Western Blot 20
p21 (SX118) P21 Santa Cruz Biotechnology, Inc. Sc-53870 Western Blot, IHC 21
MDM2 (SMP14) MDM2 Santa Cruz Biotechnology, Inc. Sc-965 Western Blot 90
Cleaved Caspase-3(Asp175)(5A1E) c-Caspase 3 Cell Signaling Technology, Inc. 9664 Western Blot 17,19
L1 (MAC387) monocyte/macrophage Monocyte/macrophage Thermo Fisher Scientific Inc. MA1-33972 IHC
Myeloperoxidase (MPO) Ab-1 MPO Thermo Fisher Scientific Inc. RB-373 IHC
Fibronectin Ab-1 (Clone HFN 7.1) Fibronectin Thermo Fisher Scientific Inc. MS-165 Western Blot 220
Integrin β6 Antibody (C-19) Integrin b6 Santa Cruz Biotechnology, Inc Sc-6632 Western Blot 97
Intracellular adhesion molecule-1 (H108) ICAM-1 Santa Cruz Biotechnology, Inc Sc-7891 Western Blot 90
E06 anti-oxidized phospholipids E06 Avanti Polar Lipids 330001 IHC
GAPDH (FL-335) GAPDH Santa Cruz Biotechnology, Inc Sc-25778 Western Blot 37
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Immunohistochemistry and quantification

Formalin-fixed paraffin-embedded (FFPE) lung tissues were cut at 5 μm thickness and mounted on positively charged glass slides. Sections were de-waxed and rehydrated using standard methods. Heat mediated antigen retrieval was performed using a Lab Vision TM PT Module (Thermo Scientific, Pittsburgh, PA). Endogenous peroxidase activity was neutralized with BloxAll solution (Vector Laboratories, Burlingame, CA). Specimens were blocked with 2.5% normal serum (Vector Laboratories, Inc.) and then incubated with diluted primary antibodies at 4°C overnight. The immunohistochemistry procedure was performed using an IMMPRESS Universal kit (Vector Laboratories, Inc.) according to the manufacturer’s guidelines. Substrate was developed using an ImmPACT DAB kit (Vector Laboratories, Inc.). Hematoxylin was used for counterstaining. Digital images of slides were obtained at 20X magnification (resolution at 0.498 microns per pixel) using a whole slide scanner (ScanScope® CS System, Aperio ePathology, Vista, CA) fitted with a 20X/0.75 Plan Apo objective lens (Olympus, Center Valley, PA) and saved and retrieved using a software interface (Spectrum™ Database, Aperio ePathology). The percentage of positively stained cells was quantified using the Nuclear V9 algorithm on the whole image (Aperio ePathology). The percentage of positively stained pixels was quantified using the Positive Pixel Count V8 algorithm on the whole image (Aperio ePathology). All staining was performed using samples from at least two lobes from each animal, with at least two technical repeats.

Eosinophil staining and quantification

The eosinophil staining was performed based on Llywellyn’s method (Meyerholz et al. 2009). Sections were stained with Harris Hematoxylin, decolored with acid alcohol, and blued with 1% sodium carbohydrate. The sections were then immersed in an alkaline (pH 8-9) Sirius red solution for 3 hours at room temperature. Sections were dehydrated in alcohol and xylene and then mounted with coverslips. Digital images of slides were obtained at 40X (resolution at 0.248 microns per pixel) using the ScanScope® CS System. Eosinophil staining was performed using samples from at least two lobes from each animal, with at least two technical repeats.

To quantify eosinophils, ten random fields (size 0.5 mm X 0.5 mm) from each lung section were selected at low power (avoiding major airways/vessels). Eosinophils in these fields were counted at high power (400X) and were averaged for each lobe.

Statistical analysis

Statistical analysis was performed using Graphpad Prism 5 (Graphpad Software, Inc.). One-way ANOVA and Bonferroni’s Multiple Comparison post-test were performed to compare the means of three groups when data had a normal distribution. Kruskal-Wallis tests and Dunn’s multiple comparison post-test were performed to compare the means of three groups when data didn’t have a normal distribution. All data were shown as mean ± standard error of mean (s.e.m). A p value of less than 0.05 was considered significant.

Results

Description of the irradiated NHP used in the current study

For the microarray analysis and qRT-PCR, lung tissues from 26 NHP exposed to 9.0 to 12.0 Gy WTLI were used. NHP were segregated into two groups based on their survival status: decedents (euthanized before the end of study) (n = 14) or survivors (n = 12). Decedent animals were exposed to WTLI doses of 10.0, 11.0 or 12.0 Gy, and their mean survival time was 101.9 days. The survivor animals were exposed to 9.0 or 11.0 Gy WTLI, and they were euthanized at or after the 180 day study duration. Five non-irradiated NHP were also included for comparison.

For the Western blot and IHC assays, samples were from the NHP used in the MCM study (frozen lung tissues were available from 12 NHP; FFPE samples were available from all 13 NHP). NHP were exposed to 11.5 Gy WTLI and then either treated with AEOL 10150 from day 1-28 (AEOL 10150 treatment group, n = 7) or saline/nothing (radiation vehicle group, n = 6). Two out of the seven NHP in the AEOL 10150 cohort (n = 7) survived 180 days after radiation. All other NHP in the AEOL 10150 treatment group and the radiation vehicle group were euthanized before the end of study.

Microarray expression data analysis

The gene expression data have been deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE70943. Using the Rhesus Gene 1.1 ST Array strips, with criteria of at least 2-fold change, ANOVA p < 0.05, and FDR < 0.05, there were 397 DEGs between the non-irradiated and the decedent group. However, with the same criteria, there were only 4 DEGs between the non-irradiated and the survivor group. Because of the limited number of DEGs between the non-irradiated and the survivor group, analysis was focused on the differences between the non-irradiated and the decedent group. Fig. 1 showed the hierarchical clustering of the DEGs between the non-irradiated and the decedent groups.

Fig. 1.

Fig. 1.

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Unsupervised hierarchical clustering analysis of mRNA expression of lung tissues from 5 non-irradiated and 14 decedent NHP. The heatmap shows the expression of 379 DEGs at the probe level. Heatmap colors represent relative mRNA expression as indicated in the color key.

Among the transcripts with the most highly elevated expression in the decedent group compared to the non-irradiated group (Table 2), the most striking observation was that several transcripts important in inflammatory reactions were overrepresented in the decedent group. These overrepresented transcripts included CCL2 (16.23-fold), CCL11 (11.08-fold), interleukin 8 (IL-8) (7.01-fold), and CD163 (6.75-fold). CCL2, CCL11 and IL-8 are members of chemokines that act as chemoattractants to guide the migration of inflammatory cells. The top 20 most down-regulated DEGs were listed in Table 3.

Table 2.

Top 20 up-regulated DEGs in the decedent group compared to the non-irradiated group.

Gene Symbol Description Fold Change ANOVA p-value FDR p-value
SERPINA3 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 34.25 4.19E-10 0.000006
MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 29.15 0.000001 0.000378
PIP prolactin-induced protein 18.78 0.000067 0.004229
CCL2 chemokine (C-C motif) ligand 2 16.23 0.000012 0.001567
AREGB amphiregulin B 14.37 0.000298 0.010161
LRRN4 leucine rich repeat neuronal 4 13.72 0.000218 0.008454
LOC711693 angiopoietin-related protein 4-like 12.95 0.000108 0.005557
CCL11 chemokine (C-C motif) ligand 11 11.08 0.000011 0.001473
SERPINE1 serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 9.66 0.000156 0.006934
DKK1 dickkopf 1 homolog (Xenopus laevis) 8.63 0.000109 0.005603
TNFRSF21 tumor necrosis factor receptor superfamily, member 21, uncharacterized LOC100425582 7.61 9.10E-08 0.000118
ADAMDEC1 ADAM-like, decysin 1 7.52 0.002943 0.042076
LOC721488 potassium-transporting ATPase alpha chain 2-like 7.41 0.000865 0.019792
KRT8 keratin 8 7.39 0.000004 0.000898
IL-8 interleukin 8 7.01 0.00088 0.019994
CD163 CD163 molecule, scavenger receptor cysteine-rich type 1 protein M160-like 6.75 0.000662 0.01661
CTSL cathepsin L1-like 6.69 0.00346 0.046484
F3 coagulation factor III (thromboplastin, tissue factor) 6.54 6.60E-07 0.000338
CLDN10 claudin 10 6.38 0.000036 0.002956
DPP4 dipeptidyl-peptidase 4 6.26 0.000204 0.008129
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Table 3.

Top 20 down-regulated mRNAs in the decedent group compared to the non-irradiated group.

Gene Symbol Description Fold Change ANOVA p-value FDR p-value
PRKCE protein kinase C, epsilon −4.34 0.000025 0.002293
LOC697047 troponin C, slow skeletal and cardiac muscles-like −4.68 0.002828 0.041151
NCKAP5 NCK-associated protein 5 −4.72 0.000758 0.018211
NCKAP5 NCK-associated protein 5 −4.76 0.000054 0.003704
TOX3 TOX high mobility group box family member 3 −4.82 2.70E-07 0.000195
LTB4DH prostaglandin reductase 1-like −5.12 0.000768 0.018347
LOC714886 muscarinic acetylcholine receptor M2-like −5.28 0.000083 0.004831
C1orf110 chromosome 1 open reading frame 110 −5.43 0.000009 0.001321
HHIP hedgehog interacting protein −5.48 0.000265 0.00953
LOC708250 olfactory receptor 5D13-like −6.34 0.000049 0.003528
ITLN1 intelectin 1 (galactofuranose binding) −6.46 0.000069 0.004301
ENPP6 ectonucleotide pyrophosphatase/phosphodiesterase 6 −6.46 0.000382 0.011716
LEPR leptin receptor −7.23 9.44E-09 0.000048
COL6A6 collagen, type VI, alpha 6 −7.31 5.24E-07 0.000298
SERPINB13 serpin peptidase inhibitor, clade B (ovalbumin), member 13 −8.51 1.32E-07 0.000127
LOC697272 uncharacterized LOC697272 −8.67 0.000044 0.003282
PHEX phosphate regulating endopeptidase homolog, X-linked −8.77 0.000017 0.001856
LOC709661 immunoglobulin superfamily member 10-like −9.54 0.000007 0.001172
MS4A15 membrane-spanning 4-domains, subfamily A, member 15 −11.17 0.000001 0.000384
SLC6A4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 −20.9 1.86E-07 0.000157
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Based on the 397 DEGs that were significantly changed in the decedent group, KEGG pathway analysis was performed to identify the enriched pathways that may play important roles in RILI.

As shown in Table 4, these pathways include apoptosis (p53 signaling pathway), inflammation (malaria, complement and coagulation cascades, cytokine-cytokine receptor interaction, Rheumatoid arthritis, and focal adhesion), heart injury (Arrhythmogenic right ventricular cardiomyopathy) and infection (Staphylococcus aureus infection). KEGG pathway analysis using the DAVID bioinformatics gave a similar result. Considering that inflammatory genes were among the most up-regulated DEGs in the transcript list, inflammation has been known to play important roles in radiation-induced tissue damage (Zhao and Robbins 2009, Ding et al. 2013), and inflammation mechanistically links to apoptosis and oxidative stress (Haanen and Vermes 1995, Zhao and Robbins 2009), we next focused on investigating the roles of innate immunity in RILI. Particularly, the expression of chemokines, inflammatory cells, complement system, and apoptosis in NHP exposed to WTLI were investigated. The effect of the mitigator AEOL 10150 on those parameters was also investigated.

Table 4.

KEGG pathway analysis of the significantly changed genes

Pathway name # of Gene Gene Symbol Ratio of Enrichement adjusted p value
Malaria 9 THBS2, CCL2, IL-8, IL18, HGF, TLR4, DARC, ICAM-1, THBS1 10.62 7.48E-06
P53 signaling pathway 10 DDB2, MDM2, SESN2, TNFRSF10B, SERPINE1, CDKN1A, ZMAT3, SFN, IFGBP3, THBS1 8.85 7.48E-06
Complement and coagulation cascades 9 SERPINA1, CIQC, F13A1, C5AR1, SERPINE1, CFB, PROS1, PLAT, F3 7.85 5.88E-05
cytokine-cytokine receptor interaction 15 PDGFC, EDA2R, CCL2, CXCL3, TNFRSF21, TNFRSF10B, IL-8, IL1R2, HGF, IL18, TNFRSF12A, LEPR, LIFR, CCL11, INHBA 3.41 7.00E-04
Arrhythmogenic right ventricular cardiomyopathy (ARVC) 8 GJA1, ITGA8, CACNB2, ITGA2, ACTN2, ITGAV, ITGB6, DSP 6.51 7.00E-04
Rheumatoid arthritis 8 ATP6V1A, CCL2, CD86, IL-8, IL18, TLR4, ICAM-1, ACP5 5.29 1.20E-03
ECM-receptor interaction 8 ITGAV, THBS2, ITGB6, ITGA8, LAMC2, ITGA2, COL6A6, THBS1 5.66 1.20E-03
Staphylococcus aureus infection 6 C5AR1, FPR3, CFB, ICAM-1, C1QC, FCAR 6.57 3.20E-03
Pathways in cancer 15 RXRG, MDM2, FZD4, DCC, MYC, ITGA2, IL-8, AXIN2, AR, HGF, ITGAV, CDKN1A, RUNX1, LAMC2, HHIP 2.77 3.80E-03
Focal adhesion 11 PDGFC, THBS2, ITGA8, ITGA2, HGF, ITGAV, ACTN2, ITGB6, LAMC2, THBS1, COL6A6 3.31 4.30E-03
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Up-regulated chemokines confirmed using qRT-PCR, Western Blot and IHC

Several significantly elevated transcripts from the microarray analysis were selected for independent validation using qRT-PCR. Chemokines CCL2, CCL11 and IL-8 were chosen for validation because they are well characterized chemo-attractants for inflammatory cells (monocytes/macrophages, eosinophil and neutrophils, respectively). Using qRT-PCR assay, all three chemokines showed a significant increase comparable to the microarray results. The fold changes of CCL2, CCL11, and IL-8 in the decedent group were 20.94 ± 3.43 (p < 0.001), 12.91 ± 2.36 (p < 0.001) and 9.17 ± 2.06 (p < 0.05) compared to the non-irradiated group (Fig. 2A). All three chemokine levels were also higher in the irradiated survivor group compared to the non-irradiated group but only the difference of CCL2 was significant (10.79 ± 2.77-fold, p<0.05).

Fig. 2.

Fig. 2.

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Elevated expression of CCL2, CCL11 and IL-8 in the irradiated lungs. Expression of CCL2, CCL11 and IL-8 were assayed in the non-irradiated and irradiated animals. (A) Real-time PCR quantification of the three cytokines. Animals were grouped in to the non-irradiated group, decedent group and the survivor group. Each dot represents one animal. n = 5, 14 and 13 animals in each group respectively. (B) Western blot of the three chemokines and densitometry analysis of the blotting. Animals from the non-irradiated (n = 5), 11.5 Gy vehicle (n = 6) and 11.5 Gy AEOL 10150 treatment (n = 6) groups were used for the analysis. The euthanasia date for each animal was shown on top of the blots. The molecular weight of each target was shown on the right. Images from the same membrane were marked using the vertical lines on the right side. (C) Representative images of lung tissue sections stained with CCL2. (D) Representative images of lung tissue sections stained with CCL11. *, p < 0.05; **, p < 0.01; ***, p < 0.001. scale bar = 50 μm.

The expression of these three chemokines were further investigated on the protein level using Western blot (Fig. 2B). All three chemokines were significantly up-regulated in both the 11.5 Gy vehicle group and AEOL 10150 treatment group compared to the non-irradiated group. The fold increases of CCL2, CCL11, and IL-8 were 22.79 ± 4.67 (p < 0.001), 3.09 ± 0.82 (p < 0.05), and 2.93 ± 0.45 (p < 0.01) in the 11.5 Gy vehicle group and 15.64 ± 3.62 (p < 0.01), 2.72 ± 0.39 (p < 0.01), and 2.62 ± 0.38 (p < 0.01) in the AEOL 10150 treatment group respectively. There was no significant difference of CCL2, CCL11, and IL-8 protein expression between the 11.5 Gy vehicle and AEOL 10150 treatment groups.

The expression of these three chemokines were further studied using IHC on lung sections. CCL2 had very low expression in the non-irradiated animals and strong expression in the irradiated lungs (Fig. 2C). CCL2 was mainly expressed in the pulmonary macrophages. CCL11 had a very low expression level in non-irradiated animals. In the irradiated NHP, CCL11 had a very strong expression in airway epithelial cells, airway smooth muscle, vascular endothelium, and alveolar macrophages (Fig. 2D). There was no significant difference of CCL2 and CCL11 between the vehicle group and AEOL 10150 treatment group. IHC for IL-8 was not successful.

Complement system activation

The complement system is an important part of the innate immune system and its excessive activation contributes to inflammation-mediated tissue damage such as ischemia/reperfusion injury and autoimmune disorders (Markiewski et al. 2007). The KEGG pathway analysis suggested the activation of complement system in the irradiated lungs. To further confirm its involvement in RILI, expression of Complement factor B (CFB) and complement component 3 (C3) in the complement system were assayed using Western blot and IHC (Fig. 3). CFB and C3 were both highly elevated in the 11.5 Gy vehicle group and the AEOL 10150 treatment group (Fold changes of CFB were 3.35 ± 0.32, p < 0.001 and 2.32 ± 0.18, p < 0.01; fold changes of C3 were 2.15 ± 0.30, p < 0.001, and 1.88 ± 0.26, p < 0.01 in the 11.5 Gy vehicle group and AEOL 10150 treatment group respectively) (Fig. 3A&B). Both CFB and C3 showed amorphous intra-alveolar staining in the irradiated lungs (Fig. 3C), which may reflect an exudative process of complement elements leaking through and accumulating in the alveolar lumen. There was no significant difference of the expression of CFB and C3 between the vehicle and the AEOL 10150 treatment groups.

Fig. 3.

Fig. 3.

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Elevated expression of the alternative activation of complement system in the irradiated lungs. (A) Western blot images of CFB and C3 in the non-irradiated (n = 5), 11.5 Gy vehicle (n = 6) and 11.5 Gy AEOL 10150 treatment (n = 6) groups. The euthanasia date for each animal was shown on top of the blots. The molecular weight of each target was shown on the right. (B) Densitometry analysis of CFB and C3 expression. (C) Representative images of lung tissue sections stained with CFB and C3 respectively. **, p < 0.01; ***, p < 0.001. scale bar = 50 μm.

Increased number of inflammatory cells in the irradiated lungs

The increased levels of chemokines and activation of the complement system may induce the migration of inflammatory cells to the irradiated lungs. To test this, IHC was performed to quantify the percentage of monocytes/macrophages and neutrophils using cell-specific antibodies; the infiltration of eosinophils was also quantified based on Llywellyn’s method (Fig. 4). Levels of monocytes/macrophages, neutrophils, and eosinophils were all significantly increased in the 11.5 Gy vehicle group compared to the non-irradiated group (18.07 ± 0.98% vs. 7.73 ± 0.81%, p < 0.001; 8.32 ± 0.54 vs. 4.93 ± 0.42%, p < 0.001; and 12.47 ± 2.03% vs. 4.26 ± 0.90%, p < 0.05 respectively). This result was in agreement with the increased chemokine and complement levels in the irradiated lungs. Treatment with AEOL 10150 significantly decreased the percentage of monocytes/macrophages in the irradiated lungs (decreased to 10.99 ± 0.69%, p < 0.01). AEOL 10150 treatment had no significant effect on the levels of neutrophils and eosinophils.

Fig. 4.

Fig. 4.

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Pulmonary inflammation in the irradiated lungs. Lung sections were stained with cell type specific markers for monocytes/macrophages in the non-irradiated (n = 5), 11.5 Gy vehicle (n = 6) and 11.5 Gy AEOL 10150 treatment (n = 7) groups. An antibody against myeloperoxidase was used as a marker for neutrophils. Eosinophils were stained using alkaline Sirius red. Quantification is shown on the right side for each cell type. scale bar = 50 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Apoptosis in the irradiated lungs

Apoptosis has been suggested to play important roles in RILI (Zhang et al. 2012). Based on the KEGG pathway analysis of the mRNA array data, the p53 pathway was a significantly changed pathway in WTLI. Western blot was performed to assay the important players in the p53 pathway (Fig. 5). p53, p21, Bax, PTEN, MDM2 and cleaved Caspase3 were all significantly up-regulated in the 11.5 Gy vehicle group and AEOL 10150 treatment group compared to the non-irradiated group. There was no significant difference between the 11.5 Gy vehicle group and the AEOL 10150 treatment group.

Fig. 5.

Fig. 5.

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Elevated expression of apoptosis-associated proteins in the irradiated lungs. Key players in apoptosis were assayed using Western Blot. (A) Western blot of p53, PTEN, Bax, p21, MDM2 and cleaved-Caspase 3 from animals of the non-irradiated (n = 5), 11.5 Gy vehicle (n = 6) and 11.5 Gy AEOL 10150 treatment (n = 6) groups. The euthanasia date for each animal was shown on the top of the blots. The molecular weight of each target was shown on the right. Images from the same membrane were marked using the vertical line on the right side. (B) Densitometry quantification of each target in (A). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Increased expression of cell adhesion molecules in the irradiated lungs

Many integrin and their ligands were significantly changed in the irradiated NHP in the mRNA array. These included integrins (Integrin β6, β-like 1, α2, αV, α8) and integrin ligands (Activated leukocyte cell adhesion molecule; Intercellular adhesion molecule 1; Laminin, gamma 2; Collagen, type XV, alpha1; Versican; and Collagen, type VI, alpha 6). Western blot was performed to validate the elevated expression of selected adhesion molecules (Fig. 6). Fibronectin and integrin β6 had significantly higher expression in the 11.5 Gy vehicle group and the AEOL 10150 treatment group compared to the non-irradiated group (fold changes of 7.91 ± 0.61 and 10.34 ± 4.55 in the 11.5 Gy vehicle group, p < 0.001 for both; and 4.73 ± 0.89 and 4.55 ± 0.79 in the AEOL 10150 treatment group, p < 0.01 for both). ICAM-1 was significantly increased in the 11.5 Gy vehicle group compared to the non-irradiated group (2.28 ± 0.41-fold change, p < 0.01). Treatment with AEOL 10150 had no significant effect on the expression of these molecules.

Fig. 6.

Fig. 6.

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Elevated expression of adhesion and integrin molecules in the irradiated lungs. (A) Western blot of fibronectin, integrin β6, and ICAM-1 from animals of the non-irradiated (n = 5), 11.5 Gy vehicle (n = 6) and 11.5 Gy AEOL 10150 treatment (n = 6) groups. The euthanasia date for each animal was shown on top of the blots. The molecular weight of each target was shown on the right. (B) Densitometry quantification of each target in (A). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

AEOL 10150 decreased radiation-induced expression of OxPL

OxPL have been suggested to play a common role in triggering acute lung injury induced by different modes of injury (Imai et al. 2008). To test whether radiation caused production of OxPL, IHC was performed on the irradiated lung tissues using a monoclonal antibody specific to the oxidized form of phospholipids (Fig. 7). There was minimal expression of OxPL in the non-irradiated animals (percent positive staining for OxPL was 0.020 ± 0.004). Radiation significantly increased the expression of OxPL (percent positive staining for OxPL was increased to 0.122 ± 0.022, p < 0.001). Treatment with AEOL 10150 significantly decreased the expression level of OxPL (percent positive staining was to 0.060 ± 0.013, p < 0.05 compared to the 11.5 Gy vehicle group). The expression of OxPL was mainly localized in the foamy macrophages based on the IHC staining.

Fig. 7.

Fig. 7.

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Elevated expression of OxPL in the irradiated lungs. IHC was performed on lung tissue slides from animals of the non-irradiated (n = 5), 11.5 Gy vehicle (n = 6) and 11.5 Gy AEOL 10150 treatment (n = 7) groups using a monoclonal E06 antibody to detect the expression of OxPL. (A) Representative images of IHC staining using E06 antibody. (B) Quantification of positive staining using the Positive Pixel Counts algorithm from Aperio. *, p < 0.05; ***, p < 0.001. scale bar = 50 μm.

Discussion

In this study, gene expression was systemically investigated in the lung tissues of NHP exposed to lethal doses of whole thoracic lung irradiation. This study revealed the involvement of a sustained innate immune response in RILI as shown by the up-regulation of chemokines, the complement system, adhesion molecules, and the increased infiltration of inflammatory cells to the irradiated lung tissues. Increased oxidative stress and apoptosis-associated proteins were also observed in the irradiated lung tissues which may have a mechanistic link to the innate immune response. The data presented here will help to understand the mechanism of RILI in a nonhuman primate model relevant to human response. The study identified components of the innate immune response which are potential therapeutic targets and may also be used as biomarkers for RILI. The data may help to explain the mechanism of AEOL 10150 alleviating RILI.

Radiation dose rate

Following a nuclear detonation, there are prompt radiation from the nuclear reaction and delayed radiation mostly from the nuclear fallout. The prompt radiation dose rate is extremely high, for example, at 1000 meter from the hypocenter, the γ radiation dose rate could be 7 × 104 Gy s−1(Ruhm et al. 2018). The delayed γ radiation dose rate could be 0.277 Gy s−1, equivalent to 16.62 Gy min−1 at 1000 meter from the hypocenter; and 3.96 × 10−3 Gy s−1, equivalent to 0.24 Gy min−1 at 2000 meter from the hypocenter (Ruhm et al. 2018). The radiation dose rate decreases rapidly with time and distance. It is well known that not only the radiation dose, but also the radiation dose rate, play important roles in radiation-induced toxicity (Williams et al. 2010, DiCarlo et al. 2011). Therefore, it is important to use the proper radiation dose rate in studying radiation-induced toxicity and countermeasure development. The National Institute of Allergy and Infectious Diseases (NIAID) Radiation and Nuclear Countermeasures Program convened a workshop in 2004 to discuss the animal models for nuclear radiation effects and medical countermeasure development. The meeting recommended that a dose rate of 0.5-1.0 Gy min−1 should be used for these studies. The Preclinical Radiobiology Lab at the University of Maryland has used the dose rate of 0.8 Gy min−1 for many NHP studies (MacVittie et al. 2012, Farese et al. 2013, Farese et al. 2014, Garofalo et al. 2014, Cui et al. 2016, MacVittie et al. 2017). At the Armed Forces Radiobiology Research Institute (AFRRI), the dose rate of 0.6 Gy min−1 is a well-established standard dose rate for animal survival studies (Koch et al. 2016).

Elevated cytokines that are important for RILI

Multiple cytokines/chemokines have been shown to be elevated after thoracic irradiation in rodent models. Cytokines such as MCP-1 (CCL2), RANTES, C10, MCP-3, MIP-1γ, IP-10 and BLC have been shown to be highly expressed in the lung tissues of C57BL/6 mice at 26 weeks after 12.5 Gy thoracic irradiation (Johnston et al. 1998, Johnston et al. 2002). Additional studies show that G-CSF, IL-6, keratinocyte-derived chemokine (KC, mouse homolog to human IL-8), MCP-1 (CCL2) and IP-10 were elevated at early time points (3-6 hour after radiation exposure) then gradually returned to baseline level at 1 week post 12 Gy thoracic irradiation in C57BL/6 and C3H mouse lung tissues (Ao et al. 2009). In our study, we showed that many cytokines/chemokines were upregulated in the irradiated NHP lungs after thoracic irradiation (Table 2 and Fig. 1 & 2). CCL2, CCL11 and IL-8 were increased in the irradiated lungs on mRNA and protein levels. We further confirmed the elevated expression of CCL2 and CCL11 using IHC in the irradiated lungs. Cytokines that are important in inflammation (such as MCP-1(CCL2), IP-10, MIP-1α, TARC, IL-17, TNF-β and IL-6) were found to be significantly increased in the lung tissues after thoracic irradiation in NHP (Cui et al. 2020). Among the significantly changed cytokines/chemokines in the current study, CCL2, CCL11 and IL-8 are well studied cytokines/chemokines important in inflammation. CCL2 is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages (Deshmane et al. 2009). CCL11 is a potent eosinophil chemoattractant that is considered a major contributor to tissue eosinophilia (Adar et al. 2014, Takemura et al. 2018). IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation(Baggiolini et al. 1995).

Radiation-induced lung fibrosis have been shown in NHP exposed to WTLI using CT imaging and trichrome staining (Garofalo et al. 2014, MacVittie et al. 2017). In agreement with our findings here, literature has shown that cytokines/chemokines play important roles in radiation-induced fibrosis. For example, it has been shown that CCL2 plays important roles in radiation-induced pulmonary fibrosis by attracting CCR2+ inflammatory cells into the lung (Groves et al. 2018). CCL11 is important in radiation-induced small intestinal fibrosis by attracting eosinophils into the submucosa of the small intestine (Takemura et al. 2018). Blocking the actions of these identified cytokines/chemokines are potentially therapeutic treatments for RILI.

Elevated complement system is associated with RILI

Although the complement system has been traditionally considered a “complement” to humoral immunity, it is now perceived as a central constituent of the innate immunity (Markiewski and Lambris 2007). The complement system is composed of membrane-bound regulators and receptors and a large number of plasma proteins that react with one another to opsonize pathogens and induce inflammatory responses to fight infection. The complement is also important in removal of waste material originating from apoptotic or necrotic cells (Fishelson et al. 2001). Excessive complement activation can cause many diseases such as atypical hemolytic uremic syndrome and ischemia/reperfusion injury. The elevated expression of the complement system was previously identified in gene expression profiling studies of rodent lung tissues after thoracic irradiation (Paun et al. 2010). A more recent study also showed that thoracic irradiation induced local complement activation in mice and humans, including Complement Factor B and Complement Component 3 (Surace et al. 2015). Herein, we identified the complement activation in pathway analysis (Table 4) and confirmed the elevated expression of Complement Factor B and Complement Component 3 in the irradiated NHP lung tissues (Fig. 3). These two are important players in the alternative activation pathway of the complement system. Inhibiting the complement pathway may be an effective method to ameliorate RILI (Schubart et al. 2019).

Elevated innate immunity are associated with RILI

Abundant circumstantial evidence indicates that innate immunity and oxidative stress play important roles in radiation-induced normal tissue injury (Zhao and Robbins 2009, Ding et al. 2013). Large numbers of inflammatory cells are recruited to the field of irradiation subsequent to thoracic radiation injury. These cells include innate immune cells such as monocytes (Johnston et al. 2004, Cappuccini et al. 2011), macrophages (Johnston et al. 2004, Chiang et al. 2005, Fleckenstein et al. 2007), neutrophil and eosinophils (Cottin et al. 2004, Cappuccini et al. 2011). Inflammatory cells are important in maintaining tissue homeostasis; however, excessive infiltration of inflammatory cells could cause tissue damage. Monocytes are precursors of macrophages and dendritic cells. Excessive recruitment of monocytes contributes to pathogenesis of inflammatory and degenerative diseases (Shi and Pamer 2011). Macrophages are shown to cause tissue damage through production of reactive oxygen and nitrogen species, proteases, proinflammatory lipid mediators and proinflammatory cytokines and chemokines (Laskin et al. 2011). Activation and recruitment of neutrophils play a key role in progression of acute lung injury and acute respiratory distress syndrome by producing serine protease, matrix metalloproteinase (MMPs), cationic polypeptides, reactive oxygen and nitrogen species (Grommes and Soehnlein 2011). Myeloperoxidase (a specific marker for neutrophils) catalyzes the production of hydroxyl radical (OH) and hypochlorous acid (HOCl). Eosinophils contain highly charged basic proteins including eosinophil granule major basic proteins, eosinophil cationic proteins, and eosinophil peroxidases. These proteins are toxic to many cell types including airway epithelial cells and may contribute to tissue damage and organ dysfunction (Fulkerson and Rothenberg 2013). In our study, elevated levels of monocytes/macrophages, neutrophils and eosinophils were observed in the lungs of irradiated NHP (Fig. 4), which is consistent with the increased pulmonary inflammation observed in rodent models. Oxidative stress has been shown to cause chronic inflammation in many chronic diseases including cancer, diabetes, cardiovascular, neurological and pulmonary diseases (Reuter et al. 2010). Mechanistically, oxidative stress can activate a variety of transcription factors which lead to expression of over 500 different genes that are important for inflammation (Reuter et al. 2010). A variety of therapeutic approaches that involve antioxidant/anti-inflammatory-mediated pathways have been shown to prevent, mitigate, and/or treat radiation-induced late effects(Zhao and Robbins 2009). Treatment with AEOL 10150 has been shown to decrease RILI in both rodent and NHP models (Rabbani et al. 2007, Garofalo et al. 2014, MacVittie et al. 2017). In the current study, we showed that treatment with AEOL 10150 decreased the number of infiltrating monocytes/macrophages in the lungs of irradiated NHP (Fig. 4). This is also in agreement with data showing that AEOL 10150 decreased the number of macrophages in the lungs of irradiated mice (Rabbani et al. 2007).

Apoptosis is associated with RILI

Apoptosis contributes to many lung diseases such as acute lung injury, pulmonary fibrosis, asthma and emphysema (Sauler et al. 2019). It was shown that apoptosis persists in the lung tissues at least 6 weeks after thoracic irradiation in a rodent model (Zhang et al. 2012). Our KEGG pathway analysis suggested that p53 mediated apoptotic pathway was active in the irradiated lungs. We further confirmed that multiple players in the apoptotic pathway were elevated in the irradiated lungs from 2 to 6 months after radiation exposure (Fig. 5). Apoptosis and inflammatory reactions are known to be linked (Simon 2003). It is suggested that apoptotic cells release intracellular macromolecules into the environment if the apoptotic cells are not cleared rapidly by phagocytes; the released contents contain pro-inflammatory components thus stimulating inflammatory reaction (Rock and Kono 2008). Our data support the idea that chronic apoptosis in the irradiated lungs may contribute to the failed resolution of inflammation.

Up-regulated adhesion molecules in RILI

The migration of inflammatory cells from blood stream to the inflammatory site requires multiple steps: slow rolling, adhesion strengthening, intraluminal crawling and paracellular and transcellular migration, and migration through the basement membrane (Ley et al. 2007). Many components are required for this complex process. These players include selectins, integrins, cytokines/chemokines, and adhesion molecules. Our data also showed the up-regulation of many cell adhesion molecules such as fibronectin, integrin β6 and ICAM-1 in the irradiated NHP lungs. The upregulation of these molecules post irradiation has been shown in rodent models. Expression of fibronectin has been shown to be significantly increased at 16 weeks in C3H/HeJ mice and at 26 weeks in C57BL/6 mice after 12.5 Gy thoracic radiation(Johnston et al. 1995). ICAM-1 interacts with integrins to enable leukocytes to adhere firmly to the vascular endothelium and subsequently migrate across the endothelial barrier (Rahman and Fazal 2009). Expression of ICAM-1 was shown to be increased at 24 hours and persisted for 7 days after 10 Gy thoracic irradiation in C3H mice; Importantly, ICAM-1 knockout mice had no increase in the inflammatory cell infiltration into the lung in response to thoracic irradiation and mice treated with an anti-ICAM-1 blocking antibody showed attenuation of inflammatory cell infiltration into the lung in response to ionizing radiation exposure (Hallahan and Virudachalam 1997). Integrin αvβ6 is a major TGF-β activator in the lung. It has been shown that β6 integrin expression increased sharply at 18-20 weeks and persisted to 27 weeks post 14 Gy thoracic irradiation; Itgb6−/− mice were completely protected from radiation-induced fibrosis and a specific inhibitor of αvβ6 prevented fibrosis for up to 12 weeks after the normal time of onset (Puthawala et al. 2008). Our data are in agreement with literature showing the elevated expression of multiple adhesion molecules after radiation exposure (Fig. 6).

Up-regulated oxidative stress in RILI

OxPL has been shown as an important trigger for acute lung injury caused by multiple lung pathogens such as virus or acid aspiration (Imai et al. 2008). OxPL induces lung tissue damage by cytokine storm (IL-6, etc) which is activated through the TLR4-NFκB pathway. The pathology of acute lung injury by virus and acid aspiration share a similar phenotype as RILI such as accumulation of inflammatory cells, edema formation, and a marked increase in cytokines. Therefore, we investigated the expression of OxPL in the irradiated lungs. OxPL level was significantly elevated in the irradiated lungs and its expression mainly localized in macrophages (Fig. 7), supporting the role of macrophages in the RILI.

Potential use of AEOL 10150 for COVID-19 patients

Similar to RILI, cytokine storm has been suggested to play important roles in COVID-19 associated severe pneumonitis and acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) (Fajgenbaum and June 2020, Rios et al. 2021). Because AEOL 10150 is effective to increase survival after lethal doses of RILI, it may be a potential effective treatment for COVID-19 patients.

Proposed mechanism of RILI in the NHP model

Based on our findings in the irradiated NHP and the vast amount of information from rodent studies, we propose the following mechanism of RILI: thoracic irradiation induces apoptotic pulmonary cells to chronically release cell contents into the adjacent tissue, which activate the complement systems and cause inflammatory cell infiltration. The inflammatory cell infiltration is facilitated by the elevated levels of chemokines such as CCL2, CCL11 and IL-8, etc. These chemokines attract innate immune cells such as monocytes/macrophages, neutrophils, and eosinophils into the injury sites. Up-regulation of adhesion molecules such as fibronectin, integrin β6 and ICAM-1 helps the transmigration of inflammatory cells from blood vessel to the lung. These inflammatory cells can cause tissue damage by releasing of oxidants, proteases, and cytokines. The resulting tissue damage will cause more inflammation at the injury sites. The chronic inflammation may cause destruction of tissue matrix, edema, or deposition of scar tissue and may finally lead to loss of organ function. Oxidized phospholipid in macrophages may be responsible for the production of proinflammaotry cytokines. AEOL 10150 treatment may significantly decrease the level of OxPL and apoptosis in the irradiated lungs. The decrease of OxPL and apoptosis may decrease the productions of proinflammatory cytokines, which translates to less infiltration of immune cells, thus causing less tissue injury.

Limitations in the current study

In the current study, AEOL 10150 treatment decreased the levels of many cytokines, complement factors, inflammatory cell infiltration, apoptosis-associated proteins, and adhesion molecules. However, the decrease was mostly not significant. One possible reason is the time difference between the AEOL 10150 treatment and the tissue collection time. The AEOL 10150 was administered for day 1-28 post irradiation. The lung tissues were harvested at necropsy which was generally 2-6 months after radiation exposure. Analysis of lung tissues during or shortly after AEOL 10150 treatments may show a more direct effect of the mitigator.

Another limitation of the current study is that the study animals were not euthanized at preselected time points. Rather they were euthanized when each animal met euthanasia criteria unless the animal survived the planned study. The results from the lung tissues of irradiated NHP may reflect the terminal stages of RILI, rather than the early response to thoracic irradiation. A temporal study of the innate immune response in the irradiated lung tissues as a function of the duration of RILI may provide further insight into the mechanism of RILI.

Acknowledgement:

We acknowledge the tremendous support and expertise of the research staff of the Preclinical Radiobiology Lab at the School of Medicine, University of Maryland. We thank Dr. Susan Ascher at Georgetown University for her careful reading of the manuscript and thoughtful comments. The views expressed herein are the private views of the authors and are not necessarily those of the Henry M. Jackson Foundation for the Advancement of Military Medicine, Armed Forces Radiobiology Research Institute, the Uniformed Services University of the Health Sciences, or the Department of Defense.

Conflicts of Interest and Source of Funding:

This work was supported from Aeolus Inc. through BARDA contract HHSO100201100007C and NIAID contract HHSN272201000046C. The funders had no role in the study design, data collection, data analysis, decision to publish or preparation of the manuscript of this study. The authors declare no financial conflict of interest.

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