Monocyte Polarization is Altered by Total-Body Irradiation in Male Rhesus Macaques: Implications for Delayed Effects of Acute Radiation Exposure

Abstract

Radiation-induced fibrosis (RIF) is a common delayed effect of acute ionizing radiation exposure (DEARE) affecting diverse tissues including the heart, lungs, liver and skin, leading to reduced tissue function and increased morbidity. Monocytes, which may be classified into classical (CD14⁺⁺, CD16^-), intermediate (CD14⁺⁺, CD16⁺) and non-classical (CD14^+/low, CD16⁺⁺) subtypes in humans and non-human primates (NHPs), and monocyte-derived macrophages may play an integral role in the pathogenesis of RIF. We tested the hypothesis that moderate to high levels of total-body exposure to radiation would alter monocyte polarization and produce phenotypes that could promote multi-organ fibrosis in a wellestablished NHP model of DEARE. Subjects were 16 young adult male rhesus macaques, ten of which were exposed to high-energy, 4 Gy X-ray total-body irradiation (TBI) and six that received sham irradiation (control). Total monocytes assessed by complete blood counts were 89% depleted in TBI animals by day 9 postirradiation (P < 0.05), but recovered by day 30 postirradiation and did not differ from control levels thereafter. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and sorted into classical, intermediate and non-classical subsets using fluorescence-activated cell sorting (FACS) prior to and at 6 months post-TBI. At 6 months postirradiation, monocyte polarization shifted towards lower classical (92% → 86%) and higher intermediate (7% → 12%) and non-classical monocyte subsets (0.6% → 2%) (all P < 0.05) in TBI animals compared to baseline. No change in monocyte subsets was observed in control animals. Transcriptional profiles in classical and intermediate monocyte subsets were assessed using RNAseq. Classical monocyte gene expression did not change significantly over time or differ cross-sectionally between TBI and control groups. In contrast, significant numbers of differentially expressed genes (DEGs) were detected in intermediate monocyte comparisons between the TBI animals and all animals at baseline (304 DEGs), and in the TBI versus control animals at 6 months postirradiation (67 DEGs). Intermediate monocytes also differed between baseline and 6 months in control animals (147 DEGs). Pathway analysis was used to identify genes within significant canonical pathways, yielding 52 DEGs that were specific to irradiated intermediate monocytes. These DEGs and significant canonical pathways were associated with pro-fibrotic and anti-inflammatory signaling pathways that have been noted to induce M2 macrophage polarization. These findings support the hypothesis that TBI may alter monocyte programming and polarization towards a profibrotic phenotype, providing a novel target opportunity for therapies to inhibit or prevent RIF.

FIG. 1.

Peripheral immune cell recovery after total-body irradiation. Panels A–D: Total white blood cell, monocyte, lymphocyte and neutrophil counts, respectively, were all acutely depleted by TBI. Monocytes and neutrophils recovered by dau 30 postirradiation while total white blood cells and lymphocytes remained significantly decreased until days 51 and 58 postirradiation, respectively. After recovery, total white blood cells, monocytes and neutrophils in irradiated animals did not differ from controls, but lymphocytes increased above controls animals at day 129 postirradiation and remained elevated thereafter. *P < 0.05, **P < 0.01 and ***P < 0.001.

FIG. 2.

Gating strategy to obtain monocyte subsets. Magnetic bead selected CD14⁺ monocytes were gated based on forward- and side-scatter parameters before final gating into classical (CD14⁺⁺, CD16⁻), intermediate (CD14⁺⁺, CD16⁺) and non-classical (CD14^+/low, CD16⁺⁺) phenotypes. Panel A: Forward scatter plot. Panel B: Side scatter plot. Panel C: Forward scatter-side scatter. Panel D: Final representation of monocyte subsets by CD14 and CD16 labeling. Profiles from a representative sample from an irradiated animal at 6 months postirradiation are shown. FSC-H = forward scatter height; FSC-A = forward scatter area; SSC-W = side scatter width; SSC-H = height; SSC-A = side scatter area.

FIG. 3.

Alterations in distribution of monocyte subtypes after total-body irradiation. Panel A: Monocyte polarization was unaltered in control animals at 6 months after sham irradiation. Panel B: In contrast, irradiated animals had significant shifts towards lower classical and higher intermediate and non-classical monocyte subsets compared to baseline. Panels C and D: Flow cytometry density plots from a representative control and irradiated animal, respectively, are shown at 6 months postirradiation. (*P < 0.05 and **P < 0.01).

FIG. 4.

Canonical pathways differentiating intermediate monocytes in irradiated subjects at 6 months (n = 10) from all subjects (n = 16) at baseline. Within each pathway red bars indicate the proportion of DEGs upregulated in irradiated subjects at 6 months, blue bars indicate the proportion of DEGs downregulated in irradiated subjects at 6 months and the overall length of the bar is the −log10 P value. The vertical line indicates the threshold for significance based on a −log10 P value of 1.3 (equal to P < 0.05).

FIG. 5.

Canonical pathways differentiating intermediate monocytes in control subjects at 6 months to all subjects (n = 16) at baseline. Within each pathway red bars indicate the proportion of DEGs upregulated in control subjects at 6 months, blue bars indicate the proportion of DEGs downregulated in control subjects at 6 months within the respective pathway and the overall length of the bar is the −log10 P value. The vertical line indicates the threshold for significance based on a −log10 P value of 1.3 (equal to P < 0.05).

FIG. 6.

Canonical pathways differentiating intermediate monocytes in irradiated subjects from control at 6 months. Within each pathway red bars indicate the proportion of DEGs upregulated in irradiated subjects, the blue bars indicate the proportion of DEGs downregulated in irradiated subjects within the respective pathway and the overall length of the bar is the −log10 P value. The vertical line indicates the threshold for significance based on a −log10 P value of 1.3 (equal to P < 0.05).

FIG. 7.

Differentiating irradiated vs. control intermediate monocyte differentially expressed genes using cross-sectional and longitudinal analyses. Intermediate monocyte DEGs affected by radiation (n = 300) were determined by removing DEGs shared with longitudinal analyses of control intermediate monocytes (n = 47).

FIG. 8.

Intermediate monocyte canonical pathways differentiating irradiated vs. control subjects using cross-sectional and longitudinal differentially expressed genes. Within each pathway red bars indicate the proportion of DEGs upregulated in irradiated monocytes at 6 months, blue bars indicate the proportion of DEGs downregulated in irradiated monocytes at 6, and the overall height of the bar is the −log10 P value. The vertical line indicates the threshold for significance based on a −log10 P value of 1.3 (equal to P < 0.05).