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.

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