26. The Role of Macrophages In Mediating Radiation-induced Fibrosis

Abstract

Purpose: Radiation-induced fibrosis (RIF) remains a clinically challenging problem in cancer patients without effective treatment or prevention. To elucidate the mechanisms of RIF, we investigated the role of macrophages as mediators of fibrosis, and the reciprocal signaling that occurs between macrophages and fibroblasts. Methods: Bone marrow-derived macrophages and dermal fibroblasts were isolated from C57BL/6 mice. Cells were co-cultured in a three-dimensional collagen gel system and subjected to radiation. Gel contracture was measured over time. Flow cytometry was utilized to analyze macrophage polarization towards inflammatory and anti-inflammatory phenotypes using cell surface markers such as CD38 and CD206, respectively. Macrophages from both radiated and non-radiated co-cultures were subjected to RNA sequencing to investigate radiation induced phenotypic and functional changes. Results: Co-culturing fibroblasts and macrophages led to pronounced collagen gel contraction compared to fibroblasts alone, highlighting the essential role of macrophages. In addition, radiated macrophages had a significantly increased effect on gel contracture compared to non-radiated controls indicating a changed phenotype promoting contraction. Since macrophage involvement was found to be critical for radiation-induced functional alteration in collagen gels, we investigated macrophage phenotypes in response to radiation. Flow cytometry analysis revealed that radiation-induced alternate activation (M2) of macrophages, as shown by increased CD206 expression. Similarly, RNA sequencing data showed increased expression of interferon response genes such as Ifi206 and chemokines including c-c motif chemokine ligand 7 (ccl7) suggesting a distinct inflammatory response to radiation. Furthermore, we analyzed the impact of radiation on macrophage plasticity. Interestingly, when macrophages were polarized to M2 phenotype and then radiated, their potential to repolarize to M1 was lost, as opposed to non-radiated macrophages suggesting potential loss of macrophage plasticity during radiation. Conclusion: Our study highlights the crucial role of macrophages in early RIF and their potential as a therapeutic target to mitigate the negative side effects of radiation therapy. We observed that radiation exposure induces an M2 phenotype, upregulates interferon response genes in macrophages, and impairs plasticity. These findings suggest that radiation may contribute to the pro-fibrotic environment by activating macrophages in a physiologically distinct manner. Our study offers insights into the underlying mechanisms of RIF and the role of macrophages in this process.