Understanding the Inflammatory Response in Radiation Damaged Salivary Glands

Abstract

Over 890,000 new cases of head and neck cancer are diagnosed worldwide, with approximately 54,000 new cases in the United States(1, 2). The 5-year survival rate for head and neck cancer (HNC) patients is increasing, with treatment commonly consisting of surgical resection of the tumor followed by ionizing radiation (IR) therapy. Salivary glands are located proximal to tumors and often damaged during radiotherapy, leading to severe side effects that result in severely diminished quality of life for patients(3-5). Loss of glandular function results in chronic dry mouth, which contributes to health complications including mucositis, dental caries, and difficulty with eating and drinking. Current treatments are ineffective and only temporarily manage symptoms; therefore, they require lifelong use by patients. Understanding the mechanisms of radiation-induced salivary gland dysfunction is necessary to develop novel drug targets to reverse salivary gland function, and therefore, to improve the quality of life for HNC patients(6-8). The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data illustrate that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous work demonstrated post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors. Interactions between the inflammatory, proliferative, and redifferentiation phases of wound healing are necessary for full recovery and restoration of tissue function. In both parotid and submandibular glands, there is a decrease in innate immune cells at acute time points, and decreased cytokine concentrations in the parotid gland; however, it is not well understood how this abnormal inflammatory response relates to the subsequent phases of healing. In the present study, we modulated inflammation via cytokine treatments in an ex vivo organ culture model in order to understand the impact of inflammation on proliferation and redifferentiation. Our data indicate that differentiation, as measured by positive Aquaporin-5 staining, is significantly reduced after IR, and culturing with IL-6 or TNF-α enriched media significantly increase differentiation in parotid gland organ cultures. Interestingly, culturing sections with media with both IL-6 and TNF-α did not significantly improve differentiation in parotid gland organ cultures. Taken together, these data indicate that modest acute inflammation can rescue differentiation in parotid glands after IR, which may correlate to improved glandular function. Previous data indicate that parotid glands have significantly increased populations of T cells at chronic timepoints. It is well established that the metabolic status of immune cells has a considerable impact on their function. We utilized bulk and single cell RNA sequencing from radiation damaged salivary glands to assess differential expression of genes involved in inflammatory and metabolic pathways and identify changes in immune cells. Bulk RNAseq shows significant upregulation of several inflammatory pathways relating to cytokine and chemokine signaling in parotid glands, and significant downregulation of amino acid signaling and oxidative phosphorylation in submandibular glands. Single cell data do not show marked changes in metabolic pathways in DPT cells, but suggest increases in genes related to cytokine signaling, T cell receptor signaling, and T cell activation in double positive T cells. Together, these data imply a chronic inflammatory phenotype in parotid glands that likely contributes to prolonged glandular dysfunction.