Parotid glands have a dysregulated immune response following radiation therapy.

Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. 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 suggest 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 data indicate that 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.

Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands.

Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.

Radiation-Induced Changes in Energy Metabolism Result in Mitochondrial Dysfunction in Salivary Glands.

Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.

Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages.

Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.

Evaluating the transcriptional landscape and cell-cell communication networks in chronically irradiated parotid glands.

Understanding the transcriptional landscape that results in chronic salivary hypofunction after irradiation will help identify injury mechanisms and develop regenerative therapies. We present scRNA-seq analysis from control and irradiated murine parotid glands collected 10 months after irradiation. We identify a population of secretory cells defined by specific expression of Etv1, which may be an acinar cell precursor. Acinar and Etv1+ secretory express Ntrk2 and Erbb3, respectively while the ligands for these receptors are expressed in myoepithelial and stromal cells. Furthermore, our data suggests that secretory cells and CD4+CD8+T-cells are the most transcriptionally affected during chronic injury with radiation, suggesting active immune involvement. Lastly, evaluation of cell-cell communication networks predicts that neurotrophin, neuregulin, ECM, and immune signaling are dysregulated after irradiation, and thus may play a role in the lack of repair. This resource will be helpful to understand cell-specific pathways that may be targeted to repair chronic damage in irradiated glands.

Recent progress toward understanding the role of ZIP14 in regulating systemic manganese homeostasis.

ZIP14 is a metal transporter essential for the regulation of body manganese homeostasis. The physiological functions of ZIP14 have been uncovered mainly through two lines of in vivo studies that examined the phenotypes of ZIP14 loss, including studies of humans with ZIP14 mutations and animals with ZIP14 deficiency. This mini review aims at presenting an updated view of the important advances made towards understanding the genetic and pathological mechanisms of brain manganese overload caused by ZIP14 deficiency.

Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E2 Signaling.

Annually, >600,000 new cases of head and neck cancer (HNC) are diagnosed worldwide with primary treatment being surgery and radiotherapy. During ionizing radiation (IR) treatment of HNC, healthy salivary glands are collaterally damaged, leading to loss of function that severely diminishes the quality of life for patients due to increased health complications, including oral infections and sores, cavities, and malnutrition, among others. Therapies for salivary hypofunction are ineffective and largely palliative, indicating a need for further research to uncover effective approaches to prevent or restore loss of salivary gland function following radiotherapy. Previous work in our lab implicated prostaglandin E2 (PGE2) as an inflammatory mediator whose release from radiation-exposed cells promotes salivary gland damage and loss of function. Deletion of the P2X7 purinergic receptor for extracellular ATP reduces PGE2 secretion in irradiated primary parotid gland cells, and salivary gland function is enhanced in irradiated P2X7R-/- mice compared to wild-type mice. However, the role of PGE2 signaling in irradiated salivary glands is unclear and understanding the mechanism of PGE2 action is a goal of this study. Results show that treatment of irradiated mice with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which reduces PGE2 production via inhibition of cyclooxygenase-1 (COX-1), improves salivary gland function compared to irradiated vehicle-treated mice. To define the signaling pathway whereby PGE2 induces salivary gland dysfunction, primary parotid gland cells treated with PGE2 have increased c-Jun N-terminal Kinase (JNK) activation and cell proliferation and reduced amylase levels and store-operated calcium entry (SOCE). The in vivo effects of blocking PGE2 production were also examined and irradiated mice receiving indomethacin injections have reduced JNK activity at 8 days post-irradiation and reduced proliferation and increased amylase levels at day 30, as compared to irradiated mice without indomethacin. Combined, these data suggest a mechanism whereby irradiation-induced PGE2 signaling to JNK blocks critical steps in saliva secretion manifested by a decrease in the quality (diminished amylase) and quantity (loss of calcium channel activity) of saliva, that can be restored with indomethacin. These findings encourage further attempts evaluating indomethacin as a viable therapeutic option to prevent damage to salivary glands caused by irradiation of HNC in humans.

Integration of metabolomics and transcriptomics reveals convergent pathways driving radiation-induced salivary gland dysfunction.

Radiation therapy for head and neck cancer causes damage to the surrounding salivary glands, resulting in salivary gland hypofunction and xerostomia. Current treatments do not provide lasting restoration of salivary gland function following radiation; therefore, a new mechanistic understanding of the radiation-induced damage response is necessary for identifying therapeutic targets. The purpose of the present study was to investigate the metabolic phenotype of radiation-induced damage in parotid salivary glands by integrating transcriptomic and metabolomic data. Integrated data were then analyzed to identify significant gene-metabolite interactions. Mice received a single 5 Gy dose of targeted head and neck radiation. Parotid tissue samples were collected 5 days following treatment for RNA sequencing and metabolomics analysis. Altered metabolites and transcripts significantly converged on a specific region in the metabolic reaction network. Both integrative pathway enrichment using rank-based statistics and network analysis highlighted significantly coordinated changes in glutathione metabolism, energy metabolism (TCA cycle and thermogenesis), peroxisomal lipid metabolism, and bile acid production with radiation. Integrated changes observed in energy metabolism suggest that radiation induces a mitochondrial dysfunction phenotype. These findings validated previous pathways involved in the radiation-damage response, such as altered energy metabolism, and identified robust signatures in salivary glands, such as reduced glutathione metabolism, that may be driving salivary gland dysfunction.

The complex role of prostaglandin E2-EP receptor signaling in wound healing.

Prostaglandins are critical lipid mediators involved in the wound healing response, with prostaglandin E2 (PGE2) being the most complex and exhibiting the most diverse physiological outputs. PGE2 signals via four G protein-coupled receptors, termed EP-receptors 1-4 that induce distinct signaling pathways upon activation and lead to an array of different outputs. Recent studies examining the role of PGE2 and EP receptor signaling in wound healing following various forms of tissue damage are discussed in this review.

Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions.

Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell-cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.

Yap activation in irradiated parotid salivary glands is regulated by ROCK activity.

Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients' quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.

The Functions of ZIP8, ZIP14, and ZnT10 in the Regulation of Systemic Manganese Homeostasis.

As an essential nutrient, manganese is required for the regulation of numerous cellular processes, including cell growth, neuronal health, immune cell function, and antioxidant defense. However, excess manganese in the body is toxic and produces symptoms of neurological and behavioral defects, clinically known as manganism. Therefore, manganese balance needs to be tightly controlled. In the past eight years, mutations of genes encoding metal transporters ZIP8 (SLC39A8), ZIP14 (SLC39A14), and ZnT10 (SLC30A10) have been identified to cause dysregulated manganese homeostasis in humans, highlighting the critical roles of these genes in manganese metabolism. This review focuses on the most recent advances in the understanding of physiological functions of these three identified manganese transporters and summarizes the molecular mechanisms underlying how the loss of functions in these genes leads to impaired manganese homeostasis and human diseases.

P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction.

Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren's syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration.

Protein Profiles of Individuals with Erosive Tooth Wear

Abstract Objective: To determine if protein profiles identified in saliva could be used to determine risk and severity of erosive tooth wear. Material and Methods: Three types of saliva sampling were performed to obtain saliva from 34 18-year old individuals that received regular dental check-ups, along with clinical status of the dentition and risk factor related to erosive tooth wear using the VEDE scale. Protein profiles in saliva were determined using electrophoresis and the calculation of the percentage of a specific band at a specific molecular weight in relationship to the total protein in that sample (% of total) using molecular weight standards. This quantification was repeated for each protein band across a range of molecular weights for each sample to test for association with erosive tooth wear status. Results: There were no differences in the number of detectable proteins sourced from the parotid gland, nor the unstimulated and stimulated whole saliva. Five out of the 34 individuals had no signs of erosive tooth wear despite an acidic diet and were more likely to have proteins with molecular weight smaller than 1 KDa (p=0.03). Conclusion: There is potential for the use of protein profiling to determine risks for erosive tooth wear.

Differences in Proteomic Profiles Between Caries Free and Caries Affected Children

Abstract Objective: To determine if there are differences in protein profiles in saliva depending if children of caries-free versus caries affected. Material and Methods: A cohort of 91 children with ages between 6 and 19 years, along clinical status of caries experience. Protein profiles in saliva were determined using electrophoresis and the calculation of the percentage of a specific band at a specific molecular weight in relationship to the total protein in that sample (% of total) using molecular weight standards. This quantification was repeated for each protein band across a range of molecular weights for each sample. Chi-square, Fisher's exact, and Student t-tests were used to compare the distributions between caries-free and caries affected children (α=0.05). Results: Histatin was more likely to be non-detectable or reduced in caries-free children (OR=7.56; 95% CI 1.62-35.13) and these children had on average one less gel band detected by the assay we used. Conclusion: We have found differences in proteins between caries affected and caries-free children, suggesting that this line of investigation holds the promise of providing new tools for caries management.

Salivary Gland Hypofunction and Xerostomia in Head and Neck Radiation Patients.

BACKGROUND: The most manifest long-term consequences of radiation therapy in the head and neck cancer patient are salivary gland hypofunction and a sensation of oral dryness (xerostomia). METHODS: This critical review addresses the consequences of radiation injury to salivary gland tissue, the clinical management of salivary gland hypofunction and xerostomia, and current and potential strategies to prevent or reduce radiation injury to salivary gland tissue or restore the function of radiation-injured salivary gland tissue. RESULTS: Salivary gland hypofunction and xerostomia have severe implications for oral functioning, maintenance of oral and general health, and quality of life. Significant progress has been made to spare salivary gland function chiefly due to advances in radiation techniques. Other strategies have also been developed, e.g., radioprotectors, identification and preservation/expansion of salivary stem cells by stimulation with cholinergic muscarinic agonists, and application of new lubricating or stimulatory agents, surgical transfer of submandibular glands, and acupuncture. CONCLUSION: Many advances to manage salivary gland hypofunction and xerostomia induced by radiation therapy still only offer partial protection since they are often of short duration, lack the protective effects of saliva, or potentially have significant adverse effects. Intensity-modulated radiation therapy (IMRT), and its next step, proton therapy, have the greatest potential as a management strategy for permanently preserving salivary gland function in head and neck cancer patients.Presently, gene transfer to supplement fluid formation and stem cell transfer to increase the regenerative potential in radiation-damaged salivary glands are promising approaches for regaining function and/or regeneration of radiation-damaged salivary gland tissue.

PKCζ and JNK signaling regulate radiation-induced compensatory proliferation in parotid salivary glands.

Radiotherapy is a common treatment option for head and neck cancer patients; however, the surrounding healthy salivary glands are often incidentally irradiated during the process. As a result, patients often experience persistent xerostomia and hyposalivation, which deceases their quality of life. Clinically, there is currently no standard of care available to restore salivary function. Repair of epithelial wounds involves cellular proliferation and establishment of polarity in order to regenerate the tissue. This process is partially mediated by protein kinase C zeta (PKCζ), an apical polarity regulator; however, its role following radiation damage is not completely understood. Using an in vivo radiation model, we show a significant decrease in active PKCζ in irradiated murine parotid glands, which correlates with increased proliferation that is sustained through 30 days post-irradiation. Additionally, salivary glands in PKCζ null mice show increased basal proliferation which radiation treatment did not further potentiate. Radiation damage also activates Jun N-terminal kinase (JNK), a proliferation-inducing mitogen-activated protein kinase normally inhibited by PKCζ. In both a PKCζ null mouse model and in primary salivary gland cell cultures treated with a PKCζ inhibitor, there was increased JNK activity and production of downstream proliferative transcripts. Collectively, these findings provide a potential molecular link by which PKCζ suppression following radiation damage promotes JNK activation and radiation-induced compensatory proliferation in the salivary gland.

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

P2X7 receptor deletion suppresses γ-radiation-induced hyposalivation.

Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R-/-) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R-/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R-/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E2 (PGE2) release from primary parotid cells of wild-type but not P2X7R-/- mice. To attempt to uncover the mechanism underlying differential PGE2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE2 release in P2X7R-/- mice. Irradiated P2X7R-/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.

Current State of Knowledge on Salivary Gland Cancers.

Salivary gland cancers (SGCs), categorized as head and neck cancers (HNCs), constitute about 6% of head and neck cancer diagnoses based on estimate by American Head and Neck Society. Salivary gland tumors originate from different glandular cell types and are thus morphologically diverse. These tumors arise from any of the three major and various minor salivary glands. The incidence of SGCs has slowly increased during the last four decades. The etiology of SGCs is mostly unknown; however, specific gene mutations are associated with certain types of salivary tumors. Treatment options include surgical resection, radiation therapy (RT), chemotherapy, and multimodality therapy. HNC patients treated with RT often develop xerostomia and salivary hypofunction due to damaged salivary glands. In this review, we discuss etiology of SGCs, present findings on the role of autophagy in salivary tumorigenesis, review adverse effects of radiation treatment, and examine remedies for restoration of salivary function.