Gene Expression Analysis Reveals Inhibition of Radiation-Induced TGFβ-Signaling by Hyperbaric Oxygen Therapy in Mouse Salivary Glands

A side effect of radiation therapy in the head and neck region is injury to surrounding healthy tissues such as irreversible impaired function of the salivary glands. Hyperbaric oxygen therapy (HBOT) is clinically used to treat radiation-induced damage but its mechanism of action is largely unknown. In this study, we investigated the molecular pathways that are affected by HBOT in mouse salivary glands two weeks after radiation therapy by microarray analysis. Interestingly, HBOT led to significant attenuation of the radiation-induced expression of a set of genes and upstream regulators that are involved in processes such as fibrosis and tissue regeneration. Our data suggest that the TGFβ-pathway, which is involved in radiation-induced fibrosis and chronic loss of function after radiation therapy, is affected by HBOT. On the longer term, HBOT reduced the expression of the fibrosis-associated factor α-smooth muscle actin in irradiated salivary glands. This study highlights the potential of HBOT to inhibit the TGFβ-pathway in irradiated salivary glands and to restrain consequential radiation induced tissue injury.     

Possible role of nitric oxide in radiation-induced salivary gland dysfunction

In this study, we developed a murine model of xerostomia to elucidate the mechanism of radiation-induced salivary gland dysfunction and determined the levels of nitric oxide (NO) in the salivary glands to assess its involvement in the salivary dysfunction induced by radiation. In addition, an inhibitor of NO synthesis was administered to the model in vivo, and its effect on saliva secretion was investigated. Salivary gland irradiation at a dose of 15 Gy caused a significant decrease in secretion compared to unirradiated salivary glands. There were no marked differences between the irradiated mice and unirradiated mice in water or food consumption or in body weight changes. The NO levels in the cultured salivary gland epithelial cells were increased by treatment with a combination of interferon gamma (Ifng), interleukin 1-beta (Il1b), and tumor necrosis factor alpha (Tnfa). Irradiation increased the NO level in the salivary gland tissue. The presence of N(G)-monomethyl-l-arginine acetate (l-NMMA), an inhibitor of NO synthesis, caused a decrease in the NO level in cultured salivary gland tissues after irradiation. Administration of l-NMMA to irradiated mice improved saliva secretion. These results suggest that excessive production of NO induced by radiation is involved in the formation of radiation-induced xerostomia. The finding that administration of an inhibitor of NO synthesis ameliorated the dysfunction of irradiated salivary glands indicates that NO plays a role as a mediator of the dry mouth symptoms that occur after irradiation.     

骨髓来源细胞治疗放射性唾液腺损伤的研究进展

罹患头颈部肿瘤的患者在接受放射治疗时往往会发生放射性唾液腺损伤。射线的照射使患者唾液腺结构破坏、功能减退,患者的生活质量严重下降。对于放射性唾液腺损伤,临床上尚无有效的治疗方式。骨髓来源细胞(bone marrow-derived cells,BMDCs)最早用于治疗血液系统疾病。随着对BMDCs认识的逐渐深入,BMDCs的应用领域日益广泛。近些年来,一些动物实验的研究结果表明,利用BMDCs治疗放射性唾液腺损伤能够有效地保护腺体内各种实质细胞,促进腺组织再生,恢复唾液腺功能。本文主要对利用BMDCs治疗放射性唾液腺损伤的治疗方式、治疗效果及其主要的治疗机制进行综述,并对该领域今后的研究方向进行了展望。     

Sox9+ cells are required for salivary gland regeneration after radiation damage via the Wnt/β-catenin pathway

Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, and oral infection. Because of the lack of long-term treatment for the symptoms of xerostomia, current research has focused on finding endogenous stem cells that can differentiate into various cell lineages to replace lost tissues and restore functions. Here, we report that Sox9⁺ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9⁺ cells, the salivary glands of irradiated mice showed more severe phenotypes and the reduced proliferative capacity. Analysis of online single-cell RNA-sequencing data reveals the enrichment of the Wnt/β-catenin pathway in the Sox9⁺ cell population. Furthermore, treatment with a Wnt/β-catenin inhibitor in irradiated mice inhibits the regenerative capability of Sox9⁺ cells. Finally, we show that Sox9⁺ cells are capable of forming organoids in vitro and that transplanting these organoids into salivary glands after radiation partially restored salivary gland functions. These results suggest that regenerative therapy targeting Sox9⁺ cells is a promising approach to treat radiation-induced salivary gland injury.     

Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree

Abstract

The salivary glands often are severely and permanently damaged by therapeutic irradiation for cancer of the head and neck. The markedly reduced quantity and quality of saliva results in greatly increased susceptibility to dental caries and infection of the oral mucosa and alveolar bone. Recently, subcapsular injection of cultured mouse salivary gland cells has achieved a significant degree of regeneration in a previously irradiated mouse salivary gland; however, the recovery was limited to one lobule. We describe here a method for delivering donor rat salivary gland cells via the main duct that distributes several thousand cells throughout the recipient rat's salivary gland. The donated cells exhibited the cytodifferentiation of the structures in which they lodged, i.e., acini, granular convoluted tubules, and the several types of ducts. This method may facilitate the simultaneous functional recovery of almost all of the lobules of irradiated rat salivary glands.     

Cutting edge: Humanized nano-proresolving medicines mimic inflammation-resolution and enhance wound healing

Endogenous microparticles (MPs) were systematically profiled during the time course of self-limited inflammation. Precursors for specialized proresolving lipid mediators were identified in MPs from inflammatory exudates using liquid chromatography tandem mass spectrometry-based metabolomics. Hence, we postulated that formation of anti-inflammatory and proresolving lipid mediators could underlie beneficial effects attributed to MPs and that this process could serve as a basis for biomimicry. Using human neutrophil-derived MPs, we constructed novel nanoparticles (NPs) containing aspirin-triggered resolvin D1 or a lipoxin A(4) analog. Enriched NPs dramatically reduced polymorphonuclear cell influx in murine peritonitis, shortened resolution intervals, and exhibited proresolving actions accelerating keratinocyte healing. The enriched NPs protected against inflammation in the temporomandibular joint. These findings indicate that humanized NPs, termed nano-proresolving medicines, are mimetics of endogenous resolving mechanisms, possess potent beneficial bioactions, can reduce nanotoxicity, and offer new therapeutic approaches.     

Salivary gland-sparing prophylactic pilocarpine treatment has no effect on tumor regrowth after irradiation

Radiotherapy of head and neck cancer frequently damages the salivary glands. Prophylactic administration of the muscarinic receptor agonist pilocarpine reduces subsequent radiation damage to the salivary glands in rats, but its effects on tumor cell radiosensitivity and tumor regrowth after irradiation had not been assessed. In the current study, we first tested the effect of pilocarpine on clonogenic cell survival in vitro. No effect of pilocarpine on radiosensitivity was observed in a panel of cell lines either with or without expression of muscarinic receptors. Second, a single dose of pilocarpine known to protect salivary gland tissue from radiation damage was given to rats transplanted with subcutaneously growing rhabdomyosarcomas 1 h prior to irradiation with a single dose of 35 Gy. No alterations in growth delay were detected (26 +/- 2 days for controls compared to 26 +/- 2 days for pilocarpine treatment). Our data indicate that pilocarpine pretreatment, which has been shown previously to protect salivary glands from radiation, does not protect tumor cells or tumors. Use of this drug therefore may lead to therapeutic gain in the treatment of head and neck cancer.     

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sj?gren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sj?gren's syndrome.     

Pharmacological Activation of the EDA/EDAR Signaling Pathway Restores Salivary Gland Function following Radiation-Induced Damage

Radiotherapy of head and neck cancers often results in collateral damage to adjacent salivary glands associated with clinically significant hyposalivation and xerostomia. Due to the reduced capacity of salivary glands to regenerate, hyposalivation is treated by substitution with artificial saliva, rather than through functional restoration of the glands. During embryogenesis, the ectodysplasin/ectodysplasin receptor (EDA/EDAR) signaling pathway is a critical element in the development and growth of salivary glands. We have assessed the effects of pharmacological activation of this pathway in a mouse model of radiation-induced salivary gland dysfunction. We report that post-irradiation administration of an EDAR-agonist monoclonal antibody (mAbEDAR1) normalizes function of radiation damaged adult salivary glands as determined by stimulated salivary flow rates. In addition, salivary gland structure and homeostasis is restored to pre-irradiation levels. These results suggest that transient activation of pathways involved in salivary gland development could facilitate regeneration and restoration of function following damage.     

NK cells are effectors for resolvin E1 in the timely resolution of allergic airway inflammation

Immune responses are pathologically sustained in several common diseases, including asthma. To determine endogenous proresolving mechanisms for adaptive immune responses, we used a murine model of self-limited allergic airway inflammation. After cessation of allergen exposure, eosinophils and T cells were cleared concomitant with the appearance of increased numbers of NK cells in the lung and mediastinal lymph nodes. The mediastinal lymph node NK cells were activated, expressing CD27, CD11b, CD69, CD107a, and IFN-γ. NK cell depletion disrupted the endogenous resolution program, leading to delayed clearance of airway eosinophils and Ag-specific CD4(+) T cells. NK cell trafficking to inflamed tissues for resolution was dependent upon CXCR3 and CD62L. During resolution, eosinophils and Ag-specific CD4(+) T cells expressed NKG2D ligands, and a blocking Ab for the NKG2D receptor delayed clearance of these leukocytes. Of interest, NK cells expressed CMKLR1, a receptor for the proresolving mediator resolvin E1, and depletion of NK cells decreased resolvin E1-mediated resolution of allergic inflammation. Resolvin E1 regulated NK cell migration in vivo and NK cell cytotoxicity in vitro. Together, these findings indicate new functions in catabasis for NK cells that can also serve as targets for proresolving mediators in the resolution of adaptive immunity.     

Receptors for the neuropeptides,myoinhibitory peptide and SIFamide,in control of the salivary glands of the blacklegged tick Ixodes scapularis

Tick salivary glands are important organs that enable the hematophagous feeding of the tick. We previously described the innervation of the salivary gland acini types II and III by a pair of protocerebral salivary gland neurons that produce both myoinhibitory peptide (MIP) and SIFamide (?imo et al., 2009b). In this study we identified authentic receptors expressed in the salivary glands for these neuropeptides. Homology-based searches for these receptors in the Ixodes scapularis genome sequence were followed by gene cloning and functional expression of the receptors. Both receptors were activated by low nanomolar concentrations of their respective ligands. The temporal expression patterns of the two ligands and their respective receptors suggest that the SIFamide signaling system pre-exists in unfed salivary glands, while the MIP system is activated upon initiation of feeding. Immunoreactivity for the SIFamide receptor in the salivary gland was detected in acini types II and III, surrounding the acinar valve and extending to the basal region of the acinar lumen. The location of the SIFamide receptor in the salivary glands suggests three potential target cell types and their probable functions: myoepithelial cell that may function in the contraction of the acini and/or the control of the valve; large, basally located dopaminergic granular cells for regulation of paracrine dopamine; and neck cells that may be involved in the control of the acinar duct and its valve.     

Smad7 attenuates TGF-β-mediated aging-related hypofunction of submandibular glands

Submandibular glands have essential functions in taste, mastication, swallowing, and digestion. Submandibular gland hypofunction is prevalent in the elderly, impairing the patients’ quality of life. Current clinical treatment strategies have not decelerated or reversed the pathological process of submandibular gland hypofunction. Therefore, novel restoration strategies should be explored. However, studies on the mechanism of aging-related submandibular gland hypofunction remain very limited. The role of the TGF-β/Smad pathway in fibrosis has been studied in other organs. Therefore, this study aimed to elucidate the role of TGF-β/Smad signaling in the aging-related submandibular gland hypofunction. The results showed that Smad7 knockout in mice decreased the salivary flow rate. H&E, Masson trichrome, and immunohistochemistry staining of MCP-1 and α-SMA showed that Smad7 knockout in mice resulted in lymphocytic infiltration, acinar cell atrophy, and interstitial fibrosis. The Western blotting of collagen I and III also confirmed extensive fibrosis. We then found that Smad7 depletion resulted in the TGF-β-mediated fibrosis via mir-21, mir-29, and np_5318, and NFκB-driven inflammation activation. This study confirmed the inhibitory role of Smad7 in the aging-related submandibular gland hypofunction. Therefore, it provided a promising treatment target for aging-related dysfunction and sialadenitis of submandibular gland.     

Lipidomics in pathogenesis,progression and treatment of nonalcoholic steatohepatitis (NASH): Recent advances

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting up to 30% of the general adult population. NAFLD encompasses a histological spectrum ranging from pure steatosis to non-alcoholic steatohepatitis (NASH). NASH can progress to cirrhosis and is becoming the most common indication for liver transplantation, as a result of increasing disease prevalence and of the absence of approved treatments. Lipidomic readouts of liver blood and urine samples from experimental models and from NASH patients disclosed an abnormal lipid composition and metabolism. Collectively, these changes impair organelle function and promote cell damage, necro-inflammation and fibrosis, a condition termed lipotoxicity. We will discuss the lipid species and metabolic pathways leading to NASH development and progression to cirrhosis, as well as and those species that can contribute to inflammation resolution and fibrosis regression. We will also focus on emerging lipid-based therapeutic opportunities, including specialized proresolving lipid molecules and macrovesicles contributing to cell-to-cell communication and NASH pathophysiology.     

Suppression of Radiation-Induced Salivary Gland Dysfunction by IGF-1

Background

Radiation is a primary or secondary therapeutic modality for treatment of head and neck cancer. A common side effect of irradiation to the neck and neck region is xerostomia caused by salivary gland dysfunction. Approximately 40,000 new cases of xerostomia result from radiation treatment in the United States each year. The ensuing salivary gland hypofunction results in significant morbidity and diminishes the effectiveness of anti-cancer therapies as well as the quality of life for these patients. Previous studies in a rat model have shown no correlation between induction of apoptosis in the salivary gland and either the immediate or chronic decrease in salivary function following γ-radiation treatment.

Methodology/Principal Finding

A significant level of apoptosis can be detected in the salivary glands of FVB mice following γ-radiation treatment of the head and neck and this apoptosis is suppressed in transgenic mice expressing an activated mutant of Akt (myr-Akt1). Importantly, this suppression of apoptosis in myr-Akt1 mice preserves salivary function, as measured by saliva output, three and thirty days after γ-radiation treatment. In order to translate these studies into a preclinal model we found that intravenous injection of IGF1 stimulated activation of endogenous Akt in the salivary glands in vivo. A single injection of IGF1 prior to exposure to γ-radiation diminishes salivary acinar cell apoptosis and completely preserves salivary gland function three and thirty days following irradiation.

Conclusions/Significance

These studies suggest that apoptosis of salivary acinar cells underlies salivary gland hypofunction occurring secondary to radiation of the head and neck region. Targeted delivery of IGF1 to the salivary gland of patients receiving head and neck irradiation may be useful in reducing or eliminating xerostomia and restoring quality of life to these patients.     

Salivary Gland Hypofunction in tyrosylprotein sulfotransferase-2 Knockout Mice Is Due to Primary Hypothyroidism

Background

Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi tyrosylprotein sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice.

Methodology/Principal Findings

Quantitative MRI imaging documented that salivary glands in Tpst2-/- females were ^≈ 30% smaller than wild type or Tpst1-/- mice and that the granular convoluted tubules in Tpst2-/- submandibular glands were less prominent and were almost completely devoid of exocrine secretory granules compared to glands from wild type or Tpst1-/- mice. In addition, pilocarpine–induced salivary flow and salivary α-amylase activity in Tpst2-/- mice of both sexes was substantially lower than in wild type and Tpst1-/- mice. Anti-sulfotyrosine Western blots of salivary gland extracts and saliva showed no differences between wild type, Tpst1-/-, and Tpst2-/- mice, suggesting that the salivary gland hypofunction is due to factor(s) extrinsic to the salivary glands. Finally, we found that all indicators of hypothyroidism (serum T4, body weight) and salivary gland hypofunction (salivary flow, salivary α-amylase activity, histological changes) were restored to normal or near normal by thyroid hormone supplementation.

Conclusions/Significance

Our findings conclusively demonstrate that low body weight and salivary gland hypofunction in Tpst2-/- mice is due solely to primary hypothyroidism.     

Resolvin E1 regulates inflammation at the cellular and tissue level and restores tissue homeostasis in vivo

Resolvin E1 (RvE1) is a potent proresolving mediator of inflammation derived from omega-3 eicosapentaenoic acid that acts locally to stop leukocyte recruitment and promote resolution. RvE1 displays potent counter-regulatory and tissue-protective actions in vitro and in vivo. Periodontal disease is a local inflammatory disease initiated by bacteria characterized by neutrophil-mediated tissue injury followed by development of a chronic immune lesion. In this study, we report the treatment of established periodontitis using RvE1 as a monotherapy in rabbits compared with structurally related lipids PGE(2) and leukotriene B(4). PGE(2) and leukotriene B(4) each enhanced development of periodontitis and worsened the severity of disease. Promotion of resolution of inflammation as a therapeutic target with RvE1 resulted in complete restoration of the local lesion, and reduction in the systemic inflammatory markers C-reactive protein and IL-1beta. This report is the first to show that resolution of inflammation by a naturally occurring endogenous lipid mediator results in complete regeneration of pathologically lost tissues, including bone.     

Calcium signaling defects underlying salivary gland dysfunction

Salivary glands secrete saliva, a mixture of proteins and fluids, which plays an extremely important role in the maintenance of oral health. Loss of salivary secretion causes a dry mouth condition, xerostomia, which has numerous deleterious consequences including opportunistic infections within the oral cavity, difficulties in eating and swallowing food, and problems with speech. Saliva secretion is regulated by stimulation of specific signaling mechanisms within the acinar cells of the gland. Neurotransmitter-stimulated increase in cytosolic [Ca²⁺] ([Ca²⁺]_i) in acinar cells is the primary trigger for salivary fluid secretion from salivary glands, the loss of which is a critical factor underlying dry mouth conditions in patients. The increase in [Ca²⁺]_i regulates multiple ion channel and transport activities that together generate the osmotic gradient which drives fluid secretion across the apical membrane. Ca²⁺ entry mediated by the Store-Operated Ca²⁺ Entry (SOCE) mechanism provides the essential [Ca²⁺]_i signals to trigger salivary gland fluid secretion. Under physiological conditions depletion of ER-Ca²⁺ stores is caused by activation of IP3R by IP3 and this provides the stimulus for SOCE. Core components of SOCE in salivary gland acinar cells are the plasma membrane Ca²⁺ channels, Orai1 and TRPC1, and STIM1, a Ca²⁺-sensor protein in the ER, which regulates both channels. In addition, STIM2 likely enhances the sensitivity of cells to ER-Ca²⁺ depletion thereby tuning the cellular response to agonist stimulation. Two major, clinically relevant, conditions which cause irreversible salivary gland dysfunction are radiation treatment for head-and-neck cancers and the autoimmune exocrinopathy, Sjögren's syndrome (pSS). However, the exact mechanism(s) that causes the loss of fluid secretion, in either condition, is not clearly understood. A number of recent studies have identified that defects in critical Ca²⁺ signaling mechanisms underlie salivary gland dysfunction caused by radiation treatment or Sjögren's syndrome (pSS). This chapter will discuss these very interesting and important studies.