Specialized pro-resolving receptors are expressed in salivary glands with Sjögren's syndrome.

Our previous studies demonstrated that resolvin D1 (RvD1) and its aspirin-trigged (AT) form AT-RvD1, are effective in decreasing inflammation while restoring saliva flow rates in a Sjögren's syndrome (SS)-like mouse model before and after disease onset. Resolvins are specialized pro-resolving mediators (SPM) that actively regulate inflammation. However, we only have extensive data within the salivary glands for RvD1 and AT-RvD1, both of which bind to the receptor ALX/FPR2. As such, the presence of other SPM receptors is unknown within salivary glands. Therefore, the goal of this study was to determine the expression of SPM receptors in non-SS and SS patients. For this purpose, six human minor salivary glands from female subjects were analyzed by H&E using the Chisholm and Mason classification to determine the degree of lymphocytic infiltration. Next, confocal immunofluorescence analysis was performed to determine the presence and distribution of different SPM receptors in mucous acini and striated ducts. We observed diffuse presence of lymphocytic infiltration and clinical data were consistent with SS diagnosis in three patients. Moreover, confocal immunofluorescence analysis indicated the presence of the receptors ALX/FPR2, BLT1 and CMKLR1 in the mucous acini and striated ducts of both non-SS and SS patients. GPR32 was absent in SS and non-SS minor salivary glands. In summary, our results showed that various SPM receptors are expressed in non-SS and SS minor salivary glands, all of which may pose as potential targets for promoting pro-epithelial and anti-inflammatory/pro-resolution signaling on SS patients.

Sex-mediated elevation of the specialized pro-resolving lipid mediator levels in a Sjögren's syndrome mouse model.

Our previous results showed that the specialized pro-resolving mediator (SPM) Resolvin D1 (RvD1) promotes resolution of inflammation in salivary glands in non-obese diabetic (NOD)/ShiLtJ, a mouse model for Sjögren's syndrome (SS). Additionally, mice lacking the RvD1 receptor ALX/FPR2 show defective innate and adaptive immune responses in salivary glands. Particularly, ALX/FPR2 KO mice exhibit exacerbated inflammation in their salivary glands in response to systemic LPS treatment. Moreover, female ALX/FPR2 KO mice show increased autoantibody production and loss of salivary gland function with age. Together, these studies suggest that an underlying SPM dysregulation could be contributing to SS progression. Therefore, we investigated whether SPM production is altered in NOD/ShiLtJ using metabololipidomics and enzyme-linked immunosorbent assay (ELISA). Our results demonstrate that SPM levels were broadly elevated in plasma collected from NOD/ShiLtJ female mice after disease onset, whereas these drastic changes did not occur in male mice. Moreover, gene expression of enzymes involved in SPM biosynthesis were altered in submandibular glands (SMG) from NOD/ShiLtJ female mice after disease onset, with 5-LOX and 12/15-LOX being downregulated and upregulated, respectively. Despite this dysregulation, the abundances of the SPM products of these enzymes (ie, RvD1 and RvD2) were unaltered in freshly isolated SMG cells suggesting that other cell populations (eg, lymphocytes) may be responsible for the overabundance of SPMs that we observed. The elevation of SPMs noted here appeared to be sex mediated, meaning that it was observed only in one sex (females). Given that SS primarily affects females (roughly 90% of diagnosed cases), these results may provide some insights into the mechanisms underlying the observed sexual dimorphism.

The G-Protein-Coupled Receptor ALX/Fpr2 Regulates Adaptive Immune Responses in Mouse Submandibular Glands.

Lipoxin receptor (ALX)/N-formyl peptide receptor (FPR)-2 is a G-protein-coupled receptor that has multiple binding partners, including the endogenous lipid mediators resolvin D1, lipoxin A4, and the Ca2+-dependent phospholipid-binding protein annexin A1. Previous studies have demonstrated that resolvin D1 activates ALX/Fpr2 to resolve salivary gland inflammation in the NOD/ShiLtJ mouse model of Sjögren syndrome. Moreover, mice lacking the ALX/Fpr2 display an exacerbated salivary gland inflammation in response to lipopolysaccharide. Additionally, activation of ALX/Fpr2 has been shown to be important for regulating antibody production in B cells. These previous studies indicate that ALX/Fpr2 promotes resolution of salivary gland inflammation while modulating adaptive immunity, suggesting the need for investigation of the role of ALX/Fpr2 in regulating antibody production and secretory function in mouse salivary glands. Our results indicate that aging female knockout mice lacking ALX/Fpr2 display a significant reduction in saliva flow rates and weight loss, an increased expression of autoimmune-associated genes, an up-regulation of autoantibody production, and increased CD20-positive B-cell population. Although not all effects were noted among the male knockout mice, the results nonetheless indicate that ALX/Fpr2 is clearly involved in the adaptive immunity and secretory function in salivary glands, with further investigation warranted to determine the cause(s) of these between-sex differences.

Aspirin Triggered Resolvin D1 reduces inflammation and restores saliva secretion in a Sjögren's syndrome mouse model.

OBJECTIVES: SS is characterized by chronic inflammation of the salivary glands leading to loss of secretory function, thereby suggesting specialized pro-resolving mediators targeting inflammation to be a viable option for treating SS. Previous studies demonstrated that aspirin-triggered resolvin D1 (AT-RvD1) prevents chronic inflammation and enhances saliva secretion in a SS-like mouse model when applied before disease onset. However, this therapy cannot be used in SS patients given that diagnosis occurs post-disease onset and no reliable screening methods exist. Therefore, we examined whether treatment with AT-RvD1 reduces SS-like features in a mouse model post-disease onset. METHODS: Tail vein injections were performed in a SS-like mouse model both with and without AT-RvD1 post-disease onset for 8 weeks, with salivary gland function and inflammatory status subsequently determined. RESULTS: Treatment of a SS-like mouse model with AT-RvD1 post-disease onset restores saliva secretion in both females and males. Moreover, although AT-RvD1 treatment does not reduce the overall submandibular gland lymphocytic infiltration, it does reduce the number of T helper 17 cells within the infiltrates in both sexes. Finally, AT-RvD1 reduces SS-associated pro-inflammatory cytokine gene and protein expression levels in submandibular glands from female but not male mice. CONCLUSION: AT-RvD1 treatment administered post-disease onset reduces T helper 17 cells and successfully restores salivary gland function in a SS mouse model with variable effects noted by sex, thus warranting further examination of both the causes for the sex differences and the mechanisms responsible for the observed treatment effect.

Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters.

Previous studies showed that mouse submandibular gland cells form three-dimensional structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is complicated due to its lack of purity. By contrast, the use of synthetic peptides derived from Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111 peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain from Laminin-111 and the YIGSR peptide corresponding to the ß1 chain from Laminin-111, which are important for cell adhesion and migration. We created three-dimensional salivary cell clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides. Results indicate that the YIGSR peptide improved morphology and lumen formation in rat parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a combination of both peptides not only allowed the formation of functional three-dimensional salivary cell clusters but also increased attachment and number of cell clusters. In summary, we demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and differentiation of salivary gland cell clusters with mature lumens.

ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands, leading to dry mouth, dry eyes, and the presence of anti-nuclear antibodies. Despite modern advances, the current therapies for SS have no permanent benefit. A potential treatment could involve the use of resolvins, which are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Our previous studies indicate that ALX/FPR2, the receptor for RvD1, is expressed and active in the rat parotid cell line Par-C10. Specifically, activation of ALX/FPR2 with RvD1 blocked inflammatory signals caused by TNF-α and enhanced salivary epithelial integrity. The goal of this study was to investigate RvD1 receptor expression and signaling pathways in primary salivary cells. Additionally, we determined the role of the aspirin-triggered 17R analog (AT-RvD1, a more chemically stable RvD1 epimeric form) in prevention of TNF-α-mediated salivary inflammation in mouse submandibular glands (mSMG). Our results indicate that ALX/FPR2 is expressed in mSMG and is able to elicit intracellular Ca2+ responses and phosphorylation of Erk1/2, as well as Akt. Given that these signaling pathways are linked to cell survival, we investigated whether AT-RvD1 was able to prevent programmed cell death in mSMG. Specifically, we determined that AT-RvD1 prevented TNF-α-mediated caspase-3 activation. Finally, we show that ALX/FPR2 is expressed in human minor salivary glands with and without SS, indicating the potential therapeutic use of AT-RvD1 for this condition.

Copper chelation reduces early collagen deposition and preserves saliva secretion in irradiated salivary glands.

Radiation therapy is a first-line treatment for head and neck cancer; however, it typically leads to hyposalivation stemming from fibrosis of the salivary gland. Current strategies to restore glandular function are dependent on the presence of residual functional salivary gland tissue, a condition commonly not met in patients with extensive fibrotic coverage of the salivary gland resulting from radiation therapy. Fibrosis is defined by the pathological accumulation of connective tissue (i.e., extracellular matrix) and excessive deposition of crosslinked (fibrillar) collagen that can impact a range of tissues and given that collagen crosslinking is necessary for fibrosis formation, inhibiting this process is a reasonable focus for developing anti-fibrotic therapies. Collagen crosslinking is catalyzed by the lysyl oxidase family of secreted copper-dependent metalloenzymes, and since that copper is an essential cofactor in all lysyl oxidase family members, we tested whether localized delivery of a copper chelator into the submandibular gland of irradiated mice could suppress collagen deposition and preserve the structure and function of this organ. Our results demonstrate that transdermal injection of tetrathiomolybdate into salivary glands significantly reduced the early deposition of fibrillar collagen in irradiated mice and preserved the integrity and function of submandibular gland epithelial tissue. Together, these studies identify copper metabolism as a novel therapeutic target to control radiation induced damage to the salivary gland and the current findings further indicate the therapeutic potential of repurposing clinically approved copper chelators as neoadjuvant treatments for radiation therapy.

Understanding resolvin signaling pathways to improve oral health.

The discovery of resolvins has been a major breakthrough for understanding the processes involved in resolution of inflammation. Resolvins belong to a family of novel lipid mediators that possess dual anti-inflammatory and pro-resolution actions. Specifically, they protect healthy tissue during immune-inflammatory responses to infection or injury, thereby aiding inflammation resolution and promoting tissue healing. One of the major concerns in modern medicine is the management and treatment of oral diseases, as they are related to systemic outcomes impacting the quality of life of many patients. This review summarizes known signaling pathways utilized by resolvins to regulate inflammatory responses associated with the oral cavity.

A combination treatment of low-dose dexamethasone and aspirin-triggered resolvin D1 reduces Sjögren syndrome-like features in a mouse model.

Background: Sjögren syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and diminished secretory function of the salivary glands. Dexamethasone (DEX) resolves dry mouth and lymphocytic infiltration; however, this treatment is difficult to maintain because of multiple adverse effects (eg, osteoporosis and skin thinning); likewise, aspirin-triggered resolvin D1 (AT-RvD1) increases saliva secretion but cannot eliminate lymphocytic infiltration. Previous studies showed that a combination of low-dose DEX with AT-RvD1 before disease onset prevents SS-like features in a mouse model; however, this is not clinically practical because there are no reliable indicators of SS before disease onset. Therefore, the authors applied the combined treatment at disease onset to show its efficacy and comparative lack of adverse effects, so that it may reasonably be maintained over a patient's lifetime. Methods: NOD/ShiLtJ mice were treated with ethanol (vehicle control), high-dose DEX alone, AT-RvD1 alone, or a combination of low-dose DEX with AT-RvD1 at disease onset for 8 weeks. Then saliva flow rates were measured, and submandibular glands were harvested for histologic analyses. Results: A combined treatment of low-dose DEX with AT-RvD1 significantly decreased mast cell degranulation and lymphocytic infiltration, increased saliva secretion, and restored apical aquaporin-5 expression in submandibular glands of NOD/ShiLtJ mice. Conclusions: Low-dose DEX combined with AT-RvD1 reduces the severity of SS-like manifestation and prevents the development of advanced and potentially irreversible damage, all in a form that can reasonably be administered indefinitely without the need to cease treatment because of secondary effects.

Fibrin hydrogels fortified with FGF-7/10 and laminin-1 peptides promote regeneration of irradiated salivary glands.

Ionizing radiation, commonly used for head and neck cancer treatment, typically damages the salivary glands, resulting in hyposalivation. The development of treatments to restore this lost function is crucial for improving the quality of life for patients suffering from this condition. To address this clinical need, we have developed an innovative hydrogel by chemically conjugating laminin-1 peptides (A99 and YIGSR) and growth factors, FGF-7 and FGF-10, to fibrin hydrogels. Our results demonstrate that FGF-7/10 and laminin-1 peptides fortified fibrin hydrogel [enhanced laminin-1 peptides fibrin hydrogel (Ep-FH)] promotes salivary gland regeneration and functionality by improving epithelial tissue organization, establishing a healthy network of blood vessels and nerves, while reducing fibrosis in a head and neck irradiated mouse model. These results indicate that fibrin hydrogel-based implantable scaffolds containing pro-regenerative signals promote sustained secretory function of irradiated salivary glands, offering a potential alternative treatment for hyposalivation in head and neck cancer patients undergoing radiation treatment. These unique findings emphasize the potential of fibrin hydrogel-based implantable scaffolds enriched with pro-regenerative signals in sustaining the secretory function of irradiated salivary glands and offer a promising alternative treatment for addressing hyposalivation in head and neck cancer patients undergoing radiation therapy. STATEMENT OF SIGNIFICANCE: Radiation therapies used to treat head and neck cancers often result in damaged salivary gland, leading to severe dryness of the oral cavity. In this study, we engineered FGF-7 and FGF-10 and immobilized them into L1p-FH. The resulting hydrogel, Ep-FH, restored irradiated salivary gland functionality by enhancing epithelial tissue organization, promoting the development of a healthy network of blood vessels and nerves as well as reduction of fibrosis.

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

Sjögren'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 Sjögren's syndrome.

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium.

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1ß- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).

Current experimental methods to investigate the impact of specialized pro-resolving lipid mediators on Sjögren's syndrome.

Sjögren's syndrome is a chronic inflammatory autoimmune disease characterized by diminished secretory function of the exocrine glands. Although extensive investigation has been done to understand Sjögren's syndrome, the causes of the disease are as yet unknown and treatments remain largely ineffective, with established therapeutic interventions being limited to use of saliva substitutes with modest effectiveness. A primary feature of Sjögren's syndrome is uncontrolled inflammation of exocrine tissues and previous studies have demonstrated that lipid-based specialized pro-resolving mediators reduce inflammation and restores tissue integrity in salivary glands. However, these studies are limited to a single specialized pro-resolving lipid mediator's family member resolvin D1 or RvD1 and its aspirin-triggered epimer, AT-RvD1. Consequently, additional studies are needed to explore the potential benefits of other members of the specialized pro-resolving lipid mediator's family and related molecules (e.g., additional resolvin subtypes as well as lipoxins, maresins and protectins). In support of this goal, the current review aims to briefly describe the range of current experimental methods to investigate the impact of specialized pro-resolving lipid mediators on Sjögren's syndrome, including both strengths and weaknesses of each approach where this information is known. With this article, the possibilities presented by specialized pro-resolving lipid mediators will be introduced to a wider audience in immunology and practical advice is given to researchers who may wish to take up this work.

Early Dry Eye Disease Onset in a NOD.H-2h4 Mouse Model of Sjögren's Syndrome.

Purpose: To develop a mouse model of human dry eye disease (DED) for investigation of sex differences in autoimmune-associated dry eye pathology. Methods: Ocular surface disease was assessed by quantifying corneal epithelial damage with lissamine green stain in the NOD.H-2h4,IFNγ-/-,CD28-/- (NOD.H-2h4 DKO) mouse model of Sjögren's syndrome (SS). Lacrimal gland function was assessed by tear volume quantification with phenol red thread and lacrimal gland inflammation (i.e., dacryoadenitis) was assessed by quantification of immune cell foci, flow cytometric analysis of immune cell composition, and expression of proinflammatory markers. Results: The NOD.H-2h4 DKO mouse model of SS exhibits greater age-dependent increases in corneal damage than in NOD.H-2h4 parental mice and demonstrates an earlier disease onset in females compared to males. The severity of ocular surface disease correlates with loss of goblet cell density, increased conjunctivitis, and dacryoadenitis that is more pronounced in NOD.H-2h4 DKO than NOD.H-2h4 mice. B cells dominate lacrimal infiltrates in 16-week-old NOD.H-2h4 and NOD.H-2h4 DKO mice, but T helper cells and macrophages are also present. Lacrimal gland expression of proinflammatory genes, including the P2X7 and P2Y2 purinergic receptors, is greater in NOD.H-2h4 DKO than NOD.H-2h4 mice and correlates with dacryoadenitis. Conclusions: Our results demonstrate for the first time that autoimmune dry eye disease occurs in both sexes of NOD.H-2h4 DKO and NOD.H-2h4 mice, with earlier onset in female NOD.H-2h4 DKO mice when compared to males of the same strain. This study demonstrates that both NOD.H-2h4 and NOD.H-2h4 DKO mice are novel models that closely resemble SS-related and sex-dependent DED.

Tuft Cells Are Present in Submandibular Glands Across Species.

Tuft cells are bottle-shaped, microvilli-projecting chemosensory cells located in the lining of a variety of epithelial tissues and, following their identification approximately 60 years ago, have been linked to immune system function in a variety of epithelia. Until recently, Tuft cells had not been convincingly demonstrated to be present in salivary glands with their detection by transmission electron microscopy only shown in a handful of earlier studies using rat salivary glands, and no follow-up work has been conducted to verify their presence in salivary glands of other species. Here, we demonstrate that Tuft cells are present in the submandibular glands of various species (i.e., mouse, pig and human) using transmission electron microscopy and confocal immunofluorescent analysis for the POU class 2 homeobox 3 (POU2F3), which is considered to be a master regulator of Tuft cell identity.

P2Y2 nucleotide receptors mediate metalloprotease-dependent phosphorylation of epidermal growth factor receptor and ErbB3 in human salivary gland cells.

The G protein-coupled receptor P2Y(2) nucleotide receptor (P2Y(2)R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y(2)Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y(2)R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-alpha protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y(2)R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y(2)R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.

Laminin-1 Peptides Conjugated to Fibrin Hydrogels Promote Salivary Gland Regeneration in Irradiated Mouse Submandibular Glands.

Previous studies demonstrated that salivary gland morphogenesis and differentiation are enhanced by modification of fibrin hydrogels chemically conjugated to Laminin-1 peptides. Specifically, Laminin-1 peptides (A99: CGGALRGDN-amide and YIGSR: CGGADPGYIGSRGAA-amide) chemically conjugated to fibrin promoted formation of newly organized salivary epithelium both in vitro (e.g., using organoids) and in vivo (e.g., in a wounded mouse model). While these studies were successful, the model's usefulness for inducing regenerative patterns after radiation therapy remains unknown. Therefore, the goal of the current study was to determine whether transdermal injection with the Laminin-1 peptides A99 and YIGSR chemically conjugated to fibrin hydrogels promotes tissue regeneration in irradiated salivary glands. Results indicate that A99 and YIGSR chemically conjugated to fibrin hydrogels promote formation of functional salivary tissue when transdermally injected to irradiated salivary glands. In contrast, when left untreated, irradiated salivary glands display a loss in structure and functionality. Together, these studies indicate that fibrin hydrogel-based implantable scaffolds containing Laminin-1 peptides promote secretory function of irradiated salivary glands.

SPM Receptor Expression and Localization in Irradiated Salivary Glands.

Radiation therapy-mediated salivary gland destruction is characterized by increased inflammatory cell infiltration and fibrosis, both of which ultimately lead to salivary gland hypofunction. However, current treatments (e.g., artificial saliva and sialagogues) only promote temporary relief of symptoms. As such, developing alternative measures against radiation damage is critical for restoring salivary gland structure and function. One promising option for managing radiation therapy-mediated damage in salivary glands is by activation of specialized proresolving lipid mediator receptors due to their demonstrated role in resolution of inflammation and fibrosis in many tissues. Nonetheless, little is known about the presence and function of these receptors in healthy and/or irradiated salivary glands. Therefore, the goal of this study was to detect whether these specialized proresolving lipid mediator receptors are expressed in healthy salivary glands and, if so, if they are maintained after radiation therapy-mediated damage. Our results indicate that specialized proresolving lipid mediator receptors are heterogeneously expressed in inflammatory as well as in acinar and ductal cells within human submandibular glands and that their expression persists after radiation therapy. These findings suggest that epithelial cells as well as resident immune cells represent potential targets for modulation of resolution of inflammation and fibrosis in irradiated salivary glands.

Predicting Resolvin D1 Pharmacokinetics in Humans with Physiologically-Based Pharmacokinetic Modeling.

Sjögren's syndrome (SS) is an autoimmune disease with no effective treatment options. Resolvin D1 (RvD1) belongs to a class of lipid-based specialized pro-resolving mediators that showed efficacy in preclinical models of SS. We developed a physiologically-based pharmacokinetic (PBPK) model of RvD1 in mice and optimized the model using plasma and salivary gland pharmacokinetic (PK) studies performed in NOD/ShiLtJ mice with SS-like features. The predictive performance of the PBPK model was also evaluated with two external datasets from the literature reporting RvD1 PKs. The PBPK model adequately captured the observed concentrations of RvD1 administered at different doses and in different species. The PKs of RvD1 in virtual humans were predicted using the verified PBPK model at various doses (0.01-10 mg/kg). The first-in-human predictions of RvD1 will be useful for the clinical trial design and translation of RvD1 as an effective treatment strategy for SS.

Tight junctions in salivary epithelium.

Epithelial cell tight junctions (TJs) consist of a narrow belt-like structure in the apical region of the lateral plasma membrane that circumferentially binds each cell to its neighbor. TJs are found in tissues that are involved in polarized secretions, absorption functions, and maintaining barriers between blood and interstitial fluids. The morphology, permeability, and ion selectivity of TJ vary among different types of tissues and species. TJs are very dynamic structures that assemble, grow, reorganize, and disassemble during physiological or pathological events. Several studies have indicated the active role of TJ in intestinal, renal, and airway epithelial function; however, the functional significance of TJ in salivary gland epithelium is poorly understood. Interactions between different combinations of the TJ family (each with their own unique regulatory proteins) define tissue specificity and functions during physiopathological processes; however, these interaction patterns have not been studied in salivary glands. The purpose of this review is to analyze some of the current data regarding the regulatory components of the TJ that could potentially affect cellular functions of the salivary epithelium.