Salivary Epithelial Cells Research Articles

AB0136 Induction of reg ia, a new auto-antigen in sjögren’s syndrome patients, in salivary duct epithelial cells by interleukin-6 and -11

BackgroundThe regenerating gene, Reg, was originally isolated from a rat regenerating islet cDNA library, and its human homologue was named REG Iα. Reg gene product acts as a growth factor...

AB0222 Monocyte-derived IL-32 expression correlates with the level of inflammation in salivary glands of sjÖgren’s syndrome

BackgroundIL-32, a recently described cytokine promoting pro-inflammatory cytokines such as IL-1β, IL-6, CXCL8 and TNFα, has emerged as a key component in the development of chronic inflammatory diseases such as...

Role of salivary epithelial toll‐like receptors 2 and 4 in modulating innate immune responses in chronic periodontitis

Chronic periodontitis is initiated by sequential colonization with a broad array of bacteria and is perpetuated by an immune-inflammatory response to the changing biofilm. Host recognition of microbes is largely mediated by toll-like receptors (TLRs), which interact with conserved pathogen-associated molecular patterns. Based on ligand recognition, TLR-2 and TLR-4 interact with most periodontal pathogens. Extracrevicular bacterial reservoirs, such as the oral epithelial cells, contribute to the persistence of periodontitis. Human saliva is a rich source of oral epithelial cells that express functional TLRs. In this study we investigated the role of salivary epithelial cell (SEC) TLR-2 and TLR-4 in patients with generalized chronic periodontitis. Unstimulated whole saliva (UWS) was collected from patients with generalized chronic periodontitis and from healthy individuals after obtaining informed consent. Epithelial cells isolated from each UWS sample were assessed for TLR-2, TLR-4, peptidoglycan recognition protein (PGRP)-3 and PGRP-4 by quantitative real-time PCR. In addition, the SECs were stimulated in vitro with microbial products for up to 24h. The culture supernatant was assessed for cytokines by ELISA. Stimulation with TLR-2- or TLR-4-specific ligands induced cytokine secretion with differential kinetics and up-regulated TLR2 and TLR4 mRNAs, respectively, in cultures of SECs from patients with periodontitis. In addition, the SECs from patients with periodontitis exhibited reduced PGRP3 and PGRP4 mRNAs, the TLR-responsive genes with antibacterial properties. SECs derived from the UWS of patients with chronic periodontitis are phenotypically distinct and could represent potential resources for assessing the epithelial responses to periodontal pathogens in the course of disease progression and persistence.

Innate immune responses of exfoliated epithelial cells. (P4165)

Abstract Cell exfoliation is a characteristic feature of epithelia covering the external surface and the internal cavities of the body. While the benefit of cytology in infections and cancer diagnosis is well recognized, the clinical use of epithelial cells obtained from body fluids is relatively unexplored. As the first cells to encounter microbes, the epithelial cells exhibit toll like receptors (TLR) that bind specific conserved microbial patterns and transduce signals to thwart deleterious host responses to commensals and pathogens. We evaluated the potential application of epithelial cells derived from human saliva in investigating host responses to microbes. The salivary epithelial cells (SEC) derived from chronic periodontitis saliva exhibited distinct TLR-2 and TLR-4 expression profile and responded differentially to protein peptide derivative and lipopolysaccharides, ligand for TLR-2 and TLR-4 respectively. Interestingly, the ability of SEC to respond was not restricted to stimulation with oral microbes. Co-culture of SEC with Staphylococcus aureus biofilm, a transient cutaneous pathogen upregulated TLR-2 expression and induced increased secretion of inflammatory cytokines IL-6, IL-8, IL-12 and IFN-γ as compared to SEC exposed to a Staphylococcus epidermidis, a skin commensal. Collectively, it is postulated that the SEC is an efficient model to investigate the epithelial:microbe interphase and may serve as personalized specimen for evaluating individual response to biofilm.

OP061: Adipose tissue-derived mesenchymal stem cells for regeneration of irradiation-induced salivary gland damage

Cell-based therapy has been reported to repair or restore damaged salivary gland (SG) tissues after irradiation. This study was aimed at determining whether systemic administration of human adipose-derived mesenchymal stem cells (hAdMSCs) could ameliorate radiation-induced SG damage following irradiation. External irradiation at a dose of 15 Gy was administered to neck fields of C3H mice. Mice were administered with hAdMSCs through the tail vein immediately after irradiation, and this infusion was repeated once a week for three consecutive weeks. The administration of hAdMSCs had improved SFR at 12 weeks after irradiation. hAdMSC-transplanted SGs showed fewer damaged and atrophied acinar cells compared with the irradiated untreated group. TUNEL and PCNA assays revealed fewer apoptotic cells and increased proliferative indices in the hAdMSC group compared with the irradiated group. The injected hAdMSCs were detected in the transplanted SGs 4 weeks after transplantation. Transdifferentiation of hAdMSCs into acinar or ductal cells in vivo was also observed. This study suggests that hAdMSCs have the potential for protection against irradiation-induced cell loss as well as for transdifferentiation into salivary epithelial cells. hAdMSCs could be a candidate for cell-based therapy for restoration of radiation-induced SG damage.

Sjögren's syndrome-like autoimmune sialadenitis in MRL-Faslpr mice is associated with expression of glucocorticoid-induced TNF receptor-related protein (GITR) ligand and 4-1BB ligand

Although costimulatory molecules have been shown to play crucial roles in the immune response, their involvement in the pathogenesis of Sjögren's syndrome is incompletely understood. In this study, we evaluated the relationship between the severity of spontaneous Sjögren's syndrome-like autoimmune sialadenitis in MRL/MpJ-lpr/lpr (MRL-Faslpr) mice and the expression of 6 costimulatory molecules that play important roles in the immune response: CD80, CD86, OX40 ligand (OX40L), 4-1BB ligand (4-1BBL), glucocorticoid-induced TNF receptor-related protein ligand (GITRL), and B cell-activating factor of the tumor necrosis factor family (BAFF). Expression of the costimulatory molecules in the submandibular salivary glands of age-matched autoimmune MRL-Faslpr mice and non-autoimmune MRL/MpJ-+/+(MRL/+) and C3H/HeJ-lpr/lpr (C3H-Faslpr) mice was examined immunohistochemically and scored on a scale of 0 to 3. The severity of sialadenitis was evaluated histologically and scored on a scale of 0 to 3. We found that all of the costimulatory molecules were expressed in duct epithelial cells of salivary glands from MRL-Faslpr mice, whereas immunoreactivity was absent or weak in the MRL/+ mice. The staining intensity for all 6 costimulatory molecules was significantly higher in the MRL-Faslpr than in the MRL/+ mice. Partial correlation analysis was performed to assess the degree of association between costimulatory molecule staining scores and disease scores, which clearly revealed a significant correlation for only GITRL and 4-1BBL. These molecules showed negligible immunoreactivity in the submandibular glands of C3H-Faslpr mice, suggesting that their expression was independent of the Faslpr mutation. In conclusion, the expression of GITRL and 4-1BBL in salivary gland duct epithelial cells is associated with background genes in the MRL strain, but not with the Faslpr mutation itself, and contributes significantly to the pathogenesis of autoimmune sialadenitis in MRL-Faslpr mice. These results suggest that GITRL and 4-1BBL may be effective targets for the development of therapies for Sjögren's syndrome.

Intraglandular transplantation of bone marrow-derived clonal mesenchymal stem cells for amelioration of post-irradiation salivary gland damage

External irradiation in head and neck cancers may induce irreversible hyposalivation and consequent xerostomia, stemming from radiation damage to salivary glands (SGs). As cell-based therapy has been reported to be able to repair or restore damaged SG tissues, we attempted to determine whether bone marrow-derived clonal mesenchymal stem cells (BM-cMSCs) can ameliorate irradiation-induced salivary gland damage via a murine model. External irradiation at a dose of 15Gy was delivered to the neck fields of C57BL/6 mice. We directly administered either homologous mouse BM-cMSCs labeled with PKH26 (treatment group) or PBS (control group) into SGs 24h after irradiation. Salivary flow rate (SFR) and lag time of salivation were measured at 12weeks after transplantation. At 4 and 12weeks post-transplantation, we performed morphological, histological, and immunofluorescent examinations. Transdifferentiation of administered BM-cMSCs into salivary epithelial cells was observed by confocal microscopy. SFR was significantly increased in BM-cMSCs-transplanted mice compared with PBS-injected mice at 12weeks after transplantation. Administration of BM-cMSCs preserved the microscopic morphologies of SGs, with more functional acini in BM-cMSC-transplanted SGs than in PBS-injected SGs. Immunofluorescent staining revealed less apoptotic cells and increased microvessel density in BM-cMSC-transplanted SGs compared with PBS-injected SGs. PKH-26 labeled BM-cMSCs were detected in transplanted SGs at 4weeks after transplantation and in vivo transdifferentiation of BM-cMSCs into acinar cells was also observed. This study suggests that BM-cMSCs can ameliorate salivary damage following irradiation and can be used as a source of cell-based therapy for restoration of irradiation-induced salivary hypofunction.

P2X7 receptor activation induces inflammatory responses in salivary gland epithelium

Inflammation of the salivary gland is a well-documented aspect of salivary gland dysfunction that occurs in Sjogren's syndrome (SS), an autoimmune disease, and in γ-radiation-induced injury during treatment of head and neck cancers. Extracellular nucleotides have gained recognition as key modulators of inflammation through activation of cell surface ionotropic and metabotropic receptors, although the contribution of extracellular nucleotides to salivary gland inflammation is not well understood. In vitro studies using submandibular gland (SMG) cell aggregates isolated from wild-type C57BL/6 mice indicate that treatment with ATP or the high affinity P2X7R agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) induces membrane blebbing and enhances caspase activity, responses that were absent in SMG cell aggregates isolated from mice lacking the P2X7R (P2X7R(-/-)). Additional studies with SMG cell aggregates indicate that activation of the P2X7R with ATP or BzATP stimulates the cleavage and release of α-fodrin, a cytoskeletal protein thought to act as an autoantigen in the development of SS. In vivo administration of BzATP to ligated SMG excretory ducts enhances immune cell infiltration into the gland and initiates apoptosis of salivary epithelial cells in wild-type, but not P2X7R(-/-), mice. These findings indicate that activation of the P2X7R contributes to salivary gland inflammation in vivo, suggesting that the P2X7R may represent a novel target for the treatment of salivary gland dysfunction.

Surface Modification of PEOT/PBT Membrane with Silk Fibroin Anchoring and its Potential Application in Artificial Salivary Gland Construct

We reported the preparation of surface modified poly (ethylene oxide terephthalate) - poly (butylene terephthalate) membrane by the method of silk fibroin anchoring, namely SF/(PEOT/PBT). Its surface properties were characterized by contact angles and XPS and the biocompatibility of the composite membrane was further evaluated by human salivary epithelial cells (HSG cells) growth in vitro. Results revealed that SF/(PEOT/PBT) possessed the low water contact angle (48.0±3.0°) and immobilized a great amount of fibroin (fibroin surface coverage: 26.39 wt%), which attributed to the formation of polar groups such as hydrosulfide group, sulfonic group, carboxyl and carbonyl ones in the process of SO2 plasma treatment. HSG cells growth in vitro indicated that the silk fibroin anchoring could significantly enhance the biocompatibility of PEOT/PBT membrane, which suggested the potential application of fibroin anchoring PEOT/PBT for clinical HSG cells transplantation in the artificial salivary gland construct.

The Salivary Gland Epithelial Cell in Sjögren’s Syndrome: What Are the Steps Involved in Wounding or Killing Their Secretory Function?

In their article in this issue of The Journal , Ping, et al used in vitro cultures of epithelial cells from salivary gland tissues of patients with Sjögren’s syndrome (SS), and from control patients lacking inflammatory infiltrates1. The basic process of salivary gland cell death (apoptosis) was similar both in SS salivary epithelial cells and in normal salivary epithelial cells. Thus, the key difference in the salivary gland dysfunction of the patient with SS in vivo appears to be the presence of focal T cell infiltrate in the SS gland that serves as a source of CD40 ligand (CD40L). Although their article is a nice exercise in molecular biology, with the expected molecular gymnastics (electrophoretic mobility shifts, Western blots, and TUNEL assays), the ultimate questions that arise are: What do these results mean for the patient with SS with dry mouth? How do these results influence the future for therapeutic selection of options? The current study by Ping, et al is an extension of their report from 2005 on the role of CD40 and Fas in SS salivary gland cells2. Subtle points in the present study include the observation that inhibition of p38 and nuclear factor-κB (NF-κB) activation does not fully prevent CD95-mediated apoptosis (see their Figure 4). So there must be other pathways that CD40 is activating. Particularly in the regulation of p38 and NF-κB, many different factors influence their canonical and noncanonical activation. Thus, additional factors in situ determine the … Address correspondence to Dr. Fox. E-mail: robertfoxmd{at}mac.com

Aryl Hydrocarbon Receptor-Mediated Induction of EBV Reactivation as a Risk Factor for Sjögren’s Syndrome

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a variety of biological effects by binding to environmental pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). Although numerous animal studies have demonstrated the harmful effects of dioxins, it remains controversial whether dioxins pose a risk to human health. Enhanced lytic replication of EBV is a risk factor for the development of autoimmune diseases and cancers. This study evaluated the possibility that ligand-activated AhR reactivates EBV. EBV reactivation and AhR transactivation were evaluated with luciferase assays. Saliva samples were collected from 19 patients with primary Sjögren's syndrome (SS). Control saliva samples were obtained from 10 healthy individuals and nine patients with severe dry mouth. TCDD enhanced BZLF1 transcription, which mediates the switch from the latent to the lytic form of EBV infection in EBV-positive B cell lines and in a salivary gland epithelial cell line. Moreover, TCDD-induced increases in BZLF1 mRNA and EBV genomic DNA levels were confirmed in the B cell lines. Saliva from SS patients activated the transcription of both CYP1A1 and BZLF1. Additionally, there was a positive correlation between CYP1A1 and BZLF1 promoter activities. AhR ligands elicited the reactivation of EBV in activated B cells and salivary epithelial cells, and these ligands are involved in SS. Our findings reveal novel aspects of the biological effects of dioxin and the AhR-dependent pathogenesis of autoimmune diseases.

P38 Mitogen-activated Protein Kinase and Nuclear Factor-κB Facilitate CD40-mediated Salivary Epithelial Cell Death

Our previous studies indicated that CD40-mediated Fas-dependent apoptosis is important for the glandular destruction of Sjögren's syndrome (SS), although other immune and nonimmune mechanisms are also involved in exocrine dysfunction. We investigated the roles of p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-κB (NF-κB) in salivary epithelial cell death in SS. Expression of p38, phosphorylated p38 (pp38), and IκB-α was examined by Western blotting upon CD40 ligation. Activity of NF-κB induced by anti-CD40 monoclonal antibody (mAb) was examined by electrophoretic mobility shift assay (EMSA) and Western blotting. Expression of Fas was analyzed by flow cytometry and Western blotting with or without the p38-specific inhibitor SB203580 or the NF-κB-specific inhibitor caffeic acid phenethyl ester (CAPE). Induction of apoptosis in salivary epithelial cells was examined by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Expression of phosphorylated p38MAPK and NF-κB was measured by immunohistochemistry. pp38MAPK and NF-κB p65 were predominantly expressed in the ductal and acinar epithelium adjacent to lymphoid infiltrates of SS salivary gland by immunohistochemistry. CD40 ligation strongly enhanced p38MAPK and NF-κB activity by EMSA and Western blotting in cultured salivary epithelial cells. Treatment of cells with anti-CD40 mAb resulted in significantly upregulated Fas expression and induction of Fas-dependent apoptosis. Inhibition of p38MAPK and NF-κB activity by SB203580 and/or CAPE reduced Fas expression and apoptosis in salivary epithelial cells, establishing p38MAPK and NF-κB as proapoptotic factors in this context. CD40 ligation plays an important role in activation of p38MAPK, NF-κB, and Fas molecules to initiate proapoptotic signaling. p38MAPK and NF-κB collaborate in regulation of proapoptotic signaling in CD40-mediated Fas-dependent apoptosis in salivary epithelial cells.

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

The effects of low-level laser therapy on xerostomia (mouth dryness)

Xerostomia is a subjective complaint of mouth/oral dryness, caused by a reduction in normal salivary secretion due to different causes. Even though there are many available treatment modalities to enhance salivary flow, the therapy often remains unsatisfactory. The low-level laser therapy (low-level laser irradiation, photo-biomodulation) has been extensively used as a new, non-invasive approach and advantageous tool for reduction of xerostomia. Therefore, the aim of this study is to give a systematic overview on the effects of low-level laser therapy on xerostomia. A systematic review of published articles in PubMed database was carried out using keywords: "low-level laser therapy", "xerostomia", "mouth dryness". In all published articles, which were considered adequate for this overview, positive effects of low-level laser therapy were reported. Low-level laser therapy could significantly enhance salivary secretion and improve antimicrobial characteristics of secreted saliva (increased level of secretory immunoglobulin A; sIgA). Furthermore, low-level laser therapy could improve salivary flow and regeneration of salivary duct epithelial cells. The current literature suggests that low-level laser therapy can be safely and effectively used as an advanced treatment modality for reduction of xerostomia. Further in vivo, in vitro and clinical studies using different irradiation parameters are suggested to determine the best laser parameters to be used.

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

Differential expression of TLR-2 and TLR-4 in the epithelial cells in oral lichen planus

ObjectiveOral lichen planus (OLP) is a chronic inflammatory condition of the mucosa mediated by a complex signalling network between the keratinocytes and the sub-epithelial lymphocytes. Since OLP occurs in constantly renewing epithelium continuously exposed to commensals, we hypothesised that the epithelial cell microflora interactions may mediate the persistent inflammation. By virtue of their ability to respond to most oral commensal microorganisms, the toll like receptor-2 (TLR-2) and TLR-4 are the most widely investigated receptors in oral diseases. The overall objective of this study was to investigate the role of TLR-2 and TLR-4 in OLP. DesignSystemically healthy OLP and control subjects were recruited after obtaining the institutional review board approval. Expression of TLR-2 and TLR-4 proteins and transcripts in the tissue epithelium and in the epithelial cells isolated from saliva were determined by immunohistochemistry and quantitative real-time polymerase chain reaction respectively. ResultsThe tissue epithelium and the salivary epithelial cells expressed reduced TLR-2 and increased TLR-4 proteins and transcripts in OLP. The salivary epithelial cells from OLP subjects secreted elevated IL-12. However, upon stimulation with bacterial lipopolysaccharide the epithelial cells from OLP exhibited a mixed Th1 (IL-12) and Th2 (IL-4) response. Presence of dexamethasone significantly reduced inflammatory cytokines in the in vitro stimulated cultures of salivary epithelial cells from OLP subjects. ConclusionCollectively, our data support a critical role for the host–microbial interactions in the OLP pathogenesis. The potential use of exfoliated oral epithelial cells in saliva for functional analysis exponentially increases its value as biological specimen for clinical research.

Targeted Expression of GLI1 in the Salivary Glands Results in an Altered Differentiation Program and Hyperplasia

Hedgehog (Hh) signaling is a regulator of salivary gland morphogenesis, but its role in postnatal glands has only recently begun to be addressed. To examine the effects of deregulated Hh signaling in the salivary gland, we expressed the Hh effector protein GLI1, in salivary epithelial cells using both cytokeratin 5 and mouse mammary tumor virus (MMTV) transgenic systems. Ectopic pathway activation resulted in restrained acinar differentiation, formation of cystic lesions, and prominent appearance of ductal structures. Moreover, induced expression of GLI1 aids the formation of hyperplastic lesions, which closely resemble GLI1-induced changes in murine skin and mammary glands, suggesting that GLI1 targets cells with similar characteristics in different tissues. Furthermore, GLI1-expressing salivary epithelial cells are actively dividing, and GLI1-induced lesions are proliferative, an incident accompanied by enhanced expression of the Hh target genes, cyclin D1, and Snail. GLI1-induced salivary lesions regress after transgene withdrawal and become histologically normalized. Taken together, our data reveal the ability of GLI1 to modulate salivary acinar differentiation and to promote proliferation of ductal epithelial cells.

Hydrophobic Oxime Ethers: A Versatile Class of pDNA and siRNA Transfection Lipids

The manipulation of the cationic lipid structures to increase polynucleotide binding and delivery properties, while also minimizing associated cytotoxicity, has been a principal strategy for developing next-generation transfection agents. The polar (DNA binding) and hydrophobic domains of transfection lipids have been extensively studied; however, the linking domain comprising the substructure used to tether the polar and hydrophobic domains has attracted considerably less attention as an optimization variable. Here, we examine the use of an oxime ether as the linking domain. Hydrophobic oxime ethers were readily assembled via click chemistry by oximation of hydrophobic aldehydes using an aminooxy salt. A facile ligation reaction delivered the desired compounds with hydrophobic domain asymmetry. Using the MCF-7 breast cancer, H1792 lung cancer and PAR C10 salivary epithelial cell lines, our findings show that lipoplexes derived from oxime ether lipids transfect in the presence of serum at higher levels than commonly used liposome formulations, based on both luciferase and green fluorescent protein (GFP) assays. Given the biological compatibility of oxime ethers and their ease of formation, this functional group should find significant application as a linking domain in future designs of transfection vectors.

Salivary Gland NK Cells Are Phenotypically and Functionally Unique

Natural killer (NK) cells and CD8+ T cells play vital roles in containing and eliminating systemic cytomegalovirus (CMV). However, CMV has a tropism for the salivary gland acinar epithelial cells and persists in this organ for several weeks after primary infection. Here we characterize a distinct NK cell population that resides in the salivary gland, uncommon to any described to date, expressing both mature and immature NK cell markers. Using RORγt reporter mice and nude mice, we also show that the salivary gland NK cells are not lymphoid tissue inducer NK-like cells and are not thymic derived. During the course of murine cytomegalovirus (MCMV) infection, we found that salivary gland NK cells detect the infection and acquire activation markers, but have limited capacity to produce IFN-γ and degranulate. Salivary gland NK cell effector functions are not regulated by iNKT or Treg cells, which are mostly absent in the salivary gland. Additionally, we demonstrate that peripheral NK cells are not recruited to this organ even after the systemic infection has been controlled. Altogether, these results indicate that viral persistence and latency in the salivary glands may be due in part to the presence of unfit NK cells and the lack of recruitment of peripheral NK cells.

Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation

Treatment for most patients with head and neck cancers includes ionizing radiation. A consequence of this treatment is irreversible damage to salivary glands (SGs), which is accompanied by a loss of fluid-secreting acinar-cells and a considerable decrease of saliva output. While there are currently no adequate conventional treatments for this condition, cell-based therapies are receiving increasing attention to regenerate SGs. In this study, we investigated whether bone marrow-derived cells (BMDCs) can differentiate into salivary epithelial cells and restore SG function in head and neck irradiated mice. BMDCs from male mice were transplanted into the tail-vein of 18 Gy-irradiated female mice. Salivary output was increased in mice that received BMDCs transplantation at week 8 and 24 post-irradiation. At 24 weeks after irradiation (IR), harvested SGs (submandibular and parotid glands) of BMDC-treated mice had greater weights than those of non-treated mice. Histological analysis shows that SGs of treated mice demonstrated an increased level of tissue regenerative activity such as blood vessel formation and cell proliferation, while apoptotic activity was increased in non-transplanted mice. The expression of stem cell markers (Sca-1 or c-kit) was detected in BMDC-treated SGs. Finally, we detected an increased ratio of acinar-cell area and approximately 9% of Y-chromosome-positive (donor-derived) salivary epithelial cells in BMDC-treated mice. We propose here that cell therapy using BMDCs can rescue the functional damage of irradiated SGs by direct differentiation of donor BMDCs into salivary epithelial cells.