Mouse Salivary Glands Research Articles

Ontogenetic development of the water channel protein AQP5 in mouse salivary gland tissue.

Aquaporins (AQP) are a family of channel proteins expressed in the cell membranes of many tissue types. As water channels, they enable the selective permeation of water molecules and thus play an important role in water transport through the plasma membrane. There are numerous AQP sub-types, among which AQP5 is expressed in the salivary glands. The expression and localization of AQP5 in different salivary gland cells of animal models during fetal development and after birth have enabled the physiological functions of AQP5 to be elucidated, but subsequent changes in the adult phase are unknown. It is known that saliva production tends to decrease with age, but it is unclear how AQP5 activity and function changes developmentally, from young to old including gender differences. In the present study, we sampled the parotid, submandibular, and sublingual glands from young (8weeks old) and aged (12months old) mice of both sexes to study the effects of age- and sex-related differences in AQP5 expression. Positive fluorescence immunostaining was detected in the membranes of cells from all gland types, and this was enhanced in juvenile mice from both sexes. Western blot analyses revealed that AQP5 expression levels tended to decrease with age in both male and female animals. Conversely, AQP5 gene expression levels did not change significantly with aging, but were found to be high in submandibular gland cells of both sexes, in parotid gland cells of older female mice, and in the sublingual gland cells of young male mice.

Immune infiltration analysis reveals immune cell signatures in salivary gland tissue of primary Sjögren's syndrome.

Mouse models are the basis for primary Sjögren's syndrome (pSS) research. However, the depth of comparisons between mice and humans in salivary gland (SG) immune cells remains limited. The gene expression profiles of SGs from normal subjects and pSS patients were downloaded from the Gene Expression Comprehensive Database. The proportion of infiltrating immune cell subsets was then assessed by cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT). An experimental Sjögren's syndrome (ESS) mouse model was successfully constructed using SG protein. Based on mouse SG tissue RNA-Seq data, the seq-ImmuCC model was used to quantitatively analyze the compositional ratios of 10 immune cells in pSS patients and mouse model SG tissues. Computed and obtained 31 human data samples using the CIBERSORT deconvolution method. The immune cell infiltration results showed that, compared to normal human SG tissue, the content of gamma delta T cells was significantly different from naive CD4+ T cells and significantly increased, while the plasma cell content decreased. Principal component analysis indicated differences in immune cell infiltration between pSS patients and normal subjects. Meanwhile, for ESS model mouse data analysis, we found that the proportion of macrophages increased, while the proportion of CD4+ T cells, B cells, and monocytes decreased. Furthermore, we found that the proportion of monocytes was decreased, while the proportion of macrophages was increased in the SG tissues of pSS patients and model mice. The infiltration of CD4+ T, CD8+ T, and B cells also showed some differences. We comprehensively analyzed SG immune infiltration in pSS patients and model mice. We demonstrated conserved and nonconserved aspects of the immune system in mice and humans at the level of immune cells to help explain the primary regulation of immune mechanisms during the development of Sjögren's syndrome.

A cellular hierarchy of Notch and Kras signaling controls cell fate specification in the developing mouse salivary gland.

The development of the mouse salivary gland involves a tip-driven process of branching morphogenesis that takes place in concert with differentiation into acinar, myoepithelial, and ductal (basal and luminal) sub-lineages. By combining clonal lineage tracing with a three-dimensional (3D) reconstruction of the branched epithelial network and single-cell RNA-seq analysis, we show that in tips, a heterogeneous population of renewing progenitors transition from a Krt14+ multipotent state to unipotent states via two transcriptionally distinct bipotent states, one restricted to the Krt14+ basal and myoepithelial lineage and the other to the Krt8+ acinar and luminal lineage. Using genetic perturbations, we show how the differential expression of Notch signaling correlates with spatial segregation, exits from multipotency, and promotes the Krt8+ lineage, whereas Kras activation promotes proacinar fate. These findings provide a mechanistic basis for how positional cues within growing tips regulate the process of lineage segregation and ductal patterning.

ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

Sappanone A Aggrandises Ionising Radiation-induced Damage in Oral Epithelial Cells.

To analyse the role played by Sappanone A, a bioactive ingredient isolated from the heartwood of Caesalpinia sappan, in the regulation of oral epithelial cell viability under radiation. Cell viability of human oral keratinocytes (HOKs) and mouse salivary gland cells under ionising radiation was analysed. Expression of Ki67 was measured by immunohistochemical staining. Fragmentation of deoxyribonucleic acid (DNA) was measured by comet assay. Cell death was analysed using trypan blue exclusion assay. Cell viability was measured using a Cell Counting Kit 8 (CCK8; Abcam, Cambridge, UK) assay. Sappanone A decreased cell viability of HOK cells and mouse salivary gland cells under ionising radiation. In addition, Sappanone A enhanced radiation-induced genomic DNA fragmentation, accompanied by impaired homologous recombination and non-homologous end joining DNA repair. Mechanistic evaluation revealed that Sappanone A counteracted radiation-induced inosine monophosphate dehydrogenase 2 (IMPDH2) activation, and that this effect could be abolished by reconstituted expression of a Sappanone A-binding defective IMPDH2 mutant. The present study highlights a novel role played by Sappanone A in the modulation of radiosensitivity of oral epithelial cells.

Genetic Study of Elf5 and Ehf in the Mouse Salivary Gland

Salivary gland (SG) development, maturation, and homeostasis require coordinated roles of transcription factors (TFs) that dictate specific cell identities and fate. The ETS family of proteins are important transcriptional drivers of diverse cell lineages, tissue development, and differentiation programs and hence are also likely to play an important role in the SG. Here we have leveraged genomic and epigenomic data of the SG to examine the expression profile of ETS genes and identified 2 closely related paralogs, Elf5 and Ehf, that are highly expressed in distinct epithelial subpopulations. By using a well-defined mouse knockout model of Elf5, we show that Elf5, despite its enriched expression in the acinar cells, is functionally dispensable for maintaining the homeostatic state of the adult SG epithelium. The lack of a discernible phenotype of the Elf5-null SG might be due to possible functional redundancy with Ehf or other ETS factors. To probe this possibility and to examine the specific consequences of Ehf loss in the SG, we used CRISPR-Cas9 to generate mice in which the DNA-binding ETS domain of Ehf is disrupted due to an insertion mutation. We demonstrate that the Ehf mutant (EhfMut) mice exhibit a distinct cellular phenotype with decreased granular convoluted tubules that are accompanied by an increased accumulation of the intercalated Sox9-positive ductal cell population. Interestingly, the ductal phenotype of the EhfMut animals is highly pronounced in males, reaffirming the established sexual dimorphism of the SG that exists in rodents. Our results show that unlike Elf5, Ehf plays a nonredundant role in directing ductal cell differentiation of the SG and highlights the phenotypic subtlety in mutant mice of closely related TFs and the importance of careful consideration of cell type–specific studies.

Ultra-High Dose Rate FLASH Effect on Head and Neck Cancer Cells and Normal Salivary Gland in Mice

<h3>Purpose/Objective(s)</h3> Ultra-high dose rate irradiation (FLASH) is a novel therapeutic modality that has been reported to spare tissues compared to conventional dose rate. We aimed to investigate the effect of FLASH on head and neck (H&N) cancer cells and salivary gland dysfunction caused by radiotherapy. <h3>Materials/Methods</h3> H&N cancer cells (HSG and FaDu) were irradiated with 10 MeV electron beam at a dose rate of 132.2Gy/s (FLASH) or 0.1Gy/s (CONV), by a modified clinic linear accelerator. The irradiated dose was confirmed with EBT-XD film, and 3, 7, and 11 Gy were irradiated. We evaluated the cell cycle and the surviving fraction of cells with clonogenic assays. We administered single dose head and neck irradiation to mice with a 6 MeV electron beam at dose rate of 105.6-126 Gy/s (FLASH) or 0.03Gy/s (CONV), using a modified linear accelerator. The irradiated dose was confirmed with EBT3 film, and 9-76 Gy and 10-30 Gy were irradiated for FLASH and CONV, respectively. Parotid and submandibular glands were obtained from mice at 3 and 30 days after irradiation. Immunohistological examination and apoptotic assays were conducted to assess the radiation response. <h3>Results</h3> The proportion of sub G0/G1 was higher in the FLASH group, and the surviving fraction was significantly decreased in FLASH through every dose range. At three days post-irradiation, both 10 Gy and 30 Gy irradiated groups showed a greater number of salivary cells and increased expression of AQP5 in FLASH compared to conventional irradiated mice. As a late toxicity (30 days of irradiation), fibrosis did not show any significant difference, although FLASH group received much higher radiation dose. There was no difference in apoptosis between groups on post 3 days, but FLASH showed significantly less degree of apoptosis on 30 days after irradiation. <h3>Conclusion</h3> This study demonstrates that FLASH irradiation strengthens the therapeutic effect for H&N cancers and attenuates radiation-induced salivary gland damage. Further research is needed on the biological mechanisms involved.

Development of a mouse salivary gland-derived mesenchymal cell line for immunological studies of murine cytomegalovirus.

The salivary glands are a crucial site of replication for human cytomegalovirus (HCMV) and its murine counterpart, murine cytomegalovirus (MCMV). Studies of MCMV often involve the use of BALB/c strain mice, but most in vitro assays are carried out in the NIH 3T3 cell line, which is derived from Swiss Albino mice. This report describes a BALB/c-derived mouse salivary gland cell line immortalized using the SV40 large T antigen. Cells stained positive for PDGFR1 and negative for E-cadherin and PECAM-1, indicating mesenchymal origin. This cell line, which has been named murine salivary gland mesenchymal (mSGM), shows promise as a tool for ex vivo immunological assays due to its MHC haplotype match with the BALB/c mouse strain. In addition, plaque assays using mSGM rather than NIH 3T3 cells are significantly more sensitive for detecting low concentrations of MCMV particles. Finally, it is demonstrated that mSGM cells express all 3 BALB/c MHC class I isotypes and are susceptible to T cell-mediated ex vivo cytotoxicity assays, leading to many possible uses in immunological studies of MCMV.

Isolation, Culture, and Characterization of Primary Salivary Gland Cells.

Primary cells are an essential tool for in vitro studies and are obtained directly from living tissues or organs. They closely mimic the physiological state and maintain in vivo functions for short periods of time under optimal conditions. Isolation and culture of salivary gland (SG) cells are useful to decipher the various mechanisms involved in salivary gland dysfunction. However, unlike some other primary cell cultures, SG cell cultures from patient-derived tissues present several challenges. They are difficult to obtain, culture, expand, and characterize due to their sensitive heterogenous cell population and limited expansion potential. In addition, the majority of saliva-secreting acinar cells fail to maintain a differentiated state ex vivo for long periods, and eventually succumb to an acinar-to-ductal metaplasia, losing their secretory phenotype and functions. Herein, we describe two detailed protocols for primary SG cell isolation, culture, and expansion from human (or mouse) salivary tissues using serum-free culture media. We also describe the growth kinetics of these primary cells along with their immunocytochemical characterization. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of SG single-cell culture from freshly obtained human or mouse SG tissues. Basic Protocol 2: Preparation of SG explant culture from freshly obtained human or mouse SG tissues.

Reversely immortalized mouse salivary gland cells presented a promising metabolic and fibrotic response upon BMP9/Gdf2 stimulation

The submandibular gland (SMG) and the sublingual gland (SLG) are two of the three major salivary glands in mammals. In mice, they are adjacent to each other and open into the oral cavity, producing saliva to lubricate the mouth and aid in food digestion. Though salivary gland dysfunction accompanied with fibrosis and metabolic disturbance is common in clinic, in-depth mechanistic research is lacking. Currently, research on how to rescue salivary function is challenging, as it must resort to using terminally differentiated acinar cells or precursor acinar cells with unknown differentiation. In this study, we established reversely immortalized mouse primary SMG cells (iSMGCs) and SLG cells (iSLGCs) on the first postnatal day (P0). The iSMGCs and iSLGCs grew well, exhibited many salivary gland characteristics, and retained the metabolism-related genes derived from the original tissue as demonstrated using transcriptome sequencing (RNA-seq) analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of these two cell lines, which overlapped with those of the SMG and SLG, were enriched in cysteine and methionine metabolism. Furthermore, we investigated the role of bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2(Gdf2), on metabolic and fibrotic functions in the SMG and SLG. We demonstrated that iSMGCs and iSLGCs presented promising adipogenic and fibrotic responses upon BMP9/Gdf2 stimulation. Thus, our findings indicate that iSMGCs and iSLGCs faithfully reproduce characteristics of SMG and SLG cells and present a promising prospect for use in future study of salivary gland metabolism and fibrosis upon BMP9/Gdf2 stimulation.

PO-1822 Ultra-high dose rate FLASH effect on head and neck cancer cells and normal salivary gland in mice

PO-1822 Ultra-high dose rate FLASH effect on head and neck cancer cells and normal salivary gland in mice

O-48 - 177Lu-labeled Lys-urea-Aad-based PSMA-targeting radiotherapeutic agents with low uptake in mouse kidneys and salivary glands

O-48 - 177Lu-labeled Lys-urea-Aad-based PSMA-targeting radiotherapeutic agents with low uptake in mouse kidneys and salivary glands

Cytotoxic granzyme C-expressing ILC1s contribute to antitumor immunity and neonatal autoimmunity.

Innate lymphocytes are integral components of the cellular immune system that can coordinate host defense against a multitude of challenges and trigger immunopathology when dysregulated. Natural killer (NK) cells and innate lymphoid cells (ILCs) are innate immune effectors postulated to functionally mirror conventional cytotoxic T lymphocytes and helper T cells, respectively. Here, we showed that the cytolytic molecule granzyme C was expressed in cells with the phenotype of type 1 ILCs (ILC1s) in mouse liver and salivary gland. Cell fate-mapping and transfer studies revealed that granzyme C-expressing innate lymphocytes could be derived from ILC progenitors and did not interconvert with NK cells, ILC2s, or ILC3s. Granzyme C defined a maturation state of ILC1s. These granzyme C-expressing ILC1s required the transcription factors T-bet and, to a lesser extent, Eomes and support from transforming growth factor-β (TGF-β) signaling for their maintenance in the salivary gland. In a transgenic mouse breast cancer model, depleting ILC1s caused accelerated tumor growth. ILC1s gained granzyme C expression following interleukin-15 (IL-15) stimulation, which enabled perforin-mediated cytotoxicity. Constitutive activation of STAT5, a transcription factor regulated by IL-15, in granzyme C-expressing ILC1s triggered lethal perforin-dependent autoimmunity in neonatal mice. Thus, granzyme C marks a cytotoxic effector state of ILC1s, broadening their function beyond "helper-like" lymphocytes.

An elemental diet protects mouse salivary glands from 5-fluorouracil-induced atrophy.

An elemental diet (ED) reduces adverse effects of chemotherapy, including oral mucositis, in patients with cancer. However, the detailed mechanism(s) of the healing effects of an ED remains unclear. In the present study, the protective effects of the ED, Elental®, were examined against 5-fluorouracil (5-FU)-induced oral mucositis and salivary gland atrophy in mice. Mucositis was induced in female ICR mice by injection of 5-FU. The mice were orally administered Elental® (ED group) or saline (control group). After treatment, the mice body weight, salivary gland weight and the histological changes in the salivary gland granular duct area were monitored. The mice body weight remained stable in the ED group, but was significantly decreased in the control group. Moreover, the salivary gland weight was higher in the ED group compared with the control group. In addition, the salivary gland granular duct area cells were larger in the ED group compared with the control group. Whole transcriptome analysis and network analysis were conducted to understand the mechanisms of action of Elental® against oral mucositis. Whole transcriptome analysis and Ingenuity Pathways Analysis data suggested that Elental® contributed to the recovery of mitochondrial function in 5-FU-damaged salivary glands. Immunohistochemical analysis of salivary gland tissue demonstrated that the expression of cytochrome c oxidase subunit 4 and epidermal growth factor were higher in the ED group compared with the control group. Next, the rate of apoptosis in the salivary glands was examined using terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assays. The number of TUNEL-positive cells in the salivary glands was lower in the ED group compared with the control group. These findings suggested that Elental® may protect mouse salivary glands from 5-FU-induced atrophic changes, which suggests that ED treatment may improve xerostomia and alleviate oral mucositis in patients with cancer receiving 5-FU-based chemotherapy.

Assessment of MEF2C as a novel myoepithelial marker using normal salivary gland and pleomorphic adenoma: An immunohistochemical study

The myoepithelium of the salivary gland demonstrates smooth muscle properties, and the myoepithelium-rich pleomorphic adenomas (PA) exhibit various cytological guises and frequently produce chondroid tissues. We recently reported that gene expression of myocyte enhancer factor 2 C (MEF2C) was up-regulated in myoepithelial cell lines established from mouse salivary glands. This transcription factor is known to regulate the development of smooth muscle cells and chondrocytes. These findings have led us to postulate that MEF2C may be a new marker of the myoepithelium and thus be of help for exploring myoepithelial participation in PA. The immunoreactivity of MEF2C was semi-quantitatively analyzed in normal salivary glands and in different types of cell in PA. The pattern of staining was also compared with that of α-smooth muscle actin (αSMA) and calponin. In the parenchyma of mouse and human submandibular glands, MEF2C expression was exclusive to the nuclei of myoepithelial cells. In the neoplastic myoepithelium of PA, epithelioid and stellate cells were consistently positive, but spindle and plasmacytoid cells exhibited selective staining. Although variability/loss of expression was evident, αSMA tended to show limited expression, whereas calponin tended to show extensive expression. Intense and uniform immunoreactivity for MEF2C was observed in lacunar cells in chondroid areas of PA. This is the first reported study to have demonstrated MEF2C immunoexpression in normal and neoplastic myoepithelial cells. Among the heterogeneously differentiated elements, MEF2C may be useful to define the myoepithelial/mesenchymal phenotypes of PA cells. Along with other relevant immunohistochemical markers, it may become another standard.

The effect and mechanism of gene Fam20a on the development and function of salivary glands in mice

ObjectiveThe influence of the knockout of gene Fam20a on mice salivary glands was studied in this research, to provide a potential gene therapeutic target for salivary gland dysfunction. DesignThe control group with genotype Fam20af/f and conditional knockout (cKO) group with Fam20af/f;K14-Cre were constructed with Cre-Loxp. The influence of Fam20a on the salivary glands was studied in terms of morphology, functionality and molecular mechanism. ResultsIn terms of morphology, the cross-sectional area ratio of ductal to the total was reduced in the cKO mice, while that of extracellular matrix to the total was increased. At the sub-microscopic level, the knockout of Fam20a led to abnormal sub-microscopic structure of the duct cells. Functionally, saliva flow rate was significantly reduced in cKO mice. The result was consistent with the change of acinar cell marker Aquaporin 5 which was abnormally diffusely expressed in the cytoplasm of acinar cells. Meanwhile, the expression of ductal cell markers Cytokeratin 7 and nerve growth factor β were significantly decreased, suggesting the abnormal development and function of the duct cells. The research on the mechanism reveals that the loss of Fam20a led to the decreased expression and varied localization of bone morphogenetic protein 4 (BMP4), and a significant decrease of the proportion of phosphorylated extracellular signal-regulated protein1/2 (ERK1/2) to total ERK1/2. These changes suggested that the loss of Fam20a attenuated the activity of the BMP/ERK signaling pathway. ConclusionsFam20a affects the morphology and function of salivary glands, probably by attenuating the activity of the BMP/ERK signaling pathway.

Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation.

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial–mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial–mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell–cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.

Morphological Changes in Major Salivary Glands in Mice Treated With a Choline and Methionine Deficient Diet.

It has been shown that the methionine-choline deficient (MCD) diet induces hepatocarcinogenesis, but not in extrahepatic organs, such as the testis, and pancreas, although may increase chemical-induced carcinogenesis in the colon, mammary gland, esophagus, and pancreas. Accumulating evidence suggests that salivary glands are very susceptible to stress conditions, such as radiation, hyperglycemia, and exposure to xenobiotics in vivo. This study aimed to analyze the histological changes on the major salivary glands (parotid, submandibular, and sublingual) after MCD diet administration. Male Swiss mice were submitted to ad libitum access to the control (AIN-76) or MCD diet for 28 days. The rebound group received the MCD diet for 24 days and the control diet for 10 days. Using the AxioImager A2 microscope, the hematoxylin-eosin (HE) stained specimens (4 mm) were evaluated for tissue degeneration, nuclear hyperchromatism and atrophy. In the parotid gland from the MCD group, tissue degeneration, pyknosis, apoptosis and atrophy were observed, which remained in the rebound group, associated with hyperchromatism. In the submandibular gland from both MCD and rebound groups, severe tissue disorganization was associated with cell pleomorphism, hyperchromatic cells, apoptosis, increased eosinophilia, and inflammatory infiltrate. Finally, in the sublingual gland, there were no histological alterations in the experimental groups compared to the control. MCD can induce pre-neoplastic changes in the mouse parotid and submandibular glands, which are not reversed by a change in the diet.

In vivo Ca2+ Imaging in Mouse Salivary Glands.

Changes in intracellular calcium drive exocrine cell activity. In the salivary gland, acetylcholine released from parasympathetic neurons mobilizes endoplasmic reticulum calcium stores in acinar cells, which consequently initiates saliva secretion. However, our understanding of the signaling cascade is mainly based on ex vivo studies performed in enzymatically isolated cells. The dissociation process likely disrupts the extracellular matrix, removes neurons as the source of signal input, and disturbs the integrity of tight and gap junctional acinar connections. These alterations may affect the spatiotemporal properties of calcium signaling events. In vivo observations of calcium signals, where tissue organization is intact, are therefore important to establish the characteristics of physiological calcium signals that are crucial for the stimulation of fluid secretion. Here, we present a detailed protocol for in vivo imaging of calcium signaling events, following nervous stimulation by multi-photon microscopy in mouse salivary gland acinar cells, expressing the genetically encoded calcium indicator GCamp6F.

The role of salivary gland macrophages in infection, disease and repair.

Macrophages are mononuclear innate immune cells which have become of increasing interest in the fields of disease and regeneration, as their non-classical functions have been elucidated in addition to their classical inflammatory functions. Macrophages can regulate tissue remodeling, by both mounting and reducing inflammatory responses; and exhibit direct communication with other cells to drive tissue turnover and cell replacement. Furthermore, macrophages have recently become an attractive therapeutic target to drive tissue regeneration. The major salivary glands are glandular tissues that are exposed to pathogens through their close connection with the oral cavity. Moreover, there are a number of diseases that preferentially destroy the salivary glands, causing irreversible injury, highlighting the need for a regenerative strategy. However, characterization of macrophages in the mouse and human salivary glands is sparse and has been mostly determined from studies in infection or autoimmune pathologies. In this review, we describe the current literature around salivary gland macrophages, and speculate about the niches they inhabit and how their role in development, regeneration and cancer may inform future therapeutic advances.