Immunomodulation of salivary gland function due to cancer therapy.

Functional salivary glands (SG) are essential for maintaining oral health, and salivary dysfunction is a persistent major clinical challenge. Several cancer therapies also have off-target effects leading to SG dysfunction. Recent advances highlight the role of SG immune populations in homeostasis, dysfunction and gland regeneration. Here, we review what is known about SG immune populations during development and postnatal homeostasis. We summarize recent findings of immune cell involvement in SG dysfunction following cancer treatments such as irradiation (IR) for head and neck cancers, immune transplant leading to graft-versus-host-disease (GVHD) and immune checkpoint inhibitor (ICI) treatment. The role of immune cells in SG in both homeostasis and disease, is an emerging field of research that may provide important clues to organ dysfunction and lead to novel therapeutic targets.

FGFR2 is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation.

Exocrine acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific populations later in development and during acinar differentiation are unknown. Here, we use scRNAseq and conditional deletion of murine FGFRs in vivo to identify essential roles for FGFRs in craniofacial, early SG development and progenitor function during duct homeostasis. Importantly, we also discover that FGFR2 via MAPK signaling is critical for seromucous acinar differentiation and secretory gene expression, while FGFR1 is dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activates the FGFR2-dependent seromucous transcriptional program. Here, we propose a model where MEC-derived FGF7 drives seromucous acinar differentiation, providing a rationale for targeting FGFR2 signaling in regenerative therapies to restore acinar function.

Neurotrophin signaling is a central mechanism of salivary dysfunction after irradiation that disrupts myoepithelial cells.

The mechanisms that prevent regeneration of irradiated (IR) salivary glands remain elusive. Bulk RNAseq of IR versus non-IR human salivary glands showed that neurotrophin signaling is highly disrupted post-radiation. Neurotrophin receptors (NTRs) were significantly upregulated in myoepithelial cells (MECs) post-IR, and single cell RNAseq revealed that MECs pericytes, and duct cells are the main sources of neurotrophin ligands. Using two ex vivo models, we show that nerve growth factor (NGF) induces expression of MEC genes during development, and upregulation of NTRs in adult MECs is associated with stress-induced plasticity and morphological abnormalities in IR human glands. As MECs are epithelial progenitors after gland damage and are required for proper acinar cell contraction and secretion, we propose that MEC-specific upregulation of NTRs post-IR disrupts MEC differentiation and potentially impedes the ability of the gland to regenerate.

FGFR2b is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation.

Exocrine secretory acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific epithelial SG populations later in development and during acinar differentiation are unknown. Here, we predicted FGFR dependence in specific populations using scRNAseq data and conditional mouse models to delete FGFRs in vivo. We identifed essential roles for FGFRs in craniofacial and early SG development, as well as progenitor function during duct homeostasis. Importantly, we discovered that FGFR2b was critical for seromucous and serous acinar cell differentiation and secretory gene expression (Bpifa2 and Lpo) via MAPK signaling, while FGFR1b was dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activated the FGFR2b-dependent seromucous transcriptional program. We propose a model where MEC-derived FGF7 drives seromucous acinar differentiaton, providing a rationale for targeting FGFR2b signaling in regenerative therapies to restore acinar function.

GZMK+CD8+ T cells Target a Specific Acinar Cell Type in Sjögren's Disease.

Sjögren's Disease (SjD) is a systemic autoimmune disease without a clear etiology or effective therapy. Utilizing unbiased single-cell and spatial transcriptomics to analyze human minor salivary glands in health and disease we developed a comprehensive understanding of the cellular landscape of healthy salivary glands and how that landscape changes in SjD patients. We identified novel seromucous acinar cell types and identified a population of PRR4+CST3+WFDC2- seromucous acinar cells that are particularly targeted in SjD. Notably, GZMK+CD8 T cells, enriched in SjD, exhibited a cytotoxic phenotype and were physically associated with immune-engaged epithelial cells in disease. These findings shed light on the immune response's impact on transitioning acinar cells with high levels of secretion and explain the loss of this specific cell population in SjD. This study explores the complex interplay of varied cell types in the salivary glands and their role in the pathology of Sjögren's Disease.

A mesenchymal to epithelial switch in Fgf10 expression specifies an evolutionary-conserved population of ionocytes in salivary glands.

Fibroblast growth factor 10 (FGF10) is well established as a mesenchyme-derived growth factor and a critical regulator of fetal organ development in mice and humans. Using a single-cell RNA sequencing (RNA-seq) atlas of salivary gland (SG) and a tamoxifen inducible Fgf10CreERT2:R26-tdTomato mouse, we show that FGF10pos cells are exclusively mesenchymal until postnatal day 5 (P5) but, after P7, there is a switch in expression and only epithelial FGF10pos cells are observed after P15. Further RNA-seq analysis of sorted mesenchymal and epithelial FGF10pos cells shows that the epithelial FGF10pos population express the hallmarks of ancient ionocyte signature Forkhead box i1 and 2 (Foxi1, Foxi2), Achaete-scute homolog 3 (Ascl3), and the cystic fibrosis transmembrane conductance regulator (Cftr). We propose that epithelial FGF10pos cells are specialized SG ionocytes located in ducts and important for the ionic modification of saliva. In addition, they maintain FGF10-dependent gland homeostasis via communication with FGFR2bpos ductal and myoepithelial cells.

Salivary gland function, development, and regeneration.

Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury, will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular, and genetic levels: the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis, and regeneration, and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.

Salivary ZG16B expression loss follows exocrine gland dysfunction related to oral chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) targets include the oral mucosa and salivary glands after allogeneic hematopoietic stem cell transplant (HSCT). Without incisional biopsy, no diagnostic test exists to confirm oral cGVHD. Consequently, therapy is often withheld until severe manifestations develop. This proteomic study examined saliva and human salivary gland for a biomarker profile at first onset of oral cGVHD prior to initiation of topical steroid therapy. Whole saliva collected at onset of biopsy-proven oral GVHD was assessed using liquid chromatography-coupled tandem mass spectrometry with identification of 569 proteins, of which 77 significantly changed in abundance. ZG16B, a secretory lectin protein, was reduced 2-fold in oral cGVHD saliva (p <0.05), and significantly decreased in salivary gland secretory cells affected by cGVHD. Single-cell RNA-seq analysis of healthy MSG localized ZG16B expression to two discrete acinar cell populations. Reduced ZG16B expression may indicate specific cGVHD activity and possibly general salivary gland dysfunction.

Generation of a Single-Cell RNAseq Atlas of Murine Salivary Gland Development.

Understanding the dynamic transcriptional landscape throughout organ development will provide a template for regenerative therapies. Here, we generated a single-cell RNA sequencing atlas of murine submandibular glands identifying transcriptional profiles that revealed cellular heterogeneity during landmark developmental events: end bud formation, branching morphogenesis, cytodifferentiation, maturation, and homeostasis. Trajectory inference analysis suggests plasticity among acinar and duct populations. We identify transcription factors correlated with acinar differentiation including Spdef, Etv1, and Xbp1, and loss of Ybx1, Eno1, Sox11, and Atf4. Furthermore, we characterize two intercalated duct populations defined by either Gfra3 and Kit, or Gstt1. This atlas can be used to investigate specific cell functions and comparative studies predicting common mechanisms involved in development of branching organs.

Stress or injury induces cellular plasticity in salivary gland acinar cells.

Salivary gland function is severely disrupted by radiation therapy used to treat patients diagnosed with head and neck cancer and by Sjögren's syndrome. The resulting condition, which results in xerostomia or dry mouth, is due to irreversible loss of the secretory acinar cells within the major salivary glands. There are presently no treatments for the resolution of xerostomia. Cell-based approaches could be employed to repopulate acinar cells in the salivary gland but investigations into potential therapeutic strategies are limited by the challenges of maintaining and expanding acinar cells in vitro. We investigate the encapsulation of salivary gland cell aggregates within PEG hydrogels as a means of culturing secretory acinar cells. Lineage tracing was used to monitor the fate of acinar cells isolated from murine submandibular gland (SMG). Upon initial formation in vitro, SMG aggregates comprise both acinar and duct cells, with the majority cells of acinar origin. With longer culture times, acinar cells significantly decreased the expression of specific markers and activated the expression of keratins normally found in duct cells. A similar acinar-to-duct cell transition was also observed in vivo, following duct ligation injury. These results indicate that under conditions of stress (mechanical and enzymatic isolation from glands) or injury (duct ligation), salivary gland acinar cells exhibit plasticity to adopt a duct cell phenotype.

Concise Review: A Critical Evaluation of Criteria Used to Define Salivary Gland Stem Cells.

In the effort to develop cell-based therapies to treat salivary gland dysfunction, many different populations of cells in the adult salivary glands have been proposed as stem cells. These cell populations vary, depending on the assay used, and are often nonoverlapping, leading to the conclusion that salivary glands harbor multiple stem cells. The goal of this review is to critically appraise the assays and properties used to identify stem cells in the adult salivary gland, and to consider the caveats of each. Re-evaluation of the defining criteria may help to reconcile the many potential stem cell populations described in the salivary gland, in order to increase comparability between studies and build consensus in the field. Stem Cells 2019;37:1144-1150.

Limited Regeneration of Adult Salivary Glands after Severe Injury Involves Cellular Plasticity.

In the adult salivary glands, the origin of replacement and regenerated acinar cells remains unclear. Although many reports describe the identification of stem cells in adult salivary glands, we have shown that differentiated acinar cells can be maintained and regenerated through self-duplication. Here, we have used genetic mouse models to further investigate acinar cell replacement and regeneration during homeostasis and after injury. Under normal conditions or after duct ligation, replacement of duct and acinar cells occurs through lineage-restricted progenitors. In contrast, after irradiation, in vivo lineage tracing shows that acinar, as well as duct, cells contribute to acinar cell regeneration, revealing that cellular plasticity is involved in salivary gland repair. Our results also indicate that even after radiation damage, several cell populations have regenerative potential for restoring salivary gland function.

Cell-Specific Cre Strains For Genetic Manipulation in Salivary Glands.

The secretory acinar cells of the salivary gland are essential for saliva secretion, but are also the cell type preferentially lost following radiation treatment for head and neck cancer. The source of replacement acinar cells is currently a matter of debate. There is evidence for the presence of adult stem cells located within specific ductal regions of the salivary glands, but our laboratory recently demonstrated that differentiated acinar cells are maintained without significant stem cell contribution. To enable further investigation of salivary gland cell lineages and their origins, we generated three cell-specific Cre driver mouse strains. For genetic manipulation in acinar cells, an inducible Cre recombinase (Cre-ER) was targeted to the prolactin-induced protein (Pip) gene locus. Targeting of the Dcpp1 gene, encoding demilune cell and parotid protein, labels intercalated duct cells, a putative site of salivary gland stem cells, and serous demilune cells of the sublingual gland. Duct cell-specific Cre expression was attempted by targeting the inducible Cre to the Tcfcp2l1 gene locus. Using the R26Tomato Red reporter mouse, we demonstrate that these strains direct inducible, cell-specific expression. Genetic tracing of acinar cells using PipGCE supports the recent finding that differentiated acinar cells clonally expand. Moreover, tracing of intercalated duct cells expressing DcppGCE confirms evidence of duct cell proliferation, but further analysis is required to establish that renewal of secretory acinar cells is dependent on stem cells within these ducts.

Salivary gland homeostasis is maintained through acinar cell self-duplication.

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

Calcium signaling and cell volume regulation are altered in Sjögren's Syndrome.

OBJECTIVE: Sjögren's Syndrome (SS) is a chronic autoimmune disease, leading to deficient secretion from salivary and lacrimal glands. Saliva production is normally increased by cholinergic innervation, giving rise to intracellular calcium signaling and water transport through water channels (aquaporins, AQPs). The aim of this study was to investigate possible pathophysiological changes in cell volume regulation, AQP expression and localization, and intracellular calcium signaling in glandular cells from SS patients compared to controls. MATERIALS AND METHODS: A total of 35 SS patients and 41 non-SS controls were included. Real time qPCR was combined with immunohistochemistry to analyze the mRNA expression and cellular distribution of AQP1, 3 and 5. Cell volume regulation and intracellular calcium signaling were examined in fresh acinar cells. RESULTS: We show for the first time a reduced mRNA expression of AQP1 and 5 in SS compared to controls, accompanied by a decrease in staining intensity of AQP1, 3 and 5 in areas adjacent to local lymphocytic infiltration. Furthermore, we observed that the SS cells' capacity for volume regulation was abnormal. Similarly, the calcium response after parasympathetic agonist (carbachol) stimulation was markedly decreased in SS cells. CONCLUSIONS: It is concluded that mRNA expression of AQP1 and 5, protein distribution of AQP1, 3 and 5, glandular cell volume regulation and intracellular calcium signaling are all altered in SS, pointing to possible pathophysiological mechanisms in SS.

Aquaporins in the adult mouse submandibular and sublingual salivary glands.

Aquaporins (AQPs) is a family of membrane bound water channels found in most tissues. In addition to contribute to transepithelial water movement, AQPs are shown to be involved in a variety of processes such as proliferation, cell migration, and apoptosis. In salivary glands, it is well known that AQP5 plays an important role in fluid secretion. In recent years, several AQPs that demonstrate specific expression trends during development have been found in the mouse submandibular gland (SMG). In this study, we wanted to further investigate the presence and localization of the AQP family in the adult mouse SMG in addition to the less studied sublingual gland. Real time PCR and Western blot demonstrated the presence of AQP3, 4, 8, 9, and 11 transcripts and proteins. AQP1 and AQP7 were shown to be localized in endothelial cells, while AQP4 was found in the satellite cells of the parasympathetic ganglia in both glands. The result from this study shows that AQPs are found in defined subpopulations of cells in salivary glands, providing novel insights to their specific roles in salivary glands.

Aquaporin 5 distribution pattern during development of the mouse sublingual salivary gland.

Aquaporin 5 (AQP5) is important in salivary fluid secretion, and has been found in acinar cells of salivary glands in several species. Recently, studies have shown the AQP5 transcript and protein expression patterns as well as the temporal-spatial protein distribution during development of the mouse submandibular salivary gland. The AQP5 distribution pattern of the closely located sublingual gland (SLG) is, however, not well known. Thus, in this study, the Aqp5 RNA expression pattern and the temporal-spatial distribution of AQP5 protein in prenatal development and in adult mouse SLG was investigated. SLGs from embryonic day 14.5 (E14.5) to 18.5 and postnatal days 0 (P0), 25, and 60 were examined using real time PCR and immunohistochemistry. The Aqp5 transcript was detected from E13.5 and was found to increase towards birth and in young adults. The protein was first detected in a scattered pattern in the canalicular stage and became more organized in the luminal membrane of the acinar cells towards birth. During all postnatal developmental stages studied, AQP5 was localized in the luminal and lateral membrane of acinar cells. AQP5 was also detected in the intercalated duct and additional apical membrane staining in the entire intralobular duct was found in the terminal bud stage. These results indicate that AQP5 plays a role during embryonic salivary gland development.

Localization of AQP5 during development of the mouse submandibular salivary gland.

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5-18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.

Intracellular Ca2+ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line.

The water channel aquaporin 5 (AQP5) seems to play a key role in salivary fluid secretion and appears to be critical in the cell volume regulation of acinar cells. Recently, the cation channel transient potential vanilloid receptor 4 (TRPV4) was shown to be functionally connected to AQP5 and also to cell volume regulation in salivary glands. We used the Simian virus 40 (SV40) immortalized cell line SMG C10 from the rat submandibular salivary gland to investigate the effect of ATP and the neurotransmitter analogue carbachol on Ca(2+) signalling and cell volume regulation, as well as the involvement of TRPV4 in the responses. We used fura-2-AM imaging, cell volume measurements, and western blotting. Both carbachol and ATP increased the concentration of intracellular Ca(2+), but no volume changes could be measured. Inhibition of TRPV4 with ruthenium red impaired both ATP- and carbachol-stimulated Ca(2+) signals. Peak Ca(2+) signalling during hyposmotic exposure was significantly decreased following inhibition of TRPV4, while the cells' ability to volume regulate appeared to be unaffected. These results show that in the SMG C10 cells, simulation of nervous stimulation did not induce cell swelling, although the cells had intact volume regulatory mechanisms. Furthermore, even though Ca(2+) signals were not needed for this volume regulation, TRPV4 seems to play a role during ATP and carbachol stimulation.

Subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 assessed by immunohistochemistry.

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.