Salivary Gland Epithelial Cells Research Articles

Hydrogels for Salivary Gland Tissue Engineering.

Mimicking the complex architecture of salivary glands (SGs) outside their native niche is challenging due their multicellular and highly branched organization. However, significant progress has been made to recapitulate the gland structure and function using several in vitro and ex vivo models. Hydrogels are polymers with the potential to retain a large volume of water inside their three-dimensional structure, thus simulating extracellular matrix properties that are essential for the cell and tissue integrity. Hydrogel-based culture of SG cells has seen a tremendous success in terms of developing platforms for cell expansion, building an artificial gland, and for use in transplantation to rescue loss of SG function. Both natural and synthetic hydrogels have been used widely in SG tissue engineering applications owing to their properties that support the proliferation, reorganization, and polarization of SG epithelial cells. While recent improvements in hydrogel properties are essential to establish more sophisticated models, the emphasis should still be made towards supporting factors such as mechanotransduction and associated signaling cues. In this concise review, we discuss considerations of an ideal hydrogel-based biomaterial for SG engineering and their associated signaling pathways. We also discuss the current advances made in natural and synthetic hydrogels for SG tissue engineering applications.

Cell-derived vesicles from adipose-derived mesenchymal stem cells ameliorate irradiation-induced salivary gland cell damage.

IntroductionSalivary gland (SG) damage is commonly caused by aging, irradiation, and some medications, and currently, no damage modifying agent is available. However, cell therapy based on mesenchymal stem cells (MSCs) has been proposed as a therapeutic modality for irradiated SGs. Therefore, we administered cell-derived vesicles (CDVs) of adipose-derived mesenchymal stem cells (ADMSCs) to irradiated SG cells to investigate their radioprotective effects in vitro.MethodsThe artificial CDVs were obtained from ADMSC by tangential flow filtration (TFF) purification and ultracentrifugation. Cultured human SG epithelial cells were exposed to 2, 5 or 15 Gy of 4 MV X-rays produced by a linear accelerator. The effects of ADMSC-CDVs on SG epithelial cells damaged by irradiation were tested by proliferation activity, transepithelial electrical resistance (TEER), and amylase activity.ResultsExposure to penetrating radiation inhibited the proliferation of SG epithelial cells, but the radiation intensity required to reduce the proliferation of human submandibular gland epithelial cells (hSMGECs) was greater than required for other SG cells. ADMSC-CDVs restored the proliferative ability of SG epithelial cells reduced by irradiation, and the proliferation capacities of irradiated human parotid gland epithelial cells (hPGECs) and human sublingual gland epithelial cells (hSLGECs) were increased by administering ADMSC-CDVs to non-irradiated SG epithelial cells. Furthermore, amylase activity in irradiated hPGECs, hSMGECs, and hSLGECs was lower than in non-irradiated controls. However, amylase ability was restored in all by ADMSC-CDV treatment. Also, TEER was diminished by irradiation in hPGECs, hSMGECs, and hSLGECs and restored by ADMSC-CDV administration.ConclusionOverall, our findings demonstrate that ADMSC-CDVs have potent radioprotective effects on irradiated SG cells.

JAK/STAT Pathway Targeting in Primary Sjögren Syndrome.

Primary Sjögren's syndrome (pSS) is an autoimmune systemic disease mainly affecting exocrine glands and resulting in disabling symptoms, as dry eye and dry mouth. Mechanisms underlying pSS pathogenesis are intricate, involving multiplanar and, at the same time, interlinked levels, e.g., genetic predisposition, epigenetic modifications and the dysregulation of both immune system and glandular-resident cellular pathways, mainly salivary gland epithelial cells. Unravelling the biological and molecular complexity of pSS is still a great challenge but much progress has been made in recent years in basic and translational research field, allowing the identification of potential novel targets for therapy development. Despite such promising novelties, however, none therapy has been specifically approved for pSS treatment until now. In recent years, growing evidence has supported the modulation of Janus kinases (JAK) - signal transducers and activators of transcription (STAT) pathways as treatment strategy immune mediated diseases. JAK-STAT pathway plays a crucial role in autoimmunity and systemic inflammation, being involved in signal pathways of many cytokines. This review aims to report the state-of-the-art about the role of JAK-STAT pathway in pSS, with particular focus on available research and clinical data regarding the use of JAK inhibitors in pSS.

Deciphering the role of cDC2s in Sjögren’s syndrome: transcriptomic profile links altered antigen processes with IFN signature and autoimmunity

ObjectiveType 2 conventional dendritic cells (cDC2s) are key orchestrators of inflammatory responses, linking innate and adaptative immunity. Here we explored the regulation of immunological pathways in cDC2s from patients with...

The Expression of Follistatin-like 1 Protein Is Associated with the Activation of the EMT Program in Sjögren's Syndrome.

Background: The activation of the epithelial to mesenchymal transition (EMT) program is a pathological response of the Sjögren’s syndrome (SS) salivary glands epithelial cells (SGEC) to chronic inflammation. Follistatin-like 1 protein (FSTL1) is a secreted glycoprotein induced by transforming growth factor-β1 (TGF-β1), actively involved in the modulation of EMT. However, the role of FSTL1 in the EMT program activation in SS has not yet been investigated. Methods: TGF-β1-stimulated healthy human SGEC, SS SGEC, and SS salivary glands (SGs) biopsies were used to assess the effect of FSTL1 on the activation of the EMT program. FSTL1 gene activity was inhibited by the siRNA gene knockdown technique. Results: Here we reported that FSTL1 is up-regulated in SS SGs tissue in a correlated manner with the inflammatory grade. Blockage of FSTL1 gene expression by siRNA negatively modulates the TGF-β1-induced EMT program in vitro. We discovered that these actions were mediated through the modulation of the SMAD2/3-dependent EMT signaling pathway. Conclusions: Our data suggest that the TGF-β1-FSTL1-SMAD2/3 regulatory circuit plays a key role in the regulation of EMT in SS and targeting FSTL1 may be a strategy for the treatment of SGs EMT-dependent fibrosis.

A TGFβR inhibitor represses keratin-7 expression in 3D cultures of human salivary gland progenitor cells

Salivary gland tissue engineering offers an attractive alternative for the treatment of radiation-induced xerostomia. Key to the success of this approach is the maintenance and expansion of secretory acinar cells in vitro. However, recent studies revealed that in vitro culture of primary salivary gland epithelial cells led to undesirable upregulation of the expression of keratin-7 (K7), a marker of ductal phenotype and frequently associated with cellular stress. We have previously shown that hyaluronic acid (HA)-based, RGDSP-decorated hydrogels support the 3D growth and assembly of primary human salivary gland stem/progenitor cells (hS/PCs). Here, we investigate whether the RGDSP culture also promotes K7 expression, and if so, what factors govern the K7 expression. Compared to hS/PCs maintained in blank HA gels, those grown in RGDSP cultures expressed a significantly higher level of K7. In other tissues, various transforming growth factor-β (TGF-β) superfamily members are reported to regulate K7 expression. Similarly, our immunoblot array and ELISA experiments confirmed the increased expression of TGF-β1 and growth/differentiation factor-15 (GDF-15) in RGDSP cultures. However, 2D model studies show that only TGF-β1 is required to induce K7 expression in hS/PCs. Immunocytochemical analysis of the intracellular effectors of TGF-β signaling, SMAD 2/3, further confirmed the elevated TGF-β signaling in RGDSP cultures. To maximize the regenerative potential of h/SPCs, cultures were treated with a pharmacological inhibitor of TGF-β receptor, A83-01. Our results show that A83-01 treatment can repress K7 expression not only in 3D RGDSP cultures but also under 2D conditions with exogenous TGF-β1. Collectively, we provide a link between TGF-β signaling and K7 expression in hS/PC cultures and demonstrate the effectiveness of TGF-β inhibition to repress K7 expression while maintaining the ability of RGDSP-conjugated HA gels to facilitate the rapid development of amylase expressing spheroids. These findings represent an important step towards regenerating salivary function with a tissue-engineered salivary gland.

Postgenomic Bioinformatic Analysis of Nucleic Acid Sequences Expressed in the Salivary Gland Epithelial Cells of Primary Sjögren's Syndrome Patients in Search of Microorganisms and Endogenous Retroviruses.

Several previous studies from our laboratory have indicated that the salivary gland epithelia of primary Sjögren's syndrome (SS) patients are not only the target of autoimmune immune responses, but also key instigators of the chronic salivary gland inflammatory infiltrates of patients. In particular, the comparative analysis of salivary gland tissue specimens and of in-vitro cultured non-neoplastic salivary gland epithelial cell lines (SGEC, of ductal type) from SS-patients and non-SS disease-controls, have unequivocally highlighted the presence of intrinsic activation in the ductal epithelia of SS-patients and of aberrant expression of inflammagenic molecules thereof, that correlate with the severity of local histopathologic changes, as well as of systemic manifestations of the disease. In the same context, we have recently shown that the ductal epithelia of SS-patients manifest cell-autonomous activation of the AIM2 inflammasome owing to the presence of aberrant cytoplasmic accumulations of damaged DNA. These findings not only provide a mechanistic explanation for the intrinsic activation and inflammatory status of SS ductal epithelia, but may also point towards the putative instigating role of an exogenous or endogenous agent (i.e., a micro-organism or an endogenous retrovirus, respectively). On this basis and to further explore the nature of epithelial cell-intrinsic activation in SS, the present proposal aims to investigate the expression of endogenous retroviral and/or non-human nucleic acid sequences of microbial origin in the ductal salivary gland epithelia of SS-patients, using metagenomic analysis of high throughput DNA and RNA genome sequencing data, which will be obtained from SGEC lines derived from SS-patients and disease-controls.

Regulation of MMP9 transcription by ETS1 in immortalized salivary gland epithelial cells of patients with salivary hypofunction and primary Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) patients exhibit enhanced degradation of the salivary epithelium initially through MMP9 overexpression. We assessed the expression of MMP9 and an associated transcription factor, ETS1, in primary salivary gland epithelial cells (SGECs) and investigated potential regulatory mechanism(s) in immortalized SGECs. SGECs and iSGECs were derived from pSS and/or xerostomic “sicca” patients. siRNA knockdown of ETS1 in iSGECs was performed to determine MMP9 mRNA (qRT-PCR) and protein expression (ELISA). ETS1 binding to MMP9 promoter was assessed by luciferase activity and binding confirmed by mutagenesis and ChIP. Effects of ETS1 overexpression on progenitor and Epithelial-Mesenchymal transition (EMT) associated markers were determined by Western blot. Expression of ETS1 and its phosphorylated form in iSGECs was determined by immunofluorescence microscopy. ETS1 and MMP9 were overexpressed in SGECs of pSS and non-pSS sicca patients with salivary gland lymphocytic infiltration compared to non-pSS sicca patients without infiltration. ETS1 siRNA knockdown reduced both MMP9 mRNA and protein levels. ETS1 overexpression affected the expression of EMT and progenitor cell markers. Lastly, ETS1 bound the MMP9 promoter within the DNA region of −296 bp to −339 bp. ETS1 may impair salivary function through direct transcriptional control of the MMP9 promoter. ETS1 upregulation may also affect other factors involved in repair of the dysfunctional pSS salivary epithelium.

Organ-specific extracellular matrix directs trans-differentiation of mesenchymal stem cells and formation of salivary gland-like organoids in vivo

BackgroundCurrent treatments for salivary gland (SG) hypofunction are palliative and do not address the underlying cause or progression of the disease. SG-derived stem cells have the potential to treat SG hypofunction, but their isolation is challenging, especially when the tissue has been damaged by disease or irradiation for head and neck cancer. In the current study, we test the hypothesis that multipotent bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model are capable of trans-differentiating to the SG epithelial cell lineage when induced by a native SG-specific extracellular matrix (SG-ECM) and thus may be a viable substitute for repairing damaged SGs.MethodsRat BM-MSCs were treated with homogenates of decellularized rat SG-ECM for one hour in cell suspension and then cultured in tissue culture plates for 7 days in growth media. By day 7, the cultures contained cell aggregates and a cell monolayer. The cell aggregates were hand-selected under a dissecting microscope, transferred to a new tissue culture dish, and cultured for an additional 7 days in epithelial cell differentiation media. Cell aggregates and cells isolated from the monolayer were evaluated for expression of SG progenitor and epithelial cell specific markers, cell morphology and ultrastructure, and ability to form SG-like organoids in vivo.ResultsThe results showed that this approach was very effective and guided the trans-differentiation of a subpopulation of CD133-positive BM-MSCs to the SG epithelial cell lineage. These cells expressed amylase, tight junction proteins (Cldn 3 and 10), and markers for SG acinar (Aqp5 and Mist 1) and ductal (Krt 14) cells at both the transcript and protein levels, produced intracellular secretory granules which were morphologically identical to those found in submandibular gland, and formed SG-like organoids when implanted in the renal capsule in vivo.ConclusionsThe results of this study suggest the feasibility of using autologous BM-MSCs as an abundant source of stem cells for treating SG hypofunction and restoring the production of saliva in these patients.

Quercetin ameliorates salivary gland apoptosis and inflammation in primary Sjögren's syndrome through regulation of the leptin/OB-R signaling.

Dry mouth is the main manifestation of Sjögren syndrome (SS). Quercetin has been reported to alleviate radiation-induced salivary gland damage, yet the effect of quercetin on SS-caused salivary gland damage remains unclear. This study aimed to investigate the effects of quercetin on SS-induced salivary gland damage and the mechanism underlying its therapeutic potential in SS. Here, NOD/Ltj mice were used to spontaneously mimic SS-induced salivary gland inflammation in vivo and salivary gland epithelial cells (SGECs) were stimulated by interferon-γ (IFN-γ) to mimic cell inflammation in vitro. Results showed that quercetin significantly reduced loss of saliva flow, salivary gland damage, cell apoptosis, and inflammatory response in NOD/Ltj mice. Quercetin treatment also significantly reduced the increased serum leptin (LP) levels in NOD/Ltj mice. Furthermore, quercetin blocked the increases in the expression of obesity receptor (OB-R) and its downstream Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling in the salivary glands. In vitro experiments confirmed that quercetin could protect SGECs from IFN-γ-induced cell apoptosis and inflammation through the LP/OB-R-activated JAK2/STAT3 signaling. Hence, quercetin might protect against SS-induced salivary gland damage by relieving cell apoptosis and inflammation by inhibiting the LP/OB-R signaling, providing a new perspective for treating SS-induced dry mouth.

Interleukin-13 promotes cellular senescence through inducing mitochondrial dysfunction in IgG4-related sialadenitis

Immunoglobulin G4-related sialadenitis (IgG4-RS) is an immune-mediated fibro-inflammatory disease and the pathogenesis is still not fully understood. The aim of this study was to explore the role and mechanism of interleukin-13 (IL-13) in the cellular senescence during the progress of IgG4-RS. We found that the expression of IL-13 and IL-13 receptor α1 (IL-13Rα1) as well as the number of senescent cells were significantly higher in the submandibular glands (SMGs) of IgG4-RS patients. IL-13 directly induced senescence as shown by the elevated activity of senescence-associated β-galactosidase (SA-β-gal), the decreased cell proliferation, and the upregulation of senescence markers (p53 and p16) and senescence-associated secretory phenotype (SASP) factors (IL-1β and IL-6) in SMG-C6 cells. Mechanistically, IL-13 increased the level of phosphorylated signal transducer and activator of transcription 6 (p-STAT6) and mitochondrial-reactive oxygen species (mtROS), while decreased the mitochondrial membrane potential, ATP level, and the expression and activity of superoxide dismutase 2 (SOD2). Notably, the IL-13-induced cellular senescence and mitochondrial dysfunction could be inhibited by pretreatment with either STAT6 inhibitor AS1517499 or mitochondria-targeted ROS scavenger MitoTEMPO. Moreover, IL-13 increased the interaction between p-STAT6 and cAMP-response element binding protein (CREB)-binding protein (CBP) and decreased the transcriptional activity of CREB on SOD2. Taken together, our findings revealed a critical role of IL-13 in the induction of salivary gland epithelial cell senescence through the elevated mitochondrial oxidative stress in a STAT6–CREB–SOD2-dependent pathway in IgG4-RS.

The IL-33/ST2 Axis Promotes Primary Sjögren's Syndrome by Enhancing Salivary Epithelial Cell Activation and Type 1 Immune Response.

The molecular mechanisms of primary Sjögren's syndrome (pSS) are poorly understood. In this study, we explored the role of the IL-33/ST2 axis in the development of pSS. In the mouse model of experimental Sjögren's syndrome, we found that the saliva flow rate at weeks 4 and 30 was preserved in IL-33-/- and ST2-/- mice, compared with that of wild-type mice. At week 30 of experimental Sjögren's syndrome induction, the histological score, anti-nuclear Ab levels, and numbers of Th1 and B cells in draining lymph nodes of the salivary gland were lower in the IL-33-/- and ST2-/- mice, whereas Th17 cells and regulatory T cells were not changed. Primary salivary gland epithelial cells expressed the IL-33 receptor ST2. After stimulation with rIL-33, salivary gland epithelial cells increased the transcriptional levels of CD86 and CCL2, accompanied by the activation of the NF-κB inflammatory pathway. There was a synergistic effect between rIL-33 and rIL-12 in augmenting the production of IFN-γ in CD4+ T cells. In the pSS patients, the expression of IL-33 was elevated in the labial salivary gland, with the number of IL-33+ cells positively correlated with the score of the EULAR (European Alliance of Associations for Rheumatology) Sjögren's syndrome disease activity index (ESSDAI). ST2 was highly expressed in the cytoplasm of ductal epithelial cells, with low levels of expression in lymphatic infiltration sites. Our data suggest that the IL-33/ST2 axis may promote the development of pSS by enhancing salivary epithelial cell activation and the type 1 immune response.

Regulation of myoepithelial differentiation.

The salivary gland can be permanently impaired by radiation treatment for head and neck cancers. Efforts at tissue regeneration have focused on saliva-producing acinar cells. However, myoepithelial cells are also critical to gland function, but mechanisms that regulate their differentiation are poorly defined. To study myoepithelial differentiation, we employed mSG-PAC1 murine salivary gland epithelial cells. We demonstrate that mSG-PAC1 spheroids exhibit phenotypic plasticity between pro-acinar and myoepithelial cell fates. Increased expression of pro-acinar/acinar or myoepithelial RNAs was identified from spheroids cultured under different media conditions by microarray followed by gene-set enrichment analysis. Spheroids cultured with different medium components expressed proteins typical of either acinar or myoepithelial cells, as detected by immunocytochemistry. We demonstrate that the pattern of TAZ expression in the epithelial compartment of the differentiating murine salivary gland correlates with the expression of the myoepithelial marker alpha-SMA, as is the case for TAZ expression in mSG-PAC1 spheroids. Our analysis also indicates that YAP/TAZ target genes are upregulated together with myoepithelial markers. Importantly, siRNA targeting of TAZ expression in mSG-PAC1 spheroids diminished the expression of myoepithelial markers. Our results in this in vitro cell model implicate TAZ signaling in myoepithelial differentiation.

POS0455 TOFACITINIB DECREASES INFLAMMATORY MARKERS AND MITOCHONDRIAL MORPHOLOGICAL DAMAGE IN SALIVARY GLANDS OF A MURINE MODEL OF SJÖGREN’S SYNDROME

BackgroundAltered homeostasis of salivary gland (SG) epithelial cells in Sjögren’s syndrome (SS) patients could be the initiating factor that leads to inflammation, as well as secretory dysfunction. Mitochondria are important organelles involved in cellular metabolism and their dysfunction can induce a loss of homeostasis and inflammation. Altered mitochondrion can release mitochondrial components that can act as damage-associated molecular patterns (DAMPs) and induce an inflammatory response via pattern recognition receptors (PRRs) such as the NLRP3 inflammasome, TLR9, cGAS/STING, and ZBP1 (1). Previously we determined that SG from SS patients showed and altered autophagy, which is associated to an increased pro-inflammatory cytokines expression. Interestingly, increased expression of pro-inflammatory markers such as IL-6, was reversed by JAK inhibitor tofacitinib in three-dimensional (3D)-acini deficient in autophagy (2). It is not clear whether the alterations in autophagy found in SG patients include alterations in mitochondrial clearance (mitophagy) that may lead to the accumulation of damaged mitochondria and enhanced inflammation. In this context, recent results of our laboratory showed, for the first time, severe ultrastructural alterations of mitochondria in SG cells from SS patients (1). However, it remains to be determined if these alterations are related to inflammation and if an anti-inflammatory agent could regulate these processes.ObjectivesTo analyze the effect of tofacitinib on the mitochondrial ultrastructure in submandibular glands of a murine model of SS. In addition, to evaluate the effect of tofacitinib on the expression and activation of some PRRs involved in the recognition of mitochondrial DAMPs in the same murine model.MethodsSix-month-old female NOD.B10Sn-H2b/J mice (Jackson Laboratories, USA) were used with 4-5 mice per group. Procedures were approved by the Universidad de Chile Animal Care and Use Committee. 30 mg/kg/day tofacitinib citrate was administered by oral gavage. After 28 days of tofacitinib or vehicle administration, their submandibular glands were obtained, which were processed to evaluate the mitochondrial ultrastructure by electron microscopy or lysed in RIPA buffer to obtain proteins. The protein levels of PRRs: NLRP3, TLR9, ZBP-1, and cGAs, as well as molecules activated downstream of cGAS and ZBP-1 such as TBK1, pTBK1, pSTING, and STING were determined by Western blotting.ResultsThe results show that the mitochondria of the glandular epithelial cells of NOD.B10Sn-H2b/J mice treated with vehicle (control) present alterations such as swelling, disruption of membranes and crest disorganization that previously were reported in patients with SS (1). Interestingly, tofacitinib treatment improves the architecture of mitochondria. On the other hand, the protein levels of PRRs such as NLRP3 and cGAS decreased in mice treated with tofacitinib, as well as pTBK1.ConclusionThe altered morphology of mitochondria together with the increased protein levels of PRRs and downstream markers of these PRRs suggests release of mitochondrial DAMPs in submandibular glands of NOD.B10Sn-H2b/J mice. The improvement in mitochondrial morphology as well as the decrease in PRRs activation under tofacitinib treatment suggest a potential use of this anti-inflammatory agent in mitochondrial alterations associated with inflammation. Many questions remain to be addressed, such as determining which mitochondrial DAMP might be being released and whether this is associated with impaired mitochondrial function in SS.

Knockdown of lncRNA ENST00000603829 Inhibits the Proliferation and Invasion of Salivary Gland Pleomorphic Adenoma through Regulating Cyclin D1.

Objective Salivary gland pleomorphic adenoma (SPA) is a benign neoplasm that can still recur even after radical surgery. To investigate its underlying pathogenesis, here, we examined the significance of lncRNA ENST00000603829 in the proliferation and invasion of SPA. Methods SPA tissues (n = 30) and adjacent normal tissues (NC; n = 30) were collected from SPA patients treated at our hospital from June 2017 to December 2019. The human normal salivary gland epithelial cell line (HSG) and SPA cells (PA30, PA37, and PA116) were cultured. qRT-PCR was used for detecting the expression of cyclin D1 and lncRNA ENST00000603829 in tissues and cells. lncRNA ENST00000603829/cyclin D1 was knocked down or overexpressed in PA116 cells. The expression of cyclin D1 and lncRNA ENST00000603829 in different cell lines was examined using qRT-PCR. Transwell assays and cell counting kit-8 (CCK-8) were used to assess cellular invasion and proliferation. The testing result regarding the apoptosis rate and cell cycle was obtained via flow cytometry. Western blot provided the measurement of cyclin D1 expression in cells. Results We observed an upregulation of lncRNA ENST00000603829 and cyclin D1 expression in SPA tissues and cells. Knockdown of lncRNA ENST00000603829 inhibited cell invasion and proliferation, promoting apoptosis and retaining the cells during the G0/G1 phase. However, such effects of lncRNA ENST00000603829 knockdown were inhibited when cyclin D1 was overexpressed. Conclusion lncRNA ENST00000603829 can promote the occurrence and development of SPA through increasing cyclin D1 expression.

The aberrant levels of decorin induce damages of human salivary gland epithelial cells and polarization of macrophages.

This study aimed to preliminarily address the levels of decorin (DCN, a critical component of extracellular matrix) and its potential roles in primary Sjögren's syndrome (pSS). DCN levels were determined in the salivary glands of experimental SS (ESS) mice and pSS patients by RNA sequencing, bioinformatics analysis, or immunohistochemical staining. Its correlation with interested genes and co-localization with a putative receptor was studied in pSS patients. In addition, its potential roles on salivary gland epithelium and macrophages were tested by exogenous administration to corresponding cell lines, followed by the evaluation of apoptosis using flow cytometry or cytokine expression using quantitative real-time polymerase chain reaction. Our data revealed a significant elevation of DCN in the salivary glands of the ESS mice model and pSS patients. In addition, the bioinformatics analysis of DCN in the GSE40611 (RNA-seq, parotid glands) dataset displayed an elevation of the DCN level in the parotid glands of pSS patients that positively correlated with several chemokines (CXCL13, CXCL9, and CCL20), Interleukin -1 β (IL1 -β), and caspase3 but negatively correlated with the proliferation relative gene MKI67. The stimulatory effects of DCN on the salivary gland epithelial cells (A253 cell line) and macrophages have been determined as they are considered active participants in the progression of SS. The data showed that DCN induced the apoptosis of A253 cells and polarization of macrophages towards the M1 phenotype, characterized by the expression of pro-inflammatory cytokines. Our study provided preliminary evidence to understand the clinical significance of DCN in pSS and broadened our horizons in understanding the mechanism of pSS.

A parasympathetic neurotransmitter induces myoepithelial cell differentiation during salivary gland development

Myoepithelial cells (MECs) are responsible for receiving stimuli from the central nervous system and translating their responses into the form of secretion into glandular tissue, including salivary glands (SG), sweet glands, and mammary glands. SG MECs cause the secretion of serous saliva by contracting of acini/ductal cells with acetylcholine (Ach) from parasympathetic nerves via muscarinic receptors. To response the parasympathetic physiological stimulation, SG epithelial cell-derived MECs are supposed to be induced and placed adjacent to parasympathetic system nerve ends in SGs by forming a neuro-myoepithelial junction. For salivary secretion to function under parasympathetic control, therefore, specific regions of salivary gland epithelial cells must be mapped and the epithelium near the nerve must differentiate into MECs in order to form a nerve-myoepithelial junction during organogenesis. We hypothesized that the epithelium near the parasympathetic nerves is induced the differentiation into MECs by which the neurotransmitter acetylcholine via muscarinic receptors. qPCR and whole-mount immunohistochemical analysis in ex vivo organ culture system revealed that SG epithelial cells near a parasympathetic nerve were found to be induced to differentiate into MECs via the cholinergic receptor muscarinic 1 by carbachol (CCh), an acetylcholine agonist. In addition, CCh stimulated ERK and Akt signaling for the induction of MEC differentiation in rat submandibular gland epithelial cells. These findings indicate that muscarinic action is required for the induction of MECs and formation of a neuro-myoepithelial junction in developing SGs. This study proposes a novel concept for tissue architecture to form a neuro-myoepithelial junction during neurofunctional organogenesis including SGs.

Association between IL-7 and primary Sjögren's syndrome: A single-center study and a systematic scoping review

BackgroundThere is a lack of single marker reflecting systemic activity of primary Sjögren's syndrome (pSS). Our aim is to determine the association between interleukin(IL)-7 and pSS by combining a single-center study and a systematic scoping review. MethodsThere were 58 patients with pSS and 30 healthy controls (HCs) included in the single-center study. The multiplex immunoassay was used for detecting concentrations of IL-7, IL-4, IL-9, IL-10, IL-17, interferon(IFN) -γ, INF-α and INF-β in sera. pSS patients were evaluated for systemic activity in accordance with the European League against Rheumatism SS Disease Activity Index (ESSDAI). In the systematic scoping review, all studies regarding association of IL-7 with pSS were included. ResultspSS patients showed higher serum IL-7 levels (P = 0.028 vs HCs) which were associated with neutrophil, neutrophil-to-lymphocyte ratio(NLR), platelet-to-lymphacyte ratio(PLR), red blood cell distribution width(RDW), erythrocyte sedimentation rate(ESR), immunoglobulin(Ig)G, C-reactive protein (CRP), fever, lymphadenopathy and ESSDAI. Serum IL-7 correlated with Th cell related cytokines. Findings of the systematic scoping review on 15 articles were as follows. Firstly, IL-7 expression in salivary glands (SGs), serum and saliva increased in pSS, while IL-7 receptor(IL-7R) expression increased in SGs but decreased in peripheral blood. Secondly, increased IL-7 was mainly from SGs of pSS patients, but whether IL-7 was mainly produced by SG epithelial cells remained to be validated. Thirdly, IL-7 could exert a key role in pSS immunopathology. Although IL-7 had no direct effect on SGECs, it could activate B and T cells and stimulate secretion of IL-17, IL-4 and IFNs. ConclusionNot only are IL-7 and soluble IL-7R potential biomarkers for monitoring pSS activity, but also targeting IL-7/IL-7R pathway may be a promising therapeutic strategy.

Small RNA Expression Profiling Reveals hsa-miR-181d-5p Downregulation Associated With TNF-α Overexpression in Sjögren's Syndrome Patients.

MicroRNAs (miRNAs) are small non-coding RNAs (sRNA), that alter gene expression by binding to target messenger RNAs (mRNAs) and repressing translation. Dysregulated miRNA expression has been implicated in the pathogenesis of autoimmune diseases such as Sjögren’s syndrome (SS). The aim of this study was to characterize the global profile of sRNAs in labial salivary glands (LSG) from SS-patients and to validate potential miRNA candidates implicated in glandular inflammation. LSG from 21 SS-patients and 9 sicca controls were analyzed. A global next generation sequencing (NGS)-based sRNA profiling approach was employed to identify direct targets whereby differentially expressed miRNAs were predicted using bioinformatics tools. miRNA levels were validated by TaqMan and target mRNA levels were determined by quantitative real-time PCR. We also performed in vitro assays using recombinant TNF-α. NGS shows that ~30% of sRNAs were miRNAs. In comparison with samples from sicca controls, four miRNAs were found differentially expressed in LSG from SS-patients with low focus score (LFS) and 18 from SS-patients with high focus score (HFS). The miRNA with the most significant changes identified by NGS was hsa-miR-181d-5p and downregulation was confirmed by TaqMan analysis. Levels of TNF-α mRNA, a direct target of hsa-miR-181d-5p, were significantly increased and negatively correlated with hsa-miR-181d-5p presence. Moreover, positive correlations between TNF-α transcript levels, focus score, ESSDAI, and autoantibody levels were also detected. Furthermore, TNF-α stimulation decreased hsa-miR-181d-5p levels in vitro. Downregulation of hsa-miR-181d-5p in LSG from SS-patients could contribute to the glandular pro-inflammatory environment by deregulation of its direct target TNF-α. Further dissection of the pathophysiological mechanisms underlying the hsa-miR-181d-5p-mediated action in inflammatory conditions could be useful to evaluate the benefits of increasing hsa-miR-181d-5p levels for restoration of salivary gland epithelial cell architecture and function.

Maladaptive Autophagy in the Pathogenesis of Autoimmune Epithelitis in Sjögren's Syndrome.

Salivary gland epithelial cells (SGECs) are key cellular drivers in the pathogenesis of primary Sjögren's syndrome (SS); however, the mechanisms sustaining SGEC activation in primary SS remain unclear. We undertook this study to determine the role of autophagy in the survival and activation of SGECs in primary SS. Primary SGECs isolated from the minor SGs of patients with primary SS or sicca syndrome were evaluated by flow cytometry, immunoblotting, and immunofluorescence to assess autophagy (autophagic flux, light chain 3 IIB [LC3-IIB], p62, LC3-IIB+/lysosome-associated membrane protein 1 [LAMP-1] staining), apoptosis (annexin V/propidium iodide [PI], caspase 3), and activation (intercellular adhesion molecule, vascular cell adhesion molecule). Focus score and germinal center presence were assessed in the SGs from the same patients to assess correlation with histologic severity. Human SG (HSG) cells were stimulated in vitro with peripheral blood mononuclear cells (PBMCs) and serum from primary SS patients in the presence or absence of autophagy inhibitors to determine changes in autophagy and epithelial cell activation. SGECs from primary SS patients (n=24) exhibited increased autophagy (autophagic flux [P=0.001]; LC3-IIB [P=0.02]; p62 [P=0.064]; and as indicated by LC3-IIB/LAMP-1+ staining), increased expression of antiapoptotic molecules (Bcl-2 [P=0.006]), and reduced apoptosis (annexin V/PI [P=0.002]; caspase 3 [P=0.057]), compared to samples from patients with sicca syndrome (n=16). Autophagy correlated with histologic disease severity. In vitro experiments on HSG cells stimulated with serum and PBMCs from primary SS patients confirmed activation of autophagy and expression of adhesion molecules, which was reverted upon pharmacologic inhibition of autophagy. In primary SS SGECs, inflammation induces autophagy and prosurvival mechanisms, which promote SGEC activation and mirror histologic severity. These findings indicate that autophagy is a central contributor to the pathogenesis of primary SS and a new therapeutic target.