RACK1 plays a critical role in mast cell secretion and Ca2+ mobilization by modulating F-actin dynamics.

Although RACK1 is known to act as a signaling hub in immune cells, its presence and role in mast cells (MCs) is undetermined. MC activation via antigen stimulation results in mediator release and is preceded by cytoskeleton reorganization and Ca2+ mobilization. In this study, we found that RACK1 was distributed throughout the MC cytoplasm both in vivo and in vitro. After RACK1 knockdown (KD), MCs were rounded, and the cortical F-actin was fragmented. Following antigen stimulation, in RACK1 KD MCs, there was a reduction in cortical F-actin, an increase in monomeric G-actin and a failure to organize F-actin. RACK1 KD also increased and accelerated degranulation. CD63+ secretory granules were localized in F-actin-free cortical regions in non-stimulated RACK1 KD MCs. Additionally, RACK1 KD increased antigen-stimulated Ca2+ mobilization, but attenuated antigen-stimulated depletion of ER Ca2+ stores and thapsigargin-induced Ca2+ entry. Following MC activation there was also an increase in interaction of RACK1 with Orai1 Ca2+-channels, ß-actin and the actin-binding proteins vinculin and MyoVa. These results show that RACK1 is a critical regulator of actin dynamics, affecting mediator secretion and Ca2+ signaling in MCs. This article has an associated First Person interview with the first author of the paper.

Association of G protein-coupled receptor 78 with salivary dysfunction in male Sjögren's patients.

OBJECTIVE: Sjögren's disease (SjD) has a strong sex bias, suggesting an association with sex hormones. Male SjD represents a distinct subset of the disease, but the pathogenic mechanisms of male SjD is poorly characterized. The aim of this study is to identify initiating events related to the development of gland hypofunction and autoimmunity in male SjD patients. MATERIALS AND METHODS: Human minor salivary glands were transcriptomically analyzed with microarrays to detect differentially expressed genes in male SjD patients. Identified genes were tested on their involvement in the disease using conditional transgenic mice and gene-overexpressing cells. RESULTS: GPR78, an orphan G protein-coupled receptor, was overexpressed in the salivary glands of male SjD patients compared with male healthy controls and female SjD patients. Male GPR78 transgenic mice developed salivary gland hypofunction with increased epithelial apoptosis, which was not seen in control or female transgenic mice. In cell culture, GPR78 overexpression decreased lysosomal integrity, leading to caspase-dependent apoptotic cell death. GPR78-induced cell death in vitro was inhibited by treatment with estradiol. CONCLUSION: GPR78 overexpression can induce apoptosis and salivary gland hypofunction in male mice through lysosomal dysfunction and increased caspase-dependent apoptosis in salivary gland epithelium, which may drive disease in humans.

Lysosome-associated membrane protein 3 misexpression in salivary glands induces a Sjögren's syndrome-like phenotype in mice.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune sialadenitis with unknown aetiology. Although extensive research implicated an abnormal immune response associated with lymphocytes, an initiating event mediated by salivary gland epithelial cell (SGEC) abnormalities causing activation is poorly characterised. Transcriptome studies have suggested alternations in lysosomal function are associated with SS, but a cause and effect linkage has not been established. In this study, we demonstrated that altered lysosome activity in SGECs by expression of lysosome-associated membrane protein 3 (LAMP3) can initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS. METHODS: Retroductal cannulation of the submandibular salivary glands with an adeno-associated virus serotype 2 vector encoding LAMP3 was used to establish a model system. Pilocarpine-stimulated salivary flow and the presence of autoantibodies were assessed at several time points post-cannulation. Salivary glands from the mice were evaluated using RNAseq and histologically. RESULTS: Following LAMP3 expression, saliva flow was significantly decreased and serum anti-Ro/SSA and La/SSB antibodies could be detected in the treated mice. Mechanistically, LAMP3 expression increased apoptosis in SGECs and decreased protein expression related to saliva secretion. Analysis of RNAseq data suggested altered lysosomal function in the transduced SGECs, and that the cellular changes can chemoattract immune cells into the salivary glands. Immune cells were activated via toll-like receptors by damage-associated molecular patterns released from LAMP3-expressing SGECs. CONCLUSIONS: These results show a critical role for lysosomal trafficking in the development of SS and establish a causal relationship between LAMP3 misexpression and the development of SS.

Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis.

BACKGROUND & AIMS: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry-associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. METHODS: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf-/- mice, Sarafzf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. RESULTS: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. CONCLUSIONS: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease that is estimated to affect 35 million people worldwide. Currently, no effective treatments exist for Sjögren's syndrome, and there is a limited understanding of the physiological mechanisms associated with xerostomia and hyposalivation. The present work revealed that aquaporin 5 expression, a water channel critical for salivary gland fluid secretion, is regulated by bone morphogenetic protein 6. Increased expression of this cytokine is strongly associated with the most common symptom of primary Sjögren's syndrome, the loss of salivary gland function. This finding led us to develop a therapy in the treatment of Sjögren's syndrome by increasing the water permeability of the gland to restore saliva flow. Our study demonstrates that the targeted increase of gland permeability not only resulted in the restoration of secretory gland function but also resolved the hallmark salivary gland inflammation and systemic inflammation associated with disease. Secretory function also increased in the lacrimal gland, suggesting this local therapy could treat the systemic symptoms associated with primary Sjögren's syndrome.

Restoration of CFTR Activity in Ducts Rescues Acinar Cell Function and Reduces Inflammation in Pancreatic and Salivary Glands of Mice.

BACKGROUND & AIMS: Sjögren's syndrome and autoimmune pancreatitis are disorders with decreased function of salivary, lacrimal glands, and the exocrine pancreas. Nonobese diabetic/ShiLTJ mice and mice transduced with the cytokine BMP6 develop Sjögren's syndrome and chronic pancreatitis and MRL/Mp mice are models of autoimmune pancreatitis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a ductal Cl- channel essential for ductal fluid and HCO3- secretion. We used these models to ask the following questions: is CFTR expression altered in these diseases, does correction of CFTR correct gland function, and most notably, does correcting ductal function correct acinar function? METHODS: We treated the mice models with the CFTR corrector C18 and the potentiator VX770. Glandular, ductal, and acinar cells damage, infiltration, immune cells and function were measured in vivo and in isolated duct/acini. RESULTS: In the disease models, CFTR expression is markedly reduced. The salivary glands and pancreas are inflamed with increased fibrosis and tissue damage. Treatment with VX770 and, in particular, C18 restored salivation, rescued CFTR expression and localization, and nearly eliminated the inflammation and tissue damage. Transgenic overexpression of CFTR exclusively in the duct had similar effects. Most notably, the markedly reduced acinar cell Ca2+ signaling, Orai1, inositol triphosphate receptors, Aquaporin 5 expression, and fluid secretion were restored by rescuing ductal CFTR. CONCLUSIONS: Our findings reveal that correcting ductal function is sufficient to rescue acinar cell function and suggests that CFTR correctors are strong candidates for the treatment of Sjögren's syndrome and pancreatitis.

Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

Store-operated Ca²+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I(SOC), activated in response to Ca²+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I(CRAC); the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³+, removal of extracellular Ca²+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²+-containing, but not Ca²+-free, medium. Consistent with this, I(CRAC) is activated in cells pretreated with thapsigargin in Ca²+-free medium while I(SOC) is activated in cells pretreated in Ca²+-containing medium. Significantly, TRPC1 function is required for sustained K(Ca) activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

Association of bone morphogenetic protein 6 with exocrine gland dysfunction in patients with Sjögren's syndrome and in mice.

OBJECTIVE: Primary Sjögren's syndrome (SS) is characterized by autoimmune activation and loss of function in secretory epithelia. The present study was undertaken to investigate and characterize changes in the epithelia associated with the loss of gland function in primary SS. METHODS: To identify changes in epithelial gene expression, custom microarrays were probed with complementary RNA (cRNA) isolated from minor salivary glands (MSGs) of female patients with primary SS who had low focus scores and low salivary flow rates, and the results were compared with those obtained using cRNA from the MSGs of sex-matched healthy volunteers. The effect of bone morphogenetic protein 6 (BMP-6) on salivary gland function was tested using adeno-associated virus-mediated gene transfer to the salivary glands of C57BL/6 mice. RESULTS: A significant increase in expression of BMP-6 was observed in RNA isolated from SS patients compared with healthy volunteers. Overexpression of BMP-6 locally in the salivary or lacrimal glands of mice resulted in the loss of fluid secretion as well as changes in the connective tissue of the salivary gland. Assessment of the fluid movement in either isolated acinar cells from mice overexpressing BMP-6 or a human salivary gland cell line cultured with BMP-6 revealed a loss in volume regulation in these cells. Lymphocytic infiltration in the submandibular gland of BMP-6 vector-treated mice was increased. No significant changes in the production of proinflammatory cytokines or autoantibodies associated with SS (anti-Ro/SSA and anti-La/SSB) were found after BMP-6 overexpression. CONCLUSION: In addition to identifying BMP-6 expression in association with xerostomia and xerophthalmia in primary SS, the present results suggest that BMP-6-induced salivary and lacrimal gland dysfunction in primary SS is independent of the autoantibodies and immune activation associated with the disease.

Extracellular Ca(2+) sensing in salivary ductal cells.

Ca(2+) is secreted from the salivary acinar cells as an ionic constituent of primary saliva. Ions such as Na(+) and Cl(-) get reabsorbed whereas primary saliva flows through the salivary ductal system. Although earlier studies have shown that salivary [Ca(2+)] decreases as it flows down the ductal tree into the oral cavity, ductal reabsorption of Ca(2+) remains enigmatic. Here we report a potential role for the G protein-coupled receptor, calcium-sensing receptor (CSR), in the regulation of Ca(2+) reabsorption by salivary gland ducts. Our data show that CSR is present in the apical region of ductal cells where it is co-localized with transient receptor potential canonical 3 (TRPC3). CSR is activated in isolated salivary gland ducts as well as a ductal cell line (SMIE) by altering extracellular [Ca(2+)] or by aromatic amino acid, L-phenylalanine (L-Phe, endogenous component of saliva), as well as neomycin. CSR activation leads to Ca(2+) influx that, in polarized cells grown on a filter support, is initiated in the luminal region. We show that TRPC3 contributes to Ca(2+) entry triggered by CSR activation. Further, stimulation of CSR in SMIE cells enhances the CSR-TRPC3 association as well as surface expression of TRPC3. Together our findings suggest that CSR could serve as a Ca(2+) sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of [Ca(2+)] from the saliva via TRPC3, thus contributing to maintenance of salivary [Ca(2+)]. CSR could therefore be a potentially important protective mechanism against formation of salivary gland stones (sialolithiasis) and infection (sialoadenitis).

Membrane-type MMPs are indispensable for placental labyrinth formation and development.

The membrane-type matrix metalloproteinases (MT-MMPs) are essential for pericellular matrix remodeling in late stages of development, as well as in growth and tissue homeostasis in postnatal life. Although early morphogenesis is perceived to involve substantial tissue remodeling, the roles of MT-MMPs in these processes are only partially characterized. Here we explore the functions of 2 prominently expressed MT-MMPs, MT1-MMP and MT2-MMP, and describe their roles in the process of placental morphogenesis. The fetal portion of the placenta, in particular the labyrinth (LA), displays strong overlapping expression of MT1-MMP and MT2-MMP, which is critical for syncytiotrophoblast formation and in turn for fetal vessels. Disruption of trophoblast syncytium formation consequently leads to developmental arrest with only a few poorly branched fetal vessels entering the LA causing embryonic death at embryonic day 11.5. Through knockdown of MMP expression, we demonstrate that either MT1-MMP or MT2-MMP is crucial specifically during development of the LA. In contrast, knockdown of MT-MMP activity after LA formation is compatible with development to term and postnatal life. Taken together these data identify essential but interchangeable roles for MT1-MMP or MT2-MMP in placental vasculogenesis and provide the first example of selective temporal and spatial MMP activity required for development of the mouse embryo.

Increased T-bet+ cytotoxic effectors and type I interferon-mediated processes in chronic graft-versus-host disease of the oral mucosa.

Although chronic graft-versus-host disease (cGVHD) is a major long-term complication of allogeneic hematopoietic stem cell transplantation, little is known of its pathogenesis. We have systematically examined oral mucosa among cGVHD patients and determined that the clinical severity of oral cGVHD was correlated with apoptotic epithelial cells, often found adjacent to infiltrating effector-memory T cells expressing markers of cytotoxicity and type I cytokine polarization. Accumulation of T-bet(+) T-cell effectors was associated with both increased proliferation and the expression of the type I chemokine receptor CXCR3. Concurrently, in both infiltrating cells and keratinocytes, we observed increased expression of the CXCR3 ligand MIG (CXCL9) and interleukin-15 (IL-15), type I interferon (IFN)-inducible factors that support the migration, type I differentiation, and expansion of alloreactive effectors. In severely affected mucosa, we observed high levels of MxA, a protein specifically induced by type I IFN, and signal transducer and activator of transcription 1 (STAT1) phosphorylation, a critical step in the IFN-signaling pathway, along with increased numbers of plasmacytoid dendritic cells. These data challenge the current paradigm of cGVHD as a type II cytokine-driven disorder and support the model that oral cGVHD results from type I IFN-driven immigration, proliferation, and differentiation of T-bet(+) type I T effectors. The clinical trials are registered at http://www.clinicaltrials.gov as NCT00331968.

Conditional overexpression of TGF-beta1 disrupts mouse salivary gland development and function.

Transforming growth factor-beta (TGF-beta) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-beta1 in the salivary gland, we created a transgenic mouse (beta1(glo)) that conditionally overexpresses active TGF-beta1 upon genomic recombination by Cre recombinase. beta1(glo) mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (beta1(glo)/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those beta1(glo)/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-beta signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-beta1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-beta in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome, or with radiation therapy given to head-and-neck cancer patients.

Transduction of Salivary Gland Acinar Cells with a Novel AAV Vector 44.9.

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.

Inhibition of bone morphogenetic protein 6 receptors ameliorates Sjögren's syndrome in mice.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease, with only palliative treatments available. Recent work has suggested that increased bone morphogenetic protein 6 (BMP6) expression could alter cell signaling in the salivary gland (SG) and result in the associated salivary hypofunction. We examined the prevalence of elevated BMP6 expression in a large cohort of pSS patients and tested the therapeutic efficacy of BMP signaling inhibitors in two pSS animal models. Increased BMP6 expression was found in the SGs of 54% of pSS patients, and this increased expression was correlated with low unstimulated whole saliva flow rate. In mouse models of SS, inhibition of BMP6 signaling reduced phosphorylation of SMAD1/5/8 in the mouse submandibular glands, and led to a recovery of SG function and a decrease in inflammatory markers in the mice. The recovery of SG function after inhibition of BMP6 signaling suggests cellular plasticity within the salivary gland and a possibility for therapeutic intervention that can reverse the loss of function in pSS.

Orai1-Mediated Antimicrobial Secretion from Pancreatic Acini Shapes the Gut Microbiome and Regulates Gut Innate Immunity.

The gut microbiome participates in numerous physiologic functions and communicates intimately with the host immune system. Antimicrobial peptides are critical components of intestinal innate immunity. We report a prominent role for antimicrobials secreted by pancreatic acini in shaping the gut microbiome that is essential for intestinal innate immunity, barrier function, and survival. Deletion of the Ca2+ channel Orai1 in pancreatic acini of adult mice resulted in 60%-70% mortality within 3 weeks. Despite robust activation of the intestinal innate immune response, mice lacking acinar Orai1 exhibited intestinal bacterial outgrowth and dysbiosis, ultimately causing systemic translocation, inflammation, and death. While digestive enzyme supplementation was ineffective, treatments constraining bacterial outgrowth (purified liquid diet, broad-spectrum antibiotics) rescued survival, feeding, and weight gain. Pancreatic levels of cathelicidin-related antimicrobial peptide (CRAMP) were reduced, and supplement of synthetic CRAMP prevented intestinal disease. These findings reveal a critical role for antimicrobial pancreatic secretion in gut innate immunity.

Radiation inhibits salivary gland function by promoting STIM1 cleavage by caspase-3 and loss of SOCE through a TRPM2-dependent pathway.

Store-operated Ca2+ entry (SOCE) is critical for salivary gland fluid secretion. We report that radiation treatment caused persistent salivary gland dysfunction by activating a TRPM2-dependent mitochondrial pathway, leading to caspase-3-mediated cleavage of stromal interaction molecule 1 (STIM1) and loss of SOCE. After irradiation, acinar cells from the submandibular glands of TRPM2+/+ , but not those from TRPM2-/- mice, displayed an increase in the concentrations of mitochondrial Ca2+ and reactive oxygen species, a decrease in mitochondrial membrane potential, and activation of caspase-3, which was associated with a sustained decrease in STIM1 abundance and attenuation of SOCE. In a salivary gland cell line, silencing the mitochondrial Ca2+ uniporter or caspase-3 or treatment with inhibitors of TRPM2 or caspase-3 prevented irradiation-induced loss of STIM1 and SOCE. Expression of exogenous STIM1 in the salivary glands of irradiated mice increased SOCE and fluid secretion. We suggest that targeting the mechanisms underlying the loss of STIM1 would be a potentially useful approach for preserving salivary gland function after radiation therapy.

Lead line in rodents: an old sign of lead intoxication turned into a new method for environmental surveillance.

The "lead line" was described by Henry Burton in 1840. Rodents are used as sentinels to monitor environmental pollution, but their teeth have not been used to determine lead. To determine whether lead deposits can be observed in the teeth of lead-exposed animals, since the gingival deposits known as "lead line" would likely have a correlate in the calcified tissue to which the gums are opposed during life. Male Wistar rats were exposed to lead in the drinking water (30 mg/L) since birth until 60 days-old. Molars and the incisors of each hemimandible were analyzed by scanning electron microscopy (SEM) on regular and backscattered electrons (BSE) mode. Elements were determined using electron dispersive spectroscopy (EDS). Clean cervical margins were observed on control teeth, as opposed to the findings of extensive deposits on lead-exposed animals, even in hemimandibles that had been exhumed after being buried for 90 days. BSE/EDS indicated that those deposits were an exogenous material compatible with lead sulfite. Presence of calcium, phosphorus, magnesium, carbon, lead, and oxygen is presented. Lead-exposed animals presented marked root resorption. The lead deposits characterized here for the first time show that the "lead line" seen in gums has a calcified tissue counterpart, that is detectable post-mortem even in animals exposed to a low dose of lead. This is likely a good method to detect undue lead exposure and will likely have wide application for pollution surveillance using sentinels.

Hepatitis Delta Virus Detected in Salivary Glands of Sjögren's Syndrome Patients and Recapitulates a Sjögren's Syndrome-Like Phenotype in Vivo.

BACKGROUND: Low-level, chronic viral infections have been suspect in the development of select autoimmune diseases, including primary Sjögren's syndrome (pSS). Multiple studies have shown stimulation of antiviral response pathways in pSS tissues suggestive of a viral infection. Yet, with this data in hand, a causal link between a viral infection and development of pSS had not been identified. Therefore, a study was designed to further define the viral landscape within pSS-affected salivary gland tissue to identify potential viral-mediated triggers in the pathogenesis of this autoimmune disease. METHODS: A viral microarray was utilized to measure viral transcripts present in salivary gland tissue from patients diagnosed with pSS compared to healthy controls. Murine models of salivary gland localized HDV antigen expression were developed to evaluate the capacity of a chronic HDV signature to trigger the development of a pSS-like phenotype. RESULTS: Through this analysis, two distinct viral profiles were identified, including the increased presence of hepatitis delta virus (HDV) in 50% of pSS patients evaluated. Presence of HDV antigen and sequence were confirmed in minor salivary gland tissue. Patients with elevated HDV levels in salivary gland tissue were negative for detectible hepatitis B virus (HBV) surface antigen and antibodies to HBV or HDV. Expression of HDV antigens in vivo resulted in reduced stimulated saliva flow, increase in focal lymphocytic infiltrates, and development of autoantibodies. CONCLUSION: Identification of HDV in pSS patients and induction of a complete pSS-like phenotype in vivo provides further support of a viral-mediated etiopathology in the development of pSS.

Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis.

Matriptase/MT-SP1 is a novel tumor-associated type II transmembrane serine protease that is highly expressed in the epidermis, thymic stroma, and other epithelia. A null mutation was introduced into the Matriptase/MT-SP1 gene of mice to determine the role of Matriptase/MT-SP1 in epidermal development and neoplasia. Matriptase/MT-SP1-deficient mice developed to term but uniformly died within 48 h of birth. All epidermal surfaces of newborn mice were grossly abnormal with a dry, red, shiny, and wrinkled appearance. Matriptase/MT-SP1-deficiency caused striking malformations of the stratum corneum, characterized by dysmorphic and pleomorphic corneocytes and the absence of vesicular bodies in transitional layer cells. This aberrant skin development seriously compromised both inward and outward epidermal barrier function, leading to the rapid and fatal dehydration of Matriptase/MT-SP1-deficient pups. Loss of Matriptase/MT-SP1 also seriously affected hair follicle development resulting in generalized follicular hypoplasia, absence of erupted vibrissae, lack of vibrissal hair canal formation, ingrown vibrissae, and wholesale abortion of vibrissal follicles. Furthermore, Matriptase/MT-SP1-deficiency resulted in dramatically increased thymocyte apoptosis, and depletion of thymocytes. This study demonstrates that Matriptase/MT-SP1 has pleiotropic functions in the development of the epidermis, hair follicles, and cellular immune system.