Andrew D. McCall

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sjögrens syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sjögrens syndrome.

Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis

Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.

ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands

Sjögrens syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands, leading to dry mouth, dry eyes, and the presence of anti-nuclear antibodies. Despite modern advances, the current therapies for SS have no permanent benefit. A potential treatment could involve the use of resolvins, which are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Our previous studies indicate that ALX/FPR2, the receptor for RvD1, is expressed and active in the rat parotid cell line Par-C10. Specifically, activation of ALX/FPR2 with RvD1 blocked inflammatory signals caused by TNF-α and enhanced salivary epithelial integrity. The goal of this study was to investigate RvD1 receptor expression and signaling pathways in primary salivary cells. Additionally, we determined the role of the aspirin-triggered 17R analog (AT-RvD1, a more chemically stable RvD1 epimeric form) in prevention of TNF-α-mediated salivary inflammation in mouse submandibular glands (mSMG). Our results indicate that ALX/FPR2 is expressed in mSMG and is able to elicit intracellular Ca2+ responses and phosphorylation of Erk1/2, as well as Akt. Given that these signaling pathways are linked to cell survival, we investigated whether AT-RvD1 was able to prevent programmed cell death in mSMG. Specifically, we determined that AT-RvD1 prevented TNF-α-mediated caspase-3 activation. Finally, we show that ALX/FPR2 is expressed in human minor salivary glands with and without SS, indicating the potential therapeutic use of AT-RvD1 for this condition.

Stem Cell–Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor–reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle–derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features.

Zonula Occludens-1, Occludin and E-cadherin Expression and Organization in Salivary Glands with Sjögren’s Syndrome

Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.

Three-dimensional cultures of mouse submandibular and parotid glands: a comparative study.

Freshly isolated salivary cells can be plated on an extracellular matrix, such as growth factor‐reduced Matrigel (GFR‐MG), to induce the formation of three‐dimensional (3D) structures. Cells grown on GFR‐MG are able to form round structures with hollow lumina, capable of sustaining amylase expression. In contrast, cells grown on plastic do not exhibit these features. Our recent studies have used mouse parotid gland (PG) cells, grown on different extracellular matrices, as a model for acinar formation. However, PG cells were not able to respond to the secretory agonist carbachol beyond 5 days and did not sustain polarity over time, regardless of the substratum. An alternative option relies in the use of mouse submandibular glands (SMG), which are more anatomically accessible and yield a larger number of cells. We compared SMG and PG cell clusters (partially dissociated glands) for their ability to form hollow round structures, sustain amylase and maintain secretory function when grown on GFR‐MG. The results were as follows: (a) SMG cell clusters formed more organized and larger structures than PG cell clusters; (b) both SMG and PG cell clusters maintained α‐amylase expression over time; (c) SMG cell clusters maintained agonist‐induced secretory responses over time; and (d) SMG cell clusters maintained secretory granules and cell–cell junctions. These results indicate that mouse SMG cell clusters are more amenable for the development of a bioengineered salivary gland than PG cell clusters, as they form more organized and functional structures. Copyright

Human Salivary Protein Histatin 5 Has Potent Bactericidal Activity against ESKAPE Pathogens

ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species) pathogens have characteristic multiple-drug resistance and cause an increasing number of nosocomial infections worldwide. Peptide-based therapeutics to treat ESKAPE infections might be an alternative to conventional antibiotics. Histatin 5 (Hst 5) is a salivary cationic histidine-rich peptide produced only in humans and higher primates. It has high antifungal activity against Candida albicans through an energy-dependent, non-lytic process; but its bactericidal effects are less known. We found Hst 5 has bactericidal activity against S. aureus (60–70% killing) and A. baumannii (85–90% killing) in 10 and 100 mM sodium phosphate buffer (NaPB), while killing of >99% of P. aeruginosa, 60–80% E. cloacae and 20–60% of E. faecium was found in 10 mM NaPB. Hst 5 killed 60% of biofilm cells of P. aeruginosa, but had reduced activity against biofilms of S. aureus and A. baumannii. Hst 5 killed 20% of K. pneumonia biofilm cells but not planktonic cells. Binding and uptake studies using FITC-labeled Hst 5 showed E. faecium and E. cloacae killing required Hst 5 internalization and was energy dependent, while bactericidal activity was rapid against P. aeruginosa and A. baumannii suggesting membrane disruption. Hst 5-mediated killing of S. aureus was both non-lytic and energy independent. Additionally, we found that spermidine conjugated Hst 5 (Hst5-Spd) had improved killing activity against E. faecium, E. cloacae, and A. baumannii. Hst 5 or its derivative has antibacterial activity against five out of six ESKAPE pathogens and may be an alternative treatment for these infections.

Characterization of Angiogenesis and Lymphangiogenesis in Human Minor Salivary Glands with Sjögren’s Syndrome

Angiogenesis has been proposed to play a role in the inflammation observed in Sjögren’s Syndrome (SS). However, no studies have validated the degree of angiogenesis in salivary glands with SS. Therefore, the goal of this study was to determine the presence and localization of angiogenesis and lymphangiogenesis in salivary glands with SS. We used frozen tissue sections from human minor salivary glands (hMSG) with and without SS in our analyses. To investigate signs of angiogenesis, hMSG tissue lysates were used to detect levels of the pro-angiogenic protein vascular endothelial growth factor (VEGF) by western blot analyses. Additionally, we labeled blood vessels using antibodies specific to platelet endothelial cell adhesion molecule-1 (PECAM-1) and von Willebrand Factor (vWF) to determine blood vessel organization and volume fraction using fluorescence microscopy. Lymphatic vessel organization and volume fraction were determined using antibodies specific to lymphatic vessel endothelial hyaluronan receptor (LYVE-1). Our results suggest that expression levels of VEGF are decreased in hMSG with SS as compared with controls. Interestingly, there were no significant differences in blood or lymphatic vessel organization or volume fraction between hMSG with and without SS, suggesting that angiogenesis and lymphangiogenesis have little impact on the progression of SS.

Real-Time Approach to Flow Cell Imaging of Candida albicans Biofilm Development

The ability of Candida albicans to form biofilms is a virulence factor that allows tissue attachment and subsequent infection of host tissues. Fungal biofilms have been particularly well studied, however the vast majority of these studies have been conducted under static conditions. Oral biofilms form in the presence of salivary flow, therefore we developed a novel flow system used for real-time imaging of fungal biofilm development. C. albicans wild-type (WT) cells readily attached to the substrate surface during the 2 h attachment phase, then formed heterogeneous biofilms after 18 h flow. Quantitative values for biomass, rates of attachment and detachment, and cell–cell adhesion events were obtained for C. albicans WT cells and for a hyperfilamentous mutant Δhog1. Attachment rates of C. albicans WT cells were nearly 2-fold higher than C. albicans Δhog1 cells, although Δhog1 cells formed 4-fold higher biomass. The reduced normalized detachment rate was the primary factor responsible for the increased biomass of Δhog1 biofilm, showing that cell detachment rates are an important predictor for ultimate biofilm mass under flow. Unlike static biofilms, C. albicans cells under constant laminar flow undergo continuous detachment and seeding that may be more representative of the development of in vivo biofilms.

Fluconazole Resistant Candida auris is Susceptible to Salivary Histatin 5 Killing and to Intrinsic Host Defenses

ABSTRACT Candida auris is a newly identified species causing invasive candidemia and candidiasis. It has broad multidrug resistance (MDR) not observed for other pathogenic Candida species. Histatin 5 (Hst 5) is a well-studied salivary cationic peptide with significant antifungal activity against Candida albicans and is an attractive candidate for treating MDR fungi, since antimicrobial peptides induce minimal drug resistance. We investigated the susceptibility of C. auris to Hst 5 and neutrophils, two first-line innate defenses in the human host. The majority of C. auris clinical isolates, including fluconazole-resistant strains, were highly sensitive to Hst 5: 55 to 90% of cells were killed by use of 7.5 μM Hst 5. Hst 5 was translocated to the cytosol and vacuole in C. auris cells; such translocation is required for the killing of C. albicans by Hst 5. The inverse relationship between fluconazole resistance and Hst 5 killing suggests different cellular targets for Hst 5 than for fluconazole. C. auris showed higher tolerance to oxidative stress than C. albicans, and higher survival within neutrophils, which correlated with resistance to oxidative stress in vitro. Thus, resistance to reactive oxygen species (ROS) is likely one, though not the only, important factor in the killing of C. auris by neutrophils. Hst 5 has broad and potent candidacidal activity, enabling it to combat MDR C. auris strains effectively.