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The aim of the present study is to investigate the role of c-Jun N-terminal kinase (JNK) and matrix metalloproteinase-2 (MMP-2) in mediating the effects of interleukin-1ß (IL-1ß) on the function of lacrimal gland myoepithelial cells (MECs). MECs isolated from an α-smooth muscle actin-green fluorescent protein (SMA-GFP) transgenic mouse were treated with IL-1ß alone or in the presence of SP600125, a JNK inhibitor, or ARP100, an MMP-2 inhibitor. The GFP intensity and the cell size/area were measured, and on day 7, the SMA, calponin, and pro-MMP-2 protein levels and the MEC contraction were assessed. At baseline, the control and treated cells showed no differences in GFP intensity or cell size. Starting on day 2 and continuing on days 4 and 7, the GFP intensity and cell size were significantly lower in the IL-1ß-treated samples, and these effects were alleviated following inhibition of either JNK or MMP-2. Compared with the control, the levels of SMA and calponin were lower in the IL-1ß-treated samples, and both the JNK and MMP-2 inhibitors reversed this trend. The pro-MMP-2 protein level was elevated in the IL-1ß-treated samples, and this effect was abolished by the JNK inhibitor. Finally, oxytocin-induced MEC contraction was diminished in the IL-1ß-treated samples, and both the JNK and MMP-2 inhibitors reversed this effect. Our data suggest that IL-1ß uses the JNK/MMP-2 pathways to alter MEC functions, which might account for the diminished tears associated with aqueous-deficient dry eye disease.
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INTRODUCTION: White spot lesions are common after orthodontic treatment. Chitosan nanoparticles (CS-NPs) have emerged as promising antibacterial agents that inhibit the growth of Streptococcus mutans. The aim of the study was to investigate the nano-effect of adhesives containing CS-NPs on S. mutans and their effects on shear bond strength. MATERIALS AND METHODS: The inhibitory effects of two sizes of CS-NPs were assessed using the disc agar diffusion method. Four wells were created in the petri dishes, and each was inoculated with broth (negative control), chlorhexidine (positive control), CS-NPs (20 nm), or CS-NPs (131 nm). An Instron machine was used to evaluate shear bond strength by allocating 24 teeth into three groups, and all measurements were recorded in megapascals. Caries progression was assessed using the International Caries Detection and Assessment System and surface profilometry. Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 27.0 (Released 2020; IBM Corp., Armonk, New York, United States) for a one-way ANOVA followed by Tukey's multiple comparison test. RESULTS: Disc agar diffusion showed a reduction in S. mutans in the CS-NP group compared to the control (p < 0.001), with no statistical significance between the sizes of 20 and 131 nm (p = 0.95). Regarding shear bond strength, no differences were recorded when adhesive-containing CS-NPs and the control were compared (p = 0.44). Additionally, no differences were found within the CS-NP groups (p = 0.91). Caries assessments showed excellent agreement, as indicated by a weighted kappa. Profilometry readings showed higher surface roughness in the control than in the CS-NP groups (p < 0.001), with no statistically significant difference between the CS-NP groups (p = 0.72). CONCLUSION: CS-NPs of both sizes tested had similar antibacterial effects. In addition, the incorporation of CS-NPs did not affect shear bond strength.
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The aim of these studies was to investigate the involvement of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.
Sujet(s)
AMP cyclique , Appareil lacrymal , Souris , Animaux , AMP cyclique/métabolisme , Adenylate Cyclase/métabolisme , Ocytocine/pharmacologie , Ocytocine/métabolisme , Xanthine(isobutyl-3 methyl-1)/pharmacologie , Appareil lacrymal/métabolisme , Protéines G/métabolisme , Muscles lisses , Facteurs d'échange de nucléotides guanyliques/génétique , Facteurs d'échange de nucléotides guanyliques/métabolismeRÉSUMÉ
The lacrimal gland undergoes significant structural and functional deterioration with aging. Marked with increased inflammation and fibrosis, the aged lacrimal gland is unable to perform its protective function. As a result, the ocular surface becomes highly susceptible to various ocular surface pathologies, including corneal epitheliopathy. We and others have previously shown that mast cells mediate tissue inflammation by recruiting other immune cells. However, despite their well-known characteristics of secreting various inflammatory mediators, whether mast cells contribute to the immune cell aggregation and activation, and acinar dystrophy of the aged lacrimal gland has not been investigated. Here, we demonstrate the role of mast cells in age-related lacrimal gland pathophysiology using mast cell-deficient (cKitw-sh) mice. Our data demonstrated a significant increase in mast cell frequencies and immune cell infiltration in the lacrimal gland of aged mice. Interestingly, mast cell deficiency resulted in a substantial reduction in inflammation and preservation of lacrimal gland structure, suggesting that mast cells mediate the aging process of the lacrimal gland.
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Purpose: Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation. Methods: Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome. Results: The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1ß or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc. Conclusions: SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.
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Inflammation , Appareil lacrymal , Ostéonectine , Animaux , Souris , Souris de lignée NOD , Microscopie de fluorescence , Ostéonectine/génétique , Facteurs âgesRÉSUMÉ
In the lacrimal gland, myoepithelial cells (MEC) express muscle contractile proteins such as alpha smooth muscle actin (SMA) and calponin and therefore can contract to help expel lacrimal fluid. In a previous study, we demonstrated that lacrimal gland MEC express the oxytocin receptor (OXTR) and they contract under oxytocin (OXT) stimulation. Using NOD and MRL/lpr mice (animal models of Sjogren's syndrome), we reported a decrease in SMA and calponin protein levels plus a decline in acini contraction after stimulation with OXT. It is known that proinflammatory cytokines, such as interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ), can affect OXTR expression and signaling capacity and inhibit MEC contraction. The aim of the current study was to investigate if proinflammatory cytokines are implicated in the loss of MEC contractile ability. Thus, lacrimal gland MEC from a SMA-GFP transgenic mouse were treated with IL-1ß (10 ng/ml) for a total of 7 days. At days 0, 2, 4 and 7, GFP intensity, cell size/area, contractile proteins amounts and MEC contraction were assessed. At day 0, control and treated cells showed no differences in GFP intensity and cell size. GFP intensity started to decrease in treated MEC at day 2 (20%; p=0.02), continuing after day 4 (25%; p=0.007) and 7 (30%; p=0.0001). Mean cell area was also reduced at day 2 (34%; p=0.0005), and after 4 (51%; p<0.0001) and 7 days (30%; p=0.0015). The contraction assay at day 2 showed a 70% decrease of contraction in treated MEC (p<0.0001), 73% (p<0.0001) at day 4 and 82% (p=0.0015) at day 7 when compared to control. Levels of contractile proteins were measured on day 7 showing a decrease in SMA and calponin amount in treated MEC compared with the control group (around 30%; p=0.0016 and p=0.0206; respectively). Similar results were observed when TNF-α and IFN-γ were added along with IL-1ß. Taken together the present data and those from our previous studies with Sjogren's syndrome mouse models, they strongly suggest that proinflammatory cytokines affect lacrimal gland MEC contractile ability that may account for the reduced tear secretion associated with Sjogren's syndrome dry eye disease.
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Objective: The aims of this study were to investigate the antibacterial and cytotoxic effects of silver diamine fluoride (SDF) on periodontal pathogens and human skin constructs, respectively. Background: SDF has been proven to have bactericidal effects on cariogenic bacteria. No studies to date evaluated the bactericidal effects of SDF on periodontal pathogens nor its effect on epithelium and fibroblasts. Methods: Streptococcus mutans, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans were cultured in monospecies biofilms, exposed to increasing concentrations of SDF and inoculated on agar plates to assess viability. Human gingival fibroblasts in 2D cultures were exposed to 1 µL of 0.394% of SDF and viewed using real-time imaging. Finally, SDF was applied to human, 3D tissue scaffolds of fibroblasts and keratinocytes, and termed human skin equivalents (HSE). A clinical dose of 38% SDF was applied, and HSE were cultured for 12 hours, 1, 3, 5, and 10 days. The tissue was observed clinically and histologically with hematoxylin and eosin staining and TUNEL. Results: S. mutans and A. actinomycetemcomitans growth was completely inhibited using all dilutions of SDF, whereas P. gingivalis was still viable with 0.197% and 0.098% of SDF. Single-layer fibroblasts experienced immediate necrosis upon contact with SDF. Application of SDF to HSE showed maturation of a whitish lesion within 24 hours, followed by pigmented, crusted tissue after 3 days. Histological evaluation of treated tissues showed apoptotic cells in the epithelium and upper half of the connective tissue. Conclusion: Our data suggest that SDF has bactericidal properties against two periodontal pathogens: P. gingivalis and A. actinomycetemcomitans. SDF caused immediate necrosis of monolayer fibroblasts, but does not extend to the full extent of layered fibroblasts in HSE.
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Purpose: We reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP3) which increases intracellular calcium concentration ([Ca2+]i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC. Methods: Tear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca2+]i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. Results: OXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10-7 M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca2+]i with a maximum response at 10-6 M. Furthermore, the activation of the IP3 receptor to increase [Ca2+]i is crucial for OXT-induced MEC contraction since blocking the IP3 receptor with 2-APB completely abrogated this response. Conclusions: We conclude that OXT uses the PLC/Ca2+ pathway to stimulate MEC contraction and increase lacrimal gland secretion.
Sujet(s)
Calcium/métabolisme , Appareil lacrymal/effets des médicaments et des substances chimiques , Contraction musculaire/physiologie , Muscles lisses/métabolisme , Ocytociques/pharmacologie , Ocytocine/pharmacologie , Type C Phospholipases/physiologie , Actines/métabolisme , Animaux , Technique de Western , Cellules cultivées , Électrophorèse sur gel de polyacrylamide , Cellules épithéliales/métabolisme , Appareil lacrymal/imagerie diagnostique , Appareil lacrymal/métabolisme , Souris , Souris de lignée BALB C , Souris de lignée C57BL , Microscopie de fluorescence multiphotonique , Réaction de polymérisation en chaine en temps réel , Larmes/physiologieRÉSUMÉ
BACKGROUND: Emergence of peri-implant diseases led to the development of various methods for implant surface decontamination. This study was designed to compare the efficacy of biofilm removal from implant-like titanium surfaces by an erbium-doped yttrium-aluminum-garnet (Er:YAG) laser, titanium brush, and carbon fiber curet. METHODS: Eight study subjects were recruited. A custom mouth appliance that held eight sandblasted and acid-etched titanium discs was fabricated for each subject. Subjects were asked to wear this appliance for 72 hours to allow for biofilm development. After retrieval, discs were removed and randomized to one of four treatment groups. The discs were stained with a two-component nucleic acid dye kit, and the residual biofilm was visualized under fluorescence microscopy. Quantification of residual biofilm was performed using an image analysis software and expressed as the percentage surface area. RESULTS: Fifty-nine titanium discs were randomized to the four treatment groups. The percentage of titanium disc area covered by residual biofilm was 74.0% ± 21.6%, 32.8% ± 24.0%, 11.8% ± 10.3%, and 20.1% ± 19.2% in the control, Er:YAG, titanium brush and carbon fiber curet groups, respectively (mean ± SD). The biofilm-covered area significantly decreased in each of the three treatment groups compared with control (P < 0.008). Comparisons between treatment groups did not reveal statistical significance. CONCLUSIONS: Er:YAG laser treatment is an effective method for reducing the bacterial biofilm on titanium discs. However, on a threadless titanium surface, Er:YAG laser does not exhibit a significantly greater efficacy in biofilm removal than commonly used titanium brushes or carbon fiber curets.
Sujet(s)
Implants dentaires , Lasers à solide , Décontamination , Erbium , Humains , Lasers à solide/usage thérapeutique , Microscopie électronique à balayage , Propriétés de surface , TitaneRÉSUMÉ
The lacrimal gland (LG) is the main source of the tear film aqueous layer and its dysfunction results in dry eye disease (DED), a chronic immune-mediated disorder of the ocular surface. The desiccating stress (DS) murine model that mimics human DED, results in LG dysfunction, immune cell infiltration, and consequently insufficient tear production. To date, the immune cell kinetics in DED are poorly understood. The purpose of this study was to develop a murine model of intravital multi-photon microscopy (IV-MPM) for the LG, and to investigate the migratory kinetics and 3D morphological properties of conventional dendritic cells (cDCs), the professional antigen presenting cells of the ocular surface, in DED. Mice were placed in a controlled environmental chamber with low humidity and increased airflow rate for 2 and 4 weeks to induce DED, while control naïve transgenic mice were housed under standard conditions. DED mice had significantly decreased tear secretion and increased fluorescein staining (p < 0.01) compared to naïve controls. Histological analysis of the LG exhibited infiltrating mononuclear and polymorphonuclear cells (p < 0.05), as well as increased LG swelling (p < 0.001) in DED mice compared to controls. Immunofluorescence staining revealed increased density of cDCs in DED mice (p < 0.001). IV-MPM of the LG demonstrated increased density of cDCs in the LGs of DED mice, compared with controls (p < 0.001). cDCs were more spherical in DED at both time points compared to controls (p < 0.001); however, differences in surface area were found at 2 weeks in DED compared with naïve controls (p < 0.001). Similarly, 3D cell volume was significantly lower at 2 weeks in DED vs. the naïve controls (p < 0.001). 3D instantaneous velocity and mean track speed were significantly higher in DED compared to naïve mice (p < 0.001). Finally, the meandering index, an index for directionality, was significant increased at 4 weeks after DED compared with controls and 2 weeks of DED (p < 0.001). Our IV-MPM study sheds light into the 3D morphological alterations and cDC kinetics in the LG during DED. While in naïve LGs, cDCs exhibit a more dendritic morphology and are less motile, they became more spherical with enhanced motility during DED. This study shows that IV-MPM represents a robust tool to study immune cell trafficking and kinetics in the LG, which might elucidate cellular alterations in immunological diseases, such as DED.
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Mouvement cellulaire , Cellules dendritiques/anatomopathologie , Syndromes de l'oeil sec/anatomopathologie , Microscopie intravitale , Kératite herpétique/anatomopathologie , Appareil lacrymal/anatomopathologie , Microscopie de fluorescence multiphotonique , Vidéomicroscopie , Animaux , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Forme de la cellule , Cellules cultivées , Techniques de coculture , Cellules dendritiques/immunologie , Cellules dendritiques/métabolisme , Cellules dendritiques/virologie , Modèles animaux de maladie humaine , Syndromes de l'oeil sec/immunologie , Syndromes de l'oeil sec/métabolisme , Herpèsvirus humain de type 1/pathogénicité , Kératite herpétique/immunologie , Kératite herpétique/métabolisme , Kératite herpétique/virologie , Cinétique , Appareil lacrymal/immunologie , Appareil lacrymal/métabolisme , Appareil lacrymal/virologie , Protéines luminescentes/génétique , Protéines luminescentes/métabolisme , Souris transgéniques , Larmes/métabolismeRÉSUMÉ
OBJECTIVES: To investigate the relationship between salivary alkaline phosphatase activity (ALP), protein concentration, and chronological age with cervical vertebral maturation stages (CVMS) as noninvasive biomarkers for skeletal maturity assessment. MATERIALS AND METHODS: This cross-sectional study included 79 subjects (48 females, 31 males; 7 to 23 years old) categorized into five CVMS based on lateral cephalographs evaluated by three examiners. ALP activity and protein concentration in unstimulated whole saliva were compared among five CVMS. The association between age and CVMS was assessed and five multinomial logistic regression models were utilized to predict CVMS based on salivary ALP activity, protein concentration, and chronological age. RESULTS: Salivary ALP reached the peak at early pubertal stage and then declined with a significant difference between CVMS I and CVMS II (P < .001) and between CVMS I and CVMS V (P = .004). A significant positive correlation between age and CVMS was found (rs = 0.763, P < .001). The models' overall correct classification rates for predicting CVMS were 32.9% using protein concentration, 35.4% using ALP activity, and 53.2% using both ALP activity and age. CONCLUSIONS: The combination of salivary ALP activity and chronological age may provide the best CVMS prediction.
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Phosphatase alcaline , Vertèbres cervicales , Adolescent , Détermination de l'âge à partir du squelette , Phosphatase alcaline/métabolisme , Marqueurs biologiques/métabolisme , Enfant , Études transversales , Femelle , Humains , Mâle , Salive/enzymologie , Jeune adulteRÉSUMÉ
The male Non-Obese Diabetic (NOD) mouse is an established model of autoimmune dacryoadenitis characteristic of Sjögren's Syndrome (SS), but development of diabetes may complicate studies. The Non-Obese Diabetes Resistant (NOR) mouse is a MHC-II matched diabetes-resistant alternative, but development of autoimmune dacryoadenitis is not well-characterized. We compare features of SS in male NOD and NOR mice at 12 and 20 weeks. Stimulated tear secretion was decreased in 12 week NOD relative to BALB/c mice (pâ¯<â¯0.05), while by 20 weeks both NOD and NOR showed decreased stimulated tear secretion relative to BALB/c mice (pâ¯<â¯0.001). Tear CTSS activity was elevated in NOD and NOR relative to BALB/c mice (pâ¯<â¯0.05) at 12 and 20 weeks. While NOD and NOR lacrimal glands (LG) showed increased LG lymphocytic infiltration at 12 and 20 weeks relative to BALB/c mouse LG (pâ¯<â¯0.05), the percentage in NOD was higher relative to NOR at each age (pâ¯<â¯0.05). Gene expression of CTSS, MHC II and IFN-γ in LG were significantly increased in NOD but not NOR relative to BALB/c at 12 and 20 weeks. Redistribution of the secretory effector, Rab3D in acinar cells was observed at both time points in NOD and NOR, but thinning of myoepithelial cells at 12 weeks in NOD and NOR mice was restored by 20 weeks in NOR mice. NOD and NOR mice share features of SS-like autoimmune dacryoadenitis, suggesting common disease etiology. Other findings suggest more pronounced lymphocytic infiltration in NOD mouse LG including increased pro-inflammatory factors that may be unique to this model.
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Dacryocystite/physiopathologie , Modèles animaux de maladie humaine , Appareil lacrymal/physiopathologie , Animaux , Glycémie/métabolisme , Dacryocystite/génétique , Dacryocystite/métabolisme , Technique d'immunofluorescence indirecte , Gènes MHC de classe II/génétique , Inflammation/génétique , Mâle , Souris , Souris de lignée BALB C , Souris de lignée NOD , Souches mutantes de souris , Microscopie confocale , Réaction de polymérisation en chaine en temps réel , Larmes/physiologie , Protéines G rab3/métabolismeRÉSUMÉ
The purpose of the present studies was to investigate the impact of chronic inflammation of the lacrimal gland, as occurs in Sjögren's syndrome, on the morphology and function of myoepithelial cells (MECs). In spite of the importance of MECs for lacrimal gland function, the effect of inflammation on MECs has not been well defined. We studied changes in MEC structure and function in two animal models of aqueous deficient dry eye, NOD and MRL/lpr mice. We found a statistically significant reduction in the size of MECs in diseased compared to control lacrimal glands. We also found that oxytocin receptor was highly expressed in MECs of mouse and human lacrimal glands and that its expression was strongly reduced in diseased glands. Furthermore, we found a significant decrease in the amount of two MEC contractile proteins, α-smooth muscle actin (SMA) and calponin. Finally, oxytocin-mediated contraction was impaired in lacrimal gland acini from diseased glands. We conclude that chronic inflammation of the lacrimal gland leads to a substantial thinning of MECs, down-regulation of contractile proteins and oxytocin receptor expression, and therefore impaired acini contraction. This is the first study highlighting the role of oxytocin mediated MEC contraction on lacrimal gland function.
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Cellules acineuses/physiologie , Appareil lacrymal/physiopathologie , Contraction musculaire , Récepteurs à l'ocytocine/métabolisme , Syndrome de Gougerot-Sjögren/physiopathologie , Cellules acineuses/métabolisme , Animaux , Modèles animaux de maladie humaine , Cellules épithéliales/métabolisme , Cellules épithéliales/physiologie , Femelle , Humains , Inflammation/métabolisme , Inflammation/physiopathologie , Appareil lacrymal/métabolisme , Mâle , Souris , Souris de lignée MRL lpr , Souris de lignée NOD , Cellules musculaires/métabolisme , Cellules musculaires/physiologie , Syndrome de Gougerot-Sjögren/métabolismeRÉSUMÉ
Purpose: Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. Methods: The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Results: Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Conclusions: Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.
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Connexines/génétique , Cellules épithéliales/transplantation , Régulation de l'expression des gènes , Appareil lacrymal/métabolisme , Protéines de tissu nerveux/génétique , Syndrome de Gougerot-Sjögren/génétique , Transplantation de cellules souches/méthodes , Cellules souches/cytologie , Sujet âgé , Sujet âgé de 80 ans ou plus , Animaux , Technique de Western , Connexines/biosynthèse , Modèles animaux de maladie humaine , Cellules épithéliales/métabolisme , Cellules épithéliales/anatomopathologie , Femelle , Humains , Immunohistochimie , Appareil lacrymal/anatomopathologie , Souris , Souris de lignée C57BL , Microscopie confocale , Adulte d'âge moyen , Protéines de tissu nerveux/biosynthèse , ARN/génétique , Réaction de polymérisation en chaine en temps réel , Syndrome de Gougerot-Sjögren/métabolisme , Syndrome de Gougerot-Sjögren/thérapie , Cellules souches/métabolismeRÉSUMÉ
The TFOS DEWS II Pathophysiology Subcommittee reviewed the mechanisms involved in the initiation and perpetuation of dry eye disease. Its central mechanism is evaporative water loss leading to hyperosmolar tissue damage. Research in human disease and in animal models has shown that this, either directly or by inducing inflammation, causes a loss of both epithelial and goblet cells. The consequent decrease in surface wettability leads to early tear film breakup and amplifies hyperosmolarity via a Vicious Circle. Pain in dry eye is caused by tear hyperosmolarity, loss of lubrication, inflammatory mediators and neurosensory factors, while visual symptoms arise from tear and ocular surface irregularity. Increased friction targets damage to the lids and ocular surface, resulting in characteristic punctate epithelial keratitis, superior limbic keratoconjunctivitis, filamentary keratitis, lid parallel conjunctival folds, and lid wiper epitheliopathy. Hybrid dry eye disease, with features of both aqueous deficiency and increased evaporation, is common and efforts should be made to determine the relative contribution of each form to the total picture. To this end, practical methods are needed to measure tear evaporation in the clinic, and similarly, methods are needed to measure osmolarity at the tissue level across the ocular surface, to better determine the severity of dry eye. Areas for future research include the role of genetic mechanisms in non-Sjögren syndrome dry eye, the targeting of the terminal duct in meibomian gland disease and the influence of gaze dynamics and the closed eye state on tear stability and ocular surface inflammation.
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Syndromes de l'oeil sec , Conjonctive , Humains , Kératoconjonctivite sèche , Glandes de Meibomius , LarmesRÉSUMÉ
The purpose of the present studies was to use CyTOF and RNA-Seq technologies to identify cells and genes involved in lacrimal gland repair that could be targeted to treat diseases of lacrimal gland dysfunction. Lacrimal glands of female BALB/c mice were experimentally injured by intra-glandular injection of interleukin 1 alpha (IL-1α). The lacrimal glands were harvested at various time points following injury (1 to 14 days) and used to either prepare single cell suspensions for CyTOF immuno-phenotyping analyses or to extract RNA for gene expression studies using RNA-Seq. CyTOF immuno-phenotyping identified monocytes and neutrophils as the major infiltrating populations 1 and 2 days post injury. Clustering of significantly differentially expressed genes identified 13 distinct molecular signatures: 3 associated with immune/inflammatory processes included genes up-regulated at days 1-2 and 3 associated with reparative processes with genes up-regulated primarily between days 4 and 5. Finally, clustering identified 65 genes which were specifically up-regulated 2 days post injury which was enriched for muscle specific genes. The expression of select muscle-related proteins was confirmed by immunohistochemistry which identified a subset of cells expressing these proteins. Double staining experiments showed that these cells are distinct from the myoepithelial cells. We conclude that experimentally induced injury to the lacrimal gland leads to massive infiltration by neutrophils and monocytes which resolved after 3 days. RNAseq and immunohistochemistry identified a group of cells, other than myoepithelial cells, that express muscle-related proteins that could play an important role in lacrimal gland repair.
Sujet(s)
Appareil lacrymal/métabolisme , Analyse de séquence d'ARN/méthodes , Animaux , Souris , Souris de lignée BALB CRÉSUMÉ
The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögren's syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n = 20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 106 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren's syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.
RÉSUMÉ
PURPOSE: Gene expression and protein analysis studies require high-quality human tissue which is a challenge and difficult to obtain through live human biopsies. Human postmortem lacrimal gland (LG) and submandibular gland (SMG) tissues have the potential to provide an invaluable source for studying the mechanisms involved in LG and SMG dysfunction. Therefore, we aimed to test the suitability of post-mortem LG and SMG for molecular, biochemical, and cell biological studies. METHODS: LG and SMG tissue from healthy donors was collected and immediately placed in RNAlater solution and then shipped overnight at 4 °C. After receipt, each gland was divided into three pieces for RNA, protein, and histological analysis, respectively. Total RNA isolated from each LG and SMG was analyzed for RNA integrity using an Agilent Bioanalyzer and reverse transcription-PCR (RT-PCR). For histology, tissues were embedded in paraffin and stained with hematoxylin and eosin. For protein analysis, lysates were prepared and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. RESULTS: When the LG and SMG samples were preserved in RNAlater, the RNA integrity number (RIN) values from the LG and SMG were >7.0 from all three donors, while the RNAs from tissue not preserved in RNAlater were of poorer quality. The gene and/or protein expression of E-cadherin, aquaporin 5, alpha-smooth muscle actin (α-SMA), ß-actin, and GAPDH was preserved in all samples. In addition, histological analyses showed normal tubuloacinar structures of all glands with serous and mucous producing acini within lobules interspersed with adipose fat. CONCLUSIONS: In this study, we determined that RNA, protein, and histological sections obtained from postmortem human LG and SMG tissue preserved in RNAlater were of high quality. This would provide a viable source of human LG and SMG tissue suitable for studies of diseases that affect these glands, such as Sjögren's syndrome.
Sujet(s)
Cryoconservation , Protéines de l'oeil/isolement et purification , Appareil lacrymal/composition chimique , Solution conservation organe , Conservation d'organe , ARN/isolement et purification , Glande submandibulaire/composition chimique , Actines/métabolisme , Sujet âgé de 80 ans ou plus , Antigènes CD , Aquaporine-5/métabolisme , Autopsie , Technique de Western , Cadhérines/métabolisme , Électrophorèse sur gel de polyacrylamide , Protéines de l'oeil/métabolisme , Femelle , Glyceraldehyde 3-phosphate dehydrogenase (NADP+)/métabolisme , Humains , Appareil lacrymal/métabolisme , Adulte d'âge moyen , ARN/métabolisme , Réaction de polymérisation en chaine en temps réel , Glande submandibulaire/métabolisme , Donneurs de tissusRÉSUMÉ
PURPOSE: Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. METHODS: The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. RESULTS: There was a 2.5- and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. CONCLUSIONS: We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands.