L. Zhang

Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.

ABSTRACT The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway. Highlighted Article: The epithelial cholinergic system activates a signaling pathway that regulates paracellular permeability by modulating the content and distribution of the tight junction protein claudin-4.

Functional Vanilloid Receptor-1 in Human Submandibular Glands

Vanilloid receptor-1 (VR1) was originally found in the nervous system. Recent evidence indicates that VR1 is also expressed in various cell types. We hypothesized that VR1 exists in the human submandibular gland (SMG) and is involved in regulating salivary secretion. VR1 mRNA and protein were expressed in human SMGs and a human salivary intercalated duct cell line. VR1 was mainly located in serous acinar and ductal cells, but not in mucous acinar cells. Capsaicin, an agonist of VR1, increased intracellular free calcium, enhanced phosphorylation of extracellular signal-regulated kinase, and induced the trafficking of aquaporin 5 (AQP5) from the cytoplasm to the plasma membrane. These effects were abolished by pre-treatment with the VR1 antagonist capsazepine. Furthermore, capsaicin cream applied to the skin covering the submandibular area increased salivary secretion. These findings indicated that a functional VR1 is expressed in the human SMG and is involved in regulating salivary secretion by mediating AQP5 trafficking.

Interleukin-17 Impairs Salivary Tight Junction Integrity in Sjögren’s Syndrome:

Sjögren’s syndrome (SS) is an inflammatory autoimmune disease that causes secretory dysfunction of the salivary glands. It has been reported that proinflammatory cytokine interleukin-17 (IL-17) was elevated and tight junction (TJ) integrity disrupted in minor salivary glands from SS patients. However, whether the elevated IL-17 in SS affects TJ integrity and thereby alters the function of salivary gland is unknown. Here, by using nonobese diabetic (NOD) mice as SS model, we found that the stimulated salivary flow rate was significantly decreased in NOD mice. Lymphocyte infiltration was mainly observed in submandibular glands (SMGs), but not parotid glands (PGs), of NOD mice. IL-17 was significantly increased and mainly located in lymphocytic-infiltrating regions in SMGs but not detectable in PGs of NOD mice. Meanwhile, the epithelial barrier function was disrupted, as evidenced by an increased paracellular tracer clearance and an enlarged acinar TJ width in SMGs of NOD mice. Furthermore, claudin-1 and -3 were elevated especially at the basolateral membranes, whereas claudin-4, occludin, and zonula occludens–1 (ZO-1) were reduced in SMGs of NOD mice. Moreover, occludin and ZO-1 were dispersed into cytoplasm in SMGs of NOD mice. However, no change in the expression and distribution of TJ proteins was found in PGs. In vitro, IL-17 significantly decreased the levels and apical staining of claudin-4 and ZO-1 proteins in the cultured SMG tissues, as well as claudin-1, occludin, and ZO-1 in PG tissues. Moreover, IL-17 activated the phosphorylation of IκBα and p65 in SMG cells, whereas pretreatment with NF-κB inhibitor pyrrolidine dithiocarbamate suppressed the IL-17-induced downregulation of claudin-4 and ZO-1 in SMG tissues. Taken together, these findings indicate that IL-17 derived from infiltrating lymphocyte impairs the integrity of TJ barrier through NF-κB signaling pathway, and thus might contribute to salivary gland dysfunction in SS.

ZO-1 and -2 Are Required for TRPV1-Modulated Paracellular Permeability

The tight junction–based paracellular pathway plays an important role in saliva secretion. Zonula occludens (ZO) proteins are submembranous proteins of tight junction complex; however, their function in salivary epithelium is poorly understood. Here, we found that activation of transient receptor potential vanilloid subtype 1 (TRPV1) by capsaicin increased rat saliva secretion both in vivo and ex vivo. Meanwhile, TRPV1 activation enlarged the width of tight junctions between neighboring acinar cells, increased the paracellular flux of 4-kDa fluorescein isothiocyanate (FITC)-dextran in submandibular gland (SMG) tissues, and decreased transepithelial electric resistance (TER) in SMG-C6 cells. ZO-1, -2, and -3 were distributed principally to the apical lateral region of acinar cells in SMG tissues and continuously encircled the peripheries of SMG-C6 cells in the untreated condition. TRPV1 activation obviously diminished ZO-1 and -2 staining, but not ZO-3 or β-catenin, at the cell-cell contacts ex vivo and in vitro. Moreover, in untreated SMG-C6 cells, ZO-1 and -2 single or double knockdown by small interfering RNA (siRNA) increased the paracellular flux of 4-kDa FITC-dextran. In capsaicin-treated cells, ZO-1 and -2 single or double knockdown abolished, whereas their re-expression restored, the capsaicin-induced increase in paracellular permeability. Furthermore, TRPV1 activation increased RhoA activity, and inhibition of either RhoA or Rho kinase (ROCK) abolished the capsaicin-induced TER decrease as well as ZO-1 and -2 redistribution. These results indicate that ZO-1 and -2 play crucial roles in both basal salivary epithelial barrier function and TRPV1-modulated paracellular transport. RhoA-ROCK signaling pathway is responsible for TRPV1-modulated paracellular permeability as well as ZO-1 and -2 redistribution.

Expression of interleukin-17 in primary Sjögren's syndrome and the correlation with disease severity: A systematic review and meta-analysis

The aberrant expression of interleukin‐17 (IL‐17) has been reported in primary Sjögrens syndrome (pSS). Abnormalities in IL‐17 can promote the production of pro‐inflammatory cytokines and aggravate autoimmune disorders. The aim of this study was to investigate alterations of IL‐17 in patients with pSS and explore the correlation between IL‐17 and disease severity. Eight databases were searched for original studies reporting the expression of IL‐17 in patients with pSS and controls. Eligible reports were included in the pooled analysis, and subgroup evaluations were performed according to different types of controls and IL‐17 measurement methods. Newcastle‐Ottawa Scale criteria were used to assess the risk of bias of the included studies. In total, 45 articles are included in the meta‐analysis. The expression of IL‐17 is significantly increased in patients with pSS compared to controls. Furthermore, patients with pSS without immunosuppressive treatment show markedly higher IL‐17 levels. In addition, patients with pSS with positive rheumatoid factors tend to express a higher level of IL‐17 than patients with negative rheumatoid factors. Negative correlations between IL‐17 levels and ocular parameters are also found in patients with pSS. The results are similar after adjustment by “trim and fill” methods. In conclusion, the expression of IL‐17 is obviously increased in patients with pSS, especially among those without immunosuppressive treatment. In addition, IL‐17 level correlates with the disease severity of pSS. These findings demonstrate the significance of IL‐17 overexpression in patients with pSS and may provide insights for the development of therapeutic interventions targeting IL‐17 for pSS.

Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sjögren's syndrome

Sjögrens syndrome (SS) is an inflammatory autoimmune disease that causes hyposecretion in salivary glands. Endothelial tight junctions (TJs) play crucial roles in salivation and barrier function of blood vessels. However, whether the alteration of endothelial TJs were involved in pathogenesis of SS was still unknown. Here, the ultrastructure and function of endothelial TJs in submandibular glands (SMGs) were detected by transmission electron microscopy and in vivo paracellular permeability assay in different aged NOD mouse model for SS. CFSE-labeled lymphocytes were injected into tail vein to trace the infiltration, while claudin-5 expression and distribution were detected by immunofluorescence, qRT-PCR, and western blot. Results showed that the stimulated salivary flow rate was gradually decreased and lymphocytic infiltration was found as age increased in 12- and 21-week-old NOD mice, but not 7-week-old NOD mice. Blood vessels were dilated, while endothelial TJ width and paracellular tracer transport were increased in 12-week-old NOD mice. Moreover, the injected CFSE-labeled lymphocytes were observed in SMGs of 12-week-old NOD mice. Claudin-5 level was increased and relocalized from the apical portion of neighboring endothelial cells to lateral membranes and cytoplasm in 12-week-old NOD mice. Additionally, the alteration of claudin-5 expression and distribution was further confirmed in labial salivary glands and bilateral parotid glands from SS patients. In cultured human microvessel endothelial cell line (HMEC-1), IFN-γ stimulation significantly increased claudin-5 expression. Taken together, we identified that the endothelial TJ barrier was disrupted and contributed to the development of salivary hyposecretion and lymphocytic infiltration in SS.

AB0144 Interleukin-17 Impairs Salivary Tight Junctions in Non-Obese Diabetic Mice

Background Sjögrens syndrome (SS) is an inflammatory autoimmune disease that causes secretory dysfunction of the salivary glands. It has been reported that pro-inflammatory cytokine interleukin-17 (IL-17) was elevated and tight junction (TJ) integrity was disrupted in minor salivary gland from SS patients. The aim of the research was to observe the alteration of TJs in salivary glands of non-obese diabetic (NOD) mice and explore the impact of IL-17 on TJs. Objectives The aim of the research was to observe the alteration of TJs in salivary glands of non-obese diabetic (NOD) mice and explore the effect of IL-17 on TJs. Methods 12-week-old female NOD mice and BALB/c mice were used. Stimulated salivary flow rate and histopathological changes were evaluated. The expression of TJ molecules and IL-17 in SMG and PG were detected by real-time PCR and western blot analysis. Immunohistochemistry was used to observe the distribution of TJ molecules and IL-17 in SMGs and PGs. In addition, the alternation of TJs under the stimulation of IL-17 (50 ng/ml) were evaluated in cultured SMG and PG tissues. Results Lymphocyte infiltration was mainly observed in submandibular glands (SMGs), but not in parotid glands (PGs) of NOD mice (Fig. A). Likewise, IL-17 was significantly increased and mainly located in lymphocytic infiltrating regions in SMGs, but not detectable in PGs of NOD mice (Fig. B-D). Claudin-1 and -3 were elevated whereas claudin-4, occludin and zonula occludens-1 (ZO-1) were reduced in SMGs of NOD mice. However, no change in the expression of TJ proteins was found in PGs. In vitro, IL-17 significantly decreased the protein levels of claudin-4 and ZO-1 in the cultured SMG tissues, as well as claudin-1, occludin and ZO-1 in PG tissues. Conclusions IL-17 derived from infiltrating lymphocyte impairs the integrity of TJ, and thus might contribute to salivary gland dysfunction in SS. References Ewert P, Aguilera S, Alliende C, Kwon YJ, Albornoz A, Molina C, Urzúa U, Quest AF, Olea N, Pérez P, et al. 2010. Disruption of tight junction structure in salivary glands from Sjogrens syndrome patients is linked to proinflammatory cytokine exposure. Arthritis Rheum. 62(5):1280–1289. Katsifis GE, Rekka S, Moutsopoulos NM, Pillemer S, Wahl SM. 2009. Systemic and local interleukin-17 and linked cytokines associated with Sjögrens syndrome immunopathogenesis. Am J Pathol. 175(3):1167–1177. Nguyen CQ, Hu MH, Li Y, Stewart C, Peck AB. 2008. Salivary gland tissue expression of interleukin-23 and interleukin-17 in Sjögrens syndrome: findings in humans and mice. Arthritis Rheum. 58(3):734–743. Nguyen CQ, Yin H, Lee BH, Carcamo WC, Chiorini JA, Peck AB. 2010. Pathogenic effect of interleukin-17A in induction of Sjögrens syndrome-like disease using adenovirus-mediated gene transfer. Arthritis Res Ther. 12(6):R220. Acknowledgement This study was supported by the National Natural Science Foundation of China [grant numbers 81371163 and 81300893]. The authors declare no competing financial interests. Disclosure of Interest L.-W. Zhang Grant/research support from: National Natural Science Foundation of China [grant numbers 81371163 and 81300893], Consultant for: no, Employee of: no, Paid instructor for: no, Speakers bureau: no, X. Cong Grant/research support from: National Natural Science Foundation of China [grant numbers 81371163 and 81300893], Consultant for: no, Employee of: no, Paid instructor for: no, Speakers bureau: no, G.-Y. Yu Grant/research support from: National Natural Science Foundation of China [grant numbers 81371163 and 81300893], Consultant for: no, Employee of: no, Paid instructor for: no, Speakers bureau: no, L.-L. Wu Grant/research support from: National Natural Science Foundation of China [grant numbers 81371163 and 81300893], Consultant for: no, Employee of: no, Paid instructor for: no, Speakers bureau: no, H. Hua Grant/research support from: National Natural Science Foundation of China [grant numbers 81371163 and 81300893], Consultant for: no, Employee of: no, Paid instructor for: no, Speakers bureau: no