Naringenin Regulates Lipopolysaccharide-Induced Abnormal Airway Surface Liquid Secretion

Airway surface liquid (ASL) is one of the key factors affecting the respiratory system's physiological function. Abnormal ASL secretion can increase the incidence of various respiratory diseases. Lipopolysaccharide (LPS) stimulation can damage the airway epithelial barrier, affect the concentration of ASL contents, and down-regulate ion channel expression, which in turn causes abnormal ASL secretion. Naringenin, which exists in many Citrus foods, has the ability to promote airway surface liquid secretion. This work is designed to investigate the regulatory mechanism of naringenin on LPS-induced abnormal ASL secretion. The effects of naringenin and LPS on the viability of Calu-3 cells were measured by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS). ASL secretion volume was measured by a micropipette on air–liquid interface cultured cells. The concentration of Cl−, Na+, lysozyme, and total protein in ASL were respectively measured by assay kits. The mRNA expressions were determined by quantitative real-time polymerase chain reaction, and proteins were measured by enzyme-linked immunosorbent assay. The results indicated that LPS could affect ASL secretion and regulate cystic fibrosis transmembrane conductance regulator (CFTR), aquaporin 1 (AQP1) and aquaporin 5 (AQP5) expression. Naringenin had the ability to regulate the ASL secretion by increasing secretion volume, and Cl− and Na+ concentrations, reducing lysozyme and total protein content, and regulating CFTR, AQP1, and AQP5 expression. This study indicated that naringenin had regulating effects to attenuate LPS-induced abnormal ASL secretion.

FoxO1 as a Regulator of Aquaporin 5 Expression in the Salivary Gland

Forkhead box O1 (FoxO1) is a multifunctional initiator, mediator, and repressor of autoimmune diseases in an organ- or disease-specific manner. However, the role of FoxO1 in the salivary gland has not yet been elucidated. In this study, we discovered that FoxO1 and aquaporin 5 (AQP5) are both significantly downregulated in the patients with primary Sjögren syndrome, an autoimmune disease accompanying salivary gland dysfunction. Pharmacologic or genetic perturbation of FoxO1 in the rat salivary gland acinar cell line, SMG-C6, induced a significant downregulation of AQP5 expression, as observed in clinical specimens. There was a strong correlation between FoxO1 and AQP5 expression because FoxO1 is a direct regulator of AQP5 expression in salivary gland acinar cells through its interaction with the promoter region of AQP5. Serial injection of a FoxO1 inhibitor into mice induced a reduction of AQP5 expression in submandibular glands and, consequently, hyposalivation, which is one of the major clinical symptoms of primary Sjögren syndrome. However, there was no sign of inflammation or cell damage in the submandibular glands harvested from mice treated with the FoxO1 inhibitor. In conclusion, our findings indicate that FoxO1 in salivary gland tissue acts as a direct regulator of AQP5 expression. Thus, downregulation of FoxO1 observed in primary Sjögren syndrome is a putative mechanism for hyposalivation without the involvement of previously reported soluble factors in primary Sjögren syndrome patient sera.

Aquaporin-5 regulation of cell-cell adhesion proteins: an elusive "tail" story

Aquaporins (AQPs) are water channels that facilitate transport of water across cellular membranes. AQPs are overexpressed in several cancers. Especially in breast cancer, AQP5 overexpression correlates with spread to lymph nodes and poor prognosis. Previously, we showed that AQP5 expression reduced cell-cell adhesion by reducing levels of adherens and tight junction proteins (e.g., ZO1, plakoglobin and β-catenin) at the actual junctions. Here, we show that when targeted to the plasma membrane, the AQP5 C-terminal tail domain regulated junctional proteins. Moreover, that AQP5 interacted with ZO1, plakoglobin, β-catenin and desmoglein-2, which were all reduced at junctions upon AQP5 overexpression. Thus, our data suggest that AQP5 mediates the effect on cell-cell adhesion via interactions with junctional protein independently of AQP5 mediated water transport. AQP5 overexpression in cancers may thus contribute to carcinogenesis and cancer spread by two independent mechanisms: reduced cell-cell adhesion, a characteristic of epithelial-mesenchymal transition, and increased cell migration capacity via water transport.

AB0133 INCREASED SOLUBLE SEMAPHORIN 4D/CD100 IN THE PLASMA OF SJÖGREN’S SYNDROME AND ITS EFFECTS ON HUMAN SALIVARY GLAND CELL AND CD4+ T CELL

Background:Semaphorin 4D (SEMA4D) / CD100, known as a subfamily of axonal guidance proteins, has also been reported to act as an immunoregulator in several infectious and inflammatory diseases [1]. Sjögren’s syndrome (SS) is a systemic autoimmune disease that primarily affects the exocrine glands by infiltrated lymphocytes resulting in dryness of mouth and eyes. IL-17 was reported to impair the integrity of tight junction barrier and attenuate the expression of aquaporin 5 (AQP5), causing salivary gland dysfunction in SS [2].Objectives:This study was aimed to evaluate the role of SEMA4D in patients with SS and investigate the effect of SEMA4D on human salivary gland epithelial cell (SGEC) and T cell.Methods:Soluble SEMA4D levels in plasma were measured by enzyme-linked immunosorbent assay (ELISA) from patients with SS, non-SS sicca and healthy controls. Immortalized human SGECs, originated from acini (NS-SV-AC) and duct (NS-SV-DC), were used to evaluate the effects of SEMA4D. CD4+T cells from human peripheral blood were isolated to determine the secretion of cytokines in response to SEMA4D. IFN-γ and IL-17 were used to determine the effects on AQP5 expression of SGEC.Results:The levels of soluble SEMA4D in plasma were increased in patients with SS (median [interquartile range]: 1221.3 [393.5] pg/mL) compared to non-SS sicca (940.2 [355.1] pg/mL,p= 0.006) or healthy controls (909.5 [108.0] pg/mL,p <0.0001). The levels of soluble SEMA4D in plasma were correlated with the levels of several autoantibodies including anti-SSA (Spearman’s rho = 0.358,p= 0.006), anti-SSB (rho = 0.350,p= 0.007), and anti-muscarinic receptor 3 (M3R) Ab (rho = 0.495,p< 0.001), and also correlated with total IgG (rho = 0.431,p= 0.002). SEMA4D-stimulated SGECs showed decreased expression of tight junctions such as occludin and Zo-1. CD4+T cells secreted IFN-γ (p= 0.025), IL-17 (p= 0.028), and IL-21 (p= 0.007) with SEMA4D stimulation. IFN-γ and IL-17 decreased AQP5 expression in SGECs.Conclusion:SEMA4D contributed to decreased expression of tight junction in SGECs. SEMA4D induced production of IFN-γ and IL-17 in CD4+T cells and these cytokine decreased AQP5 expression in SGECs.References:[1]Worzfeld T, Offermanns S. Nat Rev Drug Discov. 2014;13(8):603-21.[2]Bhattarai KR, Junjappa R, Handigund M, Kim HR, Chae HJ. Autoimmun Rev. 2018;17(4):376-90.Disclosure of Interests:None declared

Suppression of high-mobility group box 1 ameliorates xerostomia in a Sjögren syndrome-triggered mouse model

Xerostomia is a self-conscious symptom. High-mobility group box 1 (HMGB1) promotes pro-inflammatory effects in many diseases. This study aimed to clarify the role of HMGB1 in Sjögren syndrome (SS)-triggered xerostomia. Nonobese diabetic (NOD)/Ltj mice were used to establish an SS-triggered xerostomia model. The results showed that saliva production was decreased and anti-Sjögren syndrome B (anti-SSB) level was increased in SS. PCR, Western blot, and immunohistochemistry experiments indicated that the HMGB1 and aquaporin 5 (AQP5) levels were enhanced and diminished in SS compared with those in the control, respectively. While the mice were treated with anti-HMGB1, xerostomia was reversed due to the elevated saliva production and reduced anti-SSB level. In addition, it was found that the inhibition of HMGB1 restrained the toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) axis activation. The TLR4 and p-IκB levels were alleviated, while the IκBα and NF-κB p65 levels were augmented. The NF-κB p65 binding activity was attenuated via the electrophoretic mobility shift assay (EMSA) after anti-HMGB1 treatment. Moreover, the repression of HMGB1 facilitated the expression of AQP5. These findings demonstrate that suppression of HMGB1 ameliorates SS-triggered xerostomia via suppressing the HMGB1/TLR4/NF-κB signaling pathway and upregulating AQP5 expression.

Alpha-Lipoic Acid Ameliorates Radiation-Induced Salivary Gland Injury by Preserving Parasympathetic Innervation in Rats

Radiation therapy is a standard treatment for patients with head and neck cancer. However, radiation exposure to the head and neck induces salivary gland (SG) dysfunction. Alpha lipoic acid (ALA) has been reported to reduce radiation-induced toxicity in normal tissues. In this study, we investigated the effect of ALA on radiation-induced SG dysfunction. Male Sprague–Dawley rats were assigned to the following treatment groups: control, ALA only (100 mg/kg, intraperitoneally), irradiation only, and ALA administration 24 h or 30 min prior to irradiation. The neck area, including SGs, was irradiated evenly at 2 Gy/min (total dose, 18 Gy) using a photon 6 MV linear accelerator. The rats were sacrificed at 2, 6, 8, and 12 weeks after irradiation. Radiation decreased SG weight, saliva secretion, AQP5 expression, parasympathetic innervation (GFRα2 and AchE expression), regeneration potentials (Shh and Ptch expression), salivary trophic factor levels (brain-derived neurotrophic factor and neurturin), and stem cell expression (Sca-1). These features were restored by treatment with ALA. This study demonstrated that ALA can rescue radiation-induced hyposalivation by preserving parasympathetic innervation and regenerative potentials.

Dynamics of Salivary Gland AQP5 under Normal and Pathologic Conditions

Aquaporin 5 (AQP5) plays an important role in the salivary gland function. The mRNA and protein for AQP5 are expressed in the acini from embryonic days E13-16 and E17-18, respectively and for entire postnatal days. Ligation-reopening of main excretory duct induces changes in the AQP5 level which would give an insight for mechanism of regeneration/self-duplication of acinar cells. The AQP5 level in the submandibular gland (SMG) decreases by chorda tympani denervation (CTD) via activation autophagosome, suggesting that its level in the SMG under normal condition is maintained by parasympathetic nerve. Isoproterenol (IPR), a β-adrenergic agonist, raised the levels of membrane AQP5 protein and its mRNA in the parotid gland (PG), suggesting coupling of the AQP5 dynamic and amylase secretion-restoration cycle. In the PG, lipopolysaccharide (LPS) is shown to activate mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalings and potentially downregulate AQP5 expression via cross coupling of activator protein-1 (AP-1) and NF-κB. In most species, Ser-156 and Thr-259 of AQP5 are experimentally phosphorylated, which is enhanced by cAMP analogues and forskolin. cAMP-dependent phosphorylation of AQP5 does not seem to be markedly involved in regulation of its intracellular trafficking but seems to play a role in its constitutive expression and lateral diffusion in the cell membrane. Additionally, Ser-156 phosphorylation may be important for cancer development.

Pituitary Hormones (FSH, LH, PRL, and GH) Differentially Regulate AQP5 Expression in Porcine Ovarian Follicular Cells

This study aimed to examine the effect of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) on Aquaporin 5 (AQP5) expression in granulosa (Gc) and theca cells (Tc) from medium (MF) and large (LF) ovarian follicles of pigs. The results showed that GH significantly decreased the expression of AQP5 in Gc from MF in relation to the control. In the Gc of large follicles, PRL stimulated the expression of AQP5. However, the increased expression of AQP5 in the Tc of LF was indicated by GH and PRL in relation to the control. A significantly higher expression of the AQP5 protein in the Gc from MF and LF was indicated by FSH and PRL. In co-cultures, an increased expression of AQP5 was observed in the Gc from LF incubated with LH, PRL, and GH. A significantly increased expression of AQP5 was also observed in co-cultures of Tc from all type of follicles incubated with LH, whereas PRL stimulated the expression of AQP5 in Tc from MF. Moreover, AQP5 protein expression increased in the co-culture isolated from MF and LF after treatment with FSH, LH, PRL, and GH. AQP5 immunoreactivity was observed in the cytoplasm, mainly in the perinuclear region and endosomes, as well as in the cell membranes of Gc and Tc from the LF and MF.

Blockade of CD40–CD154 pathway interactions suppresses ectopic lymphoid structures and inhibits pathology in the NOD/ShiLtJ mouse model of Sjögren’s syndrome

ObjectiveTo examine the role of CD40–CD154 costimulation and effects of therapeutic pathway blockade in the non-obese diabetic (NOD/ShiLtJ) model of Sjögren’s syndrome (SS).MethodsWe assessed leucocyte infiltration in salivary glands (SGs) from NOD/ShiLtJ mice by immunohistochemistry and examined transcriptomics data of SG tissue from these animals for evidence of a CD40 pathway gene signature. Additionally, we dosed MR1 (anti-CD154 antibody) in NOD mice after the onset of SS-like disease and examined the effects of MR1 treatment on sialadenitis, autoantibody production, SG leucocyte infiltration, gene expression downstream of CD40 and acquaporin 5 (AQP5) expression.ResultsWe could detect evidence of CD40 expression and pathway activation in SG tissue from NOD mice. Additionally, therapeutic treatment with MR1 suppressed CD40 pathway genes and sialadenitis, inhibited ectopic lymphoid structure formation and autoantibody production, as well as decreased the frequency of antibody-secreting cells in SGs but had minimal effects on AQP5 expression in NOD/ShiLtJ SGs.ConclusionCD40–CD154 interactions play an important role in key pathological processes in a mouse model of SS, suggesting that blockade of this costimulatory pathway in the clinic may have beneficial therapeutic effects in patients suffering from this autoimmune exocrinopathy.