Aquaporin gene therapy corrects Sjögren's syndrome phenotype in mice

Abstract

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.

Keywords: Sjögren’s syndrome; aquaporin; gene therapy.

Fig. S1.

Correlation analysis of BMP6 expression and unstimulated saliva output. BMP6 expression in individuals with pSS (n = 12) was measured by quantitative PCR relative to GAPDH [ΔΔCt (Delta Delta Ct)] and is presented as fold change over the average expression in HVs (n = 11). Spearman rank correlation analysis indicates there is a significant negative correlation between BMP6 and salivary fluid output in patients (r = −0.78, P = 0.0031).

Fig. 1.

BMP6 inhibits AQP5 expression in patients with Sjögren’s syndrome and HSG cells. (A) HSG cells were cultured without (Upper) or with (Lower) 6 ng/mL BMP6 for 3 d. Actin expression was detected with phalloidin conjugated to TRITC (red fluorescence). AQP5 expression was detected by conjugation of a specific antibody to AQP5 with FITC (green fluorescence). (B) Expression of AQP5 in MSGs of patients with pSS with high BMP6 (n = 5) and low BMP6 (n = 2) expression compared with tissue from HVs (n = 2). (Scale bar, 50 μm.)

Fig. S2.

BMP6 and AQP5 expression in MSGs from patients with pSS and HVs. Stitched immunofluorescence images of BMP6 and AQP5 in the entire biopsied sample area are shown. (Scale bar, 300 μm.)

Fig. 2.

BMP6 inhibits water permeability of HSG cells. (A) Cells were placed in the hypotonic solution following culture without (black line) or with (blue line, 0.1 ng/mL; red line, 6 ng/mL; green line, 150 ng/mL) different concentrations of BMP6. Ft/Fo, cell volume change calculated on fluorescence intensity base on 100% recovery rate of the cell volume. (B) Dosage response curve of BMP6 induces cell volume change. The 6-ng/mL dose shows significant inhibition of recovery of cell volume change (n = 3). Data are presented as mean ± SEM.

Fig. S3.

BMP6 has a bimodal effect on gene expression in HSG cells. (A) Heat map of positive probes as well as unsupervised clustering of samples. Unsupervised clustering of treated HSG cells shows clustering of the group treated with 0.1 ng/mL with the control samples followed by the group treated with 150 ng/mL. The most significant number of gene changes was observed within the 6-ng/mL treatment group. (B) Bimodal effect of gene expression in HSG cells in response to treatment with increasing concentrations of BMP6 at dosages of 0.1 ng/mL, 6 ng/mL, and 150 ng/mL. Analysis of the patterns of gene expression at the different concentrations of BMP6 identified six distinct profiles. Brown and green lines represent genes following the mirror image pattern of each other. (C) Quantitative PCR (qPCR) detection of selected genes extracted from HSG cells after BMP6 treatment with 6 ng/mL shows agreement with the results of the microarray study on samples from patients with pSS. The results obtained using the custom microarray platform were validated by qPCR and compared with microarray data from patients with pSS, which showed a similar trend in expression. CATSPER-2, cation channel sperm associated 2.

Fig. 3.

Aquaporins restore water permeability in BMP6 treated cells. HSG cells were placed in the hypotonic solution following culture without (purple line) or with 6 ng/mL (red line) BMP6 and then transfected with increasing amounts of DNA encoding AQP5 (A and B) or AQP1 (C and D), and the recovery of RVD was measured. No increase in recovery of RVD activity was observed with transfection of pUC19 (Puc) alone (A and B, orange line). Increasing recovery of RVD was observed with increasing amounts of both AQP5 and AQP1. (B and D) Maximal percent of RVD was determined compared with HSG cells cultured with media alone. Data are presented as mean ± SEM.

Fig. 4.

AQP1 restores fluid secretion in mouse models of Sjögren’s syndrome. (A) Six- to eight-week-old female C57/B6 mice were locally treated with AAV5-BMP6 vector delivered via cannulation to the submandibular gland. After 4 wk, mice were randomly divided into two groups and treated with either an AAV2 vector encoding GFP (n = 8) or AQP1 (n = 7). Four weeks after AAV2-GFP or AQP1 transduction, the pilocarpine-stimulated SFR was measured and compared with an age- and gender-matched control group of C57/B6 mice (n = 9). SFR is adjusted to body weight due to variability at this young age. (B) AQP1 immunofluorescent staining of the submandibular gland tissue obtained from controls or mice treated with AAV2-AQP1 vector. Images are from representative mice. (Magnification, 40×.) (C) Forty-six-week-old C57BL/6.NOD-Aec1/Aec2 mice were measured for stimulated salivary gland activity 16 wk after vector delivery of an AAV2 vector encoding either GFP or AQP1 (n = 4 per group). (D) Change in pilocarpine-stimulated tear flow in C57BL/6.NOD-Aec1/Aec2 mice treated with either AAV2-GFP or AAV2-AQP1 at 18 wk after vector delivery (n = 4 per group). C57BL/6.NOD-Aec1/Aec2 mouse saliva and tear volumes are relative to the baseline value of the mouse before treatment with vector. All data are mean ± SEM and analyzed by an unpaired Student t test.

Fig. 5.

Aquaporin expression in salivary glands inhibits inflammation. The lymphocytes isolated from submandibular salivary glands of AAV2-AQP1– or AAV2-GFP–treated C57BL/6.NOD-Aec1/Aec2 mice were analyzed by flow cytometry assay for the different populations of cells (n = 2 per group). Data show one representative experiment. A statistical data analysis on all of the analyzed mice is provided in Table S3.