Fig 1.
Chloroquine deactivate cathepsin B in LAMP3-overexpressing cells.
Stably LAMP3-overexpressing A253 cells were treated with chloroquine (CQ) for 24 hours at increasing concentrations. (A) Molecular mechanism about LAMP3-induced apoptotic cell death. (B) Relative change in CTSB expression (n = 4). C: control A253 cells. (C) Representative Western blots showing expression of cathepsin B and α-tubulin (control). Bar chart shows relative change of cathepsin B expression normalized by α-tubulin expression compared to control A253 cells (n = 3). *P < 0.05, N.S. = not significant (Student’s t-tests with Dunnett’s correction).
Fig 2.
Chloroquine prevents LAMP3-induced apoptotic cell death in vitro.
Stably LAMP3-overexpressing A253 cells were treated with chloroquine (CQ) for 24 hours at increasing concentrations. (A) Representative Western blots showing expression of LAMP1 and α-tubulin (control). Bar chart shows relative change of LAMP1 expression normalized by α-tubulin expression compared to control A253 cells (n = 3). C: control A253 cells. (B) Representative immunofluorescent images showing staining for galectin-3 (magenta) (40× magnification). Bar chart shows percentage of galectin-3 (Gal3) puncta-positive cells (n = 3). (C) Number of apoptotic cells was determined by flow cytometry using APC Annexin V/7-AAD. Bar chart shows percentage of difference in Annexin V-positive cells from control (n = 5). Values shown are mean ± SD. *P < 0.05, **P < 0.01, N.S. = not significant (Student’s t-tests with Dunnett’s correction).
Fig 3.
Hydroxychloroquine treatment decreases LAMP3-induced cell death in vivo.
Submandibular glands of C57BL/6 mice were instilled with AAV2-LAMP3. After 4 months, mice were given weekly intraperitoneal injections of 60 mg/kg HCQ (n = 4) or placebo (n = 5) for 3 months. (A) Representative submandibular gland tissue sections showing staining for LAMP1 (green) and galectin-3 (magenta) (40× magnification) or terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL, red) (20× magnification). (B) Bar chart shows mean fluorescence intensity of LAMP1 expression. (C) Bar chart shows percentage of galectin-3 (Gal3) puncta-positive cells. (D) Bar chart shows percentage of TUNEL-positive epithelial cells. Values shown are mean ± SD. *P < 0.05, **P < 0.01, N.S. = not significant (Student’s t-tests).
Fig 4.
Hydroxychloroquine treatment ameliorates LAMP3-associated sialadenitis in vivo.
Submandibular glands of C57BL/6 mice were instilled with AAV2-LAMP3. After 4 months, mice were given weekly intraperitoneal injections of 60 mg/kg HCQ (n = 4) or placebo (n = 5) for 3 months. (A) Representative submandibular gland tissue sections showing hematoxylin & eosin (HE) staining. Scale bars = 1 mm (inset: 100 μm). Bar charts shows the number of lymphocytic foci and the average size of lymphocytic infiltration areas in both the submandibular glands of each mouse. (B) Graph showing pilocarpine-stimulated salivary flow rate per body weight in 20 minutes. The dashed line indicates a reference level of salivary flow rate derived from control mice (instilled with AAV2-GFP). (C) Bar charts showing serum anti-Ro/SSA or anti-La/SSB antibody levels. Values shown are mean ± SD. *P < 0.05, N.S. = not significant (Student’s t-tests).
Fig 5.
Clinical response to hydroxychloroquine in SjD patients is associated with salivary gland LAMP3 mRNA expression level.
(A) Flow diagram showing SjD patient selection. (B) Dot plot showing relative expression of LAMP3 with mean and 95% confidence interval in labial minor salivary glands from SjD patients (n = 14) and healthy volunteers (HV, n = 10). (C) Dot plot showing baseline ESSDAI and change in ESSDAI after HCQ treatment. ΔESSDAI ≥ 3 is considered as minimal clinically important improvement (MCII). Continuous line represents complete response (ESSDAI = 0) to HCQ treatment. (D) Bar chart showing patient-reported symptoms after HCQ treatment. ESSPRI < 5 is considered as satisfactory control of SjD symptoms. (E) Graphs showing serum gamma globulin levels before and after HCQ treatment. Dotted lines show upper limit of normal range. *P < 0.05, **P < 0.01, N.S. = not significant (paired Student’s t-test for quantitative variables or Chi-squared test for categorical variables).
Table 1.
Baseline profile of SjD patients treated with hydroxychloroquine, stratified by minor salivary gland LAMP3 mRNA expression.