Lacrimal gland homeostasis is maintained by the AQP5 pathway by attenuating endoplasmic reticulum stress inflammation in the lacrimal gland of AQP5 knockout mice

Abstract

Purpose: AQP5^-/- mice spontaneously exhibit dry eye symptoms. The purpose of this study was to assess the endoplasmic reticulum (ER) stress-mediated inflammation generated by a deficiency of aquaporin 5 (AQP5) in the lacrimal gland.

Methods: Hematoxylin and eosin (H&E) staining, Oil Red O staining, and transmission electron microscopy (TEM) analysis were performed to identify structural changes in lacrimal gland epithelial cells because of AQP5 deficiency. Corneal epithelial defects were assessed with sodium fluorescein staining. The expression profiles of mRNA and proteins were determined by quantitative real-time reverse transcription PCR (qRT-PCR) and western blot. Mice in the quercetin group were injected intraperitoneally with 40 mg/kg of quercetin, and the control group was injected with an equal volume of dimethyl sulfoxide (DMSO) for 4 weeks.

Results: Aqueous tear secretion fell at about 50% in 1- and 6-month-old AQP5^-/- mice compared with that of AQP5^+/+ mice. TEM showed that the ER structure was damaged. ER stress was significantly increased in the lacrimal gland of AQP5^-/- mice. Lipid droplets accumulated in the matrix and acinar cells, and changes occurred in the lipid metabolism and gene expression levels for PPARα, CPT1α, and CPT2 in the AQP5^-/- mice. Immune cell infiltration and increases in the gene expression levels of the chemokines CXCL1, CXCL2, and CCL5 were found in the lacrimal gland of AQP5^-/- mice. Quercetin partially reversed ER stress levels, inflammation, and lipid accumulation, and it inhibited tear secretion.

Conclusions: The study data indicated that a deficiency of AQP5 induced pathophysiological changes and functional decompensation of the lacrimal gland. Quercetin may improve the inflammation in the lacrimal glands of AQP5^-/- mice by regulating the ER stress levels.

Figure 1

AQP5 deficiency induced dry eye–like characteristics. A: Immunofluorescence staining showed the expression of AQP5 in the lacrimal gland cell location (green: AQP5, blue: 4’6-diamidino-2-phenylindole [DAPI]). B: Expression of AQP5 and GAPDH were examined by western blotting. C: Bodyweight (n = 10 samples). D: Lacrimal gland weight and E: appearance (n = 8 samples). F: Tear secretion of AQP5^+/+ mice and AQP5^−/− mice. G: Sodium fluorescein staining of AQP5^+/+ mice and AQP5^−/− mice. Scale bars: (A) 40 μm. * p<0.05, ** p<0.01, *** p<0.0001.

Figure 2

AQP5 deficiency induced ultrastructural abnormalities and endoplasmic reticulum (ER) stress in the lacrimal gland. A: Transmission electron microscopy (TEM) showing the ultrastructure of the acinar cells of the lacrimal gland. Data are expressed as mean ± standard deviation (SD). *p<0.05, **p<0.001. Scale bars: (A) 5 μm, 2 μm. B: The deficiency of AQP5 protein induced endoplasmic reticulum stress in lacrimal glands of mice. Western blot bands for CHOP, GRP78, Caspase12, Bax, Bcl-2, and GAPDH (n = 3 samples). C: Quantified intensities of western blot bands for CHOP, GRP78, Caspase12, Bax, and Bcl-2 compared with GAPDH (n = 3 samples). *p<0.05, **p<0.01, *** p<0.0001.

Figure 3

AQP5^−/− induced abnormal lipid metabolism in the lacrimal gland. A: Oil Red O (ORO) staining (n = 6 samples). B: ORO staining intensity analyzed by ImageJ software (n = 6 samples). C,D,E: Real-time PCR analyzed the expression of PPARα, CPT1α, and CPT2 (n = 3 samples). *p<0.05, **p<0.001, two-way analysis of variance (ANOVA). Scale bars: (A) 40 μm. * p<0.05, ** p<0.01, *** p<0.0001.

Figure 4

AQP5 deficiency induced inflammation in the lacrimal gland. A: Hematoxylin and eosin (H&E) staining showed morphology changes. CD4⁺: Immunofluorescence staining of CD4⁺ demonstrated CD4⁺ T cells (red: CD4⁺, blue: 4’6-diamidino-2-phenylindole [DAPI]). Ly6g: Immunofluorescence staining of Ly6g showed neutrophils (red: Ly6g, blue: DAPI). F4/80: Immunofluorescence staining of F4/80 showed macrophages (red: F4/80, blue: DAPI). B: ImageJ immunofluorescence profile analysis showed the CD4⁺, Ly6g, and F4/80 positive scores (n = 6 samples). C: Real-time PCR results exhibited chemokine and proinflammatory factors (n = 3 samples). *p<0.05, **p<0.001, two-way analysis of variance (ANOVA). Scale bars: (A) 40 μm. *p<0.05, **p<0.01, ***p<0.0001.

Figure 5

AQP5 deficiency induced structural damage to lacrimal gland myoepithelial cells (MECs) in mice. A: Immunofluorescence staining of alpha-smooth muscle actin (α-SMA) showed myoepithelium morphology (green: α-SMA; blue: 4’6-diamidino-2-phenylindole [DAPI]). B: ImageJ and GraphPad were used to calculate the result of A (n = 6 samples). C: Western blotting showed the expression level of α-SMA and GAPDH in lacrimal glands. D: ImageJ and GraphPad were used to compute the result of C (n = 3 samples). Data were expressed as mean ± standard deviation (SD). *p<0.05, **p<0.001 two-way analysis of variance (ANOVA). Scale bars: (A) 10 μm, 2 μm. (D) 40 μm.

Figure 6

Quercetin relieved endoplasmic reticulum (ER) stress and abnormal lipid metabolism in AQP5^−/− mice. A: Transmission electron microscopy (TEM) shows the ultrastructure of the acinar cells. B: Western blot bands for CHOP, GRP78, Caspase12, Bax, Bcl-2, and GAPDH. C: Quantified intensities of western blot bands for CHOP, GRP78, Caspase12, Bax, and Bcl-2 compared with GAPDH (n = 3 samples). D: Oil Red O (ORO) staining. E: ORO staining intensity was analyzed by ImageJ software (n = 6 samples). F: Real-time PCR analysis showed PPARα, CPT1α, and CPT2 expression (n = 3 samples). *p<0.05, **p<0.01, **” in line 10 on page 22 to “*p<0.05, **p<0.001, ***p<0.0001.

Figure 7

Quercetin alleviated lacrimal gland inflammation caused by AQP5 deficiency. A: Hematoxylin and eosin (H&E) staining showed morphological changes. CD4⁺: Immunofluorescence staining of CD4+ demonstrated CD4⁺ T cells (red: CD4⁺, blue: 4’6-diamidino-2-phenylindole [DAPI]). Ly6g: Immunofluorescence staining of Ly6g showed neutrophils (red: Ly6g, blue: DAPI). F4/80: Immunofluorescence staining of F4/80 showed macrophages (red: F4/80, blue: DAPI). B: ImageJ immunofluorescence profile analysis showed the CD4⁺ positive score, Ly6g positive score, and mean fluorescence intensity of F4/80 (n = 6 samples). C: Immunofluorescence staining of alpha-smooth muscle actin (α-SMA) showed myoepithelium morphology (green: α-SMA, blue: DAPI), and ImageJ and GraPade were used to compute the result (n = 6 samples). D: Western blotting showed the expression level of α-SMA and GAPDH (n = 3 samples). E: Real-time PCR exhibits expression of chemokine and proinflammatory factors (n = 3 samples). F: The lacrimal secretion of AQP5^−/− mice was measured using the phenol red cotton thread method. G: Sodium fluorescein staining in the cornea. *p<0.05, **p<0.001. Independent sample t test. Scale bars: (A): 100 μm, 40 μm. *p<0.05, **p<0.01, ***p<0.0001.