Chloroquine Protects Human Corneal Epithelial Cells from Desiccation Stress Induced Inflammation without Altering the Autophagy Flux

Abstract

Dry eye disease (DED) is a multifactorial ocular surface disorder affecting millions of individuals worldwide. Inflammation has been associated with dry eye and anti-inflammatory drugs are now being targeted as the alternate therapeutic approach for dry eye condition. In this study, we have explored the anti-inflammatory and autophagy modulating effect of chloroquine (CQ) in human corneal epithelial and human corneal fibroblasts cells exposed to desiccation stress, (an in-vitro model for DED). Gene and protein expression profiling of inflammatory and autophagy related molecular factors were analyzed in HCE-T and primary HCF cells exposed to desiccation stress with and without CQ treatment. HCE-T and HCF cells exposed to desiccation stress exhibited increased levels of activated p65, TNF-α, MCP-1, MMP-9, and IL-6. Further, treatment with CQ decreased the levels of active p65, TNF-α, MCP-1, and MMP-9 in cells underdesiccation stress. Increased levels of LC3B and LAMP1 markers in HCE-T cells exposed to desiccation stress suggest activation of autophagy and the addition of CQ did not alter these levels. Changes in the phosphorylation levels of MAPKinase and mTOR pathway proteins were found in HCE-T cells under desiccation stress with or without CQ treatment. Taken together, the data suggests that HCE-T cells under desiccation stress showed NFκB mediated inflammation, which was rescued through the anti-inflammatory effect of CQ without altering the autophagy flux. Therefore, CQ may be used as an alternate therapeutic management for dry eye condition.

Figures

Figure 1

Morphological and viability analysis of HCE-T, Primary HCF, and HCE cells exposed to desiccation stress. (a) Bright field images of HCE-T cells exposed to desiccation stress, with and without chloroquine (CQ) /cyclosporine (CsA) treatment under the 10X. ((A) Non-desiccated/control HCE-T cells, (B) desiccated HCE-T cells, (C) desiccated cells treated with CQ, and (D) desiccated cells treated with CsA) (Figure 1(a)). (b) Percentage of cell viability of HCE-T cells exposed to desiccation cells with and without (CQ/CsA) treatment (Figure 1(b)). (c) Bright field images of primary HCF cells exposed to desiccation stress, with and without chloroquine (CQ)/cyclosporine (CsA) treatment under the 10X objective. ((A) Non-desiccated/control HCF cells, (B) desiccated HCF cells, (C) HCF cells treated with CQ, (D) desiccated HCF cells treated with CQ, (E) HCF cells treated with CsA, and (F) desiccated cells treated with CsA) (Figure 1(c)). (d) Percentage of cell viability of HCF cells exposed to desiccation cells with and without (CQ/CsA) treatment (Figure 1(d)). (e) Bright field images of primary HCE cells exposed to desiccation stress, with and without chloroquine (CQ)/cyclosporine (CsA) treatment under the 10X objective. ((A) Non-desiccated/control primary HCE cells, (B) desiccated primary HCE cells, (C) primary HCE cells treated with CQ, (D) desiccated cells treated with CQ, (E) primary HCE cells treated with CsA, and (F) desiccated cells treated with CsA (Figure 1(e)). (f) Percentage of cell viability of primary HCE cells exposed to desiccation stress with and without (CQ/ CsA) treatment (Figure 1(f)). Data are the mean ± SD, n = 3; statistical significance was denoted (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns-nonsignificant as compared to desiccated cells). Note that --- is control, +-- are HCE/HCF cells exposed to desiccation (Des), -+- are HCE/HCF cells treated with chloroquine (CQ), ++- are desiccated HCE/HCF cells treated with chloroquine (CQ), --+ are HCE/HCF cells treated with Cyclosporine (CsA), and -++ are desiccated HCE/HCF cells treated with Cyclosporine (CsA).

Figure 2

Expression of inflammatory related genes and NFκB pathway proteins in HCE-T and HCF cells under desiccation stress. (a) The mRNA expression levels of MCP-1, MMP-9, IL-6, and TNF-α in HCE-T cells under desiccation stress (Figure 2(a)). (b) Immunoblot shows the phosphorylation status of p65 in HCE-T cells exposed to desiccation stress, with and without CQ/CsA treatment. Densitometric analysis of the blots showed the ratios of total p65 and phosphorylated p65 at (Ser536) (Figure 2(b)). (c) The mRNA expression levels of MCP-1, MMP-9, IL-6, and TNF-α in HCF cells under desiccation stress (Figure 2(c)). (d) Immunoblot shows the phosphorylation status of p65 in HCF cells exposed to desiccation with and without CQ/CsA treatment. Densitometric analysis of the blots showed the ratios of total p65 and phosphorylated p65 at (Ser536) (Figure 2(d)). Data are the mean ± SD, n = 3; statistical significance was denoted (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns- nonsignificant as compared to desiccated cells). Note that --- is control, +-- are HCE-T/HCF cells exposed to desiccation (Des), -+- are HCE-T/HCF cells treated with chloroquine (CQ), ++- are desiccated HCE-T/HCF cells treated with chloroquine (CQ), --+ are HCE-T/HCF cells treated with Cyclosporine (CsA) and -++ are desiccated HCE-T/HCF cells treated with cyclosporine (CsA).

Figure 3

Expression of p65 and IκBα proteins in HCE-T cells under desiccation stress. (a) Immunoblots of nuclear/cytoplasmic fractions shows protein expression levels of p65 (NFκB) and IκBα in HCE-T cells exposed to desiccation stress (treated with or without CQ and CsA). Densitometric analysis of the blots shows the ratios of total p65 to IκBα (Figure 3(a)). (b) GFP-RelA translocation images at 20X magnification of HCE-T cells under desiccation stress treated with/without CQ and CsA (Figure 3(b)). ((A) Non-desiccated/control HCE-T cells, (B) desiccated HCE-T cells, (C) desiccated cells treated with CQ, (D) desiccated HCE-T cells treated with CsA, (E) HCE-T cells treated with TNF-α (10 ng/ml), and (F) HCE-T cells treated with TNF-α (10 ng/ml)+CsA. Data are the mean ± SD values, n = 3, statistical significance was denoted (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns- nonsignificant as compared to levels of desiccated cells). Note that --- is control, +-- HCE-T cells exposed to desiccation (Des), -+- HCE-T cells treated with chloroquine (CQ), ++- desiccated HCE-T cells treated with chloroquine (CQ), --+ HCE-T cells treated with cyclosporine (CsA), and -++ desiccated HCE-T cells treated with cyclosporine (CsA).

Figure 4

(a)-(c) Expression of autophagy related genes and proteins in HCE-T under desiccation stress. (a) The mRNA expression levels of LC3A, LC3B, ATG7, and LAMP1 in HCE-T cells exposed to desiccation stress, treated with and without CQ/CsA treatment normalized with β-actin (Figure 4(a)). (b) Immunoblot shows the protein levels of LC3, p62, and LAMP1 HCE-T cells exposed to desiccation stress and treated with and without CQ/CsA treatment (Figure 4(b)). Densitometric analysis of the blots showed the ratios of LAMP1, LC3-II and p62 to GAPDH. (c) Westernblot shows the expression levels of Beclin-1 protein in HCE-T cells exposed to desiccation stress and treated with and without CQ/CsA treatment (Figure 4(c)). Densitometric analysis of the blots showed the ratios of Beclin-1 to GAPDH. Data are the mean ± SD values, n = 3, statistical significance denoted (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns- nonsignificant as compared to levels of desiccated cells). Note that --- is control, +-- HCE-T cells exposed to desiccation (Des), -+- HCE-T cells treated with chloroquine (CQ), ++- desiccated HCE-T cells treated with Chloroquine (CQ), --+ HCE-T cells treated with cyclosporine (CsA), and -++ desiccated HCE-T cells treated with cyclosporine (CsA).

Figure 5

(a)-(c) Quantification of autophagosomes and lysosomes in desiccated HCE-T cells. (a) Cyto-Id staining for quantification of autophagosome staining in HCE-T cells under desiccation stress, with and without CQ/CsA treatment (Figure 5(a)). (b) LTR- lysotracker red staining to measure lysosome levels staining in HCE-T cells under desiccation stress, with and without CQ/CsA treatment (Figure 5(b)). (c) AO- Lysosomal pH assessed using acridine orange (AO) staining in HCE-T cells under desiccation stress, with and without CQ/CsA treatment (Figure 5(c)). UT- control or undesiccated cells, Des- HCE-T cells exposed to desiccation, CQ- HCE-T cells treated with chloroquine (CQ), CQ+Des- desiccated HCE-T cells treated with chloroquine (CQ), CsA- HCE-T cells treated with cyclosporine (CsA), and CsA+Des- desiccated HCE-T cells treated with cyclosporine (CsA).

Figure 6

Phosphorylation levels of MAP kinase, AKT/p70S6kinase and AMPK proteins in HCE-T cells under desiccation stress. Immunoblot shows phosphorylated ERK1/2, p38, AKT and P70S6kinase, and AMPK (Figure 6). Densitometric analysis of the blots showed the ratios of phosphorylated AKT, p70s6kinase, ERK1/2 and p38 to AKT, p70s6kinase, ERK1/2, and p38. Data are the mean ± SD values, n = 3, statistical significance denoted (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns- nonsignificant as compared to desiccated cells). Note that --- is control, +-- HCE-T cells exposed to desiccation (Des), -+- HCE-T cells treated with chloroquine (CQ), ++- desiccated HCE-T cells treated with chloroquine (CQ), --+ HCE-T cells treated with cyclosporine (CsA), and -++ desiccated HCE-T cells treated with cyclosporine (CsA).