Inhibitory effect of anti-malarial agents on the expression of proinflammatory chemokines via Toll-like receptor 3 signaling in human glomerular endothelial cells

Abstract

Objective: Although anti-malarial agents, chloroquine (CQ) and hydroxychloroquine (HCQ) are currently used for the treatment of systemic lupus erythematosus, their efficacy for lupus nephritis (LN) remains unclear. Given that upregulation of glomerular Toll-like receptor 3 (TLR3) signaling plays a pivotal role in the pathogenesis of LN, we examined whether CQ and HCQ affect the expression of the TLR3 signaling-induced representative proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) in cultured human glomerular endothelial cells (GECs).

Methods: We examined the effect of polyinosinic-polycytidylic acid (poly IC), an agonist of TLR3, on MCP-1, CCL5 and interferon (IFN)-β expression in GECs. We then analyzed whether pretreatment with CQ, HCQ, or dexamethasone (DEX) inhibits poly IC-induced expression of these chemokines using real-time quantitative reverse transcriptase PCR and ELISA. Phosphorylation of signal transducers and activator of transcription protein 1 (STAT1) was examined using western blotting.

Results: Poly IC increased MCP-1 and CCL5 expression in a time- and concentration-dependent manner in GECs. Pretreating cells with CQ, but not DEX, attenuated poly IC-induced MCP-1 and CCL5 expression; however, HCQ pretreatment attenuated poly IC-induced CCL5, but not MCP-1. HCQ did not affect the expression of IFN-β and phosphorylation of STAT-1.

Conclusion: Considering that TLR3 signaling is implicated, at least in part, in LN pathogenesis, our results suggest that anti-malarial agents exert a protective effect against the development of inflammation in GECs, as postulated in LN. Interestingly, CQ is a rather powerful inhibitor compared with HCQ on TLR3 signaling-induced chemokine expression in GECs. In turn, these findings may further support the theory that the use of HCQ is safer than CQ in a clinical setting. However, further detailed studies are needed to confirm our preliminary findings.

Keywords: Chloroquine; Toll-like receptor 3; glomerular endothelial cells; hydroxychloroquine; lupus nephritis.

Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.

Poly IC induces the expression of MCP-1 and CCL5 in cultured human GECs in a concentration-dependent manner. The cells were treated with various concentrations of poly IC. After 24 h incubation, RNA was extracted and subjected to real-time RT-PCR (a,c). The concentrations of MCP-1 and CCL5 in the medium were analyzed using ELISA (b,d). Data are shown as the means ± SD (n = 3, *p < 0.01, by t-test).

Figure 2.

Poly IC induces the expression of MCP-1 and CCL5 in cultured human GECs in a time-dependent manner. The cells were treated with 30 μg/mL poly IC for up to 24 h. The conditioned medium was collected, and RNA was extracted from the cells. Real-time RT-PCR (a,c,e) and ELISA (b,d) analyses were performed. Data are shown as the means ± SD (n = 3, *p < 0.01, by t-test).

Figure 3.

Knockdown of IFN-β decreases the poly IC-induced expression of both MCP-1 and CCL5, and knockdown of p65 decreases the poly IC-induced all of IFN-β, CCL5 and MCP-1 in cultured human GECs. The cells were transfected with siRNA against IFN-β, p65 or a non-silencing negative control siRNA. After 24h incubation, the cells were treated with 30 μg/mL poly IC for an additional 24 h (a–d, f, and g) or 2 h (e). The medium was collected, and RNA was extracted from cells, after which quantitative real-time RT-PCR analysis was performed. Data are shown as the means ± SD (n = 3, *p < 0.01 by t-test).

Figure 4.

(a–d) Pretreatment with CQ inhibits the expression of both MCP-1 and CCL5 induced by poly IC. The cultured GECs were pretreated with 1 or 10 μg/mL CQ for 1 h and subsequently treated with 30 μg/mL poly IC for 16 h. The medium was collected, and RNA was extracted from cells, after which quantitative real-time RT-PCR and ELISA analyses were performed. Data are shown as the means ± SD (n = 3, *p < 0.01, by t-test).

Figure 5.

Pretreatment of GECs with HCQ inhibits the expression of CCL5, but not of MCP-1 and IFN-β. The cultured GECs were pretreated with 1 or 10 μg/mL HCQ for 1h and subsequently treated with 30 μg/mL poly IC for 16h. The medium was collected, and RNA was extracted from cells, after which quantitative real-time RT-PCR (a,c,e) and ELISA (b,d) analyses were performed. Data are shown as the means ± SD (n = 3, *p < 0.01, by t-test). (f) The cells were pretreated with 10 μg/mL HCQ for 1h, and then treated with 30 μg/mL poly IC for 6h. The cells were lysed and western blotting for phosphorylated STAT1 (p-STAT1) and STAT1 was performed.

Figure 6.

(a,b) Pretreatment of DEX does not inhibit the expression of both MCP-1 and CCL5 induced by poly IC. The cultured GECs were pretreatment with 10 μM DEX for 1h and subsequently treated with 30 μg/mL poly IC for 16h. The medium was collected, and RNA was extracted from cells, after which quantitative real-time RT-PCR analysis was performed. Data are shown as the means ± SD (n = 3, *p < 0.01, by t-test).