Chloroquine Differentially Modulates Inflammatory Cytokine Expression In RAW 264.7 Cells In Response To Inactivated Staphylococcus Aureus

Aim:With over 60 years of clinical practice, chloroquine(CQ) is an effective drug for the treatment of malaria and some autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus. The mechanism by which CQ affecting autoimmune disorders is likely to associate with its inhibitory effects on monocytes or macrophages to release tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). In this study, we aimed to explore the potential anti-inflammatory effect and underlying action mechanism of CQ by using Gram-positive bacteria(inactivated Staphylococcus aureus cells, SAC)-activated RAW 264.7 macrophages as a model. Methods:1. The expression of intracellular TNF-α, IL-6 and, and CC chemokine ligand 2(CCL2) protein was measured by intracellular cytokine staining, and TNF-α, IL-6 and CCL2 protein levels in the culture supernatant were detected by cytometric bead array.2.Quantitative RT-PCR was used to detect m RNA levels of TNF-α, IL-6 and CCL2. The levels of the pro-TNF-α and the activation of MAPKs and NF-kB pathway were evaluated by Western blotting.Results:1. The results showed that CQ treatment reduced the levels of TNF-α, IL-6, and CCL2 in culture medium but increased intracellular protein expression of TNF-α and CCL2 in SAC-activated cells.2. CQ showed differential effects on their m RNA levels: it reduced IL-6 m RNA expression, increased CCL2 m RNA levels, but had no effect on TNF-α m RNA. The SAC-activated inflammatory signaling pathways were slightly or minimally affected by CQ treatment. Additionally, CQ increased the accumulation of pro-TNF-α proteins(26 k Da and 28 k Da) within cells. Conclusion:1. CQ inhibited TNF-α secretion through suppression of the processing of membrane-bound 26 k Da and 28 k Da pro-TNF-α into soluble mature TNF-α.2. Collectively, CQ differentially modulated proinflammatory cytokine expression in Gram-positive bacterium-activated macrophages at multiple regulatory layers in the course of their biosynthesis.