Effect of Hydroxychloroquine on Natural Killer Cell Activity and Function

Background and Objectives: Inflammation is associated with different diseases ranging from autoimmunity, infections, and cancer. Immuno-modulatory drugs may have applications for treating various diseases, such as the case of anti-malarial hydroxychloroquine (HCQ). However, it remains unknown what is their direct mechanism of action on immune cells, which include natural killer (NK) cells. To investigate this issue, we examined the effects of HCQ on NK cell activity, including activating receptors’ expression, cytotoxic potential, and cytokine production. Materials and Methods: The human NK cell line NK92 was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 200 IU/ml of IL-2. HCQ was added to the cells at different concentrations (1, 5, and 10 mM) and at different time points (24 and 48 hours). Post-treatment, NK92 cell viability was evaluated using the Cell Proliferation/XTT method. Protein expressions of NKG2D and CD107a were assessed using flow cytometry after 24 hours of treatment, while the cytotoxicity of NK cells was tested using the Calcein-AM method. The NK-sensitive K562 cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin/streptomycin and were used as targets for NK92 cell-mediated killing. Also, IFN-g production by NK92 cells was measured by ELISA after 24 hours of treatment with HCQ. Statistical analysis was performed where p<0.05 was considered statistically significant. Results: Upon treatment with different concentrations of HCQ for 24 hours, NK92 cells did not show any difference in their proliferation potential. However, after 48 hours, the viability of the cells significantly decreased in a dose-dependent manner, with the lowest viability noted at 10 mM of HCQ. Additionally, the expression of the activating receptor NKG2D was significantly reduced upon treatment with 5 and 10 mM of HCQ for 24 hours. Similarly, the degranulation marker CD107a significantly decreased upon HCQ treatment at all used concentrations. Similarly, the cytotoxicity of NK92 cells against K562 cells was reduced at both 5:1 and 10:1, effector to target (E: T) cell ratios, when 10 mM HCQ was used. On the other hand, IFN-g levels were significantly increased upon treatment with 1, 5, and 10 mM HCQ. Conclusions: This study highlights the effect of HCQ on activating receptors’ expression, cytotoxicity, and IFN-g production of NK cells. Our findings suggest that high doses of HCQ might impair the activation and cytolytic potential of NK cells but may increase their IFN-g production, which may recruit other cells into the inflammatory sites. Despite being an immunomodulatory drug, more studies are needed to further understand HCQ's effects on NK cells and perhaps other effector cells. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.