Discussion
RA, autoimmunity
PROLIFERATION, AUTOIMMUNITY, AUTOIMMUNE DISEASES
Although HCQ is widely used for the treatment of autoimmune diseases, the exact mechanism behind its immunomodulatory properties remains unclear. In this study, we therefore aimed to quantify the immunosuppressive effect of HCQ by studying the endosomal TLR response and lymphocyte proliferation and activation both in in vitro experiments and in vivo in a randomized placebo-controlled trial in healthy volunteers.In our in vitro experiments, HCQ dose-dependently inhibited TLR3-, 7-, and 9-driven IL-6 and IFNα production, with profound effects at concentrations > 100 ng/mL. These findings are in line with literature on TLR signaling modulation by chloroquine [HCQ did not affect T cell activation in vitro. Although a dose-dependent inhibition of T cell proliferation by chloroquine following stimulation with anti-CD3/CD28 has been described [While HCQ had strong immunosuppressive effects in vitro, especially at high concentrations, less pronounced ex vivo effects of the compound were observed in our clinical study. Compared to placebo, 5-day HCQ treatment did not significantly suppress B cell proliferation or ex vivo TLR-driven IFNα and IL-6 secretion in PBMC cultures, except for a suppressive effect on TLR7-driven responses. The most likely explanation for this discrepancy between in vitro and ex vivo is that there was insufficient drug exposure at the evaluated HCQ dose and regimen in the clinical study. By using a 5-day dose regimen of HCQ (the recommended off-label dose for COVID-19 at the time of study conduct), an average maximum plasma concentration of 121 ng/mL was reached. This concentration is considerably lower than plasma levels found in RA patients receiving HCQ treatment of 200 mg daily for a longer time period, which ranges from 200 to 500 ng/mL [The widespread use of hydroxychloroquine following the onset of the COVID-19 pandemic was the reason to initiate our experiments. The initial off-label use of HCQ was primarily based on studies that assessed in vitro antiviral activity against SARS-CoV-2 [In conclusion, we showed extensive and profound immunomodulation by HCQ in vitro; however, in a clinical study in healthy volunteers, the overall immunomodulatory effects of a 5-day HCQ treatment regimen of 2400 mg were limited. The pharmacological activity of HCQ in autoimmunity remains to be studied in greater detail, based on the assays as presented in our studies and at a therapeutic dose and regimen relevant for the condition of interest.