Antimalarials have demonstrated beneficial effects in Systemic Lupus Erithematosus and Rheumatoid Arthritis. However, the mechanisms and the molecular players targeted by these drugs remain obscure. ...Although hydroxychloroquine (HCQ) is a known ion channel inhibitor, this property has not been linked to its anti-inflammatory effects. We aimed to study whether HCQ inhibits pro-inflammatory ion channels. Electrophysiology experiments demonstrated that HCQ inhibited Ca
-activated K
conductance in THP-1 macrophages in a dose-dependent manner. In macrophages, ATP-induced K
efflux plays a key role in activating the NLRP3 inflammasome. ATP-induced IL-1beta secretion was controlled by the KCa1.1 inhibitor iberiotoxin. NS1619 and NS309 (KCa1.1 and KCa3.1 activators respectively) induced the secretion of IL-1beta. This effect was inhibited by HCQ and also by iberiotoxin and clotrimazol (KCa3.1 inhibitor), arguing against off-target effect. In vitro, HCQ inhibited IL-1beta and caspase 1 activation induced by ATP in a dose-dependent manner. HCQ impaired K
efflux induced by ATP. In vivo, HCQ inhibited caspase 1-dependent ATP-induced neutrophil recruitment. Our results show that HCQ inhibits Ca
-activated K
channels. This effect may lead to impaired inflammasome activation. These results are the basis for i) a novel anti-inflammatory mechanism for HCQ and ii) a new strategy to target pro-rheumatic Ca
-activated K
channels.
Nitric oxide (•NO) has been implicated in multiple physiological and pathological immune processes. Different methods have been developed to detect and quantify •NO, where one of the principal ...difficulties are the accurately detection in cellular system with low levels of •NO production. The choice of the •NO detection method to be used depends on the characteristics of the experimental system and the levels of •NO production which depend on either the organism source of samples or the experimental conditions. Recently, high sensitive methods to detect and image •NO have been reported using 4,5-diaminofluorescein-based fluorescent probes (DAF) and its derivate 4,5-diaminofluorescein diacetate (DAF-2 DA). This work was aimed to adapt and optimize the use of DAF probes to detect and quantify the •NO production in systems of high, moderate and low out-put production, especially in human PBMC and their subpopulations. Here, we report an original experimental design which is useful to detect and estimate •NO fluxes in human PBMC and their subpopulations with high specificity and sensitivity.
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