Localisation and functionality of the inflammasome in neutrophils

Abstract

The ‘inflammasome’, a multi-protein complex in monocytes and granulocytes, has emerged as playing a key role in innate immunity and inflammation. Regarding monocytes, definite data are available that the inflammasome, localised in the cytosol, controls the maturation of different interleukins via caspase-1 activation. Concerning the situation in neutrophil granulocytes—which was the main focus of our research—little is currently known about the subcellular localisation and the function of the inflammasome. To precisely characterise the localisation of the inflammasome in neutrophils, we utilised subcellular fractionation and detected inflammasomal components not only in the cytoplasm but also in mobile vesicles and granules. These findings may implicate that neutrophils are able to regulate their inflammasomes dynamically between intracellular stores and the cell surface, where they could participate in pathogen recognition and uptake. To gain a better understanding about the function of the inflammasomes in neutrophils, we stimulated highly purified cells with known inflammasome activators. The results of these experiments suggest that inflammasome activation in human neutrophils triggers the IL-1β, but not the IL-18, IL-1α, or IL-33 protein secretion. Besides the inflammasome-mediated pathway, IL-1β was also produced in an inflammasome-independent fashion through the action of serine proteases. IL-18 was already secreted at baseline conditions and we could not see a further increase after stimulation. In our experimental conditions, neutrophils were unable to release detectable amounts of IL-1α or IL-33. The isolated autologous monocytes produced IL-1ß, IL-1α, and IL-18 in an inflammasome-dependent manner, whereas IL-33 was not detectable in the cell culture supernatants of the monocytes. Methodologically, our studies raised the point that contaminating monocytes have to be considered when interpreting IL-1β quantification results from traditional density gradient derived neutrophils. To investigate the effect of intrinsic inflammasome activation on neutrophils, we studied patients with MWS (Muckle-Wells syndrome), a rare disorder characterised by constitutively increased inflammasome activity. The neutrophils from patients with MWS showed a tendency towards lower IL-1ß and IL-18 levels in the stimulation assays compared to healthy control cells. We hypothesised that the constitutive intrinsic activation of the inflammasome in MWS patients limits the exogenous inflammasome responsiveness.