Microparticulate P2X7 and GSDM-D mediated regulation of functional IL-1β release

The pro-inflammatory cytokine IL-1β is a secreted protein that is cleaved by caspase-1 during inflammasome activation upon recognition of internal and external insults to cells. Purinergic receptor P2X7 has been described to be involved in the release pathway of bioactive mature IL-1β by activated immune cells. Microparticle (MP) shedding has also been recently recognized as a manner of cytokine IL-1β release. However, the understanding of purinergic receptor roles in the MP-mediated IL-1β release process is still rudimentary. Gasdermin-D (GSDM-D), a protein involved in pyroptosis and inflammasome activation, has been recently described to be involved in the release of microparticles by virtue of its pore-forming ability. Hence, our current work is aimed to study the role of P2X7 in regulating GSDM-D-mediated microparticles and thereby bioactive IL-1β release. We provide evidence that cleaved functional IL-1β release in microparticles upon LPS stimulation is regulated by GSDM-D and P2X7 in a two-step fashion. GSDM-D activation first regulates release of IL-1β and P2X7 into microparticles. Then, microparticulate active P2X7 receptor then regulates the release of bioactive IL-1β encapsulated in microparticles to be able to target other cells inducing IL-8. Using an ATP model of stimulation, we further demonstrated that extracellular ATP stimulation to IL-1β containing LPS microparticles induces release of its content, which when subjected to epithelial cells induced IL-8. This effect was blocked by P2X7 inhibitor, KN62, as well as by IL-1RA. Taken together, our findings demonstrate for the first time the synergistic critical roles of GSDM-D and purinergic receptors in the regulation of microparticulate bioactive IL-1β release and induction of target cell responses.