IL-36 gamma is secreted through an unconventional pathway using the Gasdermin D and P2X7R membrane pores

Mucosal innate immunity functions as the first line of defense against invading pathogens. Members of the IL-1 family are key cytokines upregulated in the inflamed mucosa. Inflammatory cytokines are regulated by limiting their function and availability through their activation and secretion mechanisms. IL-1 cytokines secretion is affected by the lack of a signal peptide on their sequence, which prevents them from accessing the conventional protein secretion pathway; thus, they use unconventional protein secretion pathways. Here we show in mouse macrophages that LPS/ATP stimulation induces cytokine relocalization to the plasma membrane, and conventional secretion blockade using monensin or Brefeldin A triggers no IL-36 gamma accumulation within the cell. In silico modeling indicates IL-36 gamma can pass through both the P2X7R and Gasdermin D pores, and both IL-36 gamma, P2X7R and Gasdermin D mRNA are upregulated in inflammation; further, experimental blockade of these receptors' limits IL-36 gamma release. Our results demonstrate that IL-36 gamma is secreted mainly by an unconventional pathway through membrane pores formed by P2X7R and Gasdermin D.