Temporal control of Ninj1 activation determines cell-to-cell heterogeneity in IL-33 release

Interleukin-33 (IL-33), an IL-1 family cytokine, is released during cell damage. However, the mechanism underlying IL-33 release is not fully understood. Here, we generate IL-33-mCherry-expressing mouse cell lines to monitor IL-33 release at single cell resolution. During necroptosis, SYTOX uptake that represents loss of membrane integrity, and IL-33-mCherry release occur simultaneously, indicating that Mlkl-mediated damage causes immediate membrane rupture. In contrast, some apoptotic and pyroptotic cells exhibit delayed IL-33-mCherry release relative to SYTOX uptake, whereas in other cells, both events occur simultaneously. IL-33-mCherry release is only modestly affected by loss of Gsdmd or Gsdme but is markedly reduced in Ninjurin-1 (Ninj1) knockout cells. These findings indicate that Gasdermins act upstream, whereas Ninj1-mediated membrane rupture represents the principal mechanism of IL-33 release. The delay in IL-33-mCherry release in some cells implies that the activation of Ninj1 occurs with variable timing, thus contributing to cell-to-cell heterogeneity.