Background/Purpose: Gain-of-function mutations in
NLRP3 cause a spectrum of autoinflammatory diseases
known as cryopyrin-associated periodic syndromes
(CAPS), with neonatal-onset multisystem inflammatory disease (NOMID) being the
most severe form of CAPS. These mutations cause excess production of the proinflammatory cytokine IL-β, which is responsible,
at least in part, for the phenotype. IL-1β is produced in a pro-form and
processed to a mature form by active caspase-1. NLRP3 is a key component of a multiprotein complex known as the inflammasome that
mediates the maturation and release of IL-1β, in part through caspase-1
activation, and can induce rapid cell death resulting in the release of other proinflammatory mediators such as apoptosis-associated
speck-like protein containing a CARD (ASC). Two pathways to cell death have
been implicated. One is caspase-1 dependent ‘pyroptosis‘
and the other is caspase-1-independent ‘pyronecrosis‘.
 The precise molecular mechanisms, as well as the role of NLRP3 mutations
in cell death, remain to be elucidated. Our recent data (manuscript in revision)
provide evidence that caspase-1 inhibition only partially reduces IL-1β
release and fails to abrogate cell death in cells from NOMID patients, while cathepsin B inhibition prevents both cell death and
IL-1β release. Here we investigated the consequences of caspse-1 and cathepsin B inhibition on the link between released
IL-1β and cell death.

Methods: Whole blood cells from NOMID patients and healthy controls were
studied. Cells were stimulated with LPS in the presence of inhibitors of
caspase-1 or cathepsin B, followed by addition of
ATP. Cell supernatants were collected and incubated with IL-1β-capturing
beads that bind to both pro- and processed IL-1β. Cells and beads were
evaluated by flow cytometry. Supernatants and/or cell lysates from LPS stimulated
cells were also evaluated using western blot analysis for IL-1β captured
on beads and ASC.

Results: By
flow analysis we confirmed that inhibition of cathepsin
B in NOMID and healthy control monocytes, results in a significant decrease in
IL-1β release and cell death while caspase-1 inhibition fails to inhibit
cell death and only partially abrogates IL-1β release. Analysis of
IL-1β in supernatants suggests that when
caspase-1 is inhibited, only the pro form of IL-1β is found in the
supernatant. However, cathepsin B inhibition
completely prevents the appearance of IL-1β in supernatants. Furthermore,
caspase-1 inhibition has no effect on ASC release, whereas cathepsin
B significantly reduced ASC release in both NOMID patients and healthy
controls. 

 Conclusion: These data suggest that cell death in NOMID patients and ATP
induced cell death in healthy controls is caspase-1 independent, but requires cathepsin B activation, implicating the pyronecrosis
pathway. In addition, our data provide evidence that pro-IL-1β is released
from dying/dead cells despite caspase-1 inhibition. These results place
caspase-1 activation downstream of cell death events, and disassociate
IL-1β processing and cell death in NOMID patients.