Background/Purpose: Gain of function mutations in NLRP3 cause cryopyrin-associated periodic fever syndromes (CAPS), the most severe form of which is neonatal-onset multisystem inflammatory disease (NOMID), which is characterized by recurrent episodes of systemic and organ-specific inflammation. Mutations in NLRP3 result in self-activation, promoting inflammasome-mediated IL-1beta processing and release, and can induce cell death through a process known as pyronecrosis. Many inflammatory disease manifestations are responsive to IL-1beta inhibitors, although patients often continue to have minor disease flares in the context of infections or other stressors. The hematopoietic cells that are the major producers of IL-1beta in NOMID and mechanisms mediating IL-1beta release and pyronecrosis have not been well elucidated.

Methods: Whole blood cells from NOMID patients and controls, THP-1 monocytic cells, and stably STAT3 knock down THP-1 cells were stimulated with LPS in the presence of cathepsin B and STAT3 inhibitors, followed by ATP treatment. Supernatants were collected and incubated with IL-1beta capturing beads. Cells were fixed and permeabilized and stained with IL-1beta, CD14, CD16 and CD83 antibodies, and cells and beads were evaluated by flowcytometry. LPS stimulated cells were also evaluated using immunofluorescent and electron microscopy and western blot assays.

Results: A sub-population of monocytes characterized by CD14hi/CD16low expression, produce the majority of IL-1beta in peripheral blood in response to LPS stimulation in NOMID and control PBMCs. In NOMID patients this subpopulation of monocytes also undergoes rapid cell death following LPS stimulation alone.  The cell death is temporally associated with IL-1beta release, and is consistent with pyronecrosis. In contrast, CD14hi/CD16low cells from healthy subjects only release IL-1beta after both LPS and ATP stimulation and are relatively resistant to cell death.  IL-1beta release is partially inhibited in NOMID cells by caspase-1 inhibitors, and is blocked completely by inhibitors of cathepsin B.  Cathepsin B inhibitors also prevent pyronecrosis.  Similar to cathepsin, STAT3 inhibitors significantly abrogated cell death and IL-1beta release in NOMID cells. STAT3 inhibition also abolished all ATP-dependent IL-1beta release and cell death in monocytes from healthy subjects.  Moreover, shRNA-mediated knockdown of STAT3 in THP-1 cells completely prevented ATP dependent IL-1b release and cell death. We confirm that STAT3 associates with mitochondria, and that inhibition of STAT3 but not cathepsin B, prevents mitochondrial damage suggesting that mitochondrial STAT3 activation may be upstream of cathepsin B activation.

Conclusion: These results identify the predominant IL-1beta-producing cell population in the peripheral blood of NOMID patients and healthy contorls, and identify a novel role for STAT3 in mediating NLRP3 effects on pyronecrosis and IL-1beta release. We suggest that cell death may contribute to IL-1beta release.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/stat3-plays-a-central-role-in-nlrp3-inflammasome-mediated-il-1%ce%b2-production-and-pyronecrosis/