Analysis of GSDMD-N abnormality promoting neutrophil NETs mediated RA disease through NLRP3-dependent pathway

TL;DR

This study shows that the NLRP3 inflammasome and GSDMD protein drive NET formation in rheumatoid arthritis, and blocking them reduces joint inflammation and damage.

Contribution

The study identifies the NLRP3–GSDMD pathway as a novel upstream regulator of NETosis in RA and suggests it as a therapeutic target.

Findings

RA patients and CIA mice showed elevated NLRP3/GSDMD and NET markers compared to controls.

Inhibiting NLRP3 or GSDMD reduced joint swelling, bone erosion, and synovial inflammation in CIA mice.

Transcriptomic analysis revealed 12 core genes linked to heat-shock proteins and histone variants affected by GSDMD inhibition.

Abstract

Rheumatoid arthritis (RA) is characterized by persistent synovitis and progressive joint damage. Mounting evidence implicates neutrophil extracellular traps (NETs) formation (NETosis) in RA pathogenesis, yet the upstream regulatory nodes remain incompletely defined. We aimed to elucidate the role of the NLRP3 inflammasome in regulating GSDMD-dependent NETosis and to evaluate whether inhibiting NLRP3 or Gasdermin D (GSDMD) alleviates RA pathology.

Neutrophils and synovial tissues from patients with RA and osteoarthritis (OA) were analyzed for NLRP3, GSDMD, and NET-related markers by immunofluorescence, Western blot, and qPCR. A collagen-induced arthritis (CIA) mouse model was used to test the effects of pharmacological inhibition of NLRP3 or blockade of GSDMD pore formation on joint swelling, bone destruction, and synovial inflammation. Transcriptomic sequencing was performed to identify differentially expressed genes following inhibition of GSDMD pore formation. Ionomycin was used to induce ROS-independent NETosis in vitro.

The RA group exhibited elevated NLRP3/GSDMD expression and increased NET markers relative to OA controls. In CIA mice, inhibition of NLRP3 or blockade of GSDMD pore formation mitigated joint swelling, reduced bone erosion, and attenuated synovial inflammation. Transcriptomic profiling identified 12 core genes—enriched for heat-shock proteins and histone variants—with significant differential expression after GSDMD pore inhibition. Ionomycin stimulation enhanced ROS-independent NET formation and was accompanied by increased NLRP3 expression and promoted cleavage of GSDMD into its active N-terminal fragment (GSDMD-N). Conversely, suppressing NLRP3 activation or preventing GSDMD pore formation effectively inhibited this process.

These data position the NLRP3 inflammasome as a pivotal upstream regulator of GSDMD-dependent NETosis in RA. Targeting this axis—via inhibition of NLRP3 or blockade of GSDMD pore formation—ameliorates inflammatory joint pathology in vivo and constrains NETosis in vitro. Collectively, our findings support the NLRP3–GSDMD pathway as a promising therapeutic avenue for RA.

Pathways of NLRP3 inflammasome activation and ROS-independent NETosis. Left panel: A two-signal model primes (NF-κB–dependent upregulation of NLRP3 and pro-cytokines) and activates NLRP3, which recruits ASC to activate caspase-1; caspase-1 cleaves GSDMD, whose N-terminal pores enable IL-1β/IL-18 maturation and release, initiating pyroptosis-associated inflammation. Right panel: Under ROS-independent conditions, ionomycin-induced Ca2+ influx activates PAD4, driving histone citrullination and chromatin decondensation, which synergize to cause nuclear envelope breakdown and the extrusion of DNA web-like structures (NETs). Created in BioRender. Liu, R. (2025) https://BioRender.com/hz7utj2, with permission from Liu, R.

Diagram illustrating the pathways of NLRP3 inflammasome activation and ROS-independent NETosis. On the left, inflammasome activation leads to IL-1β and IL-18 release, involving caspase-1 and GSDMD. On the right, pathways show NET formation initiated by ionomycin and calcium, mediated by PAD4 and chromatin decondensation. NF-kB plays a central role in gene expression regulation.

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