Arglabin targets a non-canonical NLRP3/CCR2 axis to suppress MDSC infiltration and potentiates anti-PD-1 immunotherapy in hepatocellular carcinoma.

Background

Hepatocellular carcinoma (HCC) develops in an immunosuppressive tumor microenvironment characterized by NLRP3 overexpression, in which myeloid-derived suppressor cells (MDSCs) are abundantly enriched and play a critical role in compromising T cell-mediated antitumor immunity. Arglabin (Arg), a natural sesquiterpene lactone derived from Artemisia glabella, shows potent anti-HCC potential, yet its direct molecular target responsible for the immunomodulatory effects in HCC remains poorly defined.

Purpose

To identify its molecular target and delineate the pharmacological mechanisms underlying Arg-mediated anti-HCC immunity.

Methods

NLRP3 and CCR2 expression in myeloid-derived cells was investigated in HCC specimens by western blot, multiplex immunofluorescence and single-cell RNA sequencing data from the GEO database. Molecular interactions between Arg and NLRP3 were assessed through CETSA, SPR, LC-MS/MS, and pull-down assays. In vivo anti-tumor efficacy was evaluated in syngeneic murine models using H22 (BALB/c) and Hepa1-6 (C57BL/6) liver cancer cells. Functional studies included CCK8 and transwell migration assays. Tumor microenvironment composition was analyzed by flow cytometry, and tumor burden was monitored using in vivo bioluminescent imaging. Mechanistically, transcriptional regulation of CCR2 by NLRP3 was investigated through ChIP-qPCR.

Results

Here, we demonstrate that Arg suppresses HCC progression in syngeneic murine models by reducing MDSC infiltration and promoting CD8+ T-cell accumulation. MDSC co-implantation and depletion assays confirmed that the therapeutic efficacy of Arg functionally depends on MDSC modulation. Mechanistically, we identified NLRP3 as the direct molecular target of Arg with binding occurring specifically at the Cys280 residue; furthermore, experiments in Nlrp3-/- mice confirmed that NLRP3 is essential for the anti-HCC activity of Arg. Crucially, we reveal that Arg's antitumor effect is independent of the classical NLRP3 inflammasome/IL-1β pathway. Instead, NLRP3 functions through a non-canonical mechanism involving its nuclear translocation and recruitment to the Ccr2 promoter, thereby driving a transcriptional program that orchestrates MDSC trafficking into the tumor microenvironment. Furthermore, NLRP3/CCR2 blockade by Arg potentiates the therapeutic effects of immune checkpoint inhibitors (ICIs) to inhibit tumor growth.

Conclusion

Our findings establish Arg as an NLRP3-targeting immunomodulator that reshapes the HCC microenvironment. Specifically, Arg binds to the Cys280 residue of NLRP3, thereby inhibiting its nuclear translocation and abrogating the transcriptional regulation of CCR2, which subsequently blocks MDSC trafficking. This effect works together with PD-1 blockade to overcome immune evasion in HCC, providing a strategy for reversing immunosuppression and potentiating immunotherapy.