DLBCL-Derived HMGB2 Inhibits Macrophage Phagocytosis By up-Regulating TRIM65 to Promote NLRP3 Ubiquitination

Background：

Diffuse large B-cell lymphoma (DLBCL), the predominant non-Hodgkin lymphoma type, exhibits significant clinical and biological heterogeneity. Despite favorable outcomes with R-CHOP therapy in most cases, approximately 30-40% of patients experience disease progression or relapse. A comprehensive understanding of DLBCL pathogenesis is crucial to improve survival rates in these patients. Single-cell sequencing of DLBCL patient tumor tissues revealed a substantial increase in HMGB2 expression in highly proliferative B lymphoma cell subpopulations. HMGB2, a member of the non-histone chromosomal high mobility group protein family, plays a key role in regulating chromatin structure and gene transcription. However, its specific role in DLBCL progression remains poorly understood. This study aims to clarify the role and mechanisms underlying the abnormal overexpression of HMGB2 in DLBCL.

Methods：

We established DLBCL-Vector and DLBCL-HMGB2-sh cell lines and conducted in vivo experiments using a subcutaneous xenograft model to assess the impact of HMGB2 knockdown on tumor growth. Flow cytometry was employed to analyze the immune cell composition in the tumor microenvironment following HMGB2 knockdown. In vitro assays, including CCK-8, cell cycle, apoptosis, migration, and macrophage phagocytosis assays, were performed to investigate HMGB2's role in DLBCL cells. Transcriptome sequencing elucidated the underlying mechanisms, which were further validated through flow cytometry, immunofluorescence, and co-immunoprecipitation assays.

Results:

In the subcutaneous transplantation tumor model, HMGB2-sh group mice exhibited slower tumor growth compared to controls. However, in vitro studies did not show significant changes in proliferation, cell cycle, or apoptosis in HMGB2-sh DLBCL cells. Flow cytometry analysis of immune cell infiltration in tumor tissues showed no notable differences in T cells and NK cells between HMGB2-sh and control groups, but an increase in M1 macrophage proportion was observed in the HMGB2-sh group. Given HMGB2's role as a cytokine with potential extracellular secretion, we treated M0 macrophages with recombinant HMGB2 protein and observed an increase in M2 polarization along with reduced phagocytic activity. Transcriptomic sequencing revealed upregulated TRIM65 expression in HMGB2-treated macrophages, known to ubiquitinate and degrade NLRP3 protein, enhancing macrophage phagocytosis. Co-immunoprecipitation experiments confirmed increased NLRP3 ubiquitination in HMGB2-treated macrophages. And activation of the NLRP3 signal pathway reversed the HMGB2 cytokine-induced decline in macrophage phagocytic activity.

Conclusions：

DLBCL cells enhance TRIM65 expression in macrophages by secreting HMGB2, thereby increasing NLRP3 ubiquitination, promoting M2 polarization of macrophages, and suppressing their phagocytic capability. This study suggests HMGB2 as a potential intervention target for DLBCL therapy, providing a theoretical basis for DLBCL treatment.

No relevant conflicts of interest to declare.