PFKFB3 decreases α-ketoglutarate production while partial PFKFB3 knockdown in macrophages ameliorates arthritis in...

Aberrant 6-phosphofructo-2kinase/fructose-2,6-bisphoshatase 3 (PFKFB3) expression is tightly correlated with multiple steps of tumorigenesis; however, the pathological significance of PFKFB3 in macrophages in patients with rheumatoid arthritis (RA) remains obscure. In this study, we examined whether PFKFB3 modulates macrophage activation and promotes RA development.

Peripheral blood mononuclear cells (PBMCs) from patients with RA, THP-1 cells, and bone marrow-derived macrophages from conditional PFKFB3-knockout mice were used to investigate the mechanism underlying PFKFB3-induced macrophage regulation of RA.

We demonstrated that patients with RA have higher PFKFB3 levels than healthy volunteers. PFKFB3 silencing suppressed M1 macrophage polarization and downregulated IL-1β, CD80, IFIT1, CCL8, and CXCL10 in macrophages of patients with RA. PFKFB3 overexpression markedly upregulated IRF5, HIF1α, IL-1β, CD80, IFI27, IFI44, IFIT1, IFIT3, CCL2, CCL8, CXCL10, CXCL11, and MMP13 in phorbol 12-myristate 13-acetate-induced THP-1 cells, although these changes were partially reversed by PFK15, an inhibitor of PFKFB3 enzyme activity. Co-immunoprecipitation assays revealed that PFKFB3 interacted with GLUD1 and decreased glutamate dehydrogenase (GDH) activity and α-ketoglutarate production. PFKFB3, TNFα, IL-6, IFNγ, CXCL9, CXCL10, CXCL11, MMP13, and MMP19 were downregulated in bone marrow-derived macrophages of conditional PFKFB3-knockout mice relative to those of wild-type mice. Partial PFKFB3 knockdown in macrophages ameliorated the clinical signs of arthritis and bone destruction, inhibited proinflammatory factor expression, and promoted GDH activity and α-ketoglutarate production in tumor necrosis factor-transgenic mice. Single-cell sequencing revealed that macrophages were the most abundant cells in the ankles of arthritic mice, and partial PFKFB3 knockdown promoted M2-like polarization and was correlated with TREM2, SPP1, APOE, and C1Q expression.

PFKFB3 is upregulated in macrophages in patients with RA. PFKFB3 aggravates arthritis by modulating macrophage activity, which may be related to decreased α-ketoglutarate production.