Inhibition of PFKFB3 in macrophages ameliorates intestinal inflammation by modulating gut microbiota in DSS-induced colitis

Phosphofructo-2-kinase/fructose-2,6-biophosphatase 3 (PFKFB3), a key glycolytic enzyme, has attracted increasing attention for its essential roles in various inflammatory responses and immune-related diseases. But the functional relevance and mechanistic basis of the PFKFB3 on ulcerative colitis (UC) remain unclear. Immunohistochemical staining and publicly available data sets were used to analyze PFKFB3 expression in healthy controls (HCs) and UC patients. The role of PFKFB3 on colitis and gut microbiota was investigated by deficiency of PFKFB3 in macrophages ( PFKFB3 fl/fl Lyz2 -Cre) mice. In silico meta- and Spearman’s correlation analysis of published high-throughput transcriptomic data analyzed the correlation between PFKFB3 and microbiome-associated genes. The expression of PFKFB3 was significantly upregulated in the colon of both human UC cohorts and colitis mice. Pharmacological inhibition of PFKFB3 by 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) diminished the severity of colitis. Single-cell RNA sequencing and flow analysis revealed that the upregulated PFKFB3 was predominantly contributed by colonic macrophages. PFKFB3 fl/fl Lyz2 -Cre mice alleviated experimental colitis in contrast to littermate control ( PFKFB3 fl/fl ). Concomitantly, PFKFB3 fl/fl Lyz2 -Cre mice exhibited a remarkably Faecalibaculum genus-enhanced microenvironment, which can be horizontally transmitted to co-housed wild-type mice, leading to an attenuation of DSS-induced colitis. However, when antibiotics were administered to PFKFB3 fl/fl Lyz2 -Cre mice, the transmission effect was lost. By analyzing the UC patient cohort, Spearman’s correlation provided additional evidence for a significant positive correlation between PFKFB3 and microbiota-associated genes expression. This study demonstrated that PFKFB3 deficiency in macrophages could effectively ameliorate colonic inflammation, providing the first evidence that gut microbiota from PFKFB3-deficient mice may represent a novel therapeutic strategy for UC. IMPORTANCE PFKFB3 expression was upregulated in the colon of both ulcerative colitis (UC) patients and colitis mice, and this differential expression was predominantly contributed by colonic lamina propria macrophages. Knockout of PFKFB3 in macrophages significantly alleviated DSS-induced colitis. Knockout of PFKFB3 in macrophage mice exhibited a remarkably Faecalibaculum genus-enhanced microenvironment, which can be horizontally transmitted to co-housed wild-type mice, leading to an attenuation of DSS-induced colitis; however, when administered to antibiotics, the transmission effect was lost. By analyzing the UC patient cohort, we demonstrated significant positive correlation between PFKFB3 and microbiota-associated gene expression. Our study first elucidates the relationship of PFKFB3 in macrophages with intestinal inflammation and gut microbiota in UC, which may provide a new strategy for the treatment.