# Nanobody Nb07 mitigates sepsis by blocking the PFKM-p53-PD-1 axis to enhance macrophage phagocytosis

## TL;DR

A nanobody called Nb07 helps fight sepsis by improving macrophage function through a new pathway involving PFKM, p53, and PD-1.

## Contribution

The discovery of a novel PFKM-p53-PD-1 axis and its inhibition by a nanobody to enhance macrophage phagocytosis in sepsis.

## Key findings

- PFKM moves to the nucleus during sepsis and impairs macrophage phagocytosis.
- PFKM enhances p53 activity, leading to increased PD-1 expression and reduced phagocytosis.
- Nanobody Nb07 blocks the PFKM-p53 interaction, restoring phagocytosis and reducing sepsis severity in mice.

## Abstract

Rationale: Macrophage phagocytosis is essential for pathogen clearance during sepsis. We previously demonstrated that the glycolytic enzyme 6-phosphofructokinase, muscle type (PFKM), modulates macrophage functions and its deficiency alleviates sepsis in mice. However, the function of PFKM in regulating macrophage phagocytosis remains unclear.

Methods: CD14+ monocytes were sorted by flow cytometry from healthy volunteers and septic patients, and the subcellular localization of PFKM was assessed by immunofluorescence. Nuclear translocation mechanisms and PFKM-p53 interaction were identified by Co-immunoprecipitation coupled with mass spectrometry (Co-IP/MS) and validated by Co-IP. Transcriptomic sequencing was used to identify the downstream target of the PFKM-p53 complex. Inflammatory cytokine levels were detected by ELISA and real-time RT-PCR, and the phagocytosis of macrophages was assessed by flow cytometry. Dual-luciferase reporter assays and ChIP were employed to investigate whether PFKM acts as a co-regulator of p53 in mediating Pdcd1 transcription. Nanobodies targeting PFKM-p53 were screened and subsequently synthesized according to the sequences. The effect of nuclear PFKM and the therapeutic effect of nanobodies were evaluated on the well-established sepsis mouse models induced by Escherichia coli or cecal ligation and puncture.

Results: PFKM translocated to the macrophage nucleus during sepsis. Nuclear accumulation of PFKM impaired phagocytosis through a non-glycolytic “moonlighting” function and exacerbated sepsis. Mechanistically, PFKM interacts with p53, which facilitates its nuclear translocation. Subsequently, PFKM promotes p53 acetylation at K120, enhancing p53 binding to the Pdcd1 promoter and driving its transcription, thereby suppressing macrophage phagocytosis. Blocking the PFKM-p53 interaction with a nanobody, Nb07, restored phagocytosis of macrophages and alleviated sepsis in mice.

Conclusion: Our data reveal the PFKM-p53-PD-1 axis that suppresses macrophage phagocytosis in sepsis and highlight the therapeutic potential of targeting this pathway with nanobody-based strategies.