USP4 controls neutrophil spreading through stabilising IQGAP1 during lung inflammation.

BACKGROUND

Protein availability critically influences innate immune reactivity and the extent of immune-mediated tissue injury. A key unresolved question is how specific protein homeostasis networks within immune cells such as neutrophils can be modulated to mitigate the detrimental outcomes of acute inflammation.

METHODS

We established an acute lung injury (ALI) model in immune-specific ubiquitin-specific peptidase 4 (USP4) knockout chimeric (BM-Usp4-/-) and control (BM-WT) mice. Using cytometry by time-of-flight (CyTOF) mass cytometry, quantitative proteomics, immunoprecipitation-mass spectrometry and neutrophil functional assays, we elucidated the molecular mechanism of USP4 in a specific neutrophil migratory behaviour-spreading-as well as the therapeutic potential of USP4 inhibitors in acute respiratory distress syndrome (ARDS).

RESULTS

USP4 expression was upregulated in blood and pulmonary leucocytes from patients with ARDS and ALI mice (p=0.049, p=0.0086), but downregulated in mouse lung parenchyma (lipopolysaccharide vs control, p=0.0019). BM-Usp4-/- mice exhibited attenuated lung injury and neutrophil infiltration (in lung tissue and bronchoalveolar lavage fluid, p<0.0001 and p=0.0056 vs BM-WT). Usp4-/- neutrophils showed a reduction in both the proportion and area of spreading (p<0.0001 vs Usp4+/+neutrophils). Mechanistically, USP4 deubiquitinated and upregulated IQGAP1, enhancing CDC42 activity (p=0.0286) and F-actin formation (p<0.0001). Notably, USP4 and IQGAP1 expression correlated positively for patients with severe pneumonia (p=0.0438, R²=0.7898). Moreover, USP4 inhibitor Vialinin A in ALI mice decreased neutrophil infiltration, improved lung function and survival and alleviated lung injury progression.

CONCLUSION

USP4 links protein homeostasis to neutrophil recruitment during inflammation. Targeting the USP4-IQGAP1 axis with Vialinin A represents a promising therapeutic strategy for patients with ARDS secondary to severe pneumonia.