EGFR phosphorylation is required for TLR4-mediated macrophage activation during sepsis. However, whether and how intracellular EGFR is transported during endotoxemia have largely been unknown. Here, we show that LPS promotes high levels cell surface expression of EGFR in macrophages through two different transport mechanisms. On one hand, Rab10 is required for EEA1-mediated the membrane translocation of EGFR from the Golgi. On the other hand, EGFR phosphorylation prevents its endocytosis in a kinase activity-dependent manner. Erlotinib, an EGFR tyrosine kinase inhibitor, significantly reduced membrane EGFR expression in LPS-activated macrophage. Mechanistically, upon LPS induced TLR4/EGFR phosphorylation, MAPK14 phosphorylated Rab7a at S72 impaired membrane receptor late endocytosis, which maintains EGFR membrane localization though blocking its lysosomal degradation. Meanwhile, Rab5a is also involved in the early endocytosis of EGFR. Subsequently, inhibition of EGFR phosphorylation switches M1 phenotype to M2 phenotype and alleviates sepsis-induced acute lung injury. Mechanistic study demonstrated that Erlotinib suppressed glycolysis-dependent M1 polarization via PKM2/HIF-1ɑ pathway and promoted M2 polarization through up-regulating PPARγ induced glutamine metabolism. Collectively, our data elucidated a more in-depth mechanism of macrophages activation, and provided stronger evidence supporting EGFR as a potential therapeutic target for the treatment of sepsis.

Elevated glycolysis in lung tissue is a hallmark of sepsis-induced acute lung injury (SI-ALI), yet the role of glycolytic reprogramming and lactate-derived protein modifications in damaging epithelial cells remains poorly understood. In this study, we reveal that PDK4-driven glycolytic reprogramming promotes excessive lactate production in lung tissue during SI-ALI. Mechanistically, AARS1 in epithelial cells selectively enhances lactylation modification at the K375 site of LPCAT2, which suppresses STAT1 acetylation and facilitates STAT1 phosphorylation, nuclear translocation, and transcriptional repression of SLC7A11. This cascade ultimately triggers epithelial cells ferroptosis. Pharmacological inhibition of PDK4 attenuates lactate accumulation and LPCAT2 lactylation, thereby restoring STAT1 acetylation and SLC7A11 expression. Furthermore, AARS1 knockdown or mutation of the LPCAT2-K375 lactylation site rescues STAT1-mediated SLC7A11 suppression and mitigates ferroptosis in vitro and septic mice. Our findings revealed that elevated expression of PDK4 is a critical factor contributing to the increased lactate production in lung tissue during sepsis, and established a novel LPCAT2-K375/STAT1/SLC7A11 axis driving epithelial cells ferroptosis in SI-ALI, highlighting the crosstalk between metabolic reprogramming, post-translational modifications (PTM), and ferroptosis. Targeting the PDK4 or LPCAT2 lactylation may offer therapeutic potential for SI-ALI.

Excessive inflammatory responses mediated by innate immunity are a significant cause of sepsis-induced acute lung injury (SI-ALI), underscoring the need for further drug development to improve therapeutic outcomes. Ginkgetin (GK), a natural flavone, has demonstrated potential anti-inflammatory and antioxidative effects. However, it remains unclear whether GK can ameliorate SI-ALI and what the underlying mechanisms might be. We aimed to investigate the therapeutic effects and mechanisms of GK on SI-ALI. We established SI-ALI models using lipopolysaccharide or cecal ligation and puncture to assess the impact of GK. An autophagy agonist and inhibitor were applied to detect the effect of GK on autophagy. Proteomics and targeted gene knockdown experiments were employed to validate lysosomal-associated transmembrane protein 5 (Laptm5) as the key protein. Co-immunoprecipitation and site mutation assays were used to identify the site where GK inhibited Laptm5 ubiquitination. Biotin pulldown coupled with mass spectroscopy, molecular docking, and drug affinity responsive target stability (DARTS) were performed to elucidate the direct target and underlying mechanisms. We demonstrated that GK activated the autophagosome-lysosome pathway by increasing the protein level of Laptm5. This process facilitated autophagy-mediated degradation of tank-binding kinase 1 (TBK1) and inhibited the signal transduction of downstream inflammatory pathways. Further, we found that GK inhibited the K48-linked ubiquitination of Laptm5 and revealed the ubiquitination sites of Laptm5 (K86 and K122) for the first time. Biotin pulldown and DARTS identified ubiquitin-protein ligase E3C (Ube3c) as a target of GK in inhibiting Laptm5 ubiquitination, with TYR707 and ASN832 being the key residues. Our findings indicate that GK exerts its anti-inflammatory effect on macrophages by promoting autophagy via suppressing the ubiquitination of Laptm5, thus offering a promising therapeutic approach for SI-ALI.

Diabetic wounds are characterized by impaired angiogenesis, persistent inflammation, and delayed tissue remodeling, yet effective cell-free therapies remain limited. Adipose-derived stem cell exosomes (ADSC-exos) possess pro-angiogenic and anti-inflammatory properties, but the precise molecular mechanisms are incompletely defined. We harvested ADSC-exos from rat adipose tissue and then evaluated their impact on high-glucose (HG)-induced human umbilical vein endothelial cells (HUVECs), using Cell Counting Kit-8, 5-ethynyl-2’-deoxyuridine incorporation, scratch wound healing, and tube formation assays. In streptozotocin-induced diabetic rats with dorsal wounds, ADSC-exos were injected subcutaneously. Wound closure, thermal hyperalgesia, collagen deposition, and Ki67/CD31 were evaluated. Transcriptomic sequencing and protein-protein interaction network analysis identified Aurora kinase B (AURKB) as a candidate mediator. AURKB knockdown, overexpression, and Ras-associated protein 1 (Rap1) inhibition (GGTI298 and si-RAP1B) were used to validate the AURKB/Rap1 axis. ADSC-exos restored HG-impaired HUVEC viability, proliferation, migration, and tube formation. In diabetic rats, ADSC-exos accelerated wound closure, reduced thermal hyperalgesia, lowered inflammatory cytokines, enhanced collagen deposition, and increased Ki67-positive proliferating cells and CD31-positive vessels. Transcriptomic analysis revealed that ADSC-exos downregulated AURKB. AURKB knockdown further improved wound healing and angiogenesis, and reduced thermal hyperalgesia. AURKB overexpression abolished the protective effects of ADSC-exos on HUVECs. Mechanistically, ADSC-exos upregulated RAP1A and RAP1B expression through AURKB suppression, and both GGTI298 and si-RAP1B reversed the protective effects of AURKB knockdown on HUVECs. In conclusion, ADSC-exos accelerate diabetic wound healing by suppressing AURKB and activating the Rap1 signaling pathway, identifying the AURKB/Rap1 axis as a novel therapeutic target and supporting ADSC-exos as a promising cell-free strategy.