Two transcriptionally and functionally distinct waves of pro-inflammatory versus pro-repair neutrophils during acute liver injury

We used the CCl 4 -induced ALI model and employed flow cytometry, microscopy imaging and qRT-PCR to examine intrahepatic neutrophil subpopulations during the necroinflammatory and early and late repair phases of the wound healing response. We also analyzed intrahepatic neutrophils for their transcriptional profiles using RNA-seq. Finally, we evaluated their protein translation, mitochondrial function and cell metabolism using the Seahorse Mito Stress Test. We detected two temporarily distinct waves of neutrophils during (i) necroinflammation (at 24 hrs post-injury) and (ii) late repair (at 72 hrs). Early neutrophils were characterized by: (i) up-regulation of inflammatory cytokines (e.g., IL-12a), (ii) activation of the non-canonical NF-κB pathway, (iii) reduction of protein translation and (iv) decreased oxidative phosphorylation. In contrast, late neutrophils exhibited an enrichment of several genes and pathways associated with tissue repair and angiogenesis. In addition, early neutrophils, identified as pro-inflammatory, show strong expression for CXCR5, while second wave neutrophils, identified as angiogenic highly expressed CXCR4. Finally, our data demonstrated that neutrophils expressing CXCR5 express a short isoform of this protein, truncated by 55 aa at the N-terminal end. Gene expression analyses of different liver neutrophil populations at time points (24h- and 72h post CCl 4) compared with splenic neutrophils (control)