SPS9-02 Dynamic Evolution of Human Neutrophils During Endotoxin-induced Acute Lung Inflammation

Rationale

Acute lung inflammation is a major cause of morbidity, yet the cellular programs that initiate and sustain pulmonary injury remain incompletely defined. Although neutrophil recruitment is central to this response, the temporal phenotypic and functional states of human neutrophils during pulmonary transmigration are poorly understood. We therefore evaluated the temporal evolution of transcriptional and proteomic changes in human neutrophils following pulmonary segmental endotoxin challenge.

Method

Healthy subjects (mean age 29 years) underwent segmental endobronchial endotoxin challenge (Escherichia coli O:113, 4ng/kg) followed by bronchoalveolar lavage (BAL) and paired blood sampling after 6 hours (n = 5) and 24 hours (n = 6). Neutrophils were enriched from blood and BAL by negative selection (EasySep Direct Human Neutrophil Isolation Kit). Cell lysates were processed for RNA extraction and subsequent RNA sequencing. Enriched neutrophils were cultured for 4 hours and supernatant were collected for secretome analysis using a proximal extension assay (Olink Proteomics Uppsala, Sweden). The differentially expressed genes and proteins were identified using DESeq2 tool.

Result

At 6 hours, circulating neutrophils exhibited a mature, effector-ready phenotype (CXCR2, SELL) with upregulation of Fc receptor genes (FCGR3A, FCGR3B, FCGR2A). These cells displayed transcriptional signatures of acute activation, including degranulation (ELANE, MPO) and neutrophil extracellular trap formation (PADI4, HIST4H4). In contrast, lung-recruited neutrophils acquired an aged, tissue-infiltrating phenotype (CXCR4, ICAM1, CD69). Lung neutrophils adopted distinct transcriptional programs enriched for interferon-stimulated genes (MX1, ISG15), antigen-presentation pathways (HLA-DRB1, CD74), and immune checkpoint molecules (PDCD1, LAG3). Although these compartment-specific differences persisted at 24 hours, they were attenuated over time. These phenotypes were supported by differential transcription factor activity with circulating neutrophils enriched for JUNB/CREB1-driven immediate-early programs, whereas lung neutrophils exhibited increased RELB-, IRF5-, and IRF7-mediated inflammatory and interferon-associated networks. Secretome analysis revealed an early IL-6-IFN-γ-TNF pro-inflammatory response in lung neutrophils at 6 hours. By 24 hours, this response evolved into a mixed inflammatory-regulatory profile characterized by increased IL-10, CXCL9, and MMP12. Concurrent increases in PD-L-1 and CD83 further supported a transition from tissue-damaging effector function toward regulation of immune homeostasis.

Conclusion

Pulmonary recruitment drives rapid, compartment-specific evolution of human neutrophils, shifting their function from early inflammatory effectors to immune-interactive and regulatory cells. These findings highlight neutrophils as dynamic modulators of pulmonary immunity and identify temporally distinct neutrophil states as potential therapeutic targets in acute lung inflammation.

This abstract is funded by: NIH Intramural Funding