Structural diversity of lipid A modulates neutrophil proteome and secretome responses

Neutrophils (PMNs) are central effector cells of the innate immune system, deploying antimicrobial functions such as degranulation, ROS production, and release of antimicrobial mediators. However, the neutrophil reprogramming in response to structurally distinct lipopolysaccharide (LPS) stimuli remains incompletely defined. Here, we integrated functional profiling with quantitative high-resolution proteomics to compare human PMN responses to two LPS preparations differing primarily in lipid A acylation: hexaacylated LPS from Escherichia coli (ECO-LPS) and tetra-acylated LPS from Francisella tularensis (FT-LPS). Principal component analysis (PCA) revealed that inter-donor variability was the dominant source of variation. ECO-LPS induced clear activation phenotypes and robust, time-dependent proteome remodeling, including early loss of L-selectin and progressive depletion of granule-associated proteins concomitant with increased abundance of inflammatory signaling proteins. In contrast, FT-LPS treated PMNs were indistinguishable from controls at the cellular proteome level. In the secretome, ECO-LPS treatment caused enrichment of neutrophil degranulation and granule lumen components, consistent with intracellular depletion of granules. FT-LPS elicited limited secretory changes, suggesting a restrained activation or primed state without coordinated intracellular remodeling. Together, our results demonstrate that lipid A acylation is a key determinant of neutrophil activation state and provide a resource for defining proteomic signatures associated with LPS sensing. Neutrophils are essential first- responder cells of the innate immune system, and their activation is classically studied using targeted sets of surface markers and functional readouts, while the proteome- and secretome-level dynamics remain less well defined. By applying quantitative DIA proteomics to both the intracellular proteome and the secretome of primary human neutrophils, this study reveals how lipid A structure dictates the depth and coordination of neutrophil activation. The data demonstrate that hexaacylated LPS drives coordinated depletion of degranulation-associated proteins with a concomitant appearance in the secretome. In contrast, tetra-acylated LPS fails to induce comparable intracellular remodeling and instead produces only subtle secretory responses consistent with restrained activation. These findings refine our mechanistic understanding of how structurally distinct endotoxins differentially regulate neutrophil effector functions, emphasize the donor-intrinsic proteomic variation, and provide a reference that can guide future studies of attenuated innate sensing and neutrophil-focused therapeutic approaches. • Hexa-acylated E. coli LPS triggers robust activation and coordinated temporal proteome remodeling, including depletion of granule-associated proteins and induction of inflammatory signaling components. • Tetra-acylated F. tularensis LPS maintains a cellular proteome indistinguishable from negative controls over time, with only limited secretory changes consistent with restrained activation/priming. • E. coli LPS treatment causes enrichment of degranulation and vesicle/granule lumen pathways in the secretome, matching the intracellular depletion of these compartments.