Macrophage Proteomic Profiling Reveals Divergent TLR4-Dependent and -Independent Responses to Kdo2-Lipid A and Lipid IVa

Macrophages harness pattern recognition receptors (PRRs) to detect conserved bacterial components and mount effective immune responses. Many Gram-negative bacteria modify their lipid A structures to limit recognition by Toll-like receptor 4 (TLR4) and cytosolic Caspase-11 lipopolysaccharide sensors. One common evasion strategy is to reduce the lipid A acylation state from hexa- to tetra-acylation. This alteration can limit binding to receptors and dampen subsequent immune signaling responses, yet the proteomic alterations associated with this altered immunogenicity remain incompletely understood. Here, we systematically profiled proteomic alterations induced by extracellular or transfected hexa-acylated Kdo2-lipid A (Kdo2) and tetra-acylated lipid-IVa (IVa) to assess TLR4-dependent, TLR4-independent, and non-canonical inflammasome activation pathways. Kdo2 elicited stronger inflammatory responses in immortalized bone-marrow-derived macrophages (iBMDMs), as evidenced by robust TNF production, Caspase-11 cleavage, and IL-1α/IL-1β release. In contrast, IVa elicited minimal TNF secretion and failed to effectively induce non-canonical inflammasome activation. Global label-free quantitative proteomic analysis of iBMDMs stimulated with a low dose of immunogenic LPS displayed route-specific immune signatures: enrichment of TNF signaling, interferon-associated pathways, and mitochondrial metabolic remodeling. Equimolar amounts of low-acylated LPS failed to effectively induce these immune signatures, supporting a threshold-dependent model in which the lipid A structure and route of exposure define inflammatory progression. Collectively, our findings provide mechanistic insight into how lipid A structural variation modulates macrophage immune programming and cytosolic inflammasome activation.