Determining the LPS structural features that influence TLR4 downstream signaling.

Upon recognizing bacterial lipopolysaccharide (LPS), human TLR4 initiates two distinct signaling pathways: the MyD88 pathway from the cell surface or the TRIF pathway following endocytosis. While the first is associated with strong pro-inflammatory responses, the latter is linked to dendritic cell maturation and T cell priming. Changes in LPS structure can influence the activation of either or both pathways. This study investigates the influence of specific structural features of LPS on these pathways: the O antigen, the number of acyl chains in lipid A and the glucosamine modification of the phosphates of the lipid A diglucosamine backbone. Systematically engineered LPS differing in one or more of these features were studied by quantifying NFκB and IRF3 activation-indicators of MyD88 and TRIF pathway activation, respectively. The findings reveal that the glucosamine modification of lipid A plays a dominant role in TLR4-mediated signaling, overriding the influence of the O antigen and lipid A acylation. The absence of glucosamine modification significantly reduced the activation of both MyD88 and TRIF pathways, underscoring its importance in promoting TLR4 dimerization. Furthermore, under-acylation of LPS (with 4 or 5 acyl chains) partially reduced NFκB activation, while completely abrogating TRIF pathway activation. In contrast, hexa-and hepta-acylated LPS equally and robustly activated both pathways. Lastly, the O antigen selectively biased signaling towards the TRIF pathway without affecting the MyD88 pathway. This study provides valuable insights into how specific LPS structural modifications can be leveraged to tailor TLR4-mediated signaling.