Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

Abstract

Sialic acids, a nine carbon acidic sugar, can be detected in Pseudomonas aeruginosa (PA) when it is cultured with any exogenous sialic acids source like serum. Subsequently, several sialoglycoproteins from PA have been identified which includes flagellin. In the current study, the sialylation status of PA flagellin has been further investigated through lectin and Siglecs binding. Siglecs are immunoglobulin-like receptors which can recognize and bind to sialic acids. The sialylated PA flagellin (Fla+Sia) was observed to bind with its cognate receptor TLR5 (Toll-like receptor 5) and also with Siglec-9 on dendritic cell (DC) surface. DCs are potent antigen-presenting phagocytic immune cells which can activate other immune cells through costimulation. Immature DCs undergo maturation upon encountering antigens; which involves changes in the cell morphology, receptors expression, cytokine secretion etc. Here, immature DCs stimulated with Fla+Sia or desialylated-Fla+Sia (ds-Fla+Sia) underwent maturation but exhibited different phenotypic and functional profiles. The Fla+Sia matured DCs showed relatively lower expression of costimulatory molecules, lower pro-inflammatory cytokines secretion, higher phagocytic activity and induced the generation of more Th (T helper) and Treg (regulatory T) cells. The impact of these two antagonistic signaling pathways-TLR5-based immune-activatory and immunosupressive Siglec-9 signaling on DC maturation was further explored in DCs generated from THP-1 cell line. Here, Fla+Sia stimulated DCs showed increased Siglec-9-SHP1 association while ds-Fla-Sia stimulation showed an increased TLR5 association with its interacting partners-MyD88/TRAF-6/IRAK-4. The TLR5/Siglec-9 signaling was disrupted through siRNA transfections or chemical inhibitor treatment which revealed that flagellin-based TLR5 stimulation was crucial for the initiation of robust cell signaling events. However, Fla+Sia treatment activated Siglec-9 signaling, which directly inhibited TLR5 pathway. Interestingly, downregulating Siglec-9 signaling resulted in enhanced TLR5 activation upon Fla+Sia stimulation, along with increased MAP kinase signaling and activation of the NF-{kappa}B pathway. Thus, Siglec-9 silenced DCs responded to Fla+Sia with increased secretion of pro-inflammatory cytokines. Therefore, the altered profile and T cell polarization ability of Fla+Sia matured DCs is caused by the Siglec-9-based disruption of TLR5 signaling.