Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3–Linked Sialic Acid Ligand Binding

Abstract

Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

α2-3 Sialic acid binding and uptake by human monocyte-derived dendritic cells alters metabolism and cytokine release and initiates tolerizing T cell programming

“…The tolerogenic program imposed by the sialic acid on the DCs is therefore different from that of dexamethasone or vitamin D3-induced tolerance. Dexamethasone exerts its tolerogenic capacity only through regulation of the NF-κB signaling cascade [ 7 , 59 , 60 ], whereas the α2-3sia-induced tolerance works through the Siglec ITIM-mediated signaling, the association of SHP-phosphatases and JAK/STAT signaling, and has the strength to modulate TLR inflammatory signaling [ 61 ]. We here investigated the tolerance a2-3sia imposes on bacterial LPS induced TLR4 triggering and it will be interesting to address whether a2-3sia also antagonize other TLR triggers, for instance, those responding to viruses.…”

The Role of Hitchhiking in Cancer Therapeutics—A Review

“…APCs, like dendritic cells (DCs), are efficient immune forerunners. [ 114,115 ] Apart from limiting T cell proliferation, the immunosuppressive tumor milieu reduces DC migration, stimulation, and antigen presentation. [ 44,111 ] Researchers have been striving to develop artificial APCs (aAPCs) as a substitute for innate APCs for the past 20 years.…”

Insights into the Role of Sialylation in Cancer Metastasis, Immunity, and Therapeutic Opportunity

“…Moreover, these tumor-expressed sialoglycan ligands interact with Siglec-5, Siglec-9, and Siglec-15 on T cells, suppressing TCR-mediated signaling pathways and corresponding effector functions [ 132 , 133 , 134 , 135 , 136 ]. The binding of α2,3-linked sialic acids to Siglecs modulates the immunosuppressive phenotype of lipopolysaccharide-matured DC by decreasing the phosphorylation of molecules in the JAK-STAT pathways [ 137 ].…”