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.
Highlights
Dendritic cells (DCs) are antigen presenting cells that continuously sense the intrinsic host environment
To obtain a global overview of the a2-3 sialic acid-induced signaling in DCs, we performed LC-MS/MS-based phosphoproteomic analysis of human DCs stimulated with sialic acid-coated dendrimers
We determined the optimal dendrimer concentration at 1 μM measured an increased IL-10:IL-12 secretion profile when the a2-3sia dendrimer was given to DCs in the presence of LPS, which was not observed with the control dendrimer (Figure 1B)
Summary
Dendritic cells (DCs) are antigen presenting cells that continuously sense the intrinsic host environment. DCs possess the extraordinary capacity to recognize internal and external danger signals and respond appropriately using pattern recognition receptors (PPRs) [1]. Recognition of self-associated molecular patterns (SAMPs), such as self-antigens, leads to the induction of a tolerogenic response [3]. Glycosylation is a common post-translational modification in eukaryotes, and glycan patterns can be recognized as PAMPs or SAMPs by DC PRRs, such as C-type lectin receptors (CLRs) and Sialic acid binding immunoglobulin type lectins (Siglecs) [6]. SAMP-associated glycan patterns predominantly refer to sialic acids [3]. This negatively charged monosaccharide decorates the terminal positions of larger polysaccharide molecules on cell surfaces. The impact of sialic acids on altering T cell differentiation is highly appealing as a target in DC-based immunotherapeutic strategies [9, 10]
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