DataSheet_1_Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3–Linked Sialic Acid Ligand Binding.docx

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.

树突状细胞（Dendritic cells, DCs）是适应性免疫的关键启动因子，在识别病原体后可诱导T细胞分化以引发恰当的免疫应答。DCs具备通过识别病原体相关分子模式（pathogen-associated molecular patterns）的受体来辨别外界信号的非凡能力，这类受体包括可识别病原体表面碳水化合物结构的聚糖结合受体（glycan-binding receptors），以及可结合此类模式的其他受体，例如Toll样受体（Toll-like receptors, TLRs）。
本研究探讨了免疫抑制性唾液酸结合免疫球蛋白型凝集素（Immune inhibitory Sialic acid binding immunoglobulin type lectins）所识别的α2-3唾液酸（α2-3 sialic acid, α2-3sia）结合DCs后引发的早期信号事件。α2-3sia常见于细菌（如B群链球菌（Group B Streptococcus））表面，也可由肿瘤细胞表达。我们研究了结合至树枝状大分子核心（dendrimeric core）的α2-3sia是否会改变DCs的信号转导特性。
通过磷酸化蛋白质组学分析（phosphoproteomic analysis），我们发现DCs在单独结合α2-3sia，或联合脂多糖（Lipopolysaccharide, LPS）与Toll样受体4（TLR4）共刺激后，其信号谱存在显著差异。α2-3sia可调控TLR4信号级联反应（signaling cascade），最终导致109种磷酸化蛋白质的表达水平发生改变。这些磷酸化蛋白质被注释到7个生物学过程，其中包括白细胞介素12（IL-12）细胞因子通路的调控。
经α2-3sia树枝状大分子过夜刺激后，DCs分泌的白细胞介素10（IL-10，IL-12产生的抑制性调节因子）水平显著上调。激酶活性分析显示，JAK-STAT信号通路（JAK-STAT signaling pathway）的特征谱发生了改变。参与IL-12通路调控的磷酸化STAT3（Ser727）与磷酸化STAT5A（Ser780）的表达水平均出现下调。流式细胞术定量分析（Flow cytometric quantification）进一步证实，经α2-3sia刺激后，上述磷酸化蛋白随时间发生去磷酸化，而α2-6唾液酸（α2-6sia）则无此效应。
在单核细胞衍生树突状细胞（monocyte-derived dendritic cells, moDCs）中同时抑制STAT3与STAT5A的活性，可得到与α2-3sia诱导的DCs相似的细胞因子分泌谱（cytokine secretion profile）。综上，本研究揭示：在α2-3sia与LPS共同刺激下，DCs内的JAK-STAT通路发生特异性改变，进而调整了IL-10与IL-12的细胞因子分泌比例，最终减轻炎症反应。因此，精准调控STAT的磷酸化状态，有望成为阻断细菌或肿瘤对宿主实施免疫逃逸（immune escape）的潜在研究方向。