Table_4_Exposure of Keratinocytes to Candida Albicans in the Context of Atopic Milieu Induces Changes in the Surface Glycosylation Pattern of Small Extracellular Vesicles to Enhance Their Propensity to Interact With Inhibitory Siglec Receptors.docx

Candida albicans (C. albicans) infection is a potential complication in the individuals with atopic dermatitis (AD) and can affect clinical course of the disease. Here, using primary keratinocytes we determined that atopic milieu promotes changes in the interaction of small extracellular vesicles (sEVs) with dendritic cells and that this is further enhanced by the presence of C. albicans. sEV uptake is largely dependent on the expression of glycans on their surface; modelling of the protein interactions indicated that recognition of this pathogen through C. albicans-relevant pattern recognition receptors (PRRs) is linked to several glycosylation enzymes which may in turn affect the expression of sEV glycans. Here, significant changes in the surface glycosylation pattern, as determined by lectin array, could be observed in sEVs upon a combined exposure of keratinocytes to AD cytokines and C. albicans. This included enhanced expression of multiple types of glycans, for which several dendritic cell receptors could be proposed as binding partners. Blocking experiments showed predominant involvement of the inhibitory Siglec-7 and -9 receptors in the sEV-cell interaction and the engagement of sialic acid-containing carbohydrate moieties on the surface of sEVs. This pointed on ST6 β-Galactoside α-2,6-Sialyltransferase 1 (ST6GAL1) and Core 1 β,3-Galactosyltransferase 1 (C1GALT1) as potential enzymes involved in the process of remodelling of the sEV surface glycans upon C. albicans exposure. Our results suggest that, in combination with atopic dermatitis milieu, C. albicans promotes alterations in the glycosylation pattern of keratinocyte-derived sEVs to interact with inhibitory Siglecs on antigen presenting cells. Hence, a strategy aiming at this pathway to enhance antifungal responses and restrict pathogen spread could offer novel therapeutic options for skin candidiasis in AD.

白色念珠菌（Candida albicans, C. albicans）感染是特应性皮炎（atopic dermatitis, AD）患者的潜在并发症，可对疾病的临床进程产生影响。本研究以原代角质形成细胞为研究对象，发现特应性微环境会促进小型细胞外囊泡（small extracellular vesicles, sEVs）与树突状细胞的相互作用发生改变，且白色念珠菌的存在可进一步增强这一效应。小型细胞外囊泡的摄取过程主要依赖于其表面聚糖的表达；蛋白质相互作用模型显示，通过白色念珠菌相关模式识别受体（pattern recognition receptors, PRRs）识别该病原体，与多种糖基化酶的功能相关，而这些酶可反过来影响小型细胞外囊泡表面聚糖的表达。通过凝集素阵列检测发现，当角质形成细胞同时暴露于特应性皮炎细胞因子与白色念珠菌时，小型细胞外囊泡的表面糖基化模式出现显著变化，具体表现为多种聚糖的表达水平上调，据此可推测多种树突状细胞受体可作为其结合伴侣。阻断实验证实，抑制性Siglec-7与Siglec-9受体在小型细胞外囊泡与细胞的相互作用中发挥主要介导作用，同时小型细胞外囊泡表面含唾液酸的碳水化合物基团也参与了这一结合过程。据此推测，ST6 β-半乳糖苷α-2,6-唾液酸转移酶1（ST6 β-Galactoside α-2,6-Sialyltransferase 1, ST6GAL1）以及核心1β-1,3-半乳糖基转移酶1（Core 1 β,3-Galactosyltransferase 1, C1GALT1）是介导白色念珠菌暴露后小型细胞外囊泡表面糖基重构过程的潜在关键酶。本研究结果表明，在特应性皮炎微环境中，白色念珠菌可促使角质形成细胞来源的小型细胞外囊泡糖基化模式发生改变，进而与抗原呈递细胞表面的抑制性Siglecs结合。因此，靶向该通路以增强抗真菌应答、限制病原体扩散的策略，可为特应性皮炎合并皮肤念珠菌病提供全新的治疗选择。