Colafrancesco Serena, Barbati Cristiana, Priori Roberta, Putro Erisa, Giardina Federico, Gattamelata Angelica, Monosi Benedetta, Colasanti Tania, Celia Alessandra Ida, Cerbelli Bruna, Giordano Carla, Scarpa Susanna, Fusconi Massimo, Cavalli Giulio, Berardicurti Onorina, Gandolfo Saviana, Nayar Saba, Barone Francesca, Giacomelli Roberto, De Vita Salvatore, Alessandri Cristiano, Conti Fabrizio
Sapienza University, Rome, Italy.
Sapienza University and Saint Camillus International University of Health Science, Rome, Italy.
Arthritis Rheumatol. 2022 Apr;74(4):654-664. doi: 10.1002/art.42018. Epub 2022 Mar 1.
Salivary gland epithelial cells (SGECs) are key cellular drivers in the pathogenesis of primary Sjögren's syndrome (SS); however, the mechanisms sustaining SGEC activation in primary SS remain unclear. We undertook this study to determine the role of autophagy in the survival and activation of SGECs in primary SS.
Primary SGECs isolated from the minor SGs of patients with primary SS or sicca syndrome were evaluated by flow cytometry, immunoblotting, and immunofluorescence to assess autophagy (autophagic flux, light chain 3 IIB [LC3-IIB], p62, LC3-IIB+/lysosome-associated membrane protein 1 [LAMP-1] staining), apoptosis (annexin V/propidium iodide [PI], caspase 3), and activation (intercellular adhesion molecule, vascular cell adhesion molecule). Focus score and germinal center presence were assessed in the SGs from the same patients to assess correlation with histologic severity. Human SG (HSG) cells were stimulated in vitro with peripheral blood mononuclear cells (PBMCs) and serum from primary SS patients in the presence or absence of autophagy inhibitors to determine changes in autophagy and epithelial cell activation.
SGECs from primary SS patients (n = 24) exhibited increased autophagy (autophagic flux [P = 0.001]; LC3-IIB [P = 0.02]; p62 [P = 0.064]; and as indicated by LC3-IIB/LAMP-1+ staining), increased expression of antiapoptotic molecules (Bcl-2 [P = 0.006]), and reduced apoptosis (annexin V/PI [P = 0.002]; caspase 3 [P = 0.057]), compared to samples from patients with sicca syndrome (n = 16). Autophagy correlated with histologic disease severity. In vitro experiments on HSG cells stimulated with serum and PBMCs from primary SS patients confirmed activation of autophagy and expression of adhesion molecules, which was reverted upon pharmacologic inhibition of autophagy.
In primary SS SGECs, inflammation induces autophagy and prosurvival mechanisms, which promote SGEC activation and mirror histologic severity. These findings indicate that autophagy is a central contributor to the pathogenesis of primary SS and a new therapeutic target.
唾液腺上皮细胞（SGECs）是原发性干燥综合征（SS）发病机制中的关键细胞驱动因素；然而，原发性SS中维持SGECs激活的机制仍不清楚。我们进行了这项研究，以确定自噬在原发性SS中SGECs存活和激活中的作用。
通过流式细胞术、免疫印迹和免疫荧光评估从原发性SS或干燥综合征患者的小唾液腺中分离出的原代SGECs，以评估自噬（自噬通量、轻链3 IIB [LC3-IIB]、p62、LC3-IIB+/溶酶体相关膜蛋白1 [LAMP-1]染色）、凋亡（膜联蛋白V/碘化丙啶[PI]、半胱天冬酶3）和激活（细胞间粘附分子、血管细胞粘附分子）。评估同一患者唾液腺中的聚焦评分和生发中心存在情况，以评估与组织学严重程度的相关性。在有或没有自噬抑制剂的情况下，用人外周血单个核细胞（PBMCs）和原发性SS患者的血清体外刺激人唾液腺（HSG）细胞，以确定自噬和上皮细胞激活的变化。
与干燥综合征患者（n = 16）的样本相比，原发性SS患者（n = 24）的SGECs表现出自噬增加（自噬通量[P = 0.001]；LC3-IIB [P = 0.02]；p62 [P = 0.064]；以及LC3-IIB/LAMP-1+染色所示）、抗凋亡分子表达增加（Bcl-2 [P = 0.006]）和凋亡减少（膜联蛋白V/PI [P = 0.002]；半胱天冬酶3 [P = 0.057]）。自噬与组织学疾病严重程度相关。对用原发性SS患者的血清和PBMCs刺激的HSG细胞进行的体外实验证实了自噬的激活和粘附分子的表达，在自噬的药理学抑制后这些表达恢复正常。
在原发性SS的SGECs中，炎症诱导自噬和促生存机制，促进SGECs激活并反映组织学严重程度。这些发现表明自噬是原发性SS发病机制的核心因素和一个新的治疗靶点。
