LTK缺乏诱导巨噬细胞M2极化，通过降低趋化因子CXCL13改善干燥综合征

IF 3.7 3区医学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Cytokine Pub Date : 2025-03-27 DOI:10.1016/j.cyto.2025.156905

Xiuyuan Feng , Junhui Lu , Wei Cheng , Ping Zhao , Xin Chang , Jian Wu

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CCK-8, flow cytometry, and transmission electron microscopy (TEM), Western blotting (WB) was employed to assess proliferation, apoptosis, autophagy, and autoimmune antigens (Ro52/SSA and La/SSB) in A253 cells. Then, macrophages were treated with 100 ng/ml of LPS to induce the polarization of macrophages towards the M1 phenotype, while macrophages were treated with IL-4 to activate the macrophage M2 phenotype. LTK-silenced A253 cells were co-cultured with macrophages. WB as well as flow cytometry were used to assess macrophage polarization markers. Furthermore, protein-antibody microarrays were utilized to analyze downstream proteins regulated by LTK. Finally, the functionality of LTK was confirmed in NOD/ShiLtJ mice.</div></div><div><h3>Results</h3><div>LTK expression in the GEO database was increased in SS patients. And LTK was also significantly increased by EGF and IFN-γ. Knockdown of LTK increased proliferation and autophagy in A253 cells. 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引用次数: 0

摘要

背景：干燥综合征（SS）是一种涉及巨噬细胞浸润外分泌腺的自身免疫性疾病。LTK是一种酪氨酸激酶受体，参与许多自身免疫性疾病，如红斑狼疮。方法利用NCBI基因表达综合数据库（GEO）筛选SS患者的差异表达基因（DEGs），并在A253细胞中应用EGF和IFN-γ进行定量反转录PCR （RT-qPCR）验证。同时，用慢病毒载体转染A253细胞，实现LTK的稳定沉默。采用CCK-8、流式细胞术、透射电镜（TEM）、Western blotting （WB）检测A253细胞的增殖、凋亡、自噬和自身免疫抗原（Ro52/SSA和La/SSB）。然后，用100 ng/ml LPS处理巨噬细胞，诱导巨噬细胞向M1表型极化，同时用IL-4处理巨噬细胞，激活巨噬细胞M2表型。ltk沉默的A253细胞与巨噬细胞共培养。WB和流式细胞术检测巨噬细胞极化标记物。此外，利用蛋白抗体微阵列分析LTK调控的下游蛋白。最后，在NOD/ShiLtJ小鼠中证实LTK的功能。结果SS患者GEO数据库中sltk表达升高。EGF和IFN-γ也显著增加了LTK。LTK的下调增加了A253细胞的增殖和自噬。而LTK缺乏抑制了A253细胞Ro52/SSA和La/SSB的表达和凋亡。此外，LTK沉默的A253细胞促进巨噬细胞向M2表型极化，这与SS的发病机制有关。LTK敲低导致CXCL13表达减少，进而引发巨噬细胞M2极化。此外，LTK缺乏改善NOD/ShiLtJ小鼠颌下腺组织损伤和抑制自身免疫抗原分泌。此外，shLTK可增加颌下腺组织中自噬标志物和M2极化标志物的表达。结论ltk可通过CXCL13促进SS进行性发病。这一发现表明靶向LTK/CXCL13可能是临床治疗SS的潜在治疗策略。

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LTK deficiency induces macrophage M2 polarization and ameliorates Sjogren's syndrome by reducing chemokine CXCL13

Background

Sjogren's syndrome (SS) is an autoimmune disease involving macrophage infiltration of the exocrine glands. LTK, a receptor tyrosine kinase, is involved in many autoimmune diseases, such as lupus erythematosus. The objectives of this study was to explore the impact of LTK on autophagy in SS.

Methods

The NCBI Gene Expression Omnibus (GEO) database was used to screen for differentially expressed genes (DEGs) in SS patients and validated by quantitative reverse transcription PCR (RT-qPCR) in A253 cells with EGF and IFN-γ. Meanwhile, lentiviral vectors were used to transfect A253 cells for stable LTK silencing. CCK-8, flow cytometry, and transmission electron microscopy (TEM), Western blotting (WB) was employed to assess proliferation, apoptosis, autophagy, and autoimmune antigens (Ro52/SSA and La/SSB) in A253 cells. Then, macrophages were treated with 100 ng/ml of LPS to induce the polarization of macrophages towards the M1 phenotype, while macrophages were treated with IL-4 to activate the macrophage M2 phenotype. LTK-silenced A253 cells were co-cultured with macrophages. WB as well as flow cytometry were used to assess macrophage polarization markers. Furthermore, protein-antibody microarrays were utilized to analyze downstream proteins regulated by LTK. Finally, the functionality of LTK was confirmed in NOD/ShiLtJ mice.

Results

LTK expression in the GEO database was increased in SS patients. And LTK was also significantly increased by EGF and IFN-γ. Knockdown of LTK increased proliferation and autophagy in A253 cells. While LTK deficiency inhibited the expression of Ro52/SSA and La/SSB, and apoptosis in A253 cells. Furthermore, LTK-silenced A253 cells promoted polarization of macrophages towards the M2 phenotype, which is associated with the pathogenesis of SS. Knockdown of LTK resulted in reduced expression of CXCL13, which in turn triggered macrophage M2 polarization. Additionally, LTK deficiency ameliorated submandibular gland tissue damage and inhibited autoimmune antigens secretion in NOD/ShiLtJ mice. In addition, the expression of autophagy markers and M2 polarization markers in the submandibular gland tissue was increased by shLTK.

Conclusion

LTK could promote progressive SS pathogenesis via CXCL13. This discovery indicates that targeting LTK/CXCL13 could be a potential therapeutic strategy for the clinical management of SS.

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来源期刊

Cytokine 医学-免疫学

CiteScore

7.60

自引率

2.60%

发文量

262

审稿时长

48 days

期刊介绍： The journal Cytokine has an open access mirror journal Cytokine: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. * Devoted exclusively to the study of the molecular biology, genetics, biochemistry, immunology, genome-wide association studies, pathobiology, diagnostic and clinical applications of all known interleukins, hematopoietic factors, growth factors, cytotoxins, interferons, new cytokines, and chemokines, Cytokine provides comprehensive coverage of cytokines and their mechanisms of actions, 12 times a year by publishing original high quality refereed scientific papers from prominent investigators in both the academic and industrial sectors. We will publish 3 major types of manuscripts: 1) Original manuscripts describing research results. 2) Basic and clinical reviews describing cytokine actions and regulation. 3) Short commentaries/perspectives on recently published aspects of cytokines, pathogenesis and clinical results.