ASO-mediated knockdown of GPNMB in mutant-GRN and Grn-deficient peripheral myeloid cells disrupts lysosomal function and immune responses

Background: Increases in GPNMB are detectable in FTD-GRN cerebrospinal fluid (CSF) and post-mortem brain, and brains of aged Grn-deficient mice. Although no upregulation of GPNMB is observed in the brains of young Grn-deficient mice, peripheral immune cells of these mice do exhibit this increase in GPNMB. Importantly, the functional significance of GPNMB upregulation in progranulin-deficient states is currently unknown. Given that GPNMB has been discussed as a potential therapeutic target in GRN-mediated neurodegeneration, it is vital for the field to determine what the normal function of GPNMB is in the immune system, and whether targeting GPNMB will elicit beneficial or deleterious effects. Methods: The effects of GPNMB knock-down via antisense oligonucleotide (ASO) were assessed in peripheral blood mononuclear cells (PBMCs) from 25 neurologically healthy controls (NHCs) and age- and sex-matched FTD-GRN patients, as well as peritoneal macrophages (pMacs) from progranulin-deficient (Grn-/-) and B6 mice. Lysosomal function, antigen presentation and MHC-II processing and recycling were assessed, as well as cytokine release and transcription. Results: We demonstrate here that ASO-mediated knockdown of GPNMB increases lysosomal burden and cytokine secretion in FTD-GRN carrier and neurologically healthy controls (NHCs) monocytes. ASO-mediated knockdown of GPNMB in Grn-deficient macrophages decreased lysosomal pan-cathepsin activity and protein degradation. In addition, ASO-mediated knockdown of GPNMB increased MHC-II surface expression, which was driven by decreased MHC-II uptake and recycling, in macrophages from Grn-deficient females. Finally, ASO-mediated knockdown of GPNMB dysregulated IFNg-stimulated cytokine transcription and secretion by mouse macrophages due to the absence of regulatory actions of the GPNMB extracellular fragment (ECF). Conclusions: Our data herein reveals that GPNMB has a regulatory effect on multiple immune effector functions, including capping inflammation and immune responses in myeloid cells via secretion of its ECF. Therefore, in progranulin-deficient states, the drastic upregulation in GPNMB transcript and protein may represent a compensatory mechanism to preserve lysosomal function in myeloid cells. These novel findings indicate that targeted depletion in FTD-GRN would not be a rational therapeutic strategy because it is likely to dysregulate important immune cell effector functions.背景：在额颞叶痴呆-GRN（FTD-GRN）患者的脑脊液（CSF）和死后脑组织，以及老龄颗粒蛋白缺失（Grn缺陷）小鼠的脑组织中，均可检测到糖蛋白非转移性黑色素瘤蛋白B（GPNMB）的表达上调。尽管年轻Grn缺陷小鼠的脑组织中未观察到GPNMB的上调，但这些小鼠的外周免疫细胞确实出现了GPNMB表达升高。目前，GPNMB上调在颗粒蛋白缺失状态下的功能意义仍不明确。鉴于GPNMB已被提出作为GRN介导的神经退行性疾病的潜在治疗靶点，明确GPNMB在免疫系统中的生理功能，以及靶向GPNMB会引发有益还是有害的效应，是该领域亟待解决的关键问题。 方法：本研究通过反义寡核苷酸（ASO）介导GPNMB敲低，分别对25名神经健康对照者（NHCs）、年龄与性别匹配的FTD-GRN患者的外周血单个核细胞（PBMCs），以及颗粒蛋白缺失（Grn-/-）和C57BL/6（B6）小鼠的腹腔巨噬细胞（pMacs）进行相关评估。检测指标包括溶酶体功能、抗原呈递及主要组织相容性复合体II类（MHC-II）的加工与回收过程，同时还评估了细胞因子释放与转录情况。 结果：本研究证实，ASO介导的GPNMB敲低会增加FTD-GRN携带者及神经健康对照者单核细胞的溶酶体负荷与细胞因子分泌。在颗粒蛋白缺失型巨噬细胞中，ASO介导的GPNMB敲低会降低全组织蛋白酶活性与蛋白质降解水平。此外，在颗粒蛋白缺失雌性小鼠的巨噬细胞中，ASO介导的GPNMB敲低会升高MHC-II表面表达，这一现象由MHC-II摄取与回收过程减弱所驱动。最后，由于GPNMB细胞外片段（ECF）的调控作用缺失，ASO介导的GPNMB敲低会干扰小鼠巨噬细胞中干扰素γ（IFNγ）刺激的细胞因子转录与分泌过程。 结论：本研究数据揭示，GPNMB对多种免疫效应功能具有调控作用，具体包括通过分泌其细胞外片段（ECF）抑制髓系细胞的炎症反应与免疫应答。因此，在颗粒蛋白缺失状态下，GPNMB转录本与蛋白质的大幅上调，可能是维持髓系细胞溶酶体功能的一种代偿机制。本研究的新发现表明，针对FTD-GRN患者的GPNMB靶向敲除并非合理的治疗策略，因为这很可能会扰乱重要免疫细胞的效应功能。