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.研究背景：GPNMB水平升高可在FTD-GRN患者的脑脊液（cerebrospinal fluid, CSF）及死后脑组织、衰老的Grn基因缺陷小鼠脑组织中检测到。尽管年轻的Grn基因缺陷小鼠脑组织中未观察到GPNMB的上调，但该小鼠的外周免疫细胞确实存在GPNMB水平升高的现象。值得注意的是，目前尚不清楚前颗粒蛋白缺陷状态下GPNMB上调的功能意义。鉴于GPNMB已被讨论作为GRN介导的神经退行性疾病的潜在治疗靶点，该领域亟需明确GPNMB在免疫系统中的正常功能，以及靶向GPNMB会带来有益还是有害的效应。 研究方法：本研究通过反义寡核苷酸（antisense oligonucleotide, ASO）介导GPNMB敲低，分别对25名神经健康对照者（neurologically healthy controls, NHCs）及年龄、性别匹配的FTD-GRN患者的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），以及前颗粒蛋白缺陷型（progranulin-deficient, Grn-/-）与B6小鼠的腹腔巨噬细胞（peritoneal macrophages, pMacs）的相关效应进行评估。评估指标包括溶酶体功能、抗原呈递及MHC-II分子的加工与回收过程，同时检测细胞因子释放与转录水平。 研究结果：本研究证实，通过ASO介导GPNMB敲低，可增加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量。在Grn基因缺陷型巨噬细胞中，ASO介导的GPNMB敲低会降低溶酶体组织蛋白酶总活性与蛋白质降解能力。此外，在Grn基因缺陷型雌性小鼠的巨噬细胞中，ASO介导的GPNMB敲低会升高MHC-II分子的表面表达水平，这一现象源于MHC-II的摄取与回收过程受到抑制。最后，由于GPNMB细胞外片段（extracellular fragment, ECF）的调控作用缺失，ASO介导的GPNMB敲低会扰乱小鼠巨噬细胞中干扰素γ（interferon gamma, IFNG）刺激下的细胞因子转录与分泌过程。 研究结论：本研究数据显示，GPNMB可对多种免疫效应功能产生调控作用，包括通过分泌其细胞外片段抑制髓系细胞的炎症与免疫应答。因此，在前颗粒蛋白缺陷状态下，GPNMB转录本与蛋白质水平的显著上调可能是一种维持髓系细胞溶酶体功能的代偿机制。上述全新研究结果表明，针对FTD-GRN患者实施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。