Table_2_Limitations of PLX3397 as a microglial investigational tool: peripheral and off-target effects dictate the response to inflammation.xlsx

Microglia, the resident macrophages of the central nervous system (CNS), play a critical role in CNS homeostasis and neuroinflammation. Pexidartinib (PLX3397), a colony-stimulating factor 1 (CSF1) receptor inhibitor, is widely used to deplete microglia, offering flexible options for both long-term depletion and highly versatile depletion-repopulation cycles. However, the potential impact of PLX3397 on peripheral (immune) cells remains controversial. Until now, the microglia-specificity of this type of compounds has not been thoroughly evaluated, particularly in the context of peripherally derived neuroinflammation. Our study addresses this gap by examining the effects of PLX3397 on immune cells in the brain, liver, circulation and bone marrow, both in homeostasis and systemic inflammation models. Intriguingly, we demonstrate that PLX3397 treatment not only influences the levels of tissue-resident macrophages, but also affects circulating and bone marrow immune cells beyond the mononuclear phagocyte system (MPS). These alterations in peripheral immune cells disrupt the response to systemic inflammation, consequently impacting the phenotype irrespective of microglial depletion. Furthermore, we observed that a lower dose of PLX3397, which does not deplete microglia, demonstrates similar (non-)MPS effects, both in the periphery and the brain, but fails to fully replicate the peripheral alterations seen in the higher doses, questioning lower doses as a ‘peripheral control’ strategy. Overall, our data highlight the need for caution when interpreting studies employing this compound, as it may not be suitable for specific investigation of microglial function in the presence of systemic inflammation.

小胶质细胞（Microglia）作为中枢神经系统（central nervous system, CNS）的常驻巨噬细胞，在中枢神经系统稳态与神经炎症过程中发挥关键作用。培西达替尼（Pexidartinib, PLX3397）作为集落刺激因子1（CSF1）受体抑制剂，被广泛应用于小胶质细胞清除，可为长期清除方案与灵活多变的清除-再增殖周期提供多样化选择。然而，PLX3397对外周（免疫）细胞的潜在影响仍存在争议。迄今为止，此类化合物的小胶质细胞特异性尚未得到全面评估，尤其是在外周源性神经炎症的研究背景下。本研究针对这一研究空白，分别在稳态模型与全身性炎症模型中，检测了PLX3397对脑、肝脏、循环系统及骨髓内免疫细胞的影响。值得注意的是，本研究证实PLX3397处理不仅会改变组织驻留巨噬细胞的水平，还会对单核吞噬细胞系统（MPS）之外的循环及骨髓免疫细胞产生调控作用。外周免疫细胞的此类改变会破坏机体对全身性炎症的应答，进而独立于小胶质细胞清除过程影响细胞表型。此外，本研究观察到，未产生小胶质细胞清除效果的低剂量PLX3397，在外周组织与脑组织中均表现出相似的（非）单核吞噬细胞系统效应，但无法完全复刻高剂量给药下的外周免疫细胞改变，这对“低剂量可作为外周对照策略”的观点提出了质疑。综上，本研究数据提示，在使用该化合物开展相关研究时需保持谨慎，因为当存在全身性炎症时，该化合物或许并不适用于小胶质细胞功能的特异性探究。