CSF1R inhibition promotes neuroinflammation and behavioural deficits during graft-versus-host disease in mice.

Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognised, where brain-infiltrating donor MHC class II+ bone marrow-derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesised that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early post-transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients where CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signalling post-transplant failed to reverse GVHD-induced behavioural changes. Moreover, we observed aberrant behaviour in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr-/- grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS. Overall design: Brains from mice with chronic GVHD following allogenic transplantation with wild-type or IFNG receptor knockout bone marrow, and transplanted non-GVHD controls, were collected at day70 post-transplant. Nuclei were isolated with the 10X Genomics Chromium Nuclei Isolation kit and further purified by sorting for DAPI+ cells.

慢性移植物抗宿主病（chronic graft-versus-host disease, cGVHD）仍是异基因造血干细胞移植的严重并发症。中枢神经系统（Central nervous system, CNS）受累的情况正日益得到学界认可，脑内浸润的供体MHC II类阳性骨髓源性巨噬细胞（bone marrow-derived macrophages, BMDM）是其病理驱动因素。BMDM同时也是皮肤和肺部cGVHD的介导因子，目前评估集落刺激因子1受体（colony-stimulating factor 1 receptor, CSF1R）抗体阻断以耗竭巨噬细胞的临床试验结果颇具前景。
我们提出假说，CSF1R抗体阻断或可成为预防或治疗中枢神经系统cGVHD的有效策略。急性GVHD（acute GVHD, aGVHD）期间血脑屏障通透性升高，有助于抗体渗透进入中枢神经系统，并可通过抗CSF1R治疗耗竭小胶质细胞。但移植后早期实施CSF1R阻断却意外加重了aGVHD的神经炎症。
在已构建的cGVHD模型中，血管改变以及抗CSF1R的治疗效果更为有限。经抗CSF1R处理的小鼠脑内仍保留供体BMDM、活化的小胶质细胞、CD8+与CD4+ T细胞，以及局部细胞因子的表达。这一实验结果在供体CX3CR1阳性BMDM中条件性敲除CSF1R的GVHD受体小鼠中得到了重复验证。
值得注意的是，移植后抑制CSF1R信号通路无法逆转GVHD诱导的行为改变。此外，我们在接受抗CSF1R阻断抗体的非GVHD对照受体小鼠，以及CX3CR1阳性细胞中缺失CSF1R的未免疫小鼠中均观察到了异常行为，这揭示了稳态小胶质细胞的全新功能，并提示当前正在进行的CSF1R抑制临床试验应评估患者的神经系统不良事件。
与之相反，转入Ifngr缺陷型（Ifngr-/-）移植物可减少MHC II类阳性BMDM的浸润，从而改善神经认知功能。
本研究结果揭示了CSF1R阻断过程中意外出现的神经免疫毒性，并为中枢神经系统内cGVHD的治疗提供了新的潜在靶点。
实验整体设计：收集异基因移植野生型或IFNG受体敲除骨髓后出现慢性GVHD的小鼠，以及移植后非GVHD对照小鼠的脑组织，采集时间为移植后第70天。使用10X Genomics Chromium细胞核分离试剂盒分离细胞核，并通过DAPI阳性细胞分选进一步纯化。