Single-cell transcriptional changes of spinal cord immune cells associated with Reg1-MO treatment during EAE neuroinflammation

Regnase-1 plays essential roles in restricting inflammation by acting as a RNase degrading mRNAs involved in immune reactions via the recognition of stem-loop structures in the 3'UTRs. Here we developed a novel therapeutic strategy to suppress inflammatory responses by upregulating Regnase-1 expression, which was enabled by the simultaneous use of two antisense morpholino oligonucleotides (MOs) to alter the stem-loop structures present in the Regnase-1 3'UTR and inhibit its auto-regulation. Intracranial Reg1-MO treatment successfully altenuated the disease severity of EAE, with significant delay in disease onset and lower incidence of paralysis. To clarify the cell types responsible for the suppression of autoimmune responses by Reg1-MOs in the CNS, we performed single-cell RNA sequencing (scRNA-seq) using the 10X Chromium platform using the spinal cord CD45+ cells derived from control and Reg1-MO groups collected at the peak of disease (D16). We found that Reg1-MO treatment not only stalled the microglial activation, but also increased the suppressive fucntion of regulatory T cells. Sorted mouse spinal cord CD45+ immune cells (D16) from animals received intracranial administration of Ctrl MO or Reg1-MOs on D9 post EAE immunization.

Regnase-1（核糖核酸酶）可通过识别信使RNA（mRNA）3'非翻译区（3'UTR）中的茎环结构，降解参与免疫反应的mRNA，从而发挥限制炎症的核心作用。本研究开发了一种全新的抗炎治疗策略，通过上调Regnase-1的表达以抑制炎症反应，该策略可通过同时使用两种反义吗啉代寡核苷酸（antisense morpholino oligonucleotides，MOs），改变Regnase-1 3'UTR中的茎环结构并阻断其自身调控通路得以实现。颅内给予Reg1-MO可有效减轻实验性自身免疫性脑脊髓炎（EAE）的疾病严重程度，显著延迟疾病发作并降低麻痹发生率。为明确中枢神经系统（CNS）中Reg1-MO抑制自身免疫反应的效应细胞类型，本研究依托10X Chromium平台，对疾病峰值期（免疫后第16天，D16）对照组与Reg1-MO组来源的脊髓CD45阳性细胞开展了单细胞RNA测序（scRNA-seq）分析。结果显示，Reg1-MO治疗不仅可阻滞小胶质细胞活化，还能增强调节性T细胞（regulatory T cells）的抑制功能。本实验中，我们于实验性自身免疫性脑脊髓炎免疫后第9天（D9）对小鼠颅内注射对照MO或Reg1-MOs，随后分选获取其脊髓CD45阳性免疫细胞（D16）。