Systemic application of IL-4 attenuates secondary local inflammation processes and enhances functional recovery after traumatic spinal cord injury in rats.

Background: Traumatic spinal cord injury (SCI) triggers local and systemic inflammation cascades, limiting neuroregeneration and impeding functional recovery. We investigated the systemic effect of immunomodulation with Interleukin-4 (IL-4) on the local immune reaction and regeneration in injured spinal cord tissue, in addition to the systemic cytokine landscape after SCI in rats. Methodology: After performing a T10 laminectomy, 120 female Wistar rats were randomized to either thoracic clip compression/contusion SCI or sham. SCI animals received intraperitoneal (i.p.) IL-4 or vehicle injections twice daily for up to 7 days post-injury (dpi). Rats underwent a battery of neurobehavioral tests and were sacrificed at 1, 3, 7, 14, and 28 dpi. Transcriptomcis, proteomics and immunohistochemistry (IHC) was used to assess macrophage polarization, cellular neurodegeneration, and astrogliosis of explanted spinal cords. High throughput seromics was applied to measure levels of 18 cytokines in rat serum, which were also compared to SCI patient serum data. Results: IL-4-treated rats showed a significantly better recovery in the Basso, Beattie, Bresnahan locomotor rating scale score and several Catwalk XT gait analysis parameters at 14 dpi. RNA-Seq and proteomics analyses revealed a significant upregulation in gene ontologies pertaining to improved axonogenisis and tissue repair along with reduced neuronal death and the pro-inflammatory TNFa signaling. This was verified via IHC, showing a higher abundance of IBA1+/ARG1+ and IBA1+/CD206+ M2-macrophages with a lower abundance of IBA1+/iNOS+ M1-macrophages compared to vehicle-treated rats at 3 dpi. Furthermore, post-traumatic cyst size was significantly reduced with IL-4 treatment at 28 dpi. While APC+ oligodendrocytes showed a significantly higher cell count in the IL-4 group, no significant difference was noted in NeuN+ neurons compared to the vehicle-treated group at 28 dpi. Seromics revealed significantly higher levels of pro-inflammatory serum cytokines in vehicle-treated rats compared to sham rats in the acute to subacute post-injury phase (1, 3 and 7 dpi), which were effectively suppressed by IL-4 treatment. Consistent with our rat model, data analysis of SCI patients revealed improved outcome of patients with low serum levels of pro-inflammatory cytokines reduced by IL-4. Conclusions: Intraperitoneal IL-4 treatment modulated local and systemic inflammation after SCI and improved functional recovery. Transcriptomic and proteomic profiling revealed activation of regenerative and neuroprotective pathways and suppression of pro-inflammatory signaling, supporting the histological and behavioral findings. These results provide mechanistic insight into IL-4’s effects and support its further preclinical evaluation in SCI.

背景：创伤性脊髓损伤（Traumatic spinal cord injury, SCI）可触发局部及全身炎症级联反应，抑制神经再生并阻碍功能恢复。本研究以大鼠为模型，探讨白细胞介素-4（Interleukin-4, IL-4）免疫调节对损伤脊髓组织局部免疫反应与再生的全身影响，同时分析SCI后全身细胞因子谱的变化。
方法：对120只雌性Wistar大鼠行T10椎板切除术后，将其随机分为胸椎夹压/挫伤型SCI组与假手术组。SCI组大鼠于损伤后1~7天内，每日两次腹腔注射（intraperitoneal, i.p.）IL-4或溶剂对照。分别于损伤后1、3、7、14、28天（days post injury, dpi）对大鼠进行一系列神经行为学测试后处死。采用转录组学、蛋白质组学及免疫组化（immunohistochemistry, IHC）评估离体脊髓组织的巨噬细胞极化、细胞神经变性及星形胶质增生情况。通过高通量血清组学检测大鼠血清中18种细胞因子的水平，并与SCI患者的血清数据进行对比分析。
结果：IL-4治疗组大鼠在损伤后14天（dpi）的巴索-比蒂-布雷斯纳汉（Basso, Beattie, Bresnahan）运动评分量表（BBB评分）及多项Catwalk XT步态分析参数均显著优于对照组。RNA测序（RNA-Seq）与蛋白质组学分析显示，与轴突发生、组织修复相关的基因本体条目显著上调，同时神经元死亡及促炎TNF-α信号通路受到抑制。免疫组化验证结果表明，损伤后3天（dpi）时，与溶剂对照组相比，IL-4治疗组的IBA1+/ARG1+和IBA1+/CD206+ M2型巨噬细胞比例更高，而IBA1+/iNOS+ M1型巨噬细胞比例更低。此外，损伤后28天（dpi）时，IL-4治疗可显著减小创伤后脊髓囊肿体积。虽然IL-4组的APC+少突胶质细胞数量显著升高，但两组在损伤后28天（dpi）的NeuN+神经元数量无显著差异。血清组学结果显示，在损伤后急性至亚急性阶段（1、3、7 dpi），溶剂对照组大鼠的促炎血清细胞因子水平显著高于假手术组，而IL-4治疗可有效抑制此类细胞因子的升高。与大鼠模型结果一致，SCI患者的数据分析显示，血清促炎细胞因子水平较低的患者预后更佳，而IL-4可降低此类细胞因子的水平。
结论：腹腔注射IL-4可调节SCI后的局部与全身炎症反应，并改善大鼠的功能恢复。转录组与蛋白质组学分析显示，再生及神经保护通路被激活，促炎信号通路受到抑制，这与组织学及行为学实验结果相符。本研究阐明了IL-4的作用机制，支持其在SCI中开展进一步的临床前评估。