Data_Sheet_1_Type 2 Innate Lymphoid Cells Accumulate in the Brain After Hypoxia-Ischemia but Do Not Contribute to the Development of Preterm Brain Injury.pdf

BackgroundThe immune system of human and mouse neonates is relatively immature. However, innate lymphoid cells (ILCs), commonly divided into the subsets ILC1, ILC2, and ILC3, are already present in the placenta and other fetal compartments and exhibit higher activity than what is seen in adulthood. Recent reports have suggested the potential role of ILCs, especially ILC2s, in spontaneous preterm labor, which is associated with brain damage and subsequent long-term neurodevelopmental deficits. Therefore, we hypothesized that ILCs, and especially ILC2s, play a role in preterm brain injury.
MethodsC57Bl/6J mice at postnatal day 6 were subjected to hypoxia-ischemia (HI) insult induced by left carotid artery ligation and subsequent exposure to 10% oxygen in nitrogen. The presence of ILCs and ILC2s in the brain was examined at different time points after HI. The contribution of ILC2s to HI-induced preterm brain damage was explored using a conditionally targeted ILC2-deficient mouse strain (Rorαfl/flIL7rCre), and gray and white-matter injury were evaluated at 7 days post-HI. The inflammatory response in the injured brain was assessed using immunoassays and immunochemistry staining.
ResultsSignificant increases in ILCs and ILC2s were observed at 24 h, 3 days, and 7 days post-HI in the injured brain hemisphere compared with the uninjured hemisphere in wild-type mice. ILC2s in the brain were predominantly located in the meninges of the injured ipsilateral hemispheres after HI but not in the brain parenchyma. Overall, we did not observe changes in cytokine/chemokine levels in the brains of Rorαfl/flIL7rCre mice compared with wild type animals apart from IL-13. Gray and white-matter tissue loss in the brain was not affected after HI in Rorαfl/flIL7rCre mice. Correspondingly, we did not find any differences in reactive microglia and astrocyte numbers in the brain in Rorαfl/flIL7rCre mice compared with wild-type mice following HI insult.
ConclusionAfter HI, ILCs and ILC2s accumulate in the injured brain hemisphere. However, ILC2s do not contribute to the development of brain damage in this mouse model of preterm brain injury.

研究背景：人类和小鼠新生儿的免疫系统相对未成熟。然而，通常分为ILC1、ILC2和ILC3亚群的固有淋巴细胞（innate lymphoid cells，ILCs）已存在于胎盘和其他胎儿组织中，且活性高于成年个体。近期研究提示，固有淋巴细胞尤其是ILC2可能在自发性早产中发挥作用，而自发性早产与脑损伤及后续长期神经发育缺陷相关。因此，本研究提出假说：固有淋巴细胞，尤其是ILC2，参与了早产脑损伤的发生发展。
实验方法：选用出生后第6天的C57Bl/6J小鼠，通过左侧颈动脉结扎并随后暴露于10%氧氮混合气构建缺氧缺血（hypoxia-ischemia，HI）损伤模型。在缺氧缺血损伤后的不同时间点，检测脑组织中固有淋巴细胞及ILC2的存在情况。使用条件性敲除ILC2的小鼠品系（Rorαfl/flIL7rCre），探究ILC2对缺氧缺血诱导的早产脑损伤的调控作用，并在缺氧缺血后7天评估小鼠脑组织灰质与白质的损伤程度。通过免疫检测及免疫组织化学染色，评估损伤脑组织的炎症反应状态。
实验结果：与野生型小鼠的未损伤脑半球相比，缺氧缺血损伤侧脑半球中的固有淋巴细胞及ILC2在损伤后24小时、3天及7天均显著升高。缺氧缺血损伤后，脑组织中的ILC2主要定位于损伤同侧半球的脑膜，而非脑实质。整体而言，与野生型小鼠相比，Rorαfl/flIL7rCre小鼠脑组织中的细胞因子/趋化因子水平除IL-13外未发生明显变化。在Rorαfl/flIL7rCre小鼠中，缺氧缺血诱导的脑组织灰质与白质丢失未受影响。相应地，与野生型小鼠相比，缺氧缺血损伤后Rorαfl/flIL7rCre小鼠脑组织中的反应性小胶质细胞与星形胶质细胞数量未出现显著差异。
研究结论：缺氧缺血损伤后，固有淋巴细胞及ILC2会在损伤侧脑半球募集富集。然而，在该早产脑损伤小鼠模型中，ILC2并未参与脑损伤的发生发展。