Vaz Ana Rita, Falcão Ana Sofia, Scarpa Eleonora, Semproni Carlotta, Brites Dora
Faculty of Pharmacy, Research Institute for Medicines (iMed.ULisboa), Universidade de Lisboa, Lisbon, Portugal.
Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal.
Front Pharmacol. 2020 Jul 14;11:1012. doi: 10.3389/fphar.2020.01012. eCollection 2020.
Increased concentrations of unconjugated bilirubin (UCB), namely its free fraction (Bf), in neonatal life may cause transient or definitive injury to neurons and glial cells. We demonstrated that UCB damages neurons and glial cells by compromising oligodendrocyte maturation and myelination, and by activating astrocytes and microglia. Immature neurons and astrocytes showed to be especially vulnerable. However, whether microglia susceptibility to UCB is also age-related was never investigated. We developed a microglia culture model in which cells at 2 days (2DIV) revealed to behave as the neonatal microglia (amoeboid/reactive cells), in contrast with those at 16DIV microglia that performed as aged cells (irresponsive/dormant cells). Here, we aimed to unveil whether UCB-induced toxicity diverged from the young to the long-cultured microglia. Cells were isolated from the cortical brain of 1- to 2-day-old CD1 mice and incubated for 24 h with 50/100 nM Bf levels, which were associated to moderate and severe neonatal hyperbilirubinemia, respectively. These concentrations of Bf induced early apoptosis and amoeboid shape in 2DIV microglia, while caused late apoptosis in 16DIV cells, without altering their morphology. CD11b staining increased in both, but more markedly in 2DIV cells. Likewise, the gene expression of HMGB1, a well-known alarmin, as well as HMGB1 and GLT-1-positive cells, were enhanced as compared to long-maturated microglia. The CX3CR1 reduction in 2DIV microglia was opposed to the 16DIV cells and suggests a preferential Bf-induced sickness response in younger cells. In conformity, increased mitochondrial mass and NO were enhanced in 2DIV cells, but unchanged or reduced, respectively, in the 16DIV microglia. However, 100 nM Bf caused iNOS gene overexpression in 2DIV and 16DIV cells. While only arginase 1/IL-1β gene expression levels increased upon 50/100 nM Bf treatment in long-maturated microglia, MHCII/arginase 1/TNF-α/IL-1β/IL-6 (>10-fold) were upregulated in the 2DIV microglia. Remarkably, enhanced inflammatory-associated microRNAs (miR-155/miR-125b/miR-21/miR-146a) and reduced anti-inflammatory miR-124 were found in young microglia by both Bf concentrations, while remained unchanged (miR/21/miR-125b) or decreased (miR-155/miR-146a/miR-124) in aged cells. Altogether, these findings support the neurodevelopmental susceptibilities to UCB-induced neurotoxicity, the most severe disabilities in premature babies, and the involvement of immune-inflammation neonatal microglia processes in poorer outcomes.
新生儿期未结合胆红素（UCB）浓度升高，即其游离部分（Bf）升高，可能会对神经元和神经胶质细胞造成短暂或永久性损伤。我们证明，UCB通过损害少突胶质细胞成熟和髓鞘形成，以及激活星形胶质细胞和小胶质细胞来损伤神经元和神经胶质细胞。未成熟的神经元和星形胶质细胞表现得尤为脆弱。然而，小胶质细胞对UCB的易感性是否也与年龄有关从未被研究过。我们建立了一种小胶质细胞培养模型，其中2天（2DIV）的细胞表现为新生儿小胶质细胞（阿米巴样/反应性细胞），而16DIV的小胶质细胞则表现为老化细胞（无反应性/静止细胞）。在这里，我们旨在揭示UCB诱导的毒性在年轻小胶质细胞和长期培养的小胶质细胞之间是否存在差异。从小鼠1至2日龄的大脑皮层中分离细胞，并用50/100 nM的Bf水平孵育24小时，这两个水平分别与中度和重度新生儿高胆红素血症相关。这些Bf浓度在2DIV小胶质细胞中诱导早期凋亡和阿米巴样形态，而在16DIV细胞中导致晚期凋亡，且不改变其形态。两种细胞中CD11b染色均增加，但在2DIV细胞中更明显。同样，与长期成熟的小胶质细胞相比，一种著名的警报素HMGB1的基因表达以及HMGB1和GLT-1阳性细胞均增加。2DIV小胶质细胞中CX3CR1的减少与16DIV细胞相反，这表明年轻细胞中存在优先的Bf诱导的疾病反应。与此一致，2DIV细胞中线粒体质量增加和NO增加，但在16DIV小胶质细胞中分别保持不变或减少。然而，100 nM的Bf导致2DIV和16DIV细胞中iNOS基因过表达。虽然在长期成熟的小胶质细胞中，仅50/100 nM Bf处理后精氨酸酶1/IL-1β基因表达水平增加，但在2DIV小胶质细胞中MHCII/精氨酸酶1/TNF-α/IL-1β/IL-6（>10倍）上调。值得注意的是，两种Bf浓度在年轻小胶质细胞中均发现炎症相关微小RNA（miR-155/miR-125b/miR-21/miR-146a）增加，抗炎性miR-124减少，而在老化细胞中保持不变（miR-21/miR-125b）或减少（miR-155/miR-146a/miR-124）。总之，这些发现支持了对UCB诱导的神经毒性的神经发育易感性，早产儿中最严重的残疾，以及免疫炎症新生儿小胶质细胞过程与较差预后的相关性。
