Maj Institute of Pharmacology, Polish Academy of Sciences, Department of Pain Pharmacology, 12 Smetna Str, 31-343, Krakow, Poland.
Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri 2, 20156, Milan, Italy.
Mol Cell Probes. 2020 Dec;54:101671. doi: 10.1016/j.mcp.2020.101671. Epub 2020 Nov 4.
Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1β mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI.
创伤性脑损伤（TBI）是全球人口死亡的主要原因。TBI 后炎症过程紊乱会加重继发性脑损伤，并导致不良后果。已经确定了伴随脑创伤的多种炎症事件，如神经胶质细胞激活、趋化因子释放或补体系统级联的启动，作为 TBI 治疗的潜在靶点。然而，趋化因子在补体激活中的参与仍不清楚。我们的研究旨在确定受伤大脑中涉及 CCL2/CCL7/CCL12/CCR2 途径的分子表达变化，以及 CCL2、CCL7 和 CCL12（10、100 和 500ng/mL）对经典和凝集素补体途径和小胶质细胞培养物中炎症因子的影响。通过控制皮质撞击（CCI）对小鼠进行脑损伤建模。我们的发现表明，在创伤后 24 小时内，CCL2、CCL7 和 CCL12 在皮质、纹状体和/或丘脑的 mRNA 和蛋白质水平上呈时间依赖性上调。对所测试趋化因子受体 CCR2 的表达分析表明，它在受伤大脑区域的主要在 mRNA 水平上大量上调。使用原代皮质小胶质细胞培养物，我们观察到 LPS（100ng/mL）处理 24 小时后 CCL2、CCL7 和 CCL12 的表达显著增加。CCL2 刺激小胶质细胞增加了 IL-1β mRNA 的水平，但不影响 IL-18、IL-6 和 IL-10 的表达。此外，CCL2 显著增加了小胶质细胞激活标志物 Iba1 的表达。CCL2 和 CCL12 上调了 C1qa 的表达，但不影响 C1ra 和 C1s1 的表达（经典途径）；此外，CCL2 增加了 ficolin A 的表达，降低了 collectin 11 的表达（凝集素途径）。此外，我们观察到 CCL2 和 CCL12 处理后 pentraxin 3（补体级联的调节剂）的下调。我们没有在小胶质细胞中检测到 ficolin B、Mbl1 和 Mbl2 的表达。我们的数据将 CCL2 确定为经典和凝集素补体途径的调节剂，表明 CCL2 可能是脑损伤后药物干预的有希望的靶点。此外，我们的研究提供了证据表明 CCL2 和另外两种 CCR2 配体可能在 TBI 变化的发展中发挥作用。
