Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
J Innate Immun. 2023;15(1):468-484. doi: 10.1159/000530012. Epub 2023 Mar 7.
Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits β-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with β-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or β-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-β-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.
补体激活和 Rab GTPase 转运在炎症反应中很常见。补体成分 5a（C5a）激活细胞表面蛋白 C5a 受体 1（C5aR1），促进固有免疫细胞向感染或损伤部位募集和炎症趋化因子的分泌。持续激活会导致多种炎症和自身免疫性疾病。在这里，我们证明 C5a 诱导人单核细胞衍生巨噬细胞（HMDM）趋化和炎症趋化因子分泌的机制受 Rab5a 控制。我们发现，C5a 激活 HMDM 表面表达的 G 蛋白偶联受体 C5aR1，通过 Rab5a 转运招募β-arrestin2，然后激活下游磷脂酰肌醇 3-激酶（PI3K）/Akt 信号通路，最终导致 HMDM 的趋化和促炎趋化因子的分泌。活细胞的高分辨率晶格光片显微镜显示，C5a 激活 C5aR1-GFP 内化，并与 Rab5a-tdTomato 共定位，但不与 HEK293 细胞中的显性负突变 Rab5a-S34N-tdTomato 共定位。我们发现 Rab5a 在分化的 HMDM 中显著上调，C5aR1 的内化依赖于 Rab5a。有趣的是，虽然 Rab5a 的敲低抑制了 C5aR1 介导的 Akt 磷酸化，但它不影响 C5aR1 介导的 ERK1/2 磷酸化或 HMDM 中的细胞内钙动员。使用 Transwell 迁移和 µ-slide 趋化测定的功能分析表明，Rab5a 调节 C5a 诱导的 HMDM 趋化。此外，在 HMDM 中，C5aR1 介导 Rab5a 与β-arrestin2 的相互作用，但不介导与 G 蛋白的相互作用。此外，Rab5a 或β-arrestin2 的敲低或用 C5aR1 拮抗剂或 PI3K 抑制剂进行药理学抑制可减弱 C5a 诱导的 HMDM 前炎症趋化因子（CCL2、CCL3）的分泌。这些发现揭示了一种 C5a-C5aR1-β-arrestin2-Rab5a-PI3K 信号通路，该通路调节 HMDM 的趋化和前炎症趋化因子的分泌，并为选择性调节 C5a 诱导的炎症反应提供了新的途径。
