Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages.

IF 4.7 3区 医学 Q2 IMMUNOLOGY
Journal of Innate Immunity Pub Date : 2023-01-01 Epub Date: 2023-03-07 DOI:10.1159/000530012
Kai-Chen Wu, Nicholas D Condon, Timothy A Hill, Robert C Reid, David P Fairlie, Junxian Lim
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引用次数: 0

Abstract

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.

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Ras相关蛋白Rab5a调节人巨噬细胞中补体C5a受体的贩运、趋化性和趋化因子的分泌。
在炎症反应中经常可以观察到补体激活和 Rab GTPase 运输。补体成分 5a(C5a)可激活细胞表面蛋白 C5a 受体 1(C5aR1),从而促进先天性免疫细胞向感染或损伤部位的招募以及炎症趋化因子的分泌。持续激活可导致多种炎症和自身免疫性疾病。在这里,我们证明了 C5a 诱导人单核细胞源性巨噬细胞(HMDMs)趋化及其分泌炎症趋化因子的机制是由 Rab5a 控制的。我们发现,C5a激活了HMDMs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,最终导致HMDMs趋化和分泌促炎趋化因子。活细胞高分辨率晶格光片显微镜显示,在 HEK293 细胞中,C5a 激活 C5aR1-GFP 内化并与 Rab5a-tdTtomato 共定位,但不与显性负突变体 Rab5a-S34N-tdTtomato 共定位。我们发现,Rab5a 在分化的 HMDMs 中明显上调,而 C5aR1 的内化依赖于 Rab5a。有趣的是,虽然Rab5a的敲除抑制了C5aR1介导的Akt磷酸化,但并不影响C5aR1介导的ERK1/2磷酸化或HMDMs细胞内的钙动员。使用经孔迁移和μ-滑动趋化试验进行的功能分析表明,Rab5a调节C5a诱导的HMDMs趋化。此外,还发现 C5aR1 在 HMDMs 中介导 Rab5a 与 β-arrestin2 的相互作用,而不是与 G 蛋白的相互作用。此外,通过敲除 Rab5a 或 β-arrestin2,或使用 C5aR1 拮抗剂或 PI3K 抑制剂进行药物抑制,可减少 C5a 诱导的 HMDMs 分泌促炎趋化因子(CCL2、CCL3)。这些发现揭示了调节 HMDMs 趋化和促炎趋化因子分泌的 C5a-C5aR1-β-arrestin2-Rab5a-PI3K 信号通路,并提出了选择性调节 C5a 诱导的炎症输出的新方法。
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来源期刊
Journal of Innate Immunity
Journal of Innate Immunity 医学-免疫学
CiteScore
10.50
自引率
1.90%
发文量
35
审稿时长
7.5 months
期刊介绍: The ''Journal of Innate Immunity'' is a bimonthly journal covering all aspects within the area of innate immunity, including evolution of the immune system, molecular biology of cells involved in innate immunity, pattern recognition and signals of ‘danger’, microbial corruption, host response and inflammation, mucosal immunity, complement and coagulation, sepsis and septic shock, molecular genomics, and development of immunotherapies. The journal publishes original research articles, short communications, reviews, commentaries and letters to the editors. In addition to regular papers, some issues feature a special section with a thematic focus.
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