Department of Microbiology and Immunology, Burns' Institute, First Hospital Affiliated to the Chinese PLA General Hospital, 51 Fucheng Road, Haidian District, Beijing, 100048, People's Republic of China.
Department of Emergency, First Hospital Affiliated to Wenzhou Medical College, Wenzhou, 325000, People's Republic of China.
Inflamm Res. 2018 Feb;67(2):157-168. doi: 10.1007/s00011-017-1104-9. Epub 2017 Oct 11.
Glucagon-like peptide-1 (GLP-1)-based therapy via G protein-coupled receptor (GPCR) GLP-1R, to attenuate hyperglycemia in critical care has attracted great attention. However, the exaggerated inflammation by GLP-1R agonist, Exendin-4, in a mouse model of burn injury was quite unexpected. Recent studies found that GPCR might elicit proinflammatory effects by switching from Gαs to Gαi signaling in the immune system. Thus, we aimed to investigate the possible Gαs to Gαi switch in GLP-1R signaling in monocyte following burn injury.
Splenic monocytes from sham and burn mice 24 h following burn injury were treated with consecutive doses of Exendin-4 alone or in combination with an inhibitor of Gαi signaling (pertussis toxin, PTX), or a blocker of protein kinase A (H89). Cell viability was assessed by CCK-8, and the supernatant was collected for cytokine measurement by ELISA. Intracellular cAMP level, phosphorylated PKA activity, and nuclear NF-κB p65 were determined by ELISA, ERK1/2 activation was analyzed by Western blot. The expression of GLP-1R downstream molecules, Gαs, Gαi and G-protein coupled receptor kinase 2 (GRK2) were examined by immunofluorescence staining and Western blot.
Exendin-4 could inhibit the viability of monocyte from sham rather than burn mice. Unexpectedly, it could also reduce TNF-α secretion from sham monocyte while increase it from burn monocyte. The increased secretion of TNF-α by Exendin-4 from burn monocyte could be reversed by pretreatment of PTX or H89. Accordingly, Exendin-4 could stimulates cAMP production dose dependently from sham instead of burn monocyte. However, the blunt cAMP production from burn monocyte was further suppressed by pretreatment of PTX or H89 after 6-h incubation. Nevertheless, phosphorylated PKA activity was significantly increased by low dose of Exendin-4 in sham monocyte, by contrast, it was enhanced by high dose of Exendin-4 in burn monocyte after 1-h incubation. Following Exendin-4 treatment for 2 h ex vivo, total nuclear NF-κB and phosphorylated NF-κB activity, as well as cytoplasmic pERK1/2 expressions were reduced in sham monocyte, however, only pERK1/2 was increased by Exendin-4 in burn monocytes. Moreover, reduced expressions of GLP-1R, GRK-2 and Gαs in contrast with increased expression of Gαi were identified in burn monocyte relative to sham monocyte.
This study presents an unexpected proinflammatory switch from Gαs to Gαi signaling in burn monocyte, which promotes ERK1/2 and NF-κB activation and the downstream TNF-α secretion. This phenomenon is most probably responsible for proinflammatory response evoked by Gαs agonist Exendin-4 following burn injury.
通过 G 蛋白偶联受体 (GPCR) GLP-1R 介导的胰高血糖素样肽-1 (GLP-1) 治疗，减轻重症监护中的高血糖已引起广泛关注。然而，GLP-1R 激动剂 Exendin-4 在烧伤小鼠模型中引起的炎症反应过度，这是出乎意料的。最近的研究发现，GPCR 可能通过在免疫系统中从 Gαs 向 Gαi 信号转导而引发促炎作用。因此，我们旨在研究烧伤后单核细胞中 GLP-1R 信号转导中可能发生的 Gαs 向 Gαi 的转换。
烧伤后 24 小时，来自假手术和烧伤小鼠的脾单核细胞分别用连续剂量的 Exendin-4 单独或与 Gαi 信号转导抑制剂（百日咳毒素，PTX）或蛋白激酶 A 阻断剂（H89）联合处理。通过 CCK-8 评估细胞活力，通过 ELISA 收集上清液以测量细胞因子。通过 ELISA 测定细胞内 cAMP 水平、磷酸化 PKA 活性和核 NF-κB p65，通过 Western blot 分析 ERK1/2 激活。通过免疫荧光染色和 Western blot 检测 GLP-1R 下游分子、Gαs、Gαi 和 G 蛋白偶联受体激酶 2 (GRK2) 的表达。
Exendin-4 可抑制来自假手术而非烧伤小鼠的单核细胞活力。出乎意料的是，它还可以降低来自假手术单核细胞的 TNF-α分泌，而增加来自烧伤单核细胞的 TNF-α分泌。来自烧伤单核细胞的 Exendin-4 增加的 TNF-α分泌可以通过 PTX 或 H89 的预处理来逆转。相应地，Exendin-4 可以从来自假手术而非烧伤的单核细胞中剂量依赖性地刺激 cAMP 的产生。然而，用 PTX 或 H89 预处理后，6 小时孵育后，烧伤单核细胞中 cAMP 的产生进一步受到抑制。然而，低剂量的 Exendin-4 在来自假手术的单核细胞中显著增加磷酸化 PKA 活性，相比之下，在烧伤单核细胞中，在 1 小时孵育后，高剂量的 Exendin-4 增强磷酸化 PKA 活性。在体外 2 小时 Exendin-4 处理后，来自假手术的单核细胞中总核 NF-κB 和磷酸化 NF-κB 活性以及细胞质 pERK1/2 表达减少，而来自烧伤的单核细胞中仅 pERK1/2 被 Exendin-4 增加。此外，与来自假手术的单核细胞相比，烧伤单核细胞中发现 GLP-1R、GRK-2 和 Gαs 的表达减少，而 Gαi 的表达增加。
本研究在烧伤单核细胞中提出了一种意想不到的从 Gαs 向 Gαi 信号转导的促炎转换，该转换促进了 ERK1/2 和 NF-κB 的激活以及下游 TNF-α 的分泌。这种现象很可能是 Gαs 激动剂 Exendin-4 引起烧伤后炎症反应的原因。
