S1P/S1PR1通过上调TAM受体和吞噬相关蛋白表达增强巨噬细胞胞葬作用

S1P/S1PR1 enhances macrophage efferocytosis by upregulating the expression of TAM receptors and phagocytosis-related proteins

摘要:

目的]探究1-磷酸鞘氨醇(S1P)是否通过其受体S1PR1促进LC3相关吞噬作用,进而增强THP-1源性巨噬细胞的胞葬作用。 [方法]以80 μg/L佛波酯诱导THP-1单核细胞分化为巨噬细胞；采用254 nm紫外线照射Jurkat细胞,诱导其发生早期凋亡,以用于细胞吞噬实验。巨噬细胞分别经0、0.5、1.0和1.5 μmol/L S1P处理12 h；或以1.5 μmol/L S1P处理,并单独或联合使用S1PR1拮抗剂W146、S1PR2拮抗剂JTE-013、S1PR3拮抗剂CAY10444及ATG4B抑制剂NSC185058进行干预。采用Western blot检测MerTK、Axl、Tyro3、LC3B及LAMP1的表达水平；通过荧光标记凋亡细胞开展吞噬实验以评估吞噬效率；利用ELISA测定细胞上清液中促炎因子(TNF-α、IL-6和IL-1β)与抗炎因子(TGF-β、IL-4和IL-10)的水平。 [结果]S1P可显著增强巨噬细胞对凋亡细胞的吞噬能力,且该作用呈浓度依赖性。具体表现为:巨噬细胞吞噬凋亡细胞的效率显著提升(P＜0.01)；胞葬受体MerTK、Axl、Tyro3的表达显著上调(P＜0.01)；同时,抗炎因子(TGF-β、IL-4和IL-10)水平显著增加(P＜0.01),促炎因子(TNF-α、IL-6和IL-1β)水平显著降低(P＜0.01)。S1P阻断实验结果显示,使用W146阻断S1PR1可逆转S1P的促胞葬作用(P＜0.05),而阻断S1PR2或S1PR3则无明显效果。S1P处理能显著提高巨噬细胞内LC3B和LAMP1的基因及蛋白表达水平(P＜0.01),并促进吞噬溶酶体成熟。使用NSC185058后,巨噬细胞的吞噬效率及抗炎因子分泌均随之下降(P＜0.05)。此外,同时阻断S1PR1与LC3相关吞噬对胞葬作用的抑制效果更为显著(P＜0.01)。 [结论]S1P通过S1PR1介导的LC3相关吞噬作用,显著增强THP-1源性巨噬细胞对凋亡细胞的吞噬清除能力,并促进巨噬细胞向抗炎表型转化。

Abstract:

Aim To investigate whether sphingosine-1-phosphate (S1P) promotes LC3-associated phagocytosis via its receptor S1PR1, thereby enhancing the efferocytosis of THP-1-derived macrophages. Methods THP-1 monocytes were induced to differentiate into macrophages using 80 μg/L phorbol ester; 254 nm ultraviolet radiation was used to induce early apoptosis in Jurkat cells for cell phagocytosis experiments. Macrophages were treated with 0,0.5,1.0 and 1.5 μmol/L S1P for 12 h, or treated with 1.5 μmol/L S1P alone or in combination with S1PR1 antagonist W146, S1PR2 antagonist JTE-013, S1PR3 antagonist CAY10444 and ATG4B inhibitor NSC185058. Western blot was used to detect the expression levels of MerTK, Axl, Tyro3, LC3B and LAMP1, fluorescence-labeled apoptotic cells were applied in phagocytosis assays to evaluate phagocytic efficiency, ELISA was employed to measure the levels of pro-inflammatory factors (TNF-α, IL-6 and IL-1β) and anti-inflammatory factors (TGF-β, IL-4 and IL-10) in the supernatant. Results S1P could significantly enhance the phagocytic capacity of macrophages for apoptotic cells, and this effect was concentration-dependent. Specifically, the phagocytic efficiency of macrophages for apoptotic cells was significantly improved (P＜0.01); the expression of cell burial receptors MerTK, Axl and Tyro3 was markedly upregulated (P＜0.01); at the same time, levels of anti-inflammatory factors (TGF-β, IL-4 and IL-10) increased (P＜0.01), while pro-inflammatory factors (TNF-α, IL-6 and IL-1β) decreased (P＜0.01). The results of the S1P blockade experiment showed that blocking S1PR1 with W146 could reverse the efferocytosis of S1P (P＜0.05), while blocking S1PR2 or S1PR3 had no significant effect. S1P treatment significantly increased the gene and protein expression levels of LC3B and LAMP1 in macrophages (P＜0.01) and promoted lysosome maturation. After NSC185058 treatment, the phagocytic efficiency and anti-inflammatory factor secretion of macrophages decreased (P＜0.05). In addition, the inhibitory effect of blocking S1PR1 and LC3-related phagocytosis on efferocytosis was more significant (P＜0.01). Conclusion S1P significantly enhances the phagocytic clearance ability of THP-1-derived macrophages towards apoptotic cells through S1PR1-mediated LC3-related phagocytosis, and promotes the transformation of macrophages into anti-inflammatory phenotypes.