UMR INSERM/uB/AGROSUP 1231, Team 3 HSP-Pathies, labellisée Ligue Nationale contre le Cancer and Laboratoire d'Excellence LipSTIC, Dijon, France.
UFR des Sciences de Santé, Université de Bourgogne, Dijon, France.
Cell Death Dis. 2021 Jan 22;12(1):114. doi: 10.1038/s41419-020-03288-x.
The role of GRP94, an endoplasmic reticulum (ER) stress protein with both pro- and anti-inflammatory functions, has not been investigated in macrophages during ER stress, whereas ER stress has been reported in many diseases involving macrophages. In this work, we studied GRP94 in M1/LPS + IFNγ and M2/IL-4 primary macrophages derived from human monocytes (isolated from buffy coats), in basal and ER stress conditions induced by thapsigargin (Tg), an inducer of ER calcium depletion and tunicamycin (Tm), an inhibitor of N-glycosylation. We found that GRP94 was expressed on the membrane of M2 but not M1 macrophages. In M2, Tg, but not Tm, while decreased GRP94 content in the membrane, it induced its secretion. This correlated with the induction of a pro-inflammatory profile, which was dependent on the UPR IRE1α arm activation and on a functional GRP94. As we previously reported that GRP94 associated with complement C3 at the extracellular level, we analyzed C3 and confirmed GRP94-C3 interaction in our experimental model. Further, Tg increased this interaction and, in these conditions, C3b and cathepsin L were detected in the extracellular medium where GRP94 co-immunoprecipitated with C3 and C3b. Finally, we showed that the C3b inactivated fragment, iC3b, only present on non-stressed M2, depended on functional GRP94, making both GRP94 and iC3b potential markers of M2 cells. In conclusion, our results show that GRP94 is co-secreted with C3 under ER stress conditions which may facilitate its cleavage by cathepsin L, thus contributing to the pro-inflammatory profile observed in stressed M2 macrophages.
GRP94 是内质网（ER）应激蛋白，具有促炎和抗炎双重功能，但其在 ER 应激期间巨噬细胞中的作用尚未得到研究，而 ER 应激已在许多涉及巨噬细胞的疾病中得到报道。在这项工作中，我们研究了来源于人单核细胞（从白细胞层中分离）的 M1/LPS+IFNγ和 M2/IL-4 原代巨噬细胞中的 GRP94，在 ER 应激条件下，用 thapsigargin（Tg，内质网钙耗竭诱导剂）和 tunicamycin（Tm，N-糖基化抑制剂）诱导。我们发现 GRP94 表达在 M2 但不表达在 M1 巨噬细胞膜上。在 M2 中，Tg 而非 Tm 降低了膜中的 GRP94 含量，但诱导其分泌。这与诱导促炎表型相关，该表型依赖于 UPR IRE1α 臂的激活和功能性 GRP94。正如我们之前报道的，GRP94 与细胞外的补体 C3 相关，我们在我们的实验模型中分析了 C3，并证实了 GRP94 和 C3 的相互作用。此外，Tg 增加了这种相互作用，并且在这些条件下，C3b 和组织蛋白酶 L 在细胞外介质中被检测到，其中 GRP94 与 C3 和 C3b 共免疫沉淀。最后，我们表明，仅在非应激的 M2 上存在的 C3 失活片段 iC3b，依赖于功能性 GRP94，使得 GRP94 和 iC3b 成为 M2 细胞的潜在标志物。总之，我们的结果表明，GRP94 在 ER 应激条件下与 C3 共分泌，这可能促进组织蛋白酶 L 对其的切割，从而有助于应激 M2 巨噬细胞中观察到的促炎表型。
