The HSP GRP94 interacts with macrophage intracellular complement C3 and impacts M2 profile during ER stress

Abstract

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.

Endoplasmic Reticulum Chaperones in Viral Infection: Therapeutic Perspectives

“…Moreover, Kwon et al ( 123 ) analyzed the interactions of HCV E2 with human monocyte-derived macrophages and showed that it induced a polarization of macrophages toward an M2-like anti-inflammatory phenotype, which is consistent with the increase in TGF-β production by hepatocytes. Although they did not study GRP94 specifically, we can hypothesize that E2 also induces AIMP1 degradation and GRP94 migration to the cell membrane in macrophages, consistent with our findings in which we showed that membrane GRP94 is a signature of M2 macrophages ( 105 ).…”

“…In these cells, functional GRP94 was secreted under ER stress conditions, and this correlated with a switch to a proinflammatory profile which was dependent on activation of the UPR through IRE1α. Under these conditions, GRP94 is cosecreted with C3 and may facilitate its cleavage by cathepsin L. We also showed that GRP94 interacts directly with cathepsin L ( 105 ). Finally, GRP94 controls regulatory T cells by chaperoning GARP (glycoprotein A repetitions predominant), the docking receptor for transforming growth factor beta (TGF-β) ( 115 ) and the integrins αvβ6 and β8 involved in its release.…”

“…GRP94 has been shown to interact with and to regulate furin in HEK293F cells, and inhibition of GRP94 with an siRNA suppresses intracellular trafficking of furin as well as its proteolytic activity on the zymogen of the metalloproteinase ADAMTS9 at the cell membrane ( 104 ). We recently found that GRP94 directly interacts with cathepsin L ( 105 ). These reported data suggest that GRP94 activity could potentially impact viral entry by modulating furin and cathepsins.…”

The GRP94 Inhibitor PU-WS13 Decreases M2-like Macrophages in Murine TNBC Tumors: A Pharmaco-Imaging Study with 99mTc-Tilmanocept SPECT

“…We first used immunofluorescence to confirm previous preliminary results showing the presence of CD206 + M2-like macrophages in 4T1 biopsies, among which, 44% highly expressed GRP94 [9]. As shown in Figure 1A, CD206 + macrophages were present in the 4T1 biopsies analyzed and 35.8% ± 6.40% co-expressed GRP94 in coherence with our previous results.…”

GARP: A Key Target to Evaluate Tumor Immunosuppressive Microenvironment

“…Consequently, sGARP could be considered a new soluble factor favoring M2 polarization, which also supports an immunosuppressive TME. Of note, we have shown that primary human M2 macrophages do not express GARP at their membranes, although they produce TGF-β [ 72 ] and, to our knowledge, M2-like macrophages in the TME have not been reported to express GARP.…”