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Background/objectives: Adipose tissue macrophages (ATM) are key actors in the pathophysiology of obesity-related diseases. They have a unique intermediate M2-M1 phenotype which has been linked to endoplasmic reticulum (ER) stress. We previously reported that human M2 macrophages treated with the ER stress inducer thapsigargin switched to a pro-inflammatory phenotype that depended on the stress protein GRP94. In these conditions, GRP94 promoted cathepsin L secretion and was co-secreted with complement C3. As cathepsin L and complement C3 have been reported to play a role in the pathophysiology of obesity, in this work we studied the involvement of GRP94 in the pro-inflammatory phenotype of ATM., Methods: GRP94, cathepsin L and C3 expression were analyzed in CD206 + ATM from mice, WT or obesity-resistant transgenic fat-1, fed a high-fat diet (HFD) or a standard diet. GRP94 colocalization with cathepsin L and C3 and its effects were analyzed in human primary macrophages using thapsigargin as a control to induce ER stress and palmitic acid (PA) as a driver of metabolic activation., Results: In WT, but not in fat-1 mice, fed a HFD, we observed an increase in crown-like structures consisting of CD206 + pSTAT1+ macrophages showing high expression of GRP94 that colocalized with cathepsin L and C3. In vitro experiments showed that PA favored a M2-M1 switch depending on GRP94. This switch was prevented by omega-3 fatty acids. PA-induced GRP94-cathepsin L colocalization and a decrease in cathepsin L enzymatic activity within the cells (while the enzymatic activity in the extracellular medium was increased). These effects were prevented by the GRP94 inhibitor PU-WS13., Conclusions: GRP94 is overexpressed in macrophages both in in vivo and in vitro conditions of obesity-associated inflammation and is involved in changing their profile towards a more pro-inflammatory profile. It colocalizes with complement C3 and cathepsin L and modulates cathepsin L activity., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

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.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancers and is not eligible for hormone and anti-HER2 therapies. Identifying therapeutic targets and associated biomarkers in TNBC is a clinical challenge to improve patients' outcome and management. High infiltration of CD206+ M2-like macrophages in the tumor microenvironment (TME) indicates poor prognosis and survival in TNBC patients. As we previously showed that membrane expression of GRP94, an endoplasmic reticulum chaperone, was associated with the anti-inflammatory profile of human PBMC-derived M2 macrophages, we hypothesized that intra-tumoral CD206+ M2 macrophages expressing GRP94 may represent innovative targets in TNBC for theranostic purposes. We demonstrate in a preclinical model of 4T1 breast tumor-bearing BALB/c mice that (i) CD206-expressing M2-like macrophages in the TME of TNBC can be specifically detected and quantified using in vivo SPECT imaging with 99mTc-Tilmanocept, and (ii) the inhibition of GRP94 with the chemical inhibitor PU-WS13 induces a decrease in CD206-expressing M2-like macrophages in TME. This result correlated with reduced tumor growth and collagen content, as well as an increase in CD8+ cells in the TME. 99mTc-Tilmanocept SPECT imaging might represent an innovative non-invasive strategy to quantify CD206+ tumor-associated macrophages as a biomarker of anti-GRP94 therapy efficacy and TNBC tumor aggressiveness. [ABSTRACT FROM AUTHOR]