Monocyte-derived macrophages (MDMs) are key innate immune cells involved in essential biological processes such as tissue repair, host defense and anti-tumor immunity. However, tumor or leukemia-associated anti-inflammatory macrophages (TAMs, LAMs) promote cancer growth by stimulating angiogenesis, inhibiting anti-tumor immunity and contributing to treatment resistance. Recently, several strategies have been developed to target these macrophages, by blocking their recruitment, reducing their population, activating them or reprogramming them. Therefore, a better understanding of the underlying mechanisms involved in the generation and polarization of anti-inflammatory MDMs is crucial for the development of new therapeutic avenues.

Our team used an ex vivo model to generate and polarize human MDMs from the peripheral blood of healthy donors. After purification, primary human monocytes are differentiated into immature macrophages (M0) in response to CSF-1. These M0 macrophages are then polarized into a pro-inflammatory phenotype (M1) with LPS + IFN-γ, or an anti-inflammatory phenotype (M2) with IL-4. Flow cytometry, RT-qPCR, RNA sequencing, ELISA and various functional assays were used to characterize these different types of macrophages.

Using this model, we demonstrated the crucial role of non-apoptotic caspase-8 in both monocyte-to-macrophage differentiation and their M2 polarization and identified a novel non-canonical activation of caspase-8 (CASP8) by cathepsin B (CTSB). Specific activation of caspase-8 by cathepsin B leads to the non-canonical cleavage and activation of caspases 3 and 7 and the cleavage of several substrate proteins at sites distinct from those observed during apoptosis. Finally, we evidenced that targeting the CTSB / CASP8 axis with pharmacological agents (CA-074, Emricasan or our own original and specific non-apoptotic caspase inhibitors) or with genetic approaches (siRNA) not only inhibits the generation of anti-inflammatory macrophages but also reprograms them towards a pro-inflammatory profile. We are currently working on characterizing and targeting of LAM populations via the CTSB / CASP8 axis in bone marrow samples from patients with acute myeloid leukaemia.

Our work identifies a novel CTSB / CASP8 axis as a key regulator of monocyte-to-macrophages differentiation and M2 polarization. By targeting this pathway, we have successfully reprogrammed anti-inflammatory macrophages to a pro-inflammatory state, providing novel therapeutic strategies to counteract immunosuppressive macrophages, reshape the tumor microenvironment and enhance anti-tumor immunity.