M2 macrophage polarization assay for in vitro assessment of drug ability to relieve immunosuppressive features

Macrophages - one of the most abundant antigen-presenting cells - represent by themselves a wide group of functionally-different subpopulations involved in several immunological functions, including inflammatory responses and tissue homeostasis maintain & repair.

Unlike M1 macrophages which promote a pro-inflammatory response, alternatively activated M2 macrophages, with diametrically opposed functions, trigger inflammation resolution and suppress T cell activation via several mechanisms including release of anti-inflammatory cytokines, immune checkpoint engagement, and metabolic activities involving arginase 1 and indoleamine 2,3-dioxygenase, for instance, which promote essential amino acid – arginine and tryptophan, respectively – depletion. Such M2-related markers are the main features shared with the so-called tumor-associated macrophages (TAMs).
Our well-characterized and validated M2 polarization assay captures all of these M2 macrophage features, and therefore provides them as a wide array of valuable surrogates to evaluate effective drugs for their potential to disrupt the M2 polarization-induced signaling and/or to reprogram M2 to M1 macrophages, thereby alleviating the M2-Mediated immunosuppression

M2 macrophage phenotype and its mediated metabolic activities are modulated by p38 MAPK inhibition. (A) HTRF-based quantification of IL6 and IL10 levels released in the supernatants of M2 macrophages, either untreated  or treated with the p38 MAPK inhibitor during their polarization. ELISA-based quantification of (B) L-Arginine, (C) L-Kynurenine and L-Tryptophan levels in the supernatants M2 macrophages that underwent treatment with a p38 MAPK inhibitor during their polarization (using ARG IS-I-0400, KYN BA-E-2200 and TRP BA-E-2700 Immusmol ELISA kits respectively).

Immunosuppressive M2 macrophage phenotype is relieved by PI3K signaling inhibition.

(A) Phenotypic IL10high IL6low M2-associated status is dose-dependently reversed by a PI3K signaling inhibitor. HTRF-based quantification of IL10 and IL6 levels released in the supernatants of M2 macrophages in untreated controls or following treatment with LY294002 (PI3K inhibitor) during M2 polarization.

(B, C) Arginine and tryptophan catabolic activities are dose-dependently lowered by a PI3K signaling inhibitor. ELISA-based quantification of L-Arginine (B, using IS-I-0400 kit), L-Tryptophan and L-Kynurenine levels (C, using BA-E-2700 and BA-E-2200 kits, respectively) in the supernatants of M2 macrophages in untreated controls or following treatment with LY294002 during M2 polarization.

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