Chemical Modulation of in Vivo Macrophage Function with Subpopulation-Specific Fluorescent Prodrug Conjugates

Abstract

Immunomodulatory agents represent one of the most promising strategies for enhancing tissue regeneration without the side effects of traditional drug-based therapies. Tissue repair depends largely on macrophages, making them ideal targets for proregenerative therapies. However, given the multiple roles of macrophages in tissue homeostasis, small molecule drugs must be only active in very specific subpopulations. In this work, we have developed the first prodrug-fluorophore conjugates able to discriminate closely related subpopulations of macrophages (i.e., proinflammatory M1 vs anti-inflammatory M2 macrophages), and employed them to deplete M1 macrophages in vivo without affecting other cell populations. Selective intracellular activation and drug release enabled simultaneous fluorescence cell tracking and ablation of M1 macrophages in vivo, with the concomitant rescue of a proregenerative phenotype. Ex vivo assays in human monocyte-derived macrophages validate the translational potential of this novel platform to develop chemical immunomodulatory agents as targeted therapies for immune-related diseases.

Figure 1

Synthesis of fluorogenic BODIPY–prodrug M1 activatable conjugates.

Figure 2

Comparative fluorescence staining of M1 macrophages using BODIPY derivatives. (A) Fluorescence intensity of compounds 2 (square, gray), 3 (cross, green), and 4 (circle, blue) at different pH values. (B) Fluorescence fold increase of compounds 3 (green, 15 μM) and 4 (blue, 15 μM) between phagosomal and neutral pH. (C, D) Flow cytometry analysis of LPS-induced M1 macrophages (LPS: 100 ng mL^–1, 18 h) and nontreated macrophages after incubation with compound 3 (10 μM). SSC: side cell scattering. QY: fluorescence quantum yield. Values represented as means ± SD. (n = 4). ** for p < 0.01.

Figure 3

Functional assays in M1 and M2 mouse macrophages. (A) Incubation of compounds 6 (10 μM, white) and 5 (5 μM, gray; 10 μM, black) in different subpopulations of macrophages confirm a dose-dependent release of doxorubicin (determined by cell viability) from 5 in M1 macrophages. (B) Flow cytometry histograms of LPS-induced (100 ng mL^–1, 18 h) M1 macrophages and IL-4-induced (100 ng mL^–1, 18 h) M2 macrophages vs nontreated macrophages (M0) after incubation with anti-CD86-APC (M1 marker, 2 μg mL^–1) or anti-CD206-APC (M2 marker, 2 μg mL^–1). (C) NO production assay in cell supernatants from nontreated, M1, and M2 macrophages. Values represented as means ± SD (n = 4). n.s. for p > 0.05, * for p < 0.05, ** for p < 0.01, *** for p < 0.001.

Figure 4

Flow cytometry analysis after treatment with compound 5 (10 μM) and Annexin V-AF647 (1:100) in (A) nonactivated mouse macrophages and (B) LPS-induced (100 ng mL^–1, 18 h) M1 mouse macrophages. (C) Histograms showing Annexin V-AF647 staining in mouse macrophages that were treated with LPS (100 ng mL^–1, 18 h) (blue), compound 5 (10 μM) (purple), and combined LPS (100 ng mL^–1, 18 h) and compound 5 (10 μM) (red). (D) Normalized quantification of fluorescence intensities for Annexin V-AF647 (gray) and BODIPY fluorescence (black). Values represented as means ± SD (n = 3). * for p < 0.05, ** for p < 0.01. (E) Fluorescence confocal microscopy of live macrophages upon treatment with green-fluorescent BODIPY hydrazone 5 (150 nM) and red-fluorescent (Texas Red) zymosan beads (0.05 mg mL^–1, 1 h). Brightfield (top), green fluorescence (center), and merged green-red fluorescence (bottom) images of macrophages without (E) or with bafilomycin A (100 nM, 1 h) preincubation (F). Yellow arrows (in E) and white arrows (in F) point at zymosan-uptaking M1 macrophages. Scale bar: 10 μm.

Figure 5

Discrimination of M1/M2 mouse macrophages and ex vivo assays in human macrophages. (A) Schematic representation of the Transwell assay with mouse macrophages, where M1 and M2 subpopulations are physically isolated through a membrane permeable to small molecules. (B) Flow cytometry analysis of both M1 and M2 mouse subpopulations after coincubation with compound 5 (10 μM) under physiological conditions and subsequent costaining with Annexin V. (C, D) Flow cytometry histograms and quantification of LPS-induced (100 ng mL^–1, 18 h) human M1 macrophages vs nontreated macrophages (M0) after incubation with anti-CD86-APC (M1 marker, 2 μg mL^–1) and compound 5 (1 μM). (E) Cytospin analysis of human M1 macrophages after treatment with LPS alone or LPS and compound 5 revealed morphological differences between viable macrophages (white arrows) and preapoptotic nonviable macrophages (red arrows) due to doxorubicin release. (F) Cell viability of human macrophages after M1 polarization (100 ng mL^–1 LPS, 18 h) and incubation with compound 5 (10 μM). Values represented as means ± SD (n = 3). ** for p < 0.01, *** for p < 0.001.

Figure 6

In vivo imaging of macrophages in a zebrafish model of tissue regeneration. Snapshot fluorescence microscopy images of macrophages recruited to the wounded edge at 1 hpt (A, Movie S1, white arrows) and 24 hpt (B, Movie S2, yellow arrows). High magnification images of macrophages at the regenerative tail fin of 5-treated zebrafish (3 μM) without (C) and with (D) LPS treatment (100 ng mL^–1) at 48 hpt. Yellow arrowheads point at rounded apoptotic and necrotic macrophages upon doxorubicin release. Scale bars: 50 μm. (E) Quantification of macrophage numbers after incubation with DMSO or compounds 3, 5, and LPS. (F) In vivo quantitative analysis of the regenerated tissue area. Zebrafish larvae were treated with DMSO or compound 5 with and without LPS pretreatment, and the regeneration area upon tail fin injury was determined at 48 hpt. Values represented as means ± SD (n ≥ 10). n.s for p > 0.05, * for p < 0.05, ** for p < 0.01.