Controlling Macrophage Polarization to Modulate Inflammatory Cues Using Immune-Switch Nanoparticles

Abstract

The persistence of inflammatory mediators in tissue niches significantly impacts regenerative outcomes and contributes to chronic diseases. Interleukin-4 (IL4) boosts pro-healing phenotypes in macrophages (Mφ) and triggers the activation of signal transducer and activator of transcription 6 (STAT6). Since the IL4/STAT6 pathway reduces Mφ responsiveness to inflammation in a targeted and precise manner, IL4 delivery offers personalized possibilities to overcome inflammatory events. Despite its therapeutic potential, the limited success of IL4-targeted delivery is hampered by inefficient vehicles. Magnetically assisted technologies offer precise and tunable nanodevices for the delivery of cytokines by combining contactless modulation, high tissue penetration, imaging features, and low interference with the biological environment. Although superparamagnetic iron oxide nanoparticles (SPION) have shown clinical applicability in imaging, SPION-based approaches have rarely been explored for targeted delivery and cell programming. Herein, we hypothesized that SPION-based carriers assist in efficient IL4 delivery to Mφ, favoring a pro-regenerative phenotype (M2φ). Our results confirmed the efficiency of SPION-IL4 and Mφ responsiveness to SPION-IL4 with evidence of STAT6-mediated polarization. SPION-IL4-treated Mφ showed increased expression of M2φ associated-mediators (IL10, ARG1, CCL2, IL1Ra) when compared to the well-established soluble IL4. The ability of SPION-IL4 to direct Mφ polarization using sophisticated magnetic nanotools is valuable for resolving inflammation and assisting innovative strategies for chronic inflammatory conditions.

Keywords: SPION; cytokines; inflammation; macrophages; magnetically assisted technologies; targeted delivery.

Figure 1

Physicochemical characterization and evaluation of the functionalization efficiency of SPION-IL4. (A) Representation of the magnetic responsiveness of SPION-IL4. The magnetite iron oxide core of SPION-IL4 gives a brown color to the solution, and when the solution is placed in an EMF generated by a permanent neodymium magnet (500 mT) positioned on the right side of the flask, SPION-IL4 develops a strong magnetization that persists over time. Owing to the superparamagnetic properties of SPION, after magnet removal, SPION lose their magnetic memory (lack of net magnetization), allowing SPION to significantly avoid magnetic aggregation, which is advantageous for their use in biomedical applications; (B) EDS spectra of SPION-IL4 (red) and non-functionalized SPION (blue) and respective scanning transmission electron microscopy (STEM) images, scale bar = 200 nm; (C) Particle hydrodynamic size distribution; (D) Full range FTIR spectrum highlighting the bands replacement at the IL4 fingerprint region; (E) Western blotting for the detection and quantification of IL4. The IL4 band represents soluble/unbounded IL4, while the absence of an IL4 band indicates SPION-bonded IL4 in SPION-IL4 and supernatants S1 and S2. SPION-IL4 (S) served as a control for SPION-IL4 binding. IL4 standard solutions (0 to 12.5 µg/mL) were used for the detection of soluble IL4. S1 represents the supernatant from the IL4 incubation solution with SPION whereas S2 represents the supernatant from the first purification step. IL4 quantification was performed using Image J 1.52o software for band quantification (Supplementary Materials).

Figure 2

Viability and cytotoxicity assessment of THP1-derived macrophages treated with SPION-IL4 at 30 µg/mL (SPION-IL4/30) or 100 µg/mL (SPION-IL4/100) at two time-points. (A) Representative images and quantification of Calcein AM-labeled (live, green) and Propidium iodide-labeled (dead, red) cells. Scale bar = 50 µm. The average number of live/dead cells per field was analyzed and is represented in the graphs; (B) Brightfield images of THP1 treated for 24 h with SPION-IL4. The SPION-IL4 on the cells are identified by the dark regions, which are not observed in the SPION-IL4-free cells (TCPs). Scale bar = 25 µm; (C) MTS assay and (D) LDH release profile. Graph bars are represented as mean ± SE. All conditions were EMF stimulated for 1 h or 24 h using a magnefect nano device (350 mT/well).

Figure 3

SPION-IL4 interplay in the IL4/STAT6 pathway: from IL4Rα interaction to IL4 production. (A) Representative images from confocal microscopy (left) and SEM (right) of THP1-derived macrophages treated with SPION-IL4/100 for 1 h. The inset in the SEM image represents the negative control (TCPs). Scale bars = 35 µm and 1 µm, respectively. The pink dotted line represents SPION-IL4/100 aligned along the EMF; (B) Representative images of IL4Rα (magenta) counterstained with DAPI (blue) 24 h after treatment with SPION-IL4/30. The inset represents Exo IL4 at 24 h. Scale bar = 50 µm; (C) STAT6 phosphorylation (pSTAT6) upon treatment with SPION-IL4/30 and SPION-IL4/100, quantified by cell-based ELISA; (D) I-gene expression and II-quantification of secreted IL4. In D-I, the expression of IL4 was normalized against GAPDH and to the positive control (Exo IL4) represented by a line at y = 1; (E) 3D reconstructed images of IL4 (green) counterstained with DAPI (blue) 24 h after treatment with SPION-IL4. Scale bar = 75 µm. Quantification of the mean fluorescence intensity of IL4Rα and IL4 was performed using ImageJ 1.52o software. Bars represent mean ± SE. Data analysis was performed using the Kruskal–Wallis test followed by Tukey’s HSD test. The symbols * and ^# indicate significant differences between groups (*^,# p < 0.05, ^## p < 0.01, and **** p < 0.0001). All conditions were EMF stimulated for 1 h or 24 h using a magnefect nano device (350 mT/well).

Figure 4

Immune mediators’ profile of THP1-derived macrophages after treatment with SPION-IL4. (A) Confocal microscopy images for immunodetection of CD11b (blue) in THP1 cells 24 h after treatment with SPION-IL4/30 and SPION-IL4/100. Insets correspond to the time-point 1 h. Scale bar = 50 µm. The flow cytometry plots indicate the percentage of CD11b⁺ FITC cells; (B) Differential expression of cytokines/chemokines associated with inflammation screened by a Proteome Profiler Human Cytokine Array; (C) ARG1 quantification in culture medium 24 h after SPION-IL4 treatment; (D) Gene expression analysis of pro-(TNFα and iNOS) and anti-(IL10 and ARG1) inflammatory markers associated with macrophage phenotypes. The expression of target genes was normalized against GAPDH and the control (Exo IL4). * indicates significant differences between groups (* p < 0.05 for 24 h). Bars represent mean ± SE. Data analysis was performed using the Kruskal–Wallis test, followed by Tukey’s HSD test. All conditions were EMF stimulated for 1 h or 24 h using a magnefect nano device (350 mT/well).