α-Gal Nanoparticles in CNS Trauma: I. In Vitro Activation of Microglia Towards a Pro-Healing State

Abstract

Background: Macrophages and microglia play critical roles after spinal cord injury (SCI), with the pro-healing, anti-inflammatory (M2) subtype being implicated in tissue repair. We hypothesize that promoting this phenotype within the post-injured cord microenvironment may provide beneficial effects for mitigating tissue damage. As a proof of concept, we propose the use of nanoparticles incorporating the carbohydrate antigen, galactose-α-1,3-galactose (α-gal epitope) as an immunomodulator to transition human microglia (HMC3) cells toward a pro-healing state.

Methods: Quiescent HMC3 cells were acutely exposed to α-gal nanoparticles in the presence of human serum and subsequently characterized for changes in cell shape, expression of anti or pro-inflammatory markers, and secretion of phenotype-specific cytokines.

Results: HMC3 cells treated with serum activated α-gal nanoparticles exhibited rapid enlargement and shape change in addition to expressing CD68. Moreover, these activated cells showed increased expression of anti-inflammatory markers like Arginase-1 and CD206 without increasing production of pro-inflammatory cytokines TNF-α or IL-6.

Conclusion: This study is the first to show that resting human microglia exposed to a complex of α-gal nanoparticles and anti-Gal (from human serum) can be activated and polarized toward a putative M2 state. The data suggests that α-gal nanoparticles may have therapeutic relevance to the CNS microenvironment, in both recruiting and polarizing macrophages/microglia at the application site. The immunomodulatory activity of these α-gal nanoparticles post-SCI is further described in the companion work (Part II).

Keywords: Anti-inflammatory; Cytokines; Immunomodulation; Microglia; Spinal cord injury; α-gal.

Fig. 1

Characteristics of typical α-gal nanoparticles. A TEM image depicting native α-gal nanoparticles. Scale: 100nm B Table of α-gal nanoparticle physical properties

Fig. 2

Binding of α-gal nanoparticles to HMC3 cells under different treatment conditions. A FITC conjugated BS-Lectin was used to fluorescently label α-gal nanoparticles. Representative flow cytometry spectrograms of HMC3 cells incubated with α-gal nanoparticles and human serum (immune complex) or B cells incubated with α-gal nanoparticles only or C cells incubated with human serum only. Also shown are respective control groups (HMC3 in media only). Fluorescence microscopy at 10X was used for visual confirmation of nanoparticle binding D. Control and E immune complex. White arrows denote nanoparticle binding of BS-lectin. Scale: 100µm. The dotted vertical line represents the median fluorescence intensity (MFI) shift for BS-lectin

Fig. 3

Immunofluorescent images of HMC3 cells in the A untreated state or B exposure to α-gal nanoparticle complex. A rapid change in cell morphology was observed after α-gal complex treatment. Morphological measurements depict a significant increase in the cell area C, elongation and a decrease in circularity D. *p < 0.05 vs. control. Scale: 100µm

Fig. 4

Flow cytometry characterization of cellular markers expressed by HMC3 cells. HMC3 cells were treated with anti-Gal/α-gal nanoparticles immune complex (3 h) and compared with untreated controls for A CD68 B CD206 or C Arginase-1 expression. Data showed a significant shift in the median fluorescence intensity (MFI) of these surface markers. The markers are expressed as live singlets using BS-lectin positive cells. Corresponding plots quantifying percent gated cells are shown (mean ± SE, *p < 0.05 vs. control, n = 3 runs). The dotted vertical line represents the median fluorescence intensity (MFI) shift for markers

Fig. 5

Expression of various cytokines in HMC3 cells. The cells were treated with IFN-γ (10 ng/mL) or with anti-Gal/α-gal nanoparticles immune complexes (10 mg/mL) for up to 48 h. The supernatants were collected at respective time points and the levels of A TNF-α B IL-6 or C VEGF were measured using ELISA. D NO generation by HMC3 cells at different times post-treatment. *p < 0.1, **p < 0.05, ***p < 0.01 n = 3 runs