Dendrimer-N-acetyl-L-cysteine modulates monophagocytic response in adrenoleukodystrophy

Abstract

Objective: X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder due to mutations in the peroxisomal very long-chain fatty acyl-CoA transporter, ABCD1, with limited therapeutic options. ALD may manifest in a slowly progressive adrenomyeloneuropathy (AMN) phenotype, or switch to rapid inflammatory demyelinating cerebral disease (cALD), in which microglia have been shown to play a pathophysiological role. The aim of this study was to determine the role of patient phenotype in the immune response of ex vivo monophagocytic cells to stimulation, and to evaluate the efficacy of polyamidoamine dendrimer conjugated to the antioxidant precursor N-acetyl-cysteine (NAC) in modulating this immune response.

Methods: Human monophagocytic cells were derived from fresh whole blood, from healthy (n = 4), heterozygote carrier (n = 4), AMN (n = 7), and cALD (n = 4) patients. Cells were exposed to very long-chain fatty acids (VLCFAs; C24:0 and C26:0) and treated with dendrimer-NAC (D-NAC).

Results: Ex vivo exposure to VLCFAs significantly increased tumor necrosis factor α (TNFα) and glutamate secretion from cALD patient macrophages. Additionally, a significant reduction in total intracellular glutathione was observed in cALD patient cells. D-NAC treatment dose-dependently reduced TNFα and glutamate secretion and replenished total intracellular glutathione levels in cALD patient macrophages, more efficiently than NAC. Similarly, D-NAC treatment decreased glutamate secretion in AMN patient cells.

Interpretation: ALD phenotypes display unique inflammatory profiles in response to VLCFA stimulation, and therefore ex vivo monophagocytic cells may provide a novel test bed for therapeutic agents. Based on our findings, D-NAC may be a viable therapeutic strategy for the treatment of cALD. Ann Neurol 2018;84:452-462.

FIGURE 1:

(A) The tumor necrosis factor α (TNFα) secretion in ex vivo human cerebral adrenoleukodystrophy (cALD) monophagocytic cells in dose response to very long-chain fatty acid (VLCFA) stimulation. The labeled doses show C24:0 levels. The C26:0 level is 1/10th of the C24:0 dose. (B) Healthy control, adrenomyeloneuropathy (AMN), and cerebral response to lipopolysaccharide (LPS) and VLCFA stimulation.

FIGURE 2:

VLCFA stimulation of ex vivo human healthy control, heterozygote female, AMN, and cALD monophagocytic cells’ (A) TNFα secretion, (B) glutamate secretion, and (C) total intracellular glutathione. GSH = reduced glutathione; GSSG = oxidized glutathione. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

FIGURE 3:

Immunofluorescent and flow cytometry analysis of dendrimer uptake. (A) D-Cy5 exposure to healthy human monophagocytic cells with and without VLCFA stimulation. D-Cy5 (red), CD11b (green), and 4,6-diamidino-2-phenylindole (blue) in both VLCFA-stimulated and nonstimulated states. (B) Representative gating of flow cytometry D-Cy5 exposure in healthy, AMN, and cALD ex vivo monophagocytic cells both with and without VLCFA stimulation. Representative gating of cell population is shown and illustrates side-scattered light area (SSC-A) versus forward-scattered light area (FSC-A). (C) Representative fluorescence intensity of healthy monophagocytic cells without D-Cy5 (black), with D-Cy5 (blue), and D-Cy5 + VLCFAs (red) after 3-hour exposure and (D) 6-hour exposure. Dendrimer was measured via FL4-A detector (640 nm). (E) Mean fluorescence intensity (MFI) of healthy control monophagocytic cells (black) after 3-hour and 6-hour exposure to D-Cy5 (blue) and D-Cy5 + VLCFAs (red). (F) MFI of AMN cells after 3-h exposure. (G) MFI of cALD cells after 6-hour exposure. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

FIGURE 4:

Dendrimer–N-acetyl-L-cysteine (D-NAC) and free NAC treatment of healthy control, heterozygote female, AMN, and cALD ex vivo human monophagocytic cells stimulated with VLCFA. (A) TNFα secretion, (B) glutamate secretion, and (C) total intracellular glutathione. The D-NAC dose indicated is the dose of NAC administered as D-NAC. GSH = reduced glutathione; GSSG = oxidized glutathione.

FIGURE 5:

Unconjugated dendrimer treatment of ex vivo human monophagocytic cells’ TNFα secretion and glutamate secretion.

FIGURE 6:

Ex vivo monophagocytic cells stimulated with VLCFA and clinical neurological function from one cALD patient over 3 time points. (A) TNFα secretion, (B) glutamate secretion, and (C) major functional disability (MFD) score.