Folate receptor-targeted positron emission tomography of experimental autoimmune encephalomyelitis in rats

Background: Folate receptor-beta (FR-beta) is a cell surface receptor that is significantly upregulated on activated macrophages during inflammation and provides a potential target for folate-based therapeutic and diagnostic agents. FR-beta expression in central nervous system inflammation remains relatively unexplored. Therefore, we used focally induced acute and chronic phases of experimental autoimmune encephalomyelitis (EAE) to study patterns of FR-beta expression and evaluated its potential as an in vivo imaging target. Methods: Focal EAE was induced in rats using heat-killed Bacillus Calmette-Guerin followed by activation with complete Freund's adjuvant supplemented with Mycobacterium tuberculosis. The rats were assessed with magnetic resonance imaging and positron emission tomography/computed tomography (PET/CT) at acute (14 days) and chronic (90 days) phases of inflammation. The animals were finally sacrificed for ex vivo autoradiography of their brains. PET studies were performed using FR-beta-targeting aluminum [F-18]fluoride-labeled 1,4,7-triazacyclononane-1,4, 7-triacetic acid conjugated folate ([F-18]AlF-NOTA-folate, F-18-FOL) and 18 kDa translocator protein (TSPO)-targeting N-acetyl-N-(2-[C-11]methoxybenzyl)-2-phenoxy-5-pyridinamine (C-11-PBR28). Post-mortem immunohistochemistry was performed using anti-FR-beta, anti-cluster of differentiation 68 (anti-CD68), anti-inducible nitric oxide synthase (antiiNOS), and anti-mannose receptor C-type 1 (anti-MRC-1) antibodies. The specificity of F-18-FOL binding was verified using in vitro brain sections with folate glucosamine used as a blocking agent. Results: Immunohistochemical evaluation of focal EAE lesions demonstrated anti-FR-beta positive cells at the lesion border in both acute and chronic phases of inflammation. We found that anti-FR-beta correlated with anti-CD68 and anti-MRC-1 immunohistochemistry; for MRC-1, the correlation was most prominent in the chronic phase of inflammation. Both F-18-FOL and C-11-PBR28 radiotracers bound to the EAE lesions. Autoradiography studies verified that this binding took place in areas of anti-FR-beta positivity. A blocking assay using folate glucosamine further verified the tracer's specificity. In the chronic phase of EAE, the lesion-to-background ratio of F-18-FOL was significantly higher than that of C-11-PBR28 (P = 0.016). Conclusion: Our EAE results imply that FR-beta may be a useful target for in vivo imaging of multiple sclerosis-related immunopathology. FR-beta-targeted PET imaging with F-18-FOL may facilitate the monitoring of lesion development and complement the information obtained from TSPO imaging by bringing more specificity to the PET imaging armamentarium for neuroinflammation.