Background/Purpose: Inflammatory macrophages (MΦs) play key roles in pathogenesis of rheumatoid arthritis (RA). Fucosylation, comprising the transfer of a fucose (6-Deoxy-L-galactose) to proteins, is regulated by fucosyltransferases (FUTs) and involved in inflammation, oncogenesis, and cell differentiation. We have observed upregulated FUTs in synovial tissues from RA compared to osteoarthritis (OA) subjects. The purpose of the study is to determine: (i) the major cell types that produce FUTs; (ii) the roles of fucosylation; (iii) the efficacy of rebuilding the immune homeostasis by using a fucose analogue in RA.

Methods: Twenty eight RA and OA subjects were recruited and the study is approved by UAB IRB.  Q-PCR was performed to determine the expression of FUTs in synovial tissues and FACS sorted cells from RA synovial fluids. Inflammatory MΦs were polarized by GM-CSF using monocytes from human PBMC, synovial fluid, and mouse bone marrow. Cells were treated with a fucose analog, 2-Deoxy-D-galactose (2-D-gal, inhibited the fucosylation mediated by FUT1/2) at different time points. Antigen presenting function was studied by using Eα-GFP peptide, DQ-Ova, and denatured bovine collagen II (CII)-FITC. Proteomics analysis was carried out by LCMS. Cytoskeleton images and video were collected using a Nikon spinning disk confocal microscope. Collagen-induced arthritis (CIA) was established in DBA/1J mice. 2-D-gal (200 mg/kg BW, every 2-3 days) was administered via I.P.. FACS and histopathology analyses were performed 6 weeks posterior primary CII immunization.

Results: There is a highly positive correlation between TNFα with FUTs 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12 (p=0.0001 for all), but not FUTs 8 (p=0.46) and 13 (p=0.47) in human RA synovia. In sorted cells from RA synovial fluid, FUTs 1, 3, 5, 7, 9 were  highly expressed in M1 inflammatory MΦ, but not in M2 MΦ, synovial fibroblasts, and  T cells (p<0.01), whereas FUTs 8 and 13 were predominately expressed in synovial fibroblasts. This highly indicated that subsets of FUTs might associate with the inflammatory M1 MΦ characteristics. A fucose analog, 2-Deoxy-D-galactose (2-D-gal), precluded the differentiation of M1 MΦ. Phalloidin staining indicated 2-D-gal disrupted MΦ actin-based cytoskeleton. Furthermore, LCMS analysis revealed that plectin-10, an actin regulatory protein, is the major target of 2-D-gal in M1 MΦ. These data suggested a potential role of fucosylation in antigen processing. Indeed, 2-D-gal treatment of fully differentiated M1 MΦ for 2 days significantly reduced the uptake, processing and presentation of GFP-Eα (from I-Edα), DQ-OVA, and FITC-Collagen II (CII) antigens (p<0.01). Additionally, 2-D-gal skewed the M1 MΦ to M2 by increasing IL-10 secretion (p<0.01). In vivo, 2-D-gal treatment dramatically blocked bovine CII-induced arthritis (scores 9.5±1.7 vs 0.5± 0.3, p<0.01) with reduced inflammatory MΦ in draining LN (1.3±0.3% vs 0.5±0.1%, p<0.05), decreased TNF-α (130 vs 39 pg/ml, p<0.05), and anti-CII in the serum.

Conclusion: Fucosylation, a hallmark of M1 MΦ, orientates MΦ polarization and function. 2-D-gal, a fucose analog restores the deranged M1 MΦ and leads to resolution of arthritis by inhibiting fucosylation of cytoskeleton molecules.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reshaping-inflammatory-macrophage-development-and-functions-by-a-monosaccharide-analogue-in-rheumatoid-arthritis/