Inflammation-induced changes in the glycosylation and metabolism of human corneal fibroblast are ameliorated by a chemical inhibitor of fucosylation

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Schofield, Jack (National University of Ireland, Galway )


Dysregulation of glycosylation and metabolism are associated with pathological inflammation across various biological tissues1. Research into this dysregulation in the cornea has indicated that inflammation can induce changes in the structure and composition of corneal glycans2. An improved understanding of these alterations and the metabolic pathways leading to glycan synthesis could help identify novel diagnostic factors and therapies for corneal inflammation. Herein, we analyzed changes in glycosylation in healthy and inflamed primary human corneal fibroblasts (HCFs) by lectin immunohistochemistry and metabolism by a Seahorse XF Mini Analyzer. We then assessed the capacity of a glycosylation inhibitor to prevent cytokine-induced hyperfucosylation and ameliorate mitochondrial dysfunction.
We developed an optimized protocol to induce inflammation in primary HCFs with 20 ng/mL of IL-1β or TNF-α for 72 hours, with or without 300 μM 2F-Peracetyl-Fucose (2F-PAF), a fluorinated inhibitor of fucosylation. After treatment, cells were fixed and stained with eight glycan-binding, fluorescein-tagged lectin proteins. These lectins bind to glycans with mannose (ConA, GNA), fucose (AAL, UEA I), sialic acid (SNA), N-Acetylgalactosamine (WFA, PNA) or N-Acetylglucosamine (WGA) residues. Cytokine with/without 2F-PAF treatments were given to the cells for Seahorse metabolic analysis. Seahorse XF Cell Mito Stress Tests were carried out on healthy cytokine with/without 2F-PAF-treated HCFs, by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to analyze mitochondrial health.
Our results demonstrated that for healthy cells, the only lectins with robust fluorescence were the α-linked mannose-binding ConA and GNA lectins, with very weak fluorescence for the other lectins in the panel. In the cytokine-stimulated HCFs however, there were increases in WGA and PNA fluorescence, along with statistically significant increases (2.28- and 2.68-fold increases for IL-1β and TNF-α, n = 3) in AAL fluorescence, which binds to α1,3 or α1,6 linked fucose. Changes for UEA I (α1,2 linked, terminal fucose) and SNA (α2,6 sialic acid) were not significant. 2F-PAF treatment inhibited cytokine-induced core fucosylation in the HCFs (77.49% and 75.96% decreases for IL-1β and TNF-α, n = 3) and did not affect terminal fucosylation. In Seahorse XF Cell Mito Stress Tests, the basal and maximal respiration rates measured in OCR, increased (1.74- and 1.57- fold respectively, n = 3) in IL-1β-treated HCFs relative to healthy cells. In cells stimulated by IL-1β with 2F-PAF, basal, and maximal respiration reverted to healthy cell levels.
In corneal inflammation, increased proinflammatory cytokines are associated with altered glycosylation. The data from our cytokine assays showed similar effects, with cytokine stimulation inducing hyperfucosylation in HCFs. 2F-PAF, a fucosylation inhibitor, prevented this hyperfucosylation. Inflammation caused mitochondrial dysfunction in the cells, which 2F-PAF could also ameliorate. Together, these data indicate that inflammation induces hyperfucosylation and mitochondrial dysfunction in corneal fibroblasts and that 2F-PAF has therapeutic potential in corneal inflammation. This work lays the foundation for a biomaterial-encapsulated fucosylation inhibitor treatment for corneal inflammation.

1. Joyce, K. et al. European cells & materials 41, 401–420 (2021).
2. Woodward, A. M. et al. The American journal of pathology 189, 283–294 (2019).


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