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Neural Engineering

Neural Engineering - Poster Session C

Poster K4 - Immunomodulatory Glycan for Attenuation of Neuroinflammation in Stroke

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Stroke is a leading cause of adult disability in the United States and lacks an effective therapeutic treatment (1). Glycosylation is a common post-translational modification where a glycan (or saccharide) is added enzymatically to a protein. It is critically implicated in multiple physiological and pathological processes including central nervous system (CNS) disorders. Previous studies have shown the role of glycans in mediating neuroinflammation and disease pathophysiology in the brain (2,3). Recent studies have shown the impact of fucose (a glycan) in attenuating neuroflammation4. However, the impact of fucose treatment on mitochondrial dysfunction, a chief contributor to neuroinflammation, has not been investigated in the context of stroke. We hypothesize that fucose attenuates neuroinflammation in ischemic stroke by rescuing mitochondrial dysfunction in the immune cells. The current study investigates the impact of fucose on modulating mitochondrial dysfunction and neuroinflammation using an in vitro model. We used lipopolysaccharides (LPS)-treated bone marrow-derived macrophages (BMDMs) to create an in vitro model of neuroinflammation and performed a dose-dependent inhibition of neuroinflammation analysis.

Materials and

Methods:  We used an LPS model of neuroinflammation, where we treat BMDMs isolated from the mice bone marrow with 100 ng/ml of LPS. Metabolic activity was analyzed using alamarBlueTM before the addition of LPS to test the response of the BMDMs toward L-fucose and after the addition of LPS to test the impact of L-fucose on the LPS-treated cells. Cell viability was tested using Live/Dead assay to test the protective effects L-fucose on the BMDMs treated with LPS. CD11b staining was used to test the morphological shifts in response to LPS and the immunomodulatory impact of fucose post-treatment. Enzyme-Linked Immunosorbent Assay (ELISA) analyzed the release of the proinflammatory cytokines (TNF-α, IL-1β, etc.) from the cells as a response to LPS-induced inflammation. Tetramethylrhodamine (TMRM) assay was used to measure the mitochondrial potential of the cells to test the underlying mechanism of immunomodulatory impact of fucose. We aim to incorporate the optimized dose of fucose into tunable hydrogel for sustained release in an in vitro stroke model.

Results, Conclusions, and Discussions: Our preliminary results show a dose-dependent inhibition of pro-inflammatory LPS-stimulation of BMDMs. alamarBlueTM and live/dead assay showed no adverse impact of higher doses of fucose on the metabolic activity and viability of BMDMs. To investigate the effects that L-fucose has on LPS-treated BMDMs, we used different concentrations of L-fucose from 0.01% to 0.5%. There was a dose-dependent increase in live cells and metabolic activity post LPS treatment with increasing concentrations of L-fucose (Figure c). There was also an observable change from pro-inflammatory morphological shift to an anti-inflammatory phenotype with increasing concentrations of L-fucose (Figure a). ELISA corroborates the phenotypic shift observed using CD11b staining where we observed a decrease in the proinflammatory cytokine release (Figure b). To investigate the mechanism of action of fucose in attenuating inflammation, we focus on mitochondrial dysfunction. Preliminary results show higher fluorescence indicative of active mitochondrial potential comparable to healthy BMDMs. We aim to characterize the dose response of fucose on the mitochondrial bioenergetics in our model of neuroinflammation model using Seahorse assay. This study is foundational in investigating the impact and therapeutic dosage of fucose to attenuate neuroinflammation by modulating mitochondrial function. Future studies include impact of fucose in rescuing the mitochondrial dysfunction in BMDMs and microglial cells (brain immune cells) subjected to oxygen-glucose deprivation, mimicking the stroke microenvironment. Ultimately, our goal is to use this data to inform the design and characterization of immunomodulatory glycosylated biomaterials to mitigate neuroinflammation in stroke.

References:

1. Martin, S. S. et al. 2024 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation 149, e347-e913 (2024). https://doi.org/10.1161/CIR.0000000000001209
2. Puigdellivol, M., Allendorf, D. H. & Brown, G. C. Sialylation and Galectin-3 in Microglia-Mediated Neuroinflammation and Neurodegeneration. Front Cell Neurosci 14, 162 (2020). https://doi.org/10.3389/fncel.2020.00162
3. Rebelo, A. L., Chevalier, M. T., Russo, L. & Pandit, A. Sweet tailoring of glyco-modulatory extracellular matrix-inspired biomaterials to target neuroinflammation. Cell Rep Phys Sci 2 (2021). https://doi.org/ARTN 100321
10.1016/j.xcrp.2021.100321
4. Xu, X. et al. Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide. J Biol Chem 300, 105513 (2024). https://doi.org/10.1016/j.jbc.2023.105513

Acknowledgements (Optional):  The authors thank the Presidential Honors Fellowship Program for supporting this research and Dr. Daniel Tyrrell for their kind donation of C57BL6 young adult mice legs for BMDM isolation. Schematics were designed using Biorender.