Abstract
Apigenin, a natural flavonoid, found in several plants, fruits, vegetables, herbs, and spices, is known to have anti-oxidant and anti-inflammatory properties that are evident in the use of these substances for centuries as medicinal approaches to treat asthma, insomnia, Parkinson’s disease, neuralgia, and shingles. However, there is a considerable dearth of information regarding its effect on immune cells, especially dendritic cells (DC) that maintain the critical balance between an immunogenic and tolerogenic immune response, in an immunospecialized location like the central nervous system (CNS). In this paper we looked at the anti-inflammatory properties of Apigenin in restoration of immune function and the resultant decrease in neuroinflammation. In vivo, a significant reduction in severity of experimental autoimmune encephalomyelitis (EAE) progression and relapse was observed in C57BL/6 (progressive) and SJL/J (relapse-remitting) mouse models of multiple sclerosis upon treatment with Apigenin. Apigenin treated EAE mice show decreased expression of α4 integrin and CLEC12A on splenic DCs and an increased retention of immune cells in the periphery compared to untreated EAE mice. This correlated consequently with immunohistochemistry findings of decreased immune cell infiltration and reduced demyelination in the CNS. These results indicate a protective role of Apigenin against the neurodegenerative effects resulting from the entry of DC stimulated pathogenic T cells into the CNS thus implicating a potential therapy for neuroinflammatory disease.
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The authors wish to acknowledge US Public Health Service/National Institutes of Health grants: R01CA054559 and R56AI077414 to PJ.
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Supplementary Figure 1
Myeloid cell quantification and characterization within the blood and lymph nodes of mice induced with relapse-remitting type of EAE. Cells isolated from the blood and lymph nodes of the EAE + vehicle and EAE + Apigenin groups in SJL/J mice were pooled in each group (n=5) and run in triplicates for cell quantification. The cells were stained for CD11c, CD8α, CD11b, CD68, CD45R, and CD4 immune cell markers. Plots represent absolute cell counts of each of the cell types from animals in each group. *p<0.05 (GIF 184 kb)
Supplementary Figure 2
Impact of Apigenin on DC stimulation and antigen presentation and T cell adhesion in SJL/J mice. Splenocytes from both the EAE + vehicle and EAE + Apigenin groups of SJL/J mice were stimulated with PLP323-339 peptides for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 hours. These cells were subsequently stained with antibodies against CD11c, CD8α, MHC II, and CD86, α4, and CLEC12A. Data represents CD11c+ dendritic cells from mice expressing MHCII+CD86+(top), and CD4+ T cells expressing α4 integrin (middle), and CLEC12A (bottom) upon stimulation with PLP323-339. Each bar is representative of the mean percentage for every marker per group. Histogram plots are representative of one animal per group. *p<0.05 (GIF 298 kb)
Supplementary Figure 3
Tregs up-regulation and Th17 down-modulation in SJL/J mice upon Apigenin treatment. Flow cytometry analysis representing CD4+ cells from lymphocytes of the SJL/J mice expressing IL-17A (top) and CD25+FOXP3 (bottom) upon stimulation with PLP323-339 peptides respectively for 3 days followed by activation with PMA, ionomycin and brefeldin A for 5 hours. Each bar is representative of the mean percentage for every marker per group. Contour plots representative of one animal per group are shown on the left. *p<0.05 (GIF 326 kb)
Supplementary Figure 4
Effect of Apigenin on IFN-γ production. Flow cytometry analysis representing CD4+ cells expressing IFNγ in SJL/J mice upon ex-vivo stimulation of lymphocytes with PLP323-339 and activation with PMA, ionomycin and brefeldin. Lymphocytes were pooled from mice in each group (n=5) and run for quantifying and stimulating procedures. (GIF 188 kb)
Supplementary Figure 5
Apigenin treatment reduces immune cell migration into the CNS. Spinal cord tissues from RR-EAE (SJL/J) mice were sectioned. Spinal cord tissue from SJL/J mice was subjected to LFB and H&E staining showing areas of myelination (blue) and cellular infiltration (reddish brown). (GIF 654 kb)
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Ginwala, R., McTish, E., Raman, C. et al. Apigenin, a Natural Flavonoid, Attenuates EAE Severity Through the Modulation of Dendritic Cell and Other Immune Cell Functions. J Neuroimmune Pharmacol 11, 36–47 (2016). https://doi.org/10.1007/s11481-015-9617-x
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DOI: https://doi.org/10.1007/s11481-015-9617-x