IFN-α and vitamin B6-induced galectin-9 promotes the immunomodulatory function of human clonal mesenchymal stem cells

Abstract

Background: Mesenchymal stem cells (MSCs) possess a variety of immunomodulatory functions that can vary depending on the MSC line. Investigating priming strategies is essential for increasing the immunomodulatory potential of MSCs.

Methods: Human clonal MSCs (cMSCs) were primed with TNF-α, IFN-γ, IL-1β, IFN-α, and vitamin B6. Their immunomodulatory functions, including T-cell proliferation and cytokine production, were analyzed. The primed cMSCs were injected intravenously into a mouse model of ovalbumin-induced atopic dermatitis (AD), and their therapeutic effects were evaluated.

Results: We identified IFN-α and vitamin B6 as promising priming agents when they are combined with TNF-α and IFN-γ. The primed cMSCs showed expression of galectin-9 (Gal-9), IL-1Ra, and PDL-1. Gal-9 facilitates the induction of regulatory T cells (Tregs) and apoptosis. Treatment with primed cMSCs significantly alleviated pathological changes in an AD mouse model. Notable improvements included a reduction in epidermal thickness (p < 0.05), a decreased number of mast cells and eosinophils in the dermis (p < 0.01), restored expression of claudin-1 in the epidermis (p < 0.0001), and lower serum levels of IgE (p < 0.05).

Conclusions: This novel combination of priming factors significantly promotes the immunomodulatory functions of cMSCs by inducing Gal-9. Consequently, Gal-9 may serve as an excellent biomarker for screening primed cMSCs for their immunomodulatory capabilities, facilitating a more accurate assessment of their therapeutic effectiveness.

Keywords: Atopic dermatitis; Clonal mesenchymal stem cell; Galectin-9; IFN-α; Priming; Vitamin B6.

Fig. 1

Graphical schematic for the AD model. A schematic diagram illustrating the cMSC injection process following the induction of AD in a mouse model

Fig. 2

Diversity of the immunomodulatory capacities of various cMSCs. A Flow cytometry analysis of T-cell proliferation. B IFN-γ levels measured by ELISA. N: negative control, P: positive control. The error bars indicate the standard deviation (SD). Two independent experiments were conducted. *p values were determined using one-way ANOVA. Significance levels: *p < 0.05, **p < 0.01, and ****p < 0.0001

Fig. 3

Screening of priming conditions to promote the immunomodulatory functions of cMSCs. A Expression of immunomodulatory genes in cMSCs after treatment with various cytokines and vitamin B6. B T-cell proliferation by flow cytometry. C–F IFN-γ, TNF-α, IL-5, and IL-17A levels measured by ELISA. Error bars represent standard deviation (SD). *p and #p values represent comparisons with nonprimed and T/I-stimulated cMSCs, respectively. All p values were determined using one-way ANOVA. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, and ####p < 0.0001

Fig. 4

Synergistic effects of IFN-α and Vit B6. A PD-L1, IL-1Ra, and Gal-9 protein levels in pcMSCs analyzed by flow cytometry and ELISA. Gal-9 MFI represents the median. B Heatmap of the 30 most differentially expressed genes in cMSCs under four priming conditions. C, D Gal-9, MDK, and CCL7 mRNA expression in pcMSCs. The error bars represent the standard deviation (SD). Three independent experiments were performed. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 5

The effects of Gal-9 secreted by pcMSCs on Treg and Th17 cells. A Flow cytometry analysis of CD25⁺Foxp3⁺ Treg cells after coculture with pcMSC2. B IL-10 and Gal-9 expression levels measured by ELISA. C Treg induction in the presence of neutralizing antibodies. D IL-17A and IL-2 levels in Th17 cell differentiation with pcMSC2. E IL-17A expression after treatment with neutralizing antibodies. The error bars represent the standard deviation (SD). Three independent experiments were performed. *p values were determined using one-way ANOVA. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001

Fig. 6

Effects of Gal-9 secreted by pcMSC2 on CD4 T-cell apoptosis. Flow cytometry analysis of late apoptotic CD4⁺ T cells after coculture with pcMSC2 under CD3/CD28 stimulation, with or without Gal-9 neutralization. The error bars represent the standard deviation (SD). Three independent experiments were performed. *p values were determined using one-way ANOVA. Significance levels: ***p < 0.001 and ****p < 0.0001

Fig. 7

Primed cMSCs alleviated OVA-induced AD. A Histological analysis of skin tissues by H&E, toluidine blue, Congo red, and IF staining for CLDN1. B, C Quantification of skin thickness, mast cells, and eosinophils. D CLDN1 expression shown as MFI per unit area. E Serum levels of IgE, IgG1, and IgG2a measured by ELISA. F Relative mRNA expression in skin lesions. Data are presented as mean ± SEM. Unpaired t tests were used for statistical analysis: *p values indicate differences from the vehicle group, #p values from the nonprimed group, and the comparison with T/I-primed cMSCs is shown as a numerical p value. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001

Fig. 7

Primed cMSCs alleviated OVA-induced AD. A Histological analysis of skin tissues by H&E, toluidine blue, Congo red, and IF staining for CLDN1. B, C Quantification of skin thickness, mast cells, and eosinophils. D CLDN1 expression shown as MFI per unit area. E Serum levels of IgE, IgG1, and IgG2a measured by ELISA. F Relative mRNA expression in skin lesions. Data are presented as mean ± SEM. Unpaired t tests were used for statistical analysis: *p values indicate differences from the vehicle group, #p values from the nonprimed group, and the comparison with T/I-primed cMSCs is shown as a numerical p value. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001