Human Wharton's Jelly-Derived Mesenchymal Stromal Cells Primed by Tumor Necrosis Factor-α and Interferon-γ Modulate the Innate and Adaptive Immune Cells of Type 1 Diabetic Patients

Abstract

The unique immunomodulation and immunosuppressive potential of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) make them a promising therapeutic approach for autoimmune diseases including type 1 diabetes (T1D). The immunomodulatory effect of MSCs is exerted either by cell-cell contact or by secretome secretion. Cell-cell contact is a critical mechanism by which MSCs regulate immune-responses and generate immune regulatory cells such as tolerogenic dendritic cells (tolDCs) and regulatory T cell (Tregs). In this study, we primed WJ-MSCs with TNF-α and IFN-γ and investigated the immunomodulatory properties of primed WJ-MSCs on mature dendritic cells (mDCs) and activated T cells differentiated from mononuclear cells (MNCs) of T1D patient's. Our findings revealed that primed WJ-MSCs impaired the antigen-mediated immunity, upregulated immune-tolerance genes and downregulated immune-response genes. We also found an increase in the production of anti-inflammatory cytokines and suppression of the production of pro-inflammatory cytokines. Significant upregulation of FOXP3, IL10 and TGFB1 augmented an immunosuppressive effect on adaptive T cell immunity which represented a strong evidence in support of the formation of Tregs. Furthermore, upregulation of many critical genes involved in the immune-tolerance mechanism (IDO1 and PTGES2/PTGS) was detected. Interestingly, upregulation of ENTPD1/NT5E genes express a strong evidence to switch immunostimulatory response toward immunoregulatory response. We conclude that WJ-MSCs primed by TNF-α and IFN-γ may represent a promising tool to treat the autoimmune disorders and can provide a new evidence to consider MSCs- based therapeutic approach for the treatment of TID.

Keywords: Immunomodulation; Wharton’s jelly-derived mesenchymal stromal cells; priming; regulatory T cells; tolerogenic dendritic cells; type 1 diabetes.

Figure 1

Characterization and differentiation potential of WJ-MSCs priming with IFN-γ and TNF-α (A) Representative flow cytometry histograms showing high expression of CD90, CD105, CD73 and CD44 and lack the expression of negative MSCs cocktail (CD34, CD11b, CD19, CD45 and HLA-DR). Gray peak corresponds with isotype control and the violet peak corresponds with the antibodies. (B) Unprimed WJ-MSCs. (C) Primed WJ-MSCs. (D) Oil red staining of primed WJ-MSCs after culture in adipogenic differentiation media. (E) Alizarin red staining of primed WJ-MSCs after culture with Osteogenic differentiation media. Data were calculated from five samples. Experiments performed in triplicate for each sample. Data were analyzed using FACS canto II. Magnification = 100x.

Figure 2

Phenotype of DCs. Mean fluorescence intensity (MFI) ± SD for the surface intensity of co-stimulatory molecules and maturation markers on iDCs (black), GAD65 pulsed mDCs (red), and GAD65 pulsed mDCs co-cultured with primed WJ-MSCs (blue). Data were calculated from three different experiments for each patient. Experiments performed in triplicate for each patient. DCs from five patients were used. Statistical significance was tested using a two-way ANOVA with Tukey’s post hoc for multiple comparisons. In all analyses *p < 0.05, ***p < 0.001 and ****p < 0.0001.

Figure 3

T cells proliferation and activation and IFN-γ ELISPOT analysis (A) Representative flow cytometry analysis and mean fluorescent intensity (MFI) the dilution of CFSE fluorescent dye (B) Percentage of T cell proliferation under the following conditions: inactivated T cells (blue), activated T cells cultured without (black) and with primed WJ-MSCs (red). Statistical significance was tested using a one-way ANOVA with Tukey’s post hoc for multiple comparisons (C) Representative flow cytometry analysis of CD4+/ CD8+ activated T cells and activated CD69+ T cells cultured with and without primed WJ-MSCs and MFI ± SD of CD69+ T cell in CD4+ and CD8+ activated T cells under the following conditions: activated T cells alone (black) and activated T cells co-cultured with primed WJ-MSCs (red). An unpaired t-test was performed to test statistical significance (D). Mean ± SD of IFN-γ spots per well (200.000 cells) under the following conditions: inactivated T cells (blue), activated T cells cultured without (black) and with primed WJ-MSCs (red). Statistical significance was tested using a one-way ANOVA with Tukey’s post hoc for multiple comparisons. Data were calculated from three different experiments for each patient. Experiments performed in triplicate for each patient. T cells from five patients were used. For all analyses *p < 0.05, and ****p < 0.0001.

Figure 4

Cytokines secretion level. (A) Mean ± SD levels of secreted IL-6, IL-10, and TGF-β in cell free culture supernatant under the following culture conditions: primed WJ-MSCs alone (blue), GAD65- pulsed mDCs co-cultured without (black) and with primed WJ-MSCs (red). (B) Mean ± SD levels of secreted IL-6, IL-10, TGF-β, IL-17 and IFN-γ in cell free culture supernatant under the following culture conditions: inactivated T cells (blue), activated T cells without (black) or with primed WJ-MSCs (red). Data were calculated from five patients. Experiments performed in duplicate for each patient. In all analyses *p < 0.05, **p < 0.01 and ****p < 0.0001. ns, not significant.

Figure 5

Relative normalized expression of immunomodulatory and immunostimulatory genes of mDCs after co-cultured with primed WJ-MSCs. (A) upregulated genes. (B) downregulated gene. Mature dendritic cells from four patients were used. Results were normalized to 18S rRNA Each sample was performed in triplicate, and a mean value was calculated. Data were analyzed according to 2−ΔΔCT method using CFX Maestro™ Software - Bio-Rad. *p ≤ 0.01 and fold change ≥ 1.5.

Figure 6

Relative normalized expression of immunomodulatory and immunostimulatory genes of activated T cells after co-cultured with primed WJ-MSCs. (A) upregulated genes. (B) downregulated genes. T cells from four patients were used. Results were normalized to 18S rRNA Each sample was performed in triplicate, and a mean value was calculated. Data were analyzed according to 2−ΔΔCT method using CFX Maestro™ Software - Bio-Rad. *p ≤ 0.01 and fold change ≥ 1.5.