Impaired immunomodulatory ability of type 2 diabetic adipose-derived mesenchymal stem cells in regulation of inflammatory condition in mixed leukocyte reaction

Abstract

The immunomodulatory properties of type 2 diabetic patients' adipose-derived mesenchymal stem cells (D-ASCs) has not been extensively studied. In this study, we compared the immunomodulatory properties of D-ASCs and non-diabetic subjects mesenchymal stem cells (ND-ASCs) in co-culture with mixed leukocyte reaction (MLR). ASCs were isolated from adipose tissue samples of type 2 diabetic and non-diabetic subjects (age: 40-55). D-ASCs and ND-ASCs were co-cultured with two-way MLR. Peripheral blood mononuclear cells (PBMCs) proliferation ratio, protein levels of IFN-γ and IL-10, mRNA expression of COX-2, TNF-α, TGF-β1 and IL-6 genes in MLR, D-ASCs and ND-ASCs co-cultures were assessed using XTT, ELISA and Real-time qRT-PCR, respectively. PBMCs proliferation on days 2 and 4 of D-ASCs co-culture was higher than ND-ASCs co-culture of the same days (p < 0.001). IFN-γ level decreased on day 4 compared to day 2 of ND-ASCs co-culture, but its level had not changed in D-ASCs co-culture. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND-ASCs co-culture of the same days (p < 0.05). The expression of TNF-α and IL-6 on days 2 and 4 of D-ASCs co-culture were higher than ND-ASCs co-culture of the same days (p < 0.001). TGF-β1 on day 4 of ND-ASCs co-culture showed a slightly higher expression than D-ASCs co-culture of the same day. Lower suppression of PBMCs proliferation, declined expression of anti-inflammatory and upregulated expression of pro-inflammatory factors in D-ASCs co-culture, indicated an impairment of these cells in modulation of the inflammatory condition.

Keywords: adipose tissue; immunomodulation; inflammation; mesenchymal stem cells; type 2 diabetes.

Table 1. Designed primers sequence, length of amplicons and annealing temperature of the β-actin, COX-2, IL-6, TNF-α and TGF-β1 genes for evaluation of their mRNA levels with real time PCR

Figure 1. Characterization of ND-ASCs and D-ASCs. (A) Representative inverted microscope image of ND-ASCs and D-ASCs at passage 4 demonstrated the spindle-shaped morphology of both cells. 40x magnification, (B) Tri-lineages differentiation of ND-ASCs and D-ASCs. Isolated cells from passage 4 were respectively induced into osteogenic, adipogenic, and chondrogenic lineages in specific differentiation media of each lineage. 40x magnification, (C) ND-ASCs and D-ASCs at passage 4 were positive for the MSC-related surface markers of CD105 (PE), CD90 (PerCP) and CD73 (FITC), while they were both negative for CD45 (FITC) and CD34 (PE), in flow cytometry analysis. Abbreviation: D-ASCs: Diabetic adipose derived stem cells; ND-ASCs: Non-diabetic adipose derived stem cells; MSC: Mesenchymal stem cell

Figure 2. Effects of ND-ASCs and D-ASCs on PBMCs proliferation in the two-way mixed leukocyte reaction (MLR). Mitotically inactivated ND-ASCs, and D-ASCs were cultured with PBMCs from two donors, PBMCs proliferation was assessed on days 0, 2 and 4 of the experiment using XTT. Data are presented as mean ± SD normalized proliferation ratio of ND-ASCs and D-ASCs on days 2 and 4 of the experiment. Normalized proliferation ratio: Proliferation ratio of day 2 or 4 × 100/proliferation ratio day 0, (the proliferation ratio on day 0 of D-ASCs/MLR and ND-ASCs/MLR were considered as 100 %). ND-ASCs and D-ASCs suppressed proliferation of PBMCs (P < 0.001). The normalized proliferation ratio of PBMCs on days 2 and 4 of the DASCs co-culture were significantly higher than the ND-ASCs co-culture of the same days (P < 0.001). ** indicates a significance level of below 0.01. Abbreviation: D-ASCs: Diabetic adipose derived stem cells; NDASCs: Non-diabetic adipose derived stem cells; PBMC: Peripheral blood mononuclear cell

Figure 3. Relative expression of COX-2, TNF-α, IL-6 and TGF-β1 genes in ND-ASCs/PBMCs and D-ASCs/PBMCs co-cultures evaluated by Real-time RT-qPCR method. To perform Real-time RT-qPCR, target genes expression in the MLR group was considered as the exogenous reference, also, β-actin gene expression was considered as endogenous reference. The genes expression was calculated using 2 ^-∆∆CT formula: ΔΔCT = ΔCT co-culture (CT target gene - CT β-actin) - ΔCT MLR (CT target gene - CT β-actin). Data are presented as mean ± SD 2 ^-∆∆CT amounts. COX-2 expression on days 2 and 4 of D-ASCs co-culture was lower than ND-ASCs co-culture on the same days (P < 0.001). TNF-α and IL-6 expressions on days 2 and 4 of D-ASCs co-culture were higher than ND-ASCs co-culture on the same days (P < 0.001). There was no significant difference in the expression of TGF-β1 between both groups (P > 0.05). * Indicates a significance level below 0.05 and ** indicates a significance level of below 0.01. Abbreviation: D-ASCs; Diabetic adipose derived stem cells, ND-ASCs: Non-diabetic adipose derived stem cells; PBMC: Peripheral blood mononuclear cell; MLR: Mixed leukocyte reaction; COX-2: Cyclooxygenase-2; TNF-α: Tumor necrosis factor alpha; IL-6: interleukin-6; TGF-β1: transforming growth factor-beta

Figure 4. Effects of ND-ASCs and D-ASCs on IL-10 and IFN-γ levels. IL-10 and IFN-γ levels were assessed in supernatant of MLR and co-cultures on days 2 and 4 of experiment using ELISA. Mean of cytokines concentrations were compared in co-cultures and MLR groups. (A) IFN-γ levels increased in ND-ASCs and D-ASCs co-cultures compared to MLR on day 2. This cytokine level declined in ND-ASCs co-culture on day 4 but didn't change in D-ASCs co-culture, compared to day 2 of the same groups. (B) IL-10 levels were higher in the ND-ASCs and D-ASCs co-cultures compared to the MLR group on both days of experiment. Also, its level decreased on day 4 of co-culture groups compared to day 2 of the same groups. However, there was no statistically significant difference between D-ASCs and ND-ASCs co-culture on both days of the experiment (p > 0.05). * Indicates a significance level below 0.05 and ** indicates a significance level of below 0.01.