Endocrine Abstracts

Our research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. The AIM of this study was to evaluate endogenous interleukin (IL1,4,6,8) production to the culture medium by the skin cells (keratinocytes and fibroblasts) cocultured with mononuclears of patients with type 2 diabetes mellitus (T2DM) and T2DM with chronic foot ulcers (DFU) in a 3D culture model.

Materials and methods: A multilayer 3D immunocompetent model of human skin comprising of keratinocytes, fibroblasts and mononuclears (peripheral blood mononuclears of 9 T2DM with DFU, 9 T2DM patients, 9 healthy controls (HC)) in an agarose-fibronectin gel was used. IL1,4,6,8 production to the culture medium was evaluated just after adding mononuclears to the system and in 24 hours of their co-cultivation with fibroblasts and keratinocytes.

Results: the initial IL1 level was significantly higher for samples with T2DM and T2DM with DFU patients mononuclears compared to HC – 54.91 [51.17;62.98] vs 51.82 [48.43; 55.92] vs 44.72 [35.37; 48.03] pg/ml, H = 10.427, P = 0.005. There was a significant increase in IL1 after 24-hour keratinocytes and fibroblasts coincubation with patients mononuclears – 48.91 [39.66; 51.96] vs 44.72 [35.37; 48.03] pg/ml, T = −2.666, P = 0.008, 73.95 [68.13; 75.53] vs 51.82 [48.43; 55.92] pg/ml, T = −2.666, P = 0.008, 85.17 [82.99; 91.51] vs 54.91 [51.17; 62.98] pg/ml, T = −2.666, P = 0.008, for HC, T2DM and T2DM with DFU in 24 hours and initially, respectively. For the samples containing HC mononuclears this increase in IL1 was significantly lower than for T2DM and T2DM with DFU – 4.24 [3.65; 5.01] vs 20.21 [18.18; 23.51] vs 30.26 [26.62; 34.95] pg/ml, H = 22.317, P < 0.001, respectively. The increase in IL1 was significantly higher for samples with mononuclears of patients with T2DM with DFU, than without DFU – P < 0.001. The initial IL6 level was slightly higher for samples with patients with T2DM and T2DM with DFU mononuclears than HC – 26.28 [20.82; 28.49] vs 27.54 [24.33; 30.03] vs 19.75 [13.57; 25.03] pg/ml, H = 8.892, P = 0.012, respectively. In samples with HC mononuclears, the IL6 was undetectable with the kits used (< 5 pg/ml). In the samples with patients with T2DM and T2DM with DFU mononuclears the secretion of IL6 didn’t change significantly – 26.28 [20.82; 28.49] vs 23.76 [22.38; 27.58] pg/ml, T = −1.125, P = 0.260 and 27.54 [24.33; 30.03] vs 26.12 [23.64; 28.90] pg/ml, T = −1.836, P = 0.066 for the group of T2DM and T2DM with DFU initially and in 24-hour coincubation, respectively. The IL4 and IL8 levels were undetectable (< 5 pg/ml) with the kits used.

Conclusion: An increase in IL1 and absence of a sufficient and significant decrease in IL6 production in 24-hour coincubation of skin cells with T2DM patients mononuclears indicates the activation of immune mechanisms and the maintenance of chronic inflammatory response.