Impact of diabetic (diabetes mellitus) patients immune factors on the skin cell viability in vitro

At the moment, a lot of scientific research focused on the role of immune mechanisms in diabetic foot ulcers development and impaired healing. A 3D skin culture system as a relevant skin model may prove valuable in investigating these mechanisms and may be a useful tool to study interactions between different cell types such as keratinocytes, fibroblasts, and immune cells. The aim of our research was to study keratinocytes and fibroblasts viability in co-culture with immune factors of patients with diabetes mellitus type 2 (DM2) and patients with diabetes and chronic foot ulcers in a 3D skin culture system. In this study, the multilayer 3D immunocompetent model of human skin comprising keratinocytes, fibroblasts, and mononuclears in an agarose-fibronectin gel was used. The human immortalized keratinocyte cell line, HaCaT, and primary fibroblast cell culture isolated from skin samples of healthy man in abdominal surgery were used for the 3D system. For the experiment 20 % serum of 9 patients with chronic diabetic foot ulcers (without active inflammation signs), 9 diabetic type 2 patients and 9 healthy people, and mononuclears of the same groups of patients were used. 9 experimental series with 3 repeats were carried out. Mononuclears of patients with DM2 and DM2 and diabetic foot syndrome (DFS) had a greater inhibitory effect on fibroblasts, significantly inhibiting their proliferation to a level of 83.78 [79.03; 89.53] % vs 70.18 [66.38; 72.10] % vs 95.40 [91.75; 99.05] %, H = 21.259, p <0.001 – DM2, DFS, and the control group, respectively. There was no significant difference in the cytoinhibitory effect of mononuclears on keratinocytes between different groups: 96.40 [92.82; 100.50] % vs 93.61 [86.80; 97.10] % vs 92.87 [85.15; 95.25] %, H = 4.459, p = 0.108 – control, DM2 and DFS group, respectively. Adding serum to the culture system influenced significantly the viability of neither keratynocytes – 99.40 [95.35; 102.05]  % vs 98.60 [90.55; 100.40] % vs 94.79 [91.65; 98.16] %, H = 3.030, p = 0.220 nor of fibroblasts – 95.61 [92.39; 100.19] % vs 95.80 [88.99; 102.15] % vs 96.20 [99.69; 88.70] %, H = 0.353, p = 0.838, control, DM2 and DFS group, respectively. It was determined that the fibroblasts vialability significantly decreases after introducing mononuclears of patients with DM and patients with DM and chronic diabetic foot ulcers to the co-culture system. Adding serum of these patient groups to the culture system doesn’t influence significantly the viability of skin cells.

3D skin culture system, diabetic foot syndrome pathogenesis, immune mechanisms, diabetes mellitus, mononuclears

1. IDF Diabetes Atlas 9th Edition. Available at: https://www.diabetesatlas.org/en/resources/ (accessed 19.04.2020).

2. Geraghty T., LaPorta G. Current health and economic burden of chronic diabetic osteomyelitis. Expert Review of Pharmacoeconomics and Outcomes Research, 2019, vol. 19, no. 3, pp. 279–286. https://doi.org/10.1080/14737167.2019.1567337

3. Ibrahim A., Jude E., Langton K., Martinez-De Jesus F. R., Harkless L. B., Gawish H. [et al.]. IDF Clinical Practice Recommendations on the Diabetic Foot – 2017. A guide for healthcare professionals. Brussels, International Diabetes Federation, 2017. 70 p.

4. The 2019 IWGDF Guidance Documents on prevention and management of foot problems in diabetes. Available at: https://iwgdfguidelines.org/guidelines/guidelines/ (accessed 19.04.2020).

5. Ud-Din S., Bayat A. Non-animal models of wound healing in cutaneous repair: In silico, in vitro, ex vivo, and in vivo models of wounds and scars in human skin. Wound Repair and Regeneration, 2017, vol. 25, no. 2, pp. 164–176. https://doi.org/10.1111/wrr.12513

6. Chau D. Y. S., Johnson C., MacNeil S., Haycock J. W., Ghaemmaghami A. M. The development of a 3D immunocompetent model of human skin. Biofabrication, 2013, vol. 5, no. 3, p. 035011. https://doi.org/10.1088/1758-5082/5/3/035011

7. Ahmed S. A., Gogal R. M., Walsh J. E. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. Journal of Immunological Methods, 1994, vol. 170, no. 2, pp. 211–224. https://doi.org/10.1016/0022-1759(94)90396-4

8. Baltzis D., Eleftheriadou I., Veves A. Pathogenesis and treatment of impaired wound healing in diabetes mellitus: new insights. Advances in Therapy, 2014, vol. 31, no. 8, pp. 817–836. https://doi.org/10.1007/s12325-014-0140-x

9. Lerman O. Z., Galiano R. D., Armour M., Levine J. P., Gurtner G. C. Cellular dysfunction in the diabetic fibroblast. Impairment in migration, vascular endothelial growth factor production, and response to hypoxia. American Journal of Pathology, 2003, vol. 162, no. 1, pp. 303–312. https://doi.org/10.1016/S0002-9440(10)63821-7

10. Maione A. G., Brudno Y., Stojadinovic O., Park L. K., Smith A., Tellechea A. [et al.]. Three-dimensional human tissue models that incorporate diabetic foot ulcer-derived fibroblasts mimic in vivo features of chronic wounds. Tissue Engineering Part C: Methods, 2015, vol. 21, no. 5, pp. 499–508. https://doi.org/10.1089/ten.TEC.2014.0414

11. Kraakman M. J., Murphy A. J., Jandeleit-Dahm K., Kammoun H. L. Macrophage polarization in obesity and type 2 diabetes: weighing down our understanding of macrophage function? Frontiers in Immunology, 2014, vol. 5, art. 470. https://doi.org/10.3389/fimmu.2014.00470

12. Khanna S., Biswas S., Shang Y., Collard E., Azad A., Kauh C., Bhasker V., Gordillo G. M., Sen C. K., Roy S. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS ONE, 2010, vol. 5, no. 3, pp. e9539. https://doi.org/10.1371/journal.pone.0009539

13. Gallagher K. A., Joshi A., Carson W. F., Schaller M., Allen R., Mukerjee S. [et al.]. Epigenetic changes in bone marrow progenitor cells influence the inflammatory phenotype and alter wound healing in type 2 diabetes. Diabetes, 2015, vol. 64, no. 4, pp. 1420–1430. https://doi.org/10.2337/db14-0872

14. Sîrbulescu R. F., Boehm C. K., Soon E., Wilks M. Q., Ilieş J., Yuan H. [et al.]. Mature B cells accelerate wound healing after acute and chronic diabetic skin lesions. Wound Repair and Regeneration, 2017, vol. 25, no. 5, pp. 774–791. https://doi.org/10.1111/wrr.12584

15. Li R., Rezk A., Healy L. M., Muirhead G., Prat A., Gommerman J. L., Bar-Or A. Cytokine-defined B cell responses as therapeutic targets in multiple sclerosis. Frontiers in Immunology, 2016, vol. 6, art. 626. https://doi.org/10.3389/fimmu.2015.00626

16. Goodchild T. T., Robinson K. A., Pang W., Tondato F., Cui J., Arrington J. [et al.]. Bone marrow-derived B cells preserve ventricular function after acute myocardial infarction. JACC: Cardiovasc Interventions, 2009, vol. 2, no. 10, pp. 1005‒1016. https://doi.org/10.1016/j.jcin.2009.08.010

17. Nishio N., Ito S., Suzuki H., Isobe K. Antibodies to wounded tissue enhance cutaneous wound healing. Immunology, 2009, vol. 128, no. 3, pp. 369‒380. https://doi.org/10.1111/j.1365-2567.2009.03119.x

18. Iwata Y., Yoshizaki A., Komura K., Shimizu K., Ogawa F., Hara T. [et al.].CD19, a response regulator of B lymphocytes, regulates wound healing through hyaluronan-induced TLR4 signaling. American Journal of Pathology, 2009, vol. 175, no. 2, pp. 649‒660. https://doi.org/10.2353/ajpath.2009.080355

19. Könnecke I., Serra A., Khassawna T., Schlundt C., Schell H., Hauser A. [et al.]. T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion. Bone, 2014, vol. 64, pp. 155‒165. https://doi.org/10.1016/j.bone.2014.03.052

20. Chen L., Mehta N. D., Zhao Y., DiPietro L. A. Absence of CD4 or CD8 lymphocytes changes infiltration of inflammatory cells and profiles of cytokine expression in skin wounds, but does not impair healing. Experimental Dermatology, 2014, vol. 23, no. 3, pp. 189–194. https://doi.org/10.1111/exd.12346

21. Moura J., Madureira P., Leal E. C., Fonseca A. C., Carvalho E. Immune aging in diabetes and its implications in wound healing. Clinical Immunology, 2019, vol. 200, pp. 43–54. https://doi.org/10.1016/j.clim.2019.02.002

22. Moura J., Rodrigues J., Gonçalves M., Amaral C., Lima M., Carvalho E. Impaired T-cell differentiation in diabetic foot ulceration. Cellular and Molecular Immunology, 2017, vol. 14, no. 9, pp. 758–769 https://doi.org/10.1038/cmi.2015.116