Background BM-MSCs are currently being studied to determine their potential role in the pathogenesis of autoimmune diseases. Experimental evidence have shown that they exhibit an anti-proliferative effect on T lymphocytes. In this setting BM-MSCs show a specific immunomodulatory function in inducing regulatory phenotype and function in T cells. T regulatory lymphocytes are a population of CD4+ CD25high Foxp3+ cells which play an important role in the induction of peripheral tolerance. SSc is a complex autoimmune disease, characterized by fibrosis of the skin and various internal organs. Our previous findings suggesting that BM-MSCs derived from SSc show a premature senescence, probably related to several pathologic stimuli which these cells encountered during their lifetimes.
Objectives The aim of this work was to demonstrate that BM-MSCs are early senescent, assessing the activity of beta-galactosidase and cell cycle inhibitors (p53 and its downstream gene, p21) and investigate whether during SSc, BM-MSCs, although senescent, maintain their immunosuppressive properties, when co-cultured with T lymphocytes.
Methods After ethical commitment approval MSCs were isolated from BM obtained from 10 SSc and 10 healthy controls (HC). At third passage, the cell cycle was analyzed by flow cytometry. The beta-galactosidase activity was measured by counting positive cells after staining with X-Gal. mRNA and proteins of p21 and p53 were measured by western blot and qRT-PCR. The treatment of the cells for 24h with doxorubicin was used to induce senescence. The immunomodulatory property of BM-MSCs was tested by co-culturing these cells with CD4+ T cells.
Results Both HC and SSc BM-MSC showed stromal phenotype, and normal multidifferentiative ability. The study of cell cycle showed that about 80% of cells were in G0-G1 phase, without significant differences between SSc and HC. Interestingly, the SSc-MSCs showed an increase in beta-galactosidase compared to HC-MSC. After treatment with doxorubicin, the percentage of beta-galactosidase positive stained cells in SSc were higher with respect to HC, thus outlining that SSc cells show greater sensitivity to drug treatment. As far as the expression of p21 was concerned, intriguingly, we found a significant increase in SSc-MSCs compared with HC-MSCs. Doxorubicin treatment abolished p21 activation and, on the other hand, elicited p53 induction both in SSc and HC BM-MSCs. This regulation of p21/p53 machinery could be an attempt of BM-MSCs to bypass the doxorubicin induced. This protective mechanism seems lesser active in SSc cells. The above results were further confirmed by qRT-PCR. We found that senescent SSc MSC showed a preserved immunomodulatory activity maintaining the immunosuppressive effect and the ability to induce a regulatory phenotype on T as HC BM-MSCs.
Conclusions SSc BM-MSC showed an increase of senescence biomarkers, suggesting premature aging, probably related to several pathologic stimuli. Although the early aging of SSc BM-MSCs, the finding that these cells showed a normal immunoregulatory activity, suggesting their potential to recover by activating a sort of adaptive mechanism.
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Disclosure of Interest None Declared