Abstract
Introduction
Human bone marrow-derived mesenchymal stem cells (hBMSCs) can adopt either an immune suppressive or stimulative phenotype in response to cytokines and pathogen-associated molecular patterns (PAMPs). It is known that the glycoprotein CD24 allows for the discrimination between PAMPs and DAMPs in dendritic cells. We were able to show previously that CD24 is expressed by hBMSCs and found that its overexpression leads to the downregulation of NF-kB-regulated genes, as well as induction of the anti-inflammatory TGF beta.
In the present study the influence of various PAMPs and cytokines on the expression of CD24 in hBMSCs was analysed. Furthermore, it was tested whether in vivo-CD24-positive (CD24+) and in vivo-CD24-negative (CD24-) hBMSCs differ in regard to classical hBMSC or immune-associated surface antigens.
Methods
hBMSCs were enriched by density gradient centrifugation, cultured in vitro until passage 3 and subsequently stimulated with PAMPs or cytokines (IFN gamma, TGF beta) before analysing the expression of CD24 via qRT-PCR. Cells expressing CD24 in vivo (CD24+ hBMSCs) were enriched from bone marrow aspirates after density gradient centrifugation by the use of magnetic-associated cell sorting (MACS). Successful enrichment was evaluated by flow cytometric analysis. The enriched cells were subsequently cultured in comparison to the CD24-depleted cell population (CD24- hBMSCs) under identical conditions. The expression of various cell surface markers was compared between these two populations using flow cytometry.
Results
All tested PAMPs, as well as IFN gamma led to the downregulation of CD24 in comparison to non-stimulated control cells. In contrast, stimulation with TGF beta resulted in an increased CD24 expression.
CD24-positive hBMSCs were successfully enriched via MACS and cultured in vitro. While there was no difference between the expression of classical hBMSC surface antigens between the two cell populations, the CD24+ population had a significantly higher expression of PD-L1 than the CD24- population.
Discussion
hBMSCs are capable of ameliorating autoimmune processes by inducing T-cell anergy. Polymorphisms in CD24 are associated with the development of autoimmune diseases. In this context it is worth of note that CD24+ hBMSCs show an elevated expression of PD-L1. PD-L1 is a molecule that can induce anergy in T cells by binding to PD-1 thereby dampening the immune response to self antigens. Therefore, hBMSCs with strong CD24-expression might be beneficial in treating autoimmune diseases such as rheumatoid arthritis.
PAMPs and IFN-gamma lead to the downregulation of CD24, which may strip hBMSCs of their ability to induce T cell anergy and to dampen immune responses to self antigens.
