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ATRA protects impaired BM MSC in ITP
revealed increased intensity of IL-1β fluorescence and number of TUNEL+CD90+ MSC in the central marrow (Online Supplementary Figures S4A and S5A). These results imply that the C5b-9/IL-1β loop contributes to the dys- function of MSC-ITP-C+.
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All-trans retinoic acid directed in vitro functional recov- ery of complement-activated mesenchymal stem cells from patients with immune thrombocytopenia
We previously reported the therapeutic efficacy of ATRA in ITP patients.27 In order to ascertain the mecha-
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Figure 4. All-trans retinoic acid directed in vitro functional recovery of mesenchymal stem cells from immune thrombocytopenia patients with complement dep- osition on their mesenchymal stem cells. (A) CCK8 proliferative assays of all-trans retinoic acid (ATRA) treated mesenchymal stem cells (MSC) from patients with immune thrombocytopenia (ITP) with complement deposition on their MSC (MSC-ITP-C+) from three independent experiments (n=12; Student t-tests). (B) Apoptosis rate of ATRA-treated MSC-ITP-C+ (n=12; Student t-tests). (C, D) Intracellular expression of interleukin-1β (IL-1β) and CXCL12 in untreated and treated MSC-ITP-C+ by immunofluorescence assays. (E, F) The levels of IL-1β and CXCL12 in MSC-ITP-C+ cell lysates at day 1 and day 7 with the administration of ATRA from two independent experiments (n=12; Student t-tests). (G) Phosphorylation of MyD88, ERK1/2, p38 MAPK and NF-κB in MSC-ITP-C+ before and after treatment with ATRA by western blotting (n=8; Student t-tests). (H, I) mRNA levels of IL-1β and CXCL12 in MSC from healthy controls (MSC-control), MSC from ITP without complement deposition on their MSC (MSC-ITP-C-) and MSC-ITP-C+ before and after treatment with ATRA. mRNA levels of IL-1β and CXCL12 were normalized to the mRNA levels of 18srRNA (n=12 independent experiments). Tukey tests for multiple comparisons among pre-treatment groups. Student t-tests for the comparisons between pre-treatment and post-treatment groups from independent experiments. (J) Promoter DNA methylation of IL-1β in MSC-control, MSC-ITP-C- and MSC-ITP-C+ before and after treat- ment with ATRA. Percentages of DNA methylation were obtained by analyzing the mean methylation of all the CpG sites present in the promoter (n=12 independent experiments). Tukey tests for multiple comparisons among pre-treatment groups. χ2 tests for the comparisons between pre-treatment and post-treatment groups from independent experiments. (K) The methylation status of -299, -256, -20 and +13 CpG sites in the IL-1β proximal promoter (n=12 independent experiments). (L) There was a linear relationship between DNA methylation of the IL-1β promoter and mRNA levels (R2 = 0.5925, P<0.001, Spearman rank correlation rho).
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