It is well known that complement activation and cytokine dysregulation are critical in the pathogenesis of immune thrombocytopenia (ITP). We previously showed impaired proliferative capacity and reduced ability to inhibit T-cell proliferation of mesenchymal stem cells (MSCs) from ITP patients (Zhang JM, et al. Stem Cell Transl Med 2016), indicating that the bone marrow (BM) niche might be involved in ITP etiology. Recent studies have defined sympathetic nerve fibers and ensheathing Schwann cells as regulators in the BM niche (Arranz L, et al. Nature 2014). Given that the initial pathogenic mechanisms underlying ITP have not yet been identified, we hypothesized that complement activation played a role in the ITP BM niche including nestin+MSCs and Schwann cells.
Thirty-one consecutive patients with primary ITP and 19 healthy donors were enrolled in this prospective study. Samples from patients with ITP were divided into the two groups according to whether complement activation product C1q, C3b, C4d or C5b-9 was detectable on the surface of nestin+MSCs: the complement+nestin+MSC-ITP group and the complement-nestin+MSC-ITP group. Here we found that the complement+nestin+MSC-ITP group had fewer nestin+MSCs, lower proliferative capacity, less Cxcl12 production and a reduced capacity for inhibiting T cell proliferation, as well as higher levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and TNF-α in bone marrow supernatant compared with the complement-nestin+MSC-ITP group and the nestin+MSC-control group, indicating that activation of the complement cascade and enhancement of the inflammatory response are involved in the ITP BM niche.
To further define the possible mechanism for the vulnerability of nestin+MSC-ITP to the complement system, messenger RNA (mRNA) from 5 samples from each group respectively were analyzed by PrimeView™ Human Gene Expression Array (Affymetrix). Diminished levels of complement factor H mRNA consistent with factor H secretion was revealed in the complement+nestin+MSC-ITP group. Furthermore, the mRNA levels of C3aR, C5aR, IL-1R and caspase1 were up-regulated, whereas mRNA levels of CD46, CD55 and CD59 showed no statistically significant differences in this group. Upon further exposure to TNF-α, the complement+nestin+MSC-ITP group insignificantly up-regulated factor H compared with the other two groups.
Interestingly, in the complement+nestin+MSC-ITP group, complement activation products were detected on the outer surface of Schwann cells. Immunostaining of glial fibrillary acidic protein (Gfap) to visualize Schwann cells showed undesirable innervation in bone marrow of the complement+nestin+MSC-ITP group. GFAP mRNA expression was concurrently reduced in this group. To characterize the supporting role of Schwann cells, Schwann cells and nestin+MSCs derived from neonatal BMCD45-CD31-Ter119-Nes-GFP+Pdgfrα-/+ cells were co-cultured in vitro with different concentrations of IL-1β for 24 h. The results indicated that sympathetic neuropathy could sensitize nestin+MSCs to cell death triggered by IL-1β. Schwann cells were additionally incubated with an antagonist of interleukin-1 receptor (IL-1Ra), which consequently reversed IL-1β mediated cell damage. Together, these data suggested that IL-1β contributes to neuroglial damage, which compromised nestin+MSC survival.
To further define potential regulators that can reverse the alternations of the ITP BM niche, we developed an ITP murine model. ITP mice were randomly assigned to two groups and treated with either prednisone or IL-1Ra. Both of these two groups exhibited improved proliferative capacity and inhibition of T cell proliferation of nestin+MSCs and recovered Schwann cells innervations. No statistically differences were observed between the two groups.
In conclusion, the impaired nestin+MSCs and Schwann cells by complement activation and IL-1β in the BM niche play a role in the pathogenesis of ITP. The effect of IL-1Ra in correcting the dysfunction of nestin+MSCs and Schwann cells suggests a potential novel therapeutic approach of IL-1Ra for patients with ITP.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.