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ATRA protects impaired BM MSC in ITP
To evaluate the regulation of MSC-ITP-C+ on cell sig- naling, we performed a canonical pathway analysis. Canonical pathway annotation enabled us to assign dif- ferentially expressed genes to 28 pathways. The first nine enriched pathways were the unfolded protein response, protein ubiquitination pathway, cell cycle, IL-1 signaling, p53 signaling, endoplasmic reticulum stress pathway, ERK5 signaling, NRF2-mediated oxidative stress response, and p38 MAPK signaling, which might convey the differences between MSC-ITP-C+, MSC-ITP-C-, and MSC-control. The first 12 enriched pathways are shown in Online Supplementary Figure S2E. Disease and functional heatmaps revealed the activated or inhibitory relation- ship between differentially expressed genes and diseases and functions (Online Supplementary Figure S2F). “Organismal death” (Z-score = 16.497) and “Morbidity or mortality” (Z-score = 16.484) were significantly activated in MSC-ITP-C+.
Complement-activated mesenchymal stem cells from immune thrombocytopenia patients showed increased apoptosis and functional impairment
Bone marrow MSC were successfully isolated. Flow cytometry analysis demonstrated that MSC from both healthy donors and ITP patients expressed CD105, CD73, and CD90 and lacked expression of CD14, CD19, CD34, CD45, and HLA-DR (Online Supplementary Figure S3). MSC-control expanded and acquired a spindle shape morphology during culture. In contrast, MSC-ITP-C+ expanded more slowly and appeared larger and flattened (Figure 1B). CCK8 proliferative assays were conducted on MSC at days 1, 3, 7 and 14 after the third passage. The growth curves showed a lower proliferative capacity of MSC-ITP-C+ (Figure 1C). We further assessed the apop- totic cell rate using annexin V. As shown in Figure 1D, the rates of both early apoptosis and late apoptosis were higher in the MSC-ITP-C+ group.
Since the complement system has been shown to be associated with the inflammatory response, we deter- mined cytokine levels in bone marrow supernatants from ITP patients and healthy controls using ELISA. The levels of the inflammatory factors IL-1β and TNF-α were both significantly higher (P<0.001) in the MSC-ITP-C+ group compared with the MSC-ITP-C- and MSC-control groups (Figure 1E), while the levels of CXCL12 were significantly lower (P<0.001) (Figure 1E). Intracellular expression of IL- 1β and CXCL12 was further confirmed by immunofluo- rescence assays (Figure 1H, I). Moreover, the levels of expression of complement activation fragments C3a and C5a were also significantly higher (P<0.001 for both) in MSC-ITP-C+ (Figure 1E). There was a positive correlation between the level of IL-1β in culture supernatant and C5b-9 deposition on MSC (R2 = 0.7426, P<0.001, Spearman rank correlation rho) (Figure 1F).
In view of the diminished expression of complement factor H found by microarray analysis in the MSC-ITP-C+ group, we performed a TNF-α stimulation test. Factor H concentrations in MSC culture supernatants 12, 24, and 48 h after co-culture with TNF-α were measured by ELISA. Upon exposure to TNF-α, no significant effect on the secretion of factor H was seen in the MSC-ITP-C+ group, which was not consistent with the significantly increased factor H secretion observed in the MSC-ITP-C- and MSC-control groups (Figure 1G).
Altered CXCL12 gradients and megakaryocyte marrow niche occupancy in immune thrombocytopenia patients with mesenchymal stem cell complement deposition
As the MSC-ITP-C+ group showed attenuated expres- sion of both CXCL12 protein and its encoding gene, we investigated whether there are changes in bone marrow CXCL12 and their consequences for megakaryocytes. Given the importance of the location of CXCL12 produc- tion for this chemokine’s chemotactic function32 and the varied injury and recovery kinetics of different bone mar- row cell populations in MSC-ITP-C+, we determined the location of CXCL12 transcripts in bone marrow sections of the MSC-ITP-C+, MSC-ITP-C- and MSC-control groups by radioactive in situ hybridization (Figure 2A-C). As expected, the MSC-ITP-C+ group showed an overall increase in the CXCL12 message in the bone-associated marrow and a decrease in the central marrow (Figure 2C). Differences in the distribution of CXCL12 in the bone marrow were quantified by comparing CXCL12 tran- script levels within the bone-associated region to the lev- els in an adjacent region within the central marrow (Figure 2E). Interestingly, we detected the development of a CXCL12 gradient toward the endosteum in the MSC- ITP-C+ group (Figure 2E). Since megakaryocytes interact with sinusoidal endothelium, we examined the effects of observed shifts of CXCL12 expression on marrow vascu- lature by immunohistochemistry (Figure 2D). A notice- able, marked decrease in megakaryocytes associated with sinusoids was observed in the MSC-ITP-C+ group (Figure 2D, F). Given the development of a CXCL12 gradient toward the endosteum in the MSC-ITP-C+ group, we also quantified megakaryocyte distribution in the bone-asso- ciated region, defined as the region within 100 μm of the diaphyseal endosteum.33 Strikingly, there was an increase in megakaryocytes in the bone-associated marrow, coin- cident with the increased CXCL12 gradient (Figure 2D, G). Altogether, these results implicated spatial and tem- poral alterations in marrow CXCL12 of the MSC-ITP-C+ group in the observed changes in megakaryocyte niche occupancy. The overall numbers of megakaryocytes and the density of vessels were not significantly different between the MSC-control, MSC-ITP-C- and MSC-ITP-C+ groups (Figure 2H, I).
The C5b-9/interleukin-1β loop regulates mesenchymal stem cells from patients with immune thrombocytope- nia
On the basis of the finding of enhanced expression of IL-1β in the MSC-ITP-C+ group, we hypothesized that IL- 1β induced by complement activation plays an important role in the dysfunction of MSC. The production of pro– IL-1β and caspase-1 was significantly higher in MSC-ITP- C+ than in MSC-ITP-C- and MSC-control (Figure 3A), indicating that complement induced IL-1β maturation and caspase-1 processing.
To obtain further insight into the mechanisms underly- ing the increased expression of IL-1β, we then analyzed the role of signaling pathways in MSC from ITP patients and healthy volunteers. Expression of the interleukin-1 receptor (IL-1R) was upregulated on MSC-ITP-C+ (Figure 3B) and increased activation of MyD88 and NF-KB (p65) was detected in MSC-ITP-C+ (Figure 3C). Simultaneously, the expression levels of p-ERK1/2 and p-p38 MAPK were significantly higher in MSC-ITP-C+ than in the other two
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