PDGFR-Mediated Cytomegalovirus Infection of Megakaryocytes Induces Thrombocytopenia Via Demethylation of AML1 and IEX-1 in the TPO/c-Mpl Signaling Pathway Following Allo-HSCT

Introduction: Human cytomegalovirus (HCMV) infection is a common complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is associated with high morbidity and mortality. Thrombocytopenia is one of the major hematological complications of HCMV infection. Possible causes include direct HCMV injury to hematopoietic progenitor cells and the microenvironment, as well as HCMV-related immune thrombocytopenia. Previous in vitro studies demonstrate that HCMV could directly infect megakaryocytes(MKs) and their progenitors, resulting in decreased CFU-MK and increased apoptosis, but the underlying mechanisms remain uncertain. It remains unknown whether HCMV can directly target MKs in vivo, how MK function changes after infection, why HCMV selectively infects certain patients and what inhibits MK maturation and results in apoptosis. It has been reported that patients with HCMV-related thrombocytopenia showed poor response to rhTPO, implying blockage of the TPO/c-Mpl signaling pathway. Our previous research indicated that PDGFR⁺CXCR4^lowCCR5^lowMKs are correlated with HCMV infection.We hypothesized that PDGFR⁺CXCR4^lowCCR5^lowMKs are more susceptible to HCMV infection. HCMV could directly target MKs both in vitro and in vivo, resulting in increased apoptosis and decreased MK ploidy. HCMV infection could possibly disturb the downstream TPO/c-Mpl signaling pathway, thereby inhibiting MK differentiation and maturation.

Methods: We collected bone marrow from HCMV DNAemia patients post allo-HSCT for in vivo study. Transmission electron microscopy(TEM) was used to detect HCMV particles inside MKs. MKs were identified as CD41⁺vWF⁺cells by flow cytometry(FCM). To analyze the susceptibility of MKs to HCMV, expression levels of PDGFR, αvβ3, TLR2, CCR5 and CXCR4 in different groups were tested. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. We also measured c-Mpl expression in MKs.In vitro study, we used plasma from HCMV-infected patients post allo-HSCT to infect MKs cultured from bone marrow CD34+ cells. We validated cell susceptibility with the same markers used in vivo. Next, inhibitors of the positive markers were co-cultured with MKs. We analyzed pp65 expression in the inhibitor-treated group and control group to explore potential prevention of HCMV infection. We investigated AML1 and IEX-1 in the downstream TPO/c-Mpl signaling pathway by PCR and Western Blot. We used bisulfite sequencing PCR (BSP) to study the methylation status in different gene expression profiles of AML1 and IEX-1. 5-ara-dC is a type of DNA methylation inhibitor. After incubation with MKs, we analyzed changes in gene expression and MKs function.

Results: Using TEM, we managed to find HCMV particles in MKs from HCMV-infected patient bone marrow samples. The proportion of apoptosis markedly increased compared with HCMV-negative MKs, whereas the mean ploidy slightly decreased. C-Mpl expression showed no significant difference between the two groups. Pp65 positive cells showed elevated expression in PDGFR and reduced expression in CXCR4 and CCR5. In vitro studies revealed similar results. After treating with the PDGFR inhibitor IMC-3G3, the pp65 positive cell population was slightly decreased, but the Gleevec-treated group showed no difference. We found a decrease in both IEX-1 and AML1 on both the molecular and protein levels. Both gene promoters were hypermethylated in the HCMV-infected group. After demethylation with 5-ara-dC, IEX-1 and AML1expression levels were both up-regulated, and cell apoptosis was reduced.

Conclusion: (1)HCMV inhibited megakaryocytic differentiation and maturation and reduced MKs polyploidy both in vivo and in vitro. (2)MKs positive for PDGFR and low in CXCR4 and CCR5 were more susceptible to HCMV infection. The PDGFR inhibitor IMC-3G3 protected MKs from HCMV infection. (3)The mechanism of HCMV-associated thrombocytopenia may be a disturbance of the TPO/c-Mpl signaling pathway in MKs through hypermethylation of the AML1 and IEX-1 promoters. Demethylation with 5-ara-dC could reverse cell apoptosis. Therefore, we illustrated the possible mechanism of HCMV-induced thrombocytopenia, highlighting new insights for future potential therapeutic approaches.