Megakaryocytes engage in emperipolesis with neutrophils and emperipolesis by the immune MK subset leads to efferocytosis and is exacerbated by CXCR1/2 activation in myelofibrosis Free

Megakaryocyte emperipolesis of neutrophils is increased in the bone marrow of myelofibrosis patients and leads to their death by para-apoptosis. However, these prior observations provide little insight into the dynamics of these interactions under normal and pathological conditions. Here, we leveraged flow cytometry, RNAseq, and time lapse microscopy to derive mechanistic insights into the normal and pathological emperipolesis occurring between MK and neutrophils, respectively, in wild type (WT) mice and in the Gata1^low model of myelofibrosis.

We found that the frequency of CD41 MK in the bone marrow from WT mice increases by 2-fold with age (p<0.05) while that of Gata1^low mice does not increase with age. Gata1^low CD41pos MK contain 2-times more CD53pos immune-MK than the WT MK (p<0.05) but the level of CD53 expression is 20% lower than that of WT MK (p<0.05). The BM of Gata1^low mice contain also greater levels of neutrophils that express levels of CD11b lower than the WT ones, an indication that the Gata1low neutrophils are more efficiently cleared of senescent cells. RNAseq of Gata1^low CD41pos MK reveal downregulation of ribosomal genes and enrichment of genes specific for the immune-MK and for the CXCR1/CXCR2 (the receptor for CXCL1) signature.

Time-lapse confocal microscopy images were taken of CD41-FITC and CD11b-PE labelled cells purified from the bone marrow of young (no myelofibrosis) and old (with myelofibrosis) Gata1^low mice and age-matched WT littermates co-cultured with thrombopoietin for up to 48h. In selected experiments, the cocultures were performed with Reparixin (10μM), the inhibitor of CXCR1/CXCR2, or the CD41pos cells were also labeled with CD53-647 which recognizes the immune-subpopulation. We analyzed a total of 469 individual MK for over 415hr. Since emperipolesis is preceded by chemotaxis and cell-cell interaction, we first focused on the type of interactions occurring between the two cell types. We observed four types of interactions which we categorize as 0=none; 1=touch and go (<90sec); 2=long (>90sec) with emperipolesis resembling the slow emperipolesis described by Huang et al (Blood Adv. 2022;6:2081) and 3=long,with slow emperipolesis leading to de-rearrangement of the MK plasma membrane, formation of a second plasma membrane and death. The features of the Type3 interactions are similar to the phagocytosis leading to cell death defined as efferocytosis (deCathelineau et al. Biochemistry, 2003;3939:105).

In the older cohorts, Type0 was detected mainly in the first 0-12h of culture and involved, respectively, 25% and 3% of WT and Gata1^low MK. Type1 and 2 were observed in the 13-14 and 25-36 time windows with similar frequency among groups. In the 37-48 hrs time window, Type2 was observed only in WT cultures while Gata1^low cultures displayed numerous Type3 interactions. By contrast, in the young Gata1^low group, we observed mostly Type1 and 3 interactions and MK were difficult to find after 24hr. Reparexin delayed or prevented Type3 interactions, respectively, in the young and old Gata1^low group (Chi-Square p<0.0001). The absence of Type3 in Gata1^low cultures with Reparixin suggests that these interactions are supported by the activation of the CXCR1/2 signaling which we had detected in these cells by RNAseq.

To assess the role of immune-MK in the emperipolesis with neutrophils, we incubated CD41 MK with CD53, and the CD53pos and CD53neg cells were imaged separately. Both the WT and the Gata1^low CD41CD53neg MK engaged only in Type1 and Type2 interactions while the wild-type CD41CD53pos MK engaged also in Type3 interactions. These results were confirmed by flow cytometry analyses which indicated that double CD11bCD41pos cells are detectable among both the CD53neg and CD53pos MK present in the WT and Gata1^low co-cultures in numbers that increase over time but that only the CD53pos MK became Annexin Vpos. In summary, we conclude that 1) all MK subpopulations engage in emperipolesis with neutrophils but only the immune-MK engage in emperipolesis associated with efferocytosis; 2) due to CXCR1/CXCR2 activation, Gata1^low immune-MK are more likely to engage in efferocytosis than the wild-type ones, and this susceptibility decrease with age. In future studies we will investigate whether, in contrast to conventional models, MK efferocytosis protects Gata1^low mice from the development of fibrosis in the bone marrow.