Session: 201. Granulocytes, Monocytes, and Macrophages: Poster II
Hematology Disease Topics & Pathways:
Diseases, Animal models, Genetic Disorders, immunodeficiency, Immune Disorders, Neutropenia, Study Population, white blood cell disorders
A proband of XLN had 1.3 x 103/mL WBC with a differential count of 8% neutrophils, 5.3% monocytes, 2.3% eosinophils, 3% basophils, and 80.4% lymphocytes. BM revealed marked myeloid hypoplasia with a slight dysplastic change and maturation arrest at the myelocyte stage, and colony forming ability of BM CD34+cells in methylcellulose containing SCF, IL3, and G-CSF demonstrated profound defects in G- and GM-colonies. The patient-derived iPS cells were developed and were induced to differentiate into HSCs by a coculture over an OP9 monolayer, supplemented with optimal cytokine cocktails. Resulting frequency of CD45+CD34+cells was rather comparable to that from cord blood (CB)-derived iPS cells. However,XLN-iPS-derived CD45+CD34+cells produced 5-fold smaller number of myeloid colonies than CB-iPS-derived thosecells in the same methylcellulose culture.
To further gain insight into the underlying abnormalities induced by WASp-I290T, we established a novel knock-in mouse model harboring WASp-I292T, which was obtained from CRISPR/Cas9-engineered mouse ES cells. Unlike XLN patients, but similar to two earlier mouse studies, the WASp-I292T mice had normal count of neutrophils, monocytes, B cells, CD4+ T cells, CD8+ T cells, and NK cells (Figure 1A). Administration of G-CSF into the WASp-I292T mice increased neutrophils to the comparable level to Wt mice. Similarly, lipopolysaccharide increased neutrophils and monocytes comparable to Wtmice. However, WASp-I292T neutrophils possessed a stronger ability of phagocytosis than Wt, when determined by the uptake of fluorescently pre-labeled E. coli(p=0.029) (Figure 1B).
Colony forming ability of the bone marrow c-kit+, sca-1+, lin-(KSL) cells in methylcellulose containing EPO, IL-3, IL-6, and SCF was significantly impaired in mut mice (p=0.003) (Figure 1C). In the bone marrow, however, the fraction of granulocyte/monocyte progenitors (GMPs; c-kit+, sca-1-, CD34+, CD16/32-) and pre-neutrophils (c-kit+, Gr-1dim, CXCR2-, CXCR4+) were marginally increased in the mut mice (p=0.052, and p=0.006, respectively), and megakaryocyte/erythroid progenitors (MEP; c-kit+, sca-1-, CD34-, CD16/32-) was slightly decreased in the mut mice (p=0.051) (Figure 1D). These results suggested that granulopoietic potential was basically maintained in the WASp-I292T mice, but the process at the end of neutrophil production or the egression from the bone marrow were impaired. We previously reported that NB4WASp-I290T cells produces significantly fewer neutrophils following ATRA-induced differentiation. In addition, analysis of myeloid-related genes revealed a premature upregulation of C/EBP-epsilon in NB4WASp-I290T cells. Because WASp contains a nuclear localization signal-like motif in the basic domain, and confocal laser microscopy suggested that WASp-I290T preferentially localizes in the nucleus rather than cytoplasm, it is likely that WASp-I290T alters myeloid-related genes by direct or indirect interactions with transcription factors in the nucleus; although detailed mechanisms still remain to be elucidated. As compared with profound defects in XLN-patients, defects in WASp-I290T mice were quite modest, suggesting some compensatory mechanisms in the murine hematopoiesis. Further studies of transcription factors and protein-protein interactions would provide a more precise explanation.
Disclosures: No relevant conflicts of interest to declare.
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