Role of Moesin in Murine Hematopoiesis
Authors: Takamatsu Hiroyuki , Espinoza Luis J. , Nakao Shinji 
Published: October 2019

Abstract
Abstract 1338

Background:
Moesin, a membrane-cytoskeleton linker protein, functions in microtubule formation, in membrane ruffling, and at sites of cell-adhesion. However, its role in hematopoiesis is unclear. Although a previous study demonstrated normal hematopoietic function in moesin knock-out (KO) mice (JBC 1999), the changes in blood cell counts observed in the mice have not been thoroughly analyzed. Moesin-specific antibodies are detected in the serum of approximately 30% of patients with acquired aplastic anemia (AA). Moesin is expressed on the surface of peripheral blood T cells and monocytes, but not on hematopoietic stem cells (HSCs). Our recent study demonstrated the involvement of anti-moesin antibodies in the development of AA by stimulating T cells and monocytes to secrete tumor necrosis factor-α and interferon-γ. Given that hematopoiesis is regulated by T cells and monocytes, the absence of moesin should impact hematopoiesis. The purpose of this study was to examine blood cell counts in moesin KO mice and WT mice transplanted with anti-moesin antibody producing B cells to clarify the role of moesin in hematopoiesis.

Methods:
Moesin KO mice (F1 heterozygous females (-/X)), which were a hybrid strain between 129/Sv and C57BL/6 (B6), were kindly provided by Professor Tsukita of Osaka University. F1 heterozygous females and B6 F1 wild-type (WT) males were interbred and the littermates were genotyped. Moesin KO mice, F2 hemizygous males (-/Y), and F3 homozygous females (−/−) were analyzed in a previous study (JBC 1999). F1 heterozygous females were backcrossed to B6 males for at least 8 generations to generate moesin KO mice in a B6 background. Moesin KO mice (12 months old) were used for hematopoietic phenotype analysis and to raise anti-moesin antibodies by immunization with moesin protein. Moesin KO male mice (-/Y) were immunized with 2×107 spleen cells containing moesin from WT B6 male mice to produce anti-moesin antibody secreting cells. Anti-moesin antibody titers were determined by Western blot analysis. To analyze the effects of anti-moesin antibodies in hematopoiesis, spleen cells (1.5×107) and bone marrow (BM) cells (1×107) containing anti-moesin antibody secreting cells from moesin KO mice were transplanted to the BM of 8-week-old WT B6 male mice conditioned with irradiation (3 Gy).

Results:
Moesin KO mice were born at expected Mendelian ratios and developed and grew normally. However, the body weight of moesin KO mice (n=7) was lower than that of WT mice (n=9) (26.63±2.47 g vs. 30.71±4.85 g; P=0.049). Complete blood counts of moesin KO mice (n=4) revealed leukocytopenia (WBC 12000±3096/μL vs. 19750±2582/μL; P=0.03) and macrocytic anemia (Hb 13.8±0.50 g/dL vs. 15.8±0.50 g/dL; P=0.06; MCV 51.7±1.7 fL vs. 47.6±1.3 fL, P=0.002) as compared with WT mice (n=4); platelet counts were comparable. Moesin KO mouse spleens (n=8) were heavier than those of WT mice (0.316±0.355 g vs. 0.091±0.034 g; P=0.0002) and follicle formation in spleens of moesin KO mice were obscure with increased CD3+ lymphocytes and reduced CD19+ lymphocytes as compared with WT mice. BM of moesin KO mice was normocellular and characterized by increased CD3+ lymphocytes and reduced CD19+ lymphocytes as seen in spleens of moesin KO mice. The number of lineage negative, Sca-1+, and c-Kit+ (LSK) mouse HSCs in moesin KO BM (n=4) tended to be lower than that of WT mice (n=3) (0.97±0.29% vs. 1.85±0.20%; P=0.06). Moesin KO spleen/BM cells capable of producing anti-moesin antibodies were infused into BM of WT B6 male mice (n=3), and moesin KO hematopoietic cells were engrafted on day 21. Transplanted mice showed leukocytopenia compared to WT B6 male mice transplanted with WT spleen/BM cells (n=3) (WBC 2667±333/μL vs. 7333±333/μL; P=0.0006), but anemia and thrombocytopenia were not observed.

Conclusions:
Moesin plays an important role in murine hematopoietic and immune system development. Anti-moesin antibody-induced leukocytopenia may result from excessive secretion of myelosuppressive cytokines from immune cells as observed in AA patients. The mechanistic details of HSC impairment in moesin KO mice are currently under investigation.

Disclosures:
No relevant conflicts of interest to declare.