Functional characterization of secreted protein isthmin-1 in hematopoietic system

Abstract

Hematopoiesis is the blood cell generation process in bone marrow involving HSC as the upmost multipotent primitive cell that possess self-renewal and differentiate into progenitors to generate lineage immune cells. HSCs are localized in bone marrow niche that are specially located near to the endosteum region. Bone marrow niche plays a crucial role through paracrine signaling molecules and proteins in regulating HSC fate and control whether they stay quiescent, self-renew, or differentiate. Secreted glycoprotein ISM1 is expressed in bone marrow and other immune organs. Several studies have examined on the function of ISM1 in a number of biological processes such as angiogenesis, kidney branching morphogenesis, metabolism, organ homeostasis. Even though ISM1 is required in various processes those are interrelated with hematopoiesis, but there is no study focusing on function of ISM1 in murine hematopoietic system. Seeing the diverse expression pattern, functions in hematopoiesis related biological processes, this secreted protein ISM1 grabbed my attention to conduct this study. In this study, I identified ISM1 as a critical regulator of HSC self-renewal and differentiation. This is because, I observed expansion of LT-HSCs and ST-HSCs along with decreased abundance of MPPs in Ism1 deficient mice. Furthermore, this differentiation defect caused myeloid skewed differentiation that was evident from reduced GMPs. In line with the myeloid suppression, significant reductions of neutrophils were exhibited in bone marrow and spleen along with peripheral blood neutropenia in Ism1 deficient mice. Neutropenia is caused by the maturation arrest of neutrophil, while I observed expansion of immature neutrophils in Ism1 deficient mice. Besides, increased lymphocytes further indicated that deficiency of Ism1 causes myeloid suppression in expense with biased expansion of lymphoid populations. Apart from reduced differentiation potential, HSCs were inadequately responsive to LPS-induced inflammation, G-CSF administration, and PMA treatment. Interestingly, increased HSC pool was also persistent even in Ism1 deficient aged mice. Moreover, transcriptomic analysis further exhibited increased HSC pool and reduced MPPs in young, adult, and aged Ism1 deficient mice. Furthermore, downregulation of TGFβ and IFNγ signaling pathway genes was observed in HSC and neutrophil cluster according to transcriptomic analysis and again both TGFβ and IFNγ signaling pathway proteins were downregulated in total bone marrow proteomic analysis. Furthermore, bone marrow niche transplantation assay demonstrated that ISM1 acts on HSCs in paracrine manner to enhance HSC differentiation and thus increased neutrophil indicated restoration of neutrophil maturation. On top of that, overexpression of Ism1 exhibited reductions of LT-HSC and increased neutrophils in adult mice and aged mice that is the opposite phenotype found in Ism1 deficient mice. Collectively, these data strongly indicate that ISM1 safeguards balanced HSC self-renewal and differentiation toward overall myeloid progenitors and neutrophils to be specific. Comprehensive mechanistic understandings of regulation of ISM1 on these major phenotypes are enigmatic yet, even though transcriptomic and proteomic analysis strongly indicated downregulation of TGFβ and IFNγ signaling pathway. However, further proteomic validation to implement novel secreted protein ISM1 as therapeutic agent in clinical manifestations of HSC-based therapy, neutropenia associated multiple cancer treatment is necessary. (Word count: 495)