Available online 16 April 2024

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Under steady-state conditions, HSPCs maintain low levels of NO and iNOS, but these levels increase in response to 5-FU-induced stress.

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iNOS deficiency led to significant changes in the HSPC compartment and their functions associated with increased mitochondrial stress, and loss of quiescence of HSCs.

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Loss of iNOS led to delayed hematopoietic recovery, heightened susceptibility to bone marrow stress, and reduced demand-derived mitochondrial activity during recovery.

Abstract:

Hematopoietic stem cells (HSCs) replenish blood cells under steady state and on demand, that exhibit therapeutic potential for Bone marrow failures and leukemia. Redox signaling plays key role in immune cells and hematopoiesis. However, the role of reactive nitrogen species in hematopoiesis remains unclear and requires further investigation. We investigated the significance of inducible nitric oxide synthase/ nitric oxide (iNOS/NO) signaling in hematopoietic stem and progenitor cells (HSPCs) and hematopoiesis under steady-state and stress conditions. HSCs contain low levels of NO and iNOS under normal conditions, but these increase upon bone marrow stress. iNOS-deficient mice showed subtle changes in peripheral blood cells but significant alterations in HSPCs, including increased HSCs and multipotent progenitors. Surprisingly, iNOS-deficient mice displayed heightened susceptibility and delayed recovery of blood progeny following 5-Fluorouracil (5-FU) induced hematopoietic stress. Loss of quiescence and increased mitochondrial stress, indicated by elevated MitoSOX and MMPhi HSCs, were observed in iNOS-deficient mice. Furthermore, pharmacological approaches to mitigate mitochondrial stress rescued 5-FU-induced HSC death. Conversely, iNOS-NO signaling was required for demand-driven mitochondrial activity and proliferation during hematopoietic recovery, as iNOS-deficient mice and NO signaling inhibitors exhibit reduced mitochondrial activity. In conclusion, our study challenges the conventional view of iNOS-derived NO as a cytotoxic molecule and highlights its intriguing role in HSPCs. Together, our findings provide insights into the crucial role of the iNOS-NO-mitochondrial axis in regulating HSPCs and hematopoiesis.

Section snippetsIntroduction:

Hematopoietic stem cells (HSCs) are rare multipotent stem cells residing in bone marrow (BM) that play a vital role in replenishing blood cells under steady-state and demand-driven conditions such as infection, injury, and stress (1). HSCs' self-renewal, differentiation, migration, and death decisions contribute to homeostasis. Self-renewal activity maintains the HSC pool, while differentiation of HSCs provides immune cells via hematopoietic progenitor cells (HPCs) 1, 2, 3. Reactive oxygen

HSPC contains low NO that increases upon stress

To understand the putative NO signaling in HSPCs in the BM, we first investigated the differential NO levels in distinct BM compartments, including hematopoietic hierarchy, i.e., mature lineage progeny (Lin+), immature progeny (Linˉ), committed progenitors identified as Linˉc-Kit+ (LK), multipotent progenitors defined as LinˉSca-1+c-Kit+ (LSK), and long-term HSCs (LT-HSC) defined as LinˉSca-1+c-Kit+CD34ˉ (referred as HSCs from hereon) in the steady state (Fig. S1). Data demonstrated low NO

Discussion:

Maintaining a functional hematopoietic system is crucial for adequate blood supply during homeostasis and response to infection or injury. Reactive species, acting as signaling molecules, exhibit biphasic effects: low levels are essential for normal function, while high levels induce cytostasis and cytotoxic stresses (33). The role of NO signaling in hematopoiesis remains unclear, as the effects of NO donors and NOS inhibitors vary depending on concentration, source, proximity, cell type, and

Materials and methods:

Animal’s studies: 3 to 6 month-old, age-matched WT and iNOS−/−(Jackson Laboratory, Bar Harbor, ME, USA; 002609) male mice on C57BL/6 background were used in the study. Mice were bred at the CDRI animal facility. Animal studies were approved by the institutional animal ethics committee and performed according to its guidelines. To induce bone marrow ablation, 5-FU (150 mg per kg body weight; Cayman Chemicals) was injected intra-peritoneally (47).

Hematological analyses: Peripheral blood was

Data-sharing statement:

All major data generated or analysed during this study are included in this article and any specific data may be made available to any researchers for non-commercial purposes.

Funding

SK acknowledges the support of ECR/2017/001274 and CRG/2022/001939 grants from Science and Engineering Research Board (SERB) Department of Science and Technology (DST), India and CSIR-CDRI in-house support. MD is supported by JBR/2020/000034. NET-CSIR fellowship to SS.

Declaration of Competing Interest

None.

Acknowledgements

We thank Mr. AL Vishwakarma and Mr. Anil Kumar Verma for their help at flow cytometry facility and Intravital-confocal microscopy facility at CSIR-CDRI. We also acknowledge HCS Cellomics and microscopy facility at the department of Pharmacology, CSIR-CDRI and thank Dr. C P Pandey at the Central Instrumental facility of the Pharmacology Division for his support. We thank the Director CSIR-CDRI for his support during the study. This manuscript is a CDRI communication number 199/2023/SK.

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© 2024 Published by Elsevier Inc.