BCL-Xl Driven Accumulation of Dysfunctional Mitochondria in Aged Stromal Cells Impairs the Haematopoietic Stem Cell Response to Stress
Authors: Hellmich Charlotte , Mistry Jayna J , Jibril Aisha , Moore Jamie A , Johnson Benjamin B , Smith James G W , Wojtowicz Edyta , Beraza Naiara , Bowles Kristian M , Rushworth Stuart A 
Published: November 2021

Abstract
The burden of infections is known to increase with age. Not only is ageing associated with greater susceptibility to infections but also an increase in subsequent morbidity and mortality. The bone marrow (BM) niche is essential for the body's response to infection. Haematopoietic stem and progenitor cells (HSPCs) heavily rely on their supporting BM microenvironment to effectively expand and differentiate in response to stress and infection (1). The role of senescent cells has been explored in a number of age-related diseases including acute myeloid leukaemia (2). Here we explore the role of senescence during natural ageing in the BM microenvironment, the mechanism which drives this and how this impacts on the metabolic health of HSPCs.
BM was isolated from aged (18-24 months) and young (8-12 weeks) C57Bl/6 mice and flow cytometry was used to compare mitochondrial membrane potential (ΔΨm) and mitochondrial ROS in HSPCs. Results show that HSPCs from aged animals increase in numbers and accumulate mitochondria with low membrane potential with lower mitochondrial ROS. Next, young and aged mice were treated with lipopolycaccharide (LPS). Metabolic analysis revealed that HSPCs from young mice increase metabolism of mitochondrial TCA cycle substrates in response to LPS whereas aged HSPCs continued to rely on glycolysis. When HSPCs from aged C57Bl/6 mice (CD45.2+) were FACS purified and adoptively transferred into young PepCboy (CD45.1+) mice, thus removing them from the aged BM microenvironment, they were able to recover their mitochondrial health and showed an improved metabolic response to treatment with LPS. Furthermore, qRT-PCR analysis of p16 and p21 expression in HSPCs and mesenchymal stromal cells (MSC) showed that MSCs, but not HSPCs, acquire a senescent phenotype in aged mice, and depletion of senescent cells in the p16-3MR mouse model (3) allowed recovery of HSPC mitochondrial function and response to LPS. Mechanistically, we found a significant upregulation of the anti-apoptotic protein BCL-XL in MSCs of aged mice. This has previously been described to drive the senescent phenotype and prevent apoptosis in senescent cells. By over-expressing GFP-tagged BCL-XL in MSCs and then co-culturing them with LSKs we were able to show that BCL-XL is transferred from MSCs to HSPCs in vitro. Finally, we demonstrated that targeting BCL-XL in vivo, using the senolytic drug ABT-263, in aged mice can restore the HSPC metabolic response to stress resulting in upregulation of TCA cycle metabolism.
In conclusion, we show that the aged BM microenvironment is responsible for the HSPC metabolic shortfall resulting in impaired response to stress. Targeting the senescent cells in the environment restored the HSPC metabolic function and their response to infection in aged mice. This suggests that manipulation of the ageing BM microenvironment can help to improve the body's response to infection.
1. Mistry JJ, Marlein CR, Moore JA, Hellmich C, Wojtowicz EE, Smith JGW, et al. ROS-mediated PI3K activation drives mitochondrial transfer from stromal cells to hematopoietic stem cells in response to infection. Proc Natl Acad Sci U S A. 2019;116(49):24610-9.
2. Abdul-Aziz AM, Sun Y, Hellmich C, Marlein CR, Mistry J, Forde E, et al. Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment. Blood. 2019;133(5):446-56.
3. Demaria M, Ohtani N, Youssef SA, Rodier F, Toussaint W, Mitchell JR, et al. An essential role for senescent cells in optimal wound healing through secretion of PDGF-AA. Dev Cell. 2014;31(6):722-33.

Disclosures
No relevant conflicts of interest to declare.