PAI-1 Mediates the Switch in Free Fatty Acid Metabolism from the Liver to the Bone Marrow to Support Hematopoietic Stem and Progenitor Cell Expansion during the Initial Stages of Infection
Authors: Hampton Katherine , Mistry Jayna J , Maynard Rebecca S , Polski-Delve Alyssa Nicole , Fowler-Shorten Dominic , Hellmich Charlotte , Beraza Naiara , Bowles Kristian , Rushworth Stuart A 
Published: December 2024

The hematopoietic stem cell (HSC) pool is tightly regulated within their hypoxic niche. Under normal conditions, quiescent HSCs sustain minimal mitochondrial activity, instead favouring glycolysis to meet their low energy requirements. However in response to infection, HSCs metabolically switch from glycolysis to oxidative phosphorylation to support their increased energy demands, enabling them to expand and differentiate into downstream progenitors to increase the immune cell pool. Recently, we have showed that during acute infection, fatty acid uptake and metabolism plays a central role in providing HSCs with sufficient energy for their expansion (Mistry et al., 2021). However, how infection drives these changes in fatty acid availability to enable this HSC expansion is not known. This study investigated the role of the liver in regulating lipid availability for HSC expansion.
We have previously shown that circulating free fatty acids (FFAs) within the serum of mice increased in response to infection using both lipopolysaccharide (LPS) (16 hours) and S. Typhimurium (72 hours) models. This correlated with an expansion of bone marrow HSCs 16 hours after LPS and 72 hours after S. Typhimurium, confirmed using flow cytometry (Mistry et al., 2021). To investigate real-time fatty-acid uptake by hematopoietic cells in response to infection, an in vivo transplant model was used. CD45.1 lineage negative, CD117-positive cells tagged with firefly luciferase (LK+FF) were transplanted into CD45.2 mice. After transplantation was confirmed, mice were injected with LPS for 16 hours. Mice were then subsequently injected with a luciferin molecule conjugated to a long-chain FFA (FFA-luc), which is visible via bioluminescent imaging if the FFA-luc is taken up into the transplanted LK+FF engrafted cells. Live animal imaging using this method confirmed that long-chain FFA is taken up by hematopoietic cells in response to LPS in vivo.
Lipidomic analysis showed LPS induced an increase in total serum FFA within 6 hours, with the most significant increase seen in long chain fatty acids including palmitoleic acid, oleic acid and docosahexaenoic acid, confirmed using liquid chromatography-mass spectrometry. As the liver is the master regulator of circulating free fatty acids in the serum, we explored the role of liver metabolism in response to LPS. Bulk RNA sequencing of whole liver lysates isolated from mice treated with LPS for 6 hours revealed significant down-regulation of genes involved in fatty acid uptake and metabolism. Further pathway analysis showed that LPS down-regulated multiple key metabolic pathways, including fatty acid degradation, transport and beta-oxidation.
To understand if this was a direct effect of LPS on hepatocytes or mediated through alternative paracrine signalling, we isolated primary hepatocytes and treated them with LPS or media conditioned with serum from control or LPS treated mice. Only the media conditioned with serum from LPS treated mice downregulated genes associated with fatty acid uptake and metabolism. To identify the factors driving this metabolic switch in hepatocytes, a cytokine array of serum from control or LPS treated mice (90 minutes) was performed. This proteome cytokine array revealed a significant upregulation of several cytokines and chemokines, including CXCL1, CXCL2, CCL5, CCL20, Interleukin 6, TNFα and plasminogen activator inhibitor 1 (PAI-1). Primary mouse hepatocytes were then treated individually with the factors identified in the array for 2 and 4 hours. RT-qPCR confirmed that only PAI-1 downregulated fatty acid uptake and metabolism genes in primary hepatocytes within 2 hours.
To study the role of PAI-1 in regulating liver fatty acid metabolism in the initial response to LPS, mice were treated with a PAI-1 inhibitor, Tiplaxtinin (PAI-039). Pre-treatment of mice with Tiplaxtinin inhibited LPS-induced downregulation of liver fatty acid uptake and metabolism genes within the early stages of infection, which lead to reversal of LPS-induced increase in serum fatty acids.
In conclusion, we have shown a role for PAI-1 in switching off fatty acid uptake and metabolism in the liver. This causes an increase in FFA in the serum, allowing FFA uptake and metabolism by hematopoietic stem and progenitor cells, enabling cell expansion and differentiation into downstream progenitors to increase the immune cell pool during the initial stages of infection.