Direct Sensing of Lipopolysaccharide Limits Hematopoietic Stem Cell Selfrenewal Via TLR4-TRIF-ROS-p38 Pathway
Authors: Takizawa Hitoshi , Saito Yasuyuki , Kovtonyuk Larisa V. , Frisch Kristin , Manz Markus G. 
Published: October 2019

Abstract
Bacterial infection results in the consumption and replenishment of mature blood cells. While the cellular response of short-lived mature hematopoietic cells in peripheral tissue is well understood in this process, little is known about its impact on function of lifelong self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM). Here we examine the functional alteration of HSC and the underlying molecular mechanism upon in vivo systemic challenge of lipopolysaccharide (LPS), a gram-negative bacterial component recognized by Toll-like receptor 4 (TLR4).
To dissect direct or indirect TLR4 activation on HSCs by LPS, we reconstituted lethally irradiated wild-type (WT) recipients with WT and TLR4 deficient (Tlr4-/-) cells in a 1:1 ratio (WT;Tlr4-/- chimera), followed by systemic LPS injections. In vivo repetitive LPS injections activated dormant HSCs to cell cycle via TLR4 activation in both hematopoietic and non-hematopoietic cells. Next, the WT;Tlr4-/- chimeric mice were treated with LPS and analyzed for long term donor chimerism between WT and Tlr4-/- cells. Monthly blood and terminal BM analysis showed that Tlr4-/- cells outcompeted WT cells over time upon LPS injections while PBS treated controls showed an equal donor contribution to peripheral blood mature cells and BM HSCs, as expected. The out-competition of WT hematopoietic repopulation and WT HSC was significantly more in Tlr4-/- primary recipients and upon serial transplantation, suggesting that direct TLR4 activation on HSCs impairs their competitive self-renewing ability. To further characterize the downstream TLR4 signals active in HSCs, mixed BM chimera with WT and Trif-/- or Myd88-/- cells (WT;Trif-/- or WT;Myd88-/- chimeric mice) were tested as described above. Following LPS injections Trif-/- but not Myd88-/- HSCs showed a competitive self-renewing advantage over WT HSCs, similarly as Tlr4-/- HSCs. In vivo pharmacological inhibition of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK) inhibited LPS-induced HSC proliferation and dysfunction, identifying ROS-p38 as the responsible TLR4 downstream signals. Since ROS is known to cause DNA damage, single cell immunocytochemistry was performed to check activation of surrogate DNA damage markers. More DNA damage responses was accumulated and sustained in dormant HSCs previously stimulated with LPS as compared to PBS controls. In contrast to the HSC response to LPS, granluopoiesis induced by systemic bacterial infection, often referred to as emergency granulopoiesis, depends on MYD88 but not TRIF-ROS-p38 axis, implying differential hematopoietic regulation by two distinct TLR4 signal axis.
These data demonstrate in vivo evidence that LPS directly activates TLR4-TRIF-ROS-p38 mediated pathways in BM HSC, and results in limiting their competitive self-renewal possibly through increased divisional history and DNA damage. Our findings point to how chronic inflammation or severe infection might lead to the increased risk for malignant HSC diseases (MDS, AML, MPN) observed in the population-based studies. Intervention in this pathway during systemic infection might prevent HSCs from hematopoietic dysfunction, while preserving emergency granulopoiesis and thus at the same time allowing efficient reestablishment of immune response against pathogenic insults.

Disclosures
No relevant conflicts of interest to declare.