PU.1 Enforces Hematopoietic Stem Cell Quiescence during Chronic Inflammation
Authors: Chavez James , Rabe Jennifer L , Higa Kelly , Loeffler Dirk , Nouraiz Ahmed , Mills Taylor , Idler Beau M , Stevens Brett M. , Fleenor Courtney , Nerlov Claus , Nakajima Hideaki , Hagman James , Schroeder Timm , Kim Hyun Min , Jordan Craig T , DeGregori James , Pietras Eric 
Published: February 2020

Hematopoietic stem cell (HSC) quiescence is crucial for maintaining lifelong blood production and preventing potentially toxic overproduction of blood cells. HSC are capable of re-entering quiescence following exposure to pro-inflammatory stimuli such as interleukin (IL)-1, implying the existence of one or more 'braking' mechanisms that limit and/or overcome the mitogenic properties of these signals to limit HSC cell cycle entry. However, mechanism(s) regulating HSC quiescence during chronic inflammation have yet to be fully elucidated. In the present study, we find that the master myeloid transcription factor PU.1 represses Myc-regulated cell cycle and protein synthesis pathways in HSC during chronic inflammation, thereby guarding HSC quiescence in this context.

To gain insight into HSC cell cycle regulation in the context of chronic inflammatory signaling, we performed cell cycle and RNA-seq analysis on purified HSC from mice injected daily for 20 days with IL-1β. Strikingly, HSC from IL-1-treated mice remained in a quiescent state, and our RNA-seq analyses uncovered a significant decrease in mRNA transcripts from pathways related to cellular proliferation, translation, and ribosome biogenesis. Also, flow cytometry analyses revealed that IL-1 significantly reduced Myc protein expression and ribosomal protein S6 phosphorylation in HSC. Altogether, these data identify an inflammation-induced cell cycle restriction gene program that limits HSC proliferation in response to inflammation.

Strikingly, ingenuity pathway analysis (IPA) predicted PU.1as a possible driver of the cell cycle restriction gene program. Indeed, PU.1 mRNA and protein levels in HSC increased significantly in PU.1-eYFP knockin reporter mice treated with IL-1. Furthermore, fractionating HSC based on reporter levels showed that IL-1-exposed HSC expressing high PU.1 levels activated the cell cycle restriction program more robustly than HSC with lower PU.1 levels or from untreated control mice. Along these lines, cell cycle analyses showed that PU.1-high HSC were quiescent following IL-1 exposure. Surprisingly, functional assays revealed that the PU.1-high fraction became enriched for functional HSC following IL-1 exposure, in contrast to a primarily PU.1-low phenotype under control conditions. Collectively, these data show that low Myc levels, a quiescent cell cycle state and functional HSC identity are all associated with high PU.1 levels during chronic inflammation.

To address the requirement for PU.1, we compared HSC following IL-1 exposure from wild-type (WT) and PU.1-knockin (KI) mice, which express ~30% of normal PU.1 levels due to a point mutation in the PU.1 upstream regulatory element (URE). Strikingly, IL-1 induced aberrant HSC expansion in the BM of PU.1 KI mice, underwritten by IL-1-induced quiescence loss in PU.1 KI HSC. Excess cell cycle activity in HSC from IL-1-treated PU.1 KI mice was associated with Myc overexpression, hyperinduction of cycle and protein synthesis genes, and an increased protein synthesis rate. Interestingly, IL-1-treated PU.1 KI mice also exhibited exuberant platelet production and aberrant activation of megakaryocyte (Mk) lineage programs in HSC. Taken together, these data show PU.1 is required to limit HSC proliferation and cell cycle activity, and that failure to upregulate PU.1 during chronic inflammation leads to aberrant HSC expansion and dysregulated Mk lineage output.

Altogether, our data identify PU.1 as a key enforcer of HSC dormancy, and as a regulator of HSC proliferation and proteostasis during chronic inflammation. As PU.1 has been shown to slow cell cycle entry to promote myeloid differentiation in actively proliferating cells, our results suggest a mechanistic conservation in which inflammation induces PU.1 expression and engages a similar cell cycle restriction program that preserves HSC quiescence. Such a mechanism can prevent excessive Mk lineage output leading to thrombosis, and/or HSC exhaustion. Importantly, these findings re-cast inflammation-induced PU.1 expression as a guardian of blood system function, rather than a pathogenic by-product. As PU.1 serves a tumor suppressor role, these findings also may provide insight into the link between inflammation and leukemogenesis, particularly in contexts where PU.1 function is impaired in leukemic stem cells (LSC) due to mutation or indirect mechanism(s).

Disclosures

No relevant conflicts of interest to declare.