Regulation of Stress Induced Hematopoiesis

Abstract

Purpose of this review:

The hematopoietic compartment is tasked with the establishment and maintenance of the entire blood program in steady-state and in response to stress. Key to this process are hematopoietic stem cells (HSCs), which possess the unique ability to self-renew and differentiate to replenish blood cells throughout an organism’s lifetime. Though tightly regulated, the hematopoietic system is vulnerable to both intrinsic and extrinsic factors that influence hematopoietic stem and progenitor cell (HSPC) fate. Here, we review recent advances in our understanding of hematopoietic regulation under stress conditions such as inflammation, aging, mitochondrial defects and damage to DNA or endoplasmic reticulum.

Recent findings:

Recent studies have illustrated the vast mechanisms involved in regulating stress-induced hematopoiesis, including cytokine-mediated lineage bias, gene signature changes in aged HSCs associated with chronic inflammation, the impact of clonal hematopoiesis and stress tolerance, characterization of the HSPC response to endoplasmic reticulum stress and of several epigenetic regulators that influence HSPC response to cell cycle stress.

Summary:

Several key recent findings have deepened our understanding of stress hematopoiesis. These studies will advance our abilities to reduce the impact of stress in disease and aging through clinical interventions to treat stress related outcomes.

Introduction

The development and maintenance of a productive hematopoietic system depends on a complex network of regulatory processes. Central to this program are HSCs, which self-renew to maintain the stem cell population and differentiate to produce a series of progenitor and precursor populations, ultimately giving rise to all mature hematopoietic lineages. Various cells produced through hematopoiesis perform a multitude of roles for the body. Red blood cells oxygenate the entire body system, myeloid-derived and lymphoid-derived white blood cells provide host defense mechanisms through the innate and adaptive immune responses, and platelets promote blood clotting and tissue repair following vessel injury. The hematopoietic compartment is tightly regulated to maintain homeostasis and promote these processes when needed, thus preserving a careful balance between HSC maintenance and differentiation under steady-state conditions.

During stress, normally quiescent HSCs and progenitor cells receive coordinated signals dictating the hematopoietic response to the given stressor. Infection, inflammation, metabolic stress, and hematopoietic regeneration all require unique, specific signaling signatures to elicit the appropriate stress response. The hematopoietic compartment is capable of identifying and responding to these signals in an elegant and organized manner. In this review, we highlight the most recent publications on stress hematopoiesis and highlight newly uncovered mechanisms that regulate this process.

Topic 1: Regulation of inflammation stress induced hematopoiesis

The identification of inflammatory signaling pathways as critical regulators of hematopoiesis, and specifically HSCs, has become a significant area of research in the stress hematopoiesis field [1–3]. Inflammatory signaling pathways regulate many aspects of hematopoiesis, including embryonic development, emergency granulopoiesis, and age-associated hematopoietic defects. Several recent publications have identified new mechanisms for regulation of inflammatory signaling in HSPCs.

During an innate immune response to pathogens, innate immune effector cells recognize pathogen-associated molecular patterns (PAMPs) directly through pattern recognition receptors (PRRs) [4]. For HSPCs, these signaling mechanisms may serve a different purpose. The NOD-like receptor (NLR) is a PRR that activates the inflammasome, a multimeric protein complex that cleaves and activates the cytokines interleukin-1β (IL-1β) and IL-18, and promotes the induction of pyroptosis through caspase activation [5]. Using zebrafish and mouse models, the Molero laboratory recently reported an evolutionary-conserved signaling pathway linking inflammasomes with HSPC fate decision, where inflammasomes can refine protein quantities of the lineage-specific transcription factor, GATA-1[6**]. Their work suggests that inflammasomes activate caspases capable of cleaving GATA-1 and inactivating it, promoting myeloid cell fate at the expense of erythropoiesis.

Skewed differentiation towards the myeloid lineage from HSPCs is a frequent stress-induced hematopoietic response [1, 7]. Cytokines are essential in regulating hematopoiesis and promoting myeloid-biased differentiation. Previous work on proinflammatory cytokine TNF-α suggest its complicated role in hematopoiesis [8–11], and an essential function in HSC specification [12]. Recently, the Passegué laboratory clarified the role of TNF-α in HSCs as a pro-survival and pro-regeneration factor. TNF-α-induced p65-NF-κB activity protected HSCs from necroptosis-mediated elimination and triggered a strong regenerative program, resulting in emergency myelopoiesis via upregulation of lineage-specific transcription factor PU.1 [13**]. Similarly, Etzrodt et al. reported TNF-α as the principal PU.1 inducing signal in HSCs [14], while the chromatin remodeling protein, Phf6, was recently identified as a modulator of TNF-α signaling [15]. Using zebrafish models of IL-6 pathway deficiencies, Tie et al. showed the necessity of IL-6 signaling for embryonic HSC specification. Myeloid cells from both the early primitive wave of hematopoiesis and the later definitive erythro-myeloid progenitors (EMPs) were revealed as major sources of IL-6. They also found that Notch1 activates IL-6R by regulating its expression in the hemogenic endothelium and HSCs [16*]. These studies demonstrate the impact of cytokines in regulating hematopoiesis and suggest the potential for unintended consequences to the hematopoietic system associated with use of TNF-α and IL-6 inhibitors in treating inflammatory diseases.

HSCs must maintain a balancing act between two inflammatory signaling extremes. On one hand, fully activated HSCs expressing little NF-κB have low regeneration potential [13**]; conversely, increased NF-κB activity is associated with decreased quiescence, rapid expansion of progenitor cells, and impaired long-term engraftment upon transplantation [17**,18**,19] (Figure 2). Ramalingam et al. showed that sustained inflammation in murine BM adversely impacted hematopoiesis via chronic activation of endothelial MAPK and NF-κB signaling. Donor BM cells from a competitive transplant displayed proliferative stress and decreased functionality. The resulting proinflammatory cytokines expressed in the BM niche drove myeloid-biased differentiation in HSCs at the expense of self-renewal [18**]. Similarly, the Scadden laboratory recently identified changes in BM stromal cells under stress conditions using protein expression mapping. Hematopoietic recovery from radiation stress appears to not rely on LeptinR+ or Nestin+ cells for niche cytokine expression, but rather a CD73+CD105-/NGFR^high stromal cell population, which express cytokines under homeostasis and have increased expression following irradiation [20*]. A redundant requirement for the PI3K isoforms p110α and p110δ was identified by the Gritsman laboratory, highlighting the importance of the PI3K pathway in responding to stress response and cytokine signals [21]. Baumgartner et al. found that activation of the PI3K and MAPK pathways results in feedback phosphorylation of MEK1, which balances the pathway activation and maintenance of HSC quiescence [22*]. These studies underscore the versatile role of cytokine signaling and inflammatory stress in regulating cell fate and hematopoiesis lineage bias.

Figure 2:

HSCs with low expressions levels of NF-κB maintain high quiescence, while high NF-κB expression is associated with decreased quiescence and differentiation.

Topic 2: Hematopoietic stress and aging

Several recent publications elucidate the emerging relationship between stress, inflammation, and aging, and their impacts on the hematopoietic system [23–26]. The rare, quiescent population of LT-HSCs contributes to blood homeostasis under different stressors. Aging permanently changes responses, functions, and regenerative ability of LT-HSCs, showing myeloid-biased hematopoietic output and poor response to infections [27**,28,29**,30*]. Age-related changes are attributed to heterogeneity within the LT-HSC compartment and a shift in the genetic landscape. Using scRNA-seq, Mann et al. identified and isolated a myeloid-biased LT-HSC (mLT-HSC) subset prevalent among aged mice. mLT-HSCs were shown to regulate myeloid versus lymphoid balance under inflammatory challenge, as an increased frequency of this subset in the aged BM was a key driver of myelopoiesis [30*]. Work from Montecino-Rodriguez et al. gives evidence that although myeloid skewing is observed in aging, it is not due to the lack of lymphoid potential in aged HSCs, but rather the production of cytokines that promote a myeloid fate [31]. To further support this, Helbling, et al. identified increased inflammatory signaling in stromal BM niche cells during aging [32].

Additionally, several studies have focused on elucidating gene signature changes in aged HSCs associated with enhanced NF-κB signaling. Through regulation of chromatin accessibility, NF-κB mediated inflammatory signaling via Rad21/cohesin activation remains elevated in old versus young mice, and contributes to the increased likelihood of HSCs to undergo inflammation-induced loss of self-renewal and differentiation toward multipotent hematopoietic progenitors (MPPs) [33]. Moreover, a gene set enrichment analysis on young and aged HSCs revealed older mice had a greater enrichment for HSC-specific TNF-α signature genes [13**].

Utilizing a short hairpin RNA-mediated screen, the Trowbridge laboratory uncovered the novel role of a histone acetyltransferase and epigenetic regulator, Kat6b, in aging. A decrease in Kat6b was observed in aging LT-HSCs, which promoted myeloid differentiation at the expense of erythroid differentiation and enhanced expression of aging- and inflammation-associated gene signatures [29**]. Szade et al. identified the cell-extrinsic factor heme-oxygenase 1 (HO-1), a heme-degrading enzyme, as an anti-aging molecule [34]. In young HO-1-deficient mice, LT-HSCs were phenotypically similar to aged wild type (WT) mice, having poor regulation of pyruvate metabolism and glycolysis, and altered expression of cell cycle genes. Transplanting HO-1-deficient LT-HSCs to a wild-type niche restored their functionality. With expansion of current knowledge regarding cross talk between inflammatory pathways and physiological aging, the possibility of manipulating both cell-intrinsic and -extrinsic factors to rescue lineage differentiation and ultimately improve immune function in age-related blood disorders such as myeloid leukemia emerges.

As with most aging cells, HSCs accumulate somatic mutations that are mainly inconsequential. However, some mutations can confer a fitness advantage on a cell, allowing for clonal hematopoiesis of indeterminate potential (CHIP). Common mutations in CHIP include the genes TP53 and DNMT3A. Chen et al. uncovered a novel mechanism by which mutant p53 drives clonal hematopoiesis [27**]. Following radiation-induced stress, p53 mutants exhibited selective HSPC expansion and a competitive advantage post-transplant, through an interaction with the epigenetic regulator EZH2 to increase levels of H3K27me3 on genes that regulate HSPC self-renewal and differentiation. Pharmacological inhibition of EZH2 decreased repopulation potential of p53 mutant HSPCs, suggesting this pathway can be therapeutically targeted to prevent CHIP progression. Chronic inflammatory insult also may serve as an environmental trigger contributing to clonal evolution of CHIP [35]. Through sequencing blood samples from ulcerative colitis patients, Zhang et al. found that the inflammatory environment promoted the distinct CHIP-associated mutations of epigenetic regulators DNMT3A and PPM1D. This research provides insight into the varying cellular stressors that shape the hematopoietic system by selecting for HSCs with mutations that enhance stress tolerance.

Topic 3: Regulation of mitochondrial stress induced hematopoiesis

Quiescent HSCs maintain low metabolic activity. When transitioning to a state of proliferation, a critical checkpoint involves surveillance of mitochondrial health [36–38]. When this checkpoint is impaired, the mitochondrial stress results in loss of stemness. Luo et al. investigated the effects of mitochondrial stress in the functional decline of HSCs. With age, the reduced expression of Sirt2, a cytosolic NAD+ dependent deacetylase, and increased mitochondrial stress leads to aberrant activation of the NLRP3 inflammasome [39]. This results in reduced HSC survival and functionality, but the authors suggest that Sirt2 overexpression can reverse the functional decline of HSC aging, increasing engraftment potential and reconstitution capacity. In related work, the Navieras group uncovered a role for the NAD+-booster nicotinamide riboside (NR) in hematopoiesis. NR reduces levels of mitochondrial activity in HSCs by activation of autophagy and increased mitochondrial clearance which results in increased asymmetrical proliferation without HSC exhaustion [40*]. These results have exciting implications for hematopoietic recovery through modulation of mitochondrial stress.

Under metabolic stress, autophagy is a molecular mechanism that HSCs employ to maintain their integrity. Autophagy is a highly conserved, lysosome-dependent degradation pathway that serves as internal quality control by removing old or damaged proteins and organelles [28, 41]. Previously, Ho et al. established autophagy as one of the essential gatekeepers of HSC quiescence, which is vital in aged HSCs [28]. Lee et al. described sustained effects of obesity-induced oxidative stress in HSCs and MPPs. They found that intracellular reactive oxygen species (ROS) were a driver for HSC dysregulation in obesity due to the concurrent upregulation of transcription factor Gfi1 in obese mice. This created skewed differentiation toward granulocyte-macrophage progenitors that remained even when the stresses associated with obesity were relieved after weight loss or transplantation into a normal recipient [42*]. Similarly, Jung et al. demonstrated that loss of the key autophagy protein, Atg5, resulted in impaired autophagy and, therefore, increased cellular metabolic stress. Accumulation of dysfunctional mitochondria inhibited cellular function and developmental impairment of progenitor cells [43*]. These works elucidate the impact of mitochondrial damage on the viability of the HSCs and deepens our understanding of the various ways HSC defects arise under stress (Figure 3).

Figure 3:

The roles of aging, obesity and loss at Atg5 in HSC function and survival.

Topic 4: Regulation of DNA damage stress induced hematopoiesis

LT-HSCs are rarely actively cycling, but under stress conditions they enter the cell cycle to restore the HSC pool [44–46]. This tightly regulated process involves several recently elucidated factors. Absence of checkpoint kinase 1 (CHK1) in hematopoietic stem and progenitor cells results in accumulation of DNA damage and activation of caspase-dependent mitochondrial apoptosis [47]. A study by the Mechali group showed that DNA repair deficient Mcm8 and Mcm9 mice had unstable DNA replication forks leading to a development of chronic DNA damage in the BM and hematopoietic defects. Here, DNA damage created myeloid-bias differentiation and increased the risk for myeloid tumors, as was evident by the dysplasia of all three myeloid lineages and development of myeloid tumors in mice [48]. These publications highlight the ability of HSCs to monitor and limit DNA damage, highlighting this important mechanism for maintenance of stem cell integrity.

Topic 5: Regulation of cell cycle stress induced hematopoiesis

Regulation of several transcriptional and epigenetic pathways contributes to hematopoiesis through influencing HSC cell cycle progression. The transcription factor Oct1 plays a role in recruitment of chromatin remodeling complexes and serves to protect blood progenitor cells from chemotoxic stress [49*]. The polymerase associated factor 1 complex, Paf1c, is essential for cell cycle progression and survival of HSPCs. Saha et al. determined that loss of cdc73, a core component of Paf1c, leads to cell cycle defects in HSPCs, increased cell death, and extensive BM failure [50]. Loss of chromatin remodeling protein and transcriptional repressor, nuclear receptor corepressor 1 (Ncor1) resulted in LT-HSCs with decreased quiescence and increased proliferation activity. Though important for maintenance of the HSC pool, Ncor1 appeared to be dispensable for HSC self-renewal [51].

Shapiro et al. established the multifunctional nuclear protein NKAP as a cell cycle regulator and critical for limiting cellular stress that can trigger cell cycle withdrawal or apoptosis [52]. Moreover, the adapter protein Cables1 was revealed to be an intrinsic cell-cycle inhibitor of hematopoietic progenitor cells in young mice. Cables1-deficient cells underwent hyperproliferation, a defect reproduced upon transplantation into WT recipient mice. Perhaps, enhanced proliferation of Cables1−/− HSCs operates preferentially during hematopoietic stress, as aged Cables1−/− mice had increased mobilization of progenitor cells, reduction of the HSC compartment, and altered peripheral blood counts [53*]. Collectively, these data highlight our recent understanding of the molecular machinery that orchestrates cell cycle regulation of HSPCs.

Topic 6: Regulation of endoplasmic reticulum stress

Several recent publications have identified the importance of the HSPC response to endoplasmic reticulum stress [54–57]. In the event of an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, a stress response is triggered through the unfolded protein response (UPR) mechanism. There are three mechanisms for UPR, inositol-requiring enzyme 1α (IRE1α encoded by ERN1), pancreatic eIF2α kinase (PERK) and activating transcription factor 6 (ATF6). Activation of these ER stress responses either restores the balance of protein homeostasis, by constraining protein synthesis and increasing protein folding and degradation, or triggers apoptosis if a cell is deemed unrepairable. Work from Hidalgo San Jose, et al shows how uniquely sensitive HSCs are to misfolded protein levels [58*]. Liu et al. uncovered a role for the IRE1α pathway in responding to ER stress [59], which had previously been shown to be downregulated in the HSC UPR response [57]. This illustrates the complex nature of the UPR and highlights the importance of this pathway for HSC maintenance.

Linking UPR, autophagy, and the ATF4 mediated integrated stress response, Xie et al. identify the importance of sphingolipid composition in HSC homeostasis and how alterations in composition can simultaneously activate all of these stress response pathways in HSPCs [60]. Leveau et al. highlights the importance of Ttc7a in regulation of HSC self-renewal and ability to regulate the stress response as Ttc7a-deficient HSCs have an increased capacity to replenish the hematopoietic system following serial transplants and higher proliferative capacity upon induced ER stress[61]. Together, these publications highlight HSPC sensitivity to the protein and lipid composition and how this contributes to stress hematopoiesis.

Conclusions and future directions

Hematopoietic stem cells have the ability to dynamically respond to adverse conditions in the BM niche including inflammation, aging, epigenetic changes, challenges from ROS and damage to DNA or proteins to ensure survival. These changes result in a spectrum of stress responses from subtle to profound shifts in quiescence and self-renewal of HSCs and alterations of HSC differentiation. The activation of myeloid-bias differentiation in young and geriatric HSCs is frequently driven by specific cytokine production. The intersection between aging and inflammation depicts the consequences of what can occur when the balance is tipped towards enhanced inflammatory signaling, such as a predisposition to myeloid leukemia in older age. An intriguing discovery regarding the heterogeneity within the LT-HSC compartment is the presence HSC subset markers, with an age-dependent myeloid bias and frequency, that become apparent upon inflammatory stimuli [30]. Both CD61 and Neogenin-1 (Neo-1) were recently identified as markers for mLT-HSCs, as LT-HSCs expressing these surface proteins also showed skewing towards myelopoiesis [30, 62]. Similarly, EPCR+/CD34- provides superior enrichment for LT-HSC activity relative to the total SLAM fraction following IL-1 exposure [63]. These findings advance our current strategies to identify HSCs under non homeostatic conditions and suggest a continued need to refine the characterization of the heterogeneity in the HSPC compartment.

Several groups have applied computational modeling to study clonal hematopoiesis [64], hematopoietic commitment [65–68], and treatment strategies for hematological disorders [69, 70]. Klose et al. developed a novel computational model of HSC response in circumstances of normal aging and homeostatic conditions, as well as stress and physiological aging [71*]. This mathematical model approach can be applied to quantify a variety of biological phenomena in a unifying context and has tremendous implications for understanding stress hematopoiesis.

Further characterization of factors and pathways that directly affect proliferation and differentiation of HSCs will lend us a clearer picture of the signaling network involved in blood formation under stress. Taken together, this review highlights the progress in the field towards unraveling the programs associated with HSC maintenance during several stress related challenges. This offers the opportunity for development of new therapies that restore HSC function and regeneration, while minimizing the consequences for healthy hematopoietic stem cells in the process.

Figure 1:

Graphic summary of several newly proposed mechanisms for regulation of inflammatory signaling in HSPCs.

Key Points.

• Under stress, the cytokines IL-6 and TNF-α instruct a myeloid-bias lineage choice

• Hematopoietic stem cell self-renewal and differentiation output is heavily influenced by the ability to balance inflammatory levels

• HSC gene expression modulated by several transcription factors and epigenetic regulators impacts cell cycle progression and cellular stress

• Long term consequences of increased mitochondrial stress result in HSC dysfunction such as loss of quiescence, cellular exhaustion and skewed differentiation

• The HSC response to ER challenge is multifaceted and can be advantageous depending on the cellular demand and intensity of the stress

• Chronic inflammatory stress contributes to the increased likelihood of loss of self-renewal and differentiation in aged HSCs