In the battle of microbes and mammalian cells, the fight or flight (die) decision is emerging as a central factor contributing to resistance to infection. Since the work of Bradley and Metcalf in the 1960s on colony-stimulating factors that regulate hematopoiesis [1,2], we have gained a deep understanding of soluble factors and downstream signaling pathways controlling hematopoiesis at steady state and following infection. Changes in the induction of inflammatory cytokines and hematopoietic growth factors in response to microbes have been intensively studied as a mechanism to explain abnormal responses to infection and the development of immune suppression. Despite this effort, few immunological studies explain the common occurrence of multilineage suppression of hematopoiesis in patients with life-threatening systemic infections.
A hematological hallmark of septic shock patients is peripheral blood cytopenia . This persistent cytopenia commonly affects myeloid, lymphoid, and erythroid lineages, resulting in immunosuppression, and is a key prognostic indicator for survival. The Pediatric Acute Lung Injury and Sepsis Investigators network revealed that nonsurvivors of ‘influenza’ infection appear to have defective emergency hematopoiesis and were therefore profoundly pancytopenic and had frequently developed a bacterial superinfection . However, a convincing mechanism to explain this failure of emergency hematopoiesis has not been proposed. Numerous viral and bacterial pathogens, including HIV, lymphocytic choriomeningitis virus, cytomegalovirus, human herpesvirus-6, human herpesvirus-7, vaccinia virus (VACV), Bartonella, and parvovirus B19, are known to infect hematopoietic stem progenitor cells (HSPCs) and in some cases, remain dormant in HSPCs [5–12]. Recently, it was revealed that abortive HIV infection of T cells induces a caspase-1-dependent cell death, known as pyroptosis [13,14]. HIV infection can infect hematopoietic progenitor cells and induce cytopenia, and numerous studies demonstrate that infection of CD34+ HSPCs with HIV induces cell death and impairs reconstitution in humanized mouse models [6,11,15–17].
One possibility to explain defects in emergency hematopoiesis during systemic infection is the inappropriate activation of cell death, a hypothesis proposed by Hotchkiss et al.[18,19] in 1999 using data collected from mice and humans. Alternatively, suppression of HSPC proliferation, differentiation, and self-renewal can also explain these clinical syndromes. Recent findings demonstrate that hematopoietic progenitor cells drive hematopoiesis at steady state, rather than long-term hematopoietic stem cells (HSC), suggesting that the response of the progenitor cell compartment to intracellular infection and inflammatory cytokines may be central to an effective immune response [20▪,21▪].
Since 1972, apoptotic and necrotic cell death has dominated the literature as two forms of cell death with distinct effects on the immune system . The discovery of genes regulating apoptosis, most notably B-cell lymphoma 2 (Bcl-2) , has driven major scientific and clinical advances in the field of cell death. Alternative nonapoptotic modes of programmed cell death have been recently recognized to exist, including pyroptosis – a caspase-1-dependent cell death – and necroptosis – a receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL)-dependent, caspase-independent cell death (Fig. 1).
There are a multitude of intracellular proteins acting as cellular sentinels that monitor for signs of infection. When triggered, they move swiftly to induce the release of inflammatory cytokines and/or to induce an inflammatory form of cell death, both of which can drive emergency hematopoiesis. During pyroptosis or necroptosis, emergency hematopoiesis can be potently influenced by the programmed release of inflammatory cytokines. The release of host-derived damage-associated molecular patterns (DAMPs) such as mitochondrial DNA  and high-mobility group protein B1 (HMGB1)  further induces cytokine production and influences emergency hematopoiesis [26▪▪,27] (Fig. 2). These forms of cell death contrast to the immunologically-silent apoptotic forms of cell death . How cells choose the fight or die option during infection remains enigmatic: is it a binary switch controlling both cytokine production and nonapoptotic cell death? Or does this depend on the cell type and the pathway recruited? What are the crucial intracellular targets of these cell death pathways that culminate in the demise of the cell? And what are the specific DAMPs that activate the surrounding immune cells to drive inflammation and emergency hematopoiesis? Here, we will focus on the role of inflammatory cell death including pyroptosis and necroptosis as key mechanisms controlling emergency hematopoiesis. We will discuss recent advances which demonstrate that nonapoptotic inflammatory cell death can regulate emergency hematopoiesis.
DEFINING THE FORMS OF INFLAMMATORY CELL DEATH
The Nomenclature Committee on Cell Death has prepared guidelines to define different forms of cell death, including intrinsic and extrinsic apoptosis, mitotic catastrophe, and necroptosis [29▪]. Here, we will extend the definitions of pyroptosis to include recent data and to acknowledge the significant differences in morphology of pyroptotic cells .
The dependence on RIPK3 and the pseudo-kinase MLKL provides the best definition of necroptosis . The common use of necrostatin-1 (Nec-1) and necrostatin-1s – chemical inhibitors of RIPK1 – to define necroptotic death can be useful; however, RIPK1-independent, RIPK3-dependent forms of death are now well recognized [32▪▪–34▪▪]. Furthermore Nec-1 may inhibit inflammatory cell responses that are not linked to cell death [35,36▪▪]. Like apoptosis, where a multitude of inputs and cellular stresses can trigger the apoptotic cascade, necroptosis can be engaged via numerous upstream pathways. These include the ligation of death receptors, Toll-like receptors (TLRs), and intracellular receptors such as DNA-dependent activator of interferon-regulatory factors (DAI) [37–39] (Fig. 1). Negative regulators are also now well defined: RIPK1, caspase-8, cellular FADD-like IL-1β-converting enzyme-inhibitory protein (cFLIP), and Fas-associated protein with Death Domain (FADD) all play key roles in restricting the activity of the necroptotic pathway [32▪▪–34▪▪,40,41▪,42,43]. Combined, these upstream regulators form an important arm of the innate immune system, acting as sentinels for pathogens or other pathophysiological processes that aim to subvert the apoptotic machinery.
Pyroptosis is defined by the dependence on caspase-1 or caspase-11 in mice and either caspase-1 or caspase-4 in humans [30,44]. Caspase-1 activation is dependent on the formation of a macromolecular complex, termed the inflammasome. Numerous inputs and cellular stresses can trigger the formation of the inflammasome complex, often via the adaptor protein apoptosis associated speck-like protein containing a CARD (ASC), which bridges the interaction of the ‘sensing’ proteins [nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), retinoic acid-inducible gene 1 (RIG-1), absent in melanoma 2 (AIM2)] and caspase-1, leading to auto-cleavage and activation of caspase-1 . Pyroptosis induces inflammation via the release of the active form of the pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, and IL-18  (Fig. 1). Necroptosis can induce inflammation and drive emergency hematopoiesis by releasing IL-1α, IL-33, and damage-associated host factors  (Fig. 1 and Fig. 2). Here, we will consider scenarios where these processes are subverted by pathogens and abnormalities in host biochemical pathways.
APOPTOSIS, NECROPTOSIS, AND PYROPTOSIS CROSS-TALK
An emerging theme in the field of inflammatory cell death is that the apoptotic and nonapoptotic cell death pathways communicate extensively. Vince et al. demonstrate that when inhibitor of apoptosis proteins (IAPs) are depleted, RIPK3 activation triggers caspase-8 and NLRP3/caspase-1 activation, leading to reactive oxygen species production and IL-1β secretion independently of RIPK3 kinase activity and MLKL [49▪]. Interestingly, cIAPs were shown to regulate cytokine production and myelopoiesis in a RIPK1 and RIPK3-dependent manner that was independent of necroptosis [36▪▪].
Dimerization of RIPK1 and RIPK3 activates cell death; however, the precise pathway taken to cell death remains unclear. Evidence suggests that RIPK3 can drive MLKL-dependent necroptosis or caspase-1-dependent pyroptosis. Mutations affecting the kinase domain of RIPK3 unexpectedly induce caspase-8-dependent lethality, but not MLKL-mediated cell death [50▪]. Subsequent studies showed that the mutation likely induced a conformational change in RIPK3, exposing the receptor interacting protein homotypic interaction motif (RHIM) domain rather than inhibiting the kinase activity per se[51▪]. Cook et al. propose that the availability of substrates, namely caspase-8, MLKL, and FADD, determine the outcome to RIPK1 and RIPK3 dimerization. A RIPK3 construct that can be induced to dimerize was used to show that the kinase domain of RIPK3 drives MLKL-mediated necroptosis in the absence of RIPK1, caspase-8, and FADD. In contrast, in the absence of MLKL, dimerized RIPK3 induces caspase-8-dependent apoptosis, and cleavage of caspase-3 and poly (ADP ribose) polymerase. This process is enhanced by RIPK1 and occurs independently of RIPK3 kinase function. These studies have revealed significant interactions between pyroptosis, necroptosis, and caspase-8-dependent apoptosis. How these interactions alter the morphological and biochemical changes associated with cell death, the kinetics of cell death, and the pathophysiological outcome of inflammatory cell death awaits further study.
CONTROVERSIAL KILLING BY MIXED LINEAGE KINASE DOMAIN-LIKE PROTEIN
MLKL has recently emerged as a central player in the execution of RIPK3-dependent necroptotic death, but its precise role in this cell death pathway is highly controversial . Phosphorylation of the activation loop of MLKL induces a conformational change disrupting an auto-inhibitory interaction between the pseudokinase domain and the four-helix bundle. This promotes an interaction with phosphatidylinositol phosphates promoting membrane localization [53,54▪]. It is controversial whether this membrane localization and pore formation represents the end of this pathway , or if additional players such as phosphoglycerate mutase family member 5 (PGAM5), the mitochondrial fission factor Drp1, or the transient receptor potential melastatin-related 7 (TRPM7) exist downstream to induce mitochondrial fragmentation and calcium current across the plasma membrane to kill the cell [55–57]. Future in-vivo studies using cells deficient in these proteins in parallel with RIPK3-deficient cells will be helpful to determine the specific nature of RIPK3-dependent cell death.
INFLAMMATORY CELL DEATH DRIVES EMERGENCY HEMATOPOIESIS
The archetypal example of a nonapoptotic form of cell death driving emergency hematopoiesis comes from work on caspase-1 and IL-1β. IL-1β, the most commonly studied product of the pyroptotic cell death pathway, is a potent inducer of granulocyte colony-stimulating factor (G-CSF) and IL-6, both of which drive granulopoiesis  (Fig. 2). The other major cytokine processed by active caspase-1 – IL-18  – can induce interferon (IFN)γ, which is known to regulate HSC self-renewal, repopulation, and proliferation during infection and aplastic anemia [60–62] (Fig. 2).
Interleukin-1α – a necroptotic  and pyroptotic DAMP  – can also influence G-CSF and IL-6 production to drive emergency hematopoiesis (Fig. 2). IL-33, which is an inflammatory DAMP released during necroptosis [34▪▪], can influence emergency hematopoiesis and eosinophil production. IL-33 can induce HSPC mobilization in a CCR2-dependent manner to fight fungal infection [64▪]. IL-33 also promotes IL-5 production and thereby can cause systemic eosinophilia in vivo (Fig. 2). Eosinophils are now known to express cell surface receptors, paired immunoglobulin-like receptor (PIR)-A and PIR-B, which modulate eosinophil cell death activation to enable IL-5-mediated expansion, demonstrating the complexity of cell death pathways operating in discrete cell types during emergency hematopoiesis [66▪]. IL-33 also promotes the differentiation of bone marrow lineage-Sca1+ c-Kit-CD25+ cells to type 2 innate lymphoid cells (ILC2) . The number of natural ILC2 (nILC2) also increases following IL-33 treatment in vivo, and these cells play key roles during Helminth infection [68▪].
NEGATIVE REGULATION OF EMERGENCY HEMATOPOIESIS BY INFLAMMATORY CELL DEATH
Two scenarios exist that can account for effects of nonapoptotic inflammatory cell death on emergency hematopoiesis: first, an indirect effect of chronic inflammation on HSPC activity; and secondly, direct killing of HSPCs (Fig. 2).
Indirect negative feedback of emergency hematopoiesis
Hematopoietic growth factors are induced to high levels during infection and they promote emergency hematopoiesis. Several inflammatory cytokines counteract the actions of hematopoietic growth factors to perturb hematopoiesis. Type I interferon drastically reduces the number of hematopoietic progenitor cells following lymphocytic choriomeningitis virus infection . Its mechanism of action is not understood, but may act by licensing these cells to undergo nonapoptotic cell death [33▪▪,69–72,73▪,74] or they may interfere with the proliferation and differentiation of HSPCs . IL-1β production during pyroptosis may interfere with emergency hematopoiesis because treatment of mice with recombinant IL-1 receptor antagonist supports hematopoiesis and reduces mortality following chemotherapy [76,77]. Consistently, G-CSF treatment increases the expression of the IL-1R antagonist and supports engraftment of donor HSC .
Another factor released from pyroptotic cells – IL-18  – synergizes with IL-12 to up-regulate IFNγ which perturbs HSC proliferation, differentiation, and self-renewal at steady state, during infection with Mycobacterium avium or Ehrlichia muris[60,61], and when chronically expressed can lead to aplastic anemia  (Fig. 2). IFNγ pretreatment of HSPCs also reduces the engraftment potential of donor cells, suggesting that these are cell-intrinsic changes .
Damage-associated molecular patterns are released during pyroptosis and necroptosis, and they have diverse effects on hematopoiesis. For example, HMGB1 and mitochondrial DNA are potent inducers of type I interferon, and they have the capacity to negatively regulate emergency hematopoiesis [24,25,80]. IL-33, also considered a DAMP, can antagonize eosinophil production by inducing GM-CSF production, in contrast to its positive role in IL-5 production  (Fig. 2). Additional studies will be required to establish the physiological role of IL-33 in the regulation of eosinophil production via its effects on IL-5 and GM-CSF.
Direct negative feedback
Killing of HSPCs is hypothesized to be an efficient means of removing infected cells, thereby eliminating the risk of spread to progeny cells . Both pyroptotic and necroptotic machinery exists in HSPCs, and both pathways have now been shown to have the capability to kill HSPCs. NLRP1-dependent caspase-1-dependent pyroptosis can kill HSPCs during hematopoietic stress induced by viral infection or chemotherapy, causing cytopenia, immunosuppression, and bone marrow failure  (Fig. 2). Recently, it was shown that deletion of caspase-1/11 improved the survival of neonatal mice following bacterial challenge. This reduction in mortality was associated with elevated numbers of HSC in the bone marrow and spleen [81▪▪]. Likewise, RIPK3-dependent necroptotic cell death limits the ‘self-renewal’ capacity of RIPK1-deficient long-term HSC in lethally-irradiated recipient mice in a tumor necrosis factor (TNF)-dependent process, suggesting that the necroptotic machinery can exert selective pressure on long-term HSC [34▪▪,82▪▪] (Fig. 2).
TARGETING CELL DEATH TO MODULATE EMERGENCY HEMATOPOIESIS
Pharmacological inhibition of pyroptosis and necroptosis may have therapeutic value in diverse clinical settings including infection, auto-immunity, and hematopoietic stress. For example, an NLRP3-specific inhibitor has been shown to protect against experimental autoimmune encephalomyelitis and murine neonatal lethality caused by mutations equivalent to human cryopyrin-associated periodic syndrome . Caspase-1 and caspase-4 inhibitors have not been tested in the clinic in the setting of infection, but extensive mouse data suggest that inhibitors will be beneficial for patients with systemic inflammatory disease and infection-triggered cytopenias [59,81▪▪,84,85].
Genetic or pharmacological inhibition of the necroptotic machinery, namely RIPK1 and RIPK3, alleviates cerulean-induced pancreatitis [86,87], TNF-induced inflammation in mice [35,50▪,88,89], atherosclerosis in Apoe and Ldlr mutant mice , retinal degeneration [91,92], ischemia-reperfusion injury of the kidneys [93,94], myocardial infarction , steatohepatitis, and hepatic injury induced by ethanol [96–98]. Furthermore, necroptosis was first described to play a role in viral defense during VACV infection, a virus that can inhibit apoptosis [99,100] and pyroptosis . Chan et al. showed that RIPK1 was required for TNF-induced necroptosis of VACV-infected cells. Subsequently, VACV-induced necroptosis was shown to require RIPK3 . Treatment with the RIPK1 inhibitor, Nec-1, or the human MLKL inhibitor, NSA, inhibits the cytopathic effects of the HIV-1 virus . HSV-1 and HSV-2 induce a RIPK3/MLKL-dependent death of mouse embryonic fibroblasts, and RIPK3-deficient mice are susceptible to HSV-1 and display high viral titers [105,106]. In contrast, work in human cell lines demonstrates that the ICP6 and ICP10 proteins from HSV-1 and HSV-2, respectively, can inhibit TNF-induced necroptosis, indicating cell type and possibly species-specific differences in necroptosis pathways [106,107]. Exacerbations in necroptosis and pyroptosis have therefore been demonstrated in a variety of mouse models, but their contribution to human disease is only now being tested in clinical trials for chronic inflammatory disease.
The lethality caused by loss of RIPK1 and the normal development of RIPK3-deficient mice suggested that RIPK3 would be ideal for drug development. However, mice with a RIPK3 D161N mutation display increased caspase-8 and RIPK1 activity leading to apoptosis. Compounds targeting the kinase activity of RIPK3 can at high concentrations also induce apoptosis by promoting a conformational change in RIPK3 that drives RHIM interactions with RIPK1 and activation of caspase-8 [51▪]. Mutations of other residues of RIPK3 (D161G, D143N, K51A) inhibit necroptosis in response to TLR3, TLR4, TNFR1, DAI, and IFNβ, but do not trigger the lethality seen in the RIPK3 D161N mutants [51▪]. These data suggest that RIPK3 antagonists may be valuable for inhibition of necroptosis, but are likely to promote induction of apoptosis in some settings, a feature that may be useful in the setting of cancer chemotherapy.
As we now appreciate the complex interplay between the nonapoptotic and apoptotic cell death cascades, and the appearance of discrete features of apoptosis in response to nonapoptotic stimuli, more rigorous genetic, biochemical, and morphological information will be required to characterize, refine, and integrate the many forms of cell death that are now known to exist. Characterization of additional DAMPs generated during nonapoptotic cell death will shed light on the indirect modulators of emergency hematopoiesis. Recent findings have also unveiled nonapoptotic cell death as a key biological process restricting HSPC ‘self-renewal’, and could have clinical implications in the setting of systemic infection, and also for improving the engraftment potential of HSPC in transplantation settings using bone marrow, G-CSF-mobilized peripheral blood stem cells, umbilical cord blood units, and for gene therapy clinical trials.
Financial support and sponsorship
The study was supported by NIH grant 1R01HL124209–01A1 (B.A.C.), and J.S. has an NHMRC fellowship (541901).
Conflicts of interest
There are no conflicts of interest.
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