Fight or Flight - Regulation of Emergency Hematopoiesis by Pyroptosis and Necroptosis

Abstract

Purpose of review

A feature of the innate immune response that is conserved across kingdoms is the induction of cell death. In this review, we discuss the direct and indirect effects of increased inflammatory cell death, including pyroptosis, a caspase-1-dependent cell death, and necroptosis, a RIPK3/MLKL-dependent, caspase-independent cell death, on emergency hematopoiesis.

Recent findings

Activation of non-apoptotic cell death pathways during infection can trigger release of cytokines and/or damage-associated molecular patterns (DAMPs) such as IL-1α, IL-1β, IL-18, IL-33, HMGB1 and mtDNA to promote emergency hematopoiesis. During systemic infection, pyroptosis and necroptosis can directly kill hematopoietic stem and progenitor cells, which results in impaired hematopoiesis, cytopenia and immunosuppression. Although originally described as discrete entities, there now appears to be more intimate connections between the non-apoptotic and death receptor signaling pathways.

Summary

The choice to undergo pyroptotic and necroptotic cell death constitutes a rapid response system serving to eliminate infected cells, including hematopoietic stem and progenitor cells. This system has the potential to be detrimental to emergency hematopoiesis during severe infection. We discuss the potential of pharmacological intervention for the pyroptosis and necroptosis pathways that may be beneficial during periods of infection and emergency hematopoiesis.

Introduction

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 1960’s 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 has 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 multi-lineage suppression of hematopoiesis in patients with life-threatening systemic infections.

A hematological hallmark of septic shock patients is peripheral blood cytopenia [3]. 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 (PALISI) network revealed that non-survivors of influenza infection appear to have defective emergency hematopoiesis and were therefore profoundly pan-cytopenic and had frequently developed a bacterial superinfection [4]. However, a convincing mechanism to explain this failure of emergency hematopoiesis has not been proposed. Numerous viral and bacterial pathogens, including HIV, LCMV, cytomegalovirus, human herpesvirus-6, human herpesvirus-7, vaccinia virus (VACV), Bartonella and parvovirus B19 are known to infect hematopoietic stem and progenitor cells (HSPC) and in some cases, remain dormant in HSPC [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+ HSPC 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 and colleagues in 1999 using data collected from mice and humans [18,19]. Alternatively, suppression of hematopoietic stem and progenitor cell proliferation, differentiation and self-renewal can also explain these clinical syndromes. Recent findings demonstrating that hematopoietic progenitor cells drive hematopoiesis at steady state, rather than long-term HSC, suggest 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 [22]. The discovery of genes regulating apoptosis, most notably Bcl-2 [23], has driven major scientific and clinical advances in the field of cell death. Alternative non-apoptotic modes of programmed cell death have been recently recognized to exist, including pyroptosis, a caspase-1-dependent cell death, and necroptosis, a RIPK3/MLKL-dependent, caspase-independent cell death (Figure 1).

Fig. 1. Apoptosis and the inflammatory cell death pathways, pyroptosis and necroptosis.

Non-apoptotic cell death pathways can be triggered by numerous factors including inflammasome activation, death receptor ligation and intracellular pathogens. When caspase activity is blocked, TLR ligation and a cytoplasmic complex comprising RIPK1 can drive necroptotic death. Phosphorylation and oligomerization of RIPK3 activates necroptosis by inducing phosphorylation of MLKL and its translocation to the cell membrane. The cellular contents released from necroptotic cells can serve as DAMPs to further induce inflammation. Pyroptosis is induced by the formation of the inflammasome complex to induce caspase-1 activation and IL-1β and IL-18 maturation. Pathogen clearance and inflammation must be balanced to prevent a chronic inflammatory response.

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 [24] and HMGB1 [25] further induces cytokine production and influences emergency hematopoiesis [26••,27] (Figure 2). These forms of cell death contrast to the immunologically-silent apoptotic forms of cell death [28]. How cells choose the fight or die option during infection remains enigmatic: is it a binary switch controlling both cytokine production and non-apoptotic cell death? Or does this depend on the cell type and 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 that demonstrate that non-apoptotic inflammatory cell death can regulate emergency hematopoiesis.

Fig. 2. Direct and indirect effects of pyroptosis and necroptosis on emergency hematopoiesis.

The non-apoptotic and inflammatory forms of cell death, pyroptosis and necroptosis, have direct and indirect effects on emergency hematopoiesis. These modalities of cell death release inflammatory cytokines and DAMPs that stimulate emergency hematopoiesis. In chronic settings, including prolonged periods of infectious stress that may be characterized by secondary infection or systemic infection, these inflammatory factors may impair hematopoiesis. Pyroptosis and necroptosis can also exert a strong influence on hematopoiesis by directly killing HSPC.

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 [30].

The dependence on receptor interacting protein kinase 3 (RIPK3) and the pseudo-kinase mixed lineage kinase domain-like protein (MLKL) provides the best definition of necroptosis [31]. 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••,33••,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 DAI [37–39] (Figure 1). Negative regulators are also now well defined: RIPK1, caspase-8, cFLIP and 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 ASC, which bridges the interaction of the ‘sensing’ proteins (NLRs, RIG, AIM2) and caspase-1, leading to auto-cleavage and activation of caspase-1 [45]. Pyroptosis induces inflammation via the release of the active form of the pro-inflammatory cytokines IL-1α, IL-1β and IL-18 [46] (Figure 1). Necroptosis can induce inflammation and drive emergency hematopoiesis by releasing IL-1α, IL-33 and damage-associated host factors [47] (Figure 1 and Figure 2). Here we will consider scenarios where these processes are subverted by pathogens and abnormalities in host biochemical pathways.

Apoptosis, necroptosis and pyroptosis crosstalk

An emerging theme in the field of inflammatory cell death is that the apoptotic and non-apoptotic cell death pathways communicate extensively. Vince and colleagues, demonstrate that when inhibitor of apoptosis proteins (IAPs) are depleted, RIPK3 activation triggers caspase-8 and NLRP3/Caspase-1 activation leading to ROS production and IL-1β secretion independently of RIPK3 kinase activity [48] and MLKL [49•]. Interestingly cIAPs where 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 RHIM domain rather than inhibiting the kinase activity per se [51•]. Cook and colleagues propose that the availability of substrates, namely caspase-8, MLKL and FADD, determine the outcome to RIPK1 and RIPK3 dimerization [52]. 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 PARP. 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 MLKL

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 [31]. 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 if this membrane localization and pore formation represents the end of this pathway [55], or if additional players such as PGAM5, the mitochondrial fission factor Drp1, or 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 non-apoptotic 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 [58] (Figure 2). The other major cytokine processed by active caspase-1, IL-18, [59••] can induce IFNγ which is known to regulate HSC self-renewal, repopulation and proliferation during infection and aplastic anemia [60–62] (Figure 2).

IL-1α, a necroptotic [47] and pyroptotic DAMP [63], can also influence G-CSF and IL-6 production to drive emergency hematopoiesis (Figure 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 [65] (Figure 2). Eosinophils are now known to express cell surface receptors, PIR-A and PIR-B, that modulate eosinophil cell death activation to enable IL-5-mediated expansion, demonstrating the complexity of cell death pathways operating 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) cells [67]. 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 non-apoptotic inflammatory cell death on emergency hematopoiesis: the first, an indirect effect of chronic inflammation on HSPC activity; and secondly, direct killing of HSPC (Figure 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 LCMV infection [5]. Its mechanism of action is not understood but may act by licensing these cells to undergo non-apoptotic cell death [33,69–72,73•,74] or they may interfere with the proliferation and differentiation of HSPC [75]. 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 hematopoietic stem cells [78].

Another factor released from pyroptotic cells, IL-18 [59], synergizes with IL-12 to upregulate 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 [62] (Figure 2). IFNγ pretreatment of HSPC also reduces the engraftment potential of donor cells suggesting that these are cell-intrinsic changes [79].

DAMPs are released during pyroptosis and necroptosis, and they have diverse effects on hematopoiesis. For example, HMGB1 and mtDNA 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 antagonise eosinophil production by inducing GM-CSF production, in contrast to its positive role in IL-5 production [65] (Figure 2). Additional studies will be required to establish the physiological role of IL-33 in regulation of eosinophil production via its effects on IL-5 and GM-CSF.

Direct negative feedback

Killing of hematopoietic stem and progenitor cells is hypothesized to be an efficient means of removing infected cells, thereby eliminating the risk of spread to progeny cells [30]. Both pyroptotic and necroptotic machinery exists in HSPC and both pathways have now been shown to have the capability to kill HSPC. NLRP1-dependent caspase-1-dependent pyroptosis can kill HSPC during hematopoietic stress induced by viral infection or chemotherapy, causing cytopenia, immunosuppression and bone marrow failure [59] (Figure 2). Recently it was shown that deletion of caspase1/11 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 LT-HSC in lethally-irradiated recipient mice in a TNF-dependent process suggesting that the necroptotic machinery can exert selective pressure on LT-HSC [34,82••] (Figure 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 (EAE) and murine neonatal lethality caused by mutations equivalent to human cryopyrin-associated periodic syndrome (CAPS) [83]. Caspase-1 and caspase-4 inhibitors have not been tested in the clinic in the setting of infection but extensive mouse data suggests 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 [90], retinal degeneration [91,92], ischemia-reperfusion injury of the kidneys [93,94], myocardial infarction [95], 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 [101]. Chan et al showed that RIPK1 was required for TNF-induced necroptosis of VACV-infected cells [102]. Subsequently VACV-induced necroptosis was shown to require RIPK3 [103]. Treatment with the RIPK1 inhibitor, Nec-1, or the human MLKL inhibitor NSA inhibits the cytopathic effects of the HIV-1 virus [104]. 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.

Conclusion

As we now appreciate the complex interplay between the non-apoptotic and apoptotic cell death cascades, and the appearance of discrete features of apoptosis in response to non-apoptotic 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 non-apoptotic cell death will shed light on the indirect modulators of emergency hematopoiesis. Recent findings have also unveiled non-apoptotic 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.

Key points.

1. Pyroptosis and necroptosis are non-apoptotic inflammatory forms of death

2. Pyroptosis and necroptosis can kill HSC and progenitor cells

3. Apoptotic and non-apoptotic pathways cross-talk

4. Systemic activation of cell death can inhibit hematopoiesis

Abbreviations

ASC

apoptosis associated speck-like protein containing a CARD

DAMPs

damage-associated molecular patterns

G-CSF

granulocyte colony-stimulating factor

GM-CSF

granulocyte/macrophage colony-stimulating factor

HMGB1

high-mobility group protein B1

HSC

hematopoietic stem cells

HSPC

hematopoietic stem and progenitor cells

IAPs

inhibitor of apoptosis proteins

IL

interleukin

ILC2

type-2 innate lymphoid cells

LT-HSC

long term HSC

MLKL

mixed lineage kinase domain-like protein

mtDNA

mitochondrial DNA

Nec-1

necrostatin-1

NLRP

nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein

NLRs

nucleotide-binding domain and leucine-rich repeat containing gene family

RHIM

receptor interacting protein homotypic interaction motif

RIPK

receptor interacting protein kinase

ROS

reactive oxygen species

TLRs

Toll-like receptors

TNFα

Tumor necrosis factor alpha