Emergency granulopoiesis.

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Nature Reviews Immunology | AOP, published online 22 April 2014; doi:10.1038/nri3660

Emergency granulopoiesis Markus G. Manz and Steffen Boettcher

Abstract | Neutrophils are a key cell type of the innate immune system. They are short-lived and need to be continuously generated in steady-state conditions from haematopoietic stem and progenitor cells in the bone marrow to ensure their immediate availability for the containment of invading pathogens. However, if microbial infection cannot be controlled locally, and consequently develops into a life-threatening condition, neutrophils are used up in large quantities and the haematopoietic system has to rapidly adapt to the increased demand by switching from steady-state to emergency granulopoiesis. This involves the markedly increased de novo production of neutrophils, which results from enhanced myeloid precursor cell proliferation in the bone marrow. In this Review, we discuss the molecular and cellular events that regulate emergency granulopoiesis, a process that is crucial for host survival. Granulocytes The generic term for neutrophils, eosinophils and basophils, which have in common a granule-rich cytoplasm. Neutrophils make up the vast majority (~90%) of granulocytes in peripheral blood.

Neutrophilia A relative or absolute increase in the number of neutrophils in the blood.

Neutrophil extracellular traps (NETs). These are mainly composed of DNA that is released from neutrophils upon pathogen encounter. NETs have microbicidal activity and bind to pathogens, thereby preventing pathogen dissemination.

Division of Hematology, University Hospital Zurich, Raemistrasse 100, CH‑8091 Zurich, Switzerland. Correspondence to M.G.M. e‑mail: [email protected] doi:10.1038/nri3660 Published online 22 April 2014

The haematopoietic system is hierarchically organized with haematopoietic stem cells (HSCs) residing at the ‘top’, from which all cell types of the haematopoietic system are derived (FIG. 1). HSC divisions can result in self-renewal or differentiation into multipotent and lineage-committed haematopoietic progenitor cells (HPCs) that lack or have limited self-renewal capacity. Although HSCs divide relatively infrequently in steadystate conditions, this basic organization of the bloodforming system enables large-scale cellular amplification to occur downstream of HSCs. Through multiple rounds of cell division and cellular specification events, HSCs give rise to all of the blood cell lineages that are required for the proper function of an organism throughout its lifetime1. Given the postmitotic nature of the majority of mature haematopoietic cells, combined with their relatively short half-life (which has been reported to range from a few hours to a few days in the case of neutrophils2), mature blood cells need to be constantly generated from upstream precursors. The cellular turnover resulting from these processes is enormous, with approximately 0.5–1 × 1011 granulocytes being generated each day during steady-state conditions in an adult human3. Of note, the haematopoietic system is capable of rapid adaptation to haematopoietic stress, such as bleeding or severe infection, by increasing cellular output several-fold above steady-state levels in order to meet the higher demand for the respective blood cell type. Severe systemic bacterial infection induces a characteristic haematopoietic response programme known as ‘emergency granulopoiesis’ that can be regarded as the prime example of demand-adapted haematopoiesis (FIG. 2).

In contrast to less severe bacterial infection that is locally contained by innate immune effector mechanisms and does not cause systemic alterations, emergency granulo poiesis is well recognized in clinical settings by systemic signs such as blood leukocytosis, neutrophilia, and the appearance of immature neutrophil precursor cells in the peripheral blood (known as left-shift), which are only present in the bone marrow during physiological steady-state conditions. At the cellular level, the response to bacterial infection involves neutrophil recruitment and extravasation into the infected tissues4. Although the bidirectional migration of neutrophils is possible — enabling them to re‑enter the vasculature5–7 — the majority of neutrophils undergo cell death in the inflamed tissue as a consequence of their antibacterial effector functions, such as phagocytosis, degranulation and the release of neutrophil extracellular traps (NETs)4. Failure to control an infection locally leads to systemic bacterial dissemination, resulting in further neutrophil ‘consumption’. Thus, to counterbalance neutrophil depletion and to meet the enormous demand for neutrophils during infection, steady-state granulopoiesis is switched to emergency granulopoiesis, which is characterized by considerably enhanced de novo generation of neutrophils, accelerated cellular turnover and the release of immature and mature neutrophils from the bone marrow into the peripheral blood. Emergency granulopoiesis is probably a crucial determinant of survival during severe systemic infection, as congenital8 or iatrogenic9 forms of neutropenia — in which individuals lack the capacity to respond to infection with emergency granulopoiesis — are associated

NATURE REVIEWS | IMMUNOLOGY

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LTHSC Haematopoietic stem cells (HSCs)

Extensive self-renewal

ITHSC STHSC

Haematopoietic progenitor cells (HPCs)

CMP MEP

Mature haematopoietic cells Platelets

MPP GMP

Limited self-renewal Extensive proliferation

LMPP CDP

Monocyte Macrophage

CLP

NK cell

NKT cell

Granulocyte

No self-renewal (except for B cells, T cells and some macrophages and DCs) Limited proliferation

DC Erythrocyte

B cell

Inﬂammatory DC

T cell

Lymphoid lineages

Myeloid lineages

Figure 1 | The haematopoietic hierarchy. The haematopoietic system is maintained by self-renewing haematopoietic stem cells (HSCs), which can be subdivided based on their temporal ability to sustain the whole spectrum of mature blood cell lineages — long-term, intermediate-term and short-term haematopoietic stem cells (LT-,Reviews IT- and ST‑HSCs, Nature | Immunology respectively). HSCs give rise to multiple types of haematopoietic progenitor cells (HPC), which have an enormous proliferative potential but only very limited (if any) self-renewal capability, and thus these cells need to be continuously replenished from the HSC pool. Recently, it has been recognized that the original concept of step-wise commitment (from HSCs to multipotent, oligopotent and eventually unipotent lineage-restricted progenitors, with an early dichotomous irreversible decision-making step between the myeloid and lymphoid lineages) may not be absolute and is probably an oversimplification. Instead, several pathways — sometimes bypassing developmental checkpoints that were previously thought to be mandatory — probably exist, although their relative importance in steady-state conditions and in response to haematopoietic challenge still needs to be determined. Nevertheless, a large number of clonal HPC subsets can be isolated that have an important function in vivo, such as multipotent progenitors (MPPs), lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte–macrophage progenitors (GMPs), megakaryocyte–erythrocyte progenitors (MEPs) and common dendritic progenitors (CDPs). Finally, the largest pool of haematopoietic cells is made up of the billions of mature blood cells of different lineages that, with some exceptions (certain B cell and T cell subsets, and tissue-resident macrophages), do not self-renew and thus need to be regenerated from the pools of upstream HSCs and HPCs. DC, dendritic cell; NK cell, natural killer cell; NKT cell, natural killer T cell.

with a considerable lethality from complications relating to infection. In this Review, we discuss the cellular and molecular mechanisms that regulate emergency granulopoiesis.

Definition of emergency granulopoiesis Here, we define emergency granulopoiesis as the wellorchestrated de novo generation of neutrophils that results from increased myeloid progenitor cell proliferation in the bone marrow in response to systemically disseminated infection. The overall goal is to enhance neutrophil output in order to meet the higher demand for neutrophils during the innate response to severe infection when these cells are consumed in large quantities. In addition to systemic infection, pathological conditions

— such as myeloablation following exposure to ionizing radiation or chemotherapeutic agents — can also trigger compensatory mechanisms with the aim of re-establishing haematopoietic homeostasis that share some of the features of emergency granulopoiesis. However, there are marked differences between such iatrogenic conditions and naturally occurring ‘emergency’ conditions, such as systemic infection, which has exerted a strong evolutionary pressure. In this Review, we focus on emergency granulopoiesis that occurs as a response to infection and inflammation. However, we also discuss studies in which the experimental approach did not fulfil the most stringent definition of emergency granulopoiesis but where the findings did provide important insights into pathways that are essential to this process.

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REVIEWS a Local bacterial infection

b Systemic bacterial infection Macrophage

Bacteria

Tissue Neutrophil

Blood

Endothelium

Bone Emergency granulopoiesis

Bone marrow Steady-state granulopoiesis

HSC

Lymphopoiesis Lymphopoiesis

Osteoblast

Figure 2 | Local versus systemic bacterial infection. a | During a localized bacterial infection in an immunocompetent Nature Reviews Immunology individual, several pre-existing antimicrobial effector mechanisms contain bacterial pathogens locally. These| include, for example, the barrier function of the body’s cell surfaces, the presence of mucus that contains antimicrobial peptides on epithelial cell layers (for reasons of simplicity not shown in the figure), tissue-resident immune cells such as macrophages, and the recruitment of neutrophils to the site of bacterial infection. In most cases, this leads to a rapid resolution of the infection, thereby preventing bacterial dissemination. As a consequence, bone marrow granulopoiesis is unaffected and does not differ from steady-state conditions. b | By contrast, in the setting of severe bacterial infection that overwhelms first-line defence mechanisms, bacterial dissemination occurs and neutrophils are consumed in large quantities, with pre-existing neutrophil pools being used (such as marginalized vascular pools; not shown in the figure). To counterbalance neutrophil depletion and to provide a supply of urgently needed neutrophils to combat systemic bacterial spread, a haematopoietic response programme termed ‘emergency granulopoiesis’ is initiated, which is characterized by the large-scale de novo generation of neutrophils from myeloid progenitors in the bone marrow. The expansion of bone marrow granulopoiesis is paralleled by a decrease in bone marrow lymphopoiesis. HSC, haematopoietic stem cell.

The emergency granulopoiesis cascade represents a regulatory loop that can be dissected into three phases. First, the presence of a pathogen needs to be effectively and rapidly sensed to alert the immune system and haematopoietic system of the emergency state. Second, the emergency state has to be translated into the molecular events that are required to stimulate enhanced neutrophil production in the bone marrow. Third, emergency granulopoiesis needs to be restrained to enable the haematopoietic system to restore homeostatic steadystate conditions after an infection has been cleared, as excessive neutrophil production might have detrimental effects such as the development of inflammatory diseases, as indicated by animal studies10. It should be emphasized that a successful neutrophil response to infection requires the complex interplay between various cascades, of which emergency granulo poiesis (the de novo production of neutrophils) is but one

component. Other components include the regulation of neutrophil egress from the bone marrow (as well as from marginalized vascular pools and from haemato–lymphatic organs), neutrophil recruitment to the site of infection and the regulation of neutrophil effector functions. These aspects are not within the scope of this Review and have been reviewed recently in this journal4.

Pathogen sensing Sensing the presence of a systemically disseminated invading pathogen is the crucial first step in the emergency granulopoiesis cascade. There are functional prerequisites for the cell type (or cell types) that could fulfil this essential function. First, the cell needs to have access to the pathogen, such that it is likely to encounter the microorganism or microbial components at the time of systemic spread. Second, the cell has to be equipped with the molecular machinery required to detect pathogens



REVIEWS Indirect activation

Direct activation

Systemically disseminated bacteria Pathogen sensing

PAMPs Bone marrow

Peripheral tissues PAMP

PAMPs

TLR Molecular translation

Haematopoietic cells

Non-haematopoietic cells

Direct pathogen sensing, molecular translation and initiation of emergency granulopoiesis

Cytokines

Cytokines Haematopoietic stem and progenitor cells Initiation Emergency granulopoiesis

Figure 3 | Direct and indirect pathways for the activation of emergency granulopoiesis. Theoretically, both direct and indirect mechanisms for the activation of emergency granulopoiesis might exist. In both cases, the switch from Naturereceptors Reviews |(PRRs), Immunology steady-state to emergency granulopoiesis requires pathogen sensing through pattern recognition such as Toll-like receptors (TLRs), followed by molecular translation and the initiation of emergency granulopoiesis. Upon systemic dissemination of bacteria, pathogen sensing could occur either in peripheral tissues or in the bone marrow where haematopoiesis takes place. Both haematopoietic cells and non-haematopoietic cells, in either peripheral tissues or bone marrow, might be the cellular sensor of pathogens as both cellular compartments express the molecular machinery that is required for this purpose (that is, they express PRRs such as TLRs). As a consequence, granulopoietic cytokines (such as granulocyte colony-stimulating factor) are released, either from the sensing cells themselves or by other cell types, and their signalling activates emergency granulopoiesis. This sequence of events represents indirect activation, as the cell type that senses the presence of a pathogen is not the cell type that generates granulocytic offspring. However, haematopoietic progenitor cells also express PRRs such as TLRs and thus might directly sense systemically spreading pathogens, resulting in enhanced proliferation and granulocytic differentiation both in an autocrine cytokine-dependent manner and in a cytokine-independent manner. It is probable that these processes are highly connected and function together. PAMPs, pathogen-associated molecular patterns.

and to translate pathogen sensing into emergency granulopoiesis. To this end, pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), detect different classes of conserved pathogen-associated molecular patterns (PAMPs) that are present on the exterior and interior of microorganisms, and signalling downstream of these receptors results in the expression of genes that are required for the host response to infection. Theoretically, there are two main ways to activate the haematopoietic system and to consequently initiate emergency granulopoiesis: either directly or indirectly (FIG. 3). The concept of indirect activation of the haematopoietic system depends on the existence of a specialized cell type that is present in the peripheral tissues or the bone marrow (the primary site of haematopoiesis) and can function as a microbial sensor. This cell type does not generate neutro phil offspring itself but stimulates myeloid progenitor cell proliferation and granulocytic differentiation indirectly through the direct or indirect release of granulopoietic cytokines, such as the master regulator granulocyte colony-stimulating factor (G‑CSF). According to the model of direct activation, HSPCs (haematopoietic stem and progenitor cells), such as myeloid progenitors, can sense microorganisms through the expression of PRRs that, upon ligation, stimulate proliferation and differentiation into neutrophils. We discuss below the experimental evidence for each of these models.

Indirect activation: sensing by mature haematopoietic cells. Strong support for a model of indirect activation of haematopoiesis during systemic infection comes from studies showing that granulopoietic cytokines and growth factors — such as G‑CSF, granulocyte–macrophage colony-stimulating factor (GM‑CSF), and the early acting cytokines interleukin‑3 (IL‑3), IL‑6 and FMS-like tyrosine kinase 3 (FLT3) ligand — reach serum concentrations of up to 100‑fold above steady-state levels in both mice and humans11–18. Together with the abundance of data showing that these cytokines stimulate HSPC proliferation and granulocytic differentiation in vitro and in vivo (discussed below), a crucial role for indirect growth factor-mediated stimulation of emergency granulopoiesis is well established. But what is the identity of the pathogen-sensing cell type? A prevailing hypothesis is that emergency granulopoiesis is initiated indirectly following pathogen sensing by monocytes and tissue-resident macrophages19,20. Indeed, monocytes and macrophages would be ideal cell types for this role; blood monocytes patrol the circulation and macrophages are present in substantial quantities in almost all tissues throughout the body. Most importantly, in addition to their phagocytic and antimicrobial activities, monocytes and macrophages express the molecular machinery — including PRRs and granulopoietic cytokines (such as G‑CSF, GM‑CSF, macrophage colony-stimulating factor



REVIEWS (M‑CSF) and IL‑6) — that is thought to be required for pathogen recognition and subsequent translation into an emergency granulopoiesis response21–24. However, this assumed role for monocytes and macrophages has not been directly proven by rigorous in vivo experimentation.

Mesenchymal stromal cells A heterogeneous group of stromal cells found in various tissues that is composed of cells with the developmental potential to generate bone, cartilage and adipose tissue (also historically known as mesenchymal stem cells). Mesenchymal stromal cells in the bone marrow are crucial constituents of the haematopoietic microenvironment, which is often termed the ‘haematopoietic stem cell niche’.

Parabiosis An experimental model system in which two animals (most often mice) are surgically joined to establish a common circulation.

Indirect activation: sensing by non-haematopoietic cells. Although PRR expression and function has been best described in haematopoietic cells, there is accumulating evidence to indicate that various non-haematopoietic cell types express PRRs and thus can participate in immune responses. For example, it has been shown that TLR4 expression by bladder epithelial cells is required for the protective immune response to uropathogenic Escherichia coli 25. Furthermore, allergic asthma that is induced by house dust mites requires TLR4‑mediated stimulation of non-haematopoietic structural airway cells26. Also, TLR4 expression by endothelial cells is crucial for the efficient recruitment of neutrophils to sites of infection27,28. Moreover, various studies have shown TLR expression on mesenchymal stromal cells29–32, which indicates that these cells might have a regulatory role during immune responses. In fact, one recent study has shown that bone marrow mesenchymal progenitor cells induce monocyte emigration from the bone marrow in response to TLR agonist stimulation33. To address the uncertainty described above regarding the identity of the cell type that might sense infection and subsequently translate this into emergency granulopoiesis, reciprocal bone marrow chimeric animals were generated in which TLR4 expression is restricted to either the haematopoietic or the non-haematopoietic system. As macrophages are able to self-renew locally within tissues34 and might survive the conditioning regimen in such chimeric mice, tissue-resident macrophage chimerism was analysed; the vast majority of tissue-resident macrophages were shown to be donor derived 3 months after lethal irradiation35, a finding that has been confirmed by other studies34. Moreover, macrophages are dispensable for the induction of emergency granulopoiesis as liposomal clodronate-treated (and consequently macrophagedepleted) wild-type mice can launch an emergency granulopoiesis response that is indistinguishable from that of control mice35. When TLR4 reciprocal bone marrow chimeric mice were injected with lipopolysaccharide (LPS), TLR4 expression by haematopoietic cells was surprisingly found to be dispensable for the stimulation of LPSinduced emergency granulopoiesis. By contrast, TLR4 expression by non-haematopoietic cells was an absolute requirement for the initiation of emergency granulopoiesis35. Preliminary data from mice with a cell type-specific deletion of myeloid differentiation primary response protein 88 (Myd88) suggest that endothelial cells might be a non-haematopoietic cell type that is important in this process (S.B. and M.G.M., unpublished observations). Direct activation: sensing by HSPCs. Recent studies have indicated that HSPCs themselves express PRRs, such as TLRs36,37. What are the functional implications of TLR expression by HSPCs in the context of emergency granulopoiesis? One obvious suggestion would be that

PRR ligation on HSPCs stimulates their survival, proliferation, differentiation and migration, and thereby contributes to the overall outcome of the immune response. Indeed, it has been shown that in vitro stimulation with TLR2 and TLR4 agonists leads to cytokine-independent proliferation and myeloid cell differentiation of mouse HSPCs37. Similarly, TLRs are expressed on human CD34+ HSPCs38–41 and in vitro TLR ligation promotes myeloid differentiation of these cells at the expense of lymphopoiesis39–41. Moreover, parabiosis experiments have shown that a small proportion of HSCs constantly leaves the bone marrow, circulates in peripheral blood and re‑enters the bone marrow 42. Although the physio logical importance of this phenomenon is not clear, it is tempting to speculate that it relates to the patrolling of tissues for the presence of pathogens. Upon encountering microorganisms, circulating HSPCs could give rise to haematopoietic offspring directly at the site of the infection, thereby being closely involved in fighting infection and promoting tissue repair in peripheral organs. Indeed, ex vivo LPS pre-stimulation followed by continued in vivo LPS stimulation of HSPCs that were implanted under the kidney capsule has been shown to lead to the generation of clusters of myeloid cells within the kidney 43. Furthermore, our group has recently shown that bone marrow common dendritic progenitors (CDPs)44 express TLRs36. In vitro TLR agonist stimulation of CDPs results in downregulation of the expression of CXC-chemokine receptor 4 (CXCR4), which is involved in bone marrow retention, and concomitant upregulation of the expression of the lymph node-homing receptor CC-chemokine receptor 7 (CCR7). When TLR agonist pre-stimulated CDPs were adoptively transferred in mice, they were preferentially recruited to TLR agonist-rich inflamed lymph nodes, where CDPs gave rise to DCs without alterations in lineage commitment or proliferative capacity 36. Interestingly, in a recent study it was reported that TLR agonist-treated early HSPCs can secrete various haematopoietic growth factors, including IL‑6, at substantial levels45. Under defined conditions of in vivo myelotoxic treatment with either 5‑fluorouracil (5‑FU) or ionizing radiation, these HSPC-derived growth factors can contribute to enhanced myeloid cell generation45. However, another recent study of acute viral infection showed that myeloid cell differentiation depends on the release of haematopoietic cytokines (including IL‑6) from non-haematopoietic cells46, which resembles the finding that non-haematopoietic cells produce myelopoietic cytokines (including G‑CSF) upon LPS stimulation35. These examples illustrate that, depending on the context, direct pathogen sensing by HSPCs can affect their proliferation, lineage differentiation, migration and ability to release growth factors. In addition to the immediate effects of LPS on HSPC proliferation, lineage differentiation and migration, as described above, we have shown that repeated systemic injection of LPS activates quiescent HSCs and induces self-renewing proliferation47. However, although LPS injection or bacterial infection leads to the expansion of phenotypically defined HSC populations, it simultaneously impairs their repopulation capacity 48–51;



REVIEWS competitive repopulation experiments of wild-type bone marrow cells with Tlr4 −/− or Tlr9 −/− bone marrow cells at a ratio of 1/1 in mixed chimaeras have shown that the TLR-deficient cells dominate in chimeric animals over time52. These findings demonstrate that the effects of emergency granulopoiesis go beyond the haematopoietic progenitor level, reaching all the way up the hierarchy to the HSCs that feed into the progenitor pool. In summary, the available data strongly indicate that HSPCs express PRRs and are thus functionally equipped to sense pathogens37,43. This is likely to have a biological relevance. However, the overall contribution of direct pathogen-sensing mechanisms to emergency granulopoiesis is negligible compared with the indirect mechanisms, at least for the immediate response, as shown by experiments using reciprocal bone marrow chimeric mice in which TLR4 expression is restricted to either haematopoietic or non-haematopoietic tissues35. This is an active area of research and several aspects remain incompletely understood, including the importance of direct pathogen sensing by HSPCs at sites of infection and their local proliferation, as well as the relevance of these processes for long-term HSC homeostasis and the response of HSCs to infection and inflammation.

Molecular translation of pathogen sensing Despite some evidence for a minor growth factorindependent mechanism of myeloid progenitor proliferation and differentiation through direct pathogen sensing by HSPCs, an overwhelming body of evidence unequivocally demonstrates that emergency granulo poiesis is stimulated in an indirect manner through the concerted action of various growth factors. We discuss these cytokine and growth factor axes, their cellular targets and the respective intracellular signalling cascades and transcriptional networks that become activated to stimulate emergency granulopoiesis. Cytokine and growth factor axes. Given the fundamental importance of emergency granulopoiesis in host defence and survival8,9, it is not surprising that multiple redundant cytokine and growth factor pathways are involved in its regulation. Undoubtedly, the best studied of these is the G‑CSF–G‑CSF receptor (G-CSFR) axis. Studies in G‑CSF-deficient (Csf3−/−)53 and G‑CSFR-deficient (Csf3r −/−) mice54 have shown an essential role for G‑CSF in steady-state granulopoiesis. Mice that lack G‑CSF or G‑CSFR have a 70–90% reduction in circulating absolute neutrophil counts53,54. This is due to defects in proliferation and granulocytic differentiation at the level of myeloid progenitors, as well as the enhanced apoptosis of mature neutrophils. However, the role of G‑CSF in the context of emergency granulopoiesis is more controversial. Whereas G‑CSF-deficient mice have a markedly impaired granulopoietic response and a more severe disease course with increased lethality during infection with Listeria monocytogenes compared with wild-type mice53,55, emergency granulopoiesis upon infection with Candida albicans in G‑CSF-deficient mice is indistinguishable from that of wild-type mice56. To our knowledge, these are the only studies that have directly assessed

the role of G‑CSF in microbial infection-induced emergency granulopoiesis. However, it is important to note that neither pathogen is an ideal model to address this issue. From a medical perspective, neither the facultative intracellular Gram-positive bacterium L. monocytogenes nor the yeast C. albicans is as clinically relevant as pathogens such as the large group of Gram-negative enterobacteria (for example, E. coli) or Gram-positive bacteria (such as Staphylococcus aureus or Enterococcus species) that account for the majority of severe systemic infections in hospitalized patients. In further support of an essential role for G‑CSF in emergency granulopoiesis, multiple studies in mice and humans have shown highly upregulated serum levels of various inflammatory cytokines including G‑CSF during severe infection12–18. Furthermore, the exogenous administration of G‑CSF accurately mimics the physiological responses that are observed during emergency granulopoiesis 35. Importantly, Myd88 −/− mice have markedly reduced levels of emergency granulopoiesis following E. coli infection, most probably as a consequence of the inability to sense the infection through MYD88‑dependent TLR signalling and to subsequently increase the production of G‑CSF and other cytokines above steady-state levels (S.B. and M.G.M., unpublished observations). Collectively, the available data strongly indicate that the G‑CSF–G‑CSFR axis has a key role in emergency granulopoiesis, despite evidence for pathogen-specific differences in the contribution of the axis to this process. GM‑CSF is another myeloid cytokine that has been regarded as a major growth factor that is required for the in vivo development of neutrophils, monocytes and macrophages. This is based on the potency of GM-CSF to stimulate the growth of these myeloid lineages in vitro57 and in vivo following overexpression58 or injection59 of the recombinant protein. Although GM‑CSF-deficient (Csf2 −/−) mice have impaired reproductive capacity, develop a lung pathology known as alveolar proteinosis owing to functionally-deficient alveolar macrophages, and have reduced long-term survival, they do not have any major defects in steady-state bone marrow haematopoietic cell development or in peripheral blood counts60–62. However, upon intraperitoneal administration of L. monocytogenes, GM‑CSF-deficient mice fail to control the infection and they develop a severe depletion of bone marrow cells in addition to having decreased neutrophil infiltration into the inflamed tissue55. This response pattern is indicative of an impairment in sustaining an appropriate granulopoietic response in GM‑CSF-deficient mice, which is corroborated by the observation that this defect only becomes evident during the later course of the infection and, importantly, following the administration of high doses of L. monocytogenes. Similarly, in the setting of chronic infection with Mycobacterium avium, GM‑CSF-deficient mice were unable to increase the number of haematopoietic colony-forming cells in the bone marrow compared with wild-type mice63. Together, these data indicate a role for GM‑CSF in neutrophil pro genitor cell homeostasis that only becomes apparent if the haematopoietic system is maximally challenged.



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Permissive model of lineage specification This model proposes that upstream multipotent precursors produce downstream lineage-specific progenitors at a fixed rate that can then be stimulated upon need. Cytokines regulate the proliferation or apoptosis of cells that are already committed to a lineage due to intrinsic developmental programmes.

Instructive model of lineage specification This model proposes that upstream multipotent precursors produce defined lineage-specific progenitors upon specific need. Cytokines trigger a molecular programme in stem and progenitor cells that induces differentiation to a specific lineage.

The observation that neither G‑CSF nor GM‑CSF is absolutely essential for steady-state or emergency granulopoiesis raised the question of whether there might be compensation between these two cytokines. Mice that are deficient in both G‑CSF and GM‑CSF (Csf3−/−Csf2−/− mice)61, as well as mice lacking all three major myeloid growth factors (G‑CSF, GM‑CSF and M‑CSF; Csf3−/−Csf2 −/−Csf1−/− mice)64, still produce neutrophils in steady-state conditions. Moreover, Csf3−/−Csf2 −/− Csf1−/− mice can mount a granulopoietic response in a sterile model of peritonitis64, which shows that even the combined lack of these growth factors does not completely prevent granulopoiesis. Therefore, there might be additional growth factors involved in these processes. On the basis of the observation that IL‑6‑deficient mice have impaired neutrophil responses after C. albicans infection65, mice lacking G‑CSF, GM‑CSF and IL‑6 were generated66. As these mice are difficult to assess directly in vivo, owing to their severely reduced postnatal survival, the authors of this study developed an in vitro assay to study the role of IL‑6 in emergency granulopoiesis. Indeed, in vitro granulopoiesis in the absence of G‑CSF and GM‑CSF was decreased by a further 50% if IL‑6 was also absent. In summary, extrinsic growth factor-mediated pathways are important for both steady-state and emergency granulopoiesis. The major granulopoietic growth factor is G‑CSF and there are further contributions from GM‑CSF and IL‑6. However, despite tremendous research efforts in this field, the additional growth factors that are involved in regulating or in compensating for deficiencies in steady-state and emergency granulopoiesis remain to be identified. Thus, one could speculate that besides the extrinsic control of granulopoiesis, additional intrinsic cytokine-independent pathways might exist that enable basal levels of granulopoiesis to be maintained even in the complete absence of key growth factors. In addition to the classical granulopoietic cytokines described above, several growth factors act on HSPCs to regulate their quiescence, self-renewal, proliferation and differentiation into committed progenitors. Although these growth factors do not directly stimulate emergency granulopoiesis, they are nevertheless important for this process, as myeloid progenitors that produce neutrophils need to be replenished from upstream HSCs. As discussed above, early HSPCs do respond to inflammatory stimuli. However, this emerging aspect of HSC biology has recently been reviewed elsewhere67–69 and is not the main focus of this Review. We discuss selected aspects that are directly connected to emergency granulopoiesis in the following section. Cellular targets of cytokines and growth factors. Receptors for the classical granulopoietic cytokines described above are expressed on various haematopoietic cells. For example, expression of G-CSFR can be robustly detected throughout the myeloid lineage from early HSPCs to mature neutrophils70,71. This reflects the pleiotropic functions of G‑CSF, which, in addition to its effects on myeloid progenitors, also promotes the survival and antimicrobial effector function of postmitotic

mature neutrophils72. Infection or exogenous administration of G‑CSF has been shown to reduce the size of the common myeloid progenitor (CMP) population and simultaneously increase the size of the granulocyte– macrophage progenitor (GMP) population, which, together with the expression pattern of G‑CSFR, strongly supports the idea that G‑CSF primarily acts on the CMP and GMP levels of the haematopoietic hierarchy during emergency granulopoiesis35,73–75. In addition to providing proliferative signals, G‑CSF directly promotes granulocytic lineage specification. Indeed, two opposing models have been proposed to address the long-standing question of how granulo cyte lineage determination is achieved: the permissive model of lineage specification and the instructive model of lineage specification76. Support for the instructive model came from studies showing that enforced expression of cytokine receptors or transcription factors in noncommitted or already committed progenitors can redirect lineage specification77–80. However, definitive proof that cytokines can instruct lineage choice was provided by elegant studies using long-term tracking of single bipotent progenitor cells (GMPs). It was shown that GMPs differentiate into granulocytes or macrophages depending on the presence of either G‑CSF or M‑CSF, respectively 81. In vivo it is probable that multipotent myeloid progenitors are selectively instructed to differentiate into cells of the lineage that is in demand, thereby contributing to emergency granulopoiesis. In summary, early myeloid progenitors provide the primary reservoir for the immediate burst of de novo-generated neutrophils. However, depending on their developmental stage, myeloid progenitors have only transient and very limited (or even no) self-renewal capacity and so they need to be replenished from upstream precursors. Given the hierarchical organization of the haematopoietic system (FIG. 1), all haematopoietic cells are derived from a pool of rare HSCs that constitute approximately 0.001% of total nucleated bone marrow cells82. This raises the question of whether HSCs generate myeloid progenitor offspring at the same constant rate in both steady-state and emergency conditions — with increased neutrophil output being achieved by cytokine-mediated enhanced survival and/or proliferation of myeloid progenitors during infection — or whether early haematopoietic progenitor compartments above the level of CMPs and GMPs (including HSCs) might be directly alerted of the emergency situation and thus increase the rate of myeloid progenitor generation. In fact, there is evidence that increased HSC proliferation and differentiation might have an important role in ensuring a constant supply of lineage-committed progenitors during emergency situations. In this light, immunoregulatory type I interferons (IFNα/β) and type II IFNs (IFNγ) — which are released from many cell types in response to viral or bacterial infection — have been shown to act directly on HSPCs, which express both type I and type II IFN receptors. IFN receptor signalling promotes the entry of quiescent HSCs into the cell cycle, and chronic and excessive activation of this pathway can even result in the exhaustion of stem cell function83–85. However, the functional effect of IFNγ



REVIEWS on HSC cycling is somewhat controversial as another group showed that IFNγ does not stimulate HSC proliferation but rather exerts a strong anti-proliferative effect 86. Similarly, G‑CSF might directly and indirectly stimulate cell cycle entry of HSCs87 and acute myelogenous leukaemia (AML) blasts88, respectively. By contrast, other cytokines, such as M‑CSF, might directly instruct HSCs to differentiate along myeloid cell lineages89. In summary, it is plausible that the regulation of demand-adapted haematopoiesis occurs via feedback loops between the pathways involved in immune system activation and early haematopoiesis, but further investigation is required. Signalling cascades and transcriptional networks. Myeloid specification in early HPCs and subsequent granulocytic differentiation during steady-state conditions require the coordinated temporal activation and function of a large number of myeloid transcription factors — such as runtrelated transcription factor 1 (RUNX1), SCL (also known as TAL1), PU.1, interferon regulatory factor 4 (IRF4), IRF8, lymphoid enhancer-binding factor 1 (LEF1), zinc finger protein GFI1, CCAAT-enhancer-binding protein-α (C/EBPα) and C/EBPε90. Given the frequently observed developmental defects in the respective knockout mice, the role of these myeloid transcription factors in the context of emergency granulopoiesis is less well understood. We discuss here the intracellular signalling cascades and transcriptional networks that are activated during emergency granulopoiesis and that contribute to the stimulation of enhanced neutrophil progenitor proliferation and differentiation. C/EBPα is a key myeloid transcription factor, as shown by the complete lack of neutrophil development in Cebpa−/− mice that occurs as a result of a differentiation block at the transition from multipotent myeloid progenitor to granulocytic progenitor91,92 (FIG. 4a). Blocked neutrophil differentiation partly results from a lack of expression of the C/EBPα target gene Csf3r 91,93, which supports the importance of the G‑CSF–G‑CSFR signalling axis in granulopoiesis. However, it has been shown that this block in neutrophil differentiation can be overcome by in vitro stimulation of Cebpa−/− HPCs with myeloid cytokines, such as IL‑3, GM‑CSF and soluble IL‑6R–IL‑6 complexes93–95. These cytokines promote neutrophil differentiation directly but also indirectly through the upregulation of Csf3r expression, which restores the G‑CSF responsiveness of Cebpa−/− HPCs94. Notably, these studies of exogenous growth factor administration not only demonstrated the existence of C/EBPα-independent neutrophil differentiation pathways but also indicated that these pathways might be important for emergency granulopoiesis, as cytokines such as GM‑CSF and IL‑6 are upregulated following infection. In this light, a seminal study investigated expression levels of other C/EBP family members in early HSPC subsets following cytokine- and C. albicansinduced emergency granulopoiesis74. Whereas the expression of Cebpa, Cebpd and Cebpe is downregulated upon cytokine stimulation, Cebpb showed sustained or upregulated expression in GMPs upon treatment with G‑CSF, GM‑CSF or IL‑3, which indicates that C/EBPβ might have a crucial role during emergency granulopoiesis (FIG. 4b).

Indeed, whereas Cebpa−/− HPCs were still able to generate neutrophils in large numbers following in vitro or in vivo cytokine treatment74,94,95 or in vivo C. albicans infection74, Cebpb−/− mice failed to mount an emergency granulopoietic response74,75,96, although steady-state granulopoiesis was unaffected. In the current model, C/EBPα and C/EBPβ exert specific but opposing functions in steadystate and emergency granulopoiesis, respectively. C/EBPα functions as the master transcriptional regulator of steady-state granulopoiesis by promoting neutrophil differentiation and simultaneously restricting cell cycle progression of myeloid progenitors through inhibition of expression of cyclin-dependent kinase 2 (Cdk2) and Cdk4 (REF. 97), and of Myc 98, thereby ensuring the homeostasis of neutrophil numbers in the steady state. By contrast, C/EBPβ functions as the main transcriptional regulator for emergency granulopoiesis as it does not inhibit Cdk2 and Cdk4, and does not repress Myc, which enables accelerated cell cycle progression in neutrophil progenitors and consequently an increased neutrophil output during emergency conditions74. The model described above provides an explanation of how emergency granulopoiesis is regulated at the transcriptional level. However, what are the signalling pathways that induce the switch from C/EBPα-dependent steady-state granulopoiesis to C/EBPβ-dependent emergency granulopoiesis? G‑CSF-induced signal transduction occurs via Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathways that involve STAT1, STAT3 and STAT5. Importantly, STAT3 has been shown to be crucial for granulopoiesis72,99, and indeed STAT3 links the external stimulus of G‑CSF with the major transcriptional regulator of emergency granulopoiesis C/EBPβ73. Upon STAT3‑mediated G‑CSFinduced signalling, both STAT3 and C/EBPβ directly bind to the proximal Myc promoter, thereby driving expression of the gene directly, as well as indirectly by suppression of C/EBPα binding to the promoter 73. As a net result, C/EBPβ–promoter interactions outweigh C/EBPα–promoter interactions, which leads to enforced Myc expression and consequently to increased cell cycle progression in myeloid progenitors. STAT3 also directly promotes the expression of Cebpb73 (FIG. 4b). As outlined above, emergency granulopoiesis can also be stimulated to a lesser extent by GM-CSF 55. The GM‑CSF receptor preferentially signals through STAT5A and STAT5B 100,101 (hereafter referred to as STAT5A/B). STAT5A/B‑deficient mice have markedly reduced steady-state neutrophil numbers. Importantly, GM‑CSF-mediated neutrophil survival and neutrophil differentiation from GMPs are abrogated in STAT5A/B‑ deficient mice 102. Although not formally proven in the setting of infection, these results are in line with the assumption that GM‑CSF-induced signalling via STAT5A/B can contribute to emergency granulopoiesis. In summary, the availability of granulopoietic cytokines and growth factors primarily determines the rate at which neutrophils are produced. To this end, G‑CSF and, to a lesser extent, GM‑CSF and IL‑6 signal through JAK–STAT pathways to stimulate neutrophil precursor proliferation and differentiation. However,



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Figure 4 | Signal transduction and transcriptional networks in steady-state and emergency granulopoiesis. a | The major transcriptional regulator of steady-state granulopoiesis is CCAAT-enhancer-binding protein-α (C/EBPα), based on the observation that knockout mice completely lack neutrophils. C/EBPα drives the expression of many genes that encode proteins that are required for myeloid progenitor proliferation and granulocytic differentiation, Nature including the granulocyte Reviews | Immunology colony-stimulating factor receptor (G‑CSFR; which is encoded by CSF3R). However, C/EBPα simultaneously restricts excessive proliferation by inhibiting the expression of genes that are required for cell cycle progression, such as the genes encoding MYC, cyclin-dependent kinase 2 (CDK2) and CDK4. Of note, the upstream signalling pathways for steady-state granulopoiesis are not well understood owing to the lack of appropriate in vivo experimental models. However, as Csf3r −/− mice have a 70% reduction in neutrophil numbers, it is likely that the G‑CSFR–Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway is involved in this process. b | During emergency granulopoiesis the levels of granulopoietic cytokines, most importantly G‑CSF, are markedly increased. G‑CSFR signalling through JAK leads to nuclear translocation of phosphorylated STAT3 (pSTAT3), which directly stimulates expression of the genes encoding MYC and the major transcriptional regulator of emergency granulopoiesis C/EBPβ. C/EBPβ directly stimulates MYC transcription and also replaces C/EBPα at the MYC promoter through competition for binding, thereby leading to the inhibition of the transcriptional repression that C/EBPα exerts on MYC expression. As a net result, the proliferative effects of C/EBPβ outweigh the anti-proliferative effects of C/EBPα, resulting in enhanced myeloid progenitor cell proliferation and neutrophil generation.

emergency granulopoiesis is not simply a condition of enhanced steady-state granulopoiesis given the data showing that these processes are differentially regulated at the transcriptional level. Whereas C/EBPα is the master regulator of steady-state granulopoiesis, C/EBPβ is crucial for the regulation of emergency granulopoiesis (FIG. 4). The signalling pathways that control the switch from C/EBPα-dependent steady-state granulopoiesis to C/EBPβ-dependent emergency granulopoiesis are poorly understood.

Negative regulation of emergency granulopoiesis A fundamental feature of the immune system is the parallel existence of pro-inflammatory and anti-inflammatory mechanisms that, through sophisticated positive and negative feedback pathways, counterbalance each other to ensure homeostasis. Disturbance of this equilibrium by external cues, such as infection, leads to a temporary shift towards a pro-inflammatory state, which simultaneously activates anti-inflammatory cascades to prevent potentially detrimental outcomes in the long term. It is likely

that emergency granulopoiesis is regulated in a similar manner. Indeed, it has been shown that STAT3‑mediated G‑CSF-induced signalling, despite being the main positive signalling pathway for emergency granulopoiesis, also activates suppressor of cytokine signalling 3 (SOCS3)103. SOCS proteins are negative feedback regulators of cytokine signalling 104. SOCS3 is recruited to tyrosine residues on G‑CSFR, thereby inhibiting signal transduction through JAK–STAT3 (REFS 105,106). SOCS3‑deficient mice have prolonged STAT3 activation and are hyperresponsive to exogenous G‑CSF administration, which leads to excessive neutrophilia, increased HSPC mobilization and splenomegaly 10,107. Notably, ageing SOCS3‑deficient mice spontaneously develop an inflammatory disease that is characterized by neutrophilia and tissue infiltration by multiple haematopoietic lineages, which leads to organ dysfunction10. Similarly, it has been shown that G‑CSF stimulation induces expression of the inhibitor of nuclear factor-κB (IκB) family member B cell lymphoma 3 protein (BCL‑3) in a STAT3‑dependent manner 108. BCL‑3 was originally identified as a proto-oncogene109, but it can also



REVIEWS function as an anti-inflammatory regulator by restraining the transcription of nuclear factor-κB (NF‑κB)‑dependent genes110. BCL‑3 inhibits the expression of tumour necrosis factor (TNF) by macrophages111,112, and also negatively regulates TLR signalling 113. Importantly, BCL‑3 is required to limit myeloid progenitor proliferation and differentiation into neutrophils in an NF‑κB p50‑dependent manner, as demonstrated by knockout mouse studies108, and BCL‑3 thereby restrains excessive emergency granulopoiesis. Interestingly, despite being the master transcriptional stimulator of steady-state granulopoiesis, C/EBPα also restricts cell cycle progression in myeloid progenitors through the inhibition of Cdk2 and Cdk4 (REF. 97), and of Myc 98, with the net result of constant steady-state neutrophil counts. Given the competition between C/EBPα and C/EBPβ for binding to the Myc promoter, the reduced G‑CSF levels that occur as a consequence of efficient pathogen clearance and the resulting decrease in levels of G‑CSF-mediated C/EBPβ induction might be a mechanism for switching emergency granulopoiesis back to steady-state granulopoiesis. Collectively, these data demonstrate the importance of keeping emergency granulopoiesis under tight control. However, further investigation is required to determine how both the activation and the suppression of emergency granulopoiesis can be achieved through pathways mediated by the same signal transduction molecule (STAT3), such that each pathway can function as and when required.

Emergency versus reactive granulopoiesis The cumulative experimental evidence regarding the regulation of emergency granulopoiesis supports a model in which the presence of pathogens is translated into enhanced neutrophil production through the increased release of granulopoietic cytokines, most importantly G‑CSF. However, there is some evidence that additional complementary mechanisms might have evolved to enable enhanced neutrophil production. As these mechanisms are independent of the presence of a disseminated microbial pathogen, they can be initiated by non-infectious stimuli such as chemical agents (for example, thioglycollate, alum or 5‑FU), physical insults (for example, trauma or ionizing radiation) or autoimmune disorders (for example, rheumatoid arthritis). Therefore, we suggest the use of the term ‘emergency granulopoiesis’ to describe a microbial infection-driven process and the term ‘reactive granulopoiesis’ to describe enhanced granulopoiesis in the absence of microbial infection. What is the rationale for making this distinction? Humans and microorganisms have co‑evolved over millions of years, with pathogens having exerted a strong evolutionary pressure on human populations. By contrast, it seems improbable that the non-infectious stimuli mentioned above, which have been used in some experimental settings to induce a granulopoiesis response, have had an impact on human evolution. Undeniably, the phenotypic effects may be similar and some molecular pathways might be shared between microbial infection-driven emergency granulopoiesis and reactive granulopoiesis, but there might also be fundamental

molecular differences. For example, the vaccine adjuvant alum induces an inflammatory response in an IL‑1 receptor 1 (IL‑1R1)‑dependent manner 114 that is phenotypically similar to emergency granulopoiesis, whereas LPS-induced emergency granulopoiesis (mimicking microbial infection) is independent of IL‑1R1 signalling 35. Moreover, the neutrophil inflammatory response that is elicited by trauma or haemorrhage is different from the response observed after endotoxaemia115. Further studies are needed to dissect the molecular pathways used during microbial infection-driven emergency granulopoiesis versus reactive granulopoiesis caused by physical or chemical insults. This might lead to specific medical strategies to modulate the inflammatory response and to improve patient survival. Availability of space and growth factors in the bone marrow. One such complementary mechanism would be the provision of bone marrow space and, consequently, access to the growth factors that promote generation of the blood cells that are in demand, based on a model whereby the various haematopoietic lineages in the bone marrow are in strong competition for common growth factors. This notion is corroborated by the observation that a decrease in the size of the B cell compartment in recombination activating gene 1 (RAG1)-deficient mice led to an increase in the number of neutrophils116. Also, inflammatory stimuli — such as infection, PAMPs and vaccine adjuvants — led to decreased expression of the lymphoid growth and retention factor CXC-chemokine ligand 12 (CXCL12)117 in the bone marrow and thus resulted in the mobilization of lymphocytes from the bone marrow to secondary peripheral lymphoid organs, such as the spleen. As a consequence, the vacated bone marrow space enables granulopoiesis to increase114,117. In addition, neutrophil recruitment to inflamed tissues might be a trigger for enhanced granulopoiesis independently from the increased production of granulopoiesis-promoting cytokines. In such a model, neutrophil egress from the bone marrow — which primarily occurs through inhibition of the CXCL12–CXCR4 axis118,119 — would generate bone marrow space that is filled by newly generated neutrophils which, in turn, would rapidly leave the bone marrow for as long as the inflammatory stimulus is present to promote migration to the affected peripheral tissue. As a net result, overall neutrophil production would be increased. However, definitive experimental evidence for this hypothetical mechanism is lacking. Cell density-dependent feedback mechanism. It has been shown that antibody-mediated or genetic depletion of mature neutrophils leads to an increase in the production of G‑CSF and consequently to enhanced granulopoiesis that is reminiscent of emergency granulopoiesis120,121 despite the absence of systemically disseminated pathogens. Similarly, increased serum G‑CSF levels have been observed in patients with afebrile neutropenia without evidence of ongoing infection13. These observations raise the question of whether the availability of granulopoietic growth factors is partly regulated by their consumption



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Figure 5 | An integrated model of emergency granulopoiesis. Gram-negative bacteria that have overcome local defence barriers gain access to the systemic Nature Reviews | Immunology circulation and are consequently sensed by Toll-like receptor 4 (TLR4)‑expressing non-haematopoietic cells through the recognition of pathogen-associated molecular patterns (PAMPs), thereby indicating an emergency state. TLR4 signal transduction in non-haematopoietic cells occurs in a myeloid differentiation primary response protein 88 (MYD88)‑dependent manner, resulting in the expression and secretion of large quantities of granulocyte colony-stimulating factor (G‑CSF). In the bone marrow, non-haematopoietic cell-derived G‑CSF acts on multiple levels of the granulocytic lineage as the G‑CSF receptor (G-CSFR) is expressed continuously from the early haematopoietic stem and progenitor cell stage to the mature neutrophil stage. In addition to its anti-apoptotic and microbicidal effects in mature neutrophils, G‑CSF also stimulates neutrophil mobilization from the bone marrow to the periphery through inhibition of the CXC-chemokine receptor 4 (CXCR4)–CXC-chemokine ligand 12 (CXCL12) chemotactic axis (not shown in the figure). However, a key function of G‑CSF is that it promotes myeloid progenitor proliferation and granulocytic differentiation, which results in the enhanced generation of neutrophils and an increased neutrophil output from the bone marrow into the circulation. Concomitant with the expansion of granulopoiesis, lymphopoiesis and probably also monocytopoiesis are decreased. Neutrophils are recruited to the site of infection where they launch their effector functions, thereby having an important role in clearing the pathogen and resolving the infection. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte–macrophage progenitor; GP, granulocyte progenitor; HSC, haematopoietic stem cell; MP, monocyte progenitor.

Conclusion and future perspectives On the basis of currently available data, we propose the following integrated model of pathogen sensing and translation into emergency granulopoiesis (FIG. 5). TLR4‑expressing non-haematopoietic cells function as the primary sensor for systemically disseminated Gram-negative bacteria. Upon MYD88‑dependent signal transduction, these non-haematopoietic cells secrete large amounts of granulopoietic growth factors, most importantly G‑CSF, which in turn stimulates G‑CSFRexpressing HSPCs and leads to the increased de novo production of neutrophils. Of note, successful, in other words life-saving, innate immune responses require the coordinated actions of several important biological cascades including the migration, de novo generation and antimicrobial effector functions of neutrophils. These processes are not only related but, in terms of their measurable outcome, are also partly overlapping. The pleiotropic role of G‑CSF in these three processes illustrates how closely they are related at the molecular level. G‑CSF induces neutrophil mobilization from the bone marrow independently of its major biological function as the primary granulopoietic growth factor, and it also promotes the phagocytic activity of neutrophils and production of reactive oxygen species required for bacterial killing. The distinct biological processes of neutrophil recruitment and effector function have recently been reviewed elsewhere4. Aside from the obvious beneficial effects of haemato poietic activation for host survival, there might be detrimental long-term effects of acute and chronic infection on the haematopoietic system. Epidemiological data from a large population-based registry in Sweden show that there is an association between chronic infection and inflammation and the development of HSPC malignancies126, but there is currently a limited mechanistic understanding of this association. However, there is accumulating evidence that the chronic activation of classical inflammatory signalling pathways might be a pathogenic trigger for neoplastic transformation. Indeed, several independent studies have identified activating mutations in MYD88 in haemato-lymphoid tumours127–130. It is tempting to speculate that a genetic lesion that results in constitutive activation of MYD88‑dependent signalling and the natural chronic activation of this inflammatory pathway could have similar pathogenic effects69. A better understanding of the key cellular players and molecular pathways that are involved in regulating demand-adapted haematopoiesis — and in particular emergency granulopoiesis — will eventually lead to new approaches for the treatment of infectious diseases. It might become possible to specifically boost emergency granulopoiesis without the often detrimental effects of generalized and sustained immune system activation.



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Acknowledgements

The work in the authors’ laboratory is supported by grants f ro m t h e S w i s s N a t i o n a l S c i e n c e Fo u n d a t i o n (310030_146528/1), the Promedica Foundation, Switzerland, and the Clinical Research Priority Program of the University of Zürich, Switzerland.

Competing interests statement

The authors declare no competing interests.


HoxA10 Terminates Emergency Granulopoiesis by Increasing Expression of Triad1.

BAFF-secreting neutrophils drive plasma cell responses during emergency granulopoiesis.

Irf8) Is Required for Termination of Emergency Granulopoiesis.

Map3k8 controls granulocyte colony-stimulating factor production and neutrophil precursor proliferation in lipopolysaccharide-induced emergency granulopoiesis.

Granulopoiesis in childhood aplastic anemia.

Dietary supplementation with Lactobacilli improves emergency granulopoiesis in protein-malnourished mice and enhances respiratory innate immune response.

Control factors of granulopoiesis in human serum.

Effect of lithium on granulopoiesis in culture.

Tamm-Horsfall Protein Regulates Granulopoiesis and Systemic Neutrophil Homeostasis.

Inhibition of diffusion chamber (DC) granulopoiesis by anti-CSF serum.

Circulating inhibitors of granulopoiesis in patients with aplastic anaemia.

Possible involvement of leukocytic endogenous mediator in granulopoiesis.

Linking stress granulopoiesis to protein synthesis through GADD34.

Identification of nuclear-enriched miRNAs during mouse granulopoiesis.

Analysis of the DNA methylome and transcriptome in granulopoiesis reveals timed changes and dynamic enhancer methylation.

macrophage-colony-stimulating factor.

A combined model of human erythropoiesis and granulopoiesis under growth factor and chemotherapy treatment.

Differential expression of granulopoiesis related genes in neutrophil subsets distinguished by membrane expression of CD177.

Control of granulopoiesis in man.III. inhibition of colony formation by dense leukocytes.

Granulopoiesis in infantile genetic agranulocytosis. In vitro cloning of marrow cells in agar culture.

TIMP-1 signaling via CD63 triggers granulopoiesis and neutrophilia in mice.

The Role of Microbiota and Immunobiotics in Granulopoiesis of Immunocompromised Hosts.

Effect of sodium salicylate on human and mouse granulopoiesis in vitro.

Neutropenia in three patients with rheumatic disorders. Suppression of granulopoiesis by control-sensitive thymus-dependent lymphocytes.