Toll-like receptor signaling in hematopoietic homeostasis and the pathogenesis of hematologic diseases

Abstract

Toll-like receptors (TLRs), which are found in innate immune cells, are essential mediators of rapid inflammatory responses and appropriate T-cell activation in response to infection and tissue damage. Accumulating evidence suggests that TLR signaling is involved in normal hematopoiesis and specific hematologic pathologies. Particular TLRs and their downstream signaling mediators are expressed not only in terminally differentiated innate immune cells but also in early hematopoietic progenitors. Sterile activation of TLR signaling is required to generate early embryonic hematopoietic progenitor cells. In adult animals, TLR signaling directly or indirectly promotes differentiation of myeloid cells at the expense of that of lymphoid cells and the self-renewal of hematopoietic stem cells during infection and tissue damage. Activating mutations of the MyD88 gene, which codes for a key adaptor involved in TLR signaling, are commonly detected in B-cell lymphomas and other B-cell hematopathologies. Dysregulated TLR signaling contributes to the pathogenesis of many hematopoietic disorders, including bone marrow failure, myelodysplastic syndrome, and acute myeloid leukemia. Complete elucidation of the molecular mechanisms by which TLR signaling mediates the regulation of both normal and pathogenic hematopoiesis will prove valuable to the development of targeted therapies and strategies for improved treatment of hematopoietic disorders.

Keywords

TLR / MyD88 / hematopoiesis / bone marrow failure / leukemia / myelodysplastic syndrome

Introduction

The innate immune system functions as the first line of immune defense in humans and other multicellular organisms. This system provides rapid protective response against numerous infections and injuries. Responses to infection or tissue damage are primarily mediated by tissue-resident immune cells (including macrophages, dendritic cells, mastocytes, and histiocytes) and circulating white blood cells (monocytes, neutrophils, and natural killer cells). Pattern recognition receptors (PRRs) expressed in immune or epithelial cells first detect the presence of invading bacteria or viruses in metazoans. PRRs are encoded in the genome and are highly conserved across a wide range of species. PRRs sense danger signals by recognizing conserved molecular patterns and then inducing appropriate responses, often involving inflammation, to eliminate danger factors or to limit factor-induced tissue damage [ 1]. During pathogenic infection, PRRs detect pathogen-associated molecular patterns (PAMPs), which are released from invading bacteria or viruses and are responsible for activating responses to limit infections and eliminate pathogens. The binding of PAMPs to specific PRRs activates downstream signaling, which stimulates innate anti-pathogen immunity by inducing a local accumulation of monocytes and neutrophils, and also facilitates pathogen-specific adaptive immune response by enhancing the activation of an antigen-specific memory T-cell response. In cases of tissue damage, PRRs recognize damage-associated molecular patterns (DAMPs), which are endogenous PRR ligands released from damaged or necrotic cells; moreover, healing and inflammatory responses are activated to clear cellular debris and repair the damaged tissue [ 1]. PRRs are divided into several receptor classes, including toll-like receptors (TLRs) and C-type lectin receptors (CLRs), both of which are transmembrane receptors. PRRs include NOD-like receptors (NLRs) and retinoic acid inducible gene (RIG)-1-like receptors (RLRs) [ 1]. Of these classes, TLRs are commonly characterized with respect to their receptor structure, localization, known ligands, and downstream signaling patterns. Accumulating evidence suggests that TLR-mediated signaling not only serves an essential function in stimulating the innate immune response, which has been summarized in many excellent reviews [ 2, 3], but also participates in the regulation of hematopoietic homeostasis and possibly hematopoietic pathologies.

Although particular tissue-resident innate immune cells can self-renew, proliferate, and expand to an extent, tissue-innate immune cells are largely dependent on replenishment by circulating monocytes derived from hematopoietic stem/progenitor cells (HSPCs) in the bone marrow (BM) niche [ 4, 5]. Most innate immune effector cells are short-lived and rapidly consumed during inflammation [ 5]. Therefore, during infection or injury, augmented hematopoiesis is required to fulfill the high demand for innate immune cells. However, the chronic, abnormal activation of TLR signaling may disrupt the homeostatic state of normal hematopoiesis and even induce hematopoietic disorders. In this paper, we review the studies that have explored TLR signaling in the regulation of both normal and pathogenic hematopoiesis. We believe that research on hematopoietic TLR signaling will enhance our ability to promote normal hematopoiesis and target pathologic processes through precise molecular modulation of this important signaling pathway.

TLRs and their ligands

TLRs are type 1 transmembrane proteins with an N-terminal extracellular domain characterized by leucine-rich repeats (LRRs), a singular transmembrane helix, and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. To date, 10 unique TLRs have been characterized in humans, whereas 12 functional murine TLRs have been described [ 2]. TLRs are divided into subtypes based on which specific PAMPs they recognize. TLR1–TLR9 are functionally conserved and expressed in both mice and humans. TLR10 is functional in humans but not expressed in mice. TLR11, TLR12, and TLR13 are found only in mice [ 6]. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 are found on cell surfaces and are associated with the detection of extracellular molecular patterns, mostly bacterial PAMPs and endogenous DAMPs. TLR3, TLR7, TLR8, and TLR9, are localized in intracellular vesicles and endosomes and can recognize molecular patterns associated with intracellular infections, such as bacterial or viral nucleic acids (Fig. 1) [ 6]. Notably, TLR4 may be found on either the plasma membrane or endosomes. Analyses of the crystal structures of TLR extracellular domains reveal that LRRs form horseshoe-like concave structures in which distinct LRR patterns confer receptor specificity for several distinct PAMPs [ 7]. TLR3, TLR4, TLR5, and TLR9 homodimers recognize double-stranded RNA (dsRNA), lipopolysaccharide (LPS), bacterial flagellin, and unmethylated CpG DNA motifs, respectively [ 6, 8- 11]. TLR1/TLR2 and TLR2/TLR6 heterodimers recognize triacylated and diacylated lipoproteins, respectively [ 12- 14]. Both TLR7 and TLR8 homodimers recognize single-stranded RNA [ 15]. Recently, TLR8 has been shown to detect uridine and RNA oligonucleotide sequences at two distinct sites in its extracellular domain, both of which are essential for receptor activation [ 16].

Depending on the TLR subtype, TLRs may exist as monomers that dimerize in the presence of their ligands or as dimers that undergo a conformational change upon exposure to a ligand. Ligand-induced dimerization has been demonstrated for TLR1/TLR2, TLR2/TLR6, TLR3, TLR4, and TLR5 [ 8, 17- 21]. In contrast, TLR7, TLR8, and TLR9 exist as dimers in the absence of their ligands. Crystal structural studies have demonstrated that the ligand stimulation of TLR7, TLR8, or TLR9 induces conformational changes [ 9, 10]. Dimerization and conformational changes induced by TLR-ligand binding are believed to bring their cytoplasmic TIR domains into close proximity with each other, thereby enabling the recruitment of downstream adaptor proteins via homotypic TIR domain interactions [ 7, 10]. Further crystallization studies, particularly those on cytoplasmic TIR domains upon ligand stimulation, are needed to confirm these mechanisms. To fully activate ligand-stimulated TLR signaling, other co-receptors or facilitators may also be required in certain cases. For instance, efficient LPS-induced TLR4 activation depends on two co-receptors of TLR4, namely, CD14 and MD-2 [ 22- 24]. Intracellular TLR3, TLR7, and TLR9 have been shown to interact with the ER protein UNC93B1, which assists in the translocation from the ER to endolysosomes, a necessary step for functional TLR7 and TLR9 signaling [ 25].

Additionally, several TLRs are believed to recognize DAMPs produced as endogenous ligands. The ligation of DAMPs may signal through separate complementary pathways unlike those of PAMPs and function in an undetermined physiologic purpose. The accumulation of DAMPs has been associated with many aging-related diseases, including atherosclerosis, gout, Parkinson’s disease, Alzheimer’s disease, and age-related macular degeneration [ 26]. Known DAMPs include extracellular matrix proteins biglycan, versican, hyaluronic acid, and heparin sulfate, as well as particular cellular components, such as high-mobility group box 1 (HMGB1) protein, hsp70, hsp72, and S100A/B [ 27]. Modified forms of HMGB1 protein have been implicated in the stimulation of TLR2, TLR4, and TLR9 [ 28, 29]. HMGB1 is released into the extracellular space during inflammation, tissue damage, or necrosis, and may consequently initiate secondary inflammation [ 30]. Heat-shock proteins hsp70 and hsp72 have also been shown to signal through TLR4 [ 31, 32].

TLR-associated signaling pathways

Upon ligand-induced TLR stimulation, cytoplasmic TIR domains mediate the recruitment of adaptor proteins to the receptors. The interaction of adaptor proteins with activated receptors is essential for the transduction signaling from activated TLRs (Fig. 1). Currently, known TLR adaptor proteins include myeloid differentiation primary response gene 88 (MyD88), TIR-domain containing adaptor protein (TIRAP, also known as Mal), TIR-domain containing adaptor inducing interferon β (TRIF), TRIF-related adaptor molecule (TRAM), and sterile-α and armadillo-motif-containing protein (SARM). Most TLRs signal exclusively through the recruitment of MyD88, with the exception of TLR3, for which signaling is dependent solely on TRIF. TLR4 utilizes both MyD88 and TRIF for signal propagation [ 33]. TIRAP and TRAM function as facilitating adaptors that mediate the recruitment of MyD88 and TRIF, respectively, to TLR4 at the plasma membrane and endosomes [ 34, 35]. TIRAP facilitates TLR4-induced MyD88-dependent signaling by localizing specifically to the plasma membrane, associating with TLR4, and recruiting MyD88 upon TLR4 ligation by LPS. Unlike other TLRs, TLR4 can undergo endocytosis following LPS ligation. Following endocytosis of the TLR4 receptor, TRAM enhances TLR4-induced TRIF-dependent signaling by associating with activated TLR4 at the early endosome and directing TRIF to interact with TLR4-TRAM in the cytoplasmic space near the endosome [ 36]. SARM has been shown to negatively regulate TRIF-dependent signaling through a direct interaction with TRIF [ 37]. Therefore, TLR signaling can be broadly divided into MyD88-dependent signaling and MyD88-independent, or TRIF-dependent, signaling. Cells with MyD88 deletion are unable to transduce signal activation of most TLRs, while TRIF-dependent signaling in such cells remains intact for TLR3 and TLR4. The deletion of both MyD88 and TRIF completely eliminates all IL1R/TLR signaling (both MyD88-dependent and MyD88-independent).

MyD88-dependent signaling is mediated by a multi-protein complex known as the myddosome, whereas MyD88-independent signaling is mediated by TRIF. After being recruited to activated TLRs, MyD88 recruits IL-1 receptor associated kinase 4 (IRAK4) and IRAK2 by homotypic death domain interactions [ 35, 38]. Crystallization study has shown that the myddosome contains six MyD88, four IRAK4, and four IRAK2 subunits arranged in a helical conformation [ 38]. Once assembled, the myddosome activates the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6), which has long been implicated in the transduction of IL-1R-mediated signaling [ 39]. TRAF6 activation results in the activation of activator protein 1 (AP-1) and nuclear factor κ-light-chain-enhancer of activated B-cell (NF-κB) pro-inflammatory signaling pathways by inducing the activation of transforming growth factor β activated kinase-1 IκB kinase (TAK1-IKK) and TANK binding kinase-1 (TBK-1) signaling cascades. These processes culminate in the release of pro-inflammatory cytokines, including TNFα, IL-6, and IL-12 [ 40]. However, in MyD88-independent signaling, the recruitment of TRIF to activated TLR3 and TLR4, via TIR–TIR interactions, induces TBK-1-IKKϵ-mediated phosphorylation through TRAF3 and downstream activation of interferon regulatory factor 3 (IRF3) and IRF7, thereby resulting in the production of IFNα and IFNβ [ 41, 42]. MyD88 is also capable of inducing the production of IFNα and IFNβ upon the activation of endosomal TLR7, TLR8, or TLR9. The activation of IRF7 was determined to be essential for the induction of IFNα/β by these TLRs [ 42]. IRF7 forms a complex with MyD88 and TRAF6, in which the E3 ubiquitin ligase activity of TRAF6 is necessary for IRF7 activation [ 43]. MyD88-dependent and MyD88-independent signals are not always clearly separated. In fact, they share several downstream pathways. For example, TLR7- and TLR9-stimulated MyD88-TRAF6 can also activate IRF5 by K63 polyubiquitination, thereby allowing it to translocate to the nucleus and initiate the transcription of type I interferons [ 44]. TLR8 stimulation also induces IRF5 activation in a TRAF6-dependent manner [ 45]. TRIF-mediated signaling is also capable of activating NF-κB and AP-1 signaling through the activation of TRAF6 via a direct interaction with TRIF, although this signal is delayed compared to MyD88-dependent signaling [ 46]. In addition, while much of the framework of TLR signaling has been well established, specific signaling interactions are still being characterized, raising new questions in the process. For example, TRIF possesses a RIP homology interaction motif (RHIM), which was recently shown to interact with RIP3, which can induce MLKL necroptotic signaling. Necroptosis is normally repressed by activated caspase 8 [ 46- 48]. Thus, the inhibition of caspase 8 concurrent with TLR2, TLR3, TLR4, TLR5, or TLR9 stimulation results in necroptosis in affected cells [ 48].

TLR signaling in normal hematopoiesis

Hematopoiesis is the continuous generation of all types of blood cells throughout the life of an organism through an ordered, hierarchical process of differentiation, originating with BM hematopoietic stem cells (HSCs). This process gives rise to more than 10 unique types of blood cells, including all of the innate immune cells in the blood. HSCs are capable of generating all types of blood cells by first differentiating into multipotent progenitors (MPPs), and then common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs), followed by lineage-committed progenitors, such as granulocyte and macrophage progenitors (GMPs), megakaryocyte and erythrocyte progenitors (MEPs), early B-cell progenitors, and early T-cell progenitors. During this process, multiple cell fate decisions need to be made, such as electing proliferation over differentiation and lineage commitment or vice versa. In addition, to ensure lifelong functional hematopoiesis and to avoid exhaustion, HSCs maintain a functional population by self-renewal, a special type of proliferation without differentiation. Under normal homeostatic conditions, the self-renewal of HSCs and the cell fate decisions of hematopoietic progenitors are tightly controlled by cell-intrinsic transcriptional and translational networks, which are further regulated by factors in the local environment and/or from the peripheral circulation, including hematopoietic growth factors and cytokines. Most HSCs are maintained in a quiescent state in a highly specialized microenvironment called the HSC niche. Only a small subset of HSCs enters the cell cycle to replenish differentiating progenitor cells. However, HSC proliferation and differentiation can be induced by specific physical or disease conditions, such as infection and tissue damage to meet the increased demand for mature immune cells. Short-term enhanced proliferation and differentiation of HSCs may lead to an overproduction of hematopoietic progenitors and mature blood cells and possibly myeloproliferative or myelodysplastic-like disorders. Long-term enhanced HSC proliferation/differentiation may result in eventual HSC exhaustion and BM failure (BMF) syndromes. TLR signaling has been implicated in the regulation of hematopoiesis, either directly or indirectly, by inducing the proliferation and myeloid lineage differentiation of HSCs in response to danger signals.

The function of TLR signaling in hematopoiesis under normal homeostatic conditions is not yet entirely clear. A low level of basal stimulation by gut microbiota-derived TLR ligands regulates granulopoiesis in the absence of infection to generate a reserve pool of myeloid cells within the BM [ 49]. This relationship between the microbiota and hematopoiesis suggests that an intestinal microbiotic environment altered by antibiotic therapy or other conditions would affect hematopoiesis in the host [ 49]. However, no published studies suggest that MyD88^-/-, TRIF ^-/-, or MyD88 ^-/--TRIF ^-/- mice suffer from hematopoietic pathologies aside from the expected deficits in innate immune responses to infection, indicating that TLR signaling might be dispensable for normal homeostatic hematopoiesis in adult mammals. Recently, several studies have demonstrated that TLR-mediated innate immune and pro-inflammatory signaling serves important functions in the emergence of embryonic HSPCs. Sterile TLR4-MyD88-induced activation of NF-κB in hemogenic endothelial cells induces the subsequent activation of Notch signaling, which is vital for HSPC emergence from the aorta/gonad/mesonephros (AGM) region of both mouse and zebrafish embryos [ 50]. In fact, embryonic hemogenic tissue in the AGM has a robust molecular signature for innate immune and pro-inflammatory pathways. Studies have demonstrated that many inflammatory cytokines, including interferon γ[ 51], interferon α[ 52], IL1β[ 53], IL3 [ 54], and TNFα[ 55], as well as their corresponding receptors, are expressed in AGM hematopoietic tissue. These cytokines collaboratively stimulate a sterile inflammatory signal that regulates HSPC formation in the vertebrate embryo [ 51- 53, 55, 56]. Inactivation of these cytokine signaling pathways leads to a significant reduction in the emergence of HSPCs from AGM endothelial cells [ 50, 51]. Whether and how TLR signaling initiates hemogenic inflammatory environments remains to be determined. Given that the developing embryo is a sterile environment, this TLR4 signaling may be initiated by the controlled production of endogenous DAMPs. DAMPs are known to accumulate with aging, and whether such hematopoietic alterations are linked to aging by DAMP signaling remains to be determined.

The function of DAMP-TLR signaling with respect to hematopoiesis in the absence of infection also remains to be determined. DAMPs may utilize different receptors or co-receptors to modulate TLR signaling responses. For example, HMGB1 is known to stimulate the receptor for advanced glycation end products (RAGE) in addition to TLRs [ 27]. Further research is needed to characterize a functional response to DAMP-TLR ligation in hematopoietic tissue and immune cells, as well as to determine the difference between DAMP signaling and PAMP-TLR signaling pathways.

TLR signaling and stress hematopoiesis

The function of TLR signaling in stress hematopoiesis has been better studied. Hematopoietic homeostasis is perturbed when the immune system is challenged, such as in cases of infection or severe injury. Peripheral innate immune cells are rapidly consumed combating pathogens as part of the immune response, and BM hematopoiesis must augment the generation of myeloid cells to replace these immune cells for a continued immune response. The increased production of innate immune cells occurs at the expense of the production of lymphoid cells and necessitates the induction of HSC proliferation. This phenomenon has been termed “emergency myelopoiesis” and has been well documented in both humans and animal models. The expanded production of myeloid cells (granulocytosis and monocytosis) during infection has been observed for decades [ 57]. Toll was first implicated in Drosophila larval myelopoiesis, and in the late 1990s it was found that the disruption of Toll/Cactus signaling resulted in altered hemocyte density [ 58]. Numerous studies have shown that bacterial, viral, or fungal infections, as well as purified or synthetic TLR ligands, can induce HSC cycling and a shift toward myeloid differentiation, as well as expansion of HSC and progenitor populations [ 5, 59- 63]. Chronic exposure of HSCs to infections or TLR ligands was shown to induce HSC cycling to the point at which HSC self-renewal becomes impaired [ 64]. Three mechanisms have been proposed to explain these observations: (1) inflammatory cytokine production by peripheral immune cells, (2) intrinsic HSPC stimulation, and (3) detection of TLR ligands by niche support cells (Fig. 2).

Previously, it was widely accepted that the hematopoietic response to infection or tissue damage is induced by the systematic increase of inflammatory cytokines, such as TNFα, IL1β, IL-6 and IFN-γ. These cytokines are primarily produced in infected tissue by innate immune cells stimulated by PAMPs or DAMPs through specific TLRs. Consequently, significant increases in systemic inflammatory cytokine levels were detected in the peripheral blood of almost all patients with infections or severe tissue damage [ 65]. These cytokines reach the BM niche through the peripheral circulation and then act on the BM niche to stimulate myelopoiesis. However, recent studies have suggested that elevated cytokine levels in the local BM niche are critical for HSPC expansion. Furthermore, the discovery of functional TLRs on HSCs and hematopoietic progenitors has led researchers to hypothesize that HSCs and progenitor cells are intrinsically capable of responding to direct stimulation by TLR agonists [ 59].

HSPCs express most TLRs, including TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and TLR9 [ 5]. In vitro culture studies have demonstrated that purified Lin-Sca1⁺c-Kit⁺ HSPCs can be induced to differentiate into myeloid cells upon stimulation with Pam3CSK4 (TLR1/2), LPS (TLR4), or R848 (TLR7/8) [ 59, 60, 66]. LPS not only induces monocyte differentiation of CMP populations but also induces dendritic cell (DC) differentiation of CLP populations, even in the absence of other myeloid differentiating cytokines [ 59]. In vivo, myeloid lineage differentiation has been demonstrated using a wide variety of infection models or by stimulation with purified TLR ligands [ 5, 59, 60, 67, 68]. HSCs can respond directly to TLR2 and TLR4 agonists in vivo, as demonstrated by transplanting wild-type HSCs into TLR2^-/- and TLR4^-/- mice [ 69]. Short-term HSCs (ST-HSCs) and MPPs are potent generators of cytokines when exposed simultaneously to LPS and Pam3CSK4. These cytokines, principally IL-6, act in an autocrine and paracrine manner to induce myelopoiesis both in vitro and in vivo [ 70]. This process may be extended to circulating HSPCs, which migrate to peripheral tissue and may give rise to myeloid cells in tissue upon TLR stimulation [ 71]. Lastly, the presence of the TLR1/2 ligand Pam3CSK4 during myeloid differentiation yields functionally distinct myeloid cells that produce decreased amounts of cytokines and reactive oxygen species upon stimulation compared to normally differentiated cells [ 72].

The stromal cell population within the HSC niche may also function in the emergency myelopoietic response to infection. Mesenchymal stem cells (MSCs) also express TLRs 1-6 and produce IL-6 and IL-1β upon stimulation [ 73, 74]. The ability of MSCs to secrete IL-6 in response to TLR stimulation suggests a highly redundant system in which TLR stimulation of mature immune cells, HSCs, or niche support cells induces emergency myelopoiesis (Fig. 2). TLR4 activation in the non-hematopoietic BM compartment is essential for an infection-induced bias toward myeloid production, while, surprisingly, TLR4 expression on hematopoietic cells is dispensable for this process [ 75]. Recently, activated cytotoxic CD8⁺ T cells have been shown to induce myelopoiesis during viral infection by migrating to the BM niche and secreting IFN-γ, which is then detected by MSCs (Fig. 2) [ 76]. Consequently, these MSCs secrete IL-6, which induces the proliferation and myeloid differentiation of MPPs [ 76]. Previously, IFN-γ has been demonstrated to induce proliferation of LT-HSCs in a Mycobacterium avium chronic infection model [ 61]. Type I IFN signaling also mediates expanded myelopoiesis in TLR7-overexpressing mice [ 68]. Thus, IFN signaling is critically involved in transmitting signals from activated peripheral innate immune cells to MSCs and HSPCs when the latter cell types cannot detect infections directly.

TLR signaling in bone marrow failure

Fanconi anemia, an autosomal recessive disorder, is the most common form of inherited bone marrow failure (BMF). This disorder is characterized by defects in DNA repair resulting from the mutation of any one of 15 FANC genes [ 77]. Patients with Fanconi anemia are predisposed to develop leukemia. TLR-mediated inflammatory pathogenesis has been demonstrated by studies involving FancA- and FancC-deficient animal models. FancC directly interacts with and modulates the activation state of TLR8 by suppressing either its ubiquitinylation or its association with another undetermined ubiquitinylated protein. Enhanced activity of TLR8 and its canonical downstream signaling mediators IRAK, IKKα/β, and p38MAPK/MK2 was observed in FancA- and FancC-deficient mononuclear phagocytes, as well as in mononuclear phagocytes from Fanconi anemia patients [ 78]. As a consequence, TNF and IL-1β production is significantly increased in FancA- and FancC-deficient macrophages in a TLR8-dependent and p38/MAPK-dependent manner [ 79- 81]. FancC-deficient HSPCs are hypersensitive to TNF-α- and IL-1β-induced apoptotic effects [ 79, 81- 87]. TNF contributes to the pathogenesis of Fanconi anemia by inducing apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinases (JNK), and p38-dependent hematopoietic repression, as well as clonal evolution of acute myeloid leukemia (AML) in FancC-deficient mice [ 82, 88].

The role of TLR signaling in acquired BMF has not been thoroughly studied. Aplastic anemia (AA) is a rare HSC disorder characterized by pancytopenia and hypocellular BM. Autoimmune-mediated destruction of HSCs is pathognomonic for acquired AA in most cases. However, the precise mechanism by which the autoimmune reaction is initiated remains unknown. Using animal models, we found that necroptosis in a small subset of hematopoietic cells can trigger an autoimmune reaction leading to BMF in mice (Xin et al., unpublished results). Necroptosis is a novel type of programmed necrotic cell death that involves the release of degraded intracellular components, such as heat-shock proteins, HMGB1, ATP, uric acid, S100 molecules, and DNA [ 89]. Such components may function as DAMPs to induce a TLR-innate immune reaction-mediated sterile inflammation and can also act as autoantigens to stimulate T-cell-mediated autoimmune responses (Fig. 3). Evidence suggests that many AA syndromes exhibit cryptic clonal genetic lesions that can evolve into clonal disorders, as observed in paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS) [ 90- 93]. Immune-inflammatory stress has been detected in almost all types of BMF syndromes, and such stress plays a critical role in disease pathogenesis and progression [ 91, 94- 99]. Whether hematopoietic cells of AA patients, possibly with genetic lesions, undergo necroptosis and trigger the autoimmune inflammatory reactions requires further intensive research.

TLR signaling in myelodysplastic syndrome

MDS is a complex group of age-dependent HSC disorders. Similar to AA patients, most MDS patients are cytopenic. However, in contrast to AA, BM cellularity in MDS is typically significantly increased. Defective BM hematopoiesis due to the premature death of blood cells stimulates elevated proliferative rates in BM HSPCs despite the reduced blood cell counts observed in MDS patients. The presence of leukemia-related mutations in BM HSPCs of MDS patients suggests a pre-malignant or malignant feature of these disorders. Approximately 30% of MDS cases progress to leukemia due to the clonal selection and expansion of malignant HSPCs [ 100]. However, the molecular pathogenesis of MDS has not yet been completely elucidated.

Recently, it was found that enhanced activation of TLR-MyD88-initiated innate immune responses showed a potential contribution to the pathogenesis of MDS. Upregulation of multiple TLRs and MyD88 has been reported in MDS patient samples [ 101, 102]. Upregulated TLR signaling disrupts hematopoiesis and promotes MDS development by (1) selectively inducing the clonal evolution of mutant HSPCs; (2) inducing apoptosis and impairing the proliferation/differentiation of healthy HSPCs; (3) stimulating a sterile inflammatory BM microenvironment; and (4) promoting the repressive activity of myeloid-derived suppressor cells (MDSCs) (Fig. 3).

Compared to CD34⁺ HSPCs from normal donors, BM CD34⁺ HSPCs isolated from most MDS patients express higher levels of TLRs, including TLR1, TLR2, TLR4, and TLR6, as well as MyD88, IRAK1, TRAF6, and TIRAP [ 101- 107]. TLR2^F217S, a recurrent genetic variant, was detected in 11% of MDS patients, which is significantly higher than its known frequency in the normal population [ 102]. Such upregulation of TLRs and TLR variants results in enhanced activation of TLR downstream signaling, including IRAK1/4, NF-κB, and p38MAPK; furthermore, TLR2 stimulation of CD34⁺ BM cells increases target gene expression of JMJD3 and IL-8 [ 102]. Overexpression of TLRs, MyD88, and other signaling mediators is observed in approximately 40%-80% of MDS patients and may well represent a subgroup of MDS with poor prognosis [ 101, 108]. TLR-MyD88 signaling contributes to the pathophysiology of MDS by negatively regulating the erythroid differentiation of BM CD34⁺ cells. Inhibition of TLR-MyD88 signaling in BM CD34⁺ cells from low-risk MDS patients improves the formation of erythroid colonies [ 102]. JMJD3 and IL-8 have been described as the key mediators of TLR-MyD88 signaling in MDS HSPCs. Inhibition of TLR signaling can restore the ability of HSPCs to form erythroid colonies. Thus, targeting TLR-MyD88-IRAK-JMJD3-IL-8 signaling may have therapeutic potential for a large subset of MDS cases.

The hemizygous, interstitial deletion of chromosome 5q [del(5q)] is the most common cytogenetic abnormality in MDS and is present in about 15% of all cases. Two commonly deleted regions (CDRs) were identified in del(5q). These CDRs result in allelic deletion of a fascinating array of genes and miRNAs, amounting to 40 genes lost within the distal CDR, thereby resulting in haploinsufficiency [ 109]. The hemizygous deletion of several of these genes and miRNAs contributes to MDS development by promoting the activation of TLR signaling. For example, allelic deletion of miRNA-145 and-146a, which were detected in patient samples, leads to an upregulation of their target genes TIRAP and TRAF6 [ 110]. DIAPH1, which encodes a RhoA GTPase effector mDia1, regulates the dynamics of the actin cytoskeleton. DIAPH1 is also deleted in del(5q) MDS and upregulates CD14 in neutrophils through an unknown mechanism [ 111]. As a result, IRAK, TRAF6, and NF-κB are constitutively activated in BM samples of most del(5q) MDS patients. Knockdown or knockout of miRNA-146a or overexpression of TRAF6 in murine HSPCs recapitulated the hematologic features of del(5q) MDS in a transplant model [ 110]. The overexpression of CD14 also exacerbates the MDS disease phenotype in mice. These findings may explain why lenalidomide, an immunomodulatory agent, elicits a therapeutic response in patients with del(5q) MDS [ 111, 112].

Immature MDSCs are classically defined in tumor tissue, in which they contribute to tumor development and progression by inducing immunosuppression and tumor-promoting inflammation [ 113, 114]. Chen et al. found that Lin^-HLA-DR^-CD33⁺ MDSCs were markedly expanded in the BM of MDS patients [ 115]. S100A8/S100A9, which are known endogenous DAMPs [ 116, 117], bind to CD33 to induce the expansion and activity of MDSCs [ 110, 111]. Clonally distinct MDSCs in BM overproduce suppressive cytokines, such as IL-10 and TGF-β as well as nitric oxide (NO) and arginase, thereby contributing to the pathogenesis of MDS by inducing ineffective hematopoiesis. Transgenic overexpression of S100A9 induces an age-related hematopoietic disorder in mice, which recapitulates the phenotype of human MDS. Blockage of S100A9-CD33 signaling or induction of the maturation of MDSCs in vivo can significantly improve hematopoiesis in MDS animals [ 115]. These findings indicate that S100A9/CD33 signaling-driven primary BM expansion of MDSCs perturbs hematopoiesis and contributes to the development of MDS. S100A8/S100A9 DAMPs are well-defined endogenous agonists of TLR4 and induce MyD88-dependent and MyD88-independent signaling in various cell types. It will be interesting to determine whether S100A8/S100A9-induced TLR4 signaling influences the activity of MDSCs in MDS samples.

TLR signaling in the pathogenesis of acute myeloid leukemia

Several studies have suggested that TLR signaling plays a critical role in the pathogenesis of cancers, especially those etiologically related to inflammation. TLRs are expressed not only in innate immune cells but also in tumor cells and resident cells of the tumor microenvironment, which complicates the study of TLR signaling in cancer development and progression [ 118, 119]. TLR-MyD88 signaling in DCs is required to induce a Th1 cell-dependent antitumor immune response, especially for cancers induced by bacterial or viral infections, as well as particular carcinogens. These studies provide a strong rationale for enhancing TLR signaling as part of antitumor immunotherapy [ 120]. However, TLR signaling promotes tumor progression in a majority of other cancers. The upregulation of TLRs and MyD88 has been reported in various human cancers, including stomach, colon, prostate, ovarian, liver, and lung cancers; such upregulation is also associated with tumor metastasis, chemotherapeutic resistance, and poor prognosis in the above tumors [ 121- 127]. In addition, polymorphisms of TLRs, which may lead to constitutive MyD88 activation, are associated with higher risk for stomach/gastric, prostate, liver, lung, breast, or cervical cancers [ 128- 131]. In several animal tumor models, such as DMBA/TPA-induced skin papilloma [ 121], methylcholanthrene (MCA)-induced sarcoma [ 132], DEN-induced hepatocarcinoma (male) [ 133, 134], APC^min spontaneous, and carcinogen azoxymethane (AOM)-enhanced intestinal tumor, as well as inflammation-driven lung cancer [ 135], the deletion of a specific TLR or MyD88 reduces and/or delays tumor development and progression, thereby implicating a tumor-promoting activity of this signaling pathway [ 136]. The tumor-promoting activity of TLR-MyD88 is mediated by (1) stimulating NF-κB/JNK/ERK-survival/proliferative signaling in tumor cells; (2) inducing the production of cytokines, such as TNF, IL-1α and IL-6, which act in an autocrine and/or paracrine positive feedback loop to promote tumor growth [ 137]; and (3) stimulating the production of chemokines, such as CCL2, to recruit tumor-promoting BM myeloid cells, including MDSCs (Fig. 3). Therefore, the inactivation of TLR-MyD88 signaling may represent an improved strategic approach to more successfully treat such cancers.

In contrast to solid tumors, AML cells are mutant progenitors for innate immune cells. They can differentiate into monocytes, macrophages, and DCs (AML-DCs hereafter) upon induction. Such induced differentiation can be enhanced by the activation of TLR4, TLR7, and TLR8. In vitro studies have demonstrated that agonists of TLR4 and TLR7/8 synergistically facilitate the differentiation of AML blasts into activated AML-DCs. These AML-DCs express DC surface markers (e.g., CD40, CD86, CD1a, CD83, CCR7), maintain immunogenic leukemia-associated antigens, and stimulate the activation of anti-AML cell-specific cytotoxic T lymphocytes [ 138- 141]. A recent study has suggested that the TLR7/TLR8 agonist R848 considerably impairs the growth of human AML cells in immunodeficient mice [ 142]. These studies suggest that the use of particular TLR agonists may be a useful treatment option for AML when combined with DC-inducing cytokines by enhancing DC differentiation. AML-DCs may further enhance anti-AML activity by activating the T-cell-mediated adaptive immune response. However, such treatments must be carefully selected and evaluated in future studies because: (1) AML-DCs may still retain some of the malignant features of AML cells. (2) Whether AML-DCs can induce an anti-AML T-cell response in vivo remains undetermined. (3) Specific TLR agonists may induce the proliferation and survival of AML cells instead of their differentiation. The effects of TLR stimulation or inhibition on AML cells should be carefully studied to determine potential therapeutic usefulness.

Although mutations of TLRs and MyD88 have not been detected in AML patients [ 143], enhanced expression and activation of TLRs-MyD88 and IRAK are commonly detected in a subset of AML cells, primarily samples from patients diagnosed with the M4 and M5 subtypes of AML (Cannova et al., unpublished data). The function of TLR-MyD88 in AML pathogenesis is still being investigated. Most TLR-expressing AML cells also express high levels of pro-inflammatory cytokines, such as TNFα and IL-1α/β. Both TLR agonists and pro-inflammatory cytokines stimulate both NF-κB and JNK-AP1, which in turn promotes survival and proliferative signaling in AML cells. However, the blockage of pro-inflammatory cytokines and TLR-stimulated signaling inhibits only JNK-AP1 signaling without the complete repression of NF-κB activity because almost all hematopoietic cytokines can stimulate NF-κB in AML cells. Interestingly, pro-inflammatory cytokines induce JNK-dependent apoptosis/necroptosis in normal HSPCs. Thus, our study suggested that the inactivation of the TNF/IL1/TLR-JNK signal, together with NF-κB inhibitor treatment, may improve therapeutic success rates for AML subtypes M4 and M5 by enhancing the killing effects on AML cells and simultaneously providing protection to HSPCs [ 144, 145]. Consistent with our results, most MDS samples from the study by Rhyasen et al. were highly sensitive to IRAK inhibitor treatment. However, most AML cells are resistant to IRAK1 inhibition due to compensation provided by the expression of BCL2, a major anti-apoptotic NF-κB target gene [ 146]. Co-treatment of MDS or AML cells with the IRAK1 inhibitor plus ABT-263 (a Bcl2 inhibitor) synergistically inhibits cell proliferation, progenitor function, and survival [ 107]. Activation of TLR signaling is primarily mediated by stimulating the production of the pro-inflammatory cytokines TNF, IL-1, and IL-6. It will be interesting to determine whether the activation of JNK and NF-κB signaling in AML cells by TLRs also depends on inflammatory cytokines.

TLR-MyD88 signaling and related pathologies in B lymphocytes

Expression of TLR1, TLR6, TLR7, TLR8, TLR9, and TLR10 can also be found in B-cells. The expression levels of these TLRs fluctuate during B-cell development, maturation, and activation. These TLRs collaborate with B-cell receptors (BCRs) to regulate downstream signaling activity and serve an important function in B-cell activation, maturation, and memory. Gain-of-function mutations of MyD88 are commonly detected in B-cell neoplasms and act as driver mutations of B-cell malignancies, especially in cases with increased BM lymphoplasmacytic cells and monoclonal IgM gammopathy [ 147]. IgM monoclonal gammopathy of undetermined significance (IgM-MGUS) is a clinically asymptomatic premalignant lymphoplasmacytic proliferative disorder which can progress to symptomatic Waldenström’s macroglobulinemia (WM), lymphoma, AL amyloidosis, or IgM multiple myeloma. This trend may represent a disease progression of clonal plasma cells from premalignant to malignant stages. A MyD88^L265P mutation occurs in 41%-56% of cases of IgM-MGUS [ 148- 152] and 90%-100% cases of WM [ 148, 153- 156]. MyD88^L265P was also detected in 69% of cases of cutaneous diffuse large B-cell lymphoma (CBCL), 38% of cases of primary central nervous system lymphoma (PCNSL) [ 157], 9% of cases of gastric MALT lymphomas, 3%-10% of cases of chronic lymphocytic leukemia [ 158, 159], 7%-13% of cases of splenic marginal zone lymphoma (SMZL) [ 160, 161], and 29% of cases of activated B-cell type diffuse large B-cell lymphoma (ABC-DLBCL) [ 162]. Furthermore, other TIR domain mutations of MyD88, such as S219C, V217F, S219C, M232T, S243N, and T294P, have been reported in activated B-cell-type diffuse large B-cell lymphoma (ABC-DLBCL) [ 162]. Such mutations are gain-of-function driver mutations resulting in constitutive activation of signaling pathways such as NF-κB. For example, MyD88^L265P shows enhanced and possibly spontaneous oligomerization with IRAK4, IRAK1, and TRAF6, which promotes the activation of both Bruton’s tyrosine kinase (BTK) [ 163] and TAK1 [ 156]. Either TAK1 or BTK can activate NF-κB independently [ 162, 163]. NF-κB promotes proliferative and survival activities in neoplastic cells by upregulating the expression of target genes and inducing the production of pro-inflammatory cytokines, such as IL-6, IL-10, and IFN-β. The production of these cytokines stimulates JAK-STAT3 signaling, which collaborates with NF-κB and plays a critical role in the pathogenesis of B-cell neoplasia by forming an autocrine positive feedback loop (Fig. 4). The above studies suggest that the inhibition of both NF-κB and JAK-STAT3 pathways may represent a useful therapeutic strategy against B-cell malignancies with activating MyD88 mutations.

Prospective

TLRs were first identified as innate immune sensors of PAMPs and DAMPs 17 years ago. The function of TLR signaling in granulocyte/monocyte differentiation and DC activation has been well studied. Multiple TLRs are also expressed in normal HSPCs, BM niche cells, and leukemic cells. In the presence of danger signals, TLRs regulate hematopoiesis by either directly activating downstream signaling in HSPCs or indirectly activating HSPCs by inducing the production of inflammatory cytokines in innate immune cells or BM niche cells. Deregulation of TLR signaling has been detected in BMF, MDS, and leukemia. In BMF and MDS patients, deregulated TLR signaling is primarily detected in innate immune cells (including granulocytes, monocytes, DCs, and/or MDSCs), which repress normal HSPCs by secreting inflammatory cytokines. In B-cell lymphomas, activating mutations of MyD88 enhance the proliferation and survival of tumor cells by upregulating downstream signaling. However, the function of TLR signaling in the pathogenesis of AML remains largely unknown. Studies on solid tumors have suggested that TLR signaling can induce either tumor-suppressing or tumor-promoting activities. TLRs promote tumor growth by stimulating proliferative and survival signaling in cancer cells, as well as inducing a tumor-promoting inflammatory environment. The tumor-suppressing activity of TLRs is mediated by DCs, which activate antitumor-specific Th1 cells. In many cases, the tumor-promoting and tumor-repressing activities may be stimulated by distinct TLRs and their downstream signaling pathways. In addition, while the role of MyD88-dependent downstream signaling in multiple tumor types has been better studied, the role, if any, of MyD88-independent signaling mediated by TRIF remains unclear and merits further study. MyD88- and TRIF-mediated signaling demonstrated contrasting effects in RAS-induced pancreatic cancers [ 121]. Therefore, the function of individual TLRs with respect to their downstream signaling pathways in antitumor DCs, growth-promoting activity in leukemic cells, and immune-repressing activity in MDSCs should be determined in future studies. Such information may facilitate the specific enhancement of the anti-leukemic effects of DCs without stimulating survival/proliferative signaling in tumor cells and MDSCs by using specific TLR agonists or to specifically inhibit cancer cell growth without attenuating the antitumor activity of DCs by using TLR antagonists [ 164]. In addition, we need to determine how to enhance the treatment effects of conventional chemotherapeutic agents, radiation therapy, and other immune-editing treatments (e.g., anti-PD1 and anti-CTLA-4) by selectively utilizing TLR agonists and antagonists. Such approaches to treatment can be utilized concurrently to synergize the antitumor effects of conventional therapies or can be used following conventional therapies in order to eliminate or suppress minimal residual disease. Finally, the expression profiles of TLRs between murine DCs and human DCs may differ. Therefore, we can use animal models to develop a conceptual framework for the application of TLR signaling modulators. Nevertheless, the actual selection of TLR agonists or antagonists for patient therapy remains subject to evaluation by clinical trials.

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