Role of innate immunological/inflammatory pathways in myelodysplastic syndromes and AML: a narrative review

TLRs are pattern recognition receptors (PRRs) and major components of the innate immune system. TLRs recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [2]. The drugs targeting TLR pathway have been discussed in Table 1.

MyD88 was originally identified as a myeloid differentiation primary response gene, which is upregulated during IL-6 induced macrophage differentiation [17]. MyD88 is an important component of the signaling pathway mediated by the IL-1 and IL-18 receptors, which are responsible for TH1 cell differentiation and Interferon γ production [18].

The NF-κB signaling pathway is involved in the production of inflammatory cytokines, chemokines, and adhesion molecules, and it also regulates apoptosis, cell proliferation and differentiation, and the activation of macrophages, granulocytes, osteoclasts, dendritic cells, and erythrocytes [27, 28]. NF-κB signaling pathway leads to myeloid differentiation by activating granulocyte macrophage colony stimulating factor (GMCSF), a cytokine that promotes differentiation of bone marrow stem cells towards granulocyte and monocytes [27, 29]. In addition, the NF-κB can also act as both anti-apoptotic or a pro-apoptotic regulatory factor based on cell type and stimuli [30].

TIRAP, also known as MyD88-adaptor Like (MAL), is a key intracellular adaptor molecule not only associated with the signaling of TLR2 and TLR4, but also involved in MYD88 independent inflammatory signaling [34]. The IFN-γ-TIRAP pathway is involved in bone marrow failure and MDS [35]. An analysis of the gene expression in the CD34 + cells of MDS showed that TIRAP expression was increased in del 5q MDS [35, 36]. Mice transplanted with TIRAP-expressing HSPCs showed an overall decreased survival rate due to bone marrow failure and significant pancytopenia [35]. The TIRAP-transduced bone marrow of transplanted mice reduced the number of viable common myeloid progenitors (CMP) and granulocyte–monocyte progenitors (GMP) resulting in pancytopenia [35]. The high mobility group box 1 (HMGB1) belongs to a non-histone protein localized in the nucleus, where it acts as a DNA chaperone to help in DNA repair and maintenance [37]. It can also be seen on an extracellular surface, where it functions as a DAMP. It plays a significant role in inflammatory diseases and cancers and acts as an alarmin in promoting AML progression [37, 38]. The overexpression of TIRAP in hematopoietic cells releases IFN-γ, which can act in two ways. IFN-γ can directly affect megakaryopoiesis and erythropoiesis in the bone marrow, and secondly, it can indirectly suppress myelopoiesis through the release of HMGB1 (alarmin), which disrupts the bone endothelium, resulting in bone marrow failure [35, 36]. In vivo experiments demonstrated that blocking HMGB1 in the presence of TIRAP expression resulted in the reversal of the bone marrow endothelial defect and the restoration of myelopoiesis [36].

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for maintaining HSC quiescence and controlling myeloid-biased differentiation through minimal NF-κB signaling via cyclin dependent kinase inhibitors, which are negative regulators of cell cycle progression [39, 40]. Myeloid-derived suppressor cells (MDSCs) are premature heterogenous group of myeloid cells derived from bone marrow, and they have the ability to suppress the immune responses against a tumor [41]. The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is activated by growth factors and cytokines, and it plays an important role in MDSCs activation, function, and expansion [42, 43]. E3 ubiquitin ligase TRAF-6 binds to STAT3, leading to the phosphorylation of STAT3, which results in the activation of MDSCs [41]. TRAF-6 knockdown in mice attenuated MDSCs role in accelerating tumor progression as the inhibitory effect of MDSCs on CD4 + T cell proliferation was significantly decreased [41]. TRAF-6 expressing bone marrow transplanted mice progressed either to bone marrow failure or developed AML [44]. Activation of the canonical pathway, i.e., TLR4–TRAF6–NF-κB activation has been reported in MDS. The 5q deletion correlated with the loss of two miRNAs (miR-145 and miR-146a), which resulted in an increased expression of TRAF-6 in MDS cells [44]. The knocking down of miRNAs in mice models resulted in significant thrombocytosis, mild neutropenia, and megakaryocytic dysplasia [44]. IL-6 production and persistent elevation seen in TRAF-6 transplanted mice is responsible for platelet survival and production [44].

IL1-receptor associated kinases (IRAK) are a family of intracellular serine threonine kinases that play a significant role as signal transduction mediators of TLR and interleukin–1 receptor signaling [45]. More recently, some groups have reported that IRAK 1 regulates the rapid NLRP3 inflammasome assembly and caspase 1 cleavage in a transcription independent fashion. NRLP3 assembly is dependent on the catalytic competency of both IRAK1 and IRAK 4 [46, 47] (Fig. 2).

Transforming growth factor beta (TGF-β) is a large superfamily of growth factors that includes activins and bone morphogenetic proteins. They play a role in maintaining the proliferation and differentiation of the HSC [70]. The TGF-β family plays an important role in embryogenesis, as alterations in the pathway are associated with developmental abnormalities, autoimmune disorders, and carcinogenesis [71]. TGF-β ligands bind to cell surface receptors which are classified as type I and type II. The majority of the pathway signaling occurs via a type I receptor activin like kinase 5 (ALK 5) or TβRII [72] (Fig. 1). Upon activation, these receptors fire the downstream SMAD signaling circuit, which functions as the major signaling pathway of the TGF-β pathway [72]. The SMAD family includes eight subtypes (SMAD 1–8) with varying functions that can be either activating (SMAD 2/3) or inhibitory (SMAD 6/7) in nature [73]. TGF-β functions as a major inhibitor of the HSCs via the upregulation of cyclin-dependent kinase inhibitors, such as p15Ink4b, p21Cip1, and p27Kip1 and, in turn, maintains the quiescent state of HSC and prevents its loss [74]. SMAD signaling also depends on other coregulatory signals in the bone marrow niche, such as the nuclear protein Transcriptional Intermediary Factor 1γ (TIF1γ) and SMAD 4. In vivo models have shown that SMAD 4 inhibits the SMAD 2/3 complex, resulting in the suppression of HSC transcription, but TIF1γ competes with SMAD 4 for binding to the phosphorylated SMAD 2/3 complex and thereby promotes erythroid differentiation [75]. The pathway is also regulated by inhibitors such as SMAD7 and Ski protein, corepressor for SMAD-4 [76, 77].

The inhibitory TGF-β pathway is normally tightly regulated, but chronic TGF- β signaling is noted early in the course of MDS. Alterations in SMAD 7 and SKI proteins result in the sustained activation of pathways and lead to ineffective erythropoiesis [78]. Human CD 34 + cell lines have also shown elevated levels of microRNA 21 (miR-21) that cause decreased levels of SMAD 7, culminating in the decreased formation of erythroid colonies as noted in a comparison of MDS patients to healthy controls [78]. Similarly, SMAD 2 is upregulated in MDS, and the in vivo pharmacologic inhibition of ALK 5 or the chemical inhibition of miR-21 increased hemoglobin levels in a mouse model [79]. Murine models have shown that the trapping of Activin and GDF11 with RAP-536 inhibits the activation of SMAD 2/3 proteins and helps relieve the ineffective erythropoiesis seen in MDS [80]. In the MEDALIST trial, Luspatercept significantly reduced transfusion burden in low-risk MDS patients with ring sideroblasts refractory or intolerant or unlikely to respond with erythropoiesis-stimulating agents [81]. Luspatercept is a recombinant fusion protein of the extracellular domain of human Activin receptor 2B and IgG1 Fc domain which binds TGF β ligands to reduce SMAD2 and SMAD3 signaling resulting in erythroid differentiation [81].

S100 proteins are calcium-binding cytosolic proteins labeled due to their solubility in 100% saturated ammonium sulfate [82, 83]. Members of the S100 family perform a wide range of functions at both the extracellular and intracellular levels, including cell proliferation, differentiation, apoptosis, migration, inflammation, calcium homeostasis, and enzyme regulation [84]. S100A8 also called the migration inhibitory factor-related protein-8 (MRP-8)/calgranulin A, and S100A9, also called the migration inhibitory factor-related protein-14 (MRP-14)/calgranulin B, act as alarmins or DAMPs [82, 84]. They are released from immunocytes and circulate in the plasma as homodimers or as S100A8/A9 heterodimers also called calprotectin. The levels of S100A8 and S100A9 are high in autoimmune conditions, inflammation, and cancer [82, 84]. They are predominantly expressed in neutrophils, monocytes, and dendritic cells. S100A8 and S100A9 constitute around 45% and 1% of the cytosolic proteins in neutrophils and monocytes, respectively. However, upon activation by cell damage/stress, they are also expressed in various other cells such as mature macrophages, fibroblasts, erythroblasts, and vascular endothelial cells [85].

When S100 proteins are released from immunocytes due to cell damage/stress, they act as danger signals and play a significant role in inflammatory response facilitating apoptosis, autophagy, chemotaxis, invasion, and differentiation [85]. S100A8/A9 proteins bind to TLRs, CD33 and the receptor for advanced glycation end products (RAGE), thereby mediating several downstream effects such as the generation of inflammatory cytokines such as IL-6, TNF alpha, and IL-1 Beta, which in turn stimulate NF-κB activation leading to activation of inflammasome and osmotic lysis [64, 85, 86] (Fig. 1), (Fig. 2).

In an in vivo study conducted by Cheng et al. (2013), it was reported that the S100A9/CD3 pathway caused the stimulation of polyclonal MDSCs (CD33+/Lin−/HLA − DR−) in the bone marrow and was responsible for hematopoietic senescence in MDS. CD3 is a member of the sialic acid-binding Ig-like superfamily of lectins (Siglec) and possesses an immunoreceptor tyrosine-based inhibition motif (ITIM) which is associated with immunosuppression [64, 87]. When S100A9 binds to CD3, this ligand-receptor complex causes ITIM mediated production of immunosuppressive cytokines like IL-10 and TGF-β, which repress erythropoiesis. Data also shows the restoration of hematopoiesis by breaking the S100A9/CD3/TLR4 circuit. Hence, S100A9 can be an initiating factor for the sequence of inflammatory responses leading to defective erythropoiesis in MDS [64, 87]. Furthermore, a study was conducted by Schneider and his colleagues to investigate the 5q deletion phenotype of MDS. This study particularly focused on the molecular consequences of ribosomal protein small subunit 14 (Rps14) haploinsufficiency that happens with 5q deletion using a conditional-knockout mouse model [88]. Data showed that the Rps14 haploinsufficient bone marrow cells caused stress leading to the overexpression of S100A8/9 proteins in monocytes, macrophages, and late-stage erythroblasts. This process in turn induced the expression of P53, mediating erythroid differentiation defects. A pharmacological intervention targeting the inactivation of S100A8/A9 could improve erythropoiesis in 5qdel MDS [88].

Interleukin-8 (IL-8) is a member of the CXC chemokine subfamily, produced by blood cells, and it acts on neutrophils, attracting them to the site of inflammation [89]. IL-8 activates multiple intracellular signaling pathways in the neutrophils, allowing their pathophysiological role such as neutrophil degranulation and chemotaxis [90]. Elevated levels of IL-8 and its receptors have been reported in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, thereby activating multiple downstream signaling pathways, such as serine/threonine kinases which increase MAPK signaling and the activation of IL-8 in ovarian and lung cancer cell lines mediates angiogenesis, cell motility, and invasion [91]. Due to the hypoxia in the bone marrow microenvironment, high levels of expression of IL-8 are noticed in AML cell lines and are associated with a poor prognosis [92]. The role of IL-8 in MDS is evolving, and the overexpression of the IL-8 receptor CXCR2 was observed in AML/MDS CD34 + stem cell lines as compared to healthy CD34 + controls. CXCR2 inhibition in these cell lines led to selective inhibition of immature hematopoietic stem cell lines, sparing the control cell lines and providing a potential therapeutic target [93]. Drugs targeting IL-8 and IL-8 receptors are mentioned in Table 1.

Innate inflammatory signaling also influences the differentiation and expansion of hematopoietic stem cells (HSCs) and can be differentially activated in low-risk and high-risk MDS phenotypes [94, 95]. Apoptotic pathways predominate in bone marrow in low-risk MDS phenotypes while high-risk MDS clones are able to evade the immune system aiding its progression to AML [96]. The most common cytogenetic abnormality in lower risk MDS, del(5q), is associated with dysregulated innate immune pathways as many genes involved in innate immune signaling, such as DIAPH1, TIFAB, MiR-146a, etc. are in close proximity to the 5q region [97]. In higher risk MDS, T regulatory cells predominate leading to suppressed immune surveillance and hence disease progression [98]. Several studies have also reported that autoimmune diseases cluster in high-risk MDS phenotypes suggesting a common inflammatory link in the pathogenesis of these diseases [99‐101]. VEXAS (Vacuoles, E1 enzyme, X linked, autoinflammatory, somatic) syndrome is an example dysregulated innate pathway that links autoimmune disease with MDS. This syndrome is due to somatic mutation of Ubiquitinin-like modifier activating enzyme 1 gene (UBA1) located on X chromosome leading to upregulation of unfolded protein accumulation and increased stress in myeloid cells causing bone marrow failure [102].