Macrophages in post-chemoradiotherapy hematopoietic injury: a double-edged sword

Abstract

Radiotherapy and chemotherapy are primary cancer treatments, yet hematopoietic damage induced by these therapies is increasingly prevalent. Under physiological conditions, macrophages serve as key regulators of hematopoietic stem cells (HSCs) and the hematopoietic microenvironment. Through bidirectional regulatory mechanisms driven by functional heterogeneity among macrophage subtypes, they dynamically modulate hematopoiesis. This includes maintaining HSCs quiescence and mobilization, ensuring HSCs quality control, regulating HSCs localization, and balancing inflammatory responses with microenvironmental remodeling within the hematopoietic niche. Following chemoradiation therapy, macrophages exert bidirectional effects on hematopoietic damage. On one hand, they exacerbate injury by intensifying HSCs damage, disrupting hematopoietic microenvironment homeostasis, and inducing myeloid bias. On the other hand, they support hematopoietic recovery through mechanisms such as maintaining HSCs function and promoting hematopoietic niche reconstruction. Furthermore, we summarize the application potential of natural products and small molecules based on macrophage regulation in improving hematopoietic damage after chemoradiotherapy, aiming to provide new insights for developing clinical treatment strategies. Future research should further elucidate the molecular mechanisms of macrophage regulation of hematopoiesis after chemoradiotherapy and explore precision therapeutic strategies targeting macrophages to optimize prevention and treatment protocols for bone marrow hematopoietic damage following chemoradiotherapy.

Graphical abstract

Introduction

The bone marrow hematopoietic microenvironment serves as the core ecosystem sustaining hematopoietic homeostasis, providing structural support and signaling regulation for HSCs self-renewal and differentiation. Within this microenvironment, distinct niches exist including the vascular niche and the endosteal niche [1], with the former further subdivided into sinusoidal and arterial vascular niches [2, 3]. As the “seed cells” of the hematopoietic system, HSCs undergo precise regulation by stromal cells, cytokines, and physical cues within this microenvironment during their quiescence, mobilization, localization, and quality control. Hematopoietic stem/progenitor cells (HSPCs) represent a broader population encompassing HSCs and their downstream progenitors at various differentiation stages. Among all cells constituting this complex regulatory network, bone marrow macrophages play indispensable multifaceted roles. bone marrow macrophages exhibit high heterogeneity, categorized into BM-resident macrophages, erythropoietic island (EBI) macrophages, bone macrophages, osteoclast precursors, and other subtypes such as CD169⁺ macrophages [4]. Among these, macrophages are not only an innate immune component of the microenvironment but also key cells involved in regulating the HSCs quiescence-mobilization balance, HSCs quality control, HSCs localization, and maintenance of bone marrow niche homeostasis [5].

Radiotherapy and chemotherapy, as potent stressors, disrupt the three-dimensional structure of the bone marrow microenvironment, leading to HSCs injury, stromal cell dysfunction, vascular system damage, and metabolic homeostasis imbalance. This systemic injury directly impairs hematopoietic capacity, triggering hematopoietic failure. Under physiological conditions, macrophage polarization exerts bidirectional effects on hematopoiesis, both promoting and suppressing it [6]. Notably, the dual role of macrophages in post-chemoradiotherapy hematopoietic recovery stems from their functional heterogeneity and microenvironmental dependency. This duality positions them as critical intervention points for mitigating hematopoietic damage—by modulating macrophage polarization or functional activity, both persistent inflammatory damage to HSCs can be reduced and microenvironmental reconstruction promoted, thereby breaking the vicious cycle of hematopoietic suppression.

Current research indicates that macrophages can alleviate or exacerbate hematopoietic damage by directly regulating HSCs or indirectly altering the hematopoietic microenvironment, though the regulatory boundaries of this process remain unclear. This paper systematically explores the mechanisms by which macrophages influence hematopoietic tissues after chemoradiotherapy, analyzes their regulatory effects on HSCs and the hematopoietic microenvironment, and proposes drug intervention strategies, offering new insights for hematopoietic function recovery.

Regulatory effects of macrophages on hematopoietic stem cells and the hematopoietic niche

Macrophage regulation of hematopoietic stem cells

HSCs quiescence and mobilization

Macrophages play a crucial role in maintaining the steady state of HSCs quiescence and mobilization. Within the bone marrow microenvironment, macrophages maintain HSCs quiescence through paracrine signaling and cell-cell contacts (Fig. 1A). Hypoxia and low levels of reactive oxygen species (ROS) jointly sustain HSCs quiescence, whereas elevated ROS levels drive HSCs differentiation and mobilization [5, 7]. Studies indicate hypoxia induces macrophages to overexpress Meteorin (Metrn), which binds to serotonin receptor 2b (Htr2b) on HSPCs surfaces, activating downstream phospholipase C (PLC) signaling pathways to reduce intracellular ROS levels [8]. Macrophages also maintain low ROS levels in the hematopoietic microenvironment by expressing cyclooxygenase-2 (COX-2) and alpha-smooth muscle actin (α-SMA), thereby contributing to HSCs quiescence [9]. Concurrently, COX-2/α-SMA⁺ bone marrow macrophages regulate HSCs quiescence and mobilization by responding to norepinephrine (NE) during the day to increase TNF/ROS levels and promote HSCs mobilization, while responding to melatonin at night to reduce ROS levels and maintain HSCs quiescence [10]. This rhythmic regulation highlights the core role of macrophages as microenvironment “orchestrators.” DARC/CD234⁺ macrophages interact with the LT-HSCs transmembrane protein CD82 to maintain LT-HSCs quiescence [11]. Further studies revealed that the DARC/ACKR1⁺ macrophage subset directly anchors LT-HSCs through physical interaction (ACKR1-CD82), overlapping with the α-SMA⁺COX-2⁺ phenotype, and synergistically suppresses ROS via Prostaglandin E₂ (PGE₂) secretion [12]. Specific knockout of macrophage ACKR1 leads to loss of LT-HSCs quiescence and myeloid hyperproliferation, confirming the functional importance of this subset. This discovery provides a novel perspective on how macrophages regulate HSCs function through protein homeostasis. Functional heterogeneity among macrophage subpopulations further manifests as M2 macrophages maintaining HSCs quiescence by consuming arginine to reduce NO production through high Arg1 expression, thereby stabilizing HIF-1α protein expression. Conversely, M1 macrophages disrupt HIF-1α stability by generating NO via high NOS₂ expression, inhibiting HSCs quiescence maintenance [13]. Moreover, endosteal macrophages support osteoblast function, thereby maintaining HSCs quiescence and the stability of their bone marrow microenvironment. Depletion of macrophages leads to osteoblast functional suppression and decreased expression of HSCs niche factors (e.g., CXCL12, KL, Ang-1), ultimately triggering HSCs migration from bone marrow into the bloodstream (i.e., “mobilization”) [14]. These mechanisms, along with the phenomenon of CD169⁺ macrophage depletion inducing HSCs mobilization, collectively reveal the crucial regulatory role of macrophages in HSCs quiescence and mobilization.

Fig. 1.

Regulatory roles of macrophages in hematopoietic stem cells and the hematopoietic niche. A HSCs quiescence and mobilization; (B) HSCs quality control; (C) HSCs localization; (D) Macrophage regulation of the bone marrow niche. Graphics created using BioRender.com

HSCs quality control

Macrophages, as key regulators of the HSCs niche, exert quality control over HSCs through multiple mechanisms (Fig. 1B). Studies reveal that approximately 20–30% of HSPCs interact with macrophages at any given time point, with data indicating that macrophages preferentially regulate active or stressed HSPCs [15]. During inflammation or mobilization states, HSCs temporarily upregulate CD47 expression on their cell membranes to evade phagocytosis by macrophages. Notably, leukemic progenitor cells also upregulate CD47 expression, enabling them to evade immune surveillance and escape clearance by macrophages [16]. Further studies reveal that HSCs with elevated ROS levels emit a “phagocytic signal” via surface calreticulin, triggering macrophage clearance. Concurrently, β-2-microglobulin (B2m) serves as a “don’t eat me” signal, assisting macrophages in distinguishing healthy from damaged HSCs [17, 18]. Studies indicate that the expression level of calreticulin on the HSCs surface is a key signal for macrophage recognition and interaction: high expression leads to clearance, while moderate expression promotes proliferation [19]. However, zebrafish embryonic macrophages lack CD47. Instead, zebrafish macrophages express LRP1ab and C1qa, which bind to calreticulin to form a “phagocytic signal complex.” Thus, calreticulin-LRP1ab/C1qa constitutes the primary pathway regulating HSCs clearance in zebrafish [15]. Further analysis reveals that macrophages secrete interleukin-1β (IL-1β), activating the ERK/MAPK pathway in HSPCs to support healthy HSCs expansion [15, 19].

HSCs localization

The close interaction between macrophages and HSCs in the perivascular region begins during the embryonic stage. During embryonic development, macrophages play a crucial role in guiding the generation of the first batch of HSCs and their colonization within the nascent vascular niche [20–22]. This core function persists and becomes specialized in adult bone marrow. Extensive past research using mouse models indicated that HSCs primarily localize to the perivascular regions of bone marrow, particularly the perivascular spaces adjacent to arterioles and sinusoids [3]. However, recent human bone marrow studies combining single-cell RNA sequencing (scRNA-Seq) and in situ protein imaging (CODEX) technology have significantly revised this understanding. In adult human bone marrow, HSPCs do not preferentially occupy the traditionally recognized periarterial or periboneal niches. Instead, they preferentially localize around adipocytes. HSPCs entering early myeloid differentiation stages migrate to proliferate in the arterial-periboneal region, ultimately maturing near sinusoids [23]. This study also robustly confirms the close spatial proximity between HSPCs and macrophages [23]. This arrangement ensures HSCs can precisely receive key cytokines provided by different microenvironment cell subpopulations. Among these, the chemokine axis CXCR4-CXCL12 serves as the core signaling pathway regulating HSCs residency [24].

Within this framework, bone marrow macrophages, as a vital component of the niche, regulate HSPCs localization and retention through multiple direct mechanisms. First, macrophages serve as physical “anchors” for HSCs. For instance, specific VCAM-1⁺ macrophage subpopulations interact with integrin α4 (ITGA4) on HSPCs via their surface adhesion molecule VCAM-1, directly anchoring HSPCs within the vascular microenvironment [25]. Second, macrophages actively “modify” HSCs chemotaxis. Studies reveal they transfer membrane proteins like CXCR4 to HSCs via trogocytosis, a process regulated by the tyrosine kinase C-Kit, thereby determining HSCs marrow residency [26]. This process, accompanied by CXCR4 transfer, significantly enhances HSCs’ chemotactic response to CXCL12, promoting precise retention in the perivascular regions of the bone marrow while inhibiting migration into peripheral blood. Third, within the bone marrow macrophage–osteoblast niche, p62 inhibits the IKK/NF-κB signaling pathway via autophagy-dependent mechanisms, promoting osteoblast differentiation and Ccl4 expression. This enhances HSPCs’ chemotaxis toward CXCL12, sustaining their retention in the bone marrow. Conversely, p62 deficiency leads to enhanced NF-κB signaling and reduced Ccl4 expression, ultimately causing short-term hematopoietic stem cells and myeloid progenitor cells to escape the bone marrow [27]. In summary, macrophages in the adult bone marrow are not passive bystanders. Instead, they play an indispensable role in maintaining the stable residency of HSCs in the perivascular niche through direct anchoring and active enhancement of chemotactic responses.

Regulatory role of macrophages in the bone marrow niche

The bone marrow niche is a dynamic microenvironment composed of niche cells such as mesenchymal stem cells (MSCs), endothelial cells, and osteoblasts, along with the extracellular matrix (ECM). Its biochemical signals and mechanical properties jointly regulate HSCs fate. Recent studies reveal that macrophages, as key regulators of the niche, play central roles both structurally and functionally (Fig. 1D). Structurally, osteal macrophages are closely juxtaposed with osteoblasts in anatomical position, forming a “canopy”-like structure over the osteogenic zone to enable close-range regulation of osteoblasts [27, 28]. Functionally, macrophages significantly support osteoblast growth, differentiation, and function by secreting soluble nutrients. They also promote osteoblast secretion of CXCL12—a key chemokine sustaining HSCs function—thereby indirectly reinforcing niche architecture [29]. Furthermore, macrophages serve as the core “energy metabolism regulatory center” of the microenvironment, specifically by undergoing metabolic shifts in response to ECM viscoelasticity. For instance, rapid stress relaxation inhibits glycolysis and enhances oxidative phosphorylation (OXPHOS) via the VASP/HIF1α signaling axis, driving macrophage polarization toward the M2 phenotype (CD206⁺ARG1⁺). This significantly upregulates TGF-β1 expression, thereby recruiting MSCs and promoting their osteogenic differentiation [29]. Beyond secreting various factors, macrophages exchange information with stromal cells by releasing nanoscale vesicles. In recent years, extracellular vesicles (EVs) have garnered significant attention as crucial information carriers. EVs represent a collective term for lipid membrane vesicles secreted by cells, primarily comprising subtypes such as exosomes and microvesicles. For instance, M1 macrophage-derived EVs (rich in exosomes) induce bone marrow MSCs to differentiate into osteoblasts by delivering microRNA-21a-5p during early inflammation [30]. More importantly, macrophages can respond to external mechanical stimuli by secreting a previously unknown mechanosensitive lipolytic factor—reticulum calcium-binding protein 2 (RCN2). Secreted RCN2 binds to a receptor complex composed of Neuropilin 2 (NRP2) and integrin β1 (ITGB1), activating the cAMP-PKA signaling pathway to efficiently mobilize bone marrow adipose tissue (BMAT) for lipolysis [31]. The lipids released during this process provide critical energy substrates for osteogenesis and the proliferation and differentiation of lymphoid progenitor cells (CLPs) [31]. Furthermore, macrophages interact with neutrophils and endothelial cells, forming a complex and finely tuned niche homeostasis regulatory network. Specifically, macrophages convert arachidonic acid (AA) supplied by neutrophils into prostaglandin H2 (PGH₂), which endothelial cells further transform into prostaglandin E (PGE₂) released into the microenvironment. This ultimately promotes HSCs expansion to maintain bone marrow niche homeostasis [32].

Under physiological homeostasis, macrophages play an indispensable central role in maintaining HSCs quiescence, implementing quality control, guiding their correct localization, and coordinating bone marrow niche homeostasis through their highly heterogeneous subpopulations and finely tuned regulatory networks [5, 7, 16, 24, 32]. However, chemoradiotherapy, as a potent exogenous stressor, completely disrupts this meticulously maintained microenvironmental homeostasis. At this juncture, the intrinsic functional programs of macrophages become abnormally activated, highlighting their “double-edged sword” nature. On one hand, mechanisms that physiologically address localized injury and clear abnormal cells become disproportionately amplified against the backdrop of systemic damage induced by chemoradiotherapy. This shift from “precise regulation” to “broad attack” exacerbates HSCs injury, disrupts microenvironmental homeostasis, and induces myeloid differentiation bias [33–35]. On the other hand, their inherent tissue repair and regenerative support functions—such as providing protective contact, clearing apoptotic debris, and secreting proangiogenic and osteogenic factors [36, 37]—become the cellular foundation for promoting hematopoietic reconstruction. Thus, the dual role of macrophages post-chemoradiation therapy fundamentally reflects the imbalance and reemergence of different facets of their physiological functions under extreme pathological conditions.

Bidirectional effects of macrophages on hematopoietic reconstruction after radiotherapy

Suppression of hematopoietic reconstruction

Exacerbation of HSCs damage

Recent studies indicate that abnormal macrophage activation in the post-radiation bone marrow microenvironment is a key mediator of HSCs damage (Fig. 2A). Ionizing radiation (IR) induces macrophage proinflammatory cytokine secretion and ROS bursts, directly disrupting the quiescent state of HSCs [33]. IR-mediated macrophage effects on HSCs occur in a time-dependent sequence, causing early and late-phase damage. Early damage arises from IR directly inducing ROS bursts within LT-HSCs while simultaneously activating CD169⁺ macrophages to acquire an M1 phenotype. These activated macrophages express iNOS and release NO, which reacts with ROS to generate ONOO⁻, leading to early HSCs apoptosis [38, 39]. However, N-acetylcysteine (NAC), a ROS scavenger, fails to completely block apoptosis, indicating the existence of ROS-independent pathways such as the mitochondrial apoptosis pathway. Furthermore, NO/ONOO⁻-induced apoptosis may be associated with p53 [40]. High concentrations of ONOO⁻ also induce protein tyrosine nitration, lipid peroxidation, and DNA single-strand breaks while inhibiting erythroid progenitor cell growth [41], further impairing hematopoietic function. Notably, the Labile Iron Pool (LIP) in macrophages possesses ONOO⁻ scavenging capacity with catalytic efficiency reaching 10⁶–10⁷ M⁻¹s⁻¹ [42]. IR may indirectly exacerbate HSCs damage by depleting macrophage LIP and thereby weakening antioxidant defenses.

Fig. 2.

Macrophage-mediated suppression of hematopoietic reconstitution following radiotherapy. A Exacerbates HSCs damage; (B) Disrupts hematopoietic microenvironment homeostasis; (C) Induces myeloid bias. Graphics created using BioRender.com

Macrophage-mediated late-stage HSCs damage involves sustained TNF-α secretion by CD169⁺ macrophages, inducing endoplasmic reticulum (ER) stress and mitochondrial dysfunction, leading to secondary ROS bursts. Late-stage HSCs hematopoietic damage is entirely ROS-dependent [38, 39]. ROS manifestation disrupts key signaling pathways including p38 MAPK, PI3K/Akt/mTOR, thereby inhibiting HSCs self-renewal and promoting differentiation or depletion [37, 43–46], and by inducing genomic DNA damage and genetic instability in HSCs via oxidative stress (OS) [47, 48]. Notably, molecules such as 27-hydroxy cholesterol (27HC) can influence HSCs fate by regulating ROS levels, offering novel insights for targeted interventions [44]. Furthermore, TNF-α forces HSCs into the cell cycle and biases myeloid differentiation by activating the PU.1 transcription factor. However, this abnormal differentiation exacerbates HSCs depletion and bone marrow suppression before TNF-α levels normalize, ultimately leading to hematopoietic failure [49]. This suggests that early blockade of macrophage activation or TNF-α signaling is crucial for preventing long-term hematopoietic dysfunction. Furthermore, the bystander effect of macrophages post-radiation therapy creates a chronic inflammatory microenvironment through the paracrine cycle, continuously releasing ROS, NO, and TNF-α [42]. ROS and NO sustain each other’s production via positive feedback loops, while TNF-α further amplifies inflammatory signaling. Research indicates that this macrophage-mediated bystander effect may exhibit genotype-dependent characteristics, with significant variations in apoptotic responses, macrophage activation, and oxidative stress capacity. These findings underscore the critical role of genetic background in the long-term consequences of radiation exposure. For example, CBA/Ca and C57BL/6 are distinct mouse strains. The genetic background of CBA/Ca mice predisposes them to leukemia following radiation exposure, whereas C57BL/6 mice exhibit greater resistance due to enhanced anti-apoptotic mechanisms and macrophage responses [33, 50, 51].

Disruption of hematopoietic microenvironment homeostasis

Radiation injury significantly impairs the host’s immune defense against bacterial pathogens, with dysfunction of macrophages—key immune effector cells—being a major mechanism (Fig. 2B). Studies indicate that radiation damage not only delays the resolution of inflammatory responses but also directly impairs phagocytic function in monocyte-derived macrophages [52]. This functional impairment may relate to the anti-radiation mechanisms of macrophages. To maintain survival, post-radiation macrophages reduce metabolic activity, manifested by decreased glucose uptake, significantly lowered ATP levels, and downregulated expression of D-3-phosphoglycerate dehydrogenase (an enzyme metabolizing glycolytic intermediates). Concurrently, downregulation of cathepsin D and upregulation of transferrin receptor 1 (TfR1/CD71) emerge as key features of macrophage radiation resistance [53]. Notably, studies reveal that monocytes differentiated into macrophages can efficiently repair radiation-induced DNA damage by upregulating repair proteins such as XRCC1 and PARP-1 [54]. Furthermore, bone marrow macrophages undergo pyroptosis via activation of the NLRP3/caspase-1 axis [55]. It has been demonstrated that miR-223-3p suppresses NLRP3 inflammasome activation in macrophages, thereby mitigating radiation-induced inflammatory responses [56]. Following ionizing radiation, impaired macrophage phagocytic function or even pyroptosis leads to diminished support for hematopoietic function. Conversely, studies reveal that the intrinsic liver X receptor (LXR) signaling pathway in macrophages serves as a critical hub regulating their post-radiation survival and inflammatory phenotype [57]. LXR deficiency or inhibition significantly exacerbates DNA damage in bone marrow-derived macrophages (BMDMs), manifested by elevated γ-H2AX and p53 levels and accelerated pyroptosis, while simultaneously driving polarization toward an M1 pro-inflammatory phenotype with marked upregulation of genes encoding interleukin-6 (IL-6), IL-1β, and iNOS [57]. Beyond transient local irradiation-induced inflammation, a second wave of inflammatory signaling was observed, including a marked increase in CCL3 one week post-targeted irradiation (TR) and significantly elevated IL-1β in the contralateral (CONT) bone marrow at three weeks. These persistent local and distant inflammatory signals likely collectively contribute to the sustained depletion of HSCs populations [34]. Inflammatory events such as IL-6 and IL-1β are key drivers of HSCs functional decline and accelerated hematopoietic aging. Under inflammatory stimulation, some HSCs become activated and proliferate; however, these proliferating HSCs do not undergo true self-renewal but instead generate functionally impaired progeny cells [58].

Post-radiation therapy red blood cell (RBC) damage and iron metabolism disruption are key drivers of bone marrow hematopoietic dysfunction. As central regulators of iron homeostasis, dysregulated macrophage function further exacerbates this vicious cycle in the hematopoietic system. Following radiotherapy, ionizing radiation directly damages red blood cells (RBCs), causing hemoglobin (HGB) denaturation and triggering hemolysis, which releases large amounts of free iron (Fe²⁺/Fe³⁺) into the circulatory system [59]. Under normal conditions, macrophages maintain hematopoietic homeostasis by phagocytosing senescent or damaged RBCs and regulating the expression of iron export proteins (FPN) [60]. When autophagy is impaired by radiation or inflammation, FPN cannot be effectively degraded, leading to uncontrolled iron leakage from macrophages. This creates the paradoxical situation of “intracellular iron deficiency with systemic iron overload” [61]. Dysfunctional macrophages fail to supply essential iron to recovering erythroid progenitor cells, resulting in “functional iron deficiency” that impedes erythroid hematopoiesis. Furthermore, studies indicate that radiation not only suppresses ferritinoportein 1 (Fpn1) expression [62] but also upregulates transferrin receptor 1 (TfR1) [53]. This dual effect impedes iron efflux from macrophages while increasing uptake, triggering abnormal intracellular iron retention [63] and subsequently inducing localized iron deficiency and systemic anemia [60]. This iron overload state further promotes ROS accumulation via the NOX4/ROS/P38 MAPK signaling pathway, impairing HSPCs function and delaying hematopoietic recovery in the BM [64, 65]. Moreover, iron deposition triggers ferroptosis in macrophages within the spleen and BM [59, 66, 67], significantly diminishing their capacity to clear senescent red blood cells and support HSPCs. Conversely, high-dose radiation induces depletion of CD169 ⁺ macrophages, directly impairing their iron recycling function [68]. Notably, ionizing radiation alone induces M1 polarization of macrophages, whereas iron synergizes with radiation to promote M2 polarization [59]. Collectively, our study reveals a radiation therapy-triggered positive feedback loop: post-radiation erythrocyte hemolysis releases iron, which induces macrophage dysfunction and ultimately exacerbates hematopoietic damage [60–65]. At the therapeutic level, this mechanism suggests iron chelators or antioxidants may hold intervention potential. The depletion of erythroid progenitor cells following radiotherapy also weakens the indirect regulatory role of erythropoietin (EPO), leading to uncontrolled clearance of transfused red blood cells by macrophages. This manifests as reduced red blood cell survival rates 24 h after transfusion [69], providing a theoretical basis for combined therapy using EPO alongside iron metabolism modulators.

At the mechanical microenvironment level, radiation may disrupt adhesion between macrophages and Ter119⁺⁺ erythroid precursor cells (R4/R5 stage) within erythropoietic islands by interfering with Palladin-mediated actin assembly, leading to anemia or erythropoietic defects [35]. while increased collagen and fibronectin deposition following macrophage activation [70] may further impair HSPC function by altering the physical properties of hematopoietic tissues. These findings offer novel insights for exploring therapeutic strategies targeting extracellular matrix remodeling.

Induction of myeloid bias

Under steady-state conditions, macrophages precisely regulate HSCs differentiation through multiple mechanisms, maintaining myeloid-lymphoid balance to ensure normal hematopoietic function (Fig. 2C). However, this regulatory network undergoes significant alterations post-irradiation. Decreased clearance capacity of aged bone marrow macrophages, coupled with elevated IL-1β levels, drives HSCs toward an aged phenotype. This is manifested by upregulation of CD41 and CD61 expression, indicating a marked increase in differentiation bias toward the megakaryocyte (Mk) lineage [71]. MS4A3 (Membrane Spanning 4-Domains A3) plays a critical role in myeloid cell differentiation, and its abnormal expression is associated with leukemia [72]. ROS has been shown to promote myeloid differentiation while suppressing lymphoid differentiation by activating the TGF-β/p38MAPK pathway. MS4A3, as an upstream regulator, modulates ROS levels to drive this process [72]. Concurrently, NO produced by macrophages inhibits CD8 + T cell proliferation and activation [73, 74], further weakening immune surveillance against abnormal clones. Persistent myeloid differentiation advantage leads to accumulation of clonal myeloid precursor cells, accompanied by DNA repair defects and epigenetic dysregulation (e.g., DNMT3A or TET2 mutations), ultimately increasing leukemia risk through genomic instability. Notably, this radiation-induced macrophage-mediated myeloid bias exhibits individual variability. For instance, bone marrow macrophages from radiation-sensitive CBA/Ca mice readily polarize toward a proinflammatory (M1) phenotype, closely associated with acute myeloid leukemia (AML) development [33, 50, 75]. Reprogramming macrophages to a arginase-1 positive phenotype using donor MSCs improves MDS/MPN-like leukemia [76], suggesting therapeutic potential in targeting macrophage polarization. Furthermore, post-radiation M1 macrophages mediate sustained p53 activation via oxidative stress, potentially promoting leukemic transformation [75]. This indicates that myeloid bias serves not only as a marker of microenvironmental disruption but also as a “pre-transformation window” for malignant leukemization.

Promoting hematopoietic reconstruction

Protecting and maintaining HSPCs

Following radiotherapy, the α-SMA⁺COX-2⁺ macrophage subpopulation in the bone marrow microenvironment establishes direct physical contact with HSCs by forming specialized bicellular structures (cytoplasmic protrusions enveloping SLAM⁺ hematopoietic stem/progenitor cells). This interaction is crucial for maintaining HSCs undifferentiated status and radiation protection [36] (Fig. 3A). Notably, this macrophage subset exhibits marked radiation resistance. Following sublethal irradiation at 600 cGy, their COX-2 expression further increases. By activating the PGE₂-Akt-ROS signaling pathway, they effectively reduce ROS accumulation within HSPCs, thereby safeguarding the stability of the LSK CD34⁻ROSlo primitive hematopoietic stem cell pool [36]. Furthermore, 15-prostaglandin dehydrogenase (15-PGDH) is highly expressed in splenic macrophages. By inhibiting PGD^Hi and elevating PGE₂ levels, it significantly promotes HSPC expansion and post-transplant hematopoietic reconstitution [77]. Moreover, during γ-irradiation, macrophage-mediated protein tyrosine phosphatase 1B (PTP1B) suppresses ROS accumulation by regulating glutathione (GSH) synthesis and antagonizes Bax/Bcl-2 pro-apoptotic signaling, thereby preventing exacerbated hematopoietic damage. Conversely, PTP1B deficiency increases macrophage radiosensitivity, manifesting as impaired GSH synthesis, ROS accumulation, DNA repair defects (abnormal γ-H2AX phosphorylation and 8-hydroxyguanosine accumulation), and cellular senescence (elevated β-galactosidase activity). This ultimately diminishes their supportive capacity for HSPCs, impairing hematopoietic regeneration [78].

Fig. 3.

Macrophages promote hematopoietic reconstitution after radiotherapy. A Protect and maintain HSPCs; (B) Support hematopoietic niche restoration. Graphics created using BioRender.com

Macrophage functional heterogeneity plays a crucial role in HSCs maintenance. EBI macrophages mediate the targeted transfer of mitochondria to early erythroid progenitor cells via the CD47-SIRPα signaling pathway to maintain their metabolic homeostasis [79]. Additionally, monocyte-derived DCs promote early erythroid progenitor proliferation by upregulating SCF expression, while CD169⁺ macrophage subpopulations support late erythroid maturation by recycling iron from senescent erythrocytes [80]. These findings demonstrate the critical role of macrophages in erythroid hematopoietic reconstitution following radiation injury.

Supporting hematopoietic niche reconstruction

Bone marrow contains two functionally distinct vascular compartments: arterial vessels that maintain HSCs quiescence and sinusoids responsible for cell migration [81]. Radiation therapy disrupts the vascular niche where HSCs reside, and restoring this vascular niche is key to hematopoietic niche reconstruction [82] (Fig. 3B). Studies indicate that certain bone marrow macrophage populations persist and activate into an M2-like phenotype after radiation therapy [83]. These radiation-resistant macrophages [84], particularly CD206⁺ macrophages, sense mechanical environment changes caused by bone marrow ablation post-radiation injury via the Piezo1 channel. This activates the calmodulin/NFAT/HIF-1α pathway, further promoting VEGF-A secretion, thereby facilitating sinus regeneration and hematopoietic recovery [83]. The latest research by Liu’s team deepens this mechanism: mechanical stress activates the Piezo1 channel in macrophages, promoting their polarization toward the CD206⁺ phenotype. Activated macrophages not only secrete VEGF-A to promote angiogenesis but also stimulate osteoblast differentiation by releasing osteogenic factors such as BMP-2, forming a “vascular regeneration-bone formation” positive feedback loop that accelerates niche repair [85]. Furthermore, studies indicate a positive correlation between macrophage numbers and microvascular density (MVD), strongly supporting macrophage involvement in vascular repair [86]. Recent studies reveal that M1/M2 macrophages, differentiated from monocytes, exert complementary and synergistic roles in neovascularization and tissue repair. Early-stage neovascularization following irradiation correlates with M1 macrophage infiltration, whereas late-stage vascular sparsification correlates with M2 macrophage infiltration [87]. This finding appears to contradict the conclusion that M2 macrophages promote hematopoietic reconstitution. However, it reflects differences in research perspectives between local vascular and bone marrow niches, or the heterogeneity of M2 subtypes. This apparent contradiction may reflect time-dependent effects [83, 87]. In broader vascular regeneration, the supportive role of macrophages primarily involves producing VEGF-A and interacting with vascular endothelial cells [37, 88], or supporting vascular regeneration by secreting M2-like phenotype exosomes (M2-Exos) [89, 90]. Given the unique niche characteristics of bone marrow vasculature, subsequent studies should investigate whether macrophages exert distinct effects on the regeneration of bone marrow arterial vessels versus sinusoidal vessels following radiotherapy.

Macrophages serve as key effector cells in post-radiation hematopoietic niche repair, with their functions finely regulated by osteoblasts and BM-MSCs. Under steady-state conditions, osteoblasts anchor HSCs via adhesion molecules such as VCAM-1 and N-cadherin, while secreting factors like CXCL12 and SCF to maintain HSCs quiescence [91]. However, high-dose radiation directly damages osteoblast precursor cells, causing loss of these supportive functions. At this point, M2 macrophage-derived EVs deliver miR-142-3p to inhibit TGF-β1, thereby promoting osteogenic differentiation and restoring the differentiation balance of BM-MSCs [37]. Conversely, M2 macrophages promote osteogenesis and HSC retention by secreting factors such as PGE₂ and IL-6 through RELA-dependent mechanisms. Yet, RELA deficiency disrupts the M1/M2 balance, resulting in reduced lymphopoiesis, abnormal myeloid cell proliferation, and enhanced HSCs mobilization—severely compromising the hematopoietic niche [92]. In vitro experiments demonstrate that inducing macrophage phenotypic conversion using BM-MSC-derived EVs represents another critical strategy for hematopoietic recovery. Research by Kink et al. confirmed that MSC-derived EVs, particularly those pretreated with LPS (LPS-EVs), effectively polarize monocytes toward M2-like characteristics. This is manifested by upregulation of anti-inflammatory factors IL-10 and IDO, along with tissue repair-related genes FGF-2 and IL-7. These EVs-educated monocytes significantly improved mouse survival in acute radiation syndrome models by promoting hematopoietic recovery [93]. This process exerts multiple protective effects on hematopoietic recovery. On one hand, M2 macrophages secrete growth factors such as VEGF, IGF-1, and monocyte chemotactic protein-1 (MCP-1/CCL2) [94], synergizing with BM-MSCs to repair vascular endothelial niches and support HSCs self-renewal and differentiation. On the other hand, particularly M2 macrophage subpopulations induced by LPS-pretreated MSC-EVs, significantly enhance FLT-3 L and IL-15 secretion, promoting peripheral blood cell recovery in mice. They generate adenosine through high CD73 expression, suppressing inflammation and promoting vascular maturation, while downregulating M1 markers CD86 and HLA-DR, and upregulate PD-L1/PD-L2 to maintain hematopoietic microenvironment homeostasis [95]. Subsequent studies further revealed that LPS-induced MSC-EVs can directly regulate monocytes, inducing macrophage-like anti-inflammatory, pro-regenerative, and hematopoietic functions [96]. The reparative effects of macrophages are further amplified through the vascular system. For instance, plasma-derived exosomes (RP-Exos) promote macrophage proliferation and upregulate angiogenesis genes, alleviating vascular dysfunction post-radiation [97]. Currently, MSC-macrophage axis-mediated hematopoietic niche repair remains largely confined to in vitro experiments, with limited in vivo studies.

The regulatory mechanisms of macrophages on the hematopoietic system are significantly influenced by variations in radiation dose. Specifically, radiation induces dual polarization of bone marrow macrophages toward M1 and M2 subtypes, but varying doses and types of radiation lead to distinct polarization preferences for different macrophage subtypes. For instance, medium-to-high radiation doses (> 5 Gy) trigger RAW 264.7 macrophages toward M1 polarization, excessively secreting inflammatory mediators like TNF-α and IL-6. This activates the NF-κB pathway via ROS/RNS bursts, exacerbating HSCs DNA damage and apoptosis, ultimately suppressing hematopoietic function [45]. A single 5 Gy dose activates residual bone marrow macrophages post-irradiation, resulting in a mixed M1/M2 and M2-like polarization pattern [83]. High-dose radiation exceeding 10 Gy causes severe damage to macrophages [98]. For instance, 10 Gy of γ-rays represents the dose causing the greatest enhancement of NO production [99]. After cumulative exposure to 10 Gy, IR induces morphological alterations in macrophages and increases their phagocytic rate, but does not affect MMP-2 and MMP-9 proteolytic activity [100]. Following short-term 18 Gy radiation, macrophage viability significantly declines, and macrophages polarize toward pro-inflammatory M1 macrophages [97]. At 80 Gy irradiation, early M1 polarization promotes angiogenesis, while late M2 polarization reduction leads to vascular sparseness and tissue atrophy [87]. Reduced macrophage numbers and dysfunction result in loss of hematopoietic microenvironment support capacity. Notably, tissue-resident macrophages—such as bone marrow CD169⁺ and splenic marginal zone macrophages—exhibit heterogeneous radiation sensitivity. This spatially specific regulatory network may explain organ-specific differences in hematopoietic injury.

Bidirectional effects of macrophages on hematopoietic reconstruction after chemotherapy

Suppression of hematopoietic reconstruction

Following chemotherapy, dysregulation of macrophage function is a key factor accelerating hematopoietic damage. Studies reveal that CD11b⁺F4/80⁺Ly-6G⁻ macrophages (including osteomacs and CD169⁺ subpopulations) exhibit significantly reduced numbers after CTX treatment, accompanied by decreased secretion of CXCL12, directly impairing their supportive role in the HSCs niche [101]. Chemotherapy drugs further suppress hematopoiesis by activating specific macrophage pathways. For instance, 5-fluorouracil (5-FU) upregulates macrophage PD-L1 expression via the cGAS-STING pathway, inducing T-cell exhaustion. More destructive than the loss of supportive function is the mechanism whereby BMDMs transform into active inflammatory attackers under chemotherapy drug stimulation. Studies confirm that doxorubicin(DOX), melphalan, cisplatin, vincristine, etoposide, paclitaxel, methotrexate, and cytarabine all induce BMDMs to process and release IL-1β. Furthermore, the combination of DOX and vincristine exhibits synergistic activation effects on BMDMs, significantly amplifying the transcription and secretion of IL-1β, IL-6, and CXCL1, directly demonstrating that combination chemotherapy is more likely to induce excessive inflammation. Mechanistically, this effect depends on ZAK kinase-mediated phosphorylation of p38 MAPK, which upstream activates the NLRP3 inflammasome, thereby promoting IL-1β maturation and release [102]. IL-1β produced by macrophages causes functional damage to the hematopoietic system. An in vivo study demonstrated that the IL-1-signal-driven inflammatory microenvironment in the bone marrow following 5-FU chemotherapy stress significantly suppressed hematopoietic regeneration; pharmacologically blocking the IL-1 signaling pathway effectively improved post-chemotherapy hematopoietic recovery [103]. Pietras et al. further elucidated this phenomenon, demonstrating that chronic IL-1 exposure drives HSCs toward premature myeloid differentiation at the expense of self-renewal. Consequently, the chronic inflammatory microenvironment sustained by persistent macrophage-derived IL-1 secretion after chemotherapy causes enduring hematopoietic damage by depleting the long-term regenerative potential of HSCs. Additionally, chemotherapy causes loss of phospholipid asymmetry in mature red blood cell membranes, resulting in phosphatidylserine (PS) exposure on the cell surface. This alteration triggers recognition and clearance of red blood cells by macrophages, leading to anemia [104]. Finally, inappropriate macrophage regulation of HSCs activity represents another critical mechanism exacerbating damage. Their regulatory role exhibits a distinct “double-edged sword” characteristic: while they possess protective potential under specific conditions, excessive or sustained suppression severely impairs the system’s regenerative capacity. Zhao et al. clearly demonstrated this: when HSCs are in a macrophage-induced deep quiescent state, the body’s hematopoietic recovery capacity is significantly impaired after combined radiation and chemotherapy, manifesting as more severe anemia and pancytopenia [105]. This suggests that persistent inhibitory signaling from macrophages post-chemoradiation impedes the activation and proliferation of surviving HSCs, thereby delaying or even blocking hematopoietic recovery. In summary, post-chemotherapy macrophages collectively constitute a major barrier to effective hematopoietic reconstruction through multiple pathways: weakening niche support, actively inducing inflammation, excessively clearing blood cells, and inappropriately suppressing HSCs activity.

Promotion of hematopoietic reconstitution

In contrast to the adverse effects of improper macrophage suppression of HSCs activation, which exacerbates hematopoietic damage, the induced quiescence of HSCs serves as a critical protective strategy for the hematopoietic system under specific conditions. The core rationale lies in inducing HSCs to enter a quiescent phase (G0 phase), thereby temporarily shielding them from 5-FU’s attack on highly proliferative cells and safeguarding the most primitive hematopoietic “seed” cell pool. Zhao et al. demonstrated that macrophages activated by specific stimuli effectively induce HSCs quiescence. Mice harboring these quiescent HSCs exhibited significantly improved survival rates after lethal 5-FU treatment [105]. Beyond this “passive protection” strategy, macrophages can also directly promote hematopoietic reconstitution under specific conditions [106].

The dynamic changes in macrophages after chemotherapy finely regulate LT-HSCs dormancy and bone marrow regeneration. For example, following 5-FU treatment, the reduced number of DARC⁺ macrophages leads to decreased CD82 expression on LT-HSCs surfaces, triggering LT-HSCs to exit dormancy and enter proliferation and differentiation phases to rebuild the bone marrow hematopoietic system [11]. Furthermore, studies reveal that endothelial cell-selective adhesion molecule (ESAM) expression in HSPCs is critical for erythroid hematopoietic recovery following 5-FU-induced bone marrow injury. BMDMs upregulate ESAM expression after 5-FU treatment; these macrophages may promote erythroid hematopoietic recovery by forming an EBI that provides a supportive microenvironment for erythroid progenitor cells [107]. Similarly, treatment with multiple doses of gemcitabine (GEM), paclitaxel (PTX), or the combination regimen PTX + DOX significantly increases macrophage numbers [108]. This change stems from chemotherapy-induced compensatory reactive myelopoiesis, aimed at replenishing the damaged hematopoietic system. The CCL2/CCR2 signaling pathway plays a crucial role in chemotherapy-induced macrophage recruitment [108]. Notably, the sympathetic nervous system modulates post-chemotherapy hematopoietic recovery by regulating macrophage involvement. Specifically, neuropeptide Y (NPY) activates the PI3K/Akt/mTOR pathway by binding to Y1 receptors on macrophages. This promotes macrophage secretion of TGF-β, thereby inhibiting chemotherapy-induced apoptosis of sympathetic nerve fibers and sustaining the survival of bone marrow endothelial cells (ECs) and MSCs, indirectly supporting HSCs regeneration [109]. Further studies revealed that the NPY derivative NPY(6–20) exhibits superior protective effects compared to full-length NPY, more effectively restoring the number of chemotherapy-damaged LT-HSCs [110]. More intricately, certain pathways that prove detrimental in the long term can play crucial promotive roles during the acute phase, with the IL-1 signaling pathway serving as a prime example. This dual role primarily depends on the “duration” and “intensity” of signal exposure. The same study by Pietras et al. revealed another facet of IL-1: following acute 5-FU injury, IL-1 directly acts on HSCs. By activating PU.1-dependent gene programs, it accelerates their proliferation and directs differentiation toward the myeloid lineage, ensuring rapid replenishment of immune cells like neutrophils during critical moments [111]. This stands in stark contrast to the chronic depletion caused by prolonged IL-1 exposure described earlier, perfectly illustrating the “double-edged sword” role of macrophages in hematopoietic regulation.

Pharmacological intervention targeting macrophages to improve hematopoietic damage after radiochemotherapy

As the “regulatory hub” of the bone marrow microenvironment, the functional state of macrophages directly determines the efficiency and quality of hematopoietic reconstitution. In recent years, macrophage-targeted drug intervention strategies have primarily focused on two approaches: first, directly activating the hematopoietic support function of macrophages through exogenous cytokines (such as Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and IL-3); second, utilizing small-molecule compounds or natural medicines to regulate macrophages and remodel their hematopoietic function (Table 1).

Table 1.

Pharmacological intervention targeting macrophages to improve hematopoietic damage following radiotherapy and chemotherapy

NO.	Interventions	Model	In Vivo / In Vitro	In Vitro Findings & Postulated Mechanism	In Vivo Phenotype
1 [112]	AstragalosideIII (AS-III)	In vivo: CTX-induced immunosuppression model in C57BL/6 mice In vitro: Hypoxia/LPS-induced inflammation model in RAW264.7 macrophages	20,40,80 mg/kg/day,10 days, in vivo ; 0–50 µM, in vitro;	In hypoxia/LPS-induced macrophages: 1. Inhibits the HIF-1α/PDHK-1 pathway to improve abnormal energy metabolism 2. Reduces release of inflammatory mediators including NO, TNF-α, and IL-1β 3. Enhances cell migration and phagocytic function	In the CTX model: 1. Reduced spleen and thymus atrophy 2. Improved decline in blood cell counts such as white blood cells 3. Inhibited activation of the HIF-1α/PDHK-1 pathway in spleen and bone marrow tissues
2 [113]	Neutral heteropolysaccharide (PNANb)	In vivo: CTX-induced immunosuppression model in C57BL/6 mice In vitro: RAW264.7 macrophages	50,100 mg/kg/days, 6 days, in vivo; 20,40,80,160 ,320 µg/mL,24 h, in vitro;	In RAW264.7 macrophages: 1. Activation of TLR4 promotes the release of NO and proinflammatory cytokines by activating the MAPK and NF-κB signaling pathways.	In the CTX model: 1. Improves spleen and thymus indices 2. Elevates serum IgG, IgM, and multiple cytokine levels 3. Its immune-enhancing effects depend on the presence of macrophages (the effect disappears after macrophage depletion)
3 [114]	Angelica sinensis polysaccharides (ASP)	In vivo: 5-FU-induced stress anemia model in C57BL/6 mice In vitro: RAW264.7 macrophages	100 mg/kg/days,7/10days, in vivo; 100 µg/mL, 6 h, in vitro;	In RAW264.7 macrophages: 1. Enhances glycolytic metabolism; 2. Induces polarization toward an M2-like phenotype (upregulates Arg-1, CD206); 3. Strengthens phagocytic function.	In the 5-FU anemia model: 1. Accelerates the recovery of red blood cells, hemoglobin, and hematocrit 2. Promotes extramedullary erythroid hematopoiesis in the spleen 3. Increases the number of EIMs in the spleen and upregulates the expression of erythroid differentiation-related genes such as Epor in splenic cells.
4 [115]	Lentinan (LNT)	In vivo: THP-induced bone marrow suppression model in Swiss mice In vitro: Mouse BMDMs	20 mg/kg/12 h, 20days, in vivo; 5–40 µM,6–72 h, in vitro;	In BMDMs: 1. LNT can directly activate BMDMs through the MAPK/NF-κB signaling pathway, promoting the release of G-CSF, GM-CSF, and M-CSF.	In the THP model: 1. Reduces bone marrow damage and accelerates recovery of white blood cells and neutrophils 2. Significantly elevates serum levels of G-CSF, GM-CSF, and M-CSF 3. Shortens the self-repair cycle of bone marrow suppression
5 [116]	Shenzhu Erkang Syrup (SZEK)	In vivo: CTX-induced hematopoietic dysfunction model in BALB/c mice	0.3 ml/25 g / day,43 days,in vivo;	(This study inferred mechanisms through microbiome and metabolomics analysis without conducting independent in vitro cell experiments.)	In the CTX model: 1. Improves pathological damage to bone marrow and spleen 2. Increases the number of HSCs, progenitor cells, and lymphocytes in bone marrow 3. Inhibits the differentiation of M2 macrophages (CD206⁺) and reduces levels of their associated factors (IL-10, TGF-β)
6 [46]	Fucoxanthin (FX)	In vitro: RAW264.7 macrophages irradiated with 6 Gy X-rays	5 µM, in vitro;	In radiation-induced macrophages: 1. Suppresses M1 polarization and promotes M2 polarization by regulating Sirtuin 1 (SIRT1) and estrogen receptor alpha (ERα) expression. 2. Inhibits IR-induced glycolysis-related genes and restores mitochondrial biogenesis gene expression.	(This study is a purely in vitro experiment.)
7 [100]	Red Ginseng saponin fraction (RGSF)	In vitro: RAW264.7 macrophages irradiated with γ-rays and stimulated with LPS	0,2.5,5,10,20 µg/mL,24 h, in vitro;	In a radiation-enhanced macrophage inflammation model: 1. Significantly suppressed NO and IL-1β production; 2. The mechanism of action involves inhibiting the Chk2/NF-κB signaling pathway.	(This study is a purely in vitro experiment.)
8 [117]	Annona muricata leaf polysaccharides(ALPS)	In vitro: RAW264.7 and BMDM cells treated with cisplatin	31.25–62.5 µg/mL, 24 h, in vitro;	In cisplatin-damaged macrophages: 1. By mitigating mitochondrial damage, suppressing ROS and apoptotic pathways (caspase signaling), protecting macrophages from chemotherapy drug toxicity, maintaining macrophage immune function, and indirectly supporting hematopoietic microenvironment stability.	(This study is a purely in vitro experiment.)
9 [118]	Nitro-oleic acid (NO2OA)	In vivo: C57BL/6 mice irradiated with ⁶⁰Co-γ rays	10 mg/kg, in vitro;	(This study was conducted as an in vivo experiment.)	In radiation injury models: 1. Promotes recovery of peripheral blood leukocytes, including significant restoration of lymphocytes, neutrophils, and monocytes; 2. Increases the number of macrophage precursors in bone marrow, stimulates G-CSF production, and modulates macrophage function, thereby helping to mitigate radiation-induced inflammatory responses and tissue damage.
10 [119]	Leukocyte cell-derived chemotaxin 2 (LECT2)	In vivo: Multiple gene knockout/conditional knockout mouse models In vitro: Primary macrophages and osteogenic cells	300 µg/kg Weight/days, 5days, in vivo; 5 µg/mL,24 h, in vitro;	In vitro cultured macrophages/osteoblasts: 1. Receptor CD209a activates macrophages and osteoblasts, suppressing TNF expression in these cells to reduce their inhibitory effect on HSCs.	In vivo: 1. By acting on macrophages and bone marrow cells to inhibit their TNF secretion, thereby promoting HSCs expansion and mobilization.
11 [120, 121]	Butyrate	In vivo: 5-FU/TBI-induced C57BL6 mouse In vitro: Macrophages and HSCs	Transferrin concentration gradient (0.5 mg/L to 500 mg/L), TPO(100 ng/mL)+ SCF༈10 ng/mL༉,18-21days, in vitro;	In macrophages: 1. Directly enhances their phagocytic capacity for red blood cells by inhibiting HDAC. In HSCs culture: 1. Demonstrates that iron concentration directly regulates HSCs self-renewal and differentiation.	In vivo: 1. Enhances erythrocyte phagocytosis by CD169⁺ bone marrow macrophages, elevates local iron concentration in the bone marrow, thereby promoting HSCs differentiation and hematopoietic regeneration; 2. Increases the number of mature blood cells such as erythrocytes and leukocytes; reduces HSCs self-renewal.
12 [122]	7, 8-diacetoxy-4-methylthiocoumarin (DAMTC)	In vivo: TBI of C57BL/6 mice	5 µg/kg (intraperitoneal injection, administered 24 h after radiation exposure);	(This study was conducted as an in vivo experiment.)	Post-radiation therapeutic administration: 1. Significantly improves survival rates in mice following lethal-dose radiation exposure. 2. Accelerates recovery of white blood cells (WBC) and lymphocytes, promoting multilineage hematopoiesis. 3. Stimulates differentiation of pro-inflammatory M1 macrophages in the spleen, enhancing antigen presentation and immune responses to alleviate radiation-induced immunosuppression.
13 [123]	Limosilactobacillus reuteri KBL346 (KBL346)	In vivo: CPA (CTX)-induced BALB/c immunosuppressed mice In vitro: RAW264.7 macrophages	108, 1010 CFU/kg, in vitro;	In RAW264.7 macrophages: 1. Enhances phagocytic activity and promotes secretion of TNF-α, IL−1β, IL−6, NO, and PGE2 by activating the TLR-MAPK/NF-κB pathway.	In the CPA model: 1. Improves spleen damage, restores blood cell counts, enhances T/B/NK cell function, boosts cellular immunity, and indirectly influences hematopoietic function.
14 [124]	Resveratrol	In vitro: LPS-induced inflammatory models in RAW264.7 and peritoneal macrophages	25,50,100 µg/mL,24 h, in vitro;	In LPS-induced macrophage inflammation: 1. Effectively suppresses the production of NO, PGE₂, and multiple pro-inflammatory cytokines (after irradiation modification). 2. Its anti-inflammatory mechanism involves inhibiting the TLR4-mediated MAPK/NF-κB pathway.	(This study was conducted entirely in vitro.)
15 [125]	High methionine diet	In vivo: TBI-induced hematopoietic injury model in C57BL/6 mice In vitro: RAW264.7 cells and BMDMs	10,20,40 g/kg/days, in vivo; 1.5,3,60,150,300,600 µg/mL in vitro;	In macrophages: 1. Methionine induces M2 polarization by upregulating S100A4 and activating the STAT3 pathway, thereby suppressing bone marrow inflammation.	In the TBI model: 1. Improves survival rates, promotes hematopoietic cell and bone marrow recovery, and enhances the proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs); 2. Promotes macrophage polarization toward the M2 phenotype and suppresses bone marrow inflammation; Its hematopoietic-promoting effects depend on macrophages.
16 [126]	Catharanthus roseus (L.) Don leaves-derived exosome-like nanovesicles (CLDENs)	In vivo: CTX-induced BALB/c immunosuppressed mice In vitro: RAW264.7 macrophages	6,20,60 mg/kg/days,14days, in vivo; Macrophages: 60–240 µg/ml ,48 h, lymphocytes: 30–240 µg/ml ,48 h, in vitro;	In RAW264.7 cells: 1. Activates macrophages via the TNF-α/NF-κB/PU.1 axis and promotes their polarization toward the M1 type.	In the CTX model: 1. Restores white blood cell counts, promotes hematopoietic stem cell differentiation and immune cell proliferation, and alleviates bone marrow cell cycle arrest; 2. Upregulates PU.1 protein expression in bone marrow.
17 [127]	Tetrahedral framework nucleic acid - MicroRNA-155 complex (T-155)	In vivo: CTX-induced C57BL/6 immunodeficient mice In vitro: Dendritic cells and macrophages	500 nM(200 µL/dose/2 DAYS 20days)in vivo;	In macrophages: 1. Induces M1 polarization by activating the NF-κB pathway. In dendritic cells (DCs): 1. Promotes maturation via the ERK1/2 pathway, enhances antigen presentation capacity, and indirectly supports T cell-mediated immune recovery.	In the CTX model: 1. Restores blood count parameters, repairs spleen and thymus damage, and promotes T-cell proliferation.

Natural products

Natural products such as single-component Chinese herbal medicines and compound formulas exert significant effects in promoting hematopoietic function repair by regulating the hematopoietic microenvironment and immune homeostasis [128]. Their mechanisms primarily involve suppressing inflammatory responses and modulating signaling pathways, thereby alleviating bone marrow suppression following radiotherapy and chemotherapy.

Polysaccharide components exert hematopoietic protective effects by targeting macrophage metabolism and secretory functions. For example, ASP enhances glycolytic activity in splenic macrophages, activating the EpoR/STAT5 signaling pathway to promote erythrocyte differentiation [114]. LNT induce BMDMs to secrete hematopoietic growth factors such as G-CSF and GM-CSF via the NF-κB/MAPK pathway, significantly alleviating THP-induced bone marrow damage [115]. PNANb activate macrophages via the TLR4-MAPK/NF-κB signaling axis, indirectly reversing immunosuppression [116]. Additionally, ALPS inhibits cisplatin-induced macrophage ROS accumulation and mitochondrial membrane potential loss, mitigating apoptosis by blocking the caspase pathway to maintain hematopoietic microenvironment homeostasis [117]. Beyond polysaccharides, saponins reduce hematopoietic damage in radiation protection due to their anti-inflammatory and metabolic regulatory properties. Experiments demonstrate that AS-III corrects hypoxia/LPS-induced abnormal macrophage energy metabolism and inflammatory responses by inhibiting the HIF-1α/PDHK-1 signaling pathway [112]. Furthermore, RGSF significantly reduces excessive post-radiation production of NO and IL-1β in macrophages, confirming its radiation protection potential [99].

For radiation-induced oxidative stress and metabolic disorders, natural products demonstrate multi-target regulatory advantages. For instance, FX inhibits IR-triggered pro-inflammatory gene expression (Il1b, Tnf, Ccl2) and glycolytic abnormalities by upregulating macrophage SIRT1/ERα activity, thereby blocking M1 polarization [45]. Compared to single active ingredients, traditional Chinese medicine (TCM) formulas possess advantages in exerting pharmacological effects through multiple components, multi-level mechanisms, diverse pathways, and multi-target actions. For instance, compound preparations like SZEK regulate M2 macrophage differentiation via the gut microbiota-serum metabolite axis, suppressing inflammatory factor secretion to promote hematopoietic recovery [116].

Other approaches

Currently, various macromolecular and small-molecule drugs promote hematopoietic recovery after radiotherapy and chemotherapy by targeting macrophage function. Specifically, NO2OA enhances hematopoietic reconstitution by increasing bone marrow macrophage precursor cell numbers and promoting G-CSF secretion [118]. Hyaluronic acid (HA), meanwhile, binds to CD44v6 and RHAMM receptors on bone marrow macrophage surfaces, inducing the release of hematopoiesis-promoting cytokines such as IL-1 and IL-6 to accelerate recovery from 5-FU-induced bone marrow suppression [129]. In vitro studies revealed that KBL346 enhances macrophage phagocytic activity, increases NO and PGE₂ secretion, and promotes production of immunomodulatory cytokines (TNF-α, IL-1β, IL-6). It also indirectly influences hematopoiesis by activating TLR-MAPK signaling pathways to modulate macrophage activation and function [123]. Furthermore, high methionine maintains bone marrow ECM composition, upregulates S100A4 expression, and subsequently promotes M2 polarization while suppressing M1 polarization in bone marrow macrophages via the STAT3 pathway. This manifests as increased CD206⁺ phenotype to alleviate bone marrow inflammation and enhance HSPC proliferation and differentiation [125].

LECT2 targets macrophages and osteoblasts via the CD209a receptor, inhibiting TNF expression while activating the SDF-1/CXCR4 axis to promote HSCs expansion and mobilization [119]. Similarly, DAMTC enhances immune responses by inducing splenic M1 macrophage generation [122], while resveratrol reduces macrophage inflammatory mediator release by inhibiting TLR4-mediated MAPK and NF-κB signaling pathways [124]. Crucially, macrophages play a central role in post-radiation hematopoietic recovery by regulating the gut microbiota-hematopoietic axis [130, 131]. For instance, the gut microbiota metabolite butyrate enhances macrophage phagocytic function by inhibiting histone deacetylases (HDACs), thereby promoting iron release to supply HSCs [120, 121]. In conditions of microbiota depletion or impaired macrophage function, insufficient iron supply inhibits HSCs differentiation toward maturation while enhancing their self-renewal capacity [120]. Furthermore, CLDENs extracted from Catharanthus roseus leaves increase expression of macrophage surface markers CD86 and MHC II while promoting TNF-α and IL-6 secretion. This activates the NF-κB signaling pathway and upregulates PU.1 expression, thereby promoting HSCs differentiation and immune cell proliferation [126]. Finally, T-155 promotes M1 polarization of macrophages via the miR-155/NF-κB axis, leading to secretion of inflammatory mediators like TNF-α and ameliorating CTX-induced bone marrow suppression and immunodeficiency [127]. These studies not only elucidate the molecular mechanisms of the compound-macrophage-hematopoiesis regulatory network but also provide potential therapeutic strategies for hematopoietic recovery after chemoradiotherapy.

Discussion

Macrophages exhibit complex bidirectional regulatory roles in the hematopoietic system following chemoradiotherapy, making their mechanisms and therapeutic strategies a current research focus. Bone marrow macrophages maintain hematopoietic homeostasis through precise subpopulation division, with specific subpopulations such as CD169⁺, VCAM-1⁺, and DARC⁺ forming distinct spatial localization patterns. These subpopulations construct a hematopoietic microenvironment regulatory network through receptor-ligand interactions. Following chemoradiotherapy, macrophages profoundly engage in both hematopoietic damage and repair. Regarding the damage mechanism, the abnormal activation of macrophages after chemoradiotherapy exhibits multidimensional injury characteristics. At the molecular level, CD169⁺ macrophage polarization is accompanied by ROS/RNS bursts, forming a mutually reinforcing vicious cycle with iron overload induced by erythroid precursor cells/senescent erythrocytes, ultimately leading to erythroid hematopoietic dysfunction and anemia. Structurally, increased collagen and fibronectin deposition disrupts HSCs anchoring structures mediated by adhesion molecules (VCAM-1, N-cadherin) and integrins, processes in which macrophages themselves participate. Furthermore, in immune regulation, macrophages promote myeloid differentiation bias in HSCs synergistically with immunosuppressive effects, collectively fostering a preleukemic microenvironment. Notably, while macrophages survive radiation exposure due to their robust radiation resistance, their function is significantly impaired. This survival-function disconnect—where cells survive but remain dysfunctional—challenges the traditional reliance on cell survival rates to assess radiation resistance. It offers a novel theoretical perspective for elucidating the mechanisms underlying persistent hematopoietic dysfunction after chemotherapy. Repair mechanisms exhibit marked spatiotemporal specificity. Spatially, the α-SMA⁺COX-2⁺ subpopulation protects HSCs through physical contact, EBI macrophages support erythroid regeneration via the unique mechanism of mitochondrial transfer, and the CD206⁺ subpopulation primarily promotes vascular regeneration. Additionally, MSCs mediate M2 macrophage repair of hematopoietic niches. Temporally, the repair process undergoes a dynamic transition from an M1-dominated inflammatory clearance phase to an M2-dominated tissue repair phase. Beyond the bone marrow, extra-medullary macrophages such as intestinal and splenic macrophages are also critically involved in post-chemoradiotherapy hematopoietic reconstitution.

Based on these mechanistic insights, multiple intervention strategies have been developed. The complexity of the bone marrow microenvironment determines significant heterogeneity in macrophage responses to chemoradiotherapy. Even at identical radiation doses (e.g., 2 Gy), macrophages in different regions may simultaneously exhibit both M1 and M2 phenotypes. This phenomenon fundamentally arises because their polarization is not solely determined by radiation exposure. Instead, it results from the integrated effects of unique signaling combinations within distinct bone marrow micro-regions—including intercellular communication, local metabolism, and the stromal environment—alongside the macrophages’ own developmental origins. Notably, when radiation and chemotherapy doses reach myeloablative levels (> 10 Gy), the core function of macrophages undergoes a significant shift post-treatment: they transition from cells maintaining a steady-state microenvironment to transient structural cells supporting tissue repair. For low-dose radiation scenarios, anti-inflammatory and iron chelation strategies targeting the CD206⁺ subpopulation are primarily employed. Conversely, in high-dose radiation environments, measures such as methionine diets promote the reparative function of the CD206⁺ subpopulation. However, these strategies face significant challenges in clinical translation, chiefly due to the difficulty of targeted therapy caused by the high heterogeneity of macrophage subpopulations. Furthermore, existing research often overlooks the fundamental differences in how radiotherapy and chemotherapy affect macrophages, an issue that urgently requires resolution.

Key challenges in current research encompass four primary areas. First, clarifying the compensatory relationships among macrophage subpopulations during tissue repair is essential. Second, establishing quantitative models linking radiation dose to polarization responses is crucial. Third, elucidating the specific mechanisms by which different chemotherapeutic agents exert their effects is necessary. Finally, addressing the phenotypic uncertainties arising from combined radiotherapy and chemotherapy is imperative. Future research should prioritize the following directions. The primary task is to utilize models such as genetic lineage tracing and specific cell subpopulation knockout to dynamically analyze in vivo whether and how other macrophage subpopulations compensate functionally after the loss of a specific subpopulation by altering their polarization state, secretory profile, or intercellular interactions. The emphasis lies on revealing the specific signaling pathways of compensatory communication and evaluating whether such compensation promotes repair or exacerbates disruption. Second, quantitative models linking radiotherapy doses to macrophage polarization responses should be established, shifting from discrete classification to continuous lineage analysis to enable predictable regulation of the immune microenvironment. Third, elucidating how different chemotherapeutic agents remodel macrophage function via specific signaling pathways will precisely define their inhibitory or promotional effects on hematopoietic reconstitution. Finally, strategies must transition from isolated studies of radiotherapy/chemotherapy to dynamic integration of their synergistic effects. The key lies in revealing how these two therapies, under specific timing and dose combinations, induce highly variable “fuzzy” states in macrophage phenotypes and functional outputs through additive or synergistic effects. Animal models simulating clinical combination regimens must be developed, and single-cell technologies employed to decipher the resulting macrophage heterogeneity and its complex impacts on niche reconstruction. This will identify critical nodes for phenotypic switching and enable targeted interventions.

Abbreviations

ACKR1

Atypical Chemokine Receptor 1

α-SMA

α-Smooth Muscle Actin

COX-2

Cyclooxygenase-2

CTX

Cyclophosphamide

CXCL12

C-X-C Motif Chemokine Ligand 12

DARC

Duffy Antigen Receptor for Chemokines

ECM

Extracellular Matrix

EpoR

Erythropoietin Receptor

5-FU

5-Fluorouracil

HIF-1α

Hypoxia-Inducible Factor-1α

HSPC

Hematopoietic Stem/Progenitor Cell

IL-1β

Interleukin-1β

IR

Ionizing radiation

LT-HSCs

Long-term hematopoietic stem cells

MSC

Mesenchymal stem cell

NF-κB

Nuclear factor-κB

NO

Nitric oxide

NOS₂

Nitric oxide synthase 2

ONOO⁻

Peroxynitrite

PGE₂

Prostaglandin E2

RNS

Reactive nitrogen species

SCF

Stem cell factor

TGF-β

Transforming growth factor-β

TNF-α

Tumor necrosis factor-α

VCAM-1

Vascular cell adhesion molecule-1

VEGF

Vascular endothelial growth factor

Authors’ contributions

Z.L.D designed the review protocol, conducted the search, drafted the manuscript, and prepared all Figures and tables. W.B.H. screened potentially eligible studies, extracted and analyzed data, and updated reference lists. X.C. and F.M.Y. were responsible for the funding acquisition. J.L. contributed to the design of the review protocol, arbitrating potentially eligible studies, and interpreting results. J.L and F.M.Y. provided feedback on the report. All authors approved the final version of the manuscript.

Funding

This study was supported by “Chongqing Technology Innovation and Application-DevelopmentSichuan-ChongqingScience and TechnologyCooperation Plan Project”(CSTB2024TIAD-CYKJCXX0037).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.