Toll-like receptor 4 signaling links cytoskeletal remodeling to lipid accumulation in macrophages

Abstract

Background

Macrophage lipid accumulation is a hallmark of atherosclerosis and other metabolic diseases. Toll-like receptor 4 (TLR4) signaling and cytoskeletal remodeling have each been implicated in this process, yet the mechanistic connections among these three elements remain incompletely defined.

Main body

This review synthesizes current evidence for pathways in which TLR4 activation, via bifurcated signaling through MyD88/TRAM, SYK/Src, and integrins, engages small GTPases (Rac, Cdc42, Rho) and downstream effectors including cofilin and paxillin to drive cytoskeletal remodeling. These cytoskeletal changes facilitate lipid internalization through multiple routes, including macropinocytosis, receptor redistribution, and lysosomal synapse formation, and regulate lipid droplet dynamics, ultimately promoting foam cell formation. We also discuss the therapeutic potential and safety challenges of targeting cytoskeletal regulators in diseases such as atherosclerosis.

Short conclusion

The convergence of TLR4 signaling, cytoskeletal remodeling, and lipid metabolism represents a compelling mechanistic nexus for understanding foam cell formation. Elucidating the molecular details of this pathway—particularly the SYK/Src-cytoskeleton axis—may reveal macrophage-selective therapeutic targets that suppress pathological lipid accumulation while preserving host defense functions.

Introduction

The formation and enrichment of lipid-laden macrophages occur in diverse pathological conditions, including atherosclerosis [1, 2], obesity [3, 4], and cancer [5, 6]. These cells are characterized by the upregulation of lipid metabolism genes (e.g., Trem2, Cd36, Lpl) and the acquisition of features such as substantial intracellular lipid accumulation and expanded lysosomal compartments [7]. Owing to their roles in disease progression, these macrophages represent promising therapeutic targets [8–10]. However, the upstream drivers that initiate and sustain lipid accumulation in macrophages remain incompletely understood. Notably, reports on the efficacy of inhibiting individual lipid transporters such as CD36 or transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ) in reducing macrophage lipid content have been inconsistent [11–14], suggesting that complementary or convergent uptake mechanisms exist.

Emerging evidence indicates that cytoskeletal remodeling actively contributes to foam cell formation [15]. The cytoskeleton, a dynamic network of actin filaments, microtubules, and intermediate filaments, maintains cellular architecture, drives motility, and coordinates intracellular transport. Beyond these canonical functions, the cytoskeleton plays an increasingly recognized role in lipid metabolism [16]. Actin reorganization facilitates lipid influx via multiple endocytic routes and modulates the cortical actin barrier that governs both entry and exit of lipid-containing cargoes. Cytoskeletal dynamics also regulate the storage, intracellular transport, and organelle tethering of lipid droplets (LDs) [17–19]. In macrophages, cytoskeletal remodeling is intimately associated with pathological lipid accumulation. For instance, early intermittent cholesterol exposure, a key factor in accelerated atherosclerosis, reshapes arterial macrophages by altering transcriptional programs related to actin filament organization [20].

Toll-like receptors (TLRs) are pattern recognition receptors that detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and they critically drive inflammatory macrophage activation in atherosclerosis, obesity, and cancer [21–23]. Beyond their canonical inflammatory functions, TLR4 activation triggers profound morphological and cytoskeletal remodeling in macrophages. This observation is particularly significant because macrophages possess a remarkable capacity for bulk internalization of lipid-containing cargoes such as exosomes [24]. Via macropinocytosis, a process so efficient that a macrophage can internalize its entire plasma membrane surface area every 30 min (reviewed in [25]).

A pivotal, unresolved question thus arises: in lipid-rich milieus, could the enhanced macropinocytic and cytoskeletal activity driven by chronic TLR4 stimulation constitute a major route for extracellular lipid internalization, thereby linking innate immune activation directly to foam cell formation? Supporting this concept, pharmacological interventions that target cytoskeletal dynamics, including macropinocytosis inhibitors and microtubule-stabilizing agents, have shown promise in reducing lipid-laden macrophage accumulation in animal models of cardiovascular disease and cancer.

This review develops a unified mechanistic framework to explain how cytoskeletal remodeling integrates TLR4 signaling with macrophage lipid homeostasis. We systematically distinguish between direct mechanistic evidence, indirect pharmacological associations, and speculative links, and we identify critical knowledge gaps that require further investigation.

Cytoskeletal control of cellular lipid homeostasis

Lipid uptake via macropinocytosis

Macropinocytosis, known as fluid-phase endocytosis, facilitates the nonspecific bulk uptake of extracellular fluid and pericellular solutes [26]. While macropinocytosis is exploited by tumor cells to scavenge nutrients for growth and invasion [27–29], it has similarly emerged as a significant contributor to macrophage lipid accumulation, particularly under pathological conditions (Fig. 1).

Fig. 1.

Cytoskeleton remodeling potentially participates in lipid accumulation in macrophages induced by TLR4 activation. TLR4 activation by LPS or modified lipoproteins (agLDL, mmLDL) triggers actin remodeling through MyD88-dependent and Syk dependent branches. Cytoskeletal outcomes include macropinocytosis (cofilin activation), and lysosomal synapse formation for ag LDL degradation (cofilin activation). Chemokines could induce CD36 redistribution to actin-sparse membrane regions by activating integrins/Rap1 signaling. Nox2/PI3K/Akt signaling contributes to uptake of nLDL into macrophages via macropinocytosis. These processes collectively promote macrophage lipid accumulation and foam cell formation. Solid arrows indicate pathways for which direct mechanistic evidence exists from genetic and/or pharmacological experiments. Dashed arrows indicate pathways supported by pharmacological evidence only or where intermediate steps remain incompletely defined

In pathophysiological contexts, macropinocytosis contributes to diverse processes relevant to macrophage lipid biology. Following spinal cord injury, macropinocytosis plays a major role in generating foamy macrophages and promoting cholesterol crystallization, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and reduces neuronal survival [30]. In the tumor micromilieu, cholesterol-rich extracellular vesicles released from cancer cells are taken up by myeloid-derived suppressor cells via macropinocytosis, driving foam cell differentiation and immunosuppression [31]. Bone marrow-derived adipose tissue macrophages internalize lipid-laden exosomes from adipose tissue through a LY294002-sensitive mechanism consistent with macropinocytosis [32].

Fluid-phase pinocytosis of native low-density lipoprotein (nLDL) was also recognized early as a key route for macrophage foam cell formation [33–36]. This process operates independently of receptor-mediated mechanisms and may be particularly important when extracellular LDL concentrations are high. In macrophages, 4β-phorbol 12-myristate 13-acetate (4β-PMA) stimulates cellular uptake of nLDL via macropinocytosis in a CD36-independent manner through a pathway involving NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS), inhibition of phosphatase and tensin homolog (PTEN), activation of PI3K/Akt signaling, and cofilin activation leading to membrane ruffling [37, 38]. Cofilin activity is regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂); hydrolysis of PI(4,5)P₂ by phospholipase C (PLC) at the plasma membrane releases cofilin, enabling it to bind and sever F-actin [39, 40]. The relationship between Nox2-derived ROS and PLC-mediated PI(4,5)P₂ hydrolysis in cofilin regulation has not been fully resolved and warrants direct investigation. In the context of minimally oxidized LDL (mmLDL) recognition, TLR4 recruits spleen tyrosine kinase (SYK). Activated SYK initiates a Vav1/Ras/Raf/MEK/ERK1/2 cascade, leading to paxillin phosphorylation and subsequent activation of Rac, Cdc42, and Rho, which collectively drive cytoskeletal rearrangements and macropinocytosis of extracellular lipids [41].

Pharmacological inhibition with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) has demonstrated that blocking macropinocytosis can significantly ameliorate atherosclerosis. EIPA treatment suppresses arterial foam cell formation in both murine and human atherosclerotic tissue [42]. However, because EIPA targets the ubiquitous Na⁺/H⁺ exchanger, these studies cannot distinguish macropinocytosis in macrophages from effects in other cell types. Macrophage-specific genetic models targeting macropinocytosis components—such as conditional deletion of Rac1 or cofilin—are needed to establish cell-type specificity.

Lipid uptake via cortical actin remodeling

The cortical actin network, which maintains cell membrane morphology, directly influences lipid internalization. Macropinosomes must traverse the dense F-actin meshwork in the cell cortex to be successfully internalized; failure to do so results in their retention at the membrane surface [43]. This spatial constraint positions cortical actin as a key gatekeeper for bulk lipid uptake.

Integrin-mediated adhesion, acting upstream of SYK activation, regulates cortical actin organization and influences receptor distribution for lipid uptake. In RAW264.7 macrophages, chemokines stimulate the integrin/Rap-GTPase signaling axis, triggering cortical actin remodeling that drives the formation of F-actin-enriched membrane ruffles alongside F-actin-depleted zones. This redistribution facilitates CD36 accumulation in actin-sparse regions, enhancing oxidized LDL (oxLDL) uptake via CD36-mediated endocytosis [44]. This represents a mechanistically well-characterized example in which cortical actin reorganization directly modulates the spatial distribution and function of a specific lipid receptor (Fig. 1).

For aggregated LDL (agLDL), a distinct actin-dependent mechanism operates. Macrophages responding to retained and aggregated LDL undergo cortical F-actin depolymerization, which supports subsequent cofilin-1-dependent actin repolymerization at the lysosomal synapse surrounding LDL aggregates. The Rho GTPase Cdc42 plays a key role in driving actin polymerization for lysosomal synapse assembly and lipid uptake [45, 46]. This specialized structure creates a sealed compartment where extracellular LDL undergoes degradation, distinct from both phagocytosis and macropinocytosis. Notably, the lysosomal synapse formed in response to agLDL requires TLR4/MyD88/PI3K/SYK/Akt-dependent signaling [47] (Fig. 1).

Lipid efflux via actin remodeling

Compared with lipid uptake, the role of the actin cytoskeleton in lipid efflux remains less understood. In macrophages, actin polymerization facilitates the translocation of scavenger receptor class B type 1 (SR-BI) from the cytosol to the membrane, a process essential for cholesterol efflux to high-density lipoprotein [48]. However, the mechanisms governing the membrane transport of lipid-containing vesicles for extracellular release are poorly characterized.

A recent cryo-electron tomography (cryo-ET) study in NS-1E β-cells provides structural insight that may be applicable to macrophages. Under basal conditions, cortical F-actin is predominantly oriented at angles of 0–45° relative to the plasma membrane, forming bundled structures that restrict vesicle exocytosis. Upon glucose stimulation, the proportion of F-actin oriented at 45–90° increases, creating F-actin-sparse regions that facilitate the release of insulin granules [49]. Whether a comparable reorientation of cortical actin occurs in macrophages during lipid efflux remains an open question requiring direct investigation. It is plausible that the dense cortical actin network in lipid-laden foam cells contributes to impaired cholesterol efflux by physically restricting the exocytosis of cholesterol-containing vesicles.

Cytoskeletal support for lipid droplet dynamics

LDs are dynamic organelles that interact extensively with the cytoskeleton (Fig. 2). Pharmacological disruption of actin polymerization reduces LD size and lipid storage in 3T3-L1 adipocytes, and genetic loss of cofilin-1 impairs adipogenesis and lipid accumulation [50]. These observations indicate that actin dynamics are required for normal LD biology, though the precise mechanisms-whether through effects on LD biogenesis, fusion, stability, or catabolism-require further clarification.

Fig. 2.

The proposed mechanisms of how the cytoskeleton orchestrates lipid droplet storage, transport, and organelle interactions. In human cardiomyocytes, LIPTER, binding PI4P on surface of LD membrane, connects LD to the MYH10-actin cytoskeleton and facilitates LD transport towards mitochondria. NMIIA-functionalized actin filaments facilitate LD dissociation by altering LD surface to volume ratio and lipase accessibility to triglycerides in human primary neutrophils. ARL8B connects LD and lysosomes, mediating LD breakdown in human macrophage. Motor proteins include kinesins and dyneins could facilitate LD transport along microtubules towards mitochondria or endoplasmic reticulum for synthesis of VLDL in rat hepatocytes

Actin-associated motors directly regulate LD catabolism. Non-muscle myosin IIA (NMIIA) depletion enlarges LDs and elevates triglyceride storage due to defective lipolysis. Formin-like 1 (FMNL1) promotes the assembly of myosin II-decorated actin filaments on LDs, facilitating their dissociation and enhancing triglyceride accessibility for hydrolysis in primary human neutrophils in vitro [17]. These findings establish a direct, experimentally demonstrated role for actomyosin contractility in lipolysis.

Microtubule-based mechanisms are also critical. The GTPase ARL8B participates in a major lipolytic pathway in human macrophages. ARL8B-GDP localizes to LDs through an N-terminal amphipathic helix, while ARL8B-GTP preferentially associates with lysosomes. Through heterotypic complex formation, ARL8B facilitates LD–lysosome contact and promotes efficient lipid transfer between these organelles [19]. This represents the predominant lipolytic mechanism in human macrophages, supported by loss-of-function genetic experiments.

Lipid droplet transport along microtubules is mediated by kinesin and dynein motors. In rat hepatocytes, kinesin-1 is recruited to LDs via the GTPase ARF1 downstream of insulin signaling, facilitating LD transport to the smooth endoplasmic reticulum for VLDL assembly [51]. This mechanism has been established through a combination of imaging, biochemical, and genetic approaches. Additionally, the lipid droplet transporter LIPTER links LDs to the actin cytoskeleton in human cardiomyocytes by binding both phosphatidic acid and PI4P on LD membranes and myosin heavy chain 10 (MYH10) [18]. Whether analogous LD–cytoskeleton linkers operate in macrophages during foam cell formation is an unresolved question.

TLR4 signaling promotes lipid accumulation via cytoskeletal remodeling

TLR4-mediated small GTPase signaling and actin remodeling

TLR4 stimulation with LPS induces membrane ruffling and enhances macropinocytosis in macrophages through Rab13 activation. LPS triggers accumulation of GTP-bound Rab13 in dorsal ruffles, associated with a doubling of macropinocytic uptake and a notable increase in macropinosome size [52]. This study provides direct evidence linking TLR4 activation to enhanced macropinocytosis through a specific small GTPase. However, the signaling intermediates between TLR4 and Rab13 activation have not been fully defined.

The TLR4 adaptor TRAM directly participates in cytoskeletal regulation by interacting with Rab11-family interacting protein 2 (FIP2), promoting activation of the actin-regulatory GTPases Rac1 and Cdc42 [53]. This interaction represents one of the more direct connections between TLR4 signaling and the core actin remodeling machinery and is supported by biochemical and genetic approaches.

Integrin activation is upstream of LPS-induced cell spreading in inflammatory macrophages. This process is mediated by β2 integrin activation controlled by a linear signaling pathway: MyD88/IRAK/p38/Rap1 [54]. Inhibition of Rap1, but not other Ras-like or Rho-like small GTPases, abolishes αMβ2 integrin activation induced by LPS or TNF-α [55]. This pathway is supported by specific pharmacological and genetic inhibition experiments (Fig. 3).

Fig. 3.

Mechanisms of TLR4 signaling in the induction of actin remodeling in macrophages. Integrin activation is upstream of LPS-stimulated actin remodeling by inducing a linear signaling pathway: Myd88/IRAK/p38/Rap1. TLR4 adaptor TRAM controls the phagocytosis of gram-negative bacteria by promoting activation of the actin-regulatory GTPases Rac1 and Cdc42. In response to bacterial infection, peroxisomes oxidation alters glycerophospholipid composition in cell membrane and induces Rho1-dependent signals, and drives cytoskeletal remodeling of macrophages. LPS activates Rab13 and enhances the accumulation of active GTP-loaded Rab13 in macrophage dorsal ruffles. TLR4 activates the de novo lipid synthesis pathway by stimulating SREBP1α, promoting the synthesis and elongation of fatty acyl chains such as phosphatidylcholine and phosphatidylethanolamine in membrane phospholipids. This enhances the interaction between the actin cytoskeleton and membrane lipid rafts through proteins such as moesin and cofilin, facilitating actin remodeling. LPS could also induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for the reorganization of actin cytoskeleton

TLR4-mediated lipid membrane remodeling and cytoskeletal effects

Remodeling of membrane lipid composition could also influence small GTPase activity. During bacterial infection, peroxisome-driven oxidation alters glycerophospholipids and induces Rho1-dependent signaling, promoting cytoskeletal reorganization [56]. Similarly, TLR4 activation influences the actin cytoskeleton indirectly through changes in membrane lipid composition. TLR4 signaling stimulates de novo lipid synthesis via sterol regulatory element-binding protein 1α (SREBP1α), increasing the synthesis and elongation of fatty acyl chains in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These lipid changes strengthen the interaction between the actin cytoskeleton and lipid rafts through proteins including moesin and cofilin, thereby facilitating actin remodeling and augmenting phagocytic function [57, 58]. This mechanism is supported by lipidomics, biochemical interaction studies, and functional phagocytosis assays. However, whether these lipid changes specifically promote foam cell formation—as opposed to phagocytosis of pathogens—has not been directly tested (Fig. 3).

Additional TLR4–cytoskeleton connections

TLR4 engages several other pathways that may contribute to cytoskeletal remodeling and lipid accumulation, though the evidence for each varies in strength. First, LPS induces Piezo1-mediated calcium influx, which activates CaMKII and the Hippo kinases Mst1/2, leading to Rac-mediated actin reorganization [59]. This pathway is supported by genetic and pharmacological experiments. Direct evidence linking it to lipid uptake rather than general actin remodeling remains to be established. Second, LPS induces extensive microtubule acetylation, which enhances p38 kinase signaling and IL-6 production [60] (Fig. 3). In microglia, LPS reorganizes microtubules into a stable, centrosomally anchored array that facilitates cytokine trafficking, with cyclin-dependent kinase 1 identified as a critical upstream regulator [61]. Despite these established connections between TLR4 and microtubule remodeling, direct evidence that microtubule changes contribute to macrophage lipid accumulation is lacking. The role of microtubules in foam cell formation remains an area requiring dedicated investigation.

The TLR4–SYK/Src axis as a bridge to cytoskeletal remodeling

Two tyrosine kinases, SYK and Src, function as critical signaling hubs connecting TLR4 activation to cytoskeletal remodeling and lipid uptake. Understanding their distinct contributions and potential interplay is essential for a comprehensive model of TLR4-driven foam cell formation (Fig. 1).

SYK is a multi-effector hub. In the context of mmLDL recognition, TLR4 recruits and activates SYK, which initiates a Vav1/Ras/Raf/MEK/ERK1/2 cascade leading to Rac, Cdc42, and Rho activation and subsequent macropinocytosis ([41]; see also Section “Lipid uptake via macropinocytosis”). Beyond this well-characterized pathway, SYK also participates in a structurally distinct mode of lipid uptake: lysosomal synapse formation in response to agLDL. As noted in Section “Lipid uptake via cortical actin remodeling”, the TLR4/MyD88/PI3K/SYK/Akt axis is required for this process [47]. These represent the two of the best-characterized direct mechanistic links between TLR activation, cytoskeletal remodeling, and foam cell formation. Thus, SYK functions as a common node through which TLR4 engages multiple actin-dependent lipid uptake mechanisms: macropinocytosis for nLDL and mmLDL, and lysosomal synapse assembly for agLDL.

It is important to note that SYK operates in both MyD88-dependent and MyD88-independent branches of TLR4 signaling. The MyD88-independent branch induces moderate cytokine release through AP-1 activation, accompanied by robust ROS generation and cytoskeletal remodeling (reviewed in [62]). Whether these two branches differentially contribute to lipid accumulation—for instance, whether MyD88-dependent SYK activation primarily drives lysosomal synapse formation while MyD88-independent SYK signaling favors macropinocytosis—remains an unresolved question requiring systematic investigation.

Src is a parallel pathway in the atherosclerotic context. In murine atherosclerotic plaques, both Src and TLR4 are upregulated in macrophages. In vitro, oxLDL promotes the physical interaction between TLR4 and Src at the plasma membrane, enhancing Src activation and downstream lipid uptake [63]. Unlike SYK, which engages defined small GTPase cascades, the downstream effectors through which Src remodels the cytoskeleton to promote lipid uptake remain poorly defined. Whether Src acts through shared effectors (e.g., cortactin, paxillin) to cooperate with SYK-dependent pathways, or operates through an independent mechanism, is an important direction for future study.

The coexistence of SYK- and Src-dependent pathways downstream of TLR4 raises the possibility that these kinases function cooperatively or sequentially in distinct lipid uptake contexts. For instance, Src-mediated phosphorylation of SYK or shared adaptor proteins could modulate the amplitude or duration of cytoskeletal remodeling. Elucidating the functional relationship between these two kinases will be critical for a complete understanding of TLR4-driven foam cell formation and may reveal therapeutic targets with greater specificity than global TLR4 blockade.

TLR4 activation amplifies lipid accumulation in a lipid-rich milieu

A critical observation demonstrates the synergistic relationship between TLR4 activation and lipid availability. In the presence of oxLDL, LPS stimulation increases foam cell formation from 29 to 60%, while a TLR4-blocking antibody reduces the differentiation of TLR4-competent cells into foam cells from 29 to 13% [64]. This bidirectional evidence—enhancement by TLR4 activation and suppression by TLR4 blockade—provides strong support for a causal role of TLR4 in foam cell formation. These findings suggest that chronic TLR4 activation in a lipid-rich milieu such as atherosclerosis may sustain a self-reinforcing cycle: modified lipoproteins activate TLR4, TLR4-driven cytoskeletal remodeling enhances lipid uptake, and accumulated lipid may further promote inflammatory signaling. Direct in vivo evidence for this cycle, however, remains limited.

Targeting the cytoskeleton in macrophage as a therapeutic strategy

Therapeutic potential

Several pharmacological interventions targeting cytoskeletal regulators have shown efficacy in preclinical atherosclerosis models. EIPA treatment effectively antagonizes arterial foam cell formation in a mouse model of atherosclerosis [42]. Macrophage-specific knockout of the small GTPase Rheb in mice reduces Western diet-induced atherosclerotic lesions by 32%, with mechanistic studies demonstrating repression of oxLDL-induced lipid uptake, inflammation, and macrophage proliferation through inhibition of mTOR signaling [65]. This genetic study provides some of the most direct evidence that targeting cytoskeleton-regulatory GTPases in macrophages can limit atherogenesis. The ROCK inhibitor fasudil attenuates weight gain and insulin resistance in mice fed a high-fat diet [66], though the cell type(s) responsible and the mechanism—whether through effects on vascular function, adipose tissue fibrosis, macrophage infiltration, or systemic metabolism—remain unclear.

Recent work has established a high-content drug screening platform that directly targets digestive exophagy, a process by which macrophages digest aggregated LDL (agLDL) within a lysosomal synapse. A screen of over 2000 repurposed drugs identified compounds that inhibit this process, including inhibitors of SYK (lanraplenib), PI3Kβ/δ (AZD-8186), and SRC (saracatinib)—kinases known to regulate digestive exophagy. Strikingly, three of the five validated hit compounds did not inhibit oxLDL-induced foam cell formation, providing direct pharmacological evidence that the agLDL-driven and oxLDL-driven pathways to foam cell formation can be targeted independently [46]. This finding has profound therapeutic implications: a strategy that selectively inhibits digestive exophagy could reduce macrophage lipid burden from agLDL without necessarily compromising the oxLDL-handling functions that may be important for host defense.

Colchicine, an inhibitor of microtubule polymerization, is FDA-approved for secondary prevention in patients with coronary disease and reduces major adverse cardiovascular events in a broad population of patients with atherosclerosis [67, 68]. However, the mechanism of this cardiovascular benefit requires careful consideration. Colchicine's established anti-inflammatory effects include inhibition of NLRP3 inflammasome activation and reduced neutrophil chemotaxis, both of which occur at concentrations achieved with low-dose therapy (0.5 mg daily). Whether colchicine also directly inhibits macrophage foam cell formation through effects on microtubule-dependent lipid trafficking has not been clinically demonstrated.

Safety challenges

The therapeutic targeting of cytoskeletal regulators also presents significant safety challenges. The cytoskeleton is essential for host defense functions in macrophages, including phagocytosis of pathogens, migration to sites of infection, efferocytosis of apoptotic cells, and cytokine secretion. Systemic cytoskeletal inhibition carries an inherent risk of immunosuppression. In the COLCOT [69] and LoDoCo2 [70] trials, colchicine was associated with a modest increase in non-cardiovascular death, including infection-related deaths, which remains incompletely explained. Whether this reflects impaired macrophage function or other effects requires continued investigation.

No currently available pharmacological agent targeting the cytoskeleton shows cell-type selectivity for macrophages, let alone for the specific macrophage subsets involved in atherogenesis. The high-content screening platform described above represents a promising tool for identifying such selective modulators of digestive exophagy. Future development of macrophage-targeted delivery systems (e.g., nanoparticle-based approaches) or isoform-specific inhibitors that spare essential immune functions will likely be necessary for clinical translation. Additionally, the optimal therapeutic window—intermittent versus continuous treatment, and stage of disease—remains to be defined.

Conclusions

The cytoskeleton functions as a dynamic signaling hub that integrates metabolic and inflammatory signals in macrophages, positioning it as a central regulator of lipid accumulation relevant to atherosclerosis. This review has organized the available evidence into a framework wherein TLR4 activation, through MyD88/TRAM and SYK/Src branches, engages small GTPases and their effectors to drive actin remodeling, which in turn facilitates lipid internalization through macropinocytosis, receptor redistribution, and lysosomal synapse formation, as well as modulating LD dynamics. However, the mechanistic completeness of this framework varies substantially across its components. The strongest evidence supports: (1) the TLR4/SYK/Vav/Ras/MEK/ERK/paxillin/Rac/Cdc42 pathway to macropinocytosis; (2) the TLR4/MyD88/PI3K/SYK/Akt pathway to lysosomal synapse formation; (3) the MyD88/IRAK/p38/Rap1 pathway to integrin activation; and (4) the role of actomyosin and ARL8B in LD catabolism. In each of these cases, intermediate signaling steps have been experimentally validated using genetic and/or pharmacological approaches.

Several critical questions remain unresolved. For example, what is the relative contribution of macropinocytosis versus receptor-mediated routes in TLR4-driven foam cell formation in vivo? Macrophage-specific deletion of key macropinocytosis components, combined with models of atherosclerosis, could possibly address this. Can cytoskeletal modulation in tissue-resident macrophages alleviate local inflammation without compromising systemic immune defense? This question is central to the therapeutic translation of the pathway discussed here. Are there macrophage-specific isoforms of cytoskeletal regulators that could be therapeutically targeted? Genetic and proteomic screens across macrophage activation states may identify such targets.

Nevertheless, the convergence of TLR signaling, cytoskeletal remodeling, and lipid metabolism in macrophages represents a compelling nexus for understanding and potentially treating atherosclerosis. Elucidating the molecular details of this pathway—through a combination of in vivo imaging to capture real-time cytoskeletal dynamics, cell-type-specific genetic models, and biochemical reconstitution—will be essential for translating these mechanistic insights into therapeutic strategies that selectively target pathological lipid accumulation while preserving host defense.

Abbreviations

ARL8B

ADP-ribosylation factor-like protein 8B

CaMKII

Calcium/calmodulin-stimulated protein kinase II

DAMP

Damage-associated molecular pattern

ERK

Extracellular signal-regulated kinase

FMNL1

Formin-like 1

FIP2

Rab11 Family-interacting protein 2

LAM

Lipid-associated macrophages

LD

Lipid droplets

LDL

Low-density lipoprotein

LIPTER

Lipid-droplet transporter

MAPK

Mitogen-activated protein kinase

MYH10

Myosin heavy chain 10

NMIIA

Non-muscle myosin IIA

Nox2

NADPH oxidase 2

PAMPs

Pathogen-associated molecular patterns

PLC

Phospholipase C

PPARγ

Peroxisome proliferator-activated receptor γ

PRRs

Pattern recognition receptors

SR-BI

Scavenger receptor class B type 1

SREBP1α

Sterol regulatory element-binding protein 1α

SYK

Spleen tyrosine kinase

TLR4

Toll-like receptor 4

Authors’ contributions

**En Wang** : Writing-original draft. **Hamizah Shahirah Hamezah** : Writing-original draft. **Rongchun Han** : Supervision; Funding acquisition; writing-review and editing. **Xiaohui Tong** : Conceptualization; Supervision; Funding acquisition; Writing-original draft; Project administration; Writing-review and editing.

Funding

This work was supported by the Excellent Young Teachers Program of Anhui Province (YQZD2024019, China) and MOE-Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials (xtcx202408).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.