Comments on ‘Macrophages form dendrite-like pseudopods to enhance bacterial ingestion’

Macrophages are key innate immune cells that clear pathogens through phagocytosis and micropinocytosis (Mylvaganam et al., 2021). While actin-based protrusions such as filopodia and lamellipodia have been well studied as typical substructures in macrophages to capture pathogens (Blake and Gallop, 2023; Pratiwi et al., 2024), their effectiveness against massive bacterial onslaughts is limited. Gram-negative bacteria, often exhibiting significant antimicrobial resistance, present serious clinical threats, including conditions such as sepsis (Holmes et al., 2021). Although it has been indicated that lipopolysaccharide (LPS)-mediated adaptations in macrophages can improve pathogen clearance by receptor diffusion (Freeman et al., 2018), the morphological strategies that macrophages employ to enable rapid bacterial uptake during overwhelming infections remain incompletely understood. In our study recently published in the EMBO Journal, we identified a previously unrecognized morphological adaptation of macrophages, characterized by the formation of dendrite-like pseudopods (DLPs) that enable macrophages to ‘fish’ distal bacteria with remarkable efficiency (Figure 1; Fan et al., 2025). To clarify the unique subcellular structure of DLPs in vivo, we employed two-photon intravital microscopy to observe the morphology of macrophages embedded in the serosal extracellular matrix in the mouse peritoneal cavity. Within 1–2 h after intraperitoneal Salmonella challenge, resident F4/80-positive macrophages rapidly elaborated the elongated, branched pseudopods, while under mock conditions, most macrophages displayed a sleek, spindle-shaped morphology with few surface protrusions. In vitro exposure of human THP-1 macrophages and murine bone marrow-derived macrophages to Gram-negative bacteria (Salmonella, Escherichia coli, and Shigella) at high multiplicity of infection (MOI ≥ 20) reproducibly generated similar DLP structures, whereas exposure to Gram-positive bacteria (Bacillus and Listeria) or sterile epithelial-cell debris did not show this response, indicating the specificity for Gram-negative stimuli. Notably, DLP formation exhibited both dose and temporal dependence: the percentage of DLP-positive macrophages increased along with bacterial burden, plateauing at ~30% by 6 h post-infection, and promptly declined after antibiotic-mediated bacterial clearance, underscoring the dynamic and reversible nature of the response. Morphometric analysis revealed that DLPs reached 30–60 μm in length, exhibited multi-level branching, and terminated in distinctive bean-shaped tips, clearly distinguishing them from classical filopodia or lamellipodia. Filopodia are thin, needle-like projections that retract within minutes (Schaks et al., 2019), and lamellipodia are broad, sheet-like ruffles that rarely exceed 10 μm in length (Sadhu et al., 2023), while DLPs are substantially wider at their tips, taper gradually, and remain extended for several hours. Sholl analysis, traditionally employed to quantify neuronal dendrite complexity, demonstrated a marked increase in the number and length of intersections between the macrophage periphery and concentric circles, confirming the acquisition of a higher-order branched architecture. Immunostaining further revealed that DLPs are enriched in linear actin bundles and vimentin intermediate filaments, whereas microtubules are distributed uniformly, suggesting a specialized cytoskeletal composition tailored for mechanical stability and long-range exploration. Mechanistically, bacterial LPS engages Toll-like receptor 4 (TLR4) to activate the NF-κB pathway (Baldwin, 1996), leading to the marked upregulation of the macrophage-enriched guanine nucleotide exchange factor ARHGEF3 (Arthur et al., 2002). Chromatin immunoprecipitation and luciferase reporter assays confirmed a functional NF-κB binding site within the ARHGEF3 promoter; mutation of this site abrogated LPS responsiveness. Upon LPS stimulation, ARHGEF3 mRNA and protein levels rose in parallel, peaking at 4–6 h, and the protein was selectively enriched at the nascent tips of DLPs, as evidenced by super-resolution microscopy and proximity ligation assays. Co-immunoprecipitation experiments demonstrated a direct interaction between ARHGEF3 and TLR4, suggesting that the receptor may serve as a scaffold to concentrate ARHGEF3 at sites of active membrane protrusion. Consistent with this model, CRISPR-mediated knockout of TLR4 or pharmacological inhibition with TAK-242 prevented both ARHGEF3 induction and DLP formation, whereas expression of constitutively active RhoA restored pseudopod outgrowth in TLR4-deficient cells. Graphical summary of the study: the role of DLPs in enhancing the uptake of Gram-negative bacteria by macrophages. This working model was modified from Fan et al. (2025). Downstream of ARHGEF3, live-cell imaging of SiR-actin revealed periodic cycles of actin assembly and disassembly that propel stepwise DLP elongation. Each cycle comprised three phases: (i) a growth phase in which linear actin bundles fill the nascent protrusion; (ii) a steady phase of several minutes during which both the length and F-actin intensity remain constant; and (iii) a collapse phase marked by rapid actin depolymerization and slight forward extension. Subsequent re-initiation of actin polymerization at the base of the collapsed structure gave rise to a new elongation event. Pharmacological disruption of formin-mediated actin nucleation (using SMIFH2) (Nishimura et al., 2021), myosin II activity (using blebbistatin) (Zhang et al., 2017), or fascin cross-linking (using NP-G2-044) (Wang et al., 2021) abolished DLP formation, whereas inhibition of Arp2/3-mediated branched actin (using CK666) (Henson et al., 2015) or microtubule depolymerization (using nocodazole) (Florian and Mitchison, 2016) had minimal effects, underscoring the specific requirement for linear actin bundles. Importantly, vimentin intermediate filaments formed a dense meshwork that co-aligned with actin bundles; CRISPR knockout of vimentin or simvastatin-mediated disassembly of the vimentin network produced unstable bleb-like protrusions that failed to achieve the length and persistence characteristic of DLPs. Thus, the synergistic interplay between ARHGEF3-driven actin dynamics and vimentin-mediated mechanical reinforcement underpins the construction and stability of DLPs. Recent work by Huang et al. (2025) has demonstrated that vimentin coordinates actin stress fibers and podosomes to control macrophage-mediated extracellular matrix degradation in tumor settings. Our findings extend this paradigm to immune surveillance by revealing that vimentin also stabilizes infection-induced protrusions. Thus, vimentin–actin crosstalk appears context-dependent: tumor-associated macrophages deploy it for matrix invasion, whereas septic macrophages synergize vimentin and actin to capture bacteria. The observation that both scenarios rely on ARHGEF3–RhoA signaling further underscores the versatility of this molecular toolkit in shaping macrophage behavior according to microenvironmental cues. Functionally, DLPs extend the macrophage search radius and double the number of internalized bacteria compared with control cells across a range of MOIs. Confocal imaging of mCherry-labelled Salmonella demonstrated that bacteria are initially trapped at the bulbous tips of DLPs and subsequently transported retrogradely toward the cell body. During transport, bacterial fluorescence intensity progressively decreased, and co-localization with the lysosomal marker LAMP1 confirmed the delivery to degradative compartments. Adoptive transfer of DLP-competent macrophages into Salmonella-infected mice significantly reduced bacterial burden in peritoneal lavage fluid and the liver at 48 h post-infection and dampened systemic expression of pro-inflammatory cytokines. Conversely, transfer of macrophages in which DLP formation was blocked by siRNA-mediated ARHGEF3 knockdown failed to confer protection, despite comparable cytokine production, demonstrating that the enhanced bacterial uptake rather than cytokine modulation is the primary mechanism of benefit. Clinically, sepsis mortality remains high despite antibiotics in use, partly because overwhelmed macrophages fail to clear the bacterial burden. In our work, ex-vivo ‘arming’ of autologous macrophages by brief LPS pulse to induce DLPs before reinfusion could represent a cell-based adjunctive therapy that augments conventional antimicrobial regimens. More broadly, the identification of ARHGEF3 as a central regulator of infection-induced morphogenesis offers a rational target for small-molecule modulation: enhancers could boost macrophage phagocytic capacity against multidrug-resistant Gram-negative pathogens, whereas inhibitors might mitigate deleterious inflammation. Notably, ARHGEF3 expression is largely restricted to macrophages and platelets, suggesting that selective modulation may be feasible with limited off-target effects. Collectively, our work uncovers a hitherto overlooked morphological adaptation that equips macrophages with a long-range bacterial ‘fishing net’. By linking TLR4–NF-κB–ARHGEF3–RhoA signaling to vimentin-stabilized actin dynamics, we provide a mechanistic framework for enhancing macrophage-mediated bacterial clearance in morphology. These findings add a new layer to the textbook view of macrophage morphological plasticity and inspire morphological therapeutic strategies that harness macrophage mechanics to combat bacterial infection. [This work was supported by grants from the National Key R&D Program of China (2022YFC2303500 and 2024YFC2310003), the National Natural Science Foundation of China (32222022 and 92354301), R&D Program of Guangzhou National Laboratory (GZNL2023A03004), and Natural Science Foundation of Shanghai (23ZR1470900) to Y.J.]

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