Introduction

Macrophages are highly plastic innate immune cells that play crucial roles in host defense, tissue homeostasis, and regulation of inflammation [1,2]. They exhibit remarkable functional diversity depending on the environmental stimuli, allowing them to polarize into different activation states [3]. Macrophages are classified into two major subsets: classically activated (M1) macrophages, which exhibit proinflammatory and microbicidal functions, and alternatively activated (M2) macrophages, which contribute to tissue repair, immune modulation, and anti-inflammatory responses [4-6]. M2 macrophages are induced by anti-inflammatory interleukins (ILs) such as IL-4 and IL-13, which drive metabolic, transcriptional, and morphological changes essential for their function [7-9]. One of the hallmarks of M2 polarization is the elongation of macrophages, which is correlated with increased anti-inflammatory activity and enhanced migration [10]. However, molecular mechanisms underlying these morphological changes remain unclear.

Gangliosides, a class of sialic acid-containing glycosphingolipids, are integral components of the plasma membrane and play crucial roles in cellular processes, such as signal transduction, cell-cell interactions, and immune modulation [11]. Among these, ganglioside monosialodihexosylganglioside (GM3) and ganglioside disialodihexosylganglioside (GD3) regulate immune responses by modulating receptor clustering and downstream signaling events [12,13]. Recent studies have suggested that gangliosides are involved in macrophage polarization and function, although their exact role in M2 macrophages remains unclear. Notably, macrophage elongation is considered a hallmark of M2 polarization and is often associated with changes in cytoskeletal organization and lipid raft composition, where gangliosides are highly enriched [2]. It has been proposed that gangliosides influence macrophage morphology by modulating cytoskeletal rearrangements and affecting key signaling pathways such as phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) [14].

Previous research has demonstrated that the elongation of M2 macrophages is functionally relevant, as elongated macrophages exhibit enhanced migratory capacity and reduced phagocytic activity, which are characteristic features of tissue-repairing macrophages [15]. The increased migration of M2 macrophages has been attributed to their response to chemotactic signals such as monocyte chemoattractant protein-1 (MCP-1), which facilitates their recruitment to sites of injury and inflammation [16]. Conversely, the reduced phagocytic activity of M2 macrophages is thought to contribute to their immunomodulatory role, as they prioritize anti-inflammatory functions over pathogen clearance [17]. The mechanisms by which gangliosides influence the functional properties of M2 macrophages remain poorly understood, prompting further investigation into their role in macrophage function and morphology following M2 polarization.

In the present study, we aimed to elucidate the role of gangliosides in the elongation, migration, and phagocytic activity of M2 macrophages. Specifically, we investigated whether ganglioside biosynthesis was required for M2-associated morphological changes and functional adaptations. We also examined the involvement of key signaling pathways, including ERK and AKT, in ganglioside-mediated macrophage elongation. By identifying the molecular mechanisms underlying ganglioside-dependent macrophage polarization, our findings provide new insights into the regulation of macrophage plasticity, and may contribute to the development of novel therapeutic strategies for immune modulation in inflammatory diseases.

Methods

Ethical statements:

This study did not involve the use of human participants, human data, human tissues, or animals. All experiments were conducted using established mouse RAW264.7 cells and primary bone marrow macrophages (BMMs) obtained from commercial or institutional sources in accordance with relevant guidelines and regulations. Therefore, ethical approval and patient consent were not required.

1. Antibodies and reagents

Recombinant murine IL-4 (214-14), IL-13 (210-13), and MCP-1 (250-10) were purchased from PeproTech. GM3 was supplied by AdipoGen (AG-CN2-9002). Anti-GD3 (MA1-25302), anti-mouse IgM Alexa Fluor 488 (A-21042), and anti-mouse IgG Alexa Fluor 488 (A-11001) antibodies were purchased from Thermo Fisher Scientific. Anti-ERK (sc-154), anti-pERK (sc-7383), anti-pSTAT6 (sc-11762-R), anti-STAT6 (sc-981), anti-ST3Gal5 (sc-67347), anti-ST8Sia1 (sc-99093), and anti-p38 (sc-535) were purchased from Santa Cruz Biotechnology. Anti-pJNK (#9255) and anti-pp38 (#9211) antibodies were obtained from Cell Signaling Technology. Anti-AKT1 (ab32505) and anti-AKT1 (phospho S473, ab81283) antibodies were obtained from Abcam. Anti-GM3 (CAC-NBT-M102-EX) antibody was purchased from COSMO BIO. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI; D9542) was provided from Sigma-Aldrich.

2. Cell culture

RAW264.7 cells were obtained from the Korean Cell Line Bank. Mouse BMMs were prepared as previously described [18]. Briefly, bone marrow cells were isolated by flushing the diaphysis of femurs and tibias with phosphate-buffered saline and incubated overnight in α-modified Eagle medium (MEM) supplemented with 10% fetal bovine serum (FBS) to remove non-hematopoietic lineage cells. Floating cells were further incubated in α-MEM supplemented with 10% FBS and macrophage colony-stimulating factor (M-CSF) (30 ng/mL). After 3 days, non-adherent cells were removed and adherent BMMs were used further incubated in α-MEM supplemented with 10% FBS and M-CSF (30 ng/mL) for 4 days more.

3. Reverse transcription-polymerase chain reaction

Total RNA was isolated with TRIzol reagent (Invitrogen) and cDNA was obtained from 2 µg of total RNA using MMLV reverse transcriptase (Promega Corp., and target genes were amplified using AccuPower PCR PreMix). The primer sequences used in this study are listed in the Supplementary Table 1.

4. Immunoblot assay

Cells were lysed in RIPA buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, phosphatase inhibitor cocktail (Sigma-Aldrich), and proteinase inhibitor cocktail (Calbiochem). Protein concentration was determined using the bicinchoninic acid assay. Lysates (20–40 µg) were resolved on polyacrylamide gels and then immunoblotted as described previously [19].

5. Ganglioside flow cytometric analysis

RAW264.7 cells and BMMs were treated with IL-4 and IL-13 for 24 hours. After cells were fixed with IC fixation buffer (eBioscience), cells were incubated with anti-GM3 mouse IgM or anti-GD3 mouse IgG antibody at 4 °C for 2 hours. Alexa Fluor 488-conjugated anti-mouse IgM and IgG antibodies were used for anti-GM3 and GD3, respectively. The analysis was conducted using Attune X (Thermofisher) and Flow Jo 10.1 software (BD Bioscience).

6. Cell migration assay

Cell migration assays were performed using 24-well transwell inserts (8 μm). RAW264.7 cells and BMMs (2×10⁵ cells/well) were placed in the upper chamber of the transwell and treated with IL-4 and IL-13, or GM3 alone. After 24 hours, MCP-1 (10 ng/mL) was added to the lower chamber. RAW264.7 cells were incubated for 12 hours, and BMMs were incubated for 6 hours. Cells that migrated to the lower part of the membrane were stained with hematoxylin and eosin (Sigma-Aldrich) and counted.

7. Phagocytosis assay

The phagocytic ability of the macrophages was estimated using a Phagocytosis Assay Kit (Cayman Chemical). RAW264.7 cells and BMMs were seeded at a density of 2×10⁵ cells/well on 24-well chamber slides. The latex beads-rabbit IgG-fluorescein isothiocyanate complex was added to the culture medium at a 1:200 dilution and incubated at 37 °C for 2 hours. After washing thrice, the cells were counterstained with DAPI to visualize the nucleus. Cells were photographed using confocal microscopy and the phagocytosis index (% of engulfed cells/total number of cells) was calculated.

8. Gene knockdown using siRNA

To knock down endogenous St3gal5, small interfering RNA (siRNA) constructs were purchased from Bioneer. RAW264.7 cells and BMMs (2×10⁵ cells/well) were seeded into 6-well plates and siRNAs (200 nM) were transfected into cells using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. The knockdown efficiency of siRNA was verified by reverse transcription-polymerase chain reaction and Western blot analysis 48 hours after transfection.

9. Statistical analysis

Values for ganglioside levels, migration rate, and phagocytosis rate were converted into fold differences compared with those in the control group. Values were expressed as mean±standard deviation. Two-tailed Students t-test for comparisons between two different groups or one-way analysis of variance followed by Tukey’s ad hoc statistical test for comparisons between multiple groups were performed using GraphPad Prism (GraphPad Software). The minimum significance level was determined at a p-value of 0.05 for all analyses. All experiments were independently conducted at least thrice.

Results

1. Gangliosides facilitate macrophage elongation during M2 polarization

First, we investigated the involvement of gangliosides in M2-polarized macrophage elongation. The expression of ganglioside synthesis-associated mRNAs and proteins (Supplementary Fig. 1) was evaluated in BMMs after IL-4/IL-13 stimulation. UDP-glucose ceramide glucosyltransferase (Ugcg), which catalyzes the first step in ganglioside biosynthesis by converting ceramide to glucosylceramide, was reduced, and beta-1,4-galactosyltransferase 6 (B4galt6), an enzyme responsible for adding galactose to glucosylceramide to form lactosylceramide, was unchanged upon IL-4/IL-13 stimulation. In contrast, sialic acid-synthesizing genes, including St3gal5 and St8sia1, which encode GM3 and GD3 synthases, respectively, were significantly upregulated under these conditions (Fig. 1A).

Immunoblot analysis confirmed the increased expression of the two ganglioside synthesis-related proteins upon M2 polarization (Fig. 1B). These findings indicate that IL-4/IL-13 stimulation of M2 polarization might enhance ganglioside synthesis, GM3 and GD3, in macrophages. Flow cytometry was conducted to confirm the increased synthesis of GM3 and GD3 in RAW264.7 cells and BMMs during M2 polarization. The mean fluorescence intensity of both gangliosides exhibited a significant increase upon IL-4/IL-13 treatment, and quantitative analysis also showed a notable fold-increase (Fig. 1C). To identify whether increased gangliosides level is related to M2 macrophage elongation, a glucosylceramide synthase inhibitors D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) was applied to M2 condition. Both of RAW264.7 cells and BMMs were successfully elongated by IL-4 and IL-13 co-stimulation, which is a well-known characteristic of M2 macrophages. However, D-PDMP markedly reduced the elongation of both types of macrophages (Fig. 1D). As the ganglioside GM3 is a precursor for GD3 synthesis, we focused on GM3 and its producing enzyme, St3gal5, which converts lactosylceramide into GM3, to examine the involvement of gangliosides in M2 macrophage elongation. Silencing of St3gal5 using siRNA was successful in RAW264.7, and BMMs (Fig. 1E), and completely abrogated M2 stimulation-induced elongation by IL-4 and IL-13 in both cell lines (Fig. 1F). Taken together, these results suggest that increased levels of the gangliosides GM3 and GD3 in M2 macrophages enhance morphological elongation and possess potential regulatory roles in M2 macrophage function.

2. GM3 mediates increased migration of M2-polarized macrophages induced by IL-4 and IL-13

Macrophage elongation is likely to be associated with motility. To assess the role of gangliosides in M2 macrophage migration, a transwell migration assay was performed using RAW264.7 cells (Fig. 2A, 2C) and BMMs (Fig. 2B, 2D). MCP-1 induced the transmigration of macrophages, and M2 polarization upon IL-4/IL-13 stimulation was significantly enhanced compared to the control. Notably, a single GM3 treatment promoted macrophage transmigration, similar to M2 stimulation (Fig. 2A, 2B). To confirm the role of gangliosides in macrophage migration, St3gal5 was knocked down in macrophages by siRNA. The MCP-1-induced transmigration of both macrophages under M2 condition was mostly diminished to normal levels by St3gal5 silencing (Fig. 2C, 2D). These data indicated that gangliosides, particularly GM3, modulate the migratory function as well as elongation in M2 macrophages.

3. GM3 reduces phagocytosis in M2-polarized macrophages

Reduced phagocytic function is a well-known phenotype of M2 macrophage [20]. Therefore, we evaluated the function of gangliosides during macrophage phagocytosis. GM3 was treated to RAW264.7 cells and BMMs, and compared to IL-4/IL-13 treatment for M2-macrophage polarization. Fluorescence imaging revealed a significant reduction in latex bead uptake in both cell types following IL-4/IL-13 stimulation. Similar to the IL-4/IL-13 treatment, only GM3 treatment significantly decreased latex bead uptake in both cells (Fig. 3A, 3B). Based on these results, we suspected that M2 macrophages reduced their phagocytic capacity via increased levels of gangliosides such as GM3 and GD3. To confirm the involvement of ganglioside synthesis, we silenced the key enzyme St3gal5 and evaluated latex bead uptake by M2-conditioned RAW264.7 cells and BMMs. In St3gal5-silenced cells, the decreased phagocytosis was almost restored to the capacity of unpolarized cells (Fig. 3C, 3D), demonstrating that gangliosides are key regulators of phagocytic function.

4. Ganglioside-mediated macrophage elongation involves ERK and AKT pathways

Next, we investigated the signaling pathways underlying GM3-induced M2-macrophage elongation. First, RAW264.7 cells were pretreated with several pathway-specific inhibitors prior to exposure to M2 conditions. LY294002 and U0126, inhibitors of the PI3K/AKT and MEK/ERK pathways, respectively, significantly prevented IL-4/IL-13-induced elongation (Fig. 4A). We further assessed whether these pathways regulate GM3-induced macrophage elongation. Similar to M2-conditioned macrophages, only LY294002 and U0126 successfully blocked GM3-induced elongation in RAW264.7 cells and BMMs (Fig. 4B), indicating that the PI3K/AKT and MEK/ERK pathways are crucial for ganglioside-mediated elongation in M2 macrophages. Finally, we confirmed GM3-induced activation of these pathways using immunoblot assays. GM3 significantly induced phosphorylation of AKT and ERK2 after 0.5 hour but no phosphorylation of STAT6 until 6 hours in RAW264.7 cells. Moreover, p38 was dephosphorylated rather than enhanced by GM3 treatment. Activation of AKT and ERK2, but not STAT6 or p38, was consistent with the prevention of elongation by specific inhibitors (Fig. 4C). Taken together, increasing levels of gangliosides, including GM3, induce M2 macrophage elongation associated with migration and phagocytosis via the PI3K/AKT and ERK signaling pathways in the IL-4 and IL-13 rich microenvironment.

Discussion

Macrophages are innate immune cells found in all tissues that play a crucial role in maintaining homeostasis. As the first line of defense, M1 macrophages are highly responsive to the environment, detect a variety of signals, and exhibit a wide range of activation states to effectively combat different pathogens [21]. M2 macrophages have anti-inflammatory functions that play an important role in wound healing. Cytokines such as IL-4 and IL-13 stimulate the induction of M2 macrophages, which results in polarization [9,22,23].

Gangliosides, glycosphingolipids containing sialic acid, are widespread components of eukaryotic cell membranes. They are also associated with regulation of cell differentiation and proliferation [24-26]. Gangliosides are essential for safeguarding host structures from the autologous immune system by shielding host cells and tissues from complement-mediated damage and autoimmune reactions [27,28]. In cells, gangliosides are concentrated in the outermost leaflets of the plasma membrane within transient structures called lipid rafts, where their hydrophobic ceramide tail is embedded in the lipid membrane and the sugar moiety protrudes outward [29,30]. Among gangliosides, GM3 is closely associated with macrophages, which play a major role in tumor suppression and neuroinflammation [31,32].

This study explored the role of gangliosides in modulating macrophage morphology and functionality, specifically focusing on their involvement in the elongation, migration, and phagocytosis of M2-polarized macrophages. IL-4/IL-13 for M2–macrophage polarization elevated GM3 and GD3 levels by increasing St3gal5 and St8sia1 expression. D-PDMP, an inhibitor of ganglioside synthesis, reduced the expression of M2 macrophage markers, including Arg1, Cd206, and Ccl2 (Supplementary Fig. 2A, 2B). However, St3gal5 silencing had little effect on M2 macrophage marker expression (Supplementary Fig. 2C). Considering that D-PDMP inhibits not only St3gal5 and St8sia1 but also their precursor synthases Ugcg and B4galt6, these results suggest that gangliosides, rather than GM3 and GD3, might play a crucial role in M2 macrophage polarization. Our data indicated that gangliosides, particularly GM3 and GD3, are crucial for macrophage elongation during IL-4/IL-13-induced M2 polarization. The characteristic morphological change, which is the elongation of macrophages, was significantly reduced upon the downregulation of ganglioside biosynthesis via St3gal5 silencing, suggesting their functional role in cytoskeletal remodelling. These findings are consistent with a previous study in which gangliosides, especially GM3, were reported to influence cellular morphology and function through various signaling pathways [33].

St3gal5 is the key enzyme that converts lactosylceramide into GM3, and only GM3 treatment successfully increased macrophage length without IL-4/IL-13 (Supplementary Fig. 2D). St3gal5 silencing in M2 macrophages impaired their migration and restored their phagocytosis Moreover, GM3 significantly enhanced transmigration and diminished phagocytosis even in unpolarized macrophages. The elongation of macrophages upon M2 polarization couples with the enhanced migration induced by IL-4/IL-13, indicating a positive correlation between cellular length and migratory activity [10]. In general, M2 macrophages possess a lower phagocytic capacity than M1 macrophages [34]. M1 macrophages typically display a rounded morphology with limited motility, but robust phagocytic activity, reflecting their role in pathogen clearance and proinflammatory responses. In contrast, M2 macrophages, induced by cytokines, such as IL-4 and IL-13, adopt an elongated morphology and show enhanced migratory behavior while exhibiting reduced phagocytic capacity, aligning with their roles in tissue repair and anti-inflammatory responses through cytokine production [35,36]. In this regard, the gangliosides GM3 and GD3 strongly mediate cytoskeleton remodelling to extend the length of M2 polarized macrophages, resulting in increased motility and reduced phagocytosis, which are characteristics of M2 macrophages. Moreover, gangliosides synthesized from GD3, such as GM2 and GD2, may also be involved in M2 macrophage elongation; however, further examination is required to elucidate the exact mechanism.

In macrophages, IL-4/IL-13 stimulation activates the STAT6, PI3K/AKT, and MAPK pathways including c-Jun N-terminal kinase, ERK, and p38, and all these pathways are essential for M2 polarization [37,38]. However, only two inhibitors, LY294002 and U0126, abrogated M2 macrophage elongation under IL-4/IL-13 rich condition by blocking the PI3K/AKT and MEK/ERK pathways, respectively. Considering macrophage elongation as a hallmark of M2 polarization, the reduced length by the inhibition of PI3K/AKT and MEK/ERK is consistent with the recent report [39]. As GM3 successfully increased macrophage length without IL-4/IL-13, we further determined that GM3-induced elongation was mediated via the PI3K/AKT and MEK/ERK signaling pathways, similar to IL-4/IL-13. Notably, this study is the first to demonstrate that GM3 specifically induces the phosphorylation of AKT and ERK2, linking ganglioside-induced signaling to cytoskeletal remodelling and functional regulation. These results suggest that gangliosides, particularly GM3 and GD3, are key modulators of macrophage plasticity, promoting elongation and migration, and suppressing phagocytosis within the IL-4/IL-13-rich microenvironment via the PI3K/AKT and MEK/ERK pathways.

Several key findings emerged from this study, suggesting that gangliosides play a crucial role in macrophage polarization, morphogenesis, and subsequent functional alterations. These findings contribute to a better understanding of the molecular mechanisms underlying macrophage plasticity and highlight the importance of ganglioside-mediated signaling in the regulation of macrophage behavior.

Supplementary material

Article information

Conflicts of interest

Jaewon Shim is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

Funding

This work was supported by a 2-year Research Grant of Pusan National University to Ki-Tae Ha.

Author contributions

Conceptualization: HJC, KTH, SJB. Data curation: SCM. Formal analysis: MC, JS. Funding acquisition: KTH. Investigation: SCM, HJC. Methodology: HJC. Project administration: KTH, SJB. Supervision: KTH, SJB. Validation: MC, JS. Visualization: HJC. Writing-original draft: SCM, SJB. Writing-review & editing: HJC, MC, JS, KTH, SJB. All authors read and approved the final manuscript.

Fig. 1.

Gangliosides associated with the elongation of an M2-polarized macrophage by IL-4 and IL-13 stimulation. (A, B) BMMs were stimulated with IL-4 (20 ng/mL) and IL-13 (20 ng/mL) for 24 hours. Total RNA and protein were extracted, and the expressions of ganglioside synthesis-associated mRNAs and proteins related to ganglioside synthesis were estimated with RT-PCR (A) and immunoblot analysis (B). (C) RAW264.7 cells and BMMs were stimulated with IL-4 and IL-13 for 24 hours. The expression of GM3 and GD3 was examined, and the fold change in their MFI was analyzed using FACS. (D) RAW264.7 cells and BMMs were M2-polarized by IL-4 and IL-13 co-stimulation with or without D-PDMP (10 μM) pretreatment for 1 hour. The morphologic change was photographed, and the length of a macrophage was measured after 24-hour incubation. (E, F) RAW264.7 cells and BMMs were transfected with 200 nM of siRNA targeting St3gal5 or siRNA (NC). (E) The expression of St3gal5 in RAW264.7 cells and BMMs was estimated by RT-PCR. (F) RAW264.7 cells and BMMs were pretreated with siRNA as indicated and further stimulated with IL-4 and IL-13. The morphologic change was photographed and the length of a macrophage was estimated after 24-hour treatment. Quantitative data are shown as mean±SD. Scale bars, 100 μm. IL, interleukin; BMMs, bone marrow-derived macrophages; RT-PCR, reverse transcription-polymerase chain reaction; GM3, ganglioside monosialodihexosylganglioside; GD3, ganglioside disialodihexosylganglioside; MFI, mean fluorescence intensity; FACS, fluorescence-activated cell sorting; D-PDMP, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; siRNA, small interfering RNA; St3gal5, ST3 beta-galactoside alpha-2,3-sialyltransferase 5; NC, negative control; SD, standard deviation. ^**p<0.01 and ^***p<0.001.

Fig. 2.

Increased migration of an M2-polarized macrophage was mediated by gangliosides. (A, B) RAW264.7 cells (A) and BMMs (B) were placed in the upper chamber of a transwell insert and incubated with IL-4 and IL-13 or GM3 (7.5 μM) only as indicated. After 24 hours, MCP-1 (10 ng/mL) was added to the lower chamber to induce transmigration of RAW264.7 cells and BMMs for 12 hours and 6 hours, respectively. Cells on the upper layer of the insert were removed, and the remaining cells on the lower layer were stained with hematoxylin and eosin for cell counting. (C, D) RAW264.7 cells (C) and BMMs (D) were transfected with 200 nM of siRNA targeting St3gal5 or siRNA (NC), and replaced in the upper chamber of the transwell insert for incubation with IL-4 and IL-13 as indicated. After 24 hours, MCP-1 was added to the lower chamber to induce transmigration of RAW264.7 cells and BMMs for 12 hours and 6 hours, respectively. Cells on the upper layer of the insert were removed and the remaining cells on the lower layer were stained with hematoxylin and eosin for cell counting. Quantitative data are shown as mean±SD. Scale bars, 100 μm. BMMs, bone marrow-derived macrophages; IL, interleukin; GM3, ganglioside monosialodihexosylganglioside; MCP-1, monocyte chemoattractant protein-1; siRNA, small interfering RNA; St3gal5, ST3 beta-galactoside alpha-2,3-sialyltransferase 5; NC, negative control; SD, standard deviation; NS, not significant. ^**p<0.01, ^***p<0.001.

Fig. 3.

Reduction of phagocytosis in elongated macrophages was mediated by gangliosides. (A, B) RAW264.7 cells (A) and BMMs (B) were stimulated with IL-4 and IL-13 or GM3 as indicated. A latex beads-rabbit IgG-FITC complex was added to the culture medium for 2 hours and counterstained with DAPI. Phagocytosed cells were visualized with confocal microscopy and the phagocytosis index (% of engulfed cells/total number of cells) was calculated. (C, D) RAW264.7 cells (C) and BMMs (D) were transfected with 200 nM of siRNA targeting St3gal5 or siRNA (NC), and incubated with IL-4 and IL-13 as indicated. A latex beads-rabbit IgG-FITC complex was added to the culture medium for 2 hours and counterstained with DAPI. Phagocytosed cells were visualized with confocal microscopy and the phagocytosis index (% of engulfed cells/total number of cells) was calculated. Quantitative data are shown as mean±SD. Scale bars, 20 μm. BMMs, bone marrow-derived macrophages; IL, interleukin; GM3, ganglioside monosialodihexosylganglioside; FITC, fluorescein isothiocyanate; DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; siRNA, small interfering RNA; St3gal5, ST3 beta-galactoside alpha-2,3-sialyltransferase 5; NC, negative control; SD, standard deviation; NS, not significant. ^*p<0.05, ^***p<0.001.

Fig. 4.

Gangliosides induce the elongation of macrophages through ERK and AKT signaling. (A) RAW264.7 cells were pretreated with the signaling pathway inhibitors ruxolitinib (100 nM) and CP690550 (100 nM) for JAK/STAT, LY294002 (15 μM) for PI3K/AKT, U0126 (10 μM) for MEK/ERK, SP600125 (10 μM) for JNK, and SB203580 (2.5 μM) for p38 as indicated for 1 hour and further incubated with IL-4 and IL-13 for 24 hours. The morphological change was photographed, and the length of a macrophage was measured. CON indicates an untreated control group. (B) RAW264.7 cells and BMMs were pretreated with ruxolitinib, CP690550, LY294002, U0126, SP600125, and SB203580 as indicated for 1 hour and further incubated with GM3 for 24 hours. The morphological change was photographed, and the length of a macrophage was measured. CON indicates untreated control groups. (C) RAW264.7 cells were incubated with GM3 as indicated. The phosphorylation of Stat6, Akt, Erk2, and p38 was detected by immunoblot analysis. Quantitative data are shown as mean±SD. Scale bars, 100 μm. ERK, extracellular signal-regulated kinase; AKT, protein kinase B; JAK, Janus kinase; STAT, signal transducer and activator of transcription; PI3K, phosphoinositide 3-kinase; MEK, MAPK (mitogen-activated protein kinase)/ERK kinase; JNK, c-Jun N-terminal kinase; IL, interleukin; CON, control; BMMs, bone marrow-derived macrophages; GM3, ganglioside monosialodihexosylganglioside; SD, standard deviation. ^***p<0.001.

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• Ganglioside-mediated macrophage plasticity
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