M2 macrophage-derived exosomes reverse TGF-β1-induced epithelial mesenchymal transformation in BEAS-2B cells via the TGF-βRI/Smad2/3 signaling pathway

Abstract

Introduction

Airway remodeling in bronchial asthma can be inhibited by disrupting the epithelial mesenchymal transition (EMT) of activated airway epithelial cells. Exosomes, as key mediators of intercellular communication, have been implicated in the pathophysiology of asthma-related airway inflammation, remodeling, and hyperresponsiveness. This study aimed to investigate the role of M2 macrophage-derived exosomes (M2φ-exos) in modulating TGF-β1-induced EMT in airway epithelial (BEAS-2B) cells and elucidate the underlying molecular mechanism, if any.

Methods

THP-1 cells were induced to differentiate into M2 macrophages via phorbol 12-myristate 13-acetate (PMA) and IL-4. Exosomes were subsequently isolated and purified via ultracentrifugation. M2φ-exos expression was characterized by protein marker levels, transmission electron microscopy imaging, and nanoparticle tracking analysis. TGF-β1-induced BEAS-2B cells were exposed to M2φ-exos to determine the latter’s effects.

Results

THP-1 cells were successfully differentiated into M2 macrophages, as confirmed by in vitro flow cytometry. The isolated exosomes presented typical cup-shaped structures and expressed CD81 and TSG101. TGF-β1 induction altered the morphological characteristics of BEAS-2B cells and activated the TGF-βRI/Smad2/3 signaling pathway, leading to increased expression of Snail, Vimentin and Collagen 1 and decreased expression of E-cadherin. After exosome or SB431542 induction, TGF-β1-induced EMT was reversed. GW4869, an exosome release inhibitor, exhibited the ability to block the beneficial effects of exosomes.

Conclusion

M2Φ-exos inhibited EMT in BEAS-2B cells through the TGF-βRI/Smad2/3 signaling pathway. This novel insight into the role of M2Φ-exos in modulating EMT may have important implications for the beneficial effects of asthma, particularly in addressing airway remodeling.

Introduction

Asthma is a chronic and highly heterogeneous inflammatory disease affecting the airways. It emerges from the complex interplay between inflammatory cells, structural cells within the lungs, and cytokines [1]. This condition is characterized by airway hyperresponsiveness and reversible airflow obstruction, leading to irreversible airway remodeling that extends the duration of the disease [2]. The mechanism of airway remodeling remains unclear, and understanding its pathophysiology may help direct future treatments and improve overall disease prognosis.

Under normal physiological conditions, airway epithelial cells serve as the primary barrier against pathogenic microorganisms and physicochemical irritants [3,4,5]. When the airway epithelium encounters harmful foreign stimuli, a cascade of inflammatory reactions ensues, which triggers airway inflammation [6]. Airway epithelial cells are typically central to the regulation of smooth muscle cell proliferation and fibroblast differentiation, rendering them potent initiators of airway remodeling [7]. Prior studies have established that epithelial‒mesenchymal transition (EMT) plays a significant role in subepithelial fibrosis, a key hallmark of airway remodeling in asthma [8, 9]. This finding implies that inhibiting EMT, which alleviates damage to airway epithelial cells, could represent a viable target for asthma prevention and treatment strategies.

Macrophages are important immune cells that participate in various physiological processes, such as inflammation, defense, and repair [10]. Alveolar macrophages promote lung homeostasis by clearing apoptotic airway cells, bacteria, and other foreign invaders [11]. They also play a very significant role in reducing lung inflammation and promoting wound healing [12]. The function of a macrophage is determined by its polarization. The classical-activated (M1) type has proinflammatory effects, whereas the alternative-activated (M2) type has anti-inflammatory effects [13]. The same studies also suggest that M2 macrophages may serve as an entry point for asthma treatment.

Exosomes are heterogeneous vesicles that arise from the inward folding of cell membranes. There are several main stages of its formation: early endosome biogenesis; maturation of intracavitary vesicles; multivesicular bodies formation and exosome release [14]. The cell of origin of an exosome determines the latter’s size, content, and final effect [15]. Exosomes exert their influence by transferring their cargo into target cells via various mechanisms, including plasma membrane fusion, endocytosis, receptor-ligand binding, intracellular transport, and cargo release [16]. These interactions facilitate intercellular communication and have been implicated in contributing to the onset and progression of numerous diseases [14]. In a study involving animals, exosomes derived from mesenchymal stem cells were found to promote the polarization of M2 macrophages, which led to a decrease in inflammation among mice with severe steroid-resistant asthma [17]. These mesenchymal stem cell-derived exosomes also suppressed the proliferation of bronchial smooth muscle cells, thereby reducing airway remodeling [18]. As such, macrophages may use exosomes to transmit intercellular messages that regulate inflammation levels in airway epithelial cells [19]. These studies suggest that exosomes may be involved in the pathogenesis of asthma-related airway inflammation and remodeling.

Current studies on exosome function in asthma have focused mostly on how these cells contribute to macrophage polarization. However, very few studies have investigated the downward effects on asthma following polarization. As such, this study explored whether M2 macrophage-derived exosomes (M2φ-exos) inhibit EMT in human bronchial epithelial (BEAS-2B) cells and, if affirmative, delves into the molecular mechanisms that underpin this process.

Materials and methods

Cells, reagents, and antibodies

The human bronchial epithelial (BEAS-2B) cell line and human monocyte (THP-1) cell line were acquired from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, and fetal bovine serum (FBS) were procured from Thermo Fisher Scientific, Inc. (Massachusetts, USA). Phorbol-12-myristate-13-acetate (PMA, cat. no. S1819) was obtained from Beyotime Biotechnology (Jiangsu, China), and IL-4 (cat. no. M9363) was obtained from Abmole Biotechnology LTD. (Texas, USA). CD68 (cat. no. 12-0689-41), CD80 (cat. no. 11-0809-41), and CD206 (cat. no. 17-2069-41) were acquired from Thermo Fisher Scientific, Inc. The PKH67 Green Fluorescent Cell Linker Kit and 4′,6-diamidino-2-phenylindole (DAPI) were acquired from Sigma‒Aldrich (Missouri, USA), while phosphotungstic acid hydrate was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). SB431542 and GW4869 were acquired from MedChemExpress, LLC. (Shanghai, China). A BCA protein quantification kit was obtained from CoWin Biosciences (Jiangsu, China), whereas polyvinylidene difluoride (PVDF) membranes were acquired from MilliporeSigma (Massachusetts, USA). Exosome-depleted FBS (Exo-FBS, cat. no. EXO-FBS-250A-1) was purchased from System Biosciences, LLC. (California, USA). The primary antibodies against Smad3 (cat. no. ab40854), phosphorylated (p)‑Smad3 (cat. no. ab52903), and Smad7 (cat. no. ab216428) were obtained from Abcam (Cambridge, United Kingdom). Anti-snail (cat. no. 3879), anti-p-Smad2 (cat. no. AP0269), anti-CD81 (cat. no. GB111073), anti-TSG101 (cat. no. GB15618), anti-Vimentin (cat. no. GB11192), anti-Collagen 1 (cat. no. GB114197), anti-E-cadherin (cat. no. GB11868), anti-TGF-βRI (cat. no. GB11271), anti-Smad2 (cat. no. GB11511), anti-GAPDH (cat. no. GB11002) and an HRP‑conjugated goat anti‑rabbit IgG secondary antibody (cat. no. GB23303) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA), ABclonal, Inc. (Massachusetts, USA), and Wuhan Servicebio Technology Co., Ltd. (Hubei, China), respectively.

Cell cultures

BEAS-2B cells were cultivated in DMEM supplemented with 10% Exo-FBS, streptomycin (100 μg/mL), and penicillin (100 U/mL) at 37 °C in a 5% carbon dioxide (CO₂) atmosphere. The cells were subsequently passaged at a 1:3 ratio every two days to maintain their proliferation. In parallel, THP-1 cells were grown in RPMI 1640 supplemented with 10% FBS, streptomycin (100 μg/mL), and penicillin (100 U/mL) under the same environmental conditions as the BEAS-2B cells. To induce differentiation, THP-1 cells were stimulated with 320 ng/mL PMA for 24 h to transform into M0 macrophages. Further differentiation into M2 macrophages was achieved by exposing the cells to 320 ng/mL PMA and an additional 20 ng/mL IL-4 for 36 h. In certain experiments, M2 macrophages were pre-incubated with 10 μM GW4869 (the inhibitor of exosome release) for 24 h. A subset of BEAS-2B cells was pretreated with or without 10 μM SB431542 (the inhibitor of TGF-βRI) for 1 h, followed by exposure to 5 ng/mL TGF-β1 at 37 °C for 4 h. Following these pretreatments, the cells were cocultured with or without M2φ-exos at 37 °C for 24 h.

Flow cytometric analysis

The surface markers CD68, CD80, and CD206 were used to identify M0 and M2 macrophages in the following manner: Macrophages were incubated in the dark with polyclonal antibodies against these surface markers at 4 °C for 30 min. Flow cytometry (BD Biosciences, USA) was subsequently performed to detect positively stained cells.

Exosome isolation and identification

M2 macrophages were cultured for 72 h. Following this period, the cell supernatants were harvested and subjected to centrifugation at 1000×g at 4 °C for 10 min to remove dead cells and cellular debris. The resulting supernatant was then filtered through a 0.22-μm filter and further ultracentrifuged at 100,000×g at 4 °C for 70 min. Subsequently, the cell supernatants were discarded, and the remaining solution was precipitated in phosphate-buffered saline (PBS) for resuspension. Exosomes were then purified by ultracentrifugation at 100,000×g at 4 °C for an additional 70 min. Finally, the precipitates containing the exosomes were resuspended in 200 μL of PBS and stored at − 80 °C for subsequent analysis. The identification of the exosomes was conducted via transmission electron microscopy (TEM; JEM‑1200EX; JEOL, Ltd.; Tokyo, Japan), following the protocol from our previous study [20]. The distribution size and concentration of the identified exosomes were assessed through nanoparticle tracking analysis (NTA; ZetaView; Particle Metrix; Inning am Ammersee, Germany), and their specific surface markers (CD81 and TSG101) were identified by western blotting.

Exosome labeling

PKH67 was used to confirm whether M2φ-exos were taken up by the BEAS-2B cells following the protocol we utilized in a previous study [20], which was performed as follows: one hundred microliters of M2φ-exos were incubated in 1 mL of diluted C solution containing 1 μL of PKH67 dye for 5 min. Subsequently, the reaction was halted by the addition of 1 mL of 1% bovine serum albumin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Next, ultracentrifugation was performed at 100,000×g at 4 °C for 70 min, and the precipitate was resuspended in 100 μL of PBS. Afterward, the BEAS-2B cells were subjected to treatment with these labeled M2φ-exos for 3 h.

Real-time quantitative reverse transcription polymerase chain reaction (qRT‒PCR)

mRNA was detected by isolating total RNA from each BEAS-2B sample via TRIzol reagent (Thermo Fisher Scientific, Inc.). cDNA synthesis was subsequently carried out with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc.). To verify the gene expression levels, RT‒qPCR was performed with SYBR Green Master Mix (Roche Diagnostics; Massachusetts, USA). The qRT‒PCR thermal cycling conditions were as follows: an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s and extension at 60 °C for 30 s. The results were quantified via the 2 (^−ΔΔCT) method and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All primer sequences utilized are detailed in Table 1.

Table 1 Primer sequences

Western blotting

The process of protein detection was performed by extracting the total protein from each BEAS-2B sample or exosome. To ensure consistency, equal volumes of protein samples were subjected to 8–10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and subsequently transferred onto PVDF membranes. The membranes were treated with a 5% nonfat milk solution at room temperature for 2 h to block nonspecific binding sites. Following the blocking step, the membranes were incubated with primary antibodies against GAPDH (1:1,000), Vimentin (1:1,000), E-cadherin (1:1000), Collagen 1 (1:1,000), Snail (1:1,000), TGF-βRI (1:1,000), Smad2 (1:1,000), p-Smad2 (1:1,000), Smad3 (1:1,000), p-Smad3 (1:1,000), Smad7 (1:1,000), TSG101 (1:1,000), and CD81 (1:1,000) at 4 °C overnight. The membranes were subsequently incubated with secondary antibodies conjugated with HRP (1:3,000) for 1 h at room temperature. Finally, the blots were detected via an enhanced chemiluminescent solution (Beijing Solarbio Science & Technology Co., Ltd.). The analysis of band densities was conducted via ImageJ software version 6.0 (National Institutes of Health; Maryland, USA). The GAPDH protein was used as a loading control throughout the entire experimental process.

Immunofluorescence staining

The BEAS-2B cells were subjected to three washes with PBS and subsequently fixed with 4% paraformaldehyde at room temperature for 30 min. Subsequently, the samples were permeabilized with 0.25% Triton X‑100 at room temperature for 10 min and then blocked with 5% goat serum at room temperature for 1 h. Subsequently, the BEAS-2B cells were incubated overnight at 4 °C with antibodies against E-cadherin (1:500; GB12083; Servicebio Technology Co.), Collagen 1 (1:200; A22349; ABclonal), and Snail (1:200; A5243; ABclonal). The cells were then incubated with Cy3-conjugated goat anti-mouse IgG (1:200; GB21301; Servicebio Technology Co.) secondary antibody at room temperature for 1 h. DAPI (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to stain the nuclei of the BEAS-2B cells, and the staining was visualized via an inverted fluorescence microscope (BX51; Olympus Corporation; Tokyo, Japan).

Statistical analysis

All the statistical analyses presented in this study were conducted with GraphPad Prism 9.0 software (GraphPad Software, Inc.; Massachusetts, USA). The values obtained are presented as the means ± standard deviations and were derived from a minimum of three independent experiments. For the purpose of multiple comparisons, one-way ANOVA was employed, followed by the Bonferroni post hoc test. A statistically significant difference was considered when the p value was less than 0.05.

Results

Identification of M2 macrophages

The THP-1 cells in this study exhibited typical circular or polygonal shapes under a light microscope. Using IL-4, the THP-1 cells were stimulated into M2 macrophages with larger, flattened, and spike-like protrusions (Fig. 1A). At the onset, flow cytometry was used to identify the surface markers CD68 (99.3%) and CD80 (98.5%) on M0 macrophages. After 24 h of IL-4 stimulation, the surface marker CD206 (77.0%) was significantly increased in M2 macrophages compared with M0 macrophages (Fig. 1B). These results indicated that M2 macrophages were successfully induced and polarized.

Fig. 1

Characterization of M2 macrophages. A Morphological changes in M2 macrophages under a light microscope (scale bar, 50 μm and 100 μm). B The positive expression rate of CD206 in M2 macrophages was detected by flow cytometry

M2Φ-exos internalization by BEAS-2B cells

The M2Φ-exos were observed to be small, spherical or cup-shaped vesicles via TEM (Fig. 2A). In parallel, these extracellular vesicles were enriched for the exosomal markers, CD81 and TSG101. However, these markers in cell lysate were not detected (Fig. 2B). Nanoparticle tracking analysis revealed that the peak particle size diameter ranged from 120–130 nm (Fig. 2C). As such, it may be concluded that the isolated particles were exosomes.

Fig. 2

Characterization of M2Φ-exos. A Morphology of M2Φ-exos under TEM (scale bar, 100 nm). B Immunoblot analysis of CD81 and TSG101 expression levels in M2Φ-exos and the cell lysate. C Distribution range and concentration of M2Φ-exos detected by NTA. D Representative image of M2Φ-exos taken by fluorescence microscopy after 3 h. BEAS-2B cells were incubated with M2Φ-exos labeled with the green-colored PKH67 fluorescent probe. Nuclei were labeled with blue-colored 4′,6-diamidino-2-phenylindole (scale bar, 200 μm)

To verify whether these exosomes were internalized by airway epithelial cells, green-colored PKH67-labeled exosomes were cocultured with BEAS-2B cells. The nuclei of the BEAS-2B cells were then stained blue with DAPI. After 3 h, fluorescence microscopy revealed that the green fluorescent-labeled exosomes were distributed in the cytoplasm of the BEAS-2B cells, which suggested that the exosomes were effectively absorbed by the BEAS-2B cells (Fig. 2D).

M2Φ-exos inhibited TGF-β1-induced EMT-related gene expression in BEAS-2B cells

To explore the effects of M2Φ-exos on TGF-β1-induced BEAS-2B cells, we conducted coculture experiments in which BEAS-2B cells were cultured with varying concentrations of exosomes to determine the expression levels of EMT-related proteins. Compared with the control group, the TGF-β1 induction group presented elongated, spindle-shaped cells accompanied by looser intercellular junctions and greater cellular spacing. As the concentration of M2Φ-exos increased, the BEAS-2B cells in the exosome group presented polygonal morphologies, increased intercellular adhesions, and tighter connections than those in the TGF-β1 induction group did (Fig. 3A). To quantify the mRNA expression levels of Vimentin, E-cadherin, Collagen 1, and Snail in each group of BEAS-2B cells, we used qRT-PCR. Our findings revealed that, in contrast to the control group, the TGF-β1 induction group presented upregulated mRNA expression of Vimentin, Collagen 1, and Snail and downregulated mRNA expression of E-cadherin (Fig. 3B). After the addition of 50 μg/mL or 100 μg/mL M2Φ-exos, the mRNA levels of Vimentin, Collagen 1 and Snail significantly decreased, whereas the mRNA level of E-cadherin significantly increased in both groups (Fig. 3B). Notably, the alterations in mRNA expression levels were particularly pronounced when the cells were subjected to treatment with 50 μg/mL M2Φ-exos.

Fig. 3

Effects of M2Φ-exos on the morphology and expression of EMT-related genes in TGF-β1-induced BEAS-2B cells. A Morphological changes in airway epithelial cells induced by different concentrations of TGF-β1 after M2Φ-exos were stimulated under light microscopy (scale bar, 100 μm). B mRNA expression of Vimentin, E-cadherin, Collagen 1, and Snail at different concentrations of M2Φ-exos through qRT‒PCR (n = 3). ^* Indicates P < 0.05; ^** Indicates P < 0.01

GW4869 inhibited the protective effect of M2Φ-exos in TGF-β1-treated BEAS-2B cells

Then, we pre-treated M2 macrophages at the presence of 10 μM GW4869 to reduce the exosomes release. As shown in Fig. 4A, compared with the M2Φ-exos group, the group treated with GW4869 presented looser cells and more cell spaces. As shown in Fig. 4B, compared with the TGF-β1 induction group, the group treated with M2Φ-exos presented lower levels of Vimentin, Collagen 1 and Snail and higher levels of E-cadherin. These changes were reversed by GW4869.

Fig. 4

Morphological alterations and EMT-associated proteins in TGF-β1-induced BEAS-2B cells after treatment with 50 μg/mL M2Φ-exos. A Morphological changes in TGF-β1-induced airway epithelial cells upon the introduction of M2Φ-exos or GW4869 (scale bar, 100 μm). B Protein levels of Vimentin, E-cadherin, Collagen 1, and Snail in BEAS-2B cells, as determined by immunoblotting, following the addition of M2Φ-exos or GW4869 (n = 3). ^* Indicates P < 0.05; ^** Indicates P < 0.01

We validated these findings through both Western blot and immunofluorescence analyses. Intriguingly, both methodologies yielded consistent results. Specifically, upon the introduction of M2Φ-exos, we observed a significant reduction in the fluorescence intensity of the Collagen 1 and Snail proteins, whereas the intensity of the E-cadherin protein markedly increased (Fig. 5A–C). Furthermore, the results demonstrated that GW4869 effectively inhibited the trends induced by M2Φ-exos. Our findings indicated that M2Φ-exos significantly inhibited morphological changes and EMT in TGF-β1-induced BEAS-2B cells.

Fig. 5

Immunofluorescence staining of E-cadherin, Collagen 1, and Snail in BEAS-2B cells after M2Φ-exos or GW4869 treatment. The red fluorescence represents E-cadherin A, Collagen 1 B, and Snail C, respectively, whereas the blue fluorescence represents the nuclei of airway epithelial cells (scale bar, 100 μm)

M2Φ-exos inhibited TGF-β1-induced TGF-βRI/Smad signaling pathway activation

We utilized qRT‒PCR and Western blot analysis to determine whether M2Φ-exos exert a therapeutic effect by inhibiting the TGF-βRI/Smad signaling pathway. The qRT‒PCR results revealed that, in comparison with the control cells, the BEAS-2B cells in the other groups presented no statistically significant alterations in the mRNA expression of Smad2 and Smad3. Compared with the control group, the TGF-β1 induction group presented increased TGF-βRI mRNA levels and a concurrent decrease in Smad7 mRNA levels. Notably, treatment with M2Φ-exos resulted in a reduction in TGF-βRI mRNA levels and an increase in Smad7 levels. Consistent with previous observations, the administration of GW4869 reversed the beneficial effect of M2Φ-exos (Fig. 6A–D). Subsequent to TGF-β1 stimulation, Western blot analysis revealed increases in the protein levels of TGF-βRI, p-Smad2/Smad2, and p-Smad3/Smad3, accompanied by dramatic decreases in Smad7 levels. Intriguingly, following the introduction of M2Φ-exos, the levels of TGF-βRI, phosphorylated Smad2 and Smad3 were significantly decreased, whereas the Smad7 levels were markedly elevated (Fig. 6E). Collectively, these findings indicate that M2Φ-exos exert their therapeutic effect by inhibiting the activation of the TGF-β1-induced TGF-βRI/Smad signaling pathway.

Fig. 6

Changes in the expression of TGFβRI, Smad2, p-Smad2, Smad3, p-Smad3, and Smad7 following treatment with M2Φ-exos or GW4869. A–D mRNA levels of TGF-βRI, Smad2, Smad3, and Smad7 detected via qRT‒PCR upon the administration of 50 μg/mL M2Φ-exos or GW4869 (n = 3). E Assessment of TGFβRI/Smad2/3 signaling pathway activation in TGF-β1-induced BEAS‑2B cells by Western blot analysis (n = 3). ^* Indicates P < 0.05; ^** Indicates P < 0.01

The combination of M2Φ-exos and SB431542 effectively reversed TGF-β1-induced EMT in BEAS-2B cells

To further investigate whether M2Φ-exos suppressed TGF-β1-induced EMT via alternative pathways, BEAS-2B cells were pretreated with 10 μM SB431542. The results demonstrated that co-culture with SB431542 led to an upregulation of E-cadherin protein expression and a downregulation of Vimentin protein expression in TGF-β1-induced BEAS-2B cells (Fig. 7A). Subsequently, we treated TGF-β1-stimulated BEAS-2B cells with a combination of M2Φ-exos and SB431542. Notably, Western blot analysis revealed that the expression levels of TGF-βRI, phosphorylated Smad2, and Smad3 were significantly reduced compared to the group treated with SB431542 (Fig. 7B). Thus, these findings indirectly demonstrate that M2Φ-exos likely reverse TGF-β1-induced airway epithelial-mesenchymal transition by targeting the TGF-βRI/Smad signaling pathway, as well as other potential mechanisms.

Fig. 7

The alterations in EMT relevant markers and TGF-βRI/Smad signaling induced by TGF-β1 were blocked by M2Φ-exos and SB431542. A Western blot analysis was performed to assess the protein levels of Vimentin and E-cadherin (n = 3). B The expression levels of TGFβRI, Smad2, p-Smad2, Smad3 and p-Smad3 were evaluated using western blot (n = 3). ^* Indicates P < 0.05; ^** Indicates P < 0.01

Discussion

Asthma is a complex and heterogeneous respiratory disorder characterized by a broad spectrum of clinical manifestations and pathological features, along with variable sensitivity to treatment interventions [21]. Repeated asthma attacks can induce the release of various inflammatory mediators, resulting in chronic airway inflammation, bronchial airway remodeling, and the development of fibrosis [22]. Airway remodeling, a pivotal aspect of asthma pathophysiology, is manifested through alterations in the structure, composition, and functional properties of airway cells. These changes arise as secondary consequences of airway inflammation, tissue damage, and subsequent excessive tissue repair processes [22]. Different asthma phenotypes induce airway remodeling through different immune mechanisms [23]. This study demonstrated that M2Φ-exos provided a protective effect on airway epithelial cells by reversing TGF-β1-induced EMT in airway epithelial cells. These findings shed light on the potential beneficial applications of M2Φ-exos in mitigating the detrimental effects of airway remodeling in asthma. Interestingly, GW4869 counteracted the protective effect of M2Φ-exos on airway epithelial cells.

Communication between cells is crucial in the immune microenvironment of the lungs, and study has shown that exosomes may be among the most important players in lung function and biology [24]. More specifically, exosomes released by structural airway cells and lung tissue cells seem to play important roles in the development of chronic respiratory diseases such as asthma [25].

Exosomes are extracellular vesicles with diameters ranging from 40 to 200 nm. They can be recognized by the presence of specific markers, including the transmembrane proteins CD63, CD9, and CD81; the heat shock proteins HSP70 and HSP90; and the tumor susceptibility gene 101 (TSG101) [15]. Our findings indicated that CD81 and TSG101 levels were positively expressed in M2Φ-exosomes, whereas these markers were undetectable in the cell lysate (Fig. 2B).

Increasing evidence has shown that macrophages serve as the primary effector cells within both the innate and adaptive immune systems [26]. M1 macrophages have been implicated as proinflammatory markers in the pathophysiology of asthma. However, the precise role of M2 macrophages in asthma remains an area of ongoing debate and controversy [26]. Emerging research indicates that, in comparison with their M1 counterparts, M2 macrophages exhibit a distinct capacity to inhibit the activation of inflammatory cells. Additionally, they are capable of reducing the expression of inflammatory cytokines and promoting tissue repair processes in asthma, as well as in other related respiratory diseases [27]. A previous study further illuminated the interplay between alveolar macrophages and airway epithelial cells is facilitated through a complex network of communication involving cytokines, membrane glycoproteins and their corresponding receptors, gap junction channels, and extracellular vesicles [28]. However, despite these advancements, a significant gap in our understanding of how exosomes specifically mediate the communication between macrophages and airway epithelial cells within the immune microenvironment of asthma. Our findings revealed that the exosomes secreted by M2 macrophages were actively taken up by airway epithelial cells and subsequently stored within their cytoplasm (Fig. 2D).

TGF-β plays a pivotal role in the intricate regulatory network governing cell proliferation. It also plays a significant role in stimulating EMT, suppressing the functional activity of immune cells, modulating immune responses, and facilitating the transformation of fibroblasts into myofibroblasts [29, 30]. TGF-β1 is considered one of the most effective inducers of EMT, and it promotes the transformation of bronchial epithelial cells into myofibroblasts, which results in excessive extracellular matrix (ECM) production and fibrosis [31]. This, in turn, underscores its indispensable role in the airway remodeling process observed in asthma pathology. In this study, airway epithelial cells were exposed to 5 ng/mL TGF-β1, which led to the induction of EMT (Fig. 3B). These findings suggested that the transition of airway epithelial cells into an interstitial phenotype contributes to the remodeling of the airway architecture. To further explore the effect of M2Φ-exos on EMT, we treated BEAS-2B cells with TGF-β1 and M2Φ-exos. Our research revealed that 50 µg/mL M2Φ-exos significantly increased the mRNA expression of E-cadherin while concurrently reducing the mRNA expression levels of Vimentin, Collagen I, and Snail (Fig. 3B). These results provide compelling evidence for the modulatory effects of M2Φ-exos on EMT. Furthermore, the results of Western blot and immunofluorescence revealed similar changes (Figs. 4B and 5).

The TGF-β signaling cascade initiates with ligand binding to the homodimeric type II TGF-β receptor (TGF-βRII), which subsequently recruits and phosphorylates the type I receptor (TGF-βRI). The activated TGF-βRI then catalyzes the phosphorylation and subsequent activation of SMAD2 and SMAD3 [32]. These receptor-regulated SMADs form a heterotrimeric complex with SMAD4, facilitating their translocation into the nucleus where they regulate the transcription of downstream target genes [33]. A recent study indicated that blocking TGF-βRI diminish the expression of genes associated with the ECM, thereby suppressing EMT [34]. It has been reported that sirtuin 7 overexpression elevated TGF-βRI, which in turn governed the proliferation and migration of airway smooth muscle cells stimulated by TGF-β1 [35]. These findings imply that TGF-βRI is involved in the process of airway remodeling.

M2Φ-exos can exert bone-protective effects through the IL-10/IL-10R signaling pathway, thereby alleviating alveolar bone resorption in mice with periodontitis [36]. Emerging evidence indicated that M2Φ-exos effectively ameliorated oxygen–glucose deprivation and reoxygenation-induced neuronal oxidative stress in vitro through activation of the Nrf2/HO-1 antioxidant signaling pathway [37]. M2Φ-exos alleviate asthma-related pulmonary fibrosis and inflammation by delivering miRNA-370 to inhibit the FGF1/MAPK/STAT1 signaling pathways [38]. A study revealed that exosomes derived from M2 alveolar macrophages contain cytokine signal transduction 3 (SOCS-3), which suppresses JAK-STAT inflammatory signaling and cytokine secretion in epithelial cells [39]. Collectively, these findings demonstrate that M2Φ-exos mediate their beneficial potential through modulation of recipient cells' biological characteristics and functional phenotypes.

However, the qRT‒PCR results demonstrated that M2Φ-exos did not affect the mRNA levels of Smad2 and Smad3, but significantly reduced TGF-βRI mRNA level while increasing Smad7 mRNA level. (Fig. 6A‒D). Smad2/3, important downstream target genes of TGF-βRI, play important roles in the human body through phosphorylation [40, 41]. We also performed Western blotting to detect the effect of M2Φ-exos on phosphorylated Smad2/3. The results indicated that M2Φ-exos increased Smad7 levels and decreased TGF-βRI and phosphorylated Smad2 and Smad3 levels (Fig. 6E). GW4869 reversed this beneficial effect. These results clearly indicate that M2Φ-exos inhibited the activation of the TGF-βRI/Smad2/3 signaling pathway, which inhibited the mesenchymal transformation of airway epithelial cells.

Exosomes can exert either beneficial or detrimental effects, depending on their cellular origin, the composition of their contents, the characteristics of recipient cells, and so on [42]. Prior studies have revealed that miRNAs encapsulated within M2 macrophage-derived exosomes promote tumor invasion, migration, and EMT [43,44,45]. In contrast, other research indicated that exosomes enriched with miR-142-3p, also derived from M2 macrophages, inhibit TGF-βRI, resulting in downregulation of ECM-associated molecules and thereby exerting an antifibrotic effect in pulmonary fibrosis [46]. Our findings provided evidence that M2Φ-exos may serve as protective mediator in airway epithelial injury by negatively regulating EMT. Consequently, M2Φ-exos play the dualistic in disease progression and tissue homeostasis. Future research should prioritize comprehensive investigations into the properties and functions of exosomes derived from diverse cell types. Given their ability to modulate multiple signaling pathways and molecular targets, exosomes may elicit previously unrecognized biological effects.

While our study provides valuable insights, several limitations warrant careful consideration. Primarily, the in vitro model employed to investigate the molecular mechanisms underlying M2Φ-exos in regulating EMT of airway epithelial cells may not fully recapitulate the complex pathophysiological microenvironment observed in vivo. Furthermore, the results from SB431542 pretreatment further suggested that M2Φ-exos may regulate the EMT in BEAS-2B cells through alternative molecular mechanisms (Fig. 7A and B). Thus, the precise mechanisms by which M2Φ-exos cargo modulates EMT processes remain incompletely elucidated. Based on our preliminary findings, we hypothesize that M2Φ-exos exert their EMT-reversing effects through protein or miRNA, potentially mediated via direct or indirect modulation of the TGF-βRI/Smads signaling pathway and other pathways in airway epithelial cells. However, future research can focus on these particular phenomena.

Conclusion

In summary, our study demonstrated that M2Φ-exos can reverse the phenotypic changes exhibited by TGF-β1-treated BEAS-2B cells. The molecular mechanism behind this reaction may be achieved through the TGF-βRI/Smad2/3 signaling pathway, as depicted in Fig. 8. Furthermore, our results also indicated that M2Φ-exos effectively participated in intercellular communication to alleviate the process of EMT. These insights offer a fresh perspective on the pathological processes of asthma and establish a novel theoretical foundation for the beneficial effect of M2Φ-exos in asthma-related airway remodeling.

Fig. 8

Schematic representation of M2Φ-exos-mediated TGF-β1-induced phenotypic transformation in BEAS-2B cells. M2Φ-exos inhibited the TGF-β1-induced mesenchymal transformation of airway epithelial cells through the TGF-βRI/Smad2/3 signaling pathway