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Mesenchymal stem cells alleviate idiopathic pneumonia syndrome by facilitating M2 polarization via CCL2/CCR2 axis and further inducing formation of regulatory CCR2 + CD4 + T cells

Stem Cell Research & Therapy volume 16, Article number: 108 (2025) Cite this article

Abstract

Background

Our previous study revealed that mesenchymal stem cells (MSCs) can secrete large amounts of the chemokine CCL2 under inflammatory conditions and alleviate idiopathic pneumonia syndrome (IPS) by promoting regulatory CCR2 + CD4 + T-cell formation through the CCL2‒CCR2 axis. Given the abundance of macrophages in lung tissue, how these macrophages are regulated by MSC-based prophylaxis via IPS and their interactions with T cells in lung tissue during allo-HSCT are still not fully understood.

Methods

An IPS mouse model was established, and MSC-based prophylaxis was administered. In vitro coculture systems and an IPS model were used to study the interactions among MSCs, macrophages and T cells.

Results

Prophylactic administration of MSCs induced M2 polarization and alleviated acute graft-versus-host disease (aGVHD) and lung injury in an IPS mouse model. In vitro coculture studies revealed that M2 polarization was induced by MSC-released CCL2 and that these M2 macrophages promoted the formation of regulatory CCR2 + CD4 + T cells. Blocking the CCL2-CCR2 interaction in vitro reversed MSC-induced M2 polarization and abolished the induction of CCR2 + CD4 + T-cell formation. Additionally, in vivo administration of a CCL2 or CCR2 antagonist in the IPS mouse model exacerbated aGVHD and lung injury, accompanied by a reduction in M2 macrophages and reduced formation of regulatory CCR2 + CD4 + T cells in lung tissue.

Conclusions

MSCs alleviate IPS by facilitating M2 polarization via the CCL2‒CCR2 axis and further inducing the formation of regulatory CCR2 + CD4 + T cells.

Background

Idiopathic pneumonia syndrome (IPS) is a widespread noninfectious alveolar injury that occurs in the acute setting after allogeneic haematopoietic stem cell transplantation (allo-HSCT). It is also considered the pulmonary manifestation of acute graft-versus-host disease (aGVHD) because of the high co-occurrence of these two diseases [1,2,3]. The mortality rate associated with IPS within the first 120 days following allo-HSCT ranges from 60 to 80%, with mortality exceeding 95% in patients who require mechanical ventilation [4]. Supportive therapy and definitive therapy, including methylprednisolone and etanercept, lack specificity, and other available options are unsatisfactory and cause significant toxicity [5]. There are still unmet needs for this clinical dilemma.

Mesenchymal stromal cells (MSCs) have been shown to possess broad immunoregulatory abilities and participate in tissue regeneration and homeostasis [6]. Some reports have shown that prophylactic administration of MSCs is an effective modality for reducing the rate and severity of both acute and chronic GVHD [7, 8]. There have also been some reports that MSCs are effective for the prophylaxis and treatment of lung injury induced by radiation [9], hypoxemia [10] and lipopolysaccharide (LPS) [11]. The mechanisms related to the immunoregulatory effects of MSCs include direct cell contact and the secretion of cytokines, chemokines, signalling molecules and growth factors, which regulate the balance of M1/M2 macrophages [12] and the polarization of T cells from Th1 to Th2 or Treg cells to control the overactivated immune response [6, 13]. MSCs are reportedly effective in various diseases, such as acute lung injury [9, 12], colitis [14], radiation injury [9], and GVHD [15]. Studies conducted by our group have shown that prophylactic administration of MSCs can effectively alleviate IPS in a mouse model following allo-HSCT [16, 17]. Our findings also indicate that MSCs attenuate lung tissue inflammation in IPS mice by reducing the release of inflammatory chemokines and cytokines [17] and inducing the formation of immunoregulatory CCR2 + CD4 + T cells via the secretion of the chemokine (C-C motif) ligand 2 (CCL2) under inflammatory conditions [16]. However, the mechanisms related to the prophylactic and therapeutic effects of MSCs on IPS are still under investigation.

There are many macrophages in lung tissue that serve as first-line defenders of the alveoli and airways [18]. Macrophages are a heterogeneous cell population whose phenotype and functions are regulated by the surrounding microenvironment [19, 20]. Macrophages can be broadly classified into two distinct subsets. M1 macrophages, which are proinflammatory, are polarized by LPS, either alone or in combination with Th1 cytokines, and secrete proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor α (TNF-α). In contrast, M2 macrophages exhibit anti-inflammatory and immunoregulatory properties and are polarized by Th2 cytokines. The balance between M1 and M2 macrophages plays a pivotal role in determining organ outcomes during inflammation or injury [20, 21]. Early reports have shown that MSCs reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages. The preferential shift of macrophages from the M1 phenotype to the M2 phenotype is related to the immune-modulating properties of MSCs, which play a key role in promoting tissue repair [6, 22, 23]. Recent reviews have further highlighted that MSCs can modulate the M1/M2 macrophage balance to improve outcomes in conditions such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [12]. The mechanisms related to regulating their functions and phenotype include MSC-derived exosomes and soluble factors [12]. Recently, studies by our group and others have shown that MSCs can secrete more CCL2 under inflammatory conditions [14, 16, 24]. However, whether MSCs can promote M2 polarization via the secretion of CCL2 to alleviate IPS-related lung injury in the allo-HSCT setting has not been fully investigated.

T-cell activation plays a central role in the development of aGVHD/IPS, and the induction of immunoregulatory T cells such as Tregs is beneficial for these immune complications in allo-HSCT [25, 26]. Macrophages provide a microenvironment for the presentation of allo-antigens to donor-derived, naïve CD4 + T cells, which subsequently differentiate into different subsets [27, 28]. When aGVHD/IPS occurs, M1 macrophages are predominant and drive CD4 + T cells to differentiate into Th1 and Th17 lineages rather than Th2 lineages [29], which are responsible for the pathogenesis of aGVHD/IPS. MSCs inhibit Th1 and Th17 activity and induce the formation of Th2 and Treg cells by cytokines or induced M2 macrophages and are responsible for the prophylaxis and treatment of GVHD [30, 31]. Recently, Milger K et al. identified a novel pulmonary CCR2 + CD4 + T-cell subset with a protective role in the development of lung fibrosis [32]. The frequencies of this T-cell subset were increased in experimental fibrosis and displayed distinct chemokine receptor and mRNA profiles, suggesting immune regulatory functions. This immunoregulatory effect was confirmed in vitro using suppressor assays. More importantly, adoptive transfer of this T-cell subset in a mouse model attenuated the development of lung fibrosis. However, the formation and regulation of this newly identified T-cell subset has not been fully investigated. A previous study by our group revealed that MSCs can induce the formation of CCR2 + CD4 + T cells in vitro and in an IPS mouse model through the CCL2‒CCR2 axis, accompanied by the inhibition of T-cell activation and alleviation of lung injury. These results also revealed that CCR2 + CD4 + T cells may be important for inhibiting inflammation and preventing lung injury in the IPS setting.

In the context of IPS prophylaxis in the allo-HSCT setting, the roles played by MSCs are not fully understood. Although many studies have shown that MSCs can regulate the polarization of macrophages to the M2 phenotype, which affects the differentiation of T cells, the production of CCL2 by MSCs and the function of the CCL2‒CCR2 axis in M2 polarization and CCR2 + CD4 + T-cell formation need further investigation. In this study, we investigated the interactions among MSCs, macrophages and T cells in an in vitro coculture system and in an IPS mouse model. We particularly highlighted the role of MSC-secreted CCL2 in the polarization of macrophages and the formation of regulatory CCR2 + CD4 + T cells. The results demonstrate that MSCs alleviate IPS by facilitating M2 polarization via the CCL2‒CCR2 axis and further inducing the formation of regulatory CCR2 + CD4 + T cells.

Methods

Animals

Female BALB/C (H-2d, 8–10 weeks old) and male C57BL/6 (B6, H-2b, 4–5 weeks old) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the mice were maintained at the Experimental Animal Center, Peking University Health Sciences Center, under specific pathogen-free (SPF) conditions. The animal protocol was carried out in strict accordance with National Institutes of Health guidelines and approved by the Ethics Committee of Animal Experiments of the School of Peking University First Hospital (J2022007). We also followed the recently published ARRIVE 2.0 guidelines.

Establishment and assessment of the IPS mouse model

The allo-HSCT procedure has been previously described [33]. This is a generally accepted method for establishing an IPS. Briefly, recipient BALB/C mice received cyclophosphamide (120 mg/kg/day) intraperitoneally on days − 3 and − 2 and received 7.5 Gy total body irradiation (TBI) by X-ray at a dose rate of 0.4 Gy/min as a pre-bone marrow transplantation (BMT) conditioning regimen on Day 0, after which 1 × 107 bone marrow (BM) cells alone or with 2 × 107 purified spleen cells as a resource of allogeneic T cells from C57BL/6 donor mice were transplanted via the caudal vein after TBI. The mice in the sham group were injected with an equivalent volume of phosphate-buffered saline (PBS) via the tail vein, which served as a control. Recipients were monitored for survival daily and scored for the degree of clinical aGVHD weekly (posture, activity, fur, skin, and weight loss), as previously described [34], or were sacrificed for laboratory testing. The mice were randomly divided into separate groups, with each group containing 10 mice. Two parallel experiments were conducted simultaneously. One group was monitored for survival and aGVHD scores, while the other group was periodically sacrificed for related laboratory analyses. Prior to all invasive procedures, the mice were anaesthetized via an intraperitoneal injection of tribromoethanol (Sigma‒Aldrich, USA) at a dose of 250 mg/kg of body weight. Euthanasia was performed using carbon dioxide asphyxiation following standard ethical protocols.

In vivo intervention

To evaluate the prophylactic effect of MSCs on GVHD/IPS, 5 × 105 MSCs were infused intravenously via the tail vein on Day 0 and Day 2 posttransplantation. To block the interaction between CCR2 and CCL2, a CCR2 antagonist (2 mg/kg, RS102895, MedChemExpress) or CCL2 antagonist (100 mg/kg, Bindarit, MedChemExpress) was given via intraperitoneal injection every other day until the recipients were sacrificed. The dosages of Bindarit and RS504393 used in this study were selected on the basis of previous literature [16]. The grouping schemes were the same as those described above.

Isolation and preparation of cells

Mesenchymal stem cells

Standard protocols for the isolation and culture of MSCs were used as previously described [35]. Briefly, the bone marrow of 72-hour-old C57BL/6 mice was flushed from the long bones, and the bone fragments were digested with collagenase type II (Sigma‒Aldrich, USA) for 2 h. The digested chips were cultured in α-MEM (Gibco, USA) supplemented with 10% FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin under 5% CO2 at 37 °C. The culture medium was changed 72 h later, and the cells were first passaged on Day 5. Subsequent passages were performed every three days. After the third passage, the MSCs were harvested and assayed by flow cytometric analysis. All experiments were performed with MSCs between passage 3 and passage 5.

Thioglycollate-elicited peritoneal macrophages (TEPMs)

BALB/C mice (6–8 weeks old) were intraperitoneally injected with 3 mL of sterile 3% sodium thioglycollate (Sigma‒Aldrich, USA). After three days, the mice were sacrificed, and 10 mL of PBS solution was injected into the peritoneal cavity to collect TEPMs. The ascitic fluid was centrifuged at 300 × g for 5 min, and the cell pellet was resuspended in DMEM (Gibco, USA) supplemented with 20% FBS and antibiotics. The cells were seeded into plates and cultured for 2 h. Nonadherent cells were removed by washing, and the medium was replaced with fresh complete medium. The adherent cells were cultured further for subsequent experiments.

Splenic T cells

The spleens of C57BL/6 mice were mashed through a 70-µm cell strainer, and mononuclear cells (MNCs) were isolated from the cell suspension with Percoll. CD3 + T cells were subsequently purified from the MNCs with a MACS CD3 + T-cell isolation kit II (Miltenyi Biotec, Germany). T cells were then activated with anti-CD3/CD28 antibodies (2 µg/mL and 3 µg/mL, BioLegend) for 8 h in RPMI 1640 culture medium (Gibco, USA) supplemented with 10% FBS.

In vitro coculture system

The coculture system was performed with 0.4-µm Transwell plates (Corning), with 6 replicates in each group. A total of 2 × 105 MSCs were seeded in the upper chamber, and 1 × 106 T cells and/or 1 × 105 macrophages were seeded in the lower chamber to avoid direct contact between the MSCs and the other two cell types. Samples were collected and detected at 24 h, 48 h, and 72 h. To clarify the importance of MSC-trained macrophages during the formation of immunoregulatory CCR2 + CD4 + T cells in vitro, we depleted MSC-trained macrophages from the lower chamber and further cultured MSCs and T cells for another 24 h.

Flow cytometry analysis

The cells in the coculture system, including the MSCs, macrophages and T cells, were harvested and washed with PBS. The samples were stained with F4/80-APC/Fire750, CD11b-PE, CD206-APC, CD86-FITC, CD86-BV510 and CCR2-BV421 fluorescent antibodies (BioLegend, CA, USA) to detect macrophages and CCR2 expression. To determine the expression of CCL2 in MSCs, the cells were stimulated with brefeldin A (BioLegend, CA, USA) for 6 h. The cells were subsequently subjected to surface marker staining with CD44-PE, CD11b-PE, CD90-BV510, and CD105-APC. Following surface staining, the cells were fixed and permeabilized using an Intrasure kit (BD Biosciences, NJ, USA) according to the instructions for intracellular staining. Finally, the intracellular marker CCL2 was detected using BV421. T cells were stained with CD3-APC, CD4-FITC, CD69-PE, and CCR2-BV421 fluorescent antibodies to detect T-cell activation and CCR2 expression. For analysis of T-cell subsets and macrophages in lung tissue, the lungs were removed and minced using scissors and then digested in α-MEM with 2% FBS and 1% collagenase and DNase for 30 min at 37 °C. The samples were stained with SiglecF-BV421, CD45-FITC, CD11C-PE, CD206-APC, CD86-PE/Cy7, CD3-APC, CD4-FITC, CD69-PE, and CCR2-BV421 (BioLegend, CA, USA).

To determine the percent chimerism of H-2Kb donor cells in the BM, the recipients were sacrificed 10 days post-HSCT, and the BM cells were harvested to determine the chimeric status. Standard flow cytometric surface staining protocols were used as previously described. Briefly, BM cells were labelled with a mouse fluorescein isothiocyanate (FITC)-conjugated anti-H-2Kb antibody and allophycocyanin (APC)-conjugated anti-H-2Kd antibody. Flow cytometry was performed using a FACS auto flow cytometer (BD Biosciences, San Jose, CA, USA), and data analysis was performed with FlowJo software (FlowJo, LLC).

Histopathology

The target tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Haematoxylin and eosin (H&E) staining was used for pathological assessment, and the results were evaluated in a blinded fashion under a light microscope. The degree of damage to the target organs was blindly assessed according to a standardized scoring system [36, 37]. Lung injury was assessed semiquantitatively on the basis of parameters such as alveolar septal thickening, haemorrhage, inflammatory cell infiltration, and consolidation. The scoring criteria were as follows: 0, normal; 1, extremely mild damage (less than 10% area of visual field); 2, mild damage (10–25% of visual field); 3, moderate damage (25–50% of visual field); 4, severe damage (50–75% of visual field); and 5, extremely severe damage (over 75% of visual field).

Immunohistochemistry

To evaluate T-cell infiltration in the lung tissue of IPS recipients, immunohistochemical analysis was performed using formalin-fixed, paraffin-embedded mouse tissue sections according to standard protocols. Briefly, the sections were deparaffinized, rehydrated through an ethanol gradient, and subjected to antigen retrieval in EDTA buffer using a pressure cooker. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, and the sections were blocked with 5% bovine serum albumin (BSA). The slides were then incubated overnight at 4 °C with the appropriate primary anti-CD3 antibody. On the following day, goat anti-rabbit IgG secondary antibodies were applied, and the signal was visualized using a DAB peroxidase substrate kit. Histological assessment was conducted by manual counting in 20 random visual fields at 10× magnification. All analyses were performed in a blinded manner to ensure objectivity.

Immunofluorescence

Immunofluorescence staining was performed to detect M2 macrophages (F4/80 + CD206 + cells) in the lung tissue. Murine lungs were harvested and fixed overnight in 4% paraformaldehyde. The fixed tissue was stained with primary antibodies, including anti-CD206 (1:100; Abcam) and anti-F4/80 (1:300; Abcam). Nuclei were stained with 4′,6-diamidino2-phenyl-indole (DAPI, abs47047616). Confocal microscopy (TCS-SP8 STED 3x) was used to visualize the slides.

ELISA

CCL2 concentrations in supernatants from the coculture system were measured by enzyme-linked immunosorbent assay (ELISA) kits (ABclonal) according to the manufacturer’s instructions.

Statistical analysis

Graphing and statistical analysis were performed with GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). Kaplan‒Meier survival curve analysis was performed using the Mantel‒Cox log-rank method for curve comparison analysis. All of the quantitative data shown in the graphs represent the mean ± SEM of each group, and the statistical comparison between two groups was analysed for significance using the nonparametric unpaired Mann‒Whitney test. Intergroup differences were assessed by one-way analysis of variance (ANOVA) followed by post hoc analysis (Dunnett’s multiple comparisons test). P values were considered statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Results

MSCs reduce mortality and alleviate the severity of lung injury in an IPS mouse model

To determine the prophylactic efficacy of MSCs on aGVHD and IPS, we first established an IPS mouse model and intravenously transferred MSCs generated in vitro as described in our previous study [16]. To ensure the successful establishment of the IPS mouse model, H-2Kb donor cells from recipient bone marrow were detected using flow cytometry, which revealed full chimaerism after Day 10 posttransplantation (Supplementary Fig. 2). To evaluate the severity of aGVHD, recipient mice were assessed daily after transplantation for classic systemic signs of aGVHD compared with those in the BMT group. The intravenous infusion of 5 × 105 MSCs into recipients on Day 0 and Day 2 posttransplant was associated with improvement in aGVHD parameters, such as increased survival time (Fig. 1B), reduced body weight loss (Fig. 1C), decreased systemic manifestations and reduced GVHD scores (Fig. 1D). Additionally, pathological examination was carried out to evaluate lung injury after transplantation. Compared with BMT, MSC infusion alleviated lung hyperemia, oedema and consolidation at the gross anatomic level (Fig. 1E) and improved lung appearance at the histopathological level (Fig. 1F-G). These data indicate that MSCs can prolong survival and alleviate lung injury in an IPS mouse model following allo-HSCT.

Fig. 1
figure 1

MSCs reduce GVHD severity and alleviate lung injury in an IPS mouse model. A GVHD/IPS mouse model was established as described in the Materials and Methods. Briefly, cyclophosphamide was administered to BALB/c recipients at 120 mg/kg/day intraperitoneally for 2 days, followed by total body irradiation at a dose of 7.5 Gy (0.4 Gy/min) for conditioning. C57BL/6 donor bone marrow cells and/or splenic cells were subsequently transplanted. MSCs were injected intravenously through the tail vein on Days 0 and + 2 for GVHD/IPS prophylaxis. (A) The study is described in the procedure chart. (B, C, D) The results showed that MSCs could reduce mortality and prolong survival, reduce body weight loss, and improve the clinical manifestations of aGVHD. (E, F) Compared with the control, the infusion of MSCs also alleviated lung hyperemia, oedema and consolidation at the gross anatomic level and improved lung appearance at the histopathological level. Scale bar = 200 μm. (G) Pooled data analysis revealed that MSCs could reduce the score of lung injury according to histopathological criteria (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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MSCs induce the M1 to M2 switch in lung tissue in the IPS mouse model

To investigate whether a switch from M1 to M2 polarization in lung tissue occurred during MSC intervention, the lungs were removed on Day 14 posttransplantation, and the tissue slides were analysed using immunofluorescence to identify the expression of the macrophage marker F4/80 and the M2 marker CD206 in the lung tissues. The results revealed that the lungs of recipients transplanted with bone marrow cells only presented moderate M2-related CD206 expression. However, CD206 expression was sharply downregulated in the lung tissue of the IPS group. After the treatment of IPS mice with MSCs, CD206 expression was more pronounced in the lung tissue of IPS mice (Fig. 2A). Merged high-power images revealed that exclusively strong CD206 expression was observed in F4/80 + macrophages in the lung tissue in the MSC-treated group. We also observed the same switch by analysing collagenase-digested cells from lung tissue using flow cytometry. The results also revealed that the mean fluorescence intensity (MFI) of CD206 in F4/80 + macrophages was significantly increased after treatment with MSCs (Fig. 2B-C). On the basis of these results, we determined that MSCs regulate the polarization of macrophages to the anti-inflammatory M2 phenotype, which may have beneficial effects on lung inflammation in vivo.

Fig. 2
figure 2

MSCs induced the M1 to M2 switch in lung tissue in the IPS mouse model. To investigate the effect of MSCs on M2 polarization in the IPS mouse model, the lungs were removed on Day 14 posttransplantation, and the tissue slides were analysed using immunofluorescence to identify the expression of the macrophage marker F4/80 (green) and the M2 marker CD206 (red). (A) CD206 was sharply downregulated in lung tissues in the IPS mouse model and upregulated after intervention with MSCs. Merged/high-power images revealed exclusively strong expression of CD206 in F4/80 + macrophages in the lung tissues of the MSC-treated group. Scale bar = 100 μm. (B) Flow cytometric analysis of gated F4/80 + cells from collagenase-digested cells in lung tissues. (C) Representative histograms and pooled data of the CD206 mean fluorescence intensity (MFI) showed that the infusion of MSCs increased the CD206 MFI of F4/80 + CD11b + macrophages. These results demonstrate that MSCs could regulate M2 polarization in lung tissue in the IPS mouse model. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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CCL2 derived from MSCs induces M2 polarization of macrophages

To investigate the mechanisms underlying MSC-induced M2 polarization, an in vitro coculture system of MSCs and TEPMs was established. We used TEPMs rather than bone marrow-derived macrophages (BMDMs) or alveolar macrophages (AMs) because the former are easy to acquire and consist mostly of M1 macrophages with inflammatory features, which are more frequently used for exploring the mechanisms related to the M2 switch from the M1 phenotype. As shown in Fig. 3A-B, following 48 h of coculture with MSCs, F4/80 + CD11b + dual-positive TEPMs presented significantly more CD206 + and fewer CD86 + macrophages than were observed in the untreated group (Fig. 3C). The MFI also revealed increased CD206 and decreased CD86 protein expression in the total macrophage population after coculture with MSCs (Fig. 3D-E). Moreover, the TEPMs could switch from M1 to M2 macrophages spontaneously and increase the ratio of M2 macrophages in the culture system over time. The addition of MSCs to the TEPM culture system further promoted the switch to the M2 phenotype (Fig. 3F-G). These in vitro results suggest that MSCs induce the polarization of macrophages from the M1 to the M2 phenotype, as they do in vivo.

Fig. 3
figure 3

MSCs induced macrophage polarization to the M2 phenotype in vitro. Thioglycollate-elicited peritoneal macrophages (TEPMs) were cocultured with MSCs for 48 h in a Transwell system. (A) Representative gating strategy for the analysis of total macrophages (F4/80 + CD11b + cells), the M2 subtype (CD206 + subsets) and the M1 subtype (CD86 + subsets) among F4/80 + CD11b + macrophages, as assessed with flow cytometry. (B) Pooled data of CD206 + macrophages in F4/80 + CD11b + macrophages are shown. (C) Pooled data of CD86 + macrophages in F4/80 + CD11b + macrophages are shown. (D) Representative histograms and pooled data of the CD206 mean fluorescent intensity (MFI) in F4/80 + CD11b + macrophages. (E) Representative histograms and pooled data of the CD86 MFI in F4/80 + CD11b + macrophages. (F, G) Time-dependent analysis of CD206 and CD86 expression on F4/80 + CD11b + macrophages in the coculture system and macrophages cultured alone as controls via flow cytometry. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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On the basis of our previous findings that MSCs can secrete more CCL2 in a coculture system with activated T lymphocytes, which affects T-cell activation and differentiation through the CCL2-CCR2 axis [16], we investigated whether the interaction between MSCs and macrophages is still affected by this axis in the present study. We first cocultured TEPMs with MSCs and found that more CCL2 was produced by detecting the intracellular CCL2 concentration in MSCs using flow cytometry (Fig. 4A) and the CCL2 concentration in the culture supernatant (Fig. 4B). Similarly, greater CCR2 expression on macrophages was detected in the coculture group than in the macrophage-only group after 72 h (Fig. 4C). To further validate the role of MSC-derived CCL2 in macrophage polarization, a CCL2 or CCR2 antagonist was added to the coculture system. After 48 h of culture, a reduction in the number of CD206 + M2 macrophages (Fig. 4D) and an increase in the number of CD86 + M1 macrophages (Fig. 4E) were detected, indicating that CCL2-CCR2 blockade reversed the effect of MSCs on M2 polarization. Therefore, CCL2 derived from MSCs in a coculture system is necessary for M2 polarization, and this effect is independent of cell contact.

Fig. 4
figure 4

CCL2 derived from MSCs induced M2 polarization. TEPMs were cocultured with MSCs for 48 h in the Transwell system. (A, B) Increased production of CCL2 in MSCs was documented via detection of intracellular CCL2 with flow cytometry, and the concentration of CCL2 in the supernatant of the coculture system was determined via ELISA. (C) CCR2 expression on macrophages was increased after coculture with MSCs, as assessed by flow cytometry. (D, E) CD206 expression was downregulated and CD86 expression was upregulated in MSC-trained macrophages after treatment with a CCL2 or CCR2 antagonist, as assessed by flow cytometry. These results demonstrated that CCL2 derived from MSCs may be responsible for M2 polarization. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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MSC-trained macrophages promote the formation of regulatory CCR2 + CD4 + T cells in vitro

Our previous study revealed that MSCs inhibit T-cell activation and induce the formation of regulatory CCR2 + CD4 + T cells, which may be one of the mechanisms responsible for effective IPS prophylaxis [16]. However, the regulation of T-cell subset formation has not been fully investigated. We propose that MSCs affect T-cell differentiation directly or via MSC-induced M2 macrophages through the CCL2‒CCR2 axis. To test this hypothesis, we first cocultured MSCs, TEPMs and activated T cells in a Transwell system by separating MSCs from macrophages and T cells. The results revealed that M2 polarization occurred, according to CD206 and CD86 expression (Fig. 5A), T-cell activation was inhibited (Fig. 5B), and the formation of CCR2 + T cells (Fig. 5C), especially CCR2 + CD4 + T cells, was significantly promoted (Fig. 5D).

To further clarify the importance of macrophages in the MSC-induced formation of CCR2 + CD4 + T cells, we depleted MSC-trained macrophages in this culture system for another 24 h. As shown in Fig. 5F-G, there was a significant reduction in the number of CCR2 + T cells, especially CCR2 + CD4 + T cells. Moreover, the inhibition of T-cell activation was also diminished (Fig. 5E). It was concluded that MSC-induced M2 polarization is responsible for the formation of regulatory CCR2 + CD4 + T cells. To gain further insight into the role of the CCL2‒CCR2 axis in this cascade immune response, we applied a CCL2 antagonist to the coculture system. The results revealed that M2 polarization was reversed (Fig. 6D-E) and CCR2 expression was downregulated in macrophages, as illustrated in Fig. 6A-C. Furthermore, the addition of the CCL2 antagonist diminished the formation of CCR2 + CD4 + T cells (Fig. 6G).

Fig. 5
figure 5

MSC-trained macrophages induced T-cell differentiation towards the CCR2 + CD4 + T-cell subset in vitro. To clarify the interactions among MSCs, macrophages and T cells, TEPMs and activated T cells in the lower chamber of the Transwell unit were cocultured with MSCs in the upper chamber for 48 h. The subsets of macrophages and T cells were analysed by flow cytometry. (A) Representative data and pooled data of the CD206 and CD86 MFI in F4/80 + CD11b + macrophages also revealed M2 polarization. (B) The inhibitory effect on T-cell activation was documented by detecting CD69 expression on T cells in this coculture system. (C, D) The formation of CCR2 + CD3 + T cells, especially CCR2 + CD4 + T cells, was induced in this coculture system. (E, F and G) MSC-trained macrophages were withdrawn from the lower chamber for another 24 h of culture, and the results showed that the removal of macrophages from the coculture system could relieve the suppressive effect on T-cell activation and diminish the formation of CCR2 + CD4 + T cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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Fig. 6
figure 6

Blockade of the CCL2-CCR2 axis reversed M2 polarization and reduced the number of CCR2 + CD4 + T cells in the MSC, macrophage and T-cell coculture system. TEPMs were cocultured with activated T cells in the lower chamber and with MSCs in the upper chamber of the Transwell system. A CCL2 antagonist was added to the culture system for 48 h to block the CCL2-CCR2 interaction. (A, B, C) CCR2 expression was reduced in total macrophages and in the CD206 + and CD86 + subsets. (D, E) Macrophages were reversed to the M1 phenotype by reducing the CD206 MFI and increasing the CD86 MFI in F4/80 + CD11b + macrophages. (F, G) T cells were reactivated, and their differentiation towards CCR2 + CD4 + T cells was inhibited after the interaction between CCL2 and CCR2 was blocked. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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Blockade of the CCL2‒CCR2 axis weakens the prophylactic effect of MSCs on GVHD and IPS

To further determine the role of the CCL2‒CCR2 axis in the prophylactic effect of MSCs on GVHD and IPS, a CCR2 or CCL2 antagonist was injected intraperitoneally every other day from Day 0 to Day 6. Notably, both antagonists markedly diminished the protective effect of MSCs, which manifested as a shortened survival time (Fig. 7A-B), increased weight loss (Fig. 7C), worse GVHD scores (Fig. 7D), and exacerbated lung tissue damage (Fig. 7E-F). Immunofluorescence analysis of lung tissue revealed that injection of a CCR2 or CCL2 antagonist reduced the expression of CD206 on F4/80 + macrophages compared with that on MSCs (Fig. 8A), which further confirmed the role of the CCL2-CCR2 axis in M2 polarization in vivo during MSC-mediated prophylaxis for IPS. Moreover, there was an increase in T-cell infiltration following the application of both antagonists (Fig. 8B). Further analysis of T-cell subsets from lung tissues revealed a reduction in the proportion of CCR2 + T cells, particularly CCR2 + CD4 + T cells, and this reduction paralleled the decrease in M2 polarization (Fig. 8C-D). Taken together, these data confirmed that in vivo administration of MSCs in an IPS mouse model facilitated M2 polarization via the CCL2-CCR2 axis and further induced the formation of CCR2 + CD4 + T cells in vitro. This may be one of the main mechanisms responsible for the protective effect against lung injury after the prophylactic use of MSCs in the allo-HSCT setting.

Fig. 7
figure 7

Blockade of the CCL2‒CCR2 axis reversed the prophylactic effect of MSCs on GVHD/IPS in a mouse model. As shown in Fig. 1, prophylactic administration of MSCs alleviated aGVHD and lung injury in the GVHD/IPS mouse model. (A) The study schedule is shown in the procedure chart. (B, C, D) CCL2 and CCR2 antagonists reversed the prophylactic effect of MSCs on GVHD. (E, F) Representative images of HE-stained histological sections of lung tissues showing that the addition of a CCL2 or CCR2 antagonist reversed the prophylactic effect of MSCs on lung injury (n = 5). Scale bar = 200 μm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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Fig. 8
figure 8

Blockade of the CCL2-CCR2 axis reversed the effect of MSCs on M2 polarization, aggravated T-cell infiltration and reduced the formation of CCR2 + CD4 + T cells in lung tissue. In the IPS model, the mice that received prophylactic MSCs also received a CCL2 or CCR2 antagonist intraperitoneally every other day posttransplantation from Day 0 to Day 6. The lung tissue on Day 7 was analysed for macrophage polarization, T-cell infiltration and T-cell subsets. (A) Representative images of immunofluorescence staining for the macrophage marker F4/80 (green) and the M2 marker CD206 (red) in lung tissue. CD206 + macrophages were diminished after treatment with both the CCL2 and CCR2 antagonist. Scale bar = 200 μm. (B) Immunohistochemical staining with a mouse CD3 monoclonal antibody revealed that T-cell infiltration was more pronounced in lung tissue after treatment with a CCL2 or CCR2 antagonist. Scale bar = 200 μm. (C, D) Analysis of collagenase-digested cells in lung tissues using flow cytometry revealed that the percentage of CCR2 + CD3 + T cells, especially CCR2 + CD4 + T cells, was decreased after treatment with a CCL2 or CCR2 antagonist. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns., no significant difference. The data are shown as the means ± SEMs

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Discussion

IPS is a major complication that commonly occurs in the early stage following allo-HSCT. It is characterized by the infiltration of donor-derived leukocytes and the release of inflammatory cytokines and is considered a pulmonary manifestation of GVHD. Pathologically, IPS is characterized by interstitial-alveolar pneumonia and interstitial fibrosis in the absence of any identifiable infections. Owing to its high mortality rate and lack of effective prophylactic and therapeutic modalities, many researchers are seeking new approaches to address this difficult problem.

MSCs are known for their wide-ranging immunoregulatory abilities and their involvement in tissue regeneration and maintaining homeostasis. They can suppress the proliferation of T and B cells, reduce NK cell cytotoxicity, and promote Treg activity [6]. MSCs expanded in vitro have also been utilized in therapeutic approaches for autoimmune diseases [38], graft rejection [39], and many inflammatory disorders, such as COVID-19 [40]. Some preclinical and clinical reports support the excellent efficacy of MSCs for improving engraftment and/or GVHD in allo-HSCT [41, 42]. Huang R et al. demonstrated in a clinical trial that early, repeated infusions of MSCs reduced the incidence and severity of aGVHD, resulting in improved GVHD-free and relapse-free survival rates in patients following haplo-HSCT. There have been few studies on the prophylaxis and treatment of lung injury, especially IPS, with MSCs, in the allo-HSCT setting, either experimentally or clinically. However, many clinical studies have been performed on a wide range of lung diseases, including ARDS [43], COVID-19-related ARDS [44], ALI [23], chronic obstructive pulmonary disease (COPD) [45], and idiopathic pulmonary fibrosis (IPF) [46], and have shown some beneficial effects. MSCs may have several advantages in treating lung inflammatory diseases. Joo et al. [47] reported that MSCs infused intravenously in a GVHD mouse model initially accumulate in the lungs before migrating to other organs. Moreover, their immunosuppressive activity can be enhanced in the presence of inflammatory cytokines [48, 49]. When MSCs first reach lung tissue after intravenous injection, the elevated level of inflammatory cytokines may amplify the immunosuppressive effect of MSCs. These observations suggest that MSCs might be more effective in preventing or treating IPS. Our previous research demonstrated that MSC administration can significantly prolong survival and alleviate lung injury in a mouse model of IPS [16]. However, the mechanisms underlying the effects on IPS have not been fully elucidated.

Macrophages are abundant in the lung microenvironment, where M1-type macrophages play a pivotal role in the proinflammatory reactions involved in host defence, and M2-type macrophages contribute to anti-inflammatory responses and tissue remodelling. These macrophages can mutually transition from one type to another, depending on the inflammatory status or steady state of the microenvironment [18, 20, 21]. Thus, the regulation of macrophage polarization is essential for balancing both proinflammatory and anti-inflammatory effects. Increasing evidence has shown that macrophage polarization, which is regulated by cytokines, chemokines, and transcription factors, is closely linked to the onset and progression of pulmonary inflammatory diseases [50]. Many studies have shown that MSCs can suppress the development of proinflammatory M1 phenotypes and promote the shift from proinflammatory to anti-inflammatory M2 phenotypes [14, 27]. MSC-trained macrophages play protective roles in various diseases, such as colitis [14], radiation-induced injury [15], and GVHD [15, 51]. MSCs regulate the transition from the M1 type to the M2 type in many ways, including through MSC-derived exosomes and soluble factors such as TGF-b, thrombospondin-1, decorin, pentraxin 3, prostaglandin (PGE2), and insulin-like growth factor [12].

In this study and our previous study [16], we showed that MSCs produce large amounts of CCL2 under inflammatory conditions. Additionally, the concentration of CCL2 increased in the BAL fluid of MSC-treated mice. Moreover, M2 polarization was noted in both the in vitro culture system and the lung tissue of IPS mice after the administration of MSCs in vivo, which was accompanied by increased expression of CCR2, the main receptor for CCL2, on the surface of macrophages. The interaction between MSC-derived CCL2 and the CCR2 receptor on macrophages may drive inflammatory M1 macrophages to transition into anti-inflammatory M2 macrophages, contributing to the immunosuppressive effect of MSCs [14, 52]. In fact, there are several clues concerning the immunoregulatory effects of CCL2 secreted by MSCs. For example, CCL2-deficient MSCs fail to establish long-lasting contact with T cells and are unable to ameliorate lupus symptoms [53]. The absence of CCL2 expression impairs the suppressive effect of MSCs on B cells in lupus [54]. Additionally, MSCs recruit CCR2 + monocytes to mitigate allergic airway inflammation [55]. This study demonstrated that the protective effect of MSCs on lung injury in an IPS mouse model was associated with M2 polarization. CCL2 and CCR2 antagonists reversed M2 polarization, and the lack of a prophylactic effect of MSCs on lung injury in IPS further confirms this conclusion.

The CCR2 + CD4 + T-cell subset was newly noted in patients with idiopathic pulmonary fibrosis [32]. The frequencies of this T-cell subset were increased in experimental fibrosis, resulting in a distinct chemokine receptor profile and unique mRNA expression patterns. In vitro suppressor assays and adoptive transfer of this T-cell subset attenuated the development of lung fibrosis, confirming this immunoregulatory effect. However, the regulation of this T-cell subset has not been fully investigated. A previous study by our group revealed that MSCs induce the formation of CCR2 + CD4 + T cells in vitro and in an IPS mouse model through the secretion of CCL2, accompanied by the inhibition of T-cell activation and alleviation of lung injury [16]. These findings and those of our previous studies suggest that MSCs induce both M2 polarization and the formation of CCR2 + CD4 + T cells. We suggest that MSCs can directly induce the formation of CCR2 + CD4 + T cells and/or indirectly induce their formation through polarized M2 macrophages, which further affects the outcome of lung injury in an IPS mouse model. In this study, MSCs did not induce the formation of CCR2 + CD4 + T cells when they were cocultured with purified T cells. However, only the addition of M2 macrophages to the above coculture system promoted the formation of CCR2 + CD4 + T cells. These results further demonstrate that MSC-trained macrophages play an important role in inducing the formation of CCR2 + CD4 + T cells. The addition of a CCR2 or CCL2 antagonist reversed M2 polarization and reduced the number of CCR2 + CD4 + T cells, further confirming the importance of the CCL2-CCR2 axis in the regulation of this immune cascade.

Our research demonstrates that macrophages trained by MSCs play a key role in the induction of CCR2 + CD4 + T cells and establishes the central role of the CCL2‒CCR2 axis in this process. Although our findings are valuable, several limitations remain. First, other potential pathways influencing this process should be further investigated. Intravenously infused MSCs are known to be sequestered in the lungs, where they release various soluble mediators. Li et al. showed that MSCs can preferentially modulate macrophages to alleviate pulmonary fibrosis by expressing CCL2 and CXCL1 [56]. Similarly, Kim et al. demonstrated that MSCs regulate immune responses and M1/M2 macrophage polarization to attenuate ALI through the suppressor of cytokine signalling (SOCS) proteins [57]. Therefore, further research should focus on exploring these additional molecules and their interactions with the CCL2‒CCR2 axis in the prophylaxis and treatment of IPS. Second, further studies are needed to evaluate the impact of different MSC administration routes, timings and dosages in IPS to optimize treatment efficacy. While early preclinical and clinical studies have demonstrated the safety and efficacy of MSC therapy, substantial variability in efficacy has been observed across different administration protocols. Thus, determining the optimal MSC administration strategy remains the main focus of preclinical studies and later clinical applications.

Conclusions

In summary, the regulation of macrophage polarization may be an effective modality for the prophylaxis and treatment of IPS in the allo-HSCT setting. The infusion of MSCs to regulate the function of macrophages and T cells can be an excellent prophylactic measure. The mechanisms for prophylaxis and treatment of IPS using MSCs involve M2 polarization via the CCL2‒CCR2 axis and promotion of immunoregulatory CCR2 + CD4 + T-cell formation. This may serve as a very early therapeutic intervention for IPS before lung damage becomes profound and irreversible.

Data availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. All data and materials relevant to this study are included within the article. Further information is available from the corresponding author upon reasonable request.

Abbreviations

MSCs:

Mesenchymal stem cells

BMT:

Bone Marrow Transplantation

IPS:

Idiopathic pneumonia syndrome

allo-HSCT:

Allogeneic hematopoietic stem cell transplantation

aGVHD:

Acute graft-versus-host disease

LPS:

Lipopolysaccharide

CCL2:

Chemokine (C-C motif) ligand 2

IL:

1β-Interleukin-1β

TBI:

Total body irradiation

TEPMs:

Thioglycollate-elicited peritoneal macrophages

MNCs:

Mononuclear cells

BMDM:

Bone marrow derived macrophages

AM:

Alveolar macrophages

ARDS:

Acute respiratory distress syndrome

ALI:

Acute lung injury

COPD:

Chronic obstructive pulmonary disease

IPF:

Idiopathic pulmonary fibrosis

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Acknowledgements

Authors thank Prof Zhi-bo Liu and Dr. Chang-lun Wang (Beijing National Laboratory for Molecular Sciences, Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, College of Chemistry and Molecular Engineering, Peking University), for their assistance on instrumental support and experimental skills.

Funding

This study was supported by the National Natural Science Foundation of China (No.81970160, No.82071757 and No.81570160).

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  1. Department of Hematology, Peking University First Hospital, 8#, Xishiku Street, Xicheng District, Beijing, 100034, PR China

    Chao Xue, Wei Liu, Yuan Li, Yue Yin, Bo Tang, Jinye Zhu, Yujun Dong, Huihui Liu & Hanyun Ren

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Contributions

H.R. conceived and designed the study. H.L. participated in the research design and reviewed the manuscript. C.X. designed the study and performed the most experiments of this study and wrote the paper. B.T. and J.Z. helped to establish aGVHD mouse models and performed part of the flow cytometric analysis. Y.D., Y.L., and Y.Y. participated in the research design.

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Correspondence to Huihui Liu or Hanyun Ren.

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All procedures involving animals were approved by the Animal Experiments Ethics Committee of Peking University First Hospital (Approval Number: J2022007). Title of the approved project: Mechanisms of MSC-Mediated Regulation of Macrophage Function via the CCR2-CCL2 Axis in Idiopathic Pneumonia Syndrome (Date of approval: March 28, 2022). All investigations were conducted according to the principles outlined in the Declaration of Helsinki. This study was reported in compliance with the ARRIVE 2.0 guidelines for the care and use of animals.

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Xue, C., Liu, W., Li, Y. et al. Mesenchymal stem cells alleviate idiopathic pneumonia syndrome by facilitating M2 polarization via CCL2/CCR2 axis and further inducing formation of regulatory CCR2 + CD4 + T cells. Stem Cell Res Ther 16, 108 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04232-6

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04232-6

Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512