Acute Respiratory Distress Syndrome (ARDS) often result from infections, trauma, or other systemic inflammatory responses. These conditions, severe and life-threatening, are characterized by extensive lung inflammation leading to respiratory failure (Wick et al., 2024). They are marked by rapid inflammation onset and increased permeability of the alveolar-capillary barrier, which causes respiratory dysfunction (Vichare and Janjic, 2022; Lim et al., 2023). Recent studies and clinical trials have proposed new treatments for ARDS, including the following: Corticosteroids, which suppress the inflammatory response (Chang et al., 2022). Specifically, steroids such as dexamethasone have shown to reduce inflammation and enhance patient survival (Monteverde-Fernandez et al., 2019). Interleukin (IL)-6 inhibitors like tocilizumab, used in COVID-19-associated ARDS, potentially offer therapeutic benefits by targeting inflammatory mediators (Nasonov and Samsonov, 2020). However, current treatments remain predominantly supportive and do not adequately address the underlying pathology.

Another treatment drawing significant interest is mesenchymal stem cells (MSCs)-based therapy which has shown promise in treating ARDS. One of the primary attributes of MSCs is their potent immunomodulatory and anti-inflammatory effects, offering valuable therapeutic potential in severe inflammatory diseases like ARDS (Zhang et al., 2022). MSCs engage various immune cells, including T cells, B cells, natural killer (NK) cells, and dendritic cells, to regulate their activity (Yan et al., 2023; Chen et al., 2024). For instance, MSCs can mitigate excessive immune responses by inhibiting T cell activation and promoting the generation of regulatory T cells (Tregs). Additionally, MSCs diminish the secretion of inflammatory cytokines (TNF-α, IL-1β, IL-6) and modulate inflammatory responses by secreting anti-inflammatory cytokines (TGF-β, IL-10) (Tsuchiya et al., 2020; Kushioka et al., 2023). The ‘priming’ process involves exposing MSCs to specific stimuli, such as inflammatory cytokines, hypoxic conditions, or chemical agents, to enhance their therapeutic potential. It is known that priming with inflammatory cytokines such as Interferon gamma (IFN-γ) or TNF-α improves their immunosuppressive abilities (Varkouhi et al., 2019; Park et al., 2023; Tolstova et al., 2023; Herger et al., 2024). Primed MSCs represent a strategic approach to optimize therapeutic effects, particularly in complex conditions like diseases, including acute lung injury (ALI) and ARDS (Varkouhi et al., 2019; Liu et al., 2022; Wang et al., 2022). The immunomodulatory and anti-inflammatory capacities of primed MSCs can suppress excessive lung inflammation, thus reducing alveolar damage and vascular leakage, which in turn helps prevent hypoxia and deterioration of lung function, key pathological features of ARDS.

Given the properties of MSCs, numerous clinical trials have been conducted to assess the efficacy of MSCs in treating ARDS triggered by various factors, including COVID-19 (Chen et al., 2020; Lanzoni et al., 2021; Yousefi Dehbidi et al., 2022). However, the mechanisms by which MSCs exert therapeutic effects on ARDS are still undefined, with specific studies on primed MSCs being notably scarce.

In this study, we aimed to elucidate the mechanisms by which primed MSCs can effectively mitigate ARDS symptoms, focusing on a condition lacking fundamental treatments. Our methodology included both in vitro and in vivo assessments to determine the clinical viability of these findings.

hUCB-MSCs culture

hUCB-MSCs were supplied by Kangstem Biotech GMP Center (Gwangmyeong, Korea) and cultured in KSB-3 Basal medium (Kangstem Biotech, Gwangmyeong, Korea) supplemented with 10% fetal bovine serum (FBS; Gibco, NY, USA) and 100 µg/mL primocin (Invitrogen, MA, USA). We conducted all experiments with hUCB-MSCs at passage 8. hUCB-MSCs were primed for 24 h with IFN-γ (Peprotech, NJ, USA) or IL-1β (Peprotech) to boost their immunomodulatory function or enhance their responsiveness in inflammatory environments. Primed MSCs were cultured for 24 h under three different conditions: first, with 20 ng/mL IFN-γ; second, with 5 ng/mL IL-1β; and third, with both 20 ng/mL IFN-γ and 5 ng/mL IL-1β. All cells were maintained at 37°C in a 5% CO₂ incubator.

Mixed lymphocyte reaction (MLR assay)

To assess the immunogenicity and immunosuppressive capacity of hUCB-MSCs, we conducted both direct and indirect co-culture assays following the ‘Advanced Biologics and Mesenchymal Stem Cell Therapy Mixed Lymphocyte Reaction Test’ established by the MFDS.

Direct co-culture assay

To assess the immunogenicity of primed MSCs, we co-cultured peripheral blood mononuclear cells (PBMCs) with Mitomycin C (MMC; Sigma-Aldrich, MO, USA)-treated MSCs. The negative control consisted of a PBMC culture to assess baseline PBMC proliferation. Specifically, 1×10⁵ Cells PBMCs were co-cultured with 1×10⁴ Cells MSCs at a 10:1 ratio (PBMCs to MSCs) in a 96-well plate for 96 h. PBMC proliferation was evaluated using a 5-bromo-2′-deoxyuridine (BrdU; Roche, BS, Switzerland) incorporation assay.

Indirect co-culture assay

To assess the immunosuppressive capacity of primed MSCs, 2.5 µg/mL Concanavalin A (Con A; Invitrogen)-activated PBMCs (1×10⁶ cells) were cultured at the bottom of a 24-well plate, while primed hUCB-MSCs (1.0×10⁵ cells) were added to the upper insert at a 10:1 ratio (PBMCs to MSCs). The negative control consisted of a PBMC culture to assess baseline PBMC proliferation. For the positive control, PBMC were stimulated with 2.5 µg/mL Con A as the mitogen. The co-culture was maintained for 96 h in incubator at 37°C and 5% CO₂. PBMC proliferation was assessed using BrdU incorporation analysis.

M1 and M2 macrophages

Human monocytes (THP-1 (TIB-202); ATCC, WV, USA) were maintained in RPMI-1640 Medium (ATCC), supplemented with 10% FBS, 50 pM ß-mercaptoethanol (Gibco), and 100 µg/mL primocin at 37°C in a 5% CO₂ incubator.

THP-1 monocytes were seeded at 1×10⁶ cells per well in a 6-well plate and cultured in RPMI medium with 150 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) and 10% FBS for 24 h to differentiate into M0 macrophages. They were then polarized into M1 macrophages by incubating in RPMI medium with 20 ng/mL IFN-γ, 100 ng/mL LPS (Sigma-Aldrich), and 10% FBS. hUCB-MSCs (1×10⁶ cells) were seeded in a transwell and co-cultured for 72 h. TNF-α levels were measured in the culture medium using a Human TNF-α Quantikine ELISA Kit (R&D Systems, MN, USA), as per the manufacturer’s instructions.

To further assess the ability of hUCB-MSCs to induce M2 differentiation and IL-10 secretion, THP-1 monocytes were cultured in RPMI medium with 10% FBS and 150 nM PMA for 24 h, followed by the addition of another 150 nM PMA for an additional 24 h to induce macrophage differentiation. Subsequently, hUCB-MSCs (1×10⁵ cells) were seeded in a transwell and co-cultured for 72 h. IL-10 levels were quantified in the culture medium using the Human IL-10 Quantikine ELISA Kit (R&D Systems) per the manufacturer’s instructions.

mRNA-sequence

Total mRNA samples from naive MSCs, IFN-γ -primed MSCs, IL-1β-primed MSCs and IFN-γ/IL-1β-primed MSCs were isolated using the Truseq stranded mRNA library prep kit (Illumina Inc., CA, USA) for RNA sequencing (RNA-seq). RNA quantity and quality were assessed using a Nanodrop device (Thermo Scientific, MA, USA). Subsequently, RNA-seq was conducted on the Novaseq 6000 system (Illumina Inc.). mRNA-Seq raw data facilitated abundance estimation with Kallisto. Differential expression analysis was executed using edgeR from R packages, categorizing differentially expressed genes (DEG) based on conditions UP (Case/Control): FDR (adjusted p-value)<0.05 & log2Fold Change≥1, DOWN (Case/Control): FDR (adjusted p-value)<0.05 & log2FoldChange≤–1. Gene ontology (GO) classification and functional distribution analysis were conducted with the ontology database (org.Hs.eg.db) from R packages, focusing on genes showing a 2-fold difference and FDR<0.05. Analyses using Kyoto Encyclopedia of Genes (KEGG) and Genomes utilized the KEGGREST package and path view from R packages. Gene set enrichment analysis (GSEA) was performed employing the GSEA 4.3.3 tool and MSigDB (hallmark gene sets and C5 ontology gene sets).

Animals and experimental drugs

The LPS -induced mouse model is a prevalent method for studying ARDS (Zhai et al., 2021; Cong et al., 2022; Tunstead et al., 2024). A sterile LPS solution (Sigma-Aldrich) was prepared in phosphate-buffered saline (PBS) at concentrations of 5 mg/kg. Eight-week-old male C57BL/6 mice (Koatech, Pyeongtaek, Korea) were divided into four groups: normal group (NC, n=8); control group (LPS-induced ARDS, n=14); naive MSCs group (naive MSCs administered post-LPS-induced ARDS, n=14); IFN-γ/IL-1β-primed MSCs group (IFN-γ/IL-1β-primed MSCs administered post-LPS-induced ARDS, n=14). All mice were anesthetized using Alfaxalone (Alfaxan^®, Jurox, NSW, Australia) and Xylazine (Rompun^®, Bayer AG, NRW, Germany). In the ARDS model, LPS was intratracheally administered at a dose of 5 mg/kg to induce lung injury. In the experimental groups, either naive or primed MSCs were injected via the tail vein at a dose of 1×10⁶ cells/200 μL. The control group received intravenous PBS. Injections were carried out twice, at 4 h and 3 days post-LPS administration. All mice were sacrificed 4 days after LPS administration.

Macroscopic examination

The lung surface was visually inspected to assess the extent of damage. Only visible damage on the lung surface was evaluated. Observations were scored based on the presence of partial mild, generalized mild, partial moderate, generalized moderate, partial severe, and generalized severe congestion. Scores ranged from 0 to 6, reflecting the severity of lesions in each category.

Histology analysis

Four days post-LPS injury, the mice were sacrificed, their lungs collected, and fixed in formalin for histological analysis. The fixed lungs were encased in paraffin and sectioned into 5-µm slices. Lung injury severity was assessed using these slices stained with hematoxylin and eosin. Four histopathological categories were evaluated: alveolar congestion, hemorrhage, inflammation, and alveolar wall thickening, with scores ranging from 0 to 4 based on lesion severity in each category, yielding a total possible score of 16 (Rhee et al., 2011).

BALF analysis

Bronchoalveolar lavage fluid (BALF) was collected from the left lung lobe four days after inducing ARDS with LPS in mice (Cong et al., 2022). The BALF was centrifuged, and the supernatant was analyzed for TNF-α and IL-6 cytokines using assays (R&D Systems).

Spatial transcriptomics analysis

Lung tissue samples from groups treated with vehicle (control), naive MSCs, and IFN-γ/IL-1β-primed MSCs were immediately frozen and embedded in OCT compound. Spatial transcriptomics were performed using the visium platform (10x Genomics, Inc., CA, USA). Data analysis was conducted using programming languages such as R (ver. 4.3.1, R Foundation, VIE, Austria) and Python (ver. 3.8.17, Python Software Foundation, OR, USA) with the pipelines Seurat (ver. 4.4.0, https://satijalab.r-universe.dev/Seurat/doc/readme), Scanpy (ver. 1.9.4, https://scanpy.readthedocs.io/en/1.9.x/release-notes/release-latest.html), Scanorama (ver. 1.7.3, https://github.com/brianhie/scanorama), and SpaceRanger (ver. 2.1.1, 10x Genomics, CA, USA). GRCh38 (Homo sapiens) and mm10 (Mus musculus) were the reference genomes used. In Scanpy, normalization was performed by adjusting counts per spot per sample. We identified top DEGs, sorting by FC >0 for p_val_adj <0.05. Additionally, 64 immune and stromal cell types were analyzed using cell type enrichment analysis (xCell) (Aran, 2020).

Statistical analysis

All data were analyzed using GraphPad Prism version 5 (GraphPad Prism, CA, USA) and are presented as mean ± standard error of the mean (SEM). One-way analysis of variance was used to compare multiple groups, followed by Tukey post hoc analysis for pairwise comparisons of groups. A Student’s t test (two-tailed unpaired) was used when comparing two values. p<0.05 indicated a statistically significant difference (*p<0.05; **p<0.01; ***p<0.001).

Immunogenicity and immunosuppression assessed by MLR assay

We investigated the immunosuppressive efficacy of hUCB-MSCs using an indirect MLR assay. In this assay, the proliferation of PBMCs was measured over 72 h following activation with Con A and co-culture with either naive MSCs or primed MSCs. Our results demonstrate that both naive and primed MSCs significantly inhibited the activation and proliferation of PBMCs. Specifically, PBMC proliferation in response to Con A stimulation over 72 h was 5.3-fold higher compared to unstimulated PBMCs. In co-culture with naive MSCs, PBMC proliferation was increased by 4.4-fold (83.4%, vs. Con A only group), by 3.5-fold with IFN-γ-primed MSCs (65.8%, vs. Con A), by 4.7-fold with IL-1β-primed MSCs (88.5%, vs. Con A only group), and by 3.2-fold with IFN-γ/IL-1β-primed MSCs (60.8%, vs. Con A only group) (Fig. 1A).The IFN-γ/IL-1β-primed MSCs demonstrated the highest suppressive efficacy, indicating their superior potential in modulating immune responses.

Figure 1. Primed MSCs exhibit enhanced immunoregulatory abilities compared to naive MSCs. (A) The immunomodulation abilities of naive and primed MSCs were confirmed through both direct and indirect MLR assays, using IFN-γ or IL-1β (n=5). (B) Assessment of TNF-α secretion in M1 macrophages (n=4), and (C) assessment of IL-10 secretion in M2 macrophages (n=4). The data were presented as the mean±SEM. *p<0.05 and ***p<0.001. ConA: 2.5 µg/mL Concanavalin A treatment, NC: negative control, PC: positive control, Naive MSCs: co-culture with naive MSCs, IL-1β-primed MSCs: co-culture with IL-1β-primed MSCs, IFN-γ-primed MSCs: co-culture with IFN-γ-primed MSCs, IFN-γ/IL-1β-primed MSCs: co-culture with IFN-γ/IL-1β-primed MSCs.

Furthermore, we investigated the immunogenicity of hUCB-MSCs. To evaluate their immunogenic potential, we performed a direct MLR assay by co-culturing PBMCs with naive MSCs, IFN-γ-primed MSCs, IL-1β-primed MSCs, and MSCs primed with both IFN-γ and IL-1β. The results confirmed the absence of immunogenicity in all MSCs groups (Supplementary Fig. 1).

Therapeutic mechanisms of primed MSCs through M1/M2 macrophage polarization and cytokine

To investigate the anti-inflammatory effects of naive MSCs or primed MSCs on macrophages, we measured the production of inflammatory cytokines in M1/M2 macrophages. We first assessed the secretion levels of TNF-α in M1 macrophages stimulated with LPS/IFN-γ and co-cultured with hUCB-MSCs for 72 h. LPS/IFN-γ treatment significantly elevated TNF-α levels to 13,846 pg/ml. However, this effect was reduced by co-culture with naive MSCs, IL-1β-primed MSCs, IFN-γ-primed MSCs, and IFN-γ/IL-1β-primed MSCs (Fig. 1B). Specifically, the concentration of TNF-α was 3,300 pg/ml (23.9%, vs. positive control (PC)) with naive MSCs, 4,270 pg/ml (30.8%, vs. PC) with IFN-γ-primed MSCs, 3,191 pg/ml (23.0%, vs. PC) with IL-1β-primed MSCs, and 2,193 pg/ml (15.8%, vs. PC) with IFN-γ/IL-1β-primed MSCs. Among these, the IFN-γ/IL-1β-primed MSCs exhibited the most significant suppression of TNF-α secretion.

In addition, we measured the secretion of IL-10 in co-cultures of naive or primed MSCs with M0 macrophages. The results showed that IFN-γ/IL-1β-primed MSCs induced higher secretion levels compared to naive MSCs, IL-1β-primed MSCs, and IFN-γ-primed MSCs (Fig. 1C). Consequently, IFN-γ/IL-1β-primed MSCs demonstrated the greatest efficacy in inflammatory modulation. Based on these findings, we selected IFN-γ/IL-1β-primed MSCs as the most effective in regulating inflammation.

Identification of inflammation/immune modulation potential of IFN-γ/IL-1β-primed MSCs through mRNA-Seq

To elucidate the molecular mechanisms and characteristics underlying the immune cell inhibitory capacity of FN-γ-, IL-1β-, and IFN-γ/IL-1β-primed MSCs compared to naive MSCs, we performed total mRNA sequencing and subsequently conducted differential expression analysis on the identified DEGs. The overall expression patterns of DEGs are shown in a heatmap (Fig. 2A), where distinct clustering of gene expression between IFN-γ-, IL-1β-, and IFN-γ/IL-1β-primed MSCs, and naive MSCs are observed. The Venn diagram (Fig. 2B) illustrates the overlap of up-regulated DEGs among IFN-γ-, IL-1β-, and IFN-γ/IL-1β-primed MSCs. Differential expression analysis revealed 851 upregulated and 352 downregulated DEGs in MSCs primed with IFN-γ/IL-1β compared to naive MSCs, as depicted in the volcano plot (Fig. 2C). Notably, many of the top 10 up-regulated GO terms in IFN-γ/IL-1β-primed MSCs are recognized as critical components of the immune system, crucial for regulating inflammation and immune responses. To further understand the biological functions of these up-regulated DEGs, we conducted GO term enrichment analysis and selected the top 10 terms for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The BP category included terms such as ‘immune response,’ ‘inflammatory response,’ and ‘defense response to virus.’ In the MF category, terms such as ‘MHC class II protein complex binding,’ ‘MHC class II receptor activity,’ ‘CXCR chemokine receptor binding,’ and ‘chemokine activity’ were highlighted (Fig. 2D). Additionally, genes with increased expression in MSCs treated with IFN-γ, IL-1β, or IFN-γ/IL-1β were identified compared to naive MSCs (Supplementary Table 1). Specifically, when treated with IFN-γ/IL-1β, the gene expression levels showed a synergistic effect compared to those treated with either IFN-γ or IL-1β alone. These genes are involved in immune and inflammation response regulation, including the CXCL/CCL family, SERPIN family, MMPs, IL-6, and VEGFC. The CXCL family includes genes such as CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, CXCL9, CXCL10, CXCL11, and CXCL16 and the CCL family includes genes like CCL2, CCL5, and CCL20. An increase in the expression of SERPIN family genes such as SERPINB2, SERPINB7, SERPING1, and SERPINA9 was also observed.

Figure 2. RNA analysis of primed MSCs compared to naive MSCs. (A) The heatmap shows the expression value changes of DEGs between high and low expression levels, with gene names omitted, and verifies group clustering. (B) The Venn diagram illustrates the shared upregulated DEGs among IFN-γ-primed MSCs vs. naive MSCs, IL-1β-primed MSCs vs. naive MSCs, and IFN-γ/IL-1β-primed MSCs vs. naive MSCs. (C) The volcano plot, a bioinformatics tool, displays gene expression distribution between two conditions by plotting UP (Case/Control): FDR (adjust p-value)<0.05 & log2FoldChange>=1, DOWN (Case/Control): FDR (adjust p-value)<0.05 & log2Fold Change<=–1 for each gene. (D) GO term analysis revealed the top 10 terms for classification and distribution analysis using up-regulated DEGs.

Enrichment of immune- and inflammation-related pathways in IFN-γ/IL-1β-primed MSCs

To investigate the statistical enrichment of genetic changes in IFN-γ/IL-1β-primed MSCs, Gene Set Enrichment Analysis (GSEA) was conducted using DEGs between IFN-γ/IL-1β-primed MSCs and naive MSCs. This analysis identified nine gene sets significantly associated with immune- and inflammation-related biological processes (Fig. 3). Among these, notable activation was observed in pathways such as ‘defense response to virus’ and ‘interferon-mediated signaling pathway,’ highlighting the enhanced antiviral defense capabilities of primed MSCs through interferon signaling. Furthermore, the enrichment of pathways related to ‘adaptive immune response’ and ‘T cell-mediated immunity’ suggests that primed MSCs regulate immunity through associated genes, with this activation playing a role in immunomodulation. These results suggest that the GSEA of DEGs between IFN-γ/IL-1β-primed MSCs and naive MSCs revealed enrichment in immune- and inflammation-related pathways, highlighting the enhanced antiviral defense and antigen presentation capabilities of the primed MSCs.

Figure 3. GSEA was performed on DEGs to identify relevant gene sets. Focusing on pathways related to inflammation and immune regulation, we selected 9 significant gene sets with p-values<0.05 and FDR q-values<0.05.

Lung repair and immune modulation by IFN-γ/IL-1β-primed MSCs in LPS-induced ARDS mouse model

The LPS-induced lung injury model is widely utilized in rodents to study ARDS (Zhai et al., 2021; Cong et al., 2022; Tunstead et al., 2024) and has demonstrated the ability to replicate the neutrophilic inflammatory response characteristic of ARDS patients (Yang et al., 2021). Direct lung injury in rodents can be modeled by administering LPS to the lungs, resulting in damage primarily to the alveolar epithelium (Sucre et al., 2023).

We administered either IFN-γ/IL-1β-primed or naive MSCs to an LPS-induced ARDS mouse model and collected lung tissue and BALF samples to evaluate therapeutic effects on pathology, neutrophil count, and the protein levels of IL-6 and TNF-α in BALF (Fig. 4A). Macroscopic examination revealed severe congestion in the vehicle group compared to the normal group. However, administration of either naive or IFN-γ/IL-1β-primed MSCs led to a partial reduction in lung congestion, suggesting that MSCs can alleviate LPS-induced lung damage (Fig. 4B, 4D). Histopathological analysis corroborated these findings, indicating severe pathological changes in the vehicle group such as congestion, necrosis, thickening of alveolar walls, and infiltration of immune cells due to LPS. Nevertheless, these pathological changes were significantly mitigated in groups receiving either IFN-γ/IL-1β-primed or naive MSCs, with IFN-γ/IL-1β-primed MSCs showing a particularly potent therapeutic effect (Fig. 4C, 4E). Further analysis of BALF demonstrated that inflammatory cytokines IL-6 and TNF-α were elevated in LPS-treated ARDS mice. However, MSCs administration significantly reduced these elevated cytokine levels, with IFN-γ/IL-1β-primed MSCs showing especially notable effects on IL-6 and TNF-α in BALF (Fig. 4F, 4G). In summary, these results affirm that IFN-γ/IL-1β-primed MSCs provide superior therapeutic effects compared to naive MSCs in the ARDS mouse model.

Figure 4. Primed MSCs mitigate the pathological effects on the lungs in ARDS induced by LPS administration. (A) Schematic of the experimental design. (B) Representative images of whole lungs from all experimental groups. (C) H&E staining of lung sections from each group. Scale bars=100 μm. (D) Quantitative analysis of the lung injury score. Lung injury severity was scored from 0 to 6, based on the presence of partial or generalized mild, moderate, or severe congestion. Higher scores indicated more severe lesions. (E) Analysis of lung pathology scores. Pathology scores was assessed using H&E-stained slices, evaluating alveolar congestion, hemorrhage, inflammation, and alveolar wall thickening. Scores ranged from 0 to 4 per category, with a total possible score of 16. (F) Measurement of TNF-α concentrations in BALF from all groups. (G) Measurement of IL-6 concentrations in BALF from all groups. Data were presented as the mean ± SEM. *p<0.05 vs. vehicle, **p<0.01 vs. vehicle, and ***p<0.001 vs. vehicle (Normal=8, Vehicle=14, Naive MSCs=14, Primed MSCs=14).

Spatial transcriptomic profile of IFN-γ/IL-1β-primed MSCs in LPS-induced ARDS mouse model

In the LPS-induced ARDS mouse model, spatial transcriptomic analyses were conducted to examine the gene expression patterns that elucidate the mechanisms of action and therapeutic effects of immune cells in naive MSCs or IFN-γ/IL-1β-primed MSCs within the injured lung tissue. Cluster analysis distinguished between human and mouse cells. Consequently, the analysis of human-specific genes revealed that both naive MSCs and IFN-γ/IL-1β-primed MSCs were present around damaged lung tissue, yet absent in the vehicle group (Fig. 5A). Notably, IFN-γ/IL-1β-primed MSCs were more prevalent than naive MSCs. Additionally, genes associated with metabolism, leukocyte migration, and blood flow were significantly increased in -primed MSCs, while immune activity decreased (Fig. 5B). Our results suggest that IFN-γ/IL-1β-primed MSCs specifically targets the lung in a LPS-induced ARDS model and holds therapeutic potential.

Figure 5. Distribution and immunosuppressive effects of naive MSCs and primed MSCs through spatial transcriptome analysis. (A) The distribution of naive and primed MSCs in the lung was assessed by analyzing human transcripts (yellow), showing a higher presence of primed MSCs. (B) Human cluster analysis revealed the immunosuppressive efficacy of both naive and primed MSCs, with primed MSCs displaying greater suppressive effects. (C) Analysis of immune and stromal cell types using cell type enrichment analysis (xCell) demonstrated an increase in Tregs and a reduction in macrophages, monocytes, and neutrophils, suggesting potential immunomodulatory effects. (D) xCell analysis compared mouse cell type enrichment across groups, with darker colors indicating higher abundance or activation of major cell types.

Next, we investigated the mouse gene patterns to identify the therapeutic mechanisms. Immune and stromal cell types were evaluated using cell type enrichment analysis via xCell. The IFN-γ/IL-1β-primed MSCs group exhibited higher gene expression related to Tregs compared to the vehicle group (Fig. 5C). In the vehicle group, genes associated with macrophages, monocytes, and neutrophils were highly expressed, but this expression diminished following the administration of either naive MSCs or IFN-γ/IL-1β-primed MSCs. A more pronounced reduction was seen in areas where human genes were expressed. The reduction in immune cells and inflammation was significantly greater in the IFN-γ/IL-1β-primed MSCs group than in the naive MSCs group. Further, cell type analysis indicated that while the vehicle group showed elevated expression of B cells, macrophages, monocytes, and neutrophils, a decrease in these cell types was observed after administering either naive MSCs or IFN-γ/IL-1β-primed MSCs (Fig. 5D). Specifically, the IFN-γ/IL-1β-primed MSCs group more effectively suppressed the activity of immune cells, including B cells and T cells, than the naive MSCs group. In summary, IFN-γ/IL-1β-primed MSCs demonstrated enhanced suppression of immune activity and inflammation-related cells in the lung injury tissues of ARDS.

Identification of key factors of IFN-γ/IL-1β-primed MSCs on the spatial transcriptomic dataset

In this study, we identified the major therapeutic effects and key mediators of IFN-γ/IL-1β-primed MSCs in treating ARDS. We highlighted the top four genes whose expression was significantly increased in the vehicle group (adjusted p-value<0.05; ranked by log fold change) and demonstrated their suppression by IFN-γ/IL-1β-primed MSCs using Spatial Feature Plot (Fig. 6A) and VlnPlot (Fig. 6B). These genes (Uba52, CXcl9, Cxcl5, and Cfb) are closely associated with inflammatory processes, immune responses, and chemokine activity (Metzemaekers et al., 2018; Callahan et al., 2021). Additionally, in our effort to pinpoint the key mediators of IFN-γ/IL-1β-primed MSCs, we noted a significant upregulation of the Eln (elastin) gene, the most highly expressed among all differentially expressed genes. Eln is an essential component of the extracellular matrix and plays a vital role in maintaining tissue elasticity and integrity (Mecham, 2018; Heinz, 2020). Elastin is essential for lung function, contributing to the structural framework for proper respiratory mechanics and playing a critical role in tissue regeneration and repair (Mecham, 2018). Given these attributes, the pronounced elevation of Eln in IFN-γ/IL-1β-primed MSCs indicates that it may be a key mediator in the regenerative and anti-inflammatory effects of these cells, especially in cases of lung injury or dysfunction.

Figure 6. Identification of key factors of naive and primed MSCs on the top 5 inflammation-related genes with elevated expression in acutely injured lungs. (A) Spatial feature plot analysis depicted the reduction in expression of the top 5 genes by both naive and primed MSCs. (B) VlnPlot analysis showed decreased expression levels of the top 5 genes due to the influence of naive and primed MSCs. Both types of MSCs suppressed the expression of inflammation-related genes, with primed MSCs demonstrating greater inhibitory efficacy.

Immune regulatory efficacy of naive MSCs and IFN-γ/IL-1β-primed MSCs on the spatial transcriptomic dataset

A Volcano plot analysis was conducted to compare DEGs in lung tissue between the IFN-γ/IL-1β-primed MSCs-treated group and the vehicle group, revealing 753 DEGs (Fig. 7A). These DEGs displayed either upregulated or downregulated expression patterns. GO analysis of these DEGs revealed significant enrichment in several pathways (Fig. 7B). Within the biological process (BP) category, genes associated with pathways such as the regulation of innate immune responses, cytokine-mediated signaling, and response to type II interferon were significantly downregulated, indicating that these immune and inflammatory response-related processes are reduced under IFN-γ/IL-1β-primed MSCs-treated conditions. In the cellular component (CC) category, genes associated with phagocytic vesicles, MHC complexes, and endocytic vesicles were significantly downregulated, indicating a potential reduction in antigen processing and immune-related vesicular functions. Additionally, the molecular function (MF) analysis showed upregulation in growth factor binding and extracellular matrix structural constituents, along with a downregulation in cytokine receptor binding and chemokine/cytokine activity, highlighting the immunosuppressive and reparative potential of IFN-γ/IL-1β-primed MSCs. These results demonstrate that IFN-γ/IL-1β-primed MSCs possess enhanced capacities in regulating immune responses and tissue repair mechanisms.

Figure 7. Immunoregulatory efficacy of primed MSCs through spatial transcriptomic analysis. (A) Volcano plot analysis contrasting the vehicle group with primed MSCs, identifying downregulated factors on the left and upregulated factors on the right. (B) Bar plot of the top 10 downregulated Gene Ontology terms for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF).

Therapeutics mechanism of naive MSCs and IFN-γ/IL-1β-primed MSCs on the spatial transcriptomic dataset

The analysis of key immune mechanisms provides valuable insights for the treatment of ARDS. The signaling pathways were explored through KEGG pathway enrichment analysis in lung tissue from an ARDS mouse model (Fig. 8A). To elucidate the therapeutic mechanisms of naive MSCs and IFN-γ/IL-1β-primed MSCs, we explored the NF-κB and TNF signaling pathways, which are critical in ARDS pathology (Fig. 8B). (Chen and Hua, 2020; Huang et al., 2024) NF-κB pathway analysis indicated that expression levels of IL-1β, TNFα, LTB, CD14, CD40, Syk, IKKβ, IκBα, p100, gadd45β, TRAF1/2, A1/Bf1-1, MIP-1 β, MIP-2, SDF-1α and ICAM were lower in the IFN-γ/IL-1β-primed MSCs group than in the vehicle group (Supplementary Fig. 2A). The IFN-γ/IL-1β-primed MSCs group showed more extensive downregulation of inflammatory genes, including IL-1β, TNFα, MIP-1 β, MIP-2, VCAM-1, ELC, SDF-1α and ICAM compared to the naive MSCs group. TNF pathway analysis indicated that levels of TNF, TACE, INF/LTA, TNFR2, TRAF1, IKKβ, IκBα, c/EBPβ, IKKs, IRF1, Ccl2, Ccl5, Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl10, IL-1β, Tnf, Nfkbia, Socs3, Traf1, Ifi47 and Icam1 were lower in the IFN-γ/IL-1β-primed MSCs group than in the vehicle group (Supplementary Fig. 2B). In the IFN-γ/IL-1β-primed MSCs group, a more significant reduction of inflammatory genes was noted, including TNF, INF/LTA, IRF1, Ccl2, Ccl5, Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl10, IL-1β, Tnf, Ifi47, Icam1, Vcam1 compared to the naive MSCs group. These findings indicate that IFN-γ/IL-1β-primed MSCs provide enhanced therapeutic potential through modulation of crucial inflammatory pathways in ARDS. Additionally, IFN-γ/IL-1β-primed MSCs exhibit inhibitory effects on MAC-1, LFA-1, PSGL1, CASP11, CASP1, Syk, gp91, p47phox, p22phox, p67phox, p40phox and Rac in the Neutrophil Extracellular Trap (NET) formation signaling pathway (Supplementary Fig. 2C). These results demonstrate that IFN-γ/IL-1β-primed MSCs effectively reduce immune cell activation and neutrophil infiltration by suppressing inflammatory genes, underscoring their potential to mitigate excessive inflammatory responses in ARDS.

Figure 8. Therapeutic mechanisms via spatial transcriptomic analysis. (A) Downregulated KEGG pathway analysis of ARDS-related genes. (B) Reduction of inflammatory factors by naive and primed MSCs in the NF-κB and TNF signaling pathways, with primed MSCs demonstrating enhanced efficacy, including targeting factors addressed by naive MSCs. Blue letters and arrows indicate downregulated factors and signals for both naive MSCs and primed MSCs injected groups compared to the ARDS control group, while green letters and arrows highlight more downregulated factors and signals in primed MSCs than in naive MSCs injected groups. TNF-a: Tumor necrosis factor-α, IDO1: Indoleamine 2,3-Dioxygenase 1, PGE2: Prostaglandin E2, Serpins: Serine protease inhibitors, CXCL: C-X-C motif chemokine ligand, CCL: C-C motif chemokine ligand, SDF-1: Stromal cell-derived factor-1, ICAM:intercellular adhesion molecule, MIP: microphage inflammatory protein.

MSCs have been widely studied for their immunomodulatory and regenerative properties in various diseases, including ARDS in COVID-19, though their clinical efficacy remains inconclusive. This study aims to ascertain whether primed MSCs show superior therapeutic efficacy compared to naive MSCs in ARDS and to elucidate the underlying mechanisms.

Initially, the immunomodulatory effects of IFN-γ and IL-1β, both individually and in combination, were explored using MLR assays in vitro. The results demonstrated that the response of mixed lymphocytes was more pronounced with IFN-γ than with IL-1β, with the combined treatment group exhibiting the highest level of immunomodulatory activity. Additionally, macrophage polarization induced by priming with both cytokines tended to enhance M2 macrophage promotion. Previous studies have shown that treatment with IFN-γ enhances the expression of IDO-1, IL-10, PD-L1, and PGE2 from MSCs, particularly IDO-1 and PGE2, which are known to effectively suppress T cell proliferation (Dunn et al., 2022). Conversely, Priming MSCs with IL-1β does not significantly enhance the suppression of T cell proliferation; however, RNA sequencing analysis demonstrates a notable increase in PTGES and IL-6 expression with IL-1β treatment compared to IFN-γ treatment alone. Additionally, co-culturing M0 macrophages with MSCs promotes M2 macrophage polarization and activity, suggesting a synergistic mechanism that drives M2 phenotype induction under combined treatment conditions (Colombini et al., 2022).

RNA sequencing analysis revealed distinct changes in gene expression profiles induced by priming, highlighting their therapeutic relevance in ARDS. Specifically, priming significantly modulated the expression of key genes, including members of the CXCL/CCL family, SERPIN family, MMPs, and ICAM, as well as immunomodulatory factors such as PGE2-related genes, IDO, and IL-6. CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 are implicated in neutrophil recruitment (Rajarathnam et al., 2019), while CXCL9, CXCL10, and CXCL11 are associated with T cell recruitment (Li et al., 2020). CCL2 and CCL5 orchestrate the recruitment of monocytes and macrophages, whereas CCL20 facilitates lymphocyte recruitment (Yoshimura et al., 2023). The expression of MMP3 and MMP13, which facilitate MSCs migration, increased alongside elevated levels of VEGFC, promoting vascular regeneration, and ICAM1, enhancing interactions with immune cells (Hass and Otte, 2012; Almalki and Agrawal, 2016; Chang et al., 2021; Zheng et al., 2021). Among the mentioned genes, SERPINB2, IL-6, and PTGS, associated with macrophage M2 polarization, exhibited greater increases when treated with both IL-1β and IFN-γ compared to treatment with IFN-γ alone.

Interestingly, these gene alterations correlated closely with efficacy evaluations and spatial transcriptome analyses in in vivo mouse models. In LPS-induced ARDS models where IFN-γ/IL-1β-primed MSCs were administered, marked improvement in histopathological lesions of severe inflammation were noted compared to naive MSCs, accompanied by a significant decrease in TNF-α and IL-6 in BALF. The spatial transcriptome analysis of the in vivo models revealed increased migration of IFN-γ/IL-1β-primed MSCs through image analysis. A general reduction in T cells, monocytes, macrophages, and neutrophils was observed, coupled with an increase in regulatory T cells as well as both endothelial and epithelial cells. This suggests that MSCs primed with IFN-γ and IL-1β effectively enhance their migration, immunomodulatory capacity, and regenerative capabilities, resulting in improved treatment outcomes for ARDS pathophysiology.

In an analysis of gene expression alterations within spatial transcriptome data, the administration of primed MSCs resulted in a pronounced decrease in genes linked with chemokines, such as CXCL5 and CXCL9, compared to the LPS-induced group. Moreover, considerable reductions were noted in alterations pertaining to complement factors like Cfb and ubiquitin-like proteins such as Uba52. These findings suggest that MSCs-mediated modulation of inflammatory responses leads to reduced secretion of chemokines by inflammatory cells, which subsequently lowers immune reactions and apoptotic events. Intriguingly, an increase in elastin expression was observed. Elastin generally decreases during the initial stages of ARDS but increases during the recovery phase, contributing to the restoration of lung elasticity. Furthermore, the observed reduction in markers of inflammation and tissue damage linked to ARDS, together with increased expression of elastin genes, primarily focusing on the distribution of human cells in mouse lung tissue, indicates that MSCs directly contribute to the amelioration of inflammation.

Through spatial transcriptome analysis, it was observed that primed MSCs showcased enhanced regulatory capabilities over NF-κB and TNF-α signaling compared to naive MSCs. NF-κB is recognized as a master regulator involved in both the initiation and resolution of inflammatory responses (Luan et al., 2024). Dysregulated NF-κB activation is a hallmark of several pulmonary disorders, including ALI/ARDS, asthma, idiopathic pulmonary fibrosis (IPF), and chronic obstructive pulmonary disease (COPD) (Alharbi et al., 2022; Millar et al., 2022). In ARDS, the severity and lethality are largely driven by an NF-κB-mediated cytokine storm, characterized by excessive PMN extravasation and cytokine release, leading to widespread inflammation and coagulation (Fajgenbaum and June, 2020; Millar et al., 2022). Recent evidence indicates that the SARS-CoV-2 spike protein activates NF-κB in endothelial cells and macrophages, exacerbating inflammation (Robles et al., 2022; Palestra et al., 2023). While NF-κB activation contributes to acute symptom deterioration, it also promotes endothelial barrier repair and tissue recovery during the resolution phase (Millar et al., 2022). Therefore, in the context of therapies targeting NF-κB, consideration of the specific target cells and timing of administration is essential. Another crucial signaling pathway is TNF signaling, a known activator of NF-κB. TNF plays a pivotal role in inflammatory pulmonary conditions, including severe pneumonia and ARDS (Lucas et al., 2021). Signaling through the TNF-TNF receptor significantly influences the integrity of the alveolar-capillary barrier, the pathophysiology of neutrophilic alveolitis, and alveolar fluid clearance (AFC) (Lipke et al., 2010; Lucas et al., 2021). The death signaling, impairment of AFC, and hyperpermeability induced by TNF-TNF receptor signaling are critically important in the pathogenesis of ARDS (Lucas et al., 2021; Widowati et al., 2023). Consequently, treatments inhibiting TNF have been suggested to reduce the severity of lung injury (Widowati et al., 2023).

This study verified the efficacy of priming MSCs with IFN-γ and IL-1β, which alleviates ARDS symptoms by enhancing the homing ability of MSCs, suppressing the immune functions of T cells and M1 macrophages, while enhancing the activities of T-reg and M2 macrophages, and promoting the regeneration of endothelial and epithelial cells. These significant alterations correlate with gene changes in MSCs, symptomatic improvement in a live ARDS model, and spatial transcriptomic modifications at the lesion sites. Additionally, the augmentation of these therapeutic mechanisms was verified by downregulating factors in the NF-κB and TNF pathways.

In summary, IFN-γ/IL-1β-primed MSCs effectively modulate immune and inflammatory responses in damaged lung tissue. Pre-stimulation with IFN-γ and IL-1β significantly enhances the therapeutic potential of hUCB-MSCs, establishing cytokine-primed MSCs as a promising approach for ARDS treatment and regenerative medicine.

This research was supported by the Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Health & Welfare) (21C0710L1).

The authors declare that they have no competing interests.

SL and KSK contributed to the conception and design of the study and supervised all experimental testing. THK and SKS contributed the design of the study, performed experiments, interpreted the data, and wrote the manuscript. YSH performed the analysis of RNA-seq and spacial transcriptomic data and interpreted the data, and wrote the manuscript. WMS performed animal experiments and interpretation of data. JEL, BKK and YJC analyzed and interpreted the data, and edited the manuscript. SL and KSK gave financial support and supervised the project. All authors read and approved the final manuscript.