Mesenchymal Stem Cells Alleviate Airway Inflammation via Modulation of T-Helper 17/Regulatory T Cells Balance in Mice with Ovalbumin-Induced Asthma
Asthma is a complex chronic inflammatory disorder of the airways characterized by dysregulated immune responses, airway hyperresponsiveness (AHR), and structural remodeling. Despite advancements in conventional therapies, long-term management of asthma remains challenging due to limited efficacy and side effects. Recent research has highlighted the imbalance between T-helper 17 (Th17) cells and regulatory T (Treg) cells as a critical factor driving asthma pathogenesis. Th17 cells promote neutrophilic and eosinophilic inflammation through interleukin (IL)-17A, while Treg cells suppress immune activation via anti-inflammatory cytokines like IL-10 and transforming growth factor beta 1 (TGF-β1). Mesenchymal stem cells (MSCs), with their immunomodulatory properties, have emerged as promising candidates for restoring this imbalance. This study investigates the therapeutic potential of MSCs in a murine model of ovalbumin (OVA)-induced asthma, focusing on their ability to modulate Th17/Treg equilibrium and mitigate airway inflammation.
Experimental Design and Methodology
Animal Model and MSC Preparation
Female BALB/c mice (6–8 weeks old, 16–24 g) were divided into three groups: (1) control group (PBS sensitization/challenge), (2) OVA group (asthma model with OVA sensitization/challenge), and (3) OVA + MSCs group (asthma model treated with 10^6 MSCs via tail vein injection). Bone marrow-derived MSCs were isolated from BALB/c mice femurs and tibias and expanded in vitro prior to transplantation. The OVA-induced asthma protocol involved intraperitoneal sensitization with OVA-alum on days 0, 7, and 14, followed by intranasal OVA challenges from days 21 to 27. MSCs were administered 24 hours after the final OVA challenge.
Assessment of Airway Hyperresponsiveness and Inflammation
Airway hyperresponsiveness was measured 24 hours post-treatment using whole-body plethysmography. Mice were exposed to increasing methacholine concentrations (3–50 mg/ml), and AHR was quantified using the enhanced pause (Penh) index. Bronchoalveolar lavage fluid (BALF) was collected post-sacrifice to analyze inflammatory cell infiltration, including eosinophils, macrophages, lymphocytes, and neutrophils. Cytospin preparations of BALF cells were stained with Diff-Quick for differential counting. Lung tissues were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) to evaluate peribronchial inflammation and goblet cell hyperplasia. Inflammation severity was scored on a 0–4 scale, while mucus production was quantified as the percentage of PAS-positive bronchial epithelium.
Cytokine Profiling and Immune Cell Analysis
Levels of IL-6, IL-10, IL-17A, and TGF-β1 in BALF were measured via enzyme-linked immunosorbent assay (ELISA). Splenic lymphocytes were isolated for flow cytometry to determine Th17 (CD4+IL-17A+) and Treg (CD4+CD25+Foxp3+) cell populations. Surface and intracellular staining protocols included fluorochrome-conjugated antibodies against CD4, CD25, IL-17A, and Foxp3.
Key Findings
MSCs Attenuate Airway Hyperresponsiveness and Inflammation
MSC treatment significantly reduced AHR in OVA-challenged mice. At 50 mg/ml methacholine, Penh values decreased from 583.74 ± 59.26% (OVA group) to 175.92 ± 21.38% (OVA + MSCs group) (P < 0.001). BALF analysis revealed a marked reduction in inflammatory cells: macrophages (33.86 ± 5.71 × 10^4/ml vs. 52.19 ± 7.12 × 10^4/ml), eosinophils (21.46 ± 3.51 × 10^4/ml vs. 73.59 ± 9.62 × 10^4/ml), lymphocytes (19.82 ± 2.94 × 10^4/ml vs. 33.59 ± 4.27 × 10^4/ml), and neutrophils (12.61 ± 0.92 × 10^4/ml vs. 17.52 ± 2.76 × 10^4/ml) (P < 0.001 for all comparisons).
Histopathological evaluation corroborated these results. H&E staining showed reduced peribronchial inflammatory infiltrates in the MSC-treated group (inflammation score: 1.97 ± 0.31 vs. 5.13 ± 0.98 in the OVA group; P < 0.001). PAS staining demonstrated diminished mucus production, with the mucus index declining from 67.16 ± 6.24% (OVA group) to 22.54 ± 3.26% (OVA + MSCs group) (P < 0.001).
Modulation of Cytokine Milieu
MSC administration shifted the BALF cytokine profile toward an anti-inflammatory state. Pro-inflammatory IL-17A and IL-6 levels decreased significantly: IL-17A (32.74 ± 4.97 pg/ml vs. 48.62 ± 7.21 pg/ml; P < 0.001) and IL-6 (14.71 ± 2.18 pg/ml vs. 22.69 ± 3.28 pg/ml; P < 0.001). Conversely, anti-inflammatory IL-10 (68.41 ± 8.22 pg/ml vs. 26.37 ± 3.13 pg/ml) and TGF-β1 (76.19 ± 7.81 pg/ml vs. 51.36 ± 6.62 pg/ml) levels rose substantially (P < 0.001).
Restoration of Th17/Treg Balance
Flow cytometry analysis of splenocytes highlighted MSC-mediated immune reprogramming. The OVA group exhibited elevated Th17 cells (26.14 ± 4.21% vs. 7.22 ± 0.74% in controls; P < 0.001), which MSC treatment reduced to 15.97 ± 2.58% (P < 0.001). Conversely, Treg populations, suppressed in asthmatic mice (3.22 ± 0.39% vs. 4.86 ± 0.49% in controls; P < 0.001), were restored to 7.81 ± 1.13% post-MSC therapy (P < 0.001).
Mechanistic Insights and Discussion
The therapeutic efficacy of MSCs in asthma stems from their dual ability to suppress pro-inflammatory pathways and enhance regulatory mechanisms. Th17 cells drive neutrophilic inflammation and airway remodeling via IL-17A, while Treg cells counterbalance these effects through IL-10 and TGF-β1. The observed Th17/Treg imbalance in asthma—characterized by elevated IL-6 and IL-17A alongside reduced IL-10 and TGF-β1—was rectified by MSC intervention.
MSCs likely modulate this equilibrium through paracrine signaling. IL-6, a key driver of Th17 differentiation, was downregulated post-MSC treatment, attenuating Th17 expansion. Simultaneously, MSC-induced IL-10 and TGF-β1 upregulation promoted Foxp3+ Treg differentiation, further suppressing Th17 activity. This cytokine shift not only reduced eosinophil and neutrophil infiltration but also mitigated goblet cell hyperplasia and AHR.
The homing of MSCs to inflamed lung tissues and their interaction with dendritic cells (DCs) may amplify these effects. Prior studies suggest MSCs inhibit DC maturation, thereby dampening T-cell activation. In this model, enhanced Treg activity and IL-10 production likely suppressed DC-mediated Th2 and Th17 polarization, creating a feedback loop that sustains anti-inflammatory responses.
Conclusion
This study demonstrates that MSC therapy alleviates OVA-induced asthma by rebalancing Th17/Treg responses. Through IL-6 suppression and IL-10/TGF-β1 induction, MSCs reduced airway inflammation, mucus hypersecretion, and bronchial hyperreactivity. These findings underscore the potential of MSC-based therapies for refractory asthma, particularly in cases driven by Th17/Treg dysregulation. Future research should explore optimal dosing regimens and long-term safety to facilitate clinical translation.
doi.org/10.1097/CM9.0000000000001699
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