Impact of one versus two doses of mesenchymal stromal cells on lung and cardiovascular repair in experimental emphysema

Abstract

Background: A single administration of mesenchymal stromal cells (MSCs) has been shown to reduce lung inflammation in experimental elastase-induced emphysema; however, effects were limited in terms of lung-tissue repair and cardiac function improvement. We hypothesized that two doses of MSCs could induce further lung and cardiovascular repair by mitigating inflammation and remodeling in a model of emphysema induced by multiple elastase instillations. We aimed to comparatively investigate the effects of one versus two doses of MSCs, administered 1 week apart, in a murine model of elastase-induced emphysema.

Methods: C57BL/6 mice were randomly divided into control (CTRL) and emphysema (E) groups. Mice in the E group received porcine pancreatic elastase (0.2 IU, 50 μL) intratracheally once weekly for four consecutive weeks; the CTRL animals received sterile saline (50 μL) using the same protocol. Three hours after the last instillation, the E group was further randomized to receive either saline (SAL) or murine MSCs (10⁵ cells) intratracheally, in one or two doses (1 week apart). Fourteen days later, mice were euthanized, and all data analyzed.

Results: Both one and two doses of MSCs improved lung mechanics, reducing keratinocyte-derived chemokine and transforming growth factor-β levels in lung homogenates, total cell and macrophage counts in bronchoalveolar lavage fluid (BALF), and collagen fiber content in airways and blood vessels, as well as increasing vascular endothelial growth factor in lung homogenates and elastic fiber content in lung parenchyma. However, only the two-dose group exhibited reductions in tumor necrosis factor-α in lung tissue, BALF neutrophil and lymphocyte count, thymus weight, and total cellularity, as well as CD8⁺ cell counts and cervical lymph node CD4⁺ and CD8⁺ T cell counts, as well as further increased elastic fiber content in the lung parenchyma and reduced severity of pulmonary arterial hypertension.

Conclusions: Two doses of MSCs enhanced lung repair and improvement in cardiac function, while inducing T cell immunosuppression, mainly of CD8⁺ cells, in elastase-induced emphysema.

Keywords: Animal models; Collagen fiber; Elastic fiber; Emphysema; Heart; Immunosuppression; Inflammation.

Fig. 1

MSC administration modulated protein levels of relevant mediators. Protein levels of a keratinocyte-derived chemokine (KC, a mouse analog of interleukin [IL]-8), b tumor necrosis factor (TNF)-α, c vascular endothelial growth factor (VEGF), and d transforming growth factor (TGF)-β in lung homogenate tissue. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group. The Kruskal–Wallis test followed by Dunn’s test was used for statistical comparison. Data are presented as median ± interquartile range. N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05)

Fig. 2

Two doses of MSCs led to further mitigation in BALF cellularity compared to a one-dose regimen. a Total leukocytes, b neutrophils, c macrophages, d lymphocytes, e CD4⁺ T cells, and f CD8⁺ T cell counts in BALF. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells, BALF bronchoalveolar lavage fluid. One-way ANOVA followed by Tukey’s test was used for statistical comparison. Data are presented as means ± SD. N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05)

Fig. 3

MSC administration did not affect BM cellularity, regardless of dose regimen. a Total leukocytes, b MN, and c PMN cell counts in BM. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells, BM bone marrow, MN mononuclear cells, PMN polymorphonuclear cells. One-way ANOVA followed by Tukey’s test was used for statistical comparison. Data are presented as means ± SD. N = 10 animals/group

Fig. 4

Two doses of MSCs, but not a one-dose regimen, led to immunosuppression in the thymus. a Thymus weight, b total cell count, c CD4⁺ T cells, d CD8⁺ T cells, and e CD4⁺CD25⁺Foxp3⁺ cell counts in thymus. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells. For statistical comparison, the following tests were used: one-way ANOVA followed by Tukey’s test in panels a, b and Kruskal–Wallis test followed by Dunn’s test in panels c–e. Data are presented as means ± SD (a, b) or median ± interquartile range (c–e). N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05). ^‡Significantly different from E-MSC-1 dose (p < 0.05)

Fig. 5

Two doses of MSCs led to immunosuppression in cervical lymph nodes, but not in mediastinal lymph nodes. a CD4⁺ T cells, b CD8⁺ T cells, and c CD4⁺CD25⁺Foxp3⁺ cell count in cLN; d CD4⁺ T cells, e CD8⁺ T cells, and f CD4⁺CD25⁺Foxp3⁺ cell count in mLN. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells, cLN cervical lymph nodes, mLN mediastinal lymph nodes. The Kruskal–Wallis test followed by Dunn’s test was used for statistical comparison. Data are presented as median ± interquartile range. N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05). ^‡Significantly different from E-MSC-1 dose (p < 0.05)

Fig. 6

One or two doses of MSCs similarly decreased collagen fiber content, but two doses led to further elastogenesis. Collagen fiber content in airways and blood vessels and elastic fiber content on lung parenchyma. Arrows indicate elastic fibers (stained black). CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells. The Kruskal–Wallis test followed by Dunn’s test was used for statistical comparison. Data are presented as median ± interquartile range. N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05). ^‡Significantly different from E-MSC-1 dose (p < 0.05)

Fig. 7

One or two doses of MSCs similarly improved lung mechanics. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells, Est,L static lung elastance. One-way ANOVA followed by Tukey’s test was used for statistical comparison. Data are presented as means ± SD. N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05)

Fig. 8

Two doses of MSCs, but not a one-dose regimen, led to improvement in cardiac function. a Diastolic right ventricle area and b PAT/PET ratio. CTRL control mice, E emphysema mice, SAL saline-treated mice, MSC MSC-treated group, MSCs mesenchymal stromal cells, PAT pulmonary artery acceleration time, PET pulmonary artery ejection time. One-way ANOVA followed by Tukey’s test was used for statistical comparison. Data are presented as means ± SD. N = 10 animals/group. *Significantly different from CTRL (p < 0.05). ^#Significantly different from E-SAL (p < 0.05). ^‡Significantly different from E-MSC-1 dose (p < 0.05)