J Korean Med Sci.  2020 Jun;35(23):e188. 10.3346/jkms.2020.35.e188.

Evaluation of Human MSCs Treatment Frequency on Airway Inflammation in a Mouse Model of Acute Asthma

• Hur J
• Kang JY
• Kim YK
• Lee SY
• Jeon S
• Kim Y
• Jung CK
• Rhee CK

• KMID: 2502923
• DOI: http://doi.org/10.3346/jkms.2020.35.e188

Abstract

Background
Studies in experimental models of allergic asthma have shown that mesenchymal stem cells (MSCs) have therapeutic potential for T-helper 2 (TH2) cell-mediated inflammation. However, the mechanisms underlying these therapeutic effects are not fully understood and their safety has not been confirmed.
Methods
Using a mouse model of experimental allergic asthma, we investigated the efficacy of human adipose-derived mesenchymal stem cells (hADSCs) or human bone marrow-derived mesenchymal stem cells (hBMSCs) according to treatment frequency and timing.
Results
Ovalbumin (OVA)-sensitized and -challenged mice exhibited airway hyperresponsiveness (AHR), airway inflammation, and significant increases in TH2 cytokine levels. Both double and single human mesenchymal stem cell (hMSC) treatments significantly decreased AHR and bronchoalveolar lavage fluid counts. In addition, single treatment with hMSCs showed significant attenuation of allergic airway inflammation. However, double treatment with hMSCs during OVA -sensitization and -challenge further increased inflammatory cell infiltration, and TH2 cytokine levels.
Conclusion
The results of treatment with hADSCs or hBMSCs suppresses AHR and airway inflammation. However, double hMSC treatment significantly induces eosinophilic airway inflammation and lung histological changes. Therefore, double hMSC treatment is ineffective against asthma and single injection frequency appears to be more important for the treatment of asthma. These results suggest that hMSC therapy can be used for treatment of asthma patients but that it should be used carefully.

Keyword

Human MSC; Frequency; Airway Inflammation; Asthma

Figure

• 

  Fig. 1 Double injection of human mesenchymal stem cells inhibited airway hyperresponsiveness and airway inflammation. (A) Schematic diagram of the mouse model of acute asthma and treatment with hADSCs or hBMSCs. Mice were sensitized on days 0 and 7 by subcutaneous injection of OVA and challenged intranasally on days 21, 23, and 25 (black dots). Aliquots of 2.5 × 107/kg hADSCs or hBMSCs were injected via the tail vein on days 7 and 27 (blue triangles). Samples were harvested on day 29. (B) Rrs was used to measure airway hyperresponsiveness with increasing concentration of nebulized methacholine. *P < 0.05, **P < 0.01 compared to the CON group; ##P < 0.01, ###P < 0.001 compared to the OVA+PBS group. (C) BALF cells were isolated by centrifugation and stained with Diff-Quik. Total numbers of macrophages, eosinophils, lymphocytes, and neutrophils in the BALF were counted. ***P < 0.001 compared to the CON group; #P < 0.05, ###P < 0.001 compared to the OVA group.hADSC = human adipose-derived mesenchymal stem cell, hBMSC = human bone marrow-derived mesenchymal stem cell, OVA = ovalbumin, Alum = aluminum, Rrs = airway resistance, CON = control, PBS = phosphate buffered saline, BAL = bronchoalveolar lavage, BALF = bronchoalveolar lavage fluid, S.c.= subcutaneous, I.n.= intranasal.

• 

  Fig. 2 Double injection of hMSCs led to more severe airway inflammation. (A–C) Representative photomicrographs of hematoxylin and eosin-stained lung sections. (A) The lungs in the mouse model of acute asthma showing airways and blood vessels surrounded by alveoli (×100). (B) Airway (×400). Black arrows indicate smooth muscle, and black stars indicate goblet cell metaplasia. (C) Blood vessel (×400). (D) Histological quantification of peribronchial and perivascular inflammation in the lung. ***P < 0.001 compared to the CON group; #P < 0.05 compared to the OVA+PBS group.CON = control, OVA = ovalbumin, PBS = phosphate buffered saline, hADSC = human adipose-derived mesenchymal stem cell, hBMSC = human bone marrow-derived mesenchymal stem cell, Br = bronchus, Bm = basement membrane, Bl = blood vessel, Eo = eosinophil, Ep = epithelium.

• 

  Fig. 3 Double injection of human mesenchymal stem cells exacerbated levels of T-helper 2 cytokine. Bronchoalveolar lavage fluid was obtained from the mouse model of acute asthma with and without treatment with hADSCs or hBMSCs and the levels of IL-4, IL-5, and IL-13 were measured.IL = interleukin, CON = control, OVA = ovalbumin, PBS = phosphate buffered saline, hADSC = human adipose-derived mesenchymal stem cell, hBMSC = human bone marrow-derived mesenchymal stem cell.*P < 0.05 compared to the CON group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the OVA+PBS group.

• 

  Fig. 4 Single injection of human mesenchymal stem cells reduced airway hyperresponsiveness and airway inflammation. (A) Schematic diagram of the mouse model of acute asthma and treatment with hADSCs or hBMSCs. Mice were sensitized on days 0 and 7 by subcutaneous injection of OVA and challenged intranasally on days 21, 23, and 25 (black dots). Aliquots of 2.5 × 107/kg hADSCs or hBMSCs were injected via the tail vein on day 27 (blue triangles). Samples were harvested on day 29. (B) Rrs was used to measure airway hyperresponsiveness with increasing concentration of nebulized methacholine. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the CON group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the OVA+PBS group. (C) BAL cells were isolated by centrifugation and stained with Diff-Quik. Total numbers of macrophages, eosinophils, lymphocytes, and neutrophils in the BAL fluid were counted. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the CON group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the OBA+PBS group.hADSC = human adipose-derived mesenchymal stem cell, hBMSC = human bone marrow-derived mesenchymal stem cell, OVA = ovalbumin, Alum = aluminum, Rrs = airway resistance, CON = control, PBS = phosphate buffered saline, BAL = bronchoalveolar lavage, S.c= subcutaneous, I.n.= intranasal.

• 

  Fig. 5 Single injection with human mesenchymal stem cells attenuated levels of T-helper 2 cytokine. Bronchoalveolar lavage fluid was obtained from the mouse model of acute asthma with and without hADSC or hBMSC treatment and levels of IL-4, IL-5, and IL-13 were measured.IL = interleukin, CON = control, OVA = ovalbumin, PBS = phosphate buffered saline, hADSC = human adipose-derived mesenchymal stem cell, hBMSC = human bone marrow-derived mesenchymal stem cell.**P < 0.01, ***P < 0.001 compared to the CON group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the OVA or OVA+PBS group.

• 

  Fig. 6 Single injection with human mesenchymal stem cells reduced eosinophil infiltration and lung inflammation. (A) Paraffin-embedded lung tissue sections were stained with hematoxylin and eosin (original magnification: ×200). (B) Airway (× 400). Black arrows indicate smooth muscle, and black stars indicate goblet cell metaplasia. (C) Blood vessel (× 400). (D) Histological quantification of peribronchial and perivascular inflammation in the lung.CON = control, OVA = ovalbumin, PBS = phosphate buffered saline, hADSC = human adipose-derived mesenchymal stem cell, hBMSC = human bone marrow-derived mesenchymal stem cell, Br = bronchus, Bm = basement membrane, Bl = blood vessel, Eo = eosinophil, Ep = epithelium.***P < 0.001 compared to the CON group; ###P < 0.001 compared to the OVA+PBS group.

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