Intratracheal Administration of Mesenchymal Stem Cells Modulates Tachykinin System, Suppresses Airway Remodeling and Reduces Airway Hyperresponsiveness in an Animal Model

Abstract

Background: The need for new options for chronic lung diseases promotes the research on stem cells for lung repair. Bone marrow-derived mesenchymal stem cells (MSCs) can modulate lung inflammation, but the data on cellular processes involved in early airway remodeling and the potential involvement of neuropeptides are scarce.

Objectives: To elucidate the mechanisms by which local administration of MSCs interferes with pathophysiological features of airway hyperresponsiveness in an animal model.

Methods: GFP-tagged mouse MSCs were intratracheally delivered in the ovalbumin mouse model with subsequent functional tests, the analysis of cytokine levels, neuropeptide expression and histological evaluation of MSCs fate and airway pathology. Additionally, MSCs were exposed to pro-inflammatory factors in vitro.

Results: Functional improvement was observed after MSC administration. Although MSCs did not adopt lung cell phenotypes, cell therapy positively affected airway remodeling reducing the hyperplastic phase of the gain in bronchial smooth muscle mass, decreasing the proliferation of epithelium in which mucus metaplasia was also lowered. Decrease of interleukin-4, interleukin-5, interleukin-13 and increase of interleukin-10 in bronchoalveolar lavage was also observed. Exposed to pro-inflammatory cytokines, MSCs upregulated indoleamine 2,3-dioxygenase. Moreover, asthma-related in vivo upregulation of pro-inflammatory neurokinin 1 and neurokinin 2 receptors was counteracted by MSCs that also determined a partial restoration of VIP, a neuropeptide with anti-inflammatory properties.

Conclusion: Intratracheally administered MSCs positively modulate airway remodeling, reduce inflammation and improve function, demonstrating their ability to promote tissue homeostasis in the course of experimental allergic asthma. Because of a limited tissue retention, the functional impact of MSCs may be attributed to their immunomodulatory response combined with the interference of neuropeptide system activation and tissue remodeling.

Fig 1. Experimental Design.

Scheme of in vivo experiments.

Fig 2. MSCs are retained within the lungs and improve function in vivo.

(A) FACS analysis of MSCs for mesenchymal, hematopoietic and endothelial markers. (B) Phase contrast and GFP fluorescence image of living MSCs after lentiviral transduction. (C) Airway reactivity to ACh as change in resistance (RL) in control, asthmatic (OVA) and MSC-treated (OVA+MSCs) asthmatic mice. (D) Detection of GFP gene by PCR in the lungs of MSC-treated mice. (E) GFP-positive cells (green, arrows) in MSCs-treated mice. (F) GFP-positive MSCs (green, arrows and inset) lack the expression of epithelial markers CK and TTF1 (red). (G) Airway reactivity to acetylcholine as change in resistance in control mice and control mice after instillation of MSCs. Scale bars 20 μm. *P<0.05 vs control; **P<0.05 vs OVA. Four to eight mice were used for airway function assessment. OVA: ovalbumin; ACh: acetylcholine; CK: pan-cytokeratin.

Fig 3. MSCs positively interfere with airway remodeling.

(A) Proliferating (Ki67, green, arrows) epithelial cells (CK, red) in vehicle-treated and MSC-treated animals. (B) The fraction of Ki67-positive epithelial cells. (C) Cycling (Ki67, green, arrows) SMCs (α-SMA, red) in the airway wall. (D) The fraction of Ki67-positive SMCs. (E) Airway smooth muscle mass in OVA and OVA+MSC mice. (F) Quantification of airway smooth muscle mass. (G) Mucin-positive cells (green) in epithelium of vehicle-treated and MSC-treated asthmatic mice. (H) The percentage of epithelial cells expressing mucin. (I) Acid mucopolysaccharides accumulation in OVA and OVA+MSCs groups. (J) Human colon as positive control for mucicarmine staining. Scale bars (A and C) 10 μm, (E and G) 50 μm, (I and J) 20 μm. Five to seven animals were used for airway remodeling data. CK: pan-cytokeratin; α-SMA: α-smooth muscle actin.

Fig 4. MSCs decrease lung inflammation.

(A) With respect to controls, massive accumulation of inflammatory cells visible in the lungs of OVA animals was reduced in MSC-treated asthmatic mice. (B) Detection of CD45 and CD3 positive cells (red) within the inflammatory milieu. (C) Mast cells (toluidine blue, arrows) in the lungs of vehicle- and MSC-treated asthmatic mice. (D) The number of mast cells per mm² of tissue. (E,F) Total cell number and differential cell count in the BALF collected from control, OVA and OVA+MSCs animals. (G) Cytokines levels measured in the BALF. Scale bars (A) 50 μm, (C) 20 μm. *P<0.05 vs control; #P<0.05 vs OVA. Four to ten animals were used for mast cell count. BALF from three to ten mice were used for cell count and cytokine assay. BALF: bronchoalveolar lavage fluid; mac: macrophages; lymph: lymphocytes; PMN: polymorphonuclear leukocytes; eos: eosinophiles.

Fig 5. Immunomodulatory properties of MSCs.

(A-C) Expression of mRNA for TGF-β, IL-10 and IDO measured by real-time RT-PCR in control (C, white bars) and MSCs (yellow bars) stimulated with IFNγ and TNFα after 3, 6, 12 and 24 h. *P<0.05 vs control cells. Expression of mRNA for (D-G) NK1-R, NK2-R, VIP and CGRP measured by real-time RT-PCR in control, OVA and OVA+MSCs groups. Three sets of MSCs were used for in vitro experiments. Four to seven animals were used for PCR analysis of neuropepetides. TGF-β: transforming growth factor-β; IDO: indoleamine 2,3-dioxygenase; CGRP: calcitonin gene-related peptide; VIP: vasoactive intestinal peptide; NK1-R; neurokinin 1 receptor; NK2-R: neurokinin 2 receptor.