Canine Stem Cell-derived Exosomes for Lung Inflammation: Efficacy of Intratracheal Versus Intravenous Administration in an Acute Lung Injury Mouse Model

Abstract

Background/aim: Acute lung injury (ALI) is an important pathological process in acute respiratory distress syndrome; however, feasible and effective treatment strategies for ALI are limited. Recent studies have suggested that stem cell-derived exosomes can ameliorate ALI; however, there remains no consensus on the protocols used, including the route of administration. This study aimed to identify the appropriate route of administration of canine stem cell-derived exosomes (cSC-Exos) in ALI. Lipopolysaccharides were used to induce ALI.

Materials and methods: Mice with ALI were treated with cSC-Exos by intratracheal instillation or intravenous injection. The efficacy of the route of administration was confirmed by determining the total cell count in the bronchoalveolar lavage fluid and histopathological changes. The treatment mechanism was confirmed by measuring cytokine levels and immune cell changes in M2 macrophages (CD206+ cells) and regulatory T cells (FOXP+ cells).

Results: When cSC-Exos were injected, inflammation was alleviated, pro-inflammatory cytokine levels were reduced, and FOXP3+ and CD206+ cells were activated. Following intratracheal instillation, an enhanced inflammation-relieving response was observed.

Conclusion: This study compared the effects of stem cell-derived exosomes on alleviating lung inflammation according to injection routes in an ALI mouse model. It was confirmed that direct injection of exosomes into the airway had a greater ability to alleviate lung inflammation than intravenous injection by polarizing M2 macrophages and increasing regulatory T cells.

Keywords: Acute lung injury; dog; exosome; inflammation; stem cell.

Figure 1

Mesenchymal stem cell-derived exosome (Exo) administration resulted in a reduction in total cells present in bronchoalveolar lavage fluid (BALF) in a mouse model of lung injury induced by lipopolysaccharide (LPS). (A) Experimental setup. The mice were subjected to intratracheal (i.t.) administration of LPS, followed by treatment with either Exo or vehicle (Dulbecco’s phosphate-buffered saline) via i.t. and intravenous routes, 3 h post-LPS exposure. (B) BALF cell counts. Total cell count was determined 72 h after injury. (C) Counts of neutrophils, lymphocytes, and monocytes in BALF were assessed 72 h after injury. n=10-15 mice per group. Results are presented as means±standard deviations.

Figure 2

The efficacy of exosomes in mitigating pathological lung effects induced by lipopolysaccharide (LPS) administration varies depending on the route of administration. (A) Lung sections were stained with hematoxylin and eosin and histologically examined 72 h after LPS instillation. The representative sections are shown at ×10 and ×40 original magnification. The phosphate-buffered saline group showed normal lung tissue. (B) Lung injury scores, including hyperemia and congestion, edema, neutrophil migration, tissue infiltration, and histological damage scores, were assessed. n=10-15 mice per group. *p<0.05, **p<0.01, ***p<0.001; ns: not significant. Results are presented as means±standard deviations.

Figure 3

Changes in inflammatory cytokine expression according to exosome administration route. n=10-15 mice per group. ***p<0.001; ns: not significant. Results are presented as means±standard deviations.

Figure 4

Changes in M2 macrophages and regulatory T cells (Tregs) after cST-Exo treatment according to route of administration. (A) The numbers of CD206+ M2 and (B) FOXP+Treg cells in the lungs were determined by immunofluorescence. n=10-15 mice per group. **p<0.01, ***p<0.001; ns: not significant. Results are presented as means±standard deviations.