Molecular and Cellular Mechanisms Responsible for Beneficial Effects of Mesenchymal Stem Cell-Derived Product "Exo-d-MAPPS" in Attenuation of Chronic Airway Inflammation

Abstract

Mesenchymal stem cells (MSCs), due to their potential for differentiation into alveolar epithelial cells and their immunosuppressive characteristics, are considered a new therapeutic agent in cell-based therapy of inflammatory lung disorders, including chronic obstructive pulmonary disease (COPD). Since most of the MSC-mediated beneficent effects were the consequence of their paracrine action, herewith, we investigated the effects of a newly designed MSC-derived product "Exosome-derived Multiple Allogeneic Protein Paracrine Signaling (Exo-d-MAPPS)" in the attenuation of chronic airway inflammation by using an animal model of COPD (induced by chronic exposure to cigarette smoke (CS)) and clinical data obtained from Exo-d-MAPPS-treated COPD patients. Exo-d-MAPPS contains a high concentration of immunomodulatory factors which are capable of attenuating chronic airway inflammation, including soluble TNF receptors I and II, IL-1 receptor antagonist, and soluble receptor for advanced glycation end products. Accordingly, Exo-d-MAPPS significantly improved respiratory function, downregulated serum levels of inflammatory cytokines (TNF-α, IL-1β, IL-12, and IFN-γ), increased serum concentration of immunosuppressive IL-10, and attenuated chronic airway inflammation in CS-exposed mice. The cellular makeup of the lungs revealed that Exo-d-MAPPS treatment attenuated the production of inflammatory cytokines in lung-infiltrated macrophages, neutrophils, and natural killer and natural killer T cells and alleviated the antigen-presenting properties of lung-infiltrated macrophages and dendritic cells (DCs). Additionally, Exo-d-MAPPS promoted the expansion of immunosuppressive IL-10-producing alternatively activated macrophages, regulatory DCs, and CD4+FoxP3+T regulatory cells in inflamed lungs which resulted in the attenuation of chronic airway inflammation. In a similar manner, as it was observed in an animal model, Exo-d-MAPPS treatment significantly improved the pulmonary status and quality of life of COPD patients. Importantly, Exo-d-MAPPS was well tolerated since none of the 30 COPD patients reported any adverse effects after Exo-d-MAPPS administration. In summing up, we believe that Exo-d-MAPPS could be considered a potentially new therapeutic agent in the treatment of chronic inflammatory lung diseases whose efficacy should be further explored in large clinical trials.

Figure 1

Exo-d-MAPPS attenuated CS-induced airway inflammation in mice. Exo-d-MAPPS significantly improved pulmonary function in CS-exposed mice, as evidenced by blood gas analysis: significantly elevated PaO₂, SaO₂, and pH and decreased PaCO₂ (a–d). Representative H&E staining of the lungs (×100 magnification) obtained from the control ((e) A and (e) B) and experimental mice ((e) C and (e) D) showing partial alveolar wall destruction, widened alveolar septa and expanded alveolar space, and capillary dilation and congestion with massive infiltration of neutrophils, lymphocytes, and monocytes in the lungs of CS-exposed mice (e-C; black arrows) and alveolar and blood vessel structures with a lower number of lung-infiltrated leucocytes in the lungs of CS+Exo-d-MAPPS-treated animals (e-D). Significantly downregulated levels of proinflammatory cytokines (TNF-α, IL-12, IFN-γ, and IL-1β) (f) and increased concentration of anti-inflammatory IL-10 (g) in serum samples of CS+Exo-d-MAPPS-treated animals. Values are presented as mean ± SEM; n = 8 mice/group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 2

Exo-d-MAPPS treatment prevented the influx of inflammatory macrophages and induced the expansion of alternatively activated, IL-10-producing macrophages in the CS-injured lungs. Representative dot plots showing isotype controls (a). The total number of lung-infiltrated F4/80+ macrophages was significantly lower in CS+Exo-d-MAPPS-treated animals, as evidenced by representative flow cytometry plots (b). A significantly decreased number of CD80-expressing (c), I-A-expressing (d), and TNF-α- (e) and IL-12-producing (f) F4/80+macrophages were noticed in the lungs of CS+Exo-d-MAPPS-treated mice, as evidenced by representative flow cytometry plots. Representative dot plots showing a significantly increased number of alternatively activated, CD206-expressing, (g) and IL-10-producing F4/80+ macrophages (h) were observed in CS-exposed animals that received Exo-d-MAPPS. Values are presented as mean ± SEM; n = 8 mice/group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 3

Exo-d-MAPPS attenuated the capacity of NK and NKT cells and neutrophils to produce inflammatory cytokines in CS-injured lungs. Representative dot plots showing isotype controls (a). The number of IL-17-producing (b, c) and IFN-γ-producing NK and NKT cells (d, e) was significantly lower in the lungs of CS+Exo-d-MAPPS-treated mice compared to CS+vehicle-treated animals, as evidenced by representative dot plots. Representative dot plots showing isotype controls gated on neutrophils (f).Exo-d-MAPPS significantly reduced the influx of TNF-α and IL-1β-producing CD45+Gr-1+ neutrophils in CS-injured lungs (g, h). Values are presented as mean ± SEM; n = 8 mice/group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

Exo-d-MAPPS reduces the influx of inflammatory DCs and promotes the expansion of regulatory DCs in CS-injured lungs. Representative dot plots showing isotype controls (a). The total number of F4/80-CD11c+I-A+ DCs was significantly lower in the lungs of CS+Exo-d-MAPPS-treated animals, as evidenced by representative flow cytometry plots (b). A significantly decreased number of CD80-expressing (c) and IL-12-producing (d) inflammatory F4/80-CD11c+I-A+ DCs were observed in the lungs of CS+Exo-d-MAPPS-treated mice, as evidenced by representative flow cytometry plots. Exo-d-MAPPS treatment significantly increased the presence of regulatory, IL-10-producing F4/80-CD11c+I-A+ DCs in the lungs of CS-exposed animals, as evidenced by representative flow cytometry plots (e). Values are presented as mean ± SEM; n = 8 mice/group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 5

Exo-d-MAPPS significantly reduced the total number of inflammatory CD4+ and CD8+ T lymphocytes and increased the presence of immunosuppressive Tregs in inflamed lungs of CS-exposed animals. Representative dot plots showing isotype controls (a). A significantly lower number of CXCR3-expressing and IFN-γ-producing CD4+Th1 cells (b), IL-17-producing CD4+Th17 cells (c), CXCR3-expressing and IFN-γ-producing CD8+CTLs (d), and IL-17- (e) and TNF-α-producing CD8+CTLs (f) were noticed in the lungs of CS-treated mice that received Exo-d-MAPPS, as evidenced by representative dot plots. Exo-d-MAPPS treatment significantly increased the presence of FoxP3-expressing, IL-10-producing CD4+ Tregs in the lungs of CS-exposed animals, as evidenced by representative flow cytometry plots (g). Values are presented as mean ± SEM; n = 8 mice/group. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 6

Exo-d-MAPPS treatment significantly improved the pulmonary status of COPD patients. Exo-d-MAPPS contains a high concentration of immunosuppressive factors (sTNFRI, sTNFRII, IL-1Ra, and sRAGE) (a). All of the 30 Exo-d-MAPPS-treated COPD patients showed marked improvement in pulmonary status, as evidenced by an increase in percentage change relative to initial value of FEV1 (%ΔFEV1), significantly higher PEF, decreased CCQ total score, and increased 6-minute walking distance (6MWD). Values are presented as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 (b e). The representative CT images showing less hyperexpanded lung, less flattened diaphragm, and reduced centrilobular and paraseptal emphysema in a COPD patient, one month after Exo-d-MAPPS treatment (f, g).