In the present study, both AD-MSCs and EVs similarly decreased collagen fiber deposition in the lung parenchyma and air passage, TGF-β levels in lung tissue, total cell matters, CD4

+

CD25

+

Foxp3

+

(Treg cells) and eosinophil percentage in BALF, IL-5 levels within lung tissue, and percentage of CD3

+

CD4

+

T cells in the thymus. In contrast, systemic administration of AD-MSCs or EVs experienced different effects on eosinophil cell counts, levels of IL-4, IL-13, and eotaxin lung tissue and CD3

+

CD4

+

T tissue in BALF, and lung mechanics (Table 

1

).

Table 1

Comparison between human adipose-derived mesenchymal stromal cells (AD-MSC) with extracellular vesicles (EV) within OVA-induced allergic asthma model

Decrease of static lung elastance

Reduction of collagen fiber content material in lung parenchyma and airway

Decrease of eosinophils in lung parenchyma

Reduction of Capital t lymphocytes in BALF

Increase of Treg cells in BALF

Reduction of eosinophils within BALF

Reduction of TGF-β in lung tissue

Reduction of eotaxin lung tissue

Reduction of IL-5 in lung tissue

Reduction of IL-4 in lung tissue

Reduction of IL-13 in lung tissue

The model of sensitive asthma used herein was developed by our team [ 15 ] and has been previously demonstrated in order to induce changes in lung mechanics, inflammation, and respiratory tract remodeling (mucous-cell hyperplasia, presence of myofibroblasts, subepithelial fibrosis and thickening of the basement membrane, smooth muscle cellular hypertrophy, and deposition of collagen fibers in the air passage and lung parenchyma) [ 15 , 16 ]. Female C57BL/6 rodents were used as they mount a good allergic response to ovalbumin [ 15 , 16 , 26 ] and are more susceptible to experimental asthma than males [ 26 ].

AD-MSCs are easily obtained in large numbers, existing high proliferation rates compared to bone marrow-MSCs [ 27 ], and have shown beneficial effects when administered systemically prior to sensitization or during challenge in OVA-induced asthma versions [ 6 9 ]. However , to date, no study had examined the therapeutic administration of human AD-MSCs and their particular EVs in experimental allergic asthma, when airway redesigning is already established. Both AD-MSCs and EVs were given intravenously, as a previous study found no differences in results after intratracheal versus we ntravenous administration of bone marrow-derived MSCs within experimental asthma [ 15 ]. Additionally , recent studies have proven that a systemic dose of 10 5 BM-MSC per mouse, similar to that used in this particular study, is effective in experimental asthma [ 15 , 28 ]. Furthermore dose of AD-MSCs, we used an equivalent dosage of EVs secreted by 10 5   AD-MSCs, since recent studies reported that human being MSC-derived EVs were as effective as their parent originate cells in mitigating lung inflammation in experimental lung injury [ 11 , 29 , 30 ]. Based on our previous studies, each AD-MSCs and EVs were administered 24  hours following the last challenge, when airway inflammation and remodeling procedures are established and lung function is already compromised [ 23 , 24 ], thus resembling human asthma.

We observed that EVs (but not human AD-MSCs) reduced static lung elastance, in comparison with other studies that reported decreased lung mechanical adjustments after administration of human AD-MSCs [ 6 , 8 , 10 ]. These types of differences could be attributed to several factors: (1) the number of AD-MSCs administered, (2) the timing of AD-MSCs systemic management (before or after allergic asthma induction), (3) the particular severity of asthma, and (4) the method used to calculate lung mechanical parameters. In the present study, in contrast to the books, human AD-MSCs were administered both in lower numbers after airway remodeling was already established. Moreover, lung mechanics had been measured by end-inflation method, but not by flexiVent or maybe the Penh system.

The model of OVA-induced allergic asthma used in this research is characterized by T helper 2 cell release associated with cytokines such as IL-4, IL-5, and IL-13, as well as the eosinophil chemoattractant eotaxin, which contribute to airway inflammation in asthma [ 23 , 24 ] Among other functions, these cytokines induce fibroblast proliferation, extracellular matrix deposition, airway hyperresponsiveness, epithelial cell apoptosis, mucus production, and eosinophil recruitment [ 31 ]. Therefore , they play important roles not just in the inflammatory process, but also in airway remodeling, and are also thus considered important therapeutic targets [ 32 , 33 ]. AD-MSCs decreased IL-5, IL-13, and eotaxin levels in lung tissue, perhaps by decreasing the number of eosinophils in BALF [ 33 , 34 ]. TGF-β also plays an important role within tissue remodeling. TGF-β is produced by epithelial cells, fibroblasts, eosinophils, and macrophages, and stimulates the production of collagen I and III, fibronectin, proteoglycans (fibroblasts) [ 35 37 ]. Both EVs and AD-MSCs similarly reduced collagen dietary fiber content and TGF-β levels. Our results suggest that AD-MSCs and EVs seem to modulate inflammation through different systems, but had similar effects on the remodeling process.

IFN-γ produced mainly by Th1 cells can counterbalance the allergic inflamed response mediated by Th2 cells, and it is present in BALF of asthmatic humans and mice [ 38 ]. IL-10 can be released by many cell types, such as Th2 tissue, Treg cells, mast cells, eosinophils, and macrophages [ 39 ]; in this study, no significant changes were noticed between groups despite the increased number of eosinophils and Treg cells in BALF. IFN-γ and IL-10 levels failed to increase after ovalbumin-induced allergic asthma in accordance with previous research [ 25 , 40 ]. The absence of changes in IFN-γ plus IL-10 may be related to the timing of analysis (7  days after the last challenge), since such modifications happen to be described during asthma resolution [ 41 ].

Human bone marrow-derived MSCs injected in mice migrate to the thymus, inhibiting growth of naive lymphocytes into CD3 + CD4 + T cells [ 42 , 43 ]. In our studies, no changes were observed in the particular CD3 + CD4 + T cell populations in mediastinal lymph client tissue after induction of asthma with ovalbumin. Each AD-MSCs and EVs decreased the number of CD3 + CD4 + T cellular material in the thymus, but only systemic administration of EVs caused a reduction of CD3 + CD4 + T cells within BALF. Both AD-MSCs and EVs reduced CD3 + CD4 + To cells in the thymus, but only EVs inhibited deposition of CD3 + CD4 + T cells in the lung. One system of MSC action may be through induction of Treg cells [ 44 ]. MSCs derived from mouse adipose cells induce Treg cells in an ovalbumin-induced asthma model [ 6 ]; however , Luz-Crawford et al. [ 45 ] demonstrated that MSCs derived from mouse bone marrow regulate Treg cell levels only when the MSCs are co-cultured with lymphocytes before the differentiation process, not when the lymphocytes are already differentiated. In experimental asthma, after sensitization plus challenge with Aspergillus hyphae, Treg cells were increased in the spleen organ and not modified after systemic administration of MSCs [ 46 ].

Likewise, there were no differences in Treg cell counts in the mediastinal lymph nodes across the groups analyzed in the present study. Treg cell counts in BALF increased in OVA pets compared to control animals, which may be associated to the timing associated with analysis, i. e., late in the course of asthma, when each inflammation and remodeling are clearly established. Systemic management of AD-MSCs and EVs reduced levels of Treg cellular material in BALF.

The current study has some limitations that should be addressed. First, nor AD-MSCs nor EVs were tracked after systemic management, limiting our knowledge concerning their delivery and homing. Second, only one dose of human AD-MSCs and EVs was evaluated; thus, we cannot rule out that increased doses may result in greater beneficial effects. Certainly, additional studies performing dose-response curves of AD-MSCs and EVs are required. In this model of allergic asthma, airway inflammation plus remodeling persists for some time after the challenges [ 17 ], which might allow ascertainment of whether the beneficial effects observed stay. This experimental study is a first step toward various other investigations to elucidate the mechanisms and time span of therapy with AD-MSCs and EVs in asthma. Lastly, a specific set of cytokines and growth factors was examined in this experiment; a wider range of mediators should be examined to further elucidate the mechanisms of action of AD-MSCs and EVs.