Regenerative medicine is a new therapeutic approach based on the use of stem cells and their derivatives (e.g. conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and to improve their function, including lung diseases [15].

Several studies have demonstrated that both mouse and human embryonic stem cells can be induced in culture to acquire phenotypic markers of type 2 alveolar epithelial cells, including expression of surfactant proteins and lamellar bodies, and formation of pseudoglandular structures. However, there are only limited available studies concerning the effects of embryonic stem cells, or of their derivatives, on in vivo lung pathologies [4, 5].

In parallel, other studies speculated that adult bone marrow-derived or cord blood-derived stem cells could engraft and differentiate in mature airway and alveolar epithelial cells, vascular endothelial cells, or interstitial lung cells. Interestingly, several recent reports suggested that both systemic and intrapulmonary administration of bone marrow-derived mature stem cells in mice models of acute lung injury resulted in decreased mortality, improved alveolar fluid clearance, and attenuated inflammation and lung injury, despite minimal, if any, stem cell engraftment in the lung [1]. To explain the action of mesenchymal stem cells (MSCs), several paracrine mechanisms have been proposed for bone marrow-derived stem cell effects, including release of anti-inflammatory mediators such as interleukin (IL)-10, angiopoietin-1, and keratinocyte growth factor (KGF) that could modulate immune responses.

Researchers are considering other sources of stem cells for airway tissue engineering. Extra-fetal-derived stem cells could represent new alternative sources for lung tissue regeneration. Indeed, recently, amniotic mesenchymal cells (AMCs) isolated from human and horse term placenta showed many characteristics of stem cells including a very low antigenicity, no tumorigenic effects, and the potential to differentiate into mesodermal and ectodermal lines [610]. Moreover, the therapeutic use of both equine AMCs and CM in tendon and ligament injuries in vivo showed that the regenerative property of stem cells is probably due either to their differentiation capacity or to production of mediators capable of activating the intrinsic reparative processes of damaged tissues [11]. In addition to soluble factors present in CM, recent studies showed that stem cells could communicate with target cells through paracrine mechanism by means of MVs [12, 13].

Among MVs, two different populations could be identified: exosomes, which originate from invaginations of the endosome membranes and which have a diameter between 30 and 120 nm, and the shedding vesicles that are more heterogeneous in size (80 nm to 1 micron) and arise directly by protrusions of the cell membrane. MVs express different surface markers, including binding receptors to the target cell surface. When such link occurs, the microvesicle membrane merges with that of the target cell releasing its content into the cell cytoplasm [1416]. It was demonstrated that MVs could be employed in vitro and in vivo for tissue repair, increasing the degree of healing [1719]. In line with these findings, new studies proposed the use of CM or purified MVs for regenerative treatment of damaged tissues in place of stem cells.

Among equine respiratory disorders, recurrent airway obstruction (RAO), due to its clinical aspects, is similar to some kinds of human asthma suggesting a common immunological basis [20]. RAO, reported as a debilitating and incurable respiratory disease affecting stabled mature horses worldwide, shares many features with human asthma, including lower airway inflammation, reversible airflow obstruction, bronchial hyper-responsiveness, mucus accumulation, and remodeling, resulting in severe clinical signs [21, 22]. Common clinical signs include coughing, respiratory distress, and increased breathing effort during periods of exacerbation, which are triggered by hypersensitivity reactions to allergens and irritants mainly from hay dust. Moreover, RAO and human severe asthma share many structural changes such as epithelial detachment and regeneration, goblet cell hyperplasia, and hyperplasia of the bronchial smooth muscles, probably related to neutrophils activity [2022]. While the immunogenetic background of RAO is still not completely understood, several studies have shown that interactions of innate and adaptive immune responses play an important role [21]. Finally, an increased number of cells expressing mRNA for IL-4 and IL-5 and a decreased expression of IFN-γ was demonstrated in bronchoalveolar lavage fluid (BALf) of RAO affected horses [23]. Some studies showed contradictory results regarding the involvement of cytokines characteristic for the Th1 or Th2 type of immune response and it has been suggested that cytokine profiles reflecting both types of Th responses are observed at different time points after antigen challenge, similar to that demonstrated in human asthma.

RAO develops over a period of years, and offers a unique model to study the respiratory system under chronic inflammatory conditions [22]. To date, similar to asthma, RAO can be controlled by the administration of corticosteroids and bronchodilators.

The aim of the present study was to evaluate the in vitro effect of AMC derivatives (CM and MVs) on the production of pro- and anti-inflammatory cytokines (tumor necrosis factor (TNF)-α, IL-6, and transforming growth factor (TGF)-β) in equine alveolar macrophages (AMs) collected by means of bronchoalveolar lavage (BAL), following stimulation with lipopolysaccharide (LPS). LPS was used according to Gupta et al. [1] and Mei et al. [2], who demonstrated its capability for inducing lung inflammation both in vivo and in vitro [3, 24].

The study was approved by the University of Milan Ethics Committee (Protocol Number 41/15) and informed client consent was obtained for inclusion in the study.