While i n administered MSCs were clearly detected in

While i.n-administered MSCs were clearly detected in the lungs of normal mice, and to a greater extent, the inflamed lungs of mice with chronic AAD 48h later, previous studies in murine models of kidney disease (Huuskes et al., 2015; Togel et al., 2008) had shown that these d2 antagonist could not be detected by bioluminescence imaging 7days after administration. These studies suggested that most of the exogenously administered MSCs had vanished after a week, regardless of the route of administration applied; but that these cells were able to induce longer-term paracrine effects that persisted long after they had been cleared. Consistent with the latter, and previous studies showing that repeated (once weekly) administration of MSCs markedly improved their protective effects against kidney injury and related fibrosis (Lee et al., 2010), our findings demonstrated that once weekly administration of human MSCs were able to ameliorate the airway/lung fibrosis associated with chronic AAD by increasing collagen-degrading MMP-9 levels in the murine model studied; confirming that they were still capable of protecting the allergic lung from adverse AWR despite progressively diminishing in numbers post-administration. Airway inflammation occurs in response to respiratory damage, as the lung attempts to eliminate the original insult by recruiting inflammatory cells to remove cellular debris to restore lost tissue and function (Holgate, 2008). In this study, AI was morphometrically assessed by peri-bronchial inflammation score and was significantly up-regulated in response to OVA-mediated chronic AAD in mice, as reported previously (Royce et al., 2014; Royce et al., 2009). Although both intranasal administration of MSCs alone, which homed to and were retained in the inflamed lung (for at least 48h), or serelaxin alone induced a trend towards reduced inflammation score, the combination of the two treatments was able to significantly reduce AI, however, not fully back to levels measured in saline-treated controls. A possible explanation for these findings may be that either treatment alone only affected the infiltration of a sub-set of OVA-induced inflammatory cells into the lung, whereas the combined effects of both treatments were able to target a broader subset of inflammatory cells. For example, studies performed with intravenous (i.v) tail vein injection or intratracheal administration of bone marrow-derived MSCs in OVA-treated mice with chronic AAD demonstrated through BAL extraction and inflammatory cell counts, that MSCs were able to significantly reduce eosinophil and lymphocytes counts (Bonfield et al., 2010). On the other hand, studies have shown that RLN primarily targets neutrophil (Masini et al., 2004), mast cell and leukocyte infiltration (Bani, Ballati, Masini, Bigazzi, and Sacchi, 1997), but not eosinophil (Royce et al., 2014; Royce et al., 2009) or macrophage (Samuel et al., 2011) infiltration. However, it appeared that the combination treatment was not able to fully reverse OVA-induced AI, perhaps due to the fact that both treatments were not able to prevent the infiltration of all inflammatory cells including monocytes, which represented a large proportion of the inflammatory cells identified in the lungs of OVA-injured mice (Royce et al., 2014; Royce et al., 2009); although RLN has been found to prevent monocyte-endothelium contact (Brecht, Bartsch, Baumann, Stangl, and Dschietzig, 2011). Along with AI, AWR can occur as injury to the lungs is the culmination of a number of factors, including allergens or mechanical insult and possible genetic disorders destroying the architecture and function of the airways. In normal lungs, lung tissue turnover and airway restructuring is a homeostatic process which may aid in preserving optimal functions of the airway (Laurent, 1986). In asthma however, the lungs have the capacity to undergo endogenous remodeling of the airways in attempt to self-repair respiratory structure and function damaged by allergens or genetic causes; with aberrant healing leading to the progressive deposition of collagen, that eventually leads to airway fibrosis, airway obstruction and a positive feedback loop resulting in AHR (Cohn, Elias, and Chupp, 2004; Holgate, 2008). In this study, AWR was assessed via epithelial thickness and goblet cell metaplasia (measures of airway epithelial damage) and airway fibrosis. As observed, MSCs alone did not affect epithelial thickness, goblet cell metaplasia and had only modest effects in reducing aberrant sub-epithelial and total collagen deposition. This is somewhat consistent with the modest effects of adipose tissue-derived MSCs in suppressing the airway contractile tissue mass that was up-regulated in a house dust mite-induced model of AAD (Marinas-Pardo et al., 2014), where the effects of those cells were found to decline under sustained allergen challenge. Conversely, RLN alone had broader anti-remodeling effects and was able to significantly reduce epithelial thickness and aberrant sub-epithelial/total collagen deposition (Table 1). The combined effects of both treatments did not further reverse epithelial thickness (compared to the effects of serelaxin alone), but fully reversed the OVA-induced increase in sub-epithelial and total collagen deposition, to a greater extent than either therapy alone.