MSC SLP attenuated mortality and weight loss induced by BLM
Intratracheal BLM has been widely used to induce ALI within 1 week in mice (3). In this study, each mouse received 2.5 mg/kg BLM intratracheally. As shown in Fig. 1a, the mice were intratracheally administered PBS, BLM, or MSC SLP plus BLM on day 0, corresponding to the control, BLM, and MSC SLP groups, respectively. Peripheral blood, BALFs, and lung tissues were collected on day 7.
The survival status of the mice in each group was recorded daily, which showed that BLM led to higher mortality (approximately 20%) on day 7, whereas co-treatment with MSC SLP significantly reduced the mortality (0%) on day 7 (Fig. 1b). Furthermore, the mouse body weights decreased dramatically after BLM instillation, which was significantly reversed by MSC SLP (Fig. 1c). No visible weight loss was found in control group. These results demonstrated the contribution of MSC SLP to survival and weight maintenance in the mice, suggesting its potential for conferring a protective effect against ALI.
MSC SLP attenuated BLM-induced alveolar injury
H&E staining demonstrated extensive morphological damage in BLM-instilled lungs, such as hemorrhaging, congestion, thickening of the alveolar walls, transparent membrane formation, and infiltration of inflammatory cells, especially neutrophils (Fig. 1d). In contrast, assessment of lung pathology revealed markedly more intact alveolar walls and decreased inflammation after MSC SLP treatment (Fig. 1d). Additionally, the lung-injury score (a relatively quantitative indicator) of MSC SLP-instilled mice was distinctly lower than that in BLM-instilled mice (Fig. 1e). BLM-induced alveolar septum thickening, alveolar-space broadening, and alveolar wall destruction were greatly counteracted by MSC SLP (Fig. 1f–h). No histological defects were visualized in the PBS-instilled lungs (Fig. 1d–h). The above results indicated that MSC SLP played a significant role in protecting alveoli from BLM-induced damage.
MSC SLP inhibited apoptosis induced by BLM
TUNEL assays were performed to estimate the number of apoptotic cells containing DNA fragments during the late stages of apoptosis (35). To clarify the effect of MSC SLP on apoptosis, we determined the degree of apoptosis in the lungs by TUNEL staining. TUNEL-positive epithelial cells dramatically increased in BLM-instilled lungs and remarkably restricted by MSC SLP (Fig. 2a). MSC SLP was competent in inhibiting apoptosis, which was possibly attributable to anti-apoptotic cytokines released by MSCs.
MSC SLP alleviated inflammatory infiltration induced by BLM
To determine the effect of MSC SLP on inflammatory infiltration, we assessed total protein levels, total cell numbers, and the profiles of inflammatory cells in BALFs. BLM instillation led to protein accumulation in BALFs and was remarkably blocked by MSC SLP (Fig. 2b). Moreover, BLM instillation significantly induced inflammatory cell infiltration, especially neutrophils, whereas MSC SLP significantly prevented lung inflammation (Fig. 2c, d). No statistically significant difference in inflammatory cell infiltration was found between the control and MSC SLP groups (Fig. 2c, d). To confirm this observation, the activity of MPO (a marker of neutrophilic aggregation) was evaluated in lung tissues to assess the level of neutrophils. BLM instillation caused a remarkable increased in the MPO levels, which was inhibited by MSC SLP (Fig. 2e).
MSC SLP modulated the imbalance of Treg and Th17 cells
Data from numerous studies have demonstrated that Treg and Th17 cells are associated with ALI and ARDS, in both humans and mice (36–38). Accordingly, we analyzed the balance of CD4+CD25+Foxp3+ Treg cells and Th17 cells on day 7 by flow cytometry. BLM administration led to a reduction of CD4+ T cells and an increased percentage of CD4+CD25+Foxp3+ Treg cells (Fig. 2f, g), whereas MSC SLP stimulated CD4+ T cell differentiation and inhibited the expansion of Treg cells in lung tissues (Fig. 2f, g). Moreover, the number of Th17 cells in the blood was significantly attenuated after MSC SLP treatment (Fig. 2h).
MSC SLP depressed IL-6 secretion induced by BLM
ALI is an acute and inflammatory disorder involving the release of numerous cytokines. Thus, multiplex cytokine-detection technology was applied. Among all cytokines detected in the plasma, the IL-6 concentration was elevated by BLM and efficiently mitigated by MSC SLP (Fig. 3a). To verify these findings, the levels of IL-6 in plasma and BALFs were also measured by performing ELISAs. MSC SLP markedly neutralized IL-6 induction by BLM in both plasma and BALFs (Fig. 3b, c). ELISA analysis also showed that MSC SLP attenuated increased IL-1β production in BALFs (Fig. 3d). MSC SLP tended to lower the levels of IL-10, IL-13, and IL-23 in the plasma, although these differences were not statistically significant (Fig. 3a).
MSC SLP activated p63 by inhibiting IL-6–p-STAT3 signaling
p63 has been shown to support self-renewal, inhibit cell apoptosis, and help maintain homeostasis in epithelia (21, 23–25). In this study, RT-qPCR and western blot analysis revealed that p63 expression dramatically decreased in BLM-instilled mice and that MSC SLP restored p63 expression (Fig. 4a, b). In agreement, IHC showed that p63 was highly expressed in the basal layers of the airways, but was not expressed in the muscle layer of airways. p63 expression decreased in BLM-instilled mice, but partially recovered after MSC SLP treatment (Fig. 4c). Furthermore, immunofluorescence staining also showed that p63 expression was re-activated by MSC SLP (Fig. 5a–c). α-SMA, a marker of airway smooth muscle cells in the lungs, did not co-localize with p63 (Fig. 5a, e).
Ki-67 (a proliferation marker) markedly co-localized with p63, especially in MSC SLP-instilled lungs (Fig. 5c–e), indicating that a considerable portion of p63+ cells was actively proliferating and repairing damage after MSC SLP instillation. Intensity profiles showed various degrees of co-localization between p63 and Ki-67 (Fig. 5d). To quantify the degree of co-localization between the fluorophores, Pearson’s correlation coefficient (PCC) and Mander’s overlap coefficient (MOC) (39) values were determined. Quantitative analysis verified that the p63 protein did not co-localize with α-SMA in the lungs, but was highly co-localized with Ki-67 (Fig. 5e).
Previous data showed that IL-6 regulates STAT3 signaling (40). Therefore, we subsequently detected STAT3 and activated STAT3 (p-STAT3) by western blotting. STAT3 was evidently activated to p-STAT3 in BLM-induced ALI (Fig. 6a, b). In contrast, MSC SLP significantly inhibited STAT3 phosphorylation (Fig. 6a, b). Considering that IL-6 appeared to promote STAT3 phosphorylation in basal cells of the airways and previous data showed that the IL-6–p-STAT3 pathway regulated p63 isoform expression in keratinocytes (40, 41), we hypothesized that a sharp rise of IL-6 boosted STAT3 phosphorylation and then restrained p63 expression in BLM-induced ALI. Our results suggested that MSC SLP activated p63 by inhibiting the IL-6–p-STAT3 pathway.
Intratracheal rh IL-6 reduced p63 expression
To clarify the role of IL-6, we instilled rh IL-6 into the airways of mice. ELISAs demonstrated that rh IL-6 was enriched in the lung tissues on day 1, after which it had been absorbed and removed on day 2 after rh IL-6 instillation (Fig. 6c). Consistently, rh IL-6 administration promoted STAT3 phosphorylation and decreased p63 expression (Fig. 6d–f). In the presence of sufficient exogenous IL-6, MSC SLP no longer re-activated p63 expression after rh IL-6 instillation. These results suggested that MSC SLP alleviated ALI by inhibiting inflammatory cell recruitment and reducing IL-6 production, without blocking IL-6 function.
MSC SLP increased JAG2 expression
To clarify the mechanism whereby MSC SLP alleviated ALI, we tested various target genes of p63, especially those linked to basal cell function. In this study, the mRNA and protein levels of JAG2 were both significantly down-regulated by BLM (Fig. 7a–c), whereas MSC SLP greatly increased JAG2 expression (Fig. 7a–c). Administration of rh IL-6 reduced JAG2 expression, which was not reversed by MSC SLP (Fig. 7d, e). A schematic model whereby MSC SLP alleviated BLM-induced ALI is proposed in Fig. 7 f.
MSC SLP regulated the lipid profile in plasma
To obtain an overview of the alterations in the plasma lipid profiles induced by BLM and MSC SLP, we determined the levels of 373 individual lipid species, including phosphoglycerolipids, sphingolipids, neutral lipids, and other lipids (Fig. 8a). Principal component analysis revealed apparent separations between the control, BLM-instilled, and MSC SLP-instilled mice on day 7 (Fig. 8b). A heatmap was generated, which demonstrated the significant differences found in the three groups (Fig. 8c). Each row and column shows individual lipids in each sample type. We performed relative quantifications of the major lipid classes (Fig. 8d). PC species were the most prominent lipids in the plasma, followed by lysoPC. The overall content of phosphatidylserine (PS) was significantly reduced by BLM and greatly increased by MSC SLP (Fig. 8d). Among these, the levels of PS 20:3/22:6 and PS 38:3 changed most dramatically. MSC SLP treatment greatly increased the levels of PS 20:3/22:6 and PS 38:3 reduced by BLM (Fig. 8e). Moreover, among other lipid subclasses, 12 lipids were altered considerably (Fig. 8f).