The Anti-asthmatic Effect of Intratracheally Treated Mesenchymal Stem Cells via Modulation of Lung Macrophage Activation

stem cells (MSCs) possess immunomodulatory properties that provide therapeutic potential for the treatment of inammatory diseases. While the therapeutic and clinical effects of MSCs are partially known, the effects of its administration to the airway in asthma, a chronic airway inammatory disease, remain unclear. The effects of cell through using


Measurement of airway hypersresponsivemenss and in ammation
On day 24, AHR was measured using a Buxco ® FinePointe system (FinePointe RC System, Buxco; Wilmington, NC). Mice were anesthetized with pentobarbital sodium (50 mg/kg), and lung resistance (R L ) was measured for 3 min. The measured R L values were subtracted from the baseline values and converted to a percentage.
On the same day, bronchoalveolar (BAL) uid was obtained, and lung excision was performed. BAL cell slides were stained with Diff-Quik (Systmex; Kobe, Japan) and more than 300 cells were counted in each sample to determine the number of macrophages, neutrophils, eosinophils, and lymphocytes.
To con rm the pathological changes and characteristics of the lung parenchyma, the left lung of the mouse was xed in 2% paraformaldehyde, embedded in para n, cut into 4 sections, and stained with hematoxylin and eosin.

Statistical analysis
All data are expressed as means ± standard error of the mean (SEM). To analyze the correlation analysis between two groups, Spearman's rank-order correlation test was performed. Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software; San Diego, CA). The Mann-Whitney test was used to compare the two groups and p-values less than 0.05 were considered statistically signi cant.

Results
Reduction of airway hyperresponsiveness and in ammation by ucMSC treatment in a murine asthma model To test the effect of ucMSCs on asthma, an OVA-induced asthma model was established, followed by intratracheal ucMSC treatment. ucMSC treatment signi cantly decreased AHR and airway in ammation enhanced by OVA challenge (Fig. 1A, B). The macrophage and eosinophil in ltration found in the OVA group around the bronchi and the blood vessels was strikingly reduced in the histological analysis of the OVA+ucMSC group (Fig. 1C). In addition, the increase in eosinophils in the lungs seen in the OVA group was signi cantly decreased in the OVA+ucMSC group, whereas the number of neutrophils did not change in the OVA+ucMSC group in ow cytometry analysis (Fig. S5).
Reduction of Th2 and ILC2 by ucMSC treatment in a murine asthma model The number of total CD4 + T cells and IL-5/IL-13-secreting CD4 + T cells was signi cantly increased in the OVA group. Intratracheal ucMSC treatment signi cantly decreased the number of IL-5/IL-13-secreting CD4 + T cells ( Fig. 2A). On the other hand, no signi cant change was observed in IFN-γ-or IL-17A-secreting CD4 + T cells or Treg cells following ucMSC treatment ( Fig. 2A).
The total number of innate lymphoid cells (ILCs) and IL-5/IL-13-secreting type 2 ILCs (ILC2s) signi cantly increased in the OVA group, and ucMSC treatment signi cantly reduced total ILCs and ILC2s. Similar to Th1 and Th17 cells, IFN-γ-secreting ILC1s and IL-17A-secreting ILC3s increased in the OVA-induced group but were not changed by intratracheal ucMSC treatment (Fig. 2B).
Changes in dendritic cells and macrophages by ucMSC treatment in a murine asthma model OVA challenge increased the number of DCs in the lung, especially those with enhanced expression of MHCII and CD86. Although ucMSC treatment did not reduce the total number of DCs, mature DCs with antigen-presenting properties (MHCII + CD86 + DCs) diminished signi cantly by ucMSC treatment. In contrast, immature DCs without MHCII and CD86 expression were reduced in the OVA group and were partially recovered by ucMSC treatment (Fig. 3A). Similarly, the increase in MHCII + CD86 + AMs in the OVA group was also reduced by treatment with ucMSC, whereas the diminished immature MHCII -CD86 -AMs in the OVA group were restored to normal levels by intratracheal treatment with ucMSCs ( Fig. 3B).
OVA challenge increased both M1 and M2 macrophages, which were decreased by ucMSC treatment. Of the two subsets, M2 macrophages were more effectively reduced by ucMSCs, considering that the M2/M1 ratio signi cantly decreased in the OVA+ucMSC group (Fig. 3C).
Among M2 subtypes, ucMSC treatment signi cantly reduced M2a and M2c populations, which showed very strong positive correlations with type II immune cells such as IL-5-or IL-13-secreting CD4 + T cells, IL-5-or IL-13-secreting ILC2s, and eosinophils ( Fig 3E and S6). However, the M2b population, which also showed a signi cant increase in the OVA group, did not change upon ucMSC treatment (Fig. 3D).
SiglecF expression was very high in CD11c + CD11bmacrophages (Fig. 4A), and this population was regarded as resident AMs. These resident AMs were signi cantly reduced in OVA-treated groups, and they were restored by ucMSC treatment (Fig. 4B). SiglecF + CD11c + CD11bmacrophages showed strong negative correlations with type 2 immune cells (Fig. 4C).

Changes in mRNA expression by ucMSC treatment in in vivo, ex vivo, and in vitro experiments
Analysis of mRNA expression levels in mouse lung homogenates con rmed that ucMSC treatment downregulated the expression levels of both M1 and M2 markers upregulated by OVA challenge. Enhanced expression of M2 markers (Arg1 and Retnlα) and Th2 cytokines (Il-13 and Il-5) in the OVA group were signi cantly reduced by ucMSC treatment (Fig. 5A). Similarly, ucMSCs also downregulated CD86, Il-12, and Tnfα, classi ed as M1 markers, upregulated by OVA challenge (Fig. 5B).
In order to directly specify the effect of ucMSCs on macrophages, BAL uid macrophages were collected and stimulated with IL-4 ex vivo to induce M2 macrophage polarization in the presence and absence of ucMSC treatment. mRNA expression of M1 markers (Il-12 and Tnfα), M2 markers (Cd206 and Arg1), and Th2 cytokines (Il-5, IL-13, and Tgfb1) were enhanced by IL-4 treatment and normalized by ucMSC treatment (Figs. 6A, 6B).
In addition, to determine whether ucMSCs directly or indirectly affect macrophages, the CRL-2019 AM cell line stimulated with IL-4 was treated with ucMSCs directly or indirectly via transwells, which suppressed Th2 cytokines in both groups (Fig. 6C).

Discussion
In this study, treating OVA-induced murine asthma models with ucMSCs resulted in anti-asthmatic effects by reducing type II airway in ammation and AHR. Particularly, ucMSC treatment decreased DCs and macrophages with antigen-presenting capacity. It also promoted the recovery of resident AMs, which contributed to the suppression of in ammation and maintenance of homeostasis in the lung.
Additionally, ucMSCs affected macrophages even without direct cell-to-cell contact, suggesting that soluble factors mediate the effect of ucMSCs.
Stem cells are undifferentiated cells that self-renew via cell division and multiply into differentiated cells specialized for tissues and organs depending on the environment. MSCs are adult stem cells derived from several sources, such as the placenta, umbilical cord, and adipose tissue, and they can differentiate into various lineages [19][20][21][22]. A recent study con rmed that transplanted MSCs retain their immunoregulatory properties even in allogenic treatment [23][24][25][26]. When mice were treated with human bmMSCs, the development and function of immune effector cells and T-cells were affected [27,28]. When bmMSCs were intravenously injected in the murine asthma model, bmMSCs were localized in the lung, resulting in downregulated airway in ammation and Th2 cytokines [25]. Compared to MSCs originating from the bone marrow or other adult organs [29,30], ucMSCs are favorable because they can be harvested non-invasively and mass-produced for in vitro experiments, and most importantly, with less ethical limitations [31,32]. In comparison with bmMSCs, ucMSCs have reportedly demonstrated superior immunosuppressive effects and have thus been identi ed as a potential treatment for patients with asthma [29,[33][34][35]. Until now, human-derived bmMSC studies account for the majority of MSC studies [36]. Recent ucMSC studies demonstrating its anti-asthmatic effect mainly proved its inhibitory effect on Th2 and eosinophilic in ammation [37,38].
Consistent with previous studies, we also successfully demonstrated the reduction of type II in ammation. In vivo experiments in this study con rmed that Th2 lymphocytes and ILC2s that produce IL-5 and IL-13 and induce type 2 in ammation in the lungs were decreased, con rming that ucMSCs affect not only adaptive immunity but also innate immunity. Studies have reported activation of Tregs as an inhibitory mechanism of the Th2 response to the effect of ucMSCs [35]. However, while this study demonstrated that Tregs, which were de ned as Foxp3 + CD4 + T cells, do indeed increase in the OVA group, there was no signi cant change in Tregs following intratracheal ucMSC administration. Therefore, it appears that when ucMSCs were intratracheally administered, the anti-asthmatic effects were not mediated by Tregs. Therefore, this study aimed to con rm the effect on lung macrophages induced by intratracheally ucMSC treatment by analyzing the macrophages and their subsets responding to external stimuli in the airway and intratracheally administered ucMSCs.
Macrophage activation is a dynamic process in which early macrophages react to environmental signals, such as cytokine signaling, and develop into functional macrophages [39]. When exposed to foreign substances, changes in tissue microenvironments cause macrophage polarization. In this experiment, the numbers of MHCII + CD86 + DCs and AMs were far higher in the OVA model than in the control group, but treatment with ucMSCs suppressed these numbers. This suggests that MSCs are involved in the alleviation of asthma by regulating the antigen-presenting ability of DCs as well as AMs. In an OVAinduced murine asthma model treated with bmMSCs, the reduction in the antigen-presenting ability of DCs suggests that bmMSCs possess anti-asthmatic properties by regulating DC activation [40].
Recently, several studies have been conducted to functionally classify macrophages, and it is suspected that macrophage subtypes are related to endotypes of asthma [41][42][43]. In asthma patients, an increase in M2 macrophages is generally observed. In particular, M2a macrophages are known to secrete IL-5 and IL-13, which induce the activation of Th2 cells and the in ltration of eosinophils into the lungs [44]. In accordance with the severity of asthma, M2a macrophages expressing CD206 and MHCII increase dramatically, suggesting that M2a macrophages are closely involved in the pathophysiology of asthma [45]. Additionally, M2c macrophages have been reported to be crucial in the development of pulmonary brosis [46,47], and in vitro experiments have con rmed that ASCs can regulate M2c macrophage activation [48]. This study further con rmed that treating an OVA-induced asthma model with ucMSCs resulted in a change in M2 macrophage subtypes. More speci cally, M2a and M2c macrophages were found to have a strong positive correlation with Th2 cells, ILC2s, and eosinophils. The reduction of M2a and M2c macrophages suggested that the anti-asthmatic effects of ucMSCs may be mediated by the regulation of these M2 macrophage subtypes.
AMs are principal immune cells that reside in the lungs and come in close contact with foreign substances through gaseous exchange. As the rst line of defense against invading respiratory pathogens, AMs suppress inappropriate immune responses to antigens [49,50]. Previous studies con rmed the immunosuppressive properties of AMs by demonstrating that AM depletion leads to enhanced antigen-presenting abilities in DCs, which then results in increased formation of secondary antibodies when an antigen is inhaled [51,52]. Additionally, AM-starved, OVA-sensitized mice demonstrated an increase in eosinophilic in ammation and Th2 response, verifying that AMs suppress the Th2 response in an OVA-induced murine asthma model [53].
In order to con rm the role of ucMSCs in airway in ammation through macrophage, this study analyzed the overall changes in macrophages due to ucMSC treatment through CD11b and CD11c gating [14,15]. CD11b, the subunit that constitutes the integrin αMβ2, was found to aid the adhesion and migration of macrophages, and thus helps regulate phagocytosis and cell activation [54][55][56]. CD11c, the subunit that constitutes the integrin αXβ2, allows macrophages to bind to lipopolysaccharide (LPS) or act as a complement to engulf opsonized bacteria [57,58]. SiglecF, a marker related to eosinophil apoptosis, is expressed in eosinophils and AMs, yet is not found in interstitial macrophages or in ammatory macrophages [16][17][18]. Although SiglecF is a marker of eosinophils, eosinophils are CD11ccells and could be excluded from this study, which analyzed CD11c + cells. Therefore, SiglecF + CD11c + CD11bmacrophages may represent typical resident AMs with homeostatic function in a steady state.
Interestingly, ucMSCs increased SiglecF + CD11c + CD11bresident AMs, and this population was strongly negatively correlated with Th2 cells, ILC2s, and eosinophils in this study. These results show that ucMSCs alleviate asthma by suppressing type 2 in ammation by recovering the depleted SiglecF + CD11c + CD11bmacrophage population. In accordance with this study, another study using OVA-induced asthma models reported that CD11c + CD11b low AMs were signi cantly reduced upon OVA challenge but were also restored upon treatment with human MSCs, and in vivo depletion of AMs abrogated the therapeutic effects of human MSCs on allergic airway in ammation and AHR [29].
Recent studies con rming the immunosuppressive properties of allogenic MSCs have proposed that soluble factors may be the main cause of their immunomodulatory effect [59][60][61][62][63][64]. Immune function regulation by human MSCs in mice may be mainly due to these immunosuppressive water-soluble agents, rather than direct cell-to-cell contact. When MSC-treated media were intratracheally administered to LPS-stimulated mice, in ammatory cells in BAL uid were decreased [59], and other studies suggested that MSCs mediate immunosuppressive functions by secreting water-soluble agents such as IL-6, IL-10, prostaglandin E2, and nitric oxide [60-64].
Through ex vivo experiments, we con rmed that the expression of M1-, M2-, and Th2-related markers decreased with ucMSC treatment. Through in vitro experiments, we found that administering ucMSCs directly or indirectly via transwell decreased the expression of both IL-5 and IL-13, with no signi cant difference between the two experimental groups. This further supports the idea that ucMSCs suppress type 2 in ammation by regulating macrophage activation via soluble mediators rather than direct cell-tocell contact. This study con rmed the effect of intratracheally administered ucMSCs on lung macrophage differentiation in a murine asthma model, suggesting that direct delivery of ucMSCs to airways are a potential treatment for asthma. Further studies are needed to show the in vivo effects of MSC-induced SiglecF + CD11c + CD11bmacrophages and to investigate the mechanisms by which ucMSCs regulate macrophage activation via soluble mediators.
In conclusion, intratracheally administered ucMSCs inhibit AHR and type II in ammation, which may be mediated by macrophage regulation in the asthmatic lung. In particular, ucMSC reduced the levels of M2a and M2c macrophages, suppressed the antigen-presenting capacity of DCs and AMs, and increased resident AMs. These regulatory properties of ucMSCs support the potential therapeutic implication of ucMSCs in allergic asthma. Authors' contributions: HK made substantial contributions to the conception and design of the study, carried out the study, was involved in all animal experiments and all data collection, performed the statistical analysis, and drafted the manuscript. YM carried out the study, was involved in data collection and drafted the manuscript. HYK, SHC made substantial contributions to the conception and design of the study and co-supervised the study. HRK designed the study, interpreted the data, supervised the conduction of the study and drafting of the manuscript, and revised it critically. All authors read and approved the nal manuscript. Availability of data and materials: The data that support the ndings of this study are available from the corresponding author upon reasonable request.
Ethics approval and consent to participate: All animal experimental procedures were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Resources at Seoul National University