Multiple doses of adipose tissue‐derived mesenchymal stromal cells induce immunosuppression in experimental asthma

Abstract In experimental house dust mite (HDM)‐induced allergic asthma, therapeutic administration of a single dose of adipose tissue‐derived mesenchymal stromal cells (MSCs) ameliorates lung inflammation but is unable to reverse remodeling. We hypothesized that multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects in experimental asthma. HDM was administered intranasally in C57BL/6 mice. After the last HDM challenge, mice received two or three doses of MSCs (105 cells per day) or saline intravenously. An additional cohort of mice received dexamethasone as a positive control for immunosuppression. Two and three doses of MSCs reduced lung inflammation, levels of interleukin (IL)‐4, IL‐13, and eotaxin; total leukocyte, CD4+ T‐cell, and eosinophil counts in bronchoalveolar lavage fluid; and total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes. Two and three doses of MSCs also reduced collagen fiber content and transforming growth factor‐β levels in lung tissue; however, the three‐dose regimen was more effective, and reduced these parameters to control levels, while also decreasing α‐actin content in lung tissue. Two and three doses of MSCs improved lung mechanics. Dexamethasone, two and three doses of MSCs similarly increased galectin levels, but only the three‐dose regimen increased CD39 levels in the thymus. Dexamethasone and the three‐dose, but not the two‐dose regimen, also increased levels of programmed death receptor‐1 and IL‐10, while reducing CD4+CD8low cell percentage in the thymus. In conclusion, multiple doses of MSCs reduced lung inflammation and remodeling while causing immunosuppression in HDM‐induced allergic asthma.

relief of symptoms, it does not reverse established remodeling, and a cohort of patients still experiences poor control of symptoms and exacerbations. 1,2 Mesenchymal stromal cell (MSC) therapy has demonstrated promising results in several lung disease models, but its impact on airway remodeling has been sometimes controversial. [3][4][5][6] In models of ovalbuminand Aspergillus hyphal extract-induced allergic asthma, a single dose of MSCs administered either locally or systemically resulted in reduction of both lung inflammation and remodeling. 3,[7][8][9][10] Conversely, in house dust mite extract (HDM)-induced allergic asthma, a single dose of MSCs reduced some inflammatory parameters but was unable to ameliorate lung function or remodeling. 4,5 Some reports have indicated that repeated cell-based therapy yielded better therapeutic effects, either by preventing disease progression or by further reducing inflammation and remodeling, in experimental silicosis and elastase-induced emphysema. 6,11 However, the impact of multiple-dose cell therapy regimens in experimental allergic asthma remains unknown.
We hypothesized that, in experimental allergic asthma, multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects. To address the therapeutic potential of a multiple-dose MSC regimen, this study aimed to comparatively investigate the effects of two and three doses of human adipose tissue-derived MSCs administered systemically in a model of HDM-induced allergic asthma. Endpoints of interest included lung mechanics, histology, inflammation, and remodeling; protein levels of selected mediators; leukocyte counts in primary and secondary lymphoid organs; as well as expression of immunosuppressionassociated markers in the thymus.

| MATERIALS AND METHODS
This study was approved by the Ethics Committee of the Federal University of Rio de Janeiro Health Sciences Center (CEUA-UFRJ: 047/17, Rio de Janeiro, Brazil). All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the U.S. National Academy of Sciences.

| Isolation and culture of MSCs
MSCs were isolated and cultured as described elsewhere. 3

| HDM-induced allergic asthma and therapeutic protocol
Allergic asthma was induced by HDM exposure as described elsewhere. 4,12 Briefly, female C57BL/6 mice (age 8-10 weeks, weight 20-25 g) were randomly divided into two groups. The HDM group was challenged by intranasal instillation with 25 μg of HDM extract (Dermatophagoides pteronyssinus; Greer Laboratories, Lenoir, North Carolina) diluted into 25 μL of PBS, three times a week, for three consecutive weeks, while control (CTRL) animals received 25 μL of sterile PBS under same conditions. One day after the last HDM challenge, the HDM group was further divided to randomly receive either two doses of MSCs (MSC-2D) (10 5 AD-MSC diluted in 50 μL of 0.9% NaCl per dose), three doses of MSCs (MSC-3D), or three doses of saline (SAL) intravenously, via jugular injection under anesthesia (5% isoflurane).
Each dose of MSCs or saline was administered once per day on three consecutive days. Seven days after the last HDM challenge, all animals were euthanized for data analysis (Supporting Information Figure S1).
All animals were weighed before and after the end of the protocol.

| Lung mechanics
Seven days after the last HDM challenge, the animals were sedated (diaz- to RR = 100 bpm, tidal volume (V T ) = 0.2 mL, and fraction of inspired oxygen (FIO 2 ) = 0.21. The anterior chest wall was surgically removed and a positive end-expiratory pressure of 2 cmH 2 O applied. Airflow and tracheal pressure (Ptr) were measured, 3 and lung mechanics were analyzed by the end-inflation occlusion method. 13 In an open chest preparation, Ptr reflects transpulmonary pressure (P L ). Briefly, after end-inspiratory occlusion, there is an initial rapid decline in P L (ΔP1,L) from the preocclusion value down to an inflection point (Pi), followed by a slow pressure decay until a plateau is reached. This plateau corresponds to the elastic recoil pressure of the lung (Pel). ΔP1,L selectively reflects the pressure used to overcome the airway resistance. Static lung elastance (Est,L) was determined by dividing Pel by V T . Lung mechanics measurements were obtained 10 times in each animal. 3 All data were analyzed using ANADAT software (RHT-InfoData, Inc, Montreal, Quebec, Canada).

| Collection and processing of BALF and tissues
Bronchoalveolar lavage fluid (BALF) was collected with a polyethylene cannula which was inserted into the trachea and through which a total volume of 1 mL of PBS containing 10 mM EDTA was instilled and subsequently aspirated. BALF was centrifuged at 300g for 10 minutes at 4 C. The supernatant was removed, and the pellet resuspended in 250 μL of PBS.
The left lung was removed, frozen immediately in liquid nitrogen, and stored at −80 C for molecular analysis, while the right lung was stored in 4% paraformaldehyde solution for histological analyses.
Mediastinal lymph nodes, femoral bone marrow, spleen, and thymus were also removed and macerated with 1 mL of PBS. Mediastinal lymph nodes and thymus were weighed before maceration. All samples were stored at 4 C.  proportion of α-actin was calculated as a ratio of points falling on actinstained vs non-stained tissue. To determine inflammation in the lung parenchyma, the slices were stained with hematoxylin and eosin. Inflammatory changes were graded according to a semiquantitative scoring system as absent (score 0), mild (score 1-2), moderate (score 3), or severe (score 4) by two blinded researchers. The following scoring criteria were adopted: 0, no evidence of inflammation; 1, few inflammatory cells in the peribronchial area; 2, a ring of inflammatory cells (one cell layer deep); 3, a ring of inflammatory cells two to four cells deep; and 4, a ring of inflammatory cells more than four cells deep. 15

| Enzyme-linked immunosorbent assay (ELISA)
Lung tissue and thymus homogenates were used for mediator quantifications. Briefly, the right lung was isolated, homogenized in lysis buffer solution, centrifuged (600g for 5 minutes and 10 000g for 10 minutes), and the resulting supernatant assayed. Total protein content was measured by Bradford's reagent (Sigma-Aldrich). Protein levels of IL-4, IL-13, eotaxin, IL-10, transforming growth factor (TGF)β (lung tissue), and programmed cell death protein-1 (PD-1) and IL-10 (thymus) were evaluated by ELISA (BioLegend, San Diego, California) using matched antibodies in accordance with manufacturer instructions. The final concentration was normalized to total protein content and expressed as pg/mg.

| Analyses of immunosuppression-associated markers
Thymic tissue was lysed for RNA extraction by the RNeasy Plus Mini Kit (Qiagen, Valencia, California) in accordance with manufacturer instructions. The total RNA concentration was measured by spectrophotometry in a Nanodrop ND1000 system, and the first-strand cDNA was synthe- experiments were performed in triplicate. The relative level of each gene was normalized to the housekeeping gene acidic ribosomal phosphoprotein P0 (36B4) and expressed as the fold change relative to the CTRL group using the 2 −ΔΔ Ct method, where ΔCt = Ct (target gene) − Ct (housekeeping gene). 16 The mRNA expression of the following genes was analyzed: indoleamine 2,3-dioxygenase (IDO)-2, CD39, galectin, cytotoxic T-lymphocyteassociated antigen (CTLA)-4, PD-1, and IL-10. The sequences of PCR primers can be found in Supporting Information Table S1.

| Statistical analysis
Differences among groups were assessed using one-way ANOVA followed by Bonferroni's test or an unpaired Student's t test. Data were expressed as mean ± SD. All tests were performed using the Prism 6.07 software package (GraphPad Software Inc, La Jolla, California), and statistical significance was established at P < .05.

| Multiple doses of MSCs reduced lung inflammation in HDM-induced allergic asthma
Protein levels of IL-4, IL-13, and eotaxin were higher in lung tissue homogenates of HDM-SAL compared to CTRL mice. Both two and three doses of MSCs significantly reduced protein levels of these inflammatory mediators. No differences were observed in protein levels of IL-10 among the experimental groups (Figure 1).
The HDM-SAL group demonstrated increased cell counts in peribronchial areas, with moderate lung inflammation scores compared to Compared to CTRL mice, the HDM-SAL group demonstrated an increase in total and differential cell counts in the BALF. Both two and three doses of MSCs were able to reduce total leukocyte, CD4 + T-cell, and eosinophil counts (Figure 3; Supporting Information Figure S2).

| Three doses of MSCs led to further reduction of lung remodeling compared to the two-dose regimen
HDM-SAL animals demonstrated an increase in TGF-β levels, as well as in the amount of collagen fibers and α-actin in alveolar septa compared to CTRL mice. Both two and three doses of MSCs reduced TGF-β levels and collagen fiber content in lung tissue; however, the three-dose regimen was more effective, reducing these parameters to CTRL-comparable levels. The three-dose regimen, but not two doses of MSCs, reduced α-actin content in alveolar septa (Figure 4).

| Multiple doses of MSCs led to improvements in lung mechanics parameters
HDM-SAL mice demonstrated higher Est,L and ΔP1,L compared to the CTRL group (1.4-and 2.5-fold increase, respectively). Both two and three doses of MSCs reduced these parameters to CTRLcomparable levels ( Figure 5). 3.4 | Multiple doses of MSCs reduced total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes, but not in the thymus Saline vs HDM challenge and treatment with saline vs multiple doses of MSCs had no effect on body weight (Supporting Information   Table S2). Nevertheless, relative thymus weight was increased in HDM-SAL compared to CTRL mice. Both the two-dose and threedose MSC regimens reduced relative thymus weight to CTRLcomparable levels. No differences were observed in relative mediastinal lymph node weight among the experimental groups (Supporting Information Figure S3).
HDM-SAL animals demonstrated increased total leukocyte counts in bone marrow, spleen, mediastinal lymph nodes, and thymus compared to CTRL mice. Both the two-dose and three-dose MSC regimens reduced total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes, but not in the thymus ( Figure 6). To rule out any hypothesis that multiple manipulations for the intravenous administration of MSCs could cause stress and itself induces an effect on lymphoid organs, a separate cohort of mice (SHAM) was administered a single sham challenge with saline, while the CTRL group was challenged with saline and treated with saline for three consecutive days in parallel to the remaining experimental groups. CTRL and SHAM animals demonstrated no significant differences in total cell counts in bone marrow (P = .4420), spleen (P = .1318), or thymus (P = .7733), nor any other physiological alteration.  Figure S4). HDM is present in almost all environments and is the most common allergen implicated in the onset of human asthma, affecting approximately 85% of asthmatic patients globally. 17,18 In experimental models, HDM closely reproduces several hallmark features of human disease, including the inflammatory process with Th2 and eosinophilic responses and remodeling of both airways and lung parenchyma. 4,12,19 In this study, MSCs were administered intravenously 24 hours after the last HDM challenge in order to reproduce a clinically relevant situation, as lung inflammation and remodeling were established and lung function was impaired. Although bone marrowderived MSCs have demonstrated therapeutic effects in this model of HDM-induced allergic asthma, 4,19 the availability of these cells is limited in the clinical scenario, since an invasive harvesting procedure is required. Therefore, herein we used cells collected from human adipose tissue, which is an interesting source of MSCs, as they can be easily obtained by liposuction. Furthermore, adipose tissue is estimated to contain a greater number of MSCs compared to bone marrow, and these cells appear to be expandable to a higher number of passages, 20,21 thus providing attractive advantages for use in a multiple-dose regimen.

| DISCUSSION
In previous studies, a single dose of MSCs administered prophylactically or therapeutically reduced lung influx of inflammatory cells, airway hyperresponsiveness, and mucus hypersecretion in models of ovalbumin-induced allergic asthma. 3,7,8,22,23 In HDM-induced allergic asthma, a single dose of MSCs administered prophylactically also prevented inflammation by modulating epithelial cell activation. 24 However, effects on the inflammatory process were only marginal, with no improvements in lung function and remodeling when MSCs were administered therapeutically. 4,5 In this study, both two and three IL-4 and IL-13 contribute not only to the inflammatory process but also to lung remodeling, as they can induce epithelial cell apoptosis, fibroblast proliferation, airway hyperresponsiveness, and mucus hypersecretion. 25,26 TGF-β also participates in the remodeling process by increasing extracellular matrix deposition and promoting differentiation of fibroblasts to myofibroblasts, as reflected by increased smooth muscle α-actin content. 26,27 Although a single dose of MSCs was unable to reverse lung remodeling in this model of HDM-induced allergic asthma, 4 our present findings demonstrate that multiple doses of MSCs can exert this effect. In fact, three doses of MSCs were even more effective than a two-dose regimen, thus reducing collagen fiber and smooth muscle α-actin content in alveolar septa, and protein levels of IL-13 and TGF-β to CTRL-comparable levels. Other studies have also demonstrated better effects on remodeling after repeated cell-based therapy in distinct models of lung injury, 6,11 indicating that more than one dose of MSCs possibly induces a more organized and effective re-epithelization. Furthermore, multiple doses of MSCs resulted in prolonged therapeutic benefits in a non-obese mouse model with severe diabetes 28 and in the SOD1 G93A mouse model of amyotrophic lateral sclerosis. 29 Although the mechanisms by which MSCs induce therapeutic actions are not entirely elucidated, these cells have the ability to promote immunomodulation. 3,9,10,23,30 In this study, multiple doses of MSCs alleviated lung inflammation, which may also correlate with the reduction in total leukocyte counts in the bone marrow, spleen, and mediastinal lymph nodes. As lymphocytes traffic between lymphoid organs during the differentiation process, MSCs may block their production and/or maturation, 31,32 thus inhibiting the recruitment of inflammatory cells into the lungs. In a model of elastase-induce emphysema, repeated administration of MSCs also resulted in immunosuppressive effects on lymphoid organs. 6 Nevertheless, HDM mice demonstrated a reduction in relative thymus weight, but not in total leukocyte counts in this organ, after multiple doses of MSCs. Therefore, we further investigated the impact of multiple doses of MSCs on lymphocyte subpopulations, as the thymus plays a critical role in T-cell differentiation and maturation. Furthermore, an additional group of HDM mice was treated with dexamethasone, a recommended therapy for asthmatic patients, as a positive control. 2 This study has some limitations that should be addressed. Two and three doses of MSCs were selected rather than four or more, since further increasing the number of MSC doses would have subjected animals to additional anesthesia procedures. Such repeated use of anesthetic agents might hinder the possible beneficial effects of MSCs. Additionally, more experimental groups would be required, thus adding more variables to the comparative statistical analysis. Further studies are warranted to comparatively evaluate the short-and long-term effects of multiple doses.

| CONCLUSION
In HDM-induced allergic asthma, multiple doses of MSCs were associated with reductions in inflammation and remodeling, while resulting in T-cell immunosuppression. These findings should be borne in mind for future clinical trials.