Human mesenchymal stem cells suppress chronic airway inflammation in the murine ovalbumin asthma model

Abstract

Allogeneic human mesenchymal stem cells (hMSCs) introduced intravenously can have profound anti-inflammatory activity resulting in suppression of graft vs. host disease as well as regenerative events in the case of stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of these diseases. hMSCs produce bioactive factors that provide molecular cuing for: 1) immunosuppression of T cells; 2) antiscarring; 3) angiogenesis; 4) antiapoptosis; and 5) regeneration (i.e., mitotic for host-derived progenitor cells). Studies have shown that hMSCs have profound effects on the immune system and are well-tolerated and therapeutically active in immunocompetent rodent models of multiple sclerosis and stroke. Furthermore, intravenous administration of MSCs results in pulmonary localization. Asthma is a major debilitating pulmonary disease that impacts in excess of 150 million people in the world with uncontrolled asthma potentially leading to death. In addition, the socioeconomic impact of asthma-associated illnesses at the pediatric and adult level are in the millions of dollars in healthcare costs and lost days of work. hMSCs may provide a viable multiaction therapeutic for this inflammatory lung disease by secreting bioactive factors or directing cellular activity. Our studies show the effectiveness and specificity of the hMSCs on decreasing chronic airway inflammation associated with the murine ovalbumin model of asthma. In addition, the results from these studies verify the in vivo immunoeffectiveness of hMSCs in rodents and support the potential therapeutic use of hMSCs for the treatment of airway inflammation associated with chronic asthma.

Figures

Fig. 1.

Development of chronic model of murine asthma. Mice were sensitized with ovalbumin (Ova), rested for 14 days, and then were challenged with Ova or saline every other day for 4 wk. Mice were killed 7 days later, and the lungs were processed for inflammation by bronchoalveolar lavage (BAL; A). Ovalbumin-challenged mice had a significant increase in lymphocytes (L; P ≤ 0.05) and eosinophils (Eos; n = 4, 4–6 animals for each study; P ≤ 0.05). The ovalbumin mice also had a significant decrease in macrophages (Mac; n = 4; P < 0.05). The remaining animals were processed for pathology without BAL (B, saline challenge; C, ovalbumin challenge; trichrome stain, ×40). Histology is representative of 8 different experiments [different batches of human mesenchymal stem cells (hMSCs)] with 2–3 mice in each group. Blue color shows the collagen/extracellular matrix deposition. PMN, polymorphonuclear neutrophils.

Fig. 2.

Xenographic effect of hMSCs on total cell recruitment, inflammation, and pathology. Animals were sensitized with ovalbumin and challenged with saline. The differential between these animals and naïve controls is negligible (data not shown). hMSC therapy did not significantly change the total cell count (A), inflammatory phenotype (B), or lung pathology (×40; C and D, untreated and hMSC-injected, respectively) of the saline control of the chronic murine model. Treating the saline-challenged mice with human hMSCs did not alter the lung inflammatory environment in the chronic murine model (n = 4 studies, 4–6 animals/study). Histology is representative of 8 different experiments (different batches of hMSCs) with 2–3 mice in each group.

Fig. 3.

hMSCs decrease lung inflammation in the chronic asthma lung model. hMSCs or bone marrow-derived macrophages (BMMs) were given by tail vein injection. Mice were evaluated after 7 days of challenge for total cell count (A) and inflammation (B). Treatment of the ovalbumin-challenged mice with hMSCs resulted in a significant decrease in total cell recruitment into the lung (A; n = 4; P < 0.05). This was not observed with the BMM treatment (n = 3 different preparations). The differential showed that treatment with hMSCs resulted in a modest decline in macrophages and significant decrease in eosinophils (n = 4; P ≤ 0.05) but with a robust increase in neutrophils (B; n = 4; P = 0.05). There was no statistical change between ovalbumin-challenged mice and ovalbumin-challenged mice treated with BMM (data not shown).

Fig. 4.

The specificity of the hMSC resolution of lung pathology. The specificity of the hMSC response was further shown by evaluating the histology from ovalbumin-challenged mice treated with hMSC and BMM and not treated compared with the saline-challenged controls treated in the same manner. Three different hMSCs and BMM preparations were utilized for these studies with 2 mice each being evaluated for pathology without BAL to preserve architecture. A is the ovalbumin-sensitized, ovalbumin-challenged disease group (×40). B is ovalbumin-challenged group with hMSC therapy (×40), and C is ovalbumin-challenged group with BMM therapy (×40). These images are representative of n = 3 sets of hMSCs and BMM, with n = 3–4 animals for pathology for each study group of ovalbumin, ovalbumin + hMSC, and ovalbumin + BMM. Image analysis quantified inflammatory nuclei post-hematoxylin and eosin (H&E) staining (D) using color-based pixel analysis. Each section was analyzed for over 196 images using an automated microscope-driven software system (Image-Pro Plus 7.0). Each point is the mean analysis of each section evaluated (3 sections/slide). Ova challenge induced an increase in numbers of nuclei (P < 0.001), which is consistent with the histology images and increase in total cell count. The inflammation was effectively decreased by hMSC therapy but not bone marrow therapy.

Fig. 5.

Treatment of the ovalbumin-challenged mice with hMSCs results in decreased serum IgE. Animals sensitized and challenged with ovalbumin had significantly elevated levels of IgE relative to animals sensitized with ovalbumin but challenged with saline (A; P < 0.05; n = 8 studies, 4–6 animals/study). Animals sensitized with ovalbumin and challenged with ovalbumin followed by hMSC therapy had significantly less IgE relative to the ovalbumin-challenged mice not treated with hMSCs (A; P < 0.04; n = 17). BMM-treated chronic asthma had levels of systemic IgE comparable with the ovalbumin-untreated mice (P = 0.4321; n = 18). MSCs attenuated weight loss in the ovalbumin-treated mice, whereas BMM-treated mice and ovalbumin controls continued to lose weight (B; n = 3 studies, 10–18 mice/study; P = 0.05 at day 4). Sal, saline.

Fig. 6.

hMSC attenuates inflammation in a tissue-specific manner. BAL fluid was obtained from the chronic asthma model with and without treatment with hMSCs and evaluated for IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13. The chronic models of asthma showed elevated IFN-γ (49 ± 3 pg/ml) relative to untreated mice (23 ± 7 pg/ml; n = 4; P = 0.03), consistent with the literature (48). IL-5 (13.5 ± 1.2 pg/ml) and IL-13 (63 ± 6.7 pg/ml) levels were also significantly elevated (n = 4, 4–6 animals/study; P ≤ 0.001) compared with baseline, which had no detectable levels of IL-5 or IL-13 (data not shown). Treatment of the animals with intravenous hMSCs resulted in a statistical decrease in BAL IFN-γ, IL-5, and IL-13 levels (A; n = 4, 4–6 animals/study; P = 0.05). hMSCs increased macrophage inflammatory protein-1α (MIP-1α; P < 0.05; n = 4, 4–6 animals/group) and keratinocyte-derived chemokine (KC; n = 4; P = 0.01). There was no change in MIP-1α, IL-6, or TNF-α, and IL-10 and IL-4 were not detectable in the BAL regardless of hMSC treatment. Cardiac puncture to obtain adequate serum samples was performed on the chronic asthma models after the treatment with hMSCs. The chronic asthma model had significantly elevated levels of systemic IL-1β (B; n = 8; P < 0.001), which was decreased significantly with hMSC treatment (n = 7; P = 0.05). Systemic IFN-γ concentrations increased in the chronic asthma model after MSC treatment (n = 8; P = 0.043). This was dependent on the presence of in vivo inflammation since saline animals given hMSCs had no detectable serum IFN-γ.

Fig. 7.

hMSCs decreased inducible nitric oxide synthase (iNOS) expression in BAL cell pellets of ovalbumin-challenged mice. BAL cell pellets were obtained from mice sensitized with ovalbumin and challenged with either saline or ovalbumin in the presence and absence of hMSC therapy and evaluated for iNOS gene expression. BAL cell pellets from ovalbumin-challenged mice had significantly elevated iNOS gene expression (A; n = 6; P < 0.05) compared with saline-challenged animals (n = 6). iNOS gene expression was significantly decreased after hMSC therapy in the ovalbumin-challenged mice (n = 6; P < 0.05). In the 2nd part of these studies, hMSC supernatant was cultured on the murine macrophage cell line (RAW 264.7), which was stimulated for 24 h with 0.01 μg/ml LPS for 24 h with and without (US) the addition of hMSC supernatants. At 24 h, the hMSC supernatant decreased iNOS gene expression by the macrophages (B; P ≤ 0.05; n = 3 different supernatants). Ct, threshold cycle.