Classical monocyte-derived macrophages as therapeutic targets of umbilical cord mesenchymal stem cells: comparison of intratracheal and intravenous administration in a mouse model of pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease that has no cure. Although mesenchymal stem cells (MSCs) have been reported to ameliorate lung inflammation and fibrosis in mouse models, their mechanisms of action remain unknown. Therefore, we aimed to determine the changes in various immune cells, especially macrophages and monocytes, involved in the effects of MSC treatment on pulmonary fibrosis. METHODS: We collected and analyzed explanted lung tissues and blood from patients with IPF who underwent lung transplantation. After establishing a pulmonary fibrosis model via the intratracheal administration of bleomycin (BLM) to 8-week-old mice, MSCs derived from human umbilical cords were administered intravenously or intratracheally on day 10 and the lungs were immunologically analyzed on days 14 and 21. Flow cytometry was performed to analyze the immune cell characteristics, and gene expression levels were examined using quantitative reverse transcription-polymerase chain reaction. RESULTS: In the histological analysis of explanted human lung tissues, the terminally fibrotic areas contained a larger number of macrophages and monocytes than the early fibrotic areas of the lungs. When human monocyte-derived macrophages (MoMs) were stimulated with interleukin-13 in vitro, the expression of type 2 macrophage (M2) markers was more prominent in MoMs from the classical monocyte subset than in those from intermediate or non-classical monocyte subsets, and MSCs suppressed M2 marker expression independent of MoM subsets. In the mouse model, the increased number of inflammatory cells in the bronchoalveolar lavage fluid and the degree of lung fibrosis observed in BLM-treated mice were significantly reduced by MSC treatment, which tended to be more prominent with intravenous administration than intratracheal administration. Both M1 and M2 MoMs were upregulated in BLM-treated mice. The M2c subset of M2 MoMs was significantly reduced by MSC treatment. Among M2 MoMs, M2 MoMs derived from Ly6C+ monocytes were most effectively regulated by the intravenous administration, not intratracheal administration, of MSCs. CONCLUSIONS: Inflammatory classical monocytes may play a role in lung fibrosis in human IPF and BLM-induced pulmonary fibrosis. Intravenous rather than intratracheal administration of MSCs may ameliorate pulmonary fibrosis by inhibiting monocyte differentiation into M2 macrophages.

Intravenous Mesenchymal Stem Cell Administration Modulates Monocytes/Macrophages and Ameliorates Asthmatic Airway Inflammation in a Murine Asthma Model.

Although asthma is a common chronic airway disease that responds well to anti-inflammatory agents, some patients with asthma are unresponsive to conventional treatment. Mesenchymal stem cells (MSCs) have therapeutic potential for the treatment of inflammatory diseases owing to their immunomodulatory properties. However, the target cells of MSCs are not yet clearly known. This study aimed to determine the effect of human umbilical cord-derived MSCs (hUC-MSCs) on asthmatic lungs by modulating innate immune cells and effector T cells using a murine asthmatic model. Intravenously administered hUC-MSCs reduced airway resistance, mucus production, and inflammation in the murine asthma model. hUC-MSCs attenuated not only T helper (Th) 2 cells and Th17 cells but also augmented regulatory T cells (Tregs). As for innate lymphoid cells (ILC), hUC-MSCs effectively suppressed ILC2s by downregulating master regulators of ILC2s, such as Gata3 and Tcf7. Finally, regarding lung macrophages, hUC-MSCs reduced the total number of macrophages, particularly the proportion of the enhanced monocyte-derived macrophage population. In a closer examination of monocyte-derived macrophages, hUC-MSCs reduced the M2a and M2c populations. In conclusion, hUC-MSCs can be considered as a potential anti- asthmatic treatment given their therapeutic effect on the asthmatic airway inflammation in a murine asthma model by modulating innate immune cells, such as ILC2s, M2a, and M2c macrophages, as well as affecting Tregs and effector T cells.

Mesenchymal Stem Cells Attenuate Asthmatic Inflammation and Airway Remodeling by Modulating Macrophages/Monocytes in the IL-13-Overexpressing Mouse Model.

Mesenchymal stem cells (MSCs) are attractive alternatives to conventional anti-asthmatic drugs for severe asthma. Mechanisms underlying the anti-asthmatic effects of MSCs have not yet been elucidated. This study evaluated the anti-asthmatic effects of intravenously administered MSCs, focusing on macrophages and monocytes. Seven-week-old transgenic (Tg) mice with lung-specific overexpression of IL-13 were used to simulate chronic asthma. MSCs were intravenously administered four days before sampling. We examined changes in immune cell subpopulations, gene expression, and histological phenotypes. IL-13 Tg mice exhibited diverse features of chronic asthma, including severe type 2 inflammation, airway fibrosis, and mucus metaplasia. Intravenous administration of MSCs attenuated these asthmatic features just four days after a single treatment. MSC treatment significantly reduced SiglecF-CD11c-CD11b+ monocyte-derived macrophages (MoMs) and inhibited the polarization of MoMs into M2 macrophages, especially M2a and M2c. Furthermore, MSCs downregulated the excessive accumulation of Ly6c- monocytes in the lungs. While an intravenous adoptive transfer of Ly6c- monocytes promoted the infiltration of MoM and Th2 inflammation, that of MSC-exposed Ly6c- monocytes did not. Ex vivo Ly6c- MoMs upregulated M2-related genes, which were reduced by MSC treatment. Molecules secreted by Ly6c- MoMs from IL-13 Tg mice lungs upregulated the expression of fibrosis-related genes in fibroblasts, which were also suppressed by MSC treatment. In conclusion, intravenously administered MSCs attenuate asthma phenotypes of chronic asthma by modulating macrophages. Identifying M2 macrophage subtypes revealed that exposure to MSCs transforms the phenotype and function of macrophages. We suggest that Ly6c- monocytes could be a therapeutic target for asthma management.

Intratracheal administration of mesenchymal stem cells modulates lung macrophage polarization and exerts anti-asthmatic effects.

Mesenchymal stem cells (MSCs) possess immunomodulatory properties that have therapeutic potential for the treatment of inflammatory diseases. This study investigates the effects of direct MSC administration on asthmatic airways. Umbilical cord MSCs (ucMSCs) were intratracheally administered to six-week-old female BALB/c mice sensitized and challenged with ovalbumin; airway hyperresponsiveness (AHR), analyses of airway inflammatory cells, lung histology, flow cytometry, and quantitative real-time PCR were performed. Furthermore, ex vivo and in vitro experiments were performed to assess the effects of ucMSC on M2 activation. Intratracheally administered ucMSCs decreased degree of airway resistance and the number of inflammatory cells such as T helper 2 (Th2) cells, type 2 innate lymphoid cells (ILC2), and macrophages in the murine asthma model. Particularly, MHCII and CD86 expression diminished in dendritic cells and alveolar macrophages (AMs) following ucMSC treatment. SiglecF+CD11c+CD11b- AMs show a negative correlation with type II inflammatory cells including Th2 cells, ILC2, and eosinophils in asthmatic mice and were restored following intratracheal ucMSCs treatment. In addition, ucMSCs decreased the macrophage polarization to M2, particularly M2a. The expression levels of markers associated with M2 polarization and Th2 inflammation were also decreased. ucMSC reduced Il-12 and Tnfa expression as well as that of M2 markers such as Cd206 and Retnla ex vivo. Furthermore, the in vitro study using IL-4 treated macrophages confirmed that both direct and indirect MSC treatment significantly reduced the expression of Il-5 and Il-13. In conclusion, ucMSCs appear to suppress type II inflammation by regulating lung macrophages via soluble mediators.

Mesenchymal stem cells exert their anti-asthmatic effects through macrophage modulation in a murine chronic asthma model.

Despite numerous previous studies, the full action mechanism of the pathogenesis of asthma remains undiscovered, and the need for further investigation is increasing in order to identify more effective target molecules. Recent attempts to develop more efficacious treatments for asthma have incorporated mesenchymal stem cell (MSC)-based cell therapies. This study aimed to evaluate the anti-asthmatic effects of MSCs primed with Liproxstatin-1, a potent ferroptosis inhibitor. In addition, we sought to examine the changes within macrophage populations and their characteristics in asthmatic conditions. Seven-week-old transgenic mice, constitutively overexpressing lung-specific interleukin (IL)-13, were used to simulate chronic asthma. Human umbilical cord-derived MSCs (hUC-MSCs) primed with Liproxstatin-1 were intratracheally administered four days prior to sampling. IL-13 transgenic mice demonstrated phenotypes of chronic asthma, including severe inflammation, goblet cell hyperplasia, and subepithelial fibrosis. Ly6C+M2 macrophages, found within the pro-inflammatory CD11c+CD11b+ macrophages, were upregulated and showed a strong correlation with lung eosinophil counts. Liproxstatin-1-primed hUC-MSCs showed enhanced ability to downregulate the activation of T helper type 2 cells compared to naïve MSCs in vitro and reduced airway inflammation, particularly Ly6C+M2 macrophages population, and fibrosis in vivo. In conclusion, intratracheal administration is an effective method of MSC delivery, and macrophages hold great potential as an additional therapeutic target for asthma.

Effect of Acinetobacter lwoffii on the modulation of macrophage activation and asthmatic inflammation.

BACKGROUND: Although lung macrophages are directly exposed to external stimuli, their exact immunologic roles in asthma are still largely unknown. The aim of this study was to investigate the anti-asthmatic effect of Acinetobacter lwoffii in terms of lung macrophage modulation. METHODS: Six-week-old female BALB/c mice were sensitized and challenged with ovalbumin (OVA) with or without intranasal administration of A. lwoffii during the sensitization period. Airway hyperresponsiveness and inflammation were evaluated. Using flow cytometry, macrophages were subclassified according to their activation status. In the in vitro study, a murine alveolar macrophage cell line (MH-S) treated with or without A. lwoffii before IL-13 stimulation were analysed by quantitative RT-PCR. RESULTS: In a murine asthma model, the number of inflammatory cells, including macrophages and eosinophils, decreased in mice treated with A. lwoffii (A. lwoffii/OVA group) compared with untreated mice (OVA group). The enhanced expression of MHCII in macrophages in the OVA group was decreased by A. lwoffii treatment. M2 macrophage subtypes were significantly altered. A. lwoffii treatment decreased CD11b+ M2a and CD11b+ M2c macrophages, which showed strong positive correlations with Th2 cells, ILC2 and eosinophils. In contrast, CD11b+ M2b macrophages were significantly increased by A. lwoffii treatment and showed strong positive correlations with ILC1 and ILC3. In vitro, A. lwoffii down-regulated the expression of M2 markers related but up-regulated those related to M2b macrophages. CONCLUSIONS AND CLINICAL RELEVANCE: Intranasal A. lwoffii exposure suppresses asthma development by suppressing the type 2 response via modulating lung macrophage activation, shifting M2a and M2c macrophages to M2b macrophages.