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. 2017 Aug;7(9):400.
doi: 10.4172/2157-7633.1000400. Epub 2017 Sep 22.

Mesenchymal Stem Cell Soluble Mediators and Cystic Fibrosis

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Free PMC article

Mesenchymal Stem Cell Soluble Mediators and Cystic Fibrosis

Morgan T Sutton et al. J Stem Cell Res Ther. .
Free PMC article

Abstract

Human Mesenchymal stem cells (hMSCs) secrete products (supernatants) that are anti-inflammatory and antimicrobial. We have previously shown that hMSCs decrease inflammation and Pseudomonas aeruginosa infection in the in vivo murine model of Cystic Fibrosis (CF). Cystic Fibrosis (CF) is a genetic disease in which pulmonary infection and inflammation becomes the major cause of morbidity and mortality. Our studies focus on determining how MSCs contribute to improved outcomes in the CF mouse model centering on how the MSCs impact the inflammatory response to pathogenic organisms. We hypothesize that MSCs secrete products that are anti-inflammatory in scenarios of chronic pulmonary infections using the murine model of infection and inflammation with a specific interest in Pseudomonas aeruginosa (gram negative). Further, our studies will identify whether the MSCs are impacting this inflammatory response through the regulation of peroxisome proliferator activator receptor gamma (PPARγ) which aides in decreasing inflammation.

Keywords: Anti-Inflammation; Anti-Inflammatory Cytokines; Chemokines; Cytokines; Mesenchymal stem cells; PPARγ; chemotaxis; cystic fibrosis.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The author has no conflicts of interest to report.

Figures

Figure 1
Figure 1. In Vivo Potency and Efficacy
Cftrtm1Kth (CF) and C57BL/6 (WT) mice were infected with either 106 CFUs of Pseudomonas aeruginosa or Staphylococcus aureus and monitored for 10 days. At day 10 the total cell count (1A and 1B) were determined by bronchoalveolar lavage (BAL) using cytospin and H&E staining. These studies were done 4 times with n=10 animals in each group using 4 different hMSC donors. Figure 1A and 1B show the total cell count obtained by BAL, Figure 1C and 1D focus on the Pseudomonas aeruginosa model since it is prototypic of CF lung infection and inflammation. These data demonstrate that part of the impact of hMSCs is on on redirecting the inflammatory infiltrate away from neutrophils toward having more macrophages.
Figure 2
Figure 2. hMSCs and Cell Recruitment Studies
hMSCs supernatants (n=4) were cultured in a transwell with peripheral blood monocytes from healthy volunteers. After 24 hours the cells were counted in both the bottom and top chambers to monitor the cell recruitment process. Data is the mean number of cells recruited in response to the MSC supernatants.
Figure 3
Figure 3. hMSC Cytokine Array
The same hMSC supernatants that were used for the chemotaxis studies were evaluated in a Luminex multiplex assay. Data are Mean±SEM pg/ml, for n=4 different MSC donors.
Figure 4
Figure 4. hMSCs IL-8 response defined by gene expression, secretion with and without CFTR activity and LPS exposure
hMSCs (n=10) were cultured in the presence and absence of the CFTR inhibitor I-172 for 48 hours followed by stimulation with LPS. Cells were harvested and processed for gene expression and supernatants were evaluated for IL-8 protein.
Figure 5
Figure 5. hMSCs IL-6 response defined by gene expression, secretion with and without CFTR activity and LPS exposure
hMSCs (n=10) were cultured in the presence and absence of the CFTR inhibitor I-172 for 48 hours followed by stimulation with LPS. Cells were harvested and processed for gene expression and supernatants were evaluated for IL-6 protein.
Figure 6
Figure 6. hMSCs CCL2 response defined by gene expression, secretion with and without CFTR activity and LPS exposure
hMSCs (n=10) were cultured in the presence and absence of the CFTR inhibitor I-172 for 48 hours followed by stimulation with LPS. Cells were harvested and processed for gene expression and supernatants were evaluated for CCL2 protein.
Figure 7
Figure 7. hMSCs CCL20 response defined by gene expression, secretion with and without CFTR activity and LPS exposure
hMSCs (n=10) were cultured in the presence and absence of the CFTR inhibitor I-172 for 48 hours followed by stimulation with LPS. Cells were harvested and processed for gene expression and supernatants were evaluated for CCL20 protein.
Figure 8
Figure 8. PBMCs and hMSCs-Suppression of Response to LPS Exposure Ex Vivo
Peripheral blood mononuclear cells were obtained from healthy volunteers (n=4) and cultured with and without hMSCs after being stimulated for 24 hours with LPS. Data is the change in cytokine expression post-hMSC treatment as compared to PBMCs without hMSCs.
Figure 9
Figure 9. CFTR Activity and Expression Impact PPARγ Expression which can be reversed by hMSCs Therapeutics
Bone marrow derived macrophages from CF and WT mice were processed for cDNA and monitored for PPARγ gene expression as compared to GAPDH (A, n=4). Bone marrow derived macrophages treated with the CFTR inhibitor I-172 become deficient in PPARγ by 24 hours (p<0.05) which suggest CFTR function is associated with PPARγ expression (A, n=4). The deficiency in PPARγ correlates with increased TNFα (n, p<0.05) hMSCs cultured in scenarios of deficient PPARγ express increased TNFα, but less TLR-4. The deficient PPARγ can be rescued with hMSCs (p<0.05, B, n=4) contributing to less TNFα. There was also a trend towards decreased TLR-4 with the hMSCs (p=0.06, n=4).
Figure 10
Figure 10. Theoretical Schematic of hMSC Down Regulating Inflammation and Managing Infection
Infection with or without inflammation in CF is poorly regulated and contributes to disease pathogenesis. hMSCs produce soluble mediators which redefine the tissue niche, enhancing macrophage recruitment which may can participate in host immunity. In CF, hMSCs have altered levels of soluble mediators relative to the control suggesting that the immune function of hMSCs in CF maybe alters again re-inforcing the notion of allogeneic therapeutics in the context of genetic diseases.

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