View Abstract

A3871 - Cystic Fibrosis Bronchoalveolar Lavage Fluid Lead to Human Mesenchymal Stromal Cell Death
Author Block: S. Abreu1, J. Dearborn2, N. Gasek2, C. C. Dos Santos3, P. R. Rocco4, M. J. Wargo5, M. J. Wargo5, D. J. Weiss6; 1Carlos Chagas Filho Institute of Biophysics, Laboratory of Pulmonary Investigation, Federal Univesity of Rio de Janeiro, Rio de Janeiro, Brazil, 2Department of Medicine, University of Vermont, Burlington, VT, United States, 3University of Toronto, Toronto, ON, Canada, 4Instituto de Biofisica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio De Janeiro, Brazil, 5Department of Microbiology and Molecular Genetics and Vermont Lung Center, University of Vermont, Burlington, VT, United States, 6Department of Medicine, Univ of Vermont/Hlth Sci Rsch Facility, Burlington, VT, United States.
RATIONALE. Systemic or intratracheal administration of human mesenchymal stem cells (hMSC) have potent paracrine immunomodulatory properties that are being investigated in a wide range of lung diseases, including cystic fibrosis (CF). MSCs detect specific inflammatory environments present in different diseases, tailoring their anti-inflammatory responses accordingly. Understanding MSC anti-inflammatory mechanisms in different clinical inflammatory environments is crucial to refine future MSC-based cell therapies. The aim of this study is to determine the effects of CF human bronchoalveolar lavage fluid (BALF) on hMSC behavior.METHODS. We developed an in vitro immunomodulatory phenotype-based assay approach to quantify and qualify measures of hMSC immunomodulatory actions relevant to the CF treatment. Clinically utilized hMSC were cultured and stimulated for 1, 5 or 24 hours with BALF obtained from normal volunteers or CF patients in serum free media. Conditioned media was collected for evaluation of the BALF cytotoxicity (LDH assay), protease activity, and levels of immunomodulatory cytokines. Gene expression in stimulated hMSCs was analyzed by RT-PCR. RESULTS. We observed that stimulation of hMSCs with CF BALF led to a progressive cytotoxicity as demonstrated by increased LDH percentage and cellular death over time. Furthermore, the increase in the cytotoxicity was positively correlated with higher levels of activated proteases in the CF-BALF, though the causal relationship between these measures is currently unknown. hMSCs exposed to CF-BALF present lower levels of IL-6, IL-10, TNF-α and TGF-β in cells that died throughout the stimulation. CF-BALF that did not induce cellular death present opposite behavior; the gene expression of TGF-β, IL-6, TSG-6 and SCT-1 increase while FGF-7 and ANGPT are reduced. CONCLUSION. We conclude that CF-BALF environment is detrimental to hMSC survival; the disease microenvironment elicits increased production of different mediators by the hMSCs that induced their death. Further studies are necessary to evaluate the in vivo effects stimulated hMSCs and the mechanisms that led to cellular death.