Human Mesenchymal Stem Cells Overexpressing the IL-33 Antagonist Soluble IL-1 Receptor–Like–1 Attenuate Endotoxin-Induced Acute Lung Injury

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by pulmonary edema attributable to alveolar epithelial-interstitial-endothelial injury, associated with profound inflammation and respiratory dysfunction. The IL-33/IL-1 receptor-like-1 (ST2) axis plays a key role in the development of immune-inflammatory responses in the lung. Cell-based therapy has been recently proposed as an effective alternative for the treatment of ALI and ARDS. Here, we engineered human adipose tissue-derived mesenchymal stem cells (hASCs) overexpressing soluble IL-1 receptor-like-1 (sST2), a decoy receptor for IL-33, in order to enhance their immunoregulatory and anti-inflammatory properties when applied in a murine ALI model. We administered both hASCs and hASC-sST2 systemically at 6 hours after intranasal LPS instillation, when pathological changes had already occurred. Bioluminescence imaging, immunohistochemistry, and focused transcriptional profiling confirmed the increased presence of hASCs in the injured lungs and the activation of an immunoregulatory program (CXCR-4, tumor necrosis factor-stimulated gene 6 protein, and indoleamine 2,3-dioxygenase up-regulation) in these cells, 48 hours after endotoxin challenge. A comparative evaluation of hASCs and the actions of hASC-sST2 revealed that local sST2 overproduction by hASC-sST2 further prevented IL-33, Toll-like receptor-4, IL-1β, and IFN-γ induction, but increased IL-10 expression in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in protein content, differential neutrophil counts, and proinflammatory cytokine (TNF-α, IL-6, and macrophage inflammatory protein 2) concentrations in bronchoalveolar lavage fluid. In addition, hASC-sST2-treated ALI lungs showed preserved alveolar architecture, an absence of apoptosis, and minimal inflammatory cell infiltration. These results suggest that hASCs genetically engineered to produce sST2 could become a promising therapeutic strategy for ALI/ARDS management.

Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, and Histopathology in a Mouse Model of Occupational Asthma

“…Discarded human adipose tissue from healthy adult women undergoing elective lipoaspiration was used as the source of hASCs, under the Institutional Ethics Committee approval as described elsewhere [17,19]. The raw lipoaspirate (200 mL) was washed extensively in phosphatebuffered saline (PBS) and incubated in PBS containing 0.075% type II collagenase (Life Technologies) at 37°C for 1 h to digest the extracellular matrix.…”

“…Mice were administered, via the lateral tail vein, 1 · 10 6 hASCs transduced with a bicistronic lentiviral vector (LV-T2A) encoding the firefly luciferase (fLuc) and the EGFP under the control of a cytomegalovirus promoter [17]. To track fLuc-expressing hASCs, mice were intraperitoneally injected with D-luciferin (150 mg/kg) (Promega) dissolved in PBS and imaged at different times (ranging from 10 min to 5 days) in an IVIS Ò Spectrum Imaging System (Caliper Life Sciences).…”

“…The synthesis and production of VSV-G-pseudotyped, high-titer lentiviral particles from the bicistronic expression vector pWPT-sST2FLAG2AEGFP (pWPT-sST2-EGFP) and its corresponding control pWPT-EGFP have been previously described [17]. pWPT-sST2-EGFP encodes, in a 5¢ to 3¢ orientation, the C-terminus FLAG-tagged murine sST2 cDNA, the picornaviral 2A sequence for cotranslational cleavage, and the reporter EGFP cDNA.…”

“…At 48 h after transduction, the EGFP expression was analyzed by both flow cytometry and fluorescence microscopy. The recombinant mouse sST2 protein expression was assessed by a specific mouse T1/ST2 sandwich enzyme-linked immunosorbent assay (ELISA; mdbioproducts) according to the manufacturer's instructions, and by western blot analysis using an anti-FLAG antibody [17]. The functional activity of the secreted sST2-FLAG decoy was evaluated by assessing its ability to interfere with proinflammatory IL-33-T1/ST2 signaling in P815 mouse mastocytoma cells [17].…”

“…In the present study we have evaluated the therapeutic potential of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or genetically engineered to overexpress sST2 [17]-a natural decoy receptor to IL-33 [18], key alarmin in innate eosinophilic airway inflammation-resolving the underlying immune-inflammatory process and the chronic airway hyperreactivity (AHR) inherent in the validated ammonium persulfate [(NH 4 ) 2 S 2 O 8 ; AP]-induced OA model.…”