Cindy Cen1, Monowar Aziz2, Weng-Lang Yang2, Jeffrey Nicastro1, Gene F Coppa1, Ping Wang3. 1. Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY. 2. Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY; Center for Translational Research, The Feinstein Institute for Medical Research, Manhasset, NY. 3. Department of Surgery, Hofstra North Shore-LIJ School of Medicine, Manhasset, NY; Center for Translational Research, The Feinstein Institute for Medical Research, Manhasset, NY. Electronic address: pwang@nshs.edu.
Abstract
BACKGROUND: Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in inflammation. In sepsis, interleukin (IL)-17 acts as a proinflammatory cytokine to exaggerate systemic inflammation. We hypothesize that MFG-E8 downregulates IL-17 expression in sepsis. METHODS: Sepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). Recombinant mouse MFG-E8 (rmMFG-E8) at a dosage of 20 μg/kg body weight or phosphate-buffered saline was concurrently injected. After 10 hours, blood and spleen samples were harvested for analysis. For in vitro studies, splenocytes isolated from healthy mice pretreated with rmMFG-E8 and splenocytes from MFG-E8 knockout (mfge8(-/-)) mice were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, followed by measurement of IL-17 expression with either quantitative PCR or enzyme-linked immunosorbent assay. RESULTS: At 10 hours after CLP, rmMFG-E8 inhibited the elevated levels of IL-17 protein in serum by 31%, compared with the vehicle. In the spleen, rmMFG-E8 reduced the upregulated IL-17 mRNA and protein levels by 81% and 51%, respectively. This correlated with a significant reduction in organ injury markers AST and ALT in sepsis after administration of rmMFG-E8. In vitro treatment of splenocytes isolated from healthy mice with rmMFG-E8 showed significant downregulation in PMA/ionomycin-induced IL-17 expression. In contrast, CD4 T-cells from mfge8(-/-) mice showed significant upregulation of IL-17 compared with wild-type mice. The phosphorylated level of signal transducer and activator of transcription 3 (STAT3) was downregulated in spleen tissue of septic mice treated with rmMFG-E8. Conversely, mfge8(-/-) mice showed increased phosphorylated STAT3 compared with wild-type mice after sepsis. CONCLUSION: Our findings demonstrate MFG-E8-mediated downregulation of IL-17 expression, implicating its potential as a novel therapeutic agent against sepsis.
BACKGROUND: Milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is a secretory glycoprotein with a known role in inflammation. In sepsis, interleukin (IL)-17 acts as a proinflammatory cytokine to exaggerate systemic inflammation. We hypothesize that MFG-E8 downregulates IL-17 expression in sepsis. METHODS:Sepsis was induced in 8-week-old male C57BL/6 mice by cecal ligation and puncture (CLP). Recombinant mouseMFG-E8 (rmMFG-E8) at a dosage of 20 μg/kg body weight or phosphate-buffered saline was concurrently injected. After 10 hours, blood and spleen samples were harvested for analysis. For in vitro studies, splenocytes isolated from healthy mice pretreated with rmMFG-E8 and splenocytes from MFG-E8 knockout (mfge8(-/-)) mice were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, followed by measurement of IL-17 expression with either quantitative PCR or enzyme-linked immunosorbent assay. RESULTS: At 10 hours after CLP, rmMFG-E8 inhibited the elevated levels of IL-17 protein in serum by 31%, compared with the vehicle. In the spleen, rmMFG-E8 reduced the upregulated IL-17 mRNA and protein levels by 81% and 51%, respectively. This correlated with a significant reduction in organ injury markers AST and ALT in sepsis after administration of rmMFG-E8. In vitro treatment of splenocytes isolated from healthy mice with rmMFG-E8 showed significant downregulation in PMA/ionomycin-induced IL-17 expression. In contrast, CD4 T-cells from mfge8(-/-) mice showed significant upregulation of IL-17 compared with wild-type mice. The phosphorylated level of signal transducer and activator of transcription 3 (STAT3) was downregulated in spleen tissue of septic mice treated with rmMFG-E8. Conversely, mfge8(-/-) mice showed increased phosphorylated STAT3 compared with wild-type mice after sepsis. CONCLUSION: Our findings demonstrate MFG-E8-mediated downregulation of IL-17 expression, implicating its potential as a novel therapeutic agent against sepsis.
Authors: P Ye; F H Rodriguez; S Kanaly; K L Stocking; J Schurr; P Schwarzenberger; P Oliver; W Huang; P Zhang; J Zhang; J E Shellito; G J Bagby; S Nelson; K Charrier; J J Peschon; J K Kolls Journal: J Exp Med Date: 2001-08-20 Impact factor: 14.307
Authors: Michael A Flierl; Daniel Rittirsch; Hongwei Gao; Laszlo M Hoesel; Brian A Nadeau; Danielle E Day; Firas S Zetoune; J Vidya Sarma; Markus S Huber-Lang; James L M Ferrara; Peter A Ward Journal: FASEB J Date: 2008-02-25 Impact factor: 5.834