Milk fat globule-epidermal growth factor-factor VIII downregulates interleukin-17 expression in sepsis by modulating STAT3 activation

Abstract

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. 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 20μg/kg body weight or phosphate-buffered saline was concurrently injected. After 10hours, blood and spleen samples were harvested for analysis. For invitro 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. At 10hours 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. Invitro 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. Our findings demonstrate MFG-E8-mediated downregulation of IL-17 expression, implicating its potential as a novel therapeutic agent against sepsis.