IL-11-mTORC1-STAT3 Signaling Axis Regulates The Development Of M-MDSC

Objective:To illuminate the mechanism involved in the process of MDSC induced by IL-11 plus GM-CSF.Our previous studies have demonstrated that IL-11 can obviously improve clinical symptoms in sepsis’ patients and prolong the life-span of mice with sepsis.MDSC were found significantly increased in liver of septic mice.Based of these phenomenon,we speculated that IL-11 has the potential to induce the development of MDSC.IL-11 and IL-6 have similar signal transduction pathway.It was declared that IL-6 could induce the differentiation of MDSC and STAT3 was involved in its signal transduction,but the detailed mechanism has not yet elucidated.The bioinformation analysis suggested that STAT3 possess TOS Motif which implied that mTORC1 targets on STAT3 and regulated its activity during the process of induction of MDSC with IL-11.Methods:IL-11 plus GM-CSF was used to induce the development of MDSC for 4 days from bone marrow cells mimicking the system of MDSC induction with IL-6 plus GM-CSF in vitro.The CD11b+Gr-1+ cells were detected by flow cytometry.The percentage of CD11b+Gr-1+ cells,induced by GM-CSF,IL-6 plus GM-CSF or IL-11 add with GM-CSF respectively,were compared to prove whether IL-11 has the potential to induce the development of MDSC.CD11b+Gr-1+ cells,induced by above methods,were cocultured with OVA257-264 peptide pulsed antigen presenting cells activated CD8+T cells from OT-1 mice to test the immune suppression since the defining characteristics of MDSC is immune suppressive ability.In order to detect the role of mTORC1 and STAT3 in the development of IL-11-induced MDSC,the mTORC1 inhibitor Rapamycin or STAT3 inhibitor Stattic was added to the MDSC induction system respecitvely.The percentage of MDSC and the subgroups of M-MDSC and G-MDSC were detected by flow cytometry after four days.To further verify the hypothesis,the effect of IL-11 on mTORC1 and STAT3 and the relationship between mTORC1 and STAT3 were tested by Western blot.At the end,the immunoprecipitation was performed to determine whether mTORC1 directly interact with STAT3.Results:The percentage and immune suppressive ability of CD11b+Gr-1+ cells,induced by IL-11 plus GM-CSF,were higher and more efficient than those of GM-CSF and similar to IL-6 plus GM-CSF induced MDSC.and against OVA257-264 activated CD8+ T cell proliferation.Rapamycin had no significant effect on the total numbers of MDSC,but inhibited the development of M-MDSC subpopulation significantly and promoted the development of G-MDSC subgroup.Stattic inhibited the differentiation of M-MDSC subpopulation completely and inhibited the differentiation of G-MDSC subgroup mostly.These dates indicated that mTORC1 promoted the differentiation of M-MDSC but inhibited the G-MDSC.STAT3 absolutely controlled the differentiation of M-MDSC and partially controlled the G-MDSC.Western blot analysis revealed that IL-11 promoted mTORC1 activation and up-regulated STAT3 phosphorylation.Rapamycin and mTORC1/2 inhibitor Torin1 down-regulated the activation of STAT3 induced by IL-11,and mTORC1 could further promoted the phosphorylation of STAT3 in MEF(TSC2-/-)cells.These results indicated that IL-11 regulated the activity of mTORC1 and STAT3,and mTORC1 promoted phosphorylation of STAT3.Finally,immunoprecipitation technique confirmed that Raptor can bind to STAT3 directly.This result demonstrated that mTORC1 could promote the phosphorylation of STAT3 directly.Conclusions:IL-11 induced the development of M-MDSC via IL-11/IL-11R/gp130-mTORC1-STAT3 signaling.