Effect Of Endogenous HMGB1 On Immunosuppression Of MDSCs In DSS-induced Conlitis Mice

High mobility group protein 1 (HMGB1), a conservative inflammatory molecule, exists in all most eukaryotic cells. Intranuclear HMGB1 can interact with DNA or protein-DNA complexes directly, enhances the interaction between transcription factors (such as NF-Kappa B) and their target genes. HMGB1 can be released into the extracellular milieu as a typical injury-molecule, promotes the development of inflammation. Myeloid-derived suppressor cells (MDSCs) are a heterogeneity group of immature bone marrow-derived cells, and play a vital role in various tumorous and inflammatory diseases. Some studies have reported that MDSCs accumulated significantly in inflammation and suppressed inflammatory response. Earlier research showed that extracellular HMGB1 could increase the suppressive activity of MDSCs through activating NF-Kappa B signal transduction pathway, however the role of endogenous HMGB1 on MDSCs is still unclear, so we intend to investigate the effect and related molecular mechanisms of endogenous HMGB1 on suppressive activity of MDSCs. Our research can provide new theories and clues for colitis treatment.Part 1 The effects of endogenous HMGB1 on the immunosuppression of MDSCs and related molecular mechanismsMethods1. Colitis was induced by 2.5% dextran sulfate sodium (DSS) in C57BL/6 mice. MDSCs were separated and purified from spleen tissues at 19th day after DSS drinking. Secretion of HMGB1 from MDSCs was confirmed and qualified by ELISA, and supernatant of MDSCs were used to stimulate RAW 264.7 cells to verify the pro-inflammatory activity of secretory HMGB1;2. HMGB1 siRNA sequences were designed and synthesized to knockdown the HMGB1 expression in MDSCs;3. CFSE staining T-cell proliferation essay was used to measure the suppressive activity of MDSCs, and the expression of IL-10, TGF-?,iNOS, Arg-1 and ROS were detected by ELISA, flow cytometry and RT-PCR;4. The expression of MDSCs differentiation marker molecules:CD80, CD86 and MHC-?(IA/IE) were detected by flow cytometry;5. The expression of NF-Kappa B related molecules (p-p65 and I?B-?) in MDSCs were measured by Western Blot, and extracellular HMGB1 was used to stimulate MDSCs to validate the association between immunosuppression of MDSCs and activation of NF-Kappa B.Results1. We had observed the significant inflammatory symptoms and high DAI scores of colitis mice at 8th day after DSS drinking, and the symptoms disappeared at 19th day. The proportion of MDSCs in spleen and mesenteric lymph nodes gradually increased;2. By using Percoll density gradient centrifugation and magnetic beads technologies, the rate of purity of MDSCs could reach up to 90%. The secretory HMGB1 had been detected in MDSCs from DSS-induced mice at 19th day, meanwhile it had pro-inflammatory activity and could stimulate RAW264.7 cells to increase the expression of pro-inflammatory cytokines;3. Transfecting HMGB1 siRNA sequences decreased the mRNA and protein expression of HMGB1 in MDSCs;4. Knockdown of HMGB1 in MDSCs reduced the suppressive activity of MDSCs on T cell proliferation, and decreased IL-10, iNOS expression;5. Knockdown of HMGB1 in MDSCs increased the expression of differentiation-related molecules such as CD80 and MHC-?(IA/IE) in MDSCs;6. We found that Knockdown of HMGB1 in MDSCs could reduce p-p65 expression and I?B-a degradation. Meanwhile, we observed the suppressive activity on T cells proliferation was recovered and the expression of IL-10 and iNOS were increased by using extracellular HMGB1 to activate NF-Kappa B in MDSCs.These results showed that Knockdown of HMGB1 in MDSCs could decrease the suppressive activity of MDSCs via inhibiting NF-Kappa B activity and promoting MDSCs' differentiation and maturation.Part 2 The effects of siHMGB1 on anti-inflammatory function of MDSCs in vivoMethods1. MDSCs were transfused to colitis mice via tail vein at 2th,4th and 6th day after drinking 2.5% DSS;2. The inflammatory symptoms, local pathology and colon lengths of colitis mice were observed and recorded at 8th day after drinking 2.5% DSS;3. Cytokines (IL-1?, IFN-y, IL-10 and NO) from supernatant of colon tissues and serums were detected by ELISA.Results1. Comparing to blank and negative control MDSCs groups, more severe inflammatory symptoms and local pathology features were observed from siHMGB1 MDSCs group;2. Comparing to control MDSCs groups, the expression of IL-10 and NO in siHMGB1 MDSCs group decreased, while the expression of IL-1? and IFN-y increased.These results suggested that knockdown of HMGB1 in MDSCs reduced its anti-inflammatory funtion in vivo.ConclusionsThese results suggest that endogenous HMGB1 is essential for the maintenance of suppressive activity and immature state of MDSCs. Knockdown of HMGB1 in MDSCs can significantly decrease the activity of NF-Kappa B and accelerate MDSCs differentiation, thus reduce the suppressive activity of MDSCs and further weaken anti-inflammatory funtion of MDSCs in vivo.