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The Potential Effect and Mechanism of High Mobility Group Box 1 Protein on Regulatory T Cell-mediated Immunosuppression in Mice

Author: ZhangYing
Tutor: ChangQing
School: Third Military Medical University
Course: Obstetrics and Gynecology
Keywords: High mobility group protein B1 Regulatory T cells Immunosuppressive T lymphocyte the toxicity associated antigen 4 Forkhead winged helix transcription factor Anti-CD3 Interleukin-2 Interleukin-10 Interleukin-4 IFN-γ Polarization of cell function
CLC: R392
Type: Master's thesis
Year: 2007
Downloads: 71
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Abstract

Objective: high mobility group protein B1 (HMGB1) as a late inflammatory mediators mediated sepsis and other systemic inflammatory fatal. This experiment to be observed in vitro stimulation of regulatory T cells in mice indicators expression variation, and illustrates that HMGB1 affect the cellular immune function and its mechanism discussed. Designed to provide new ideas for further in vivo experiments and the late stage of infection prevention and treatment of severe sepsis. Methods: Mice were sacrificed sterile spleen mononuclear cells were isolated. ① using immunomagnetic beads separation to obtain normal BALB / c mouse spleen CD4 ~~ T CD4 to CD25 ~-T cells and CD4 ~ CD25 ~ Treg, identification of cell purity. ② respectively, a plant lectin (PHA), concanavalin A (Con A) and solid phase coated with anti-CD3 in three different stimulant-induced activation of Treg observed T lymphocyte toxicity associated antigen 4 (cytotoxic Tlymphocyte-associated antigen 4, CTLA-4) and forkhead wing helix transcription factor (forkhead / wingedhelix transcription factor, Foxp3) expression of the time - effect relationship. (3) will be of Treg in accordance with HMGB1 (100ng/ml) stimulation time is set at 24 h, 48 h group, 72 h group observed HMGB1 stimulation of CTLA-4 and Foxp3 expression time - effect relationship. Treg according to HMGB1 concentration is set to 0ng/ml group (control group), 10ng/ml, 100ng/ml group, 1000ng/ml, 72h with flow cytometry analysis of CTLA-4, Foxp3 expression and IL -10 secretion in rats (dose - response relationship). ④ were extracted from CD4 ~ CD25 ~ Treg cells and CD4 ~ CD25 ~-T set simple CD4 ~ CD25 ~ - control group, and the other in accordance with the CD25 ~: CD25 ~ - the proportion of divided 1:1 1:5, 1:10 group 1:20 by MTT colorimetric assay CD4 ~ CD25 ~ Treg cell ratio between CD4 ~ CD25 ~-T cells suppress. ⑤ HMGB1 stimulate Treg 72h. The stimulated cells according to CD25 ~~: CD25 ~~ - ratio of 1:1 and 1:5 group set. Each group is divided into CD25 ~ - group of non-stimulated the Treg group and HMGB1-Treg group. HMGB1 stimulate suppressive function of Treg cells by MTT colorimetric test analysis. ⑥ SYBR GREEN determination of HMGB1 stimulation with Foxp3 gene expression time, the dose - response relationship. ⑦ respectively specimens from the cell culture supernatant ELISA assay different cytokine changes. Results: 1. Mice CD4 ~ CD25 ~ Treg purity analysis: multiple cell-sorting experiments confirmed that normal mouse spleen mononuclear cells after MACS two points after the election, CD4 ~ CD25 ~ T cell purity 91.74% to 98.14 % CD25 cells is lower than 3%; CD4 ~ CD25 ~ - T cell purity of 88.73% to 93.85%, less than 1% of CD25 ~ cells. CD4 ~ CD25 ~ T cells obtained by Trypan Blue detect activity greater than 97%. 2.PHA no significant activation of CD4 ~ CD25 ~ Treg cells, CTLA-4 and Foxp3 expression compared with control group mean fluorescence intensity was no significant difference (P> 0.05). Con A-stimulated Treg CTLA-4 expression increase was transient, the duration of action of not more than 48 h, and no significant effect on Foxp3 expression. Anti-CD3 better activated Treg, performance CTLA-4 and Foxp3 expression in 24 ~ 72 h were significantly increased (P <0.05 or P <0.01), the 24,48 h upregulated more obvious (P < 0.01), and continues to stimulate after 72 h. 3.HMGB1 stimulate Treg of CTLA-4 expression in 24 ~ 72 h were decreased (P <0.05 or P <0.01), in which the role of 48,72 h downregulation is particularly evident (P <0.01); different doses of HMGB1 ( 10ng/ml, 100ng/ml, 1000ng/ml) stimulation can induce the expression of CTLA-4 decreased (P <0.05 or P <0.01) HMGB1 concentration in 1000ng/ml when and decreased expression of the most obvious. Foxp3 expression of CTLA-4 showing the same trends. The cell culture supernatant IL-10 levels presented HMGB1 dose dependency that HMGB1 stimulate the higher the concentration of IL-10 levels decreased more obvious. The 4.CD4 ~ CD25 ~-T cells showed activation proliferative response, but with the increase in Treg proportion of CD4 to CD25 ~-T cell proliferative response inhibition. When the the cell ratio reaches 1:1 performance for its inhibitory effect of about 90%. When joining HMGB1 (1000ng/ml) stimulation of Treg, CD4 ~ CD25 ~-T cell proliferation inhibition lessened. This phenomenon in the cell ratio of 1:1 and 1:5 groups showed the same trend. HMGB1 stimulation of Treg of Foxp3 mRNA expression was significantly lowered (P <0.05 or P <0.01), respectively, at 24 ~ 72h, to the role of 48,72 h decreased expression is particularly significant (P <0.01); give HMGB1 stimulation 72 h after, 10ng/ml, 100ng/ml at 1000ng/ml HMGB1 stimulation can induce Foxp3 expression was decreased (P <0.0 or P <0.01), of HMGB1 concentration of 1000 ng / ml Foxp3 downregulation of the most obvious. Compared with the normal control group, different time and dose HMGB1 in vitro stimulation of Treg cell culture supernatant IL-2 levels were not statistically different (P> 0.05). The cell culture supernatant IL-10 levels with HMGB1 stimulate prolonged concentration increased and decreased significantly (P <0.05 or P <0.01). The CD4 ~ CD25 ~-T cells Treg co-cultured CD4 to CD25 ~-T cell supernatants of IL-2 and IFN-γ levels decreased, while the addition of HMGB1 to stimulate the Treg increase with HMGB1 stimulate concentration both generate the degree of suppression was significantly reversed (P <0.05 or P <0.01). Compared with the control group, Treg group IL-4/IL-10 production was significantly increased IL-4/IL-10 levels of HMGB1-Treg group and a significant difference (P <0.05 or P <0.01), and in the HMGB1 concentration of 10ng/ml and 100ng/ml decreased more significantly (P <0.01). Conclusion: a two-step immunomagnetic beads separation CD4 ~ CD25 ~ Treg cells, high purity of the resulting cells had no effect on cell viability, and fully meet the requirements for further experiments. 2 in a variety of cell stimulating agent, anti-CD3 may be preferably activated Treg, offers the possibility for further experiments. 3.HMGB1 possible by downregulating cell surface molecules inhibit the expression and inhibit the secretion of cytokines are two ways to cut Treg function of Foxp3 gene and protein expression Conformance Statement HMGB1 by inhibiting key genes Foxp3 expression mechanism thereby affecting cell immune function. Treg immune nonreactive 4.HMGB1 vitro stimulation can not be changed, of Treg mediated Th1 to Th2 drift, but HMGB1 weakened Treg induce T lymphocyte proliferation, IL-2 generation and function of the polarization process.

The Potential Effect and Mechanism of High Mobility Group Box 1 Protein on Regulatory T Cell-mediated Immunosuppression in Mice

Abstract

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