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The Effect of High Mobility Group Box-1 Protein on Regulatory T Cells after Major Burns: Experimental and Clinical Studies

Author: HuangLiFeng
Tutor: ShengZhiYong;YaoYongMing
School: PLA Postgraduate Medical School
Course: Burn Surgery
Keywords: High mobility group box-1 protein Regulatory T cell Dendritic cell T lymphocyte Ethyl pyruvate Thermal injury Sepsis
CLC: R644
Type: PhD thesis
Year: 2008
Downloads: 221
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Abstract


Objective:Part 1. The present study was performed: (1) to investigate in vivo the changes in high mobility group box 1 protein (HMGB1) and its effect on the maturation of regulatory T cell (Treg) after severe thermal injury; (2) to identify if RAGE is the possible receptor on surface of Treg to bind HMGB1; (3) to study in vivo the effect of HMGB1 stimulated Treg on splenic dendritic cell (DC) and its potential mechanisms after severe thermal injury; (4) to investigate the effect of the HMGB1 stimulated Treg on splenic T lymphocyte-mediated immunity and its potential mechanism; and (5) to observe the protective effect of ethyl pyruvate (EP) on immune function and organ function in burn rats with delayed resuscitation.Part 2. The clinical study was performed to investigate the systemic release and kinetics of HMGB1, and to observe the potential roles of the maturation of regulatory T cell and T lymphocyte-mediated immunity in severely burned patients. Meanwhile, the significance of changes in plasma HMGB1 levels, Treg and T lymphocyte immunity and their relationship with sepsis as well as outcome of the patients were investigated in patients after major burns.Methods:1. A widely used technique for induction of full-thickness scald injury was used in the present study. For rats with burn injury, and the dorsal and lateral surfaces of animals were shaved under anesthesia. After being secured in a protective template with an opening corresponding to 30% of the total body surface area, and the exposed skin of the back was immersed in 99°C water for 12 s. Sham-injured rats were subjected to the same procedure except the temperature of the bath was of room temperature. 40 ml/kg lactated Ringer’s solution was administered i.p 6 hours after the injury for delayed resuscitation, followed by i.p injection of 4 ml at 12,24,36 and 48 h after burn injury, respectively.One hundred and thirty six male Wistar rats were randomly divided into five groups as follows: normal control group (8 rats), sham burn group (32 rats), burn group (32 rats), burn with ethyl pyruvate (EP) treatment group (32 rats), and burn with anti-RAGE (advanced glycation end-products) antibody treatment group (32 rats), and the later four groups were further divided into four subgroups of 8 rats each, which were sacrificed on postburn days (PBD) 1, 3, 5 and 7 respectively. EP was added to lactated Ringer’s solution (EP 28 mM) in EP treatment group.. Anti-RAGE antibody lmg/kg was given via dorsal penile vein at 6 h and 24 h respectively after burn injury in anti-RAGE antibody treatment group. Animals of all groups were sacrificed at designated time points, and blood as well as spleen samples were harvested aseptically to determine organ damage related variables and levels of various cytokines. Spleen was divided into two portions as following: one portion was used to detect gene and protein expression levels of HMGB1 and IL-10, and the other portion was used to procure Treg and DC by MACS microbeads and T cell by using column of nylon wool. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue, as well as activated NF-kB of T cell were determined by ELISA kits for rats. Gene expression was measured by real-time quantitative PCR taken GAPDH as the internal standard.2. One hundred and six patients with total burn surface area larger than 30% were included in the present study, and they were divided into three burn size groups: 30%-49% total body surface area (TBSA) burn (group I, n=41), 50%-69% TBSA burn (group II, n=34), and >70% TBSA burn (groupIII, n=31). According to wheather there was development of sepsis or not, patients were divided into sepsis group (n=59) and none-sepsis group (n=47); then the patients with sepsis were further divided into non-survival group (n=17) and survival group (n=42). Healthy volunteers served as normal controls (n=25). The periphery blood samples were collected on PBD 1,3,7,14, and 21. The blood samples were divided into two portions as following: one portion was used to procure T cell and Treg by MACS microbeads, and the other portion was used to detect levels of HMGB1 in serum. Cells were cultured, and phenotypes were analyzed by flow cytometry and the contents of cytokines released into supernatants were also determined. All cytokines in blood, supernatant and tissue were determined by ELISA kits for human. Gene expression was assessed by real-time quantitative PCR taken GAPDH as the internal standard.Results:Part 1. (1) The significantly elevated expression levels of splenic HMGB1 gene and protein and levels of serum HMGB1 were detected on PBD 1-7, and the elevation was significantly inhibited by the treatment of EP, but not by anti-RAGE antibody. (2) The expression levels of CTLA-4 on PBD 1-5 and levels of Foxp3 in Treg on PBD 1 -7 were significantly enhanced in burn rats in comparison to Treg from sham-injured rats. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly decrease the expression levels of CTLA-4 and Foxp3 of Treg. (3) The expression of RAGE on the surface of Treg from burned rats was found to be markedly elevated on PBD 1-7 compared with sham-injured rats, and it was not influenced by treatment of EP, but was blocked by treatment of anti-RAGE antibody. (4) The expression levels of splenic IL-10 mRNA and levels of IL-10 produced by Treg in supernatant were found to be significantly enhanced on PBD 1-7, and they were significantly inhibited by treatment of EP, but not by anti-RAGE antibody. (5) It was noted that DC expressed similar levels of CD80, strongly enhanced levels of CD86 and slightly enhanced levels of MHC class II on PBD 1-7 compared with DC from sham-injured rats. The capacity of DC to engulf dextran was decreased markedly after burn injury. Treatment with EP or anti-RAGE antibody to inhibit HMGB1 could significantly raise the expression levels of CD80, MHC class II of DC, but not the capacity of DC to engulf dextran. (6) The T cell proliferative activity in response to ConA in burn-injured rats was significantly suppressed on PBD 1-7 as compared with sham-injured rats, and gene/protein expressions of IL-2 and expressions of IL-2Ra of T cells in burn-injured rats were simultaneously suppressed after burn injury to different extent. EP or anti-RAGE antibody treatment could restore T cell proliferative activity response to Con A, gene/protein expressions of IL-2, and expression of IL-2Ra after burn injury. (7) Levels of IL-4 produced by T cell as a response to Con A increased markedly after burn injury, whereas levels of IFN-γlowered markedly, indicating that Th cells might shift into Th2 cells. Intervention with EP or anti-RAGE antibody could significantly inhibit the release of IL-4 and enhance the production of IFN-γby T cell response to Con A after thermal injury, indicating that EP or anti-RAGE antibody intervention might influence the polarization of T cells in animals subjected to thermal injury and induced Th cells to drift to Th1 cells. (8) The NF-kB activation of splenic T cell was downregulated significantly on PBD 1-7. Treatment with EP or anti-RAGE antibody could completely restored the NF-kB activity of splenic T cell after burn injury. (9) The serum ALT, AST, Cr, BUN and CK-MB levels were significantly elevated after burns, and treatment with EP or anti-RAGE antibody could inhibit these increase to different extent. (10) The levels of splenic HMGB1 were positively correlated with the levels of CTLA-4, Foxp3, RAGE, IL-10, sIL-2R, IL-4, and were negatively correlated with the levels of IL-2Rα, IL-2, IFN-γand T cell proliferative activity. Significant positive correlations were also found between the levels of serum HMGB1 and levels of serum ALT, AST, Cr, BUN and CK-MB.Part 2. (1) The gene/protein levels of blood HMGB1 were significantly elevated on PBD 1-21 in patients with various burn sizes compared with normal controls, and there were obvious differences between group I and group III. The blood HMGB1 mRNA and plasma HMGB1 levels were significantly higher in septic patients than those without sepsis on PBD 7-21. Among septic patients, the HMGB1 levels in the survival group were markedly lower than those with fatal outcome on PBD 3-21. (2) Increased expressions of CTLA-4 and Foxp3 on the surface of Treg from burned patients were found on PBD 1-21 compared with normal control group, and there were obvious differences among patients with various burn sizes. The expressions of CTLA-4 and Foxp3 were significantly higher in patients with serious burns at all time points, and they were even higher septic patients than those without sepsis on PBD 3-21. Among septic patients, the expressions of CTLA-4 and Foxp3 in the survival group were obviously lower than those with fatal outcome on PBD 3-21. (3) Elevated gene/protein expression of IL-10 and TGF-β1 in Treg from burned patients were detected on PBD 1-21 in comparison to normal controls, and there was obvious difference among patients with different extent of burn injury. The gene/protein expression of IL-10 and TGF-β1 in Treg were significantly higher in septic patients than those without sepsis on PBD 3-21. Among septic patients, expression levels of IL-10 and TGF-β1 in the survival group were obviously lower than those with fatal outcome on PBD 3-21. (4) The T cell proliferative activity in response to PHA and gene/protein expression of IL-2 of T cell in burn-injured patients were significantly suppressed on PBD 1-21 compared with normal control group, and there were obvious differences between group I and group III. The T cell proliferative activity in response to PHA and gene/protein expression of IL-2 of T cell were significantly lower in septic patients than those without sepsis on PBD 3-21. Among septic patients, the T cell proliferative activity in response to PHA and gene/protein expression of IL-2 in the survival group were markedly higher than those with fatal outcome on PBD 3-21. (5) Levels of IL-4 produced by T cell response to PHA increased markedly after burn injury, whereas levels of IFN-γdecreased markedly as compared with normal control group, and there were obvious differences between group I and groupIII, indicating that Th cells might have shifted to Th2 cells. The same results were found in non-septic patients and the survival group as compared with those with sepsis and the non-survival group on PBD 3-21. (6) No significant correlations were found between serum HMGB1 levels and the immune indexes of Treg and T cells. In addition, the immune indexes of Treg were positively correlated with levels of IL-4 produced by T cells, but were negatively correlated with levels of IL-2, IFN-γand T cell proliferative activity.Conclusion:Part 1. Serum and splenic HMGB1 were markedly up-regulated for a prolonged period but delayed after severe thermal injury. HMGB1 might play an important role in the development of excessive inflammatory response and subsequent multiple organ dysfunction syndrome. The excessive release of HMGB1 might stimulate splenic Treg to mature (which might mainly induced by binding RAGE on the surface of Treg), and further stimulate splenic DC to mature abnormally, thereby inducing suppression of proliferative activity of T lymphocytes and drifting of Th1 to Th2 after burn injury. NF-κB signaling might be involved in the normal Treg mediating suppression of T lymphocyte associated with excessive release of HMGB1. EP has a therapeutic potential for suppressing HMGB1-induced immune dysfunction and ameliorating multiple organ injury.Part 2. (1) Plasma HMGB1 was markedly elevated for a delayed but prolonged period after severe thermal injury in patients. Extensive burn injury could result in increased serum HMGB1 levels, and this phemenon was found to be associated with the development of sepsis and fatal outcome of burned patients.. (2) Severe burn injury per se could lead to activation and maturation of Treg cells, thus invoking its immunodepressive activity to full extent, resulting in immunosuppression. Similar to HMGB1, different degree of elevated levels of cytokines produced by Treg and activation markers on Treg surface might also take a role involved in the pathogenesis of sepsis and mortality in burn patients. (3) Suppression of T lymphocyte immune function and drifting of Th1 to Th2 were induced by severe burn injury. The immunosuppressive state of T lymphocytes was related to the extent of burn injury, development of sepsis and poor outcome in burned patients. (4) Plasma HMGB1 levels showed no significant correlations with the immune indexes of Treg and T lympholeukocyte, but the immune indexes of Treg were significantly correlated with T lymphocyte immune function, suggesting that Treg might have a potential effect on suppression of the proliferative activity, cytokine release of T cells and drifting Th1 to Th2 following major burns.

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CLC: > Medicine, health > Surgery > Traumatology > Burns and scalds ( burns )
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