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Tremella Polysaccharides attenuated sepsis through inhibiting abnormal CD4+CD25high regulatory T cells in mice
Cellular Immunology 288 (2014) 60–65
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Tremella Polysaccharides attenuated sepsis through inhibiting abnormal CD4+CD25high regulatory T cells in mice Zhen-wei Shi a,1, Yi Liu a,b,1, Yan Xu a, Yu-rong Hong a, Qi Liu a, Xiao-lu Li a, Zhi-gang Wang a,⇑ a b
Department of Nephrology, China Meitan General Hospital, Beijing 100028, PR China Hebei United University, Tangshan, Hebei 063000, PR China
a r t i c l e
i n f o
Article history: Received 22 October 2013 Accepted 14 February 2014 Available online 26 February 2014 Keywords: Tremella Polysaccharides CD4+CD25highTregs Sepsis Burn IL-10
a b s t r a c t Tremella Polysaccharides (TPS) have been reported to play an important role in regulating immune responses. Tregs are widely identified as the critical reason for immune dysfunction during sepsis. However, whether TPS could influence the immunomodulatory activities of Tregs in post-burn sepsis mice remains unclear. In this experiment, we researched the effects of TPS on peripheral blood Tregs in sepsis mouse induced by burn plus Pseudomonas aeruginosa infection. Results showed that TPS reversed the influences of Tregs on CD4+T cells proliferation and polarization and declined the level of IL-10 in burn plus P. aeruginosa infection mice. In addition, TPS notably reduced the mortality of post-burn sepsis mice. Therefore, TPS could inhibit the abnormal activities of CD4+CD25high Tregs in burn with P. aeruginosa infection mice, at least in part via inhibiting IL-10 secretion, and trigger a shift of Th2 to Th1 with activation of CD4+T cells in burn with P. aeruginosa infection mice. Ó 2014 Published by Elsevier Inc.
1. Introduction Tremella fuciformis Berk, also known as one tonic medicine, contains carbohydrates, proteins, vitamins and a variety of amino acids. According to pharmacological studies at home and abroad, the polysaccharides of Tremella (TPS) have varieties of bioactivities, such as immune regulation, anti-tumor, anti-oxidation of aging, reducing blood sugar, anticoagulant thrombosis, anti ulcer, promoting protein synthesis, anti-viral, promoting the growth of nerve cells and improving memory. Recently, studies of bioactivity of regulatory T cells and their potential mechanisms underlying immune disorders induced by acute injury, infection and severe sepsis or septic shock have aroused increasing interest [1,2]. Many investigators identified the fraction of circulating CD4+T cells that express high level of cell-surface CD25 as the human Treg population [3–5]. And the main function of Tregs is to inhibit T cell proliferation after injury in trauma patients and disrupt protective Th1-type cytokines production. The immune response to sepsis presents a complex balance between successful induction of pro-inflammatory responses and anti-inflammatory responses required to limit damage to host tissues. Tregs certainly play a vital role in controlling this
balance, but if the negative immunoregulation of Tregs was abnormally dominant, the balance would be disappeared, resulting in incurable immunosuppression would emerge. It is known to us all that late sepsis also presents an uncontrolled immunosuppression, which is the main promoter of multiple organ dysfunction syndrome (MODS) and even death. Therefore, it is perhaps the abnormal activities of Tregs that contributed to the severe sepsis and septic shock. In addition, it was reported that the suppressive activities of CD4+CD25high Tregs are partially through generating anti-inflammatory cytokines such as IL-10 [6]. Since TPS can improve the response function of T lymphocytes, and thereby promoting the cellular immune function and antagonizing immune suppression caused by cyclophosphamide [7]. We presumed that TPS could also attenuate the immune suppression during post -burn sepsis through inhibiting abnormal CD4+CD25high regulatory T cells in mice. As for the possible mechanisms involved in this process, one hypothesis that TPS may inhibit the abnormal activities of Tregs partially via downregulating IL-10 secretion was proposed in this study.
2. Materials and methods 2.1. Mice
⇑ Corresponding author. 1
E-mail address: [email protected] (Z.-g. Wang). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cellimm.2014.02.002 0008-8749/Ó 2014 Published by Elsevier Inc.
Male BALB/c mice employed in our experiment were purchased from Laboratory Animal Center of Chinese Academy of Medical
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Sciences, Beijing. They were 6-8 weeks old, weighing 20 ± 1 g. All mice were separated into cages and raised in a temperaturecontrolled room with 12 hour (h) light and 12 h darkness to acclimatize for at least 7 days before using. All experimental manipulations were strictly according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.2. Drugs Body of Tremella was purchased from a local market in Beijing City (China). Tremella Polysaccharides were extracted as previously described [8]. The aqueous extract was hydrolyzed with 0.1 mol/L hydrochloric acid at 90 °C for 3 h to obtain six hydrolysates. After hydrolysis, 20% NaOH was added to adjust the pH to 7.0, then they were centrifuged at 3000 rpm for 10 min, and the supernatants forced through a Cellulose Superfiltration System to give the high molecular weight fractions, which were then lyophilised. The dry samples were separately applied into a Sephadex G-150 column, and eluted with water (800 mL). Thus, TPS were obtained and used for further study. 2.3. Burn with Pseudomonas aeruginosa infection model A full-thickness dorsal scald burn injury was induced in mice. At the time of injury, the animals were randomized into control, burn, or burn plus infection groups. All mice were anesthetized with 60 mg/kg intraperitoneal sodium pentobarbital. The dorsal hair was clipped and the animals in the latter two groups were placed in a polystyrene template and subjected to a 10% full-thickness scald burn by immersion of the dorsal skin in a 100 °C water bath for 7 seconds. All animals received a 2-ml intraperitoneal injection of 0.9% NaCl for resuscitation. In all experiments except the dose response experiment, the burn plus infection mice received a topical inoculation of 1000 colony-forming units (CFU) P. aeruginosa (1 106 CFU/ml) on postburn day (PBD) 1. Mice were injected with TPS (50, 100, and 200 mg/kg) at PBD 1, 2, and 3, respectively. Mice that received anesthetic served as sham burn controls. 2.4. Experimental design Two hundred mice were separated into five groups as follows: sham burn group (40 mice), burn plus P. aeruginosa infection group (40 mice), burn plus P. aeruginosa infection with TPS (50 mg/kg) treatment group (40 mice), burn plus P. aeruginosa infection with TPS (100 mg/kg) treatment group (40 mice), and burn plus P. aeruginosa infection with TPS (200 mg/kg) treatment group (40 mice). The groups were further divided into four subgroups with eight mice in each, and mice were sacrificed on PBD1, 2, 3 and 4, respectively. Moreover, ten mice were taken as normal controls(the main parameters determined in the current study were found to be highly constant in eight mice in sham burn animals on PBD 0, thus the results were shown as sham burned mice)died on PBD 0. All mice were sacrificed at designated time points, then, peripheral blood samples were collected to obtain Tregs and T cells immediately. Another 72 mice were used to assess the effect of TPS on the mortality rate of mice after burn with P. aeruginosa infection. They were evenly divided into four groups as followed: burn plus P. aeruginosa infection group, burn plus P. aeruginosa infection with TPS (50, 100, 200 mg/kg) treatment group. 2.5. Reagents and kits RPMI 1640, fetal calf serum (FCS), glutamine, penicillin, streptomycin, and HEPES were purchased from TianRunShanda Biotech Co. Ltd, Beijing, China. Mouse CD4+CD25+Treg MicroBeads were
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purchased from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. Thiazolyl blue (MTT) and TritonX-100 were purchased from Sigma, St. Louis, MO. Antibodies used for flow cytometric analysis, including fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, phycoerythrin and (PE)-conjugated anti-mouse CD25, were purchased from BD/PharMingen, San Diego, CA. Functional purified grade anti-mouse CD3 and CD28 were purchased from BD/PharMingen, San Diego, CA. Enzyme-linked immunosorbent assay (ELISA) kits of murine IL-2, IL-4 and interferon (IFN)-c were purchased from Biosource, Worcester, MA. 2.6. Isolation of CD4+CD25highT cells from peripheral blood Peripheral blood was obtained in 1–2 ml of RPMI 1640 and treated with erythrocytolysin. After centrifugation, the sedimentary cells were collected. With the anti-Treg (CD4/CD25) MicroBeads and a MiniMACS™ separator, the cells were isolated according to manufacturer’s protocols. The cells expressing CD8a, CD11b, CD45R, CD49b and Ter-119 were wiped off from peripheral blood by a CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), then, enriched CD4+T cells were obtained. According to the expression of CD25, they were further divided into CD4+CD25+T cells and CD4+CD25 T cells. The flow cytometric analysis with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 staining was used to determine the purity of isolated CD4+CD25+T cells. And then, the CD4+CD25high T cells and CD4+CD25low T cells were separated by BD Aria II. 2.7. Depuration of CD4+T cells from peripheral blood The CD4+T Cell Isolation Kit (Miltenyi Biotec GmbH, Germany) was an indirect magnetic labeling system for the separation of untouched CD4+T cells from suspensions of murine peripheral blood cells. The cocktail of biotin-conjugated antibodies against CD8a (Ly-2), CD45R (B220), DX5, CD11b (Mac-1) and Ter-119, and anti-biotin MicroBeads can make non-CD4+T cells, i.e., cytotoxic T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages, granulocytes and erythroid cells have magnetic mark indirectly. So we achieved highly pure CD4+T cells by the depletion of magnetically labeled cells. 2.8. Cell culture Cells were cultured in RPMI 1640 supplemented with 2 nM 5 mM HEPES, and 100 U/ml penicillin/mg/ml streptomycin (all from BioWhittaker, Walkersville, MD), 0.5 mM sodium pyruvate, 0.05 mM nonessential amino acids (both from Life Technologies, Gaithersburg, MD), and 5% human AB serum (Gemini Bio-Products, Woodland, CA) in 96-well U-bottom plates (Costar, Corning, NY). The anti-CD3 (clone UCHT1 for plate-bound assays and clone Hit3a for soluble conditions) and antiCD28 (clone 28.2, at 5 mg/ml) Abs were purchased from BD PharMingen (San Diego, CA). The anti-CD28 (clone 3D10) was provided by Genetics Institute (Cambridge, MA). (In subsequent assays, the UCHT1 antiCD3 mAb gave the same results as the Hit3a mAb when tested in soluble form; data not shown.) For plate-bound anti-CD3 stimulation, 50 ml of the antiCD3 Ab diluted into PBS (Life Technologies) at the indicated concentration of 5.0 or 0.05 mg/ml were added to the every culture well, placed at 37 °C for 4 h, and then washed twice with PBS. The anti-PD-L1 Ab (2A3) (16) was used at 10 mg/ml. The mouse anti-human CTLA-4 Fab (mAb 20A) was provided by Genetics Institute and has been shown to functionally block interaction with B7-1/2 (17). Recombinant human IL-2 (IL-2; Teceleukin, obtained from the National Cancer Institute, Bethesda, MD) was added to those indicated cultures to a final concentration of 50 U/ml.
L-glutamine,
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2.9. Cytokine analyses by ELISA The supernatants that were removed before addition of [3H]thymidine and were diluted and analyzed on Immulon 4 ELISA plates (Dynex Technologies, Chantilly, VA) using the Ab pairs IFN-c (M-700A, M-701-B biotin; Endogen, Woburn, MA), IL-10 (18551D, 18562D-biotinylated; BD PharMingen), IL-2 (Biosource, Worcester, MA), and IL-4 (Biosource, Worcester, MA) developed with an avidin-peroxidase conjugate (1/10,000 dilution) (Sigma, St. Louis, MO) and tetramethylbenzidine peroxide substrate (Kirkegaard & Perry Laboratories, Gaithersburg MD). All samples were run in quadruplicates. 2.10. CD4+CD25highTregs proliferation/suppression study CD4+T cells were suspended in RPMI 1640 culture medium complemented with 10% FCS, 100 U/ml penicillin, 100 lg/ml streptomycin, and placed in 48-well round bottom plates for proliferation, giving the final cell density of 2 106/ml with CD4+CD25high Tregs in ratio of 1:10 (Tregs to CD4+T cells). They were treated with 1 lg/ml anti-CD3 for 68 h at 37 °C in 5% CO2/100% humidified air. 100 ll supernatant was achieved from each well, and 10 ll thiazolyl blue (MTT, 5 mg/ml) was added. After cultured for another 4 h, 100 ll Triton-Isop (10% TritonX-100, 50% Isopropanol, 0.01 mol/L HCl) were added into each well, then, the proliferation of CD4+T cells was evaluated by measuring optical density with a microplate reader (Spectra MR, Dynex, Chantilly, VA) at the wavelength of 540 nm (OD540 nm). All samples were run in quadruplicates. After being stimulated for 72 h with 200 lg/ml TPS, the proliferative activity of CD4+T cells was analyzed by above MTT test. In order to determine the effects of CD4+CD25high Tregs on effector cells, the cytokines of Th responses were measured. CD4+CD25high Tregs and CD4+T cells were cultured in cell-ratio of 1:10 for 72 h, after that we got the supernatants to measure various cytokines, including IL-4 and IFN-c with ELISA. All samples were run in quadruplicates. 2.11. Flow cytometry To observe the expression of CD4 and CD25 on the surface of Tregs, cells were stained with hamster anti-mouse CD4-FITC antibody and CD25-PE antibody for 30 min at 4 °C in darkness, washed twice, then analyzed by flow cytometry using a FACScan (BD Biosciences, Mountain View, CA). 2.12. Statistical analysis Data were expressed as mean ± standard deviation (SD). With software of SPSS14.0, data were analyzed and tested by analysis of a one-way variance (ANOVA) test and log-rank test. P values less than 0.05 were deemed to be significant. 3. Results 3.1. The changes of IL-10 secreted by CD4+CD25high Tregs induced by TPS in model mice CD4+CD25high Tregs isolated from peripheral blood of each group were cultured in vitro. The level of IL-10 in the culture supernatant was detected during PBD 0 to 4 and recorded in Fig. 1B. The level of IL-10 in the burn group was abnormally increased. However, TPS played a great role in decreasing the level of IL-10 during PBD 1 to 4 in a dose-dependent way (P < 0.05).
3.2. The influences of TPS on the activation and differentiation of CD4+T cell collected from sepsis mice To clarify the mechanism of how TPS affected the activities of effector T cells in burn plus infection mice, CD4+T-cell proliferation and polarization which can be described through detecting cytokines were analyzed ex vivo. Compared with burn plus P. aeruginosa infection group, the proliferative activity of CD4+T cell in burn plus P. aeruginosa infection with TPS treatment group showed a notably rebound during PBD 0 to 4 (P < 0.05). And the promoted effects of TPS manifested a dose-dependent fashion (P < 0.05). The levels of IFN-c which represents Th1 cytokines and IL-4 which belongs to Th2 cytokines were detected in order to identify the differentiation of CD4+T cells. After burn injury with P. aeruginosa infection, the levels of IL-4 produced by CD4+T cells were significantly increased (P < 0.05), in contrast, the levels of IFN-c were remarkably decreased (P < 0.05) compared with sham burn group, demonstrating that CD4+T cells had evolved into Th2 response. TPS could suppress the increase of IL-4 (P < 0.05) significantly as well as the reduction of IFN-c (P < 0.05) in thermal injury with P. aeruginosa infection mice, showing that TPS induce CD4+T cells to shift to Th1 cells. At the same time, the level of IL-2 secreted by CD4+T cells was also inhibited to a certain extent following burn injury with P. aeruginosa infection (P < 0.05), while restored by TPS treatment (P < 0.05; Fig. 2). 3.3. TPS promoted CD4+T cells proliferation and oriented CD4+T cells to Th1 differentiation through suppressing CD4+CD25high Tregs which works partially through secreting IL-10 in vitro To explore whether Tregs involved in the variation of CD4+T cells in sepsis mice induced by burn plus P. aeruginosa infection, mixed lymphocyte reaction (MLR) in terms of CD4+CD25high T cells isolated from sepsis mice and normal CD4+T cells was performed. It was found that the proliferation of CD4+T cells was inhibited, and the level of IL-4 (Th2 cytokine) was increased while the level of IFN-c (Th1 cytokine) was decreased. Adding anti-10 antibody to the MLR partially countervailed the variations of CD4+T cells proliferation as well as the cytokines resulted from Tregs (P < 0.05). Furthermore, to verify the role of TPS in regulating the immune activities of Tregs, CD4+CD25highT cells were stimulated with TPS (200 lg/ml) before MLR. We found that TPS significantly reversed the changes of CD4+T cells proliferation and differentiation induced by Tregs as shown in Fig. 3 (P < 0.05). 3.4. TPS could improve the lethality of mice suffered from burn plus P. aeruginosa infection The abnormal activities of Tregs played a critical role in sever sepsis, and TPS could reverse the abnormal reactions of Tregs, we assumed that TPS could decrease the mortality rate of scald mice with intraperitoneal inoculation of P. aeruginosa. Results showed that by intraperitoneal injection of TPS (50 mg/kg) on PBD 1, 2, and 3, the mortality rate was slightly declined (Fig. 4). In contrast, given TPS (100 and 200 mg/kg) on PBD 2, 3 and 4 after P. aeruginosa injection to 10% TBSA scald mice can notably reduce the mortality rates, thus the protective effect of TPS was manifested in a dosedependent pattern (Fig. 4). 4. Discussion Even though lots of progresses in antibiotic and other supportive therapies were achieved over the past 20 years, sepsis remains to be one leading cause of death in the intensive care unit. It is now believed that those patients have features consistent with
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Fig. 1. The phenotypic characters of Tregs and the variation of IL-10 produced by Tregs after treatment with TPS. (A) Lymphocytes gated according to forward and side scatter from peripheral blood were screened by flow cytometry for the presence of CD4 and CD25 molecules. Representative FACS staining from one control subject is shown and gates corresponding to naive. R1 show that CD4+CD25high Tregs were isolated from lymphocytes. Data presented the results of a representative one out of seven experiments performed under the same conditions. (B) The level of IL-10 secreted by CD4+CD25high Tregs was reduced by administration of TPS in comparison to burn plus infection group. In addition, the down-regulatory effect of TPS on IL-10 was dose-dependent. ⁄P < 0.05, compared to burn sepsis; P < 0.05 compared to TPS (50 mg/kg).
Fig. 2. The effects of TPS on the changes of CD4+ T cells polarization and proliferation. CD4+ T cells isolated from each group were cultured in vitro. OD value was detected for describe the proliferation of CD4+T cells. At the same time, the levels of IL-2, IFN-c and IL-4 were determined from culture supernatants by ELISA, which could demonstrate the Th1/Th2 polarization. The proliferative activity of CD4+ T cells obtained from sepsis mice with administration of TPS were restored compared with burn plus infection group. Meanwhile, TPS reversed the Th2 polarization in sepsis mice. Furthermore, the effects of TPS also showed to have a dose-dependency. Data were expressed as mean ± SD of samples. ⁄P < 0.05, compared to burn sepsis; P < 0.05 compared to TPS (50 mg/kg).
immunosuppression [9,10]. Immunosuppression in sepsis, sometimes referred to as ‘‘immunoparalysis’’, is characterized by a number of factors, including the monocyte deactivation, tolerance to endotoxin, impairment of neutrophil function, lymphocyte dysfunction, and apoptosis [11]. Recent experiments have focused on understanding the presence and function of Tregs during experimental and clinical sepsis [12,13]. Moreover, an increased percentage of Tregs has been observed in the circulation of patients with septic shock, and the persistence of increased Tregs has been associated with a poor long-term prognosis [14]. Thus, the abnormal changes of Tregs during sepsis may be an important pathogenic
factor. In addition, the precise mechanisms of suppression by Tregs remain to be determined, these cells can suppress immune cells functions either directly through cell-cell contact or indirectly through the secretion of anti-inflammatory mediators, such as IL-10 [15]. IL-10 as one kind of anti-inflammatory cytokine could suppress the production of pro-inflammatory cytokines such as IL-12 required for Th1 development by dendritic cells and macrophages [16]. In this investigation, IL-10 produced by Tregs isolated from post-burn sepsis mice was significantly increased. Moreover, the proliferation of CD4+T cells was notably inhibited in burn plus infection mice, and the results in vitro verified that Tregs isolated
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Fig. 3. The effects of TPS on the changes of activation and polarization of CD4+ T cells induced by Tregs which works partially via secreting IL-10 in vitro. Compared to the effects of CD4+CD25high Tregs isolated from sepsis mice on inhibiting CD4+T cells activation and Th1 polarization, while anti-IL-10 antibody weakened the effects of CD4+CD25high Tregs on CD4+ T cells. Meanwhile, TPS reversed the inhibited role of Tregs on CD4+ T cells and triggered a shift from Th2 to Th1. Data were expressed as mean ± SD of samples. ⁄P < 0.05, compared to CD4+CD25high T cells+CD4+T cells.
from scald mice with infection indeed significantly inhibited the proliferation of CD4+T cells. Furthermore, anti-IL-10 antibody partially blocked the inhibiting effects of Tregs on CD4+T cells. Meanwhile, the Th2 cytokine-IL-4 detected in this study was abnormally enhanced after burn plus P. aeruginosa infection, demonstrating that the balance between Th1 and Th2 responses was destroyed in late sepsis. Thus, the abnormal activities of Tregs actually played a harmful role in the immunosuppression occurred in late sepsis. In Traditional Chinese Medicine, co-administration of immunomodulatory agents and chemotherapy drugs is typically used to improve the immunity potential. In many oriental countries, several immunoceuticals composed of polysaccharides are widely used, such as lentinan, schizophyllan and krestin. Those immunomodulatory agents often work by inducing lymphocyte proliferation and cytokine production, and they have protective effects toward the hematopoietic function of bone marrow and immune organs [17]. TPS was showed to have immunoregulatory functions and could improve immunosuppression resulted from some chemotherapeutics. While, it is unclear that whether TPS could play roles in the abnormal activities of Tregs that improved sepsis. Herein, we found that TPS could suppress the function of CD4+CD25highTregs, thus regulating immune functions of T lymphocyte and shift of Th2 to Th1, which were isolated from mice suffered
Fig. 4. The effect of TPS improve survival rate in a murine sepsis model in dosedependent fashion. These mice received intraperitoneal injection of TPS (50, 100, or 200 mg/kg) on day 1, 2 and 3 after burn. ⁄P < 0.05 compared with the sepsis group by the log-rank test. P < 0.05 compared with mice treated by TPS (50 mg/kg) by the log-rank test.
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from burn and P. aeruginosa infection. Meanwhile, TPS significantly lowered the level of IL-10 produced by Tregs, and thereby weakened the excessively suppressed effects of Tregs. Now that TPS could exert such effects on Tregs which is closely associated with sepsis, we assumed that TPS could improve sepsis in the burn plus infection model. Fortunately, we found that the mortality was inhibited significantly by TPS, and the therapeutic effect of TPS manifested in a dose-dependent manner (Fig. 4). TPS have been shown to have great potential value in clinic especially in patients with sepsis in the intensive care unit. In conclusion, TPS could attenuate sepsis via inhibiting the abnormal activity of CD4+CD25highTreg, at least in part through inhibiting IL-10 production, and inducing a new balance of Th2/Th1 by activating CD4+T cell-mediated immunity in burned mice plus P. aeruginosa infection. Acknowledgment This study was supported, in part, by Grants from China Meitan General Hospital-Level Project in China Meitan general hospital (No. MT 2010-16). References [1] K.H.G. Mills, Regulatory T cells: friend or foe in immunity to infection?, Nat Rev. Immunol. 4 (2004) 841–855. [2] Y. Belkaid, B.T. Rouse, Natural regulatory T cells in infectious disease, Nat. Immunol. 6 (2005) 353–360. [3] C. Baecher-Allan, J.A. Brown, G.J. Freeman, et al., CD4+ CD25high regulatory cells in human peripheral blood, J. Immunol. 167 (2001) 1245–1253.
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[4] K. Wing, E. Suri-Payer, A. Rudin, CD4+ CD25+-regulatory T cells from mouse to man, Scand. J. Immunol. 62 (2005) 1–15. [5] C. Baecher-Allan, V. Viglietta, D.A. Hafler, Human CD4+ CD25+ regulatory T cells, Seminars in Immunology, 16, Academic Press, 2004, pp. 89–98. [6] C.L. Maynard, L.E. Harrington, K.M. Janowski, et al., Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3 precursor cells in the absence of interleukin 10, Nat. Immunol. 8 (2007) 931–941. [7] N.Y. Chen, T.H. Hsu, F.Y. Lin, et al., Effects on cytokine-stimulating activities of EPS from Tremella mesenterica with various carbon sources, Food Chem. 99 (2006) 92–97. [8] R.Z. Jiang, Y. Wang, H.M. Luo, et al., Effect of the molecular mass of Tremella polysaccharides on accelerated recovery from cyclophosphamide-induced leucopenia in rats, Molecules 17 (2012) 3609–3617. [9] R.S. Hotchkiss, I.E. Karl, The pathophysiology and treatment of sepsis, N. Engl. J. Med. 348 (2003) 138–150. [10] R.S. Munford, J. Pugin, Normal responses to injury prevent systemic inflammation and can be immunosuppressive, Am. J. Respir. Crit. Care Med. 163 (2001) 316–321. [11] T.S. Wang, J.C. Deng, Molecular and cellular aspects of sepsis-induced immunosuppression, J. Mol. Med. 86 (2008) 495–506. [12] P.O. Scumpia, M.J. Delano, K.M. Kelly, et al., Increased natural CD4+ CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis, J. Immunol. 177 (2006) 7943–7949. [13] F. Venet, C.S. Chung, G. Monneret, et al., Regulatory T cell populations in sepsis and trauma, J. Leukocyte Biol. 83 (2008) 523–535. [14] K.A. Cavassani, W.F. Carson, A.P. Moreira, et al., The post sepsis-induced expansion and enhanced function of regulatory T cells create an environment to potentiate tumor growth, Blood 115 (2010) 4403–4411. [15] A.M. Miller, K. Lundberg, V. Özenci, et al., CD4+ CD25high T cells are enriched in the tumor and peripheral blood of prostate cancer patients, J. Immunol. 177 (2006) 7398–7405. [16] A. O’Garra, P. Vieira, Regulatory T cells and mechanisms of immune system control, Nat. Med. 10 (2004) 801–805. [17] H. Wang, M. Wang, J. Chen, et al., A polysaccharide from Strongylocentrotus nudus eggs protects against myelosuppression and immunosuppression in cyclophosphamide-treated mice, Int. Immunopharmacol. 11 (2011) 1946– 1953.
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Tremella Polysaccharides attenuated sepsis through inhibiting abnormal CD4+CD25high regulatory T cells in mice Zhen-wei Shi a,1, Yi Liu a,b,1, Yan Xu a, Yu-rong Hong a, Qi Liu a, Xiao-lu Li a, Zhi-gang Wang a,⇑ a b
Department of Nephrology, China Meitan General Hospital, Beijing 100028, PR China Hebei United University, Tangshan, Hebei 063000, PR China
a r t i c l e
i n f o
Article history: Received 22 October 2013 Accepted 14 February 2014 Available online 26 February 2014 Keywords: Tremella Polysaccharides CD4+CD25highTregs Sepsis Burn IL-10
a b s t r a c t Tremella Polysaccharides (TPS) have been reported to play an important role in regulating immune responses. Tregs are widely identified as the critical reason for immune dysfunction during sepsis. However, whether TPS could influence the immunomodulatory activities of Tregs in post-burn sepsis mice remains unclear. In this experiment, we researched the effects of TPS on peripheral blood Tregs in sepsis mouse induced by burn plus Pseudomonas aeruginosa infection. Results showed that TPS reversed the influences of Tregs on CD4+T cells proliferation and polarization and declined the level of IL-10 in burn plus P. aeruginosa infection mice. In addition, TPS notably reduced the mortality of post-burn sepsis mice. Therefore, TPS could inhibit the abnormal activities of CD4+CD25high Tregs in burn with P. aeruginosa infection mice, at least in part via inhibiting IL-10 secretion, and trigger a shift of Th2 to Th1 with activation of CD4+T cells in burn with P. aeruginosa infection mice. Ó 2014 Published by Elsevier Inc.
1. Introduction Tremella fuciformis Berk, also known as one tonic medicine, contains carbohydrates, proteins, vitamins and a variety of amino acids. According to pharmacological studies at home and abroad, the polysaccharides of Tremella (TPS) have varieties of bioactivities, such as immune regulation, anti-tumor, anti-oxidation of aging, reducing blood sugar, anticoagulant thrombosis, anti ulcer, promoting protein synthesis, anti-viral, promoting the growth of nerve cells and improving memory. Recently, studies of bioactivity of regulatory T cells and their potential mechanisms underlying immune disorders induced by acute injury, infection and severe sepsis or septic shock have aroused increasing interest [1,2]. Many investigators identified the fraction of circulating CD4+T cells that express high level of cell-surface CD25 as the human Treg population [3–5]. And the main function of Tregs is to inhibit T cell proliferation after injury in trauma patients and disrupt protective Th1-type cytokines production. The immune response to sepsis presents a complex balance between successful induction of pro-inflammatory responses and anti-inflammatory responses required to limit damage to host tissues. Tregs certainly play a vital role in controlling this
balance, but if the negative immunoregulation of Tregs was abnormally dominant, the balance would be disappeared, resulting in incurable immunosuppression would emerge. It is known to us all that late sepsis also presents an uncontrolled immunosuppression, which is the main promoter of multiple organ dysfunction syndrome (MODS) and even death. Therefore, it is perhaps the abnormal activities of Tregs that contributed to the severe sepsis and septic shock. In addition, it was reported that the suppressive activities of CD4+CD25high Tregs are partially through generating anti-inflammatory cytokines such as IL-10 [6]. Since TPS can improve the response function of T lymphocytes, and thereby promoting the cellular immune function and antagonizing immune suppression caused by cyclophosphamide [7]. We presumed that TPS could also attenuate the immune suppression during post -burn sepsis through inhibiting abnormal CD4+CD25high regulatory T cells in mice. As for the possible mechanisms involved in this process, one hypothesis that TPS may inhibit the abnormal activities of Tregs partially via downregulating IL-10 secretion was proposed in this study.
2. Materials and methods 2.1. Mice
⇑ Corresponding author. 1
E-mail address: [email protected] (Z.-g. Wang). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.cellimm.2014.02.002 0008-8749/Ó 2014 Published by Elsevier Inc.
Male BALB/c mice employed in our experiment were purchased from Laboratory Animal Center of Chinese Academy of Medical
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Sciences, Beijing. They were 6-8 weeks old, weighing 20 ± 1 g. All mice were separated into cages and raised in a temperaturecontrolled room with 12 hour (h) light and 12 h darkness to acclimatize for at least 7 days before using. All experimental manipulations were strictly according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. 2.2. Drugs Body of Tremella was purchased from a local market in Beijing City (China). Tremella Polysaccharides were extracted as previously described [8]. The aqueous extract was hydrolyzed with 0.1 mol/L hydrochloric acid at 90 °C for 3 h to obtain six hydrolysates. After hydrolysis, 20% NaOH was added to adjust the pH to 7.0, then they were centrifuged at 3000 rpm for 10 min, and the supernatants forced through a Cellulose Superfiltration System to give the high molecular weight fractions, which were then lyophilised. The dry samples were separately applied into a Sephadex G-150 column, and eluted with water (800 mL). Thus, TPS were obtained and used for further study. 2.3. Burn with Pseudomonas aeruginosa infection model A full-thickness dorsal scald burn injury was induced in mice. At the time of injury, the animals were randomized into control, burn, or burn plus infection groups. All mice were anesthetized with 60 mg/kg intraperitoneal sodium pentobarbital. The dorsal hair was clipped and the animals in the latter two groups were placed in a polystyrene template and subjected to a 10% full-thickness scald burn by immersion of the dorsal skin in a 100 °C water bath for 7 seconds. All animals received a 2-ml intraperitoneal injection of 0.9% NaCl for resuscitation. In all experiments except the dose response experiment, the burn plus infection mice received a topical inoculation of 1000 colony-forming units (CFU) P. aeruginosa (1 106 CFU/ml) on postburn day (PBD) 1. Mice were injected with TPS (50, 100, and 200 mg/kg) at PBD 1, 2, and 3, respectively. Mice that received anesthetic served as sham burn controls. 2.4. Experimental design Two hundred mice were separated into five groups as follows: sham burn group (40 mice), burn plus P. aeruginosa infection group (40 mice), burn plus P. aeruginosa infection with TPS (50 mg/kg) treatment group (40 mice), burn plus P. aeruginosa infection with TPS (100 mg/kg) treatment group (40 mice), and burn plus P. aeruginosa infection with TPS (200 mg/kg) treatment group (40 mice). The groups were further divided into four subgroups with eight mice in each, and mice were sacrificed on PBD1, 2, 3 and 4, respectively. Moreover, ten mice were taken as normal controls(the main parameters determined in the current study were found to be highly constant in eight mice in sham burn animals on PBD 0, thus the results were shown as sham burned mice)died on PBD 0. All mice were sacrificed at designated time points, then, peripheral blood samples were collected to obtain Tregs and T cells immediately. Another 72 mice were used to assess the effect of TPS on the mortality rate of mice after burn with P. aeruginosa infection. They were evenly divided into four groups as followed: burn plus P. aeruginosa infection group, burn plus P. aeruginosa infection with TPS (50, 100, 200 mg/kg) treatment group. 2.5. Reagents and kits RPMI 1640, fetal calf serum (FCS), glutamine, penicillin, streptomycin, and HEPES were purchased from TianRunShanda Biotech Co. Ltd, Beijing, China. Mouse CD4+CD25+Treg MicroBeads were
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purchased from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. Thiazolyl blue (MTT) and TritonX-100 were purchased from Sigma, St. Louis, MO. Antibodies used for flow cytometric analysis, including fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, phycoerythrin and (PE)-conjugated anti-mouse CD25, were purchased from BD/PharMingen, San Diego, CA. Functional purified grade anti-mouse CD3 and CD28 were purchased from BD/PharMingen, San Diego, CA. Enzyme-linked immunosorbent assay (ELISA) kits of murine IL-2, IL-4 and interferon (IFN)-c were purchased from Biosource, Worcester, MA. 2.6. Isolation of CD4+CD25highT cells from peripheral blood Peripheral blood was obtained in 1–2 ml of RPMI 1640 and treated with erythrocytolysin. After centrifugation, the sedimentary cells were collected. With the anti-Treg (CD4/CD25) MicroBeads and a MiniMACS™ separator, the cells were isolated according to manufacturer’s protocols. The cells expressing CD8a, CD11b, CD45R, CD49b and Ter-119 were wiped off from peripheral blood by a CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), then, enriched CD4+T cells were obtained. According to the expression of CD25, they were further divided into CD4+CD25+T cells and CD4+CD25 T cells. The flow cytometric analysis with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 staining was used to determine the purity of isolated CD4+CD25+T cells. And then, the CD4+CD25high T cells and CD4+CD25low T cells were separated by BD Aria II. 2.7. Depuration of CD4+T cells from peripheral blood The CD4+T Cell Isolation Kit (Miltenyi Biotec GmbH, Germany) was an indirect magnetic labeling system for the separation of untouched CD4+T cells from suspensions of murine peripheral blood cells. The cocktail of biotin-conjugated antibodies against CD8a (Ly-2), CD45R (B220), DX5, CD11b (Mac-1) and Ter-119, and anti-biotin MicroBeads can make non-CD4+T cells, i.e., cytotoxic T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages, granulocytes and erythroid cells have magnetic mark indirectly. So we achieved highly pure CD4+T cells by the depletion of magnetically labeled cells. 2.8. Cell culture Cells were cultured in RPMI 1640 supplemented with 2 nM 5 mM HEPES, and 100 U/ml penicillin/mg/ml streptomycin (all from BioWhittaker, Walkersville, MD), 0.5 mM sodium pyruvate, 0.05 mM nonessential amino acids (both from Life Technologies, Gaithersburg, MD), and 5% human AB serum (Gemini Bio-Products, Woodland, CA) in 96-well U-bottom plates (Costar, Corning, NY). The anti-CD3 (clone UCHT1 for plate-bound assays and clone Hit3a for soluble conditions) and antiCD28 (clone 28.2, at 5 mg/ml) Abs were purchased from BD PharMingen (San Diego, CA). The anti-CD28 (clone 3D10) was provided by Genetics Institute (Cambridge, MA). (In subsequent assays, the UCHT1 antiCD3 mAb gave the same results as the Hit3a mAb when tested in soluble form; data not shown.) For plate-bound anti-CD3 stimulation, 50 ml of the antiCD3 Ab diluted into PBS (Life Technologies) at the indicated concentration of 5.0 or 0.05 mg/ml were added to the every culture well, placed at 37 °C for 4 h, and then washed twice with PBS. The anti-PD-L1 Ab (2A3) (16) was used at 10 mg/ml. The mouse anti-human CTLA-4 Fab (mAb 20A) was provided by Genetics Institute and has been shown to functionally block interaction with B7-1/2 (17). Recombinant human IL-2 (IL-2; Teceleukin, obtained from the National Cancer Institute, Bethesda, MD) was added to those indicated cultures to a final concentration of 50 U/ml.
L-glutamine,
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2.9. Cytokine analyses by ELISA The supernatants that were removed before addition of [3H]thymidine and were diluted and analyzed on Immulon 4 ELISA plates (Dynex Technologies, Chantilly, VA) using the Ab pairs IFN-c (M-700A, M-701-B biotin; Endogen, Woburn, MA), IL-10 (18551D, 18562D-biotinylated; BD PharMingen), IL-2 (Biosource, Worcester, MA), and IL-4 (Biosource, Worcester, MA) developed with an avidin-peroxidase conjugate (1/10,000 dilution) (Sigma, St. Louis, MO) and tetramethylbenzidine peroxide substrate (Kirkegaard & Perry Laboratories, Gaithersburg MD). All samples were run in quadruplicates. 2.10. CD4+CD25highTregs proliferation/suppression study CD4+T cells were suspended in RPMI 1640 culture medium complemented with 10% FCS, 100 U/ml penicillin, 100 lg/ml streptomycin, and placed in 48-well round bottom plates for proliferation, giving the final cell density of 2 106/ml with CD4+CD25high Tregs in ratio of 1:10 (Tregs to CD4+T cells). They were treated with 1 lg/ml anti-CD3 for 68 h at 37 °C in 5% CO2/100% humidified air. 100 ll supernatant was achieved from each well, and 10 ll thiazolyl blue (MTT, 5 mg/ml) was added. After cultured for another 4 h, 100 ll Triton-Isop (10% TritonX-100, 50% Isopropanol, 0.01 mol/L HCl) were added into each well, then, the proliferation of CD4+T cells was evaluated by measuring optical density with a microplate reader (Spectra MR, Dynex, Chantilly, VA) at the wavelength of 540 nm (OD540 nm). All samples were run in quadruplicates. After being stimulated for 72 h with 200 lg/ml TPS, the proliferative activity of CD4+T cells was analyzed by above MTT test. In order to determine the effects of CD4+CD25high Tregs on effector cells, the cytokines of Th responses were measured. CD4+CD25high Tregs and CD4+T cells were cultured in cell-ratio of 1:10 for 72 h, after that we got the supernatants to measure various cytokines, including IL-4 and IFN-c with ELISA. All samples were run in quadruplicates. 2.11. Flow cytometry To observe the expression of CD4 and CD25 on the surface of Tregs, cells were stained with hamster anti-mouse CD4-FITC antibody and CD25-PE antibody for 30 min at 4 °C in darkness, washed twice, then analyzed by flow cytometry using a FACScan (BD Biosciences, Mountain View, CA). 2.12. Statistical analysis Data were expressed as mean ± standard deviation (SD). With software of SPSS14.0, data were analyzed and tested by analysis of a one-way variance (ANOVA) test and log-rank test. P values less than 0.05 were deemed to be significant. 3. Results 3.1. The changes of IL-10 secreted by CD4+CD25high Tregs induced by TPS in model mice CD4+CD25high Tregs isolated from peripheral blood of each group were cultured in vitro. The level of IL-10 in the culture supernatant was detected during PBD 0 to 4 and recorded in Fig. 1B. The level of IL-10 in the burn group was abnormally increased. However, TPS played a great role in decreasing the level of IL-10 during PBD 1 to 4 in a dose-dependent way (P < 0.05).
3.2. The influences of TPS on the activation and differentiation of CD4+T cell collected from sepsis mice To clarify the mechanism of how TPS affected the activities of effector T cells in burn plus infection mice, CD4+T-cell proliferation and polarization which can be described through detecting cytokines were analyzed ex vivo. Compared with burn plus P. aeruginosa infection group, the proliferative activity of CD4+T cell in burn plus P. aeruginosa infection with TPS treatment group showed a notably rebound during PBD 0 to 4 (P < 0.05). And the promoted effects of TPS manifested a dose-dependent fashion (P < 0.05). The levels of IFN-c which represents Th1 cytokines and IL-4 which belongs to Th2 cytokines were detected in order to identify the differentiation of CD4+T cells. After burn injury with P. aeruginosa infection, the levels of IL-4 produced by CD4+T cells were significantly increased (P < 0.05), in contrast, the levels of IFN-c were remarkably decreased (P < 0.05) compared with sham burn group, demonstrating that CD4+T cells had evolved into Th2 response. TPS could suppress the increase of IL-4 (P < 0.05) significantly as well as the reduction of IFN-c (P < 0.05) in thermal injury with P. aeruginosa infection mice, showing that TPS induce CD4+T cells to shift to Th1 cells. At the same time, the level of IL-2 secreted by CD4+T cells was also inhibited to a certain extent following burn injury with P. aeruginosa infection (P < 0.05), while restored by TPS treatment (P < 0.05; Fig. 2). 3.3. TPS promoted CD4+T cells proliferation and oriented CD4+T cells to Th1 differentiation through suppressing CD4+CD25high Tregs which works partially through secreting IL-10 in vitro To explore whether Tregs involved in the variation of CD4+T cells in sepsis mice induced by burn plus P. aeruginosa infection, mixed lymphocyte reaction (MLR) in terms of CD4+CD25high T cells isolated from sepsis mice and normal CD4+T cells was performed. It was found that the proliferation of CD4+T cells was inhibited, and the level of IL-4 (Th2 cytokine) was increased while the level of IFN-c (Th1 cytokine) was decreased. Adding anti-10 antibody to the MLR partially countervailed the variations of CD4+T cells proliferation as well as the cytokines resulted from Tregs (P < 0.05). Furthermore, to verify the role of TPS in regulating the immune activities of Tregs, CD4+CD25highT cells were stimulated with TPS (200 lg/ml) before MLR. We found that TPS significantly reversed the changes of CD4+T cells proliferation and differentiation induced by Tregs as shown in Fig. 3 (P < 0.05). 3.4. TPS could improve the lethality of mice suffered from burn plus P. aeruginosa infection The abnormal activities of Tregs played a critical role in sever sepsis, and TPS could reverse the abnormal reactions of Tregs, we assumed that TPS could decrease the mortality rate of scald mice with intraperitoneal inoculation of P. aeruginosa. Results showed that by intraperitoneal injection of TPS (50 mg/kg) on PBD 1, 2, and 3, the mortality rate was slightly declined (Fig. 4). In contrast, given TPS (100 and 200 mg/kg) on PBD 2, 3 and 4 after P. aeruginosa injection to 10% TBSA scald mice can notably reduce the mortality rates, thus the protective effect of TPS was manifested in a dosedependent pattern (Fig. 4). 4. Discussion Even though lots of progresses in antibiotic and other supportive therapies were achieved over the past 20 years, sepsis remains to be one leading cause of death in the intensive care unit. It is now believed that those patients have features consistent with
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Fig. 1. The phenotypic characters of Tregs and the variation of IL-10 produced by Tregs after treatment with TPS. (A) Lymphocytes gated according to forward and side scatter from peripheral blood were screened by flow cytometry for the presence of CD4 and CD25 molecules. Representative FACS staining from one control subject is shown and gates corresponding to naive. R1 show that CD4+CD25high Tregs were isolated from lymphocytes. Data presented the results of a representative one out of seven experiments performed under the same conditions. (B) The level of IL-10 secreted by CD4+CD25high Tregs was reduced by administration of TPS in comparison to burn plus infection group. In addition, the down-regulatory effect of TPS on IL-10 was dose-dependent. ⁄P < 0.05, compared to burn sepsis; P < 0.05 compared to TPS (50 mg/kg).
Fig. 2. The effects of TPS on the changes of CD4+ T cells polarization and proliferation. CD4+ T cells isolated from each group were cultured in vitro. OD value was detected for describe the proliferation of CD4+T cells. At the same time, the levels of IL-2, IFN-c and IL-4 were determined from culture supernatants by ELISA, which could demonstrate the Th1/Th2 polarization. The proliferative activity of CD4+ T cells obtained from sepsis mice with administration of TPS were restored compared with burn plus infection group. Meanwhile, TPS reversed the Th2 polarization in sepsis mice. Furthermore, the effects of TPS also showed to have a dose-dependency. Data were expressed as mean ± SD of samples. ⁄P < 0.05, compared to burn sepsis; P < 0.05 compared to TPS (50 mg/kg).
immunosuppression [9,10]. Immunosuppression in sepsis, sometimes referred to as ‘‘immunoparalysis’’, is characterized by a number of factors, including the monocyte deactivation, tolerance to endotoxin, impairment of neutrophil function, lymphocyte dysfunction, and apoptosis [11]. Recent experiments have focused on understanding the presence and function of Tregs during experimental and clinical sepsis [12,13]. Moreover, an increased percentage of Tregs has been observed in the circulation of patients with septic shock, and the persistence of increased Tregs has been associated with a poor long-term prognosis [14]. Thus, the abnormal changes of Tregs during sepsis may be an important pathogenic
factor. In addition, the precise mechanisms of suppression by Tregs remain to be determined, these cells can suppress immune cells functions either directly through cell-cell contact or indirectly through the secretion of anti-inflammatory mediators, such as IL-10 [15]. IL-10 as one kind of anti-inflammatory cytokine could suppress the production of pro-inflammatory cytokines such as IL-12 required for Th1 development by dendritic cells and macrophages [16]. In this investigation, IL-10 produced by Tregs isolated from post-burn sepsis mice was significantly increased. Moreover, the proliferation of CD4+T cells was notably inhibited in burn plus infection mice, and the results in vitro verified that Tregs isolated
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Fig. 3. The effects of TPS on the changes of activation and polarization of CD4+ T cells induced by Tregs which works partially via secreting IL-10 in vitro. Compared to the effects of CD4+CD25high Tregs isolated from sepsis mice on inhibiting CD4+T cells activation and Th1 polarization, while anti-IL-10 antibody weakened the effects of CD4+CD25high Tregs on CD4+ T cells. Meanwhile, TPS reversed the inhibited role of Tregs on CD4+ T cells and triggered a shift from Th2 to Th1. Data were expressed as mean ± SD of samples. ⁄P < 0.05, compared to CD4+CD25high T cells+CD4+T cells.
from scald mice with infection indeed significantly inhibited the proliferation of CD4+T cells. Furthermore, anti-IL-10 antibody partially blocked the inhibiting effects of Tregs on CD4+T cells. Meanwhile, the Th2 cytokine-IL-4 detected in this study was abnormally enhanced after burn plus P. aeruginosa infection, demonstrating that the balance between Th1 and Th2 responses was destroyed in late sepsis. Thus, the abnormal activities of Tregs actually played a harmful role in the immunosuppression occurred in late sepsis. In Traditional Chinese Medicine, co-administration of immunomodulatory agents and chemotherapy drugs is typically used to improve the immunity potential. In many oriental countries, several immunoceuticals composed of polysaccharides are widely used, such as lentinan, schizophyllan and krestin. Those immunomodulatory agents often work by inducing lymphocyte proliferation and cytokine production, and they have protective effects toward the hematopoietic function of bone marrow and immune organs [17]. TPS was showed to have immunoregulatory functions and could improve immunosuppression resulted from some chemotherapeutics. While, it is unclear that whether TPS could play roles in the abnormal activities of Tregs that improved sepsis. Herein, we found that TPS could suppress the function of CD4+CD25highTregs, thus regulating immune functions of T lymphocyte and shift of Th2 to Th1, which were isolated from mice suffered
Fig. 4. The effect of TPS improve survival rate in a murine sepsis model in dosedependent fashion. These mice received intraperitoneal injection of TPS (50, 100, or 200 mg/kg) on day 1, 2 and 3 after burn. ⁄P < 0.05 compared with the sepsis group by the log-rank test. P < 0.05 compared with mice treated by TPS (50 mg/kg) by the log-rank test.
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from burn and P. aeruginosa infection. Meanwhile, TPS significantly lowered the level of IL-10 produced by Tregs, and thereby weakened the excessively suppressed effects of Tregs. Now that TPS could exert such effects on Tregs which is closely associated with sepsis, we assumed that TPS could improve sepsis in the burn plus infection model. Fortunately, we found that the mortality was inhibited significantly by TPS, and the therapeutic effect of TPS manifested in a dose-dependent manner (Fig. 4). TPS have been shown to have great potential value in clinic especially in patients with sepsis in the intensive care unit. In conclusion, TPS could attenuate sepsis via inhibiting the abnormal activity of CD4+CD25highTreg, at least in part through inhibiting IL-10 production, and inducing a new balance of Th2/Th1 by activating CD4+T cell-mediated immunity in burned mice plus P. aeruginosa infection. Acknowledgment This study was supported, in part, by Grants from China Meitan General Hospital-Level Project in China Meitan general hospital (No. MT 2010-16). References [1] K.H.G. Mills, Regulatory T cells: friend or foe in immunity to infection?, Nat Rev. Immunol. 4 (2004) 841–855. [2] Y. Belkaid, B.T. Rouse, Natural regulatory T cells in infectious disease, Nat. Immunol. 6 (2005) 353–360. [3] C. Baecher-Allan, J.A. Brown, G.J. Freeman, et al., CD4+ CD25high regulatory cells in human peripheral blood, J. Immunol. 167 (2001) 1245–1253.
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