Role Of Regulatory T Cell-mediating Apoptosis Of Effector T Cells In Immune Suppression In Sepsis And Its Potential Mechanism

BACKGROUND AND OBJECTIVESepsis with subsquent multiple organ dysfunction syndrome is still the leading cause of death in patients with severe trauma or burns. Accumulating evidences have proved that immunosuppression plays an important role in sepsis. Multiple mechanisms have been proposed to explain septic immunosuppression, including a shift in T cell cytokine responses favoring a helper T cell (Th)2, rather than Thl phenotype response; a great deal loss of macrophage expression of major-histocompatibility-complex class II and costimulatory molecules; a substantial apoptosis-induced loss of lymphocytes and dendritic cells. Increased understanding of the pathophysiology and pathogenesis of sepsis and insight into the mechanisms of immunosuppression are key means to look for effective intervention strategies.Regulatory T cells (Treg), a small subset of CD4+CD25+T cells that constitutively express the transcription factor forkhead box p3(Foxp3), play an important role in maintaining immune tolerance and immune balance. Treg can function as suppressors of CD4+and CD8+effector T cell, resulting in negative regulation of both innate and acquired immune responses. In recent years, the central role of Treg in the persistent immunosuppressive states in septic patients has aroused great interests. Studies have shown that Treg may exert its immunosuppressive effect through inducing T lymphocyte apoptosis, down-regulation the expression level of dendritic cell surface costimulatory molecues, inhibiting the function of CD4+/CD8+T lymphocytes, mediating the shift from Thl to Th2response. Immunosuppressive effects of Treg are mainly mediated by both cell-cell contact and through the release of soluble mediators.Apoptosis of T cells induced by Treg was proved to contribute to the immune dysfunction and multiple organ failure observed during sepsis. The underlying mechanisms were complicated, including down-regulation of expression of interleukin (IL)-2mRNA; consumption of IL-2, resulting in a form of apoptosis dependent on cytokine deprivation; through Fas/Fasl pathway as well as granzyme depended manner. Transforming growth factor beta (TGF-β)1, a pleiotropic cytokine secreted by Th2or Treg, was proved to be able to regulate cell growth and differentiation. Treg produced both membrane-bound (TGFβ1m+) and secreted TGFβ1under certain conditions. Experiments in vitro demonstrate that Treg could suppress the proliferation of CD4+CD25-Teff via cell surface TGFβ1m+in a cell contact dependent fashion. We supposed that TGF-β1m+might be critically involved in the apoptosis of Teff induced by Treg in septic response.TGFβ1mediates its biological function mainly through a heteromeric cell-surface complex of two types of transmembrane serine/threonline kinases, named "type I" and "type Ⅱ (TβRβ)" receptors. TβRⅡ kinase is thought to be constitutively active and, within an established receptor complex, phosphorylates the type Ⅰ receptor at several Ser and Thr residues in a GS-domain. Phosphorylation of the GS-domain enables the recruitment of its downstream signal transducer:the Smad2/Smad3. The active type I receptor then phosphorylates the Smad2/Smad3on two Ser residues at their extreme C termini SSxS motif, which allows them to form complexes with the common mediator Smad4and also to form homomeric complexes and translocate into nuclear. The accumulation of the complexes in nuclear directly regulate gene transcription or indirectly regulate gene transcription by conjunction with other DNA binding proteins.The intrinsic pathways of apoptosis are regulated by members of the Bcl-2family. This family is composed of pro-apoptotic members (Bax, Bak, Bim, Bmf, Bad, Bid) and anti-apoptotic members (Bcl-2, Bcl-w, Bcl-xL, Mcl-1, Bfl-1/Al). In resting cells, pro-apoptotic proteins were neutralized endogenously by its anti-apoptotic counterparts. Bim is a member of Bcl-2with only the BH3domain. Upon apoptotic stimulus, Bim functions as death signal sensors in the cell and play a major role in transducing signals from the cytosol to the mitochondria by activating of Bax/Bak. Bax and Bak reside in the cytosol or on mitochondria and are kept in an inactive form in healthy cells. Thus following Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), ion distribution imbalance, the decline of mitochondrial potential, the release of cytochrome C, activation of caspase (cysteine-dependent, aspartate-specific proteases)-9and cell death happened.The major executioners in the apoptotic program are proteases known as caspases. The caspases are made up of two subgroups according to their functions in cell death. Initiator caspases (caspase-2,8,9, and10) are activated through the apoptosis-signaling pathways and activate the caspase-3,6and7named effector caspases, which carry out programmed cell death. There are two well-characterized pathways of caspases activation:the extrinsic pathway and intrinsic pathway. The extrinsic pathway is initiated by death receptor signaling (TNF-R family) and formation of death inducing signaling complexes (DISCs), which mediates activation of caspase-8. The intrinsic pathway is activated by stress signals and is regulated by the interplay of both pro-and anti-apoptotic members of Bcl-2family proteins and involves the release of cytochrome C as well as the activation of caspase-9. The finally activated caspase-8and caspase-9allow for the catalytic maturation of caspase-3and other caspases and eventually mediate the biochemical and morphological features of apoptosis.The objective of present study was to determine whether the apoptosis of CD4+CD25-Teff is associated with the activation of Treg in sepsis, and the involvement of TGF-β1signaling in this process. Furthermore, the effect of Treg on activation of apoptotic signaling pathways in Teff was assessed.METHODS1. Model preparation:A widely used technique for cecal ligation and puncture (CLP) operation was used in this study. Specific pathogen-free male BALB/c mice were randomly divided into5groups (20mice in each group):normal group, sham group, CLP group, CLP+PC61group, CLP+HRPN group. PC61(the specific antagonist for Treg) and HRPN (rat immunoglobulin G1) were injected intraperitoneally to mice5days prior to CLP operation, respectively in CLP+PC61group and in CLP+HRPN group.2. Animals were sacrificed24h after CLP. Sample of blood of mice was collected by extirpating eyeballs for detecting serum IL-2, IL-4, IL-10, IFN-y and TGFP levels by ELISA.3. CD4+CD25+Tregs were isolated from murine spleens with CD4+CD25+Regulatory T Cell Isolation kits. The purity of isolated CD4+CD25+Tregs and CD4+CD25-Teff was analyzed by flow cytometry. The viability of cells was assessed by Trypan blue exclusion assay. Expressions of cytotoxic T lymphocyte associated atigen-4(CTLA-4), Foxp3, TGFβ1m+on CD4+CD25+Treg as well as CD25proportion were assessed by flow cytometry. Expression of TGFβ was assessed with PT-PCR. The rate of apoptotic CD4+CD25-Teff was analyzed by flow cytometry and correlation between the level of TGFβ1m+and the apoptotic rate of CD4+CD25-Teff was analysis by statistical software.4. Expressions of Smad2/Smad3, P-Smad2/Smad3and Bcl-2superfamily members of Bcl-2/Bim in CD4+CD25-Teff were determined by Western blot analysis. The mitochondrial membrane potential in CD4+CD25-Teff was analyzed by flow cytometry. Alteration of cytochrome C in CD4+CD25"Teff was detected by confocal microscopy. Acticities of caspase-3, caspase-8, caspase-9in CD4+CD25-Teff were analyzed by chemical colorimetric assay.5. Statistical methods:SPSS13.0software was adopted for statistical analysis. Data were expressed as means±standard deviation (SD). The significance of the differences in mean values between groups was evaluated by one-way ANOVA. A p value of0.05or less was considered to indicate statistical significance.RESULTS1. Mice in CLP group performed a series of manifestations of septic symptoms, and death occurred after12h. The mortality of CLP at72h was about50%, and the immune dysfunction occurred24h after CLP, indicating that the sepsis model was stable and could be used for the current experiments.2. Pro-inflammatory cytokines including IL-2, IFN-y levels in blood in CLP group after24h were significantly higher than that of normal group and sham group (P<0.01), while the levels of IL-2and IFN-y in CLP+PC61group were much higher than CLP group (P<0.01). Only IL-4level increased significantly in CLP group, compared with normal group and sham group (P<0.01), while the levels of IL-10and TGFβ were not elevated obviously. IL-4, IL-10, TGFβ in CLP+PC61group showed a different magnitude of decline in comparison to CLP group (P<0.05, or P<0.01).3. The purity of CD4+CD25+Treg and CD4+CD25-Teff was95%and90%after isolated twice by magnetic beads, respectively. The viability of the cells was exceeding95%.4. There was no significantly difference in the expression levels of Foxp3and CTLA-4in Treg when compared sham and normal groups, while significant elevation in CLP group (P<0.05or P<0.01). The levels of Foxp3and CTLA-4in CLP+PC61group returned to the normal range, with a significant difference when compared to CLP group (P<0.05or P<0.01). TGFβ1m+on Treg was significantly enhanced in CLP group, while markedly decreased in the CLP+PC61group, even lower than that of the normal and sham grousp (P<0.05). Changes in the proportion of CD25in CD4+T cells were consistent with the TGFβ1m+, and they were much lower in normal controls as well al sham group compared to CLP+PC61group (P<0.05).5. The mRNA expression of TGFβ1in normal and sham groups had no statistical significance; while in CLP group it was obviously increased (P<0.01). TGFβ1mRNA expression in CLP+PC61group was markedly lower than that in normal controls, sham group and CLP group (P<0.01).6. Apoptotic rate of Teff in CLP group was the highest among groups, up to23.98%, the apoptotic rate in CLP+HRPN (21.74%) had no statistically significant differences when compared with CLP group (P>0.05). The apoptotic rates in CLP+PC61group, normal group and sham group showed no significant differences, but obviously lower than that in CLP group (P<0.01).7. Through the use of SPSS13.0statistics software, it was revealed that the expression rate of TGFβ1m+and apoptotic rate of Teff cells was significantly positively correlated (Pearson correlation coefficient r=0.791, P=0.000). 8. There were no significant differences of Smad2/Smad3expressions in CD4+CD25-Teff among various groups, while the expressions of P-smad2/P-Smad3in CLP group were significantly higher than that in normal and sham groups (P<0.01), P-smad2/P-Smad3expression in CLP+PC61group had no significant difference with both normal and sham groups, nevertheless it markedly lower than that in CLP group (P<0.01). Expression of anti-apoptotic protein Bcl-2in CD4+CD25-Teff in CLP group was decreased significantly (P<0.01) compared with the normal and sham groups, while the expression of apoptosis protein Bim was enhanced obviously (P<0.01). The expression of the anti-apoptotic protein Bcl-2in CLP+PC61group, compared with the normal group and sham group, was significantly reduced (P<0.01), but significantly higher than that in CLP group (P<0.01). Pro-apoptoticprotein Bim expression in CLP+PC61group, compared with normal control group and sham group, was increased significantly (P<0.01), but still lower than that in CLP group (P<0.01).9. Compared with both normal and sham groups, the mitochondrial membrane potential in CD4+CD25"Teff in CLP group was decreased significantly (P<0.01), there was no significant difference among the CLP+PC61group and the normal group as well as sham group, while it significantly higher than that in CLP group (P<0.01). Change in cytochrome C was contrast with mitochondrial membrane potential, expression of cytochrome C in CLP group was significantly higher than that in normal control and sham groups (P<0.01). While the expression of cytochrome C in CLP+PC61group, compared with control group and sham group, was not statistically significant but lower than that in CLP group.10. Activities of caspase-3, caspase-8, caspase-9in CD4+CD25-Teff in CLP group were significantly increased than that of the normal group and sham group (P<0.01). However, in CLP+PC61group, activities of caspase-3and caspase-9were not statistically significant compared with normal and sham groups, but lower than that in CLP group (P<0.01). The activity of caspase-8was significantly higher than normal and sham groups, but it was still lowered compared to CLP group (P<0.01).CONCLUSIONS1. The septic model reproduced by CLP operation appears to be stable, reliable and suitable for experimental studies.2. The CD4+CD25+Treg isolated by magnetic beads is pure and viable, suitable for the subsequent experiments.3. CLP-induced sepsis can markedly activate the negative regulation of Treg that promotes the apoptosis of CD4+CD25-Teff, and signals mediated by TGFβ1m+might be involved in this process. From the current study together with other reports, we might suppose the potential mechanisms to be as follows:combination of TGFβ1m+on Treg surface with TβR. on Teff could activate the downstream signaling molecules of TGF(3, such as Smad2/Smad3. Activated Smad2/Smad3translocate to the nucleus to regulate the expression of Bcl-2superfamily members as enhancing expression of pro-apoptotic protein like Bim and reducing the expression of anti-apoptotic protein like Bcl-2. Bax, Bak, the apoptotic executive, are then freed and translocate to the mitochondria outer membrane, leading to an increase in mitochondrial permeability and a reduction in membrane potential, and then promote the release of cytochrome C, which can activate caspase-9by combination of Apaf-1. Activation of caspase-9and the subsequent activation of caspase-3then lead to the apoptosis of Teff.