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Exogenous C3 protein enhances the adaptive immune response to polymicrobial sepsis through down-regulation of regulatory T cells
International Immunopharmacology 12 (2012) 271–277
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Exogenous C3 protein enhances the adaptive immune response to polymicrobial sepsis through down-regulation of regulatory T cells Yujie Yuan, Jianan Ren ⁎, Shougen Cao, Weiwei Zhang, Jieshou Li Department of Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, PR China
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Article history: Received 10 September 2011 Received in revised form 25 November 2011 Accepted 29 November 2011 Available online 13 December 2011 Keywords: Complement C3 Sepsis Adaptive immune Th cells T regulatory cells NF-κB
a b s t r a c t Background: The role of complement system in bridging innate and adaptive immunity has been confirmed in various invasive pathogens. It is still obscure how complement proteins promote T cell-mediated immune response during sepsis. The aim of this study is to investigate the role of exogenous C3 protein in the T-cell responses to sepsis. Methods: Sepsis was induced by colon ascendens stent peritonitis (CASP) in wild-type C57BL/6 mice, shamoperated mice for control. Human purified C3 protein (HuC3, 1 mg) was intraperitoneally injected at 6 h post-surgery, with 200 μl phosphate-buffered saline as control. The levels of C3 and cytokines, the expression of FOXP3 and NF-κB, and the percentages of CD4 + T-cell subsets were compared among the groups at given time points. Results: The polymicrobial sepsis produced considerable release of TNF-α and IL-10, and caused complement C3 exhaustion. Exogenous C3 administration markedly improved the 48 h survival rate, as compared with nontreatment (40% vs. 5%, P b 0.01). The expression of FOXP3 protein was increased during sepsis, but can be suppressed by HuC3 administration. A single injection of HuC3 postponed the decline of differentiated Th1 cells, and depressed the activation of Th2/Th17 cells. Besides, the Th1–Th2 shift in late stage of sepsis can be controlled under C3 supplementation. The suppression of NF-κB pathway might be related to the appearance of immunocompromise. Conclusions: The study confirmed the important role of exogenous C3 in up-regulation of adaptive immune response to sepsis. The complement pathway would be a pivotal target for severe sepsis management. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The complement cascade reaction to the pathogenic invasion is an important part of innate immune response. During the process, considerable proenzymes are indispensable and become activated sequentially [1]. Complement system can be activated by different pathways: classical, alternative, lectin, and the fourth way via C4/C2 bypass mechanism [2]. All pathways converge on a common route, complement C3, to the formation of membrane-attack complex. Activation of complement fulfills numerous biological effects, such as killing target cells with the membrane attack complex, promoting pathogenic phagocytosis by leukocytes and macrophages, and amplifying pro-inflammatory attributes by the release of anaphylatoxins (C3a, C4a, C5a, etc.). And the C3 is the pivotal molecule in complement cascade [3]. Sepsis, defined as a systemic immune response syndrome with confirmed evidence of infection, would lead to the robust release of cytokines from phagocytes to defend the invasion of pathogens [4]. ⁎ Corresponding author at: Jinling Hospital, 305 East Zhongshan Road, Nanjing, 210002, PR China. Tel.: + 86 13605169808; fax: + 86 2584803956. E-mail address: [email protected] (J. Ren). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.11.022
Various studies in humans have shown that excessive complement activation occurs during sepsis. In addition, the subsequent immunocompromise would be unavoidable if severe sepsis is poorly controlled [5]. However, it is still obscure whether the coming immunosuppression is related to the complement depletion in sepsis. The T-helper-type (Th) cells are considered major players in an effective immune response to many pathogenic organisms [6]. Those cells can be subdivided into three different types according to secreted cytokine signature: IFN-γ for Th1; IL-4 and IL-5 for Th2 [7]; and IL-17 for Th17 cells [8]. An important result of T-cell activation is functional polarization into Th1, Th2, or Th17 phenotypes. All three fully differentiated effector Th cell types would be suppressed by polyclonal CD4 +CD25 + regulatory T cells (Tregs) in vivo, although each type had a little different susceptibility to Tregs [6]. The effects of complement system on adaptive immunity, particularly T-cell-dependent responses, were diverse in various invasive pathogens [9–11]. For instance, C3a has been reported to favor Th1 differentiation by up-regulating the secretion of IL-12 in antigen presenting cells (APCs) [12]. Although several hypotheses have been proposed, there is not yet to be a consensus on the precise mechanism by which complement regulates T-cell immunity. We herein hypothesize that exogenous C3 protein administration during sepsis
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would enhance the differentiation of effector Th cells by inhibiting the expansion of Tregs. Up to the present, it is obscure how complement C3 promotes CD4+ T-cell responses during sepsis. In a murine model of influenza, the lack of C3 would lead to an overwhelming failure of virus clearance due to it being unable to promote cytotoxic T cells [13]. Additionally, the potential role of C3 in Th cell differentiation during sepsis has not been thoroughly understood. The aim of this study is to investigate its role in the adaptive immune response, particularly T-cell responses, to sepsis. 2. Materials and methods 2.1. Mice Wild-type 8- to 10-week-old female C57BL/6 mice with a weight between 20 and 25 g were purchased from Jisan Model Animal Center (Xi'an, Shanxi province, China). Mice were bred in our laboratory under specific pathogen-free conditions at 20–23 °C. Prior to the experiment, all mice were kept for at least two weeks to recover from transport. All experimental procedures were performed according to the guidelines on the care and use of laboratory animals set out by the animal research committee of Nanjing University. 2.2. Colon ascendens stent peritonitis (CASP) surgery The surgical procedure of CASP was performed as previously described [14]. Briefly, 14-gauge venous catheters (2.0 mm 2, Venflon; BOC, Ohmeda AB, Sweden) were cut to similar segments with 0.5–0.6 cm length each. Under complete anesthesia (ketamine, 80–100 mg/kg, i.p.) and after disinfection of the abdomen (75% alcohol), the abdominal wall was opened through a 1-cm midline incision. After exposure of the ascending colon, the prepared stent was stitched through the antimesenteric wall into the proximal lumen of the ascending colon, fixed with two stitches (7/0 Ethilon thread; Ethicon, Nordestedt, Germany) placed approximately 1 cm from the ileocecal valve. Afterward, the inner needle of the stent was removed. Stool was milked from the cecum into the ascending colon and the stent until a drop of stool (1 mm in diameter) appeared. For control purposes, sham operation was performed in similar fashion except that the stent was fixed outside the ascending colon. Fluid resuscitation was performed by flushing 0.5 ml sterile saline into the peritoneal cavity before abdominal wall closure (two layers, muscle and skin; 5/0 Ethilon thread). The operated mice were brought back in cages to recover, with free access to food and water. 2.3. Treatment with human purified C3 protein A single dosage of 1 mg human purified C3 protein (HuC3), diluted in 200 μl PBS + 0.1% bovine serum albumin (BSA) in sterile, was intraperitoneally injected at 6 h post CASP surgery in the HuC3+ group. In the HuC3− group, only 200 μl of PBS + 0.1% BSA was injected at 6 h post CASP surgery. The HuC3 functional activity was estimated as >95% based on hemolytic activity, as determined by the supplier (MyBioSource, San Diego, CA, USA). 2.4. Cytokines and complement protein measurement At 24 h and 48 h post-surgery, blood samples from each group were collected by cardiac puncture for further analysis. The amount of C3, IL-10, and TNF-α in plasma was determined with a murine ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. The levels of sensitivity were 1.0 μg/ml for C3, 12.0 pg/ml for IL-10, and 5.0 pg/ml for TNF-α.
2.5. Flow cytometry assay for T cell subsets At given time points, mice were sacrificed via cervical dislocation. Spleen was harvested and gently ground to obtain single-cell suspensions, which then were subjected to continuous density gradient centrifugation (2000 rpm, 25 min) with Percoll (GE Healthcare, Uppsala, Sweden). Splenocytes were collected and washed twice with RPMI1640. Then, cell counts were performed with a hemocytometer (Trypan blue exclusion) and re-suspended to a concentration of 106 cells/ml of medium for T cell subset analysis. Intracellular cytokine staining was performed with PE- or allophycocyanin-labeled mAbs as described [6]. Briefly, separated cells were stimulated for 5 h with PMA (50 ng/ml) and ionomycin (1 μg/ml) with GolgiStop added after 2 h. Cell stimulation was terminated by fixing in 4% formyl saline. Fixed cells were stained in 0.1% saponin permeabilization buffer for 1 h. Mouse Th1/Th2/Th17 Phenotyping Cocktail purchased from BD Pharmingen was used to stain cells in the dark for 30 min. Finally, flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences). 2.6. FOXP3 immunohistochemistry and Western blot analysis Tissues from spleen were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned according to the standard procedure. Immunochemistry analysis was performed as previously described [15]. In brief, multiple 5 μm serial sections from selected formalin-fixed, paraffin-embedded blocks were made and reacted with affinity-purified anti-FOXP3 polyclonal Ab (ab22510, ABCAM, Cambridge, UK). The slides were washed in PBS and subsequently incubated with the second Ab, HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After being washed in PBS, slides were developed with 3,3′-diaminobenzidine, and counterstained with hematoxylin. Coverslips were applied with DPX mountant (CV5000 Coverslipper; Leica Instruments). The numbers of immunophenotyped cells in the stained sections were counted by visual inspection. For each section, photographic images of each microscopic field were obtained (magnification, × 6100). The FOXP3 protein expression was analyzed as described previously [16]. Briefly, T cell subsets harvested from spleen in various time points were washed and lysed using RIPA buffer (Boston BioProducts, Worcester, MA) supplemented with protease inhibitor. Cell lysates were subsequently homogenized using QIAshredder Homogenizer columns (Qiagen, Valencia, CA). Soluble fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and transferred to nitrocellulose membrane. Membranes were saturated for 1 h at room temperature in TBST supplemented with 5% nonfat dry milk and immunoblotted overnight at 4 °C with anti-FOXP3 monoclonal antibody (clone 236/E7; ABCAM, Cambridge, MA) or α-tubulin (clone B-5-1-2; Sigma, St Louis, MO). Membranes were then washed, probed with HRP-conjugated goat anti-mouse polyclonal antibodies (Zymed Laboratories, San Francisco, CA), and revealed with enhanced chemiluminescence (ECL; Amersham Biosciences, Uppsala, Sweden). 2.7. Reverse transcription-PCR analysis and phosphorylation of NF-κB Total RNA was isolated from the purified T cell subset suspension by using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), treated with RNase free-DNase (Promega, Madison, WI, USA) and reverse transcribed to cDNA with random primer by Superscript II (Invitrogen, Carlsbad, CA, USA). The cDNA was then amplified by PCR. The forward and reverse primers were as follows: F: 5′-CCT CTC TCG TCT TCC TCC AC-3′; R: 5′-CAG TGG GCT GTC TCC AGT AA-3′ for NF-κB (132 bp), and F: 5′-ACG ACC CCT TCA TTG ACC TCA A-3′; R: 5′-GCA GTG ATG GCA TGG ACT GTG-3′ for GAPDH. PCR amplification was performed for 40 cycles at 95 °C for 20 s and 60 °C for 1 min. The mRNA
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level for each gene for each sample was normalized to GAPDH mRNA and was presented as 2 · [(Ct/ GAPDH− Ct / gene of interest)] as described. The purified T cell subsets were lysed in 500 μl of lysis buffer [50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 1 mM PMSF, aprotinin (1 μg/ml), leupeptin (1 μg/ml), and pepstatin (1 μg/ml)]. The supernatants were obtained via centrifugation at 12,000 rpm for 10 min at 4 °C, and the protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA). Afterward, the 0.5–0.8 mg of protein diluted in lysis buffer was immunoprecipitated with polyclonal p65 antibody (catalog #18-785-210321, GenWay Biotech Inc., San Diego, CA) and protein G-Sepharose at 4 °C overnight. The immunoprecipitates were subjected to 10% SDSPAGE and Western blotting with antibody to p65 (1:1000; Rockland Immunochemicals). 2.8. Electrophoretic mobility shift assays (EMSA) of NF-κB The nuclear extracts were extracted from purified T cell subsets at 24 h and 48 h post CASP procedure was isolated and quantified as previously described [17]. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, Ill, USA). EMSA was performed using a commercial kit (Gel Shift Assay System, Promega, Madison, WI). The DNA probe that consisted of the NF-κB consensus sequence (5′-AGT TGA GGG GAC TTT CCCAGG C-3′) was end-labeled with rhodamine (Amersham Pharmacia Bio., Tokyo, Japan). The nuclear extracts (5 μg/ml) were incubated with rhodamine-labeled NF-κB probe in the binding buffer (20 mM HEPES pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 30 mM KCl, and 0.2% (w/v) Tween 20) at room temperature for 15 min. The nuclear protein and oligonucleotide complexes were separated from free probes on native 5% polyacrylamide gel (BioRad, Richmond, CA) in 0.25× TBE buffer (Trisborate–EDTA) at 390 V for 1 h at 4 °C. The gel was exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan) at − 70 °C with an intensifying screed. Levels of NF-κB DNA binding activity were quantified by computer-assisted densitometric analysis. 2.9. Statistical analysis Measurement data were expressed as mean± SEM, if not stated otherwise. Statistical analyses of most data were performed using the Student's t-test, with two-sample and assuming unequal variances, where indicated. Comparison between the HuC3− group and the HuC3+ group was made by unpaired t-test. Pearson and Spearman correlation coefficients were calculated for variables following or not following Gaussian distribution, respectively. Comparisons between multiple groups were analyzed with one-way ANOVA. All statistical analyses were performed by using GraphPad Prism Software (version 5.01; GraphPad, San Diego, CA). P value below 0.05 was considered significant.
Fig. 1. Survival of CASP-operated mice after exogenous C3 treatment. Groups are depicted as indicated. Survival was 100% through the observational period in the sham group, 40% in the HuC3− group at 24 h but 5% at 48 h post-operation. Exogenous C3 injection at 6 h post-operation improved survival, with 60% at 24 h and 40% at 48 h, respectively. ⁎⁎P b .01 vs. the HuC3− group.
3.1. CASP-induced sepsis results in an expansion of regulatory T cells in vivo while HuC3 administration could suppress the expansion To investigate the activity of Tregs (CD25 +FOXP3+) in response to the CASP-induced sepsis, immunohistochemistry staining of FOXP3 was performed as mentioned above. The expression of FOXP3 protein in spleen at 24 h post-surgery was quite different among three groups (Fig. 2). The polymicrobial sepsis also led to a significant expansion of Tregs in the HuC3− group compared with the sham-operated group. Whereas, HuC3 administration at 6 h post CASP surgery greatly inhibited the activation of Tregs in the HuC3+ group, as compared to the HuC3− group (P b 0.01). Furthermore, Western blot analysis was performed to detect the effect of exogenous C3 on the expression of FOXP3 in T cell subsets, which were obtained from the spleen (Fig. 3). The Western blot results showed low expression of FOXP3 protein in the sham group at 24 h and 48 h post-surgery. Nevertheless, the protein levels were significantly increased in the HuC3− group compared with the sham group (P b 0.001). After the exogenous C3 administration, the increased expression of FOXP3 protein was remarkably suppressed compared with nontreatment (P b 0.01), but with no significant difference to the sham group. 3.2. During the process of sepsis, the Th1–Th2 shift at the late stage could be controlled under HuC3 protein administration The differentiation of T helper cells during sepsis was investigated via flow cytometry assay as described in the Materials and methods. All sham-operated mice had low but stable overall percentage of differentiated Th1, Th2, and Th17 T effectors. In contrast, CASP-induced
3. Results After the surgery with 14-G catheter, all CASP-operated mice developed severe sepsis rapidly, whereas sham-operated mice remained normal and survived through the whole observation period. To be specific, the 48 h survival rate was markedly increased in mice with exogenous C3 administration at 6 h post-surgery (HuC3+ group), as compared with CASP-operated mice without C3 treatment (40% vs. 5%, P b 0.01). The Kaplan–Meier method survival curves are shown in Fig. 1. Additionally, this polymicrobial sepsis led to a quick depletion of C3 protein and increased release of inflammatory cytokines, such as TNF-α and IL-10. Early supplementation of exogenous C3 protein during sepsis could improve the plasma levels of C3, with a decreased production of cytokines in the meanwhile (Table 1).
Table 1 The plasma levels of C3 and cytokines in late stage of severe sepsis.
C3 (μg/ml) TNF-α (pg/ml) IL-10 (pg/ml)
24 h 48 h 24 h 48 h 24 h 48 h
CASP, sham
CASP, HuC3−
186.3 171.9 225.9 186.8 300.0 171.9
112.8 81.8 1520.0 1120.0 12,820.0 11,250
(15.4) (18.2) (20.4) (21.8) (14.1) (18.2)
(10.3)a (12.9)a,c (41.2)a (48.8)a,c (505.9)a (363.4)a
CASP, HuC3+ 216.4 179.2 1240.0 1040.0 13,950.0 10,720.0
(19.2)b (21.3)b (26.6)a,b (46.8)a (365.1)a,b (526.3)a
All values are expressed as mean (SD). n = 5 for each group; three times repeated experiment. a P b .05 vs. the sham group. b P b .05 vs. the HuC3− group. c P b .05 vs. the same column at 24 h post-surgery.
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Fig. 2. Expression of FOXP3 in the spleen tissue after 24 h of CASP surgery via immunohistochemistry staining method. The nuclear FOXP3 single-staining cells were considered as Tregs (CD25+FOXP3+). Immunohistochemistry staining showed that FOXP3 mainly distributed in the nucleus with brown staining color. Few FOXP3 positive cells were observed in the sham group (A). CASP-induced sepsis produced an enhanced expression of FOXP3 in the HuC3− group (B), while the expression was relatively decreased in the HuC3+ group (C). Arrows indicated representatively FOXP3 positive cells in the surrounding region of contusion cortex. Scale bars: 100 μm. The big black box indicates the amplification of staining zone marked with small box; scale bars: 10 μm. (D) Quantification of FOXP3 positive cells in each group. Data are presented as mean± SEM of n = 5 per group; three times repeated experiment. ⁎⁎⁎P b .001 vs. the sham group; ##P b .01 vs. the HuC3− group.
sepsis resulted in a remarkable increase in the overall percentage of differentiated Th1 cells in early stage, but followed by a rapid decline after 24 h post-surgery (Fig. 4). Compared with the HuC3− group, exogenous C3 protein injection with a single dose postponed the decline for the differentiated Th1 cells and suppressed the increase of Th2 T effectors after 24 h post-surgery. In addition, the shift of T cell subsets from Th1 to Th2 effectors was observed in the HuC3− group, with a significant decline in Th1/Th2 ratio at 48 h post-surgery (P b 0.01; Fig. 4B). However, the shift could be effectively controlled under HuC3 protein administration, with higher ratio of Th1/Th2 effectors in the HuC3+ group than that in the HuC3− group (P b 0.05 at 24 h, P b 0.001 at 48 h postsurgery). Moreover, the overexpression of differentiated Th17 T effectors due to severe sepsis can be reversed by HuC3 supplementation in early stage of sepsis (Fig. 4). 3.3. CASP-induced sepsis results in the decreased expression of NF-κB in the late phase that could be improved with exogenous C3 protein supplementation
Fig. 3. Expression of FOXP3 protein in spleen from the sham group, HuC3− group and HuC3+ group at 24 h and 48 h post-surgery. Levels of FOXP3 protein were assessed by Western blot analysis with α-tubulin for control blot (A). The quantitative analysis of the Western blot results for FOXP3 protein expression is shown above (B). Bars represent as mean ± SEM; n = 5 per group; three times repeated experiment. ⁎⁎⁎P b .001 vs. the sham group, ##P b .01 vs. the HuC3− group.
To detect the expression of NF-κB in T cell subsets during sepsis, RT-PCR was performed at 24 h and 48 h post-surgery. In the sham group, the expression of NF-κB mRNA in T cells was mild but stable during the whole experiment. Nevertheless, the expression in the HuC3− group was remarkably increased at 24 h post-surgery compared to the sham group (P b 0.001), but followed by a rapid decline at 48 h post-surgery (P b 0.001, Fig. 5). Administration of HuC3 protein could inhibit the NF-κB mRNA expression significantly in comparison with the HuC3− group at 24 h post-surgery (P b 0.01). However, a single injection of exogenous C3 cannot reverse the
Y. Yuan et al. / International Immunopharmacology 12 (2012) 271–277 Fig. 4. The percentages of Th1, Th2, and Th17 cells during the process of CASP-induced sepsis. All T cell subsets were purified from the spleen. Mice were sacrificed at given time points, with five mice used at each time point in each group. Cytokine production by the Th1, Th2, and Th17 cell lines upon re-stimulation with PMA and ionomycin. Specifically, IFN-γ for Th1, IL-4 for Th2, and IL-17A for Th17, filtered in CD4+ gate cells. (A) Typical flow cytograms of Th cells at 48 h post-surgery in the HuC3−/+ groups. The counts of CD4+ T cells were compared via histogram. (B) The changes and ratio of Th differentiated cells during sepsis. Data are presented as mean ± SEM of three times repeated experiment. ⁎⁎P b .01, ⁎⁎⁎P b .001 vs. the sham group; #P b .05, ###P b .001 vs. the HuC3− group; †††P b .001 the HuC3− group at 24 h vs. the HuC3− group at 48 h post-surgery.
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DNA binding activity in both CASP-operated groups was significantly enhanced at 24 h post-surgery (P b 0.001). After 48 h of CASP surgery, the binding activity in the HuC3− group decreased quite fast, which was distinctly lower than that at 24 h post-surgery (P b 0.001). Importantly, the decline of NF-κB binding activity also could be improved with exogenous C3 protein administration. 4. Discussion
Fig. 5. Expression of NF-κB in purified T cell subsets from the spleen tissue. RT-PCR bands (A) show the NF-κB mRNA expression at 24 h and 48 h after surgery in the sham group, HuC3− group, and HuC3+ group. The density of mRNA (B) in each group is quantitatively analyzed. The phosphorylation of NF-κB subunit p65 (C) by Western blotting indicates the activity of NF-κB during sepsis. Data represent as mean ± SEM; n = 5 per group; three times repeated experiment. ⁎⁎⁎P b .001 vs. the sham group; ##P b .01 vs. the HuC3− group; †††P b .001 indicates the HuC3− group at 24 h vs. the HuC3− group at 48 h after surgery.
decline of the expression in the late phase of sepsis. In the HuC3− group, the increased phosphorylation of the NF-κB subunit p65 at 24 h post-surgery also indicated that enhanced activation of NF-κBdependent transcription occurred in early stage of sepsis. Besides, the declined p65 phosphorylation at 48 h post-surgery could be suppressed if the exogenous C3 was injected at 6 h post-surgery (Fig. 5C). On the other hand, NF-κB DNA binding activity was examined by EMSA autoradiography. A persistently low NF-κB binding activity (weak autoradiography) was found in the sham group within the period of experiment (Fig. 6). Compared with the sham group, NF-κB
In this study, CASP-induced sepsis model was utilized to investigate the changes of adaptive immunity in response to the persistent intra-abdominal infection. Tregs, as a pivotal component in inflammatory conditions, could be marked in tissue by antibodies combining to FOXP3 protein through immunohistochemistry or Western blot method [15]. Based on the available results, an extensive expansion of Tregs was observed at the late stage of sepsis, which was correlated to the subsequent Th1–Th2 shift and eventual immunosuppression. Importantly, exogenous C3 protein administration in early stage of sepsis could remarkably inhibit the activation of Tregs and delay the shift, associated with improved outcomes. It is recognized that innate immune responses, particularly those mediated by neutrophil- and macrophage-phagocytosis, are an essential part in early stage of bacterial infection [18]. Complement C3 plays an important role in this stage. It is quite helpful to enhance the phagocytosis of bacterial pathogens for both macrophages and other polymorphonuclear leukocytes. However, the complement depletion during sepsis, particularly C3 protein exhaustion, would make this positive biological effect impaired and increase the risk of recurrent infection and immunocompromise [19]. Besides, it has been confirmed that decreased complement levels would reduce priming of both CD4 + and CD8 + T cells, which suggested a more generalized role of complement [11,13]. In the present study, the differentiation of Th1 effectors was attenuated in late stage of sepsis, with enhanced differentiation of Th2/Th17 cells in the meanwhile. The observed Th1–Th2 shift indicated that the adaptive immunity against the polymicrobial sepsis had been impaired due to the complement depletion. This deviation toward Th2 differentiation could inhibit potentially protective cell-mediated immunity (CMI) responses and produce antibodies associated with antibody-dependent cell-mediated cytotoxicity (ADCC). The imbalanced deviation would further result in the elimination of Th cell subsets and the appearance of immunosuppression. Attenuated T-cell responses in late stage of sepsis may also be a consequence of decreased levels of C5a [20]. The persistently low level of C5a is a specific characteristic of complement exhaustion.
Fig. 6. NF-κB DNA binding activity in the spleen tissue after surgery. (A) ESMA autoradiography of NF-κB DNA binding activity (black arrow) at 24 h and 48 h post-surgery in the sham group, HuC3− group, and HuC3+ group. (B) Quantification of NF-κB DNA binding activity. Data represent as mean ± SEM; n = 5 per group; three times repeated experiment. ⁎P b .05, ⁎⁎P b .01, ⁎⁎⁎P b .001 vs. the sham group; #P b .05 vs. the HuC3− group; †††P b .001 means the HuC3− group at 24 h vs. the HuC3− group at 48 h after operation.
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The C5a receptors are extensively distributed on surface of T cells, as a specific epitope to C5a [13]. However, the role of the anaphylatoxin C5a is still not clearly understood. Besides, the enhanced expansion of CD25 +FOXP3 + Tregs has also been associated with the reduced CMI during sepsis [21]. To investigate the potential mechanism of T-cell responses in sepsis, the expression of NF-κB mRNA and its binding activity were assessed. The decreased expression of NF-κB mRNA with the attenuated T-cell responses might indicate that the negative control of NF-κB signaling is related to the Th1/Th2 shift and the expansion of Tregs, which is also consistent with the decreased phosphorylation of p65 subunit in NF-κB. Toscano et al. have demonstrated that NF-κB can control the expression of immunoregulatory galectin-1 protein on T cells [22]. T cells responding to presented antigen would express IL-10 in the presence of IL-12 [23]. Moreover, the enhanced expression of IL-10 is believed to be dependent upon the activation of NF-κB pathway based on previous study [24]. However, the concrete mechanism of this effect is still uncertain. Several limitations of current study should be noted. This study failed to investigate the role of C3 in regulation of B cells and antibody responses during the process of sepsis. Further studies should identify whether exogenous C3 administration co-stimulates B cells through the regulation of Th cells, although some studies have confirmed its role in enhancing the adaptive immunity in sepsis [25,26]. Besides, dendritic cells (DCs) also play an important role in bridging innate and adaptive immunity. We did not monitor its activity or investigate its relationship to complement exhaustion during sepsis. Moreover, several groups have shown that the expansion of natural CD4+CD25+ Tregs does not contribute to mortality in murine polymicrobial sepsis [27,28]. Therefore, Tregs seems likely to be an indirect mediator, which bridges the complement system and T-cell responses during sepsis. Some other populations, such as myeloid-derived suppressor cells (MDSC), and Natural Killer-T-cells, should be investigated in further studies. At last, we just confirmed the potential effect of NF-κB p65 activation on T cells. The other upstream signaling pathways of NF-κB, such as ERK1/2, IκBα kinase, and p-ERK1/2 should be studied with knockout mice in further steps. In conclusion, our findings provide strong evidence that complement C3 plays an important role in enhancing T cell responses to conditional bacterial infection during the polymicrobial sepsis. This potent effect is believed to work by depressing the expansion of regulatory T cells through NF-κB pathway. Exogenous C3 administration delays the complement depletion in late stage of sepsis, attenuates the Th1–Th2 shift, and enhances the differentiation of Th17 cells in the meantime. The findings have implications in rational design of effective treatment of sepsis-induced immunocompromise for humans. Acknowledgment This study was supported by grants from the National Natural Science Foundation of China (No. 30872456) and the Climbing Program in Natural Science Foundation of Jiangsu Province for Distinguished Scholars (No. BK2010017). References [1] Yalcindag A, He R, Laouini D, Alenius H, Carroll M, Oettgen HC, et al. The complement component C3 plays a critical role in both Th1 and Th2 responses to antigen. J Allergy Clin Immunol 2006;117:1455–61.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Exogenous C3 protein enhances the adaptive immune response to polymicrobial sepsis through down-regulation of regulatory T cells Yujie Yuan, Jianan Ren ⁎, Shougen Cao, Weiwei Zhang, Jieshou Li Department of Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, PR China
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Article history: Received 10 September 2011 Received in revised form 25 November 2011 Accepted 29 November 2011 Available online 13 December 2011 Keywords: Complement C3 Sepsis Adaptive immune Th cells T regulatory cells NF-κB
a b s t r a c t Background: The role of complement system in bridging innate and adaptive immunity has been confirmed in various invasive pathogens. It is still obscure how complement proteins promote T cell-mediated immune response during sepsis. The aim of this study is to investigate the role of exogenous C3 protein in the T-cell responses to sepsis. Methods: Sepsis was induced by colon ascendens stent peritonitis (CASP) in wild-type C57BL/6 mice, shamoperated mice for control. Human purified C3 protein (HuC3, 1 mg) was intraperitoneally injected at 6 h post-surgery, with 200 μl phosphate-buffered saline as control. The levels of C3 and cytokines, the expression of FOXP3 and NF-κB, and the percentages of CD4 + T-cell subsets were compared among the groups at given time points. Results: The polymicrobial sepsis produced considerable release of TNF-α and IL-10, and caused complement C3 exhaustion. Exogenous C3 administration markedly improved the 48 h survival rate, as compared with nontreatment (40% vs. 5%, P b 0.01). The expression of FOXP3 protein was increased during sepsis, but can be suppressed by HuC3 administration. A single injection of HuC3 postponed the decline of differentiated Th1 cells, and depressed the activation of Th2/Th17 cells. Besides, the Th1–Th2 shift in late stage of sepsis can be controlled under C3 supplementation. The suppression of NF-κB pathway might be related to the appearance of immunocompromise. Conclusions: The study confirmed the important role of exogenous C3 in up-regulation of adaptive immune response to sepsis. The complement pathway would be a pivotal target for severe sepsis management. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The complement cascade reaction to the pathogenic invasion is an important part of innate immune response. During the process, considerable proenzymes are indispensable and become activated sequentially [1]. Complement system can be activated by different pathways: classical, alternative, lectin, and the fourth way via C4/C2 bypass mechanism [2]. All pathways converge on a common route, complement C3, to the formation of membrane-attack complex. Activation of complement fulfills numerous biological effects, such as killing target cells with the membrane attack complex, promoting pathogenic phagocytosis by leukocytes and macrophages, and amplifying pro-inflammatory attributes by the release of anaphylatoxins (C3a, C4a, C5a, etc.). And the C3 is the pivotal molecule in complement cascade [3]. Sepsis, defined as a systemic immune response syndrome with confirmed evidence of infection, would lead to the robust release of cytokines from phagocytes to defend the invasion of pathogens [4]. ⁎ Corresponding author at: Jinling Hospital, 305 East Zhongshan Road, Nanjing, 210002, PR China. Tel.: + 86 13605169808; fax: + 86 2584803956. E-mail address: [email protected] (J. Ren). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.11.022
Various studies in humans have shown that excessive complement activation occurs during sepsis. In addition, the subsequent immunocompromise would be unavoidable if severe sepsis is poorly controlled [5]. However, it is still obscure whether the coming immunosuppression is related to the complement depletion in sepsis. The T-helper-type (Th) cells are considered major players in an effective immune response to many pathogenic organisms [6]. Those cells can be subdivided into three different types according to secreted cytokine signature: IFN-γ for Th1; IL-4 and IL-5 for Th2 [7]; and IL-17 for Th17 cells [8]. An important result of T-cell activation is functional polarization into Th1, Th2, or Th17 phenotypes. All three fully differentiated effector Th cell types would be suppressed by polyclonal CD4 +CD25 + regulatory T cells (Tregs) in vivo, although each type had a little different susceptibility to Tregs [6]. The effects of complement system on adaptive immunity, particularly T-cell-dependent responses, were diverse in various invasive pathogens [9–11]. For instance, C3a has been reported to favor Th1 differentiation by up-regulating the secretion of IL-12 in antigen presenting cells (APCs) [12]. Although several hypotheses have been proposed, there is not yet to be a consensus on the precise mechanism by which complement regulates T-cell immunity. We herein hypothesize that exogenous C3 protein administration during sepsis
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would enhance the differentiation of effector Th cells by inhibiting the expansion of Tregs. Up to the present, it is obscure how complement C3 promotes CD4+ T-cell responses during sepsis. In a murine model of influenza, the lack of C3 would lead to an overwhelming failure of virus clearance due to it being unable to promote cytotoxic T cells [13]. Additionally, the potential role of C3 in Th cell differentiation during sepsis has not been thoroughly understood. The aim of this study is to investigate its role in the adaptive immune response, particularly T-cell responses, to sepsis. 2. Materials and methods 2.1. Mice Wild-type 8- to 10-week-old female C57BL/6 mice with a weight between 20 and 25 g were purchased from Jisan Model Animal Center (Xi'an, Shanxi province, China). Mice were bred in our laboratory under specific pathogen-free conditions at 20–23 °C. Prior to the experiment, all mice were kept for at least two weeks to recover from transport. All experimental procedures were performed according to the guidelines on the care and use of laboratory animals set out by the animal research committee of Nanjing University. 2.2. Colon ascendens stent peritonitis (CASP) surgery The surgical procedure of CASP was performed as previously described [14]. Briefly, 14-gauge venous catheters (2.0 mm 2, Venflon; BOC, Ohmeda AB, Sweden) were cut to similar segments with 0.5–0.6 cm length each. Under complete anesthesia (ketamine, 80–100 mg/kg, i.p.) and after disinfection of the abdomen (75% alcohol), the abdominal wall was opened through a 1-cm midline incision. After exposure of the ascending colon, the prepared stent was stitched through the antimesenteric wall into the proximal lumen of the ascending colon, fixed with two stitches (7/0 Ethilon thread; Ethicon, Nordestedt, Germany) placed approximately 1 cm from the ileocecal valve. Afterward, the inner needle of the stent was removed. Stool was milked from the cecum into the ascending colon and the stent until a drop of stool (1 mm in diameter) appeared. For control purposes, sham operation was performed in similar fashion except that the stent was fixed outside the ascending colon. Fluid resuscitation was performed by flushing 0.5 ml sterile saline into the peritoneal cavity before abdominal wall closure (two layers, muscle and skin; 5/0 Ethilon thread). The operated mice were brought back in cages to recover, with free access to food and water. 2.3. Treatment with human purified C3 protein A single dosage of 1 mg human purified C3 protein (HuC3), diluted in 200 μl PBS + 0.1% bovine serum albumin (BSA) in sterile, was intraperitoneally injected at 6 h post CASP surgery in the HuC3+ group. In the HuC3− group, only 200 μl of PBS + 0.1% BSA was injected at 6 h post CASP surgery. The HuC3 functional activity was estimated as >95% based on hemolytic activity, as determined by the supplier (MyBioSource, San Diego, CA, USA). 2.4. Cytokines and complement protein measurement At 24 h and 48 h post-surgery, blood samples from each group were collected by cardiac puncture for further analysis. The amount of C3, IL-10, and TNF-α in plasma was determined with a murine ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. The levels of sensitivity were 1.0 μg/ml for C3, 12.0 pg/ml for IL-10, and 5.0 pg/ml for TNF-α.
2.5. Flow cytometry assay for T cell subsets At given time points, mice were sacrificed via cervical dislocation. Spleen was harvested and gently ground to obtain single-cell suspensions, which then were subjected to continuous density gradient centrifugation (2000 rpm, 25 min) with Percoll (GE Healthcare, Uppsala, Sweden). Splenocytes were collected and washed twice with RPMI1640. Then, cell counts were performed with a hemocytometer (Trypan blue exclusion) and re-suspended to a concentration of 106 cells/ml of medium for T cell subset analysis. Intracellular cytokine staining was performed with PE- or allophycocyanin-labeled mAbs as described [6]. Briefly, separated cells were stimulated for 5 h with PMA (50 ng/ml) and ionomycin (1 μg/ml) with GolgiStop added after 2 h. Cell stimulation was terminated by fixing in 4% formyl saline. Fixed cells were stained in 0.1% saponin permeabilization buffer for 1 h. Mouse Th1/Th2/Th17 Phenotyping Cocktail purchased from BD Pharmingen was used to stain cells in the dark for 30 min. Finally, flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences). 2.6. FOXP3 immunohistochemistry and Western blot analysis Tissues from spleen were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned according to the standard procedure. Immunochemistry analysis was performed as previously described [15]. In brief, multiple 5 μm serial sections from selected formalin-fixed, paraffin-embedded blocks were made and reacted with affinity-purified anti-FOXP3 polyclonal Ab (ab22510, ABCAM, Cambridge, UK). The slides were washed in PBS and subsequently incubated with the second Ab, HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After being washed in PBS, slides were developed with 3,3′-diaminobenzidine, and counterstained with hematoxylin. Coverslips were applied with DPX mountant (CV5000 Coverslipper; Leica Instruments). The numbers of immunophenotyped cells in the stained sections were counted by visual inspection. For each section, photographic images of each microscopic field were obtained (magnification, × 6100). The FOXP3 protein expression was analyzed as described previously [16]. Briefly, T cell subsets harvested from spleen in various time points were washed and lysed using RIPA buffer (Boston BioProducts, Worcester, MA) supplemented with protease inhibitor. Cell lysates were subsequently homogenized using QIAshredder Homogenizer columns (Qiagen, Valencia, CA). Soluble fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and transferred to nitrocellulose membrane. Membranes were saturated for 1 h at room temperature in TBST supplemented with 5% nonfat dry milk and immunoblotted overnight at 4 °C with anti-FOXP3 monoclonal antibody (clone 236/E7; ABCAM, Cambridge, MA) or α-tubulin (clone B-5-1-2; Sigma, St Louis, MO). Membranes were then washed, probed with HRP-conjugated goat anti-mouse polyclonal antibodies (Zymed Laboratories, San Francisco, CA), and revealed with enhanced chemiluminescence (ECL; Amersham Biosciences, Uppsala, Sweden). 2.7. Reverse transcription-PCR analysis and phosphorylation of NF-κB Total RNA was isolated from the purified T cell subset suspension by using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), treated with RNase free-DNase (Promega, Madison, WI, USA) and reverse transcribed to cDNA with random primer by Superscript II (Invitrogen, Carlsbad, CA, USA). The cDNA was then amplified by PCR. The forward and reverse primers were as follows: F: 5′-CCT CTC TCG TCT TCC TCC AC-3′; R: 5′-CAG TGG GCT GTC TCC AGT AA-3′ for NF-κB (132 bp), and F: 5′-ACG ACC CCT TCA TTG ACC TCA A-3′; R: 5′-GCA GTG ATG GCA TGG ACT GTG-3′ for GAPDH. PCR amplification was performed for 40 cycles at 95 °C for 20 s and 60 °C for 1 min. The mRNA
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level for each gene for each sample was normalized to GAPDH mRNA and was presented as 2 · [(Ct/ GAPDH− Ct / gene of interest)] as described. The purified T cell subsets were lysed in 500 μl of lysis buffer [50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 1 mM PMSF, aprotinin (1 μg/ml), leupeptin (1 μg/ml), and pepstatin (1 μg/ml)]. The supernatants were obtained via centrifugation at 12,000 rpm for 10 min at 4 °C, and the protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA). Afterward, the 0.5–0.8 mg of protein diluted in lysis buffer was immunoprecipitated with polyclonal p65 antibody (catalog #18-785-210321, GenWay Biotech Inc., San Diego, CA) and protein G-Sepharose at 4 °C overnight. The immunoprecipitates were subjected to 10% SDSPAGE and Western blotting with antibody to p65 (1:1000; Rockland Immunochemicals). 2.8. Electrophoretic mobility shift assays (EMSA) of NF-κB The nuclear extracts were extracted from purified T cell subsets at 24 h and 48 h post CASP procedure was isolated and quantified as previously described [17]. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, Ill, USA). EMSA was performed using a commercial kit (Gel Shift Assay System, Promega, Madison, WI). The DNA probe that consisted of the NF-κB consensus sequence (5′-AGT TGA GGG GAC TTT CCCAGG C-3′) was end-labeled with rhodamine (Amersham Pharmacia Bio., Tokyo, Japan). The nuclear extracts (5 μg/ml) were incubated with rhodamine-labeled NF-κB probe in the binding buffer (20 mM HEPES pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 30 mM KCl, and 0.2% (w/v) Tween 20) at room temperature for 15 min. The nuclear protein and oligonucleotide complexes were separated from free probes on native 5% polyacrylamide gel (BioRad, Richmond, CA) in 0.25× TBE buffer (Trisborate–EDTA) at 390 V for 1 h at 4 °C. The gel was exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan) at − 70 °C with an intensifying screed. Levels of NF-κB DNA binding activity were quantified by computer-assisted densitometric analysis. 2.9. Statistical analysis Measurement data were expressed as mean± SEM, if not stated otherwise. Statistical analyses of most data were performed using the Student's t-test, with two-sample and assuming unequal variances, where indicated. Comparison between the HuC3− group and the HuC3+ group was made by unpaired t-test. Pearson and Spearman correlation coefficients were calculated for variables following or not following Gaussian distribution, respectively. Comparisons between multiple groups were analyzed with one-way ANOVA. All statistical analyses were performed by using GraphPad Prism Software (version 5.01; GraphPad, San Diego, CA). P value below 0.05 was considered significant.
Fig. 1. Survival of CASP-operated mice after exogenous C3 treatment. Groups are depicted as indicated. Survival was 100% through the observational period in the sham group, 40% in the HuC3− group at 24 h but 5% at 48 h post-operation. Exogenous C3 injection at 6 h post-operation improved survival, with 60% at 24 h and 40% at 48 h, respectively. ⁎⁎P b .01 vs. the HuC3− group.
3.1. CASP-induced sepsis results in an expansion of regulatory T cells in vivo while HuC3 administration could suppress the expansion To investigate the activity of Tregs (CD25 +FOXP3+) in response to the CASP-induced sepsis, immunohistochemistry staining of FOXP3 was performed as mentioned above. The expression of FOXP3 protein in spleen at 24 h post-surgery was quite different among three groups (Fig. 2). The polymicrobial sepsis also led to a significant expansion of Tregs in the HuC3− group compared with the sham-operated group. Whereas, HuC3 administration at 6 h post CASP surgery greatly inhibited the activation of Tregs in the HuC3+ group, as compared to the HuC3− group (P b 0.01). Furthermore, Western blot analysis was performed to detect the effect of exogenous C3 on the expression of FOXP3 in T cell subsets, which were obtained from the spleen (Fig. 3). The Western blot results showed low expression of FOXP3 protein in the sham group at 24 h and 48 h post-surgery. Nevertheless, the protein levels were significantly increased in the HuC3− group compared with the sham group (P b 0.001). After the exogenous C3 administration, the increased expression of FOXP3 protein was remarkably suppressed compared with nontreatment (P b 0.01), but with no significant difference to the sham group. 3.2. During the process of sepsis, the Th1–Th2 shift at the late stage could be controlled under HuC3 protein administration The differentiation of T helper cells during sepsis was investigated via flow cytometry assay as described in the Materials and methods. All sham-operated mice had low but stable overall percentage of differentiated Th1, Th2, and Th17 T effectors. In contrast, CASP-induced
3. Results After the surgery with 14-G catheter, all CASP-operated mice developed severe sepsis rapidly, whereas sham-operated mice remained normal and survived through the whole observation period. To be specific, the 48 h survival rate was markedly increased in mice with exogenous C3 administration at 6 h post-surgery (HuC3+ group), as compared with CASP-operated mice without C3 treatment (40% vs. 5%, P b 0.01). The Kaplan–Meier method survival curves are shown in Fig. 1. Additionally, this polymicrobial sepsis led to a quick depletion of C3 protein and increased release of inflammatory cytokines, such as TNF-α and IL-10. Early supplementation of exogenous C3 protein during sepsis could improve the plasma levels of C3, with a decreased production of cytokines in the meanwhile (Table 1).
Table 1 The plasma levels of C3 and cytokines in late stage of severe sepsis.
C3 (μg/ml) TNF-α (pg/ml) IL-10 (pg/ml)
24 h 48 h 24 h 48 h 24 h 48 h
CASP, sham
CASP, HuC3−
186.3 171.9 225.9 186.8 300.0 171.9
112.8 81.8 1520.0 1120.0 12,820.0 11,250
(15.4) (18.2) (20.4) (21.8) (14.1) (18.2)
(10.3)a (12.9)a,c (41.2)a (48.8)a,c (505.9)a (363.4)a
CASP, HuC3+ 216.4 179.2 1240.0 1040.0 13,950.0 10,720.0
(19.2)b (21.3)b (26.6)a,b (46.8)a (365.1)a,b (526.3)a
All values are expressed as mean (SD). n = 5 for each group; three times repeated experiment. a P b .05 vs. the sham group. b P b .05 vs. the HuC3− group. c P b .05 vs. the same column at 24 h post-surgery.
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Fig. 2. Expression of FOXP3 in the spleen tissue after 24 h of CASP surgery via immunohistochemistry staining method. The nuclear FOXP3 single-staining cells were considered as Tregs (CD25+FOXP3+). Immunohistochemistry staining showed that FOXP3 mainly distributed in the nucleus with brown staining color. Few FOXP3 positive cells were observed in the sham group (A). CASP-induced sepsis produced an enhanced expression of FOXP3 in the HuC3− group (B), while the expression was relatively decreased in the HuC3+ group (C). Arrows indicated representatively FOXP3 positive cells in the surrounding region of contusion cortex. Scale bars: 100 μm. The big black box indicates the amplification of staining zone marked with small box; scale bars: 10 μm. (D) Quantification of FOXP3 positive cells in each group. Data are presented as mean± SEM of n = 5 per group; three times repeated experiment. ⁎⁎⁎P b .001 vs. the sham group; ##P b .01 vs. the HuC3− group.
sepsis resulted in a remarkable increase in the overall percentage of differentiated Th1 cells in early stage, but followed by a rapid decline after 24 h post-surgery (Fig. 4). Compared with the HuC3− group, exogenous C3 protein injection with a single dose postponed the decline for the differentiated Th1 cells and suppressed the increase of Th2 T effectors after 24 h post-surgery. In addition, the shift of T cell subsets from Th1 to Th2 effectors was observed in the HuC3− group, with a significant decline in Th1/Th2 ratio at 48 h post-surgery (P b 0.01; Fig. 4B). However, the shift could be effectively controlled under HuC3 protein administration, with higher ratio of Th1/Th2 effectors in the HuC3+ group than that in the HuC3− group (P b 0.05 at 24 h, P b 0.001 at 48 h postsurgery). Moreover, the overexpression of differentiated Th17 T effectors due to severe sepsis can be reversed by HuC3 supplementation in early stage of sepsis (Fig. 4). 3.3. CASP-induced sepsis results in the decreased expression of NF-κB in the late phase that could be improved with exogenous C3 protein supplementation
Fig. 3. Expression of FOXP3 protein in spleen from the sham group, HuC3− group and HuC3+ group at 24 h and 48 h post-surgery. Levels of FOXP3 protein were assessed by Western blot analysis with α-tubulin for control blot (A). The quantitative analysis of the Western blot results for FOXP3 protein expression is shown above (B). Bars represent as mean ± SEM; n = 5 per group; three times repeated experiment. ⁎⁎⁎P b .001 vs. the sham group, ##P b .01 vs. the HuC3− group.
To detect the expression of NF-κB in T cell subsets during sepsis, RT-PCR was performed at 24 h and 48 h post-surgery. In the sham group, the expression of NF-κB mRNA in T cells was mild but stable during the whole experiment. Nevertheless, the expression in the HuC3− group was remarkably increased at 24 h post-surgery compared to the sham group (P b 0.001), but followed by a rapid decline at 48 h post-surgery (P b 0.001, Fig. 5). Administration of HuC3 protein could inhibit the NF-κB mRNA expression significantly in comparison with the HuC3− group at 24 h post-surgery (P b 0.01). However, a single injection of exogenous C3 cannot reverse the
Y. Yuan et al. / International Immunopharmacology 12 (2012) 271–277 Fig. 4. The percentages of Th1, Th2, and Th17 cells during the process of CASP-induced sepsis. All T cell subsets were purified from the spleen. Mice were sacrificed at given time points, with five mice used at each time point in each group. Cytokine production by the Th1, Th2, and Th17 cell lines upon re-stimulation with PMA and ionomycin. Specifically, IFN-γ for Th1, IL-4 for Th2, and IL-17A for Th17, filtered in CD4+ gate cells. (A) Typical flow cytograms of Th cells at 48 h post-surgery in the HuC3−/+ groups. The counts of CD4+ T cells were compared via histogram. (B) The changes and ratio of Th differentiated cells during sepsis. Data are presented as mean ± SEM of three times repeated experiment. ⁎⁎P b .01, ⁎⁎⁎P b .001 vs. the sham group; #P b .05, ###P b .001 vs. the HuC3− group; †††P b .001 the HuC3− group at 24 h vs. the HuC3− group at 48 h post-surgery.
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DNA binding activity in both CASP-operated groups was significantly enhanced at 24 h post-surgery (P b 0.001). After 48 h of CASP surgery, the binding activity in the HuC3− group decreased quite fast, which was distinctly lower than that at 24 h post-surgery (P b 0.001). Importantly, the decline of NF-κB binding activity also could be improved with exogenous C3 protein administration. 4. Discussion
Fig. 5. Expression of NF-κB in purified T cell subsets from the spleen tissue. RT-PCR bands (A) show the NF-κB mRNA expression at 24 h and 48 h after surgery in the sham group, HuC3− group, and HuC3+ group. The density of mRNA (B) in each group is quantitatively analyzed. The phosphorylation of NF-κB subunit p65 (C) by Western blotting indicates the activity of NF-κB during sepsis. Data represent as mean ± SEM; n = 5 per group; three times repeated experiment. ⁎⁎⁎P b .001 vs. the sham group; ##P b .01 vs. the HuC3− group; †††P b .001 indicates the HuC3− group at 24 h vs. the HuC3− group at 48 h after surgery.
decline of the expression in the late phase of sepsis. In the HuC3− group, the increased phosphorylation of the NF-κB subunit p65 at 24 h post-surgery also indicated that enhanced activation of NF-κBdependent transcription occurred in early stage of sepsis. Besides, the declined p65 phosphorylation at 48 h post-surgery could be suppressed if the exogenous C3 was injected at 6 h post-surgery (Fig. 5C). On the other hand, NF-κB DNA binding activity was examined by EMSA autoradiography. A persistently low NF-κB binding activity (weak autoradiography) was found in the sham group within the period of experiment (Fig. 6). Compared with the sham group, NF-κB
In this study, CASP-induced sepsis model was utilized to investigate the changes of adaptive immunity in response to the persistent intra-abdominal infection. Tregs, as a pivotal component in inflammatory conditions, could be marked in tissue by antibodies combining to FOXP3 protein through immunohistochemistry or Western blot method [15]. Based on the available results, an extensive expansion of Tregs was observed at the late stage of sepsis, which was correlated to the subsequent Th1–Th2 shift and eventual immunosuppression. Importantly, exogenous C3 protein administration in early stage of sepsis could remarkably inhibit the activation of Tregs and delay the shift, associated with improved outcomes. It is recognized that innate immune responses, particularly those mediated by neutrophil- and macrophage-phagocytosis, are an essential part in early stage of bacterial infection [18]. Complement C3 plays an important role in this stage. It is quite helpful to enhance the phagocytosis of bacterial pathogens for both macrophages and other polymorphonuclear leukocytes. However, the complement depletion during sepsis, particularly C3 protein exhaustion, would make this positive biological effect impaired and increase the risk of recurrent infection and immunocompromise [19]. Besides, it has been confirmed that decreased complement levels would reduce priming of both CD4 + and CD8 + T cells, which suggested a more generalized role of complement [11,13]. In the present study, the differentiation of Th1 effectors was attenuated in late stage of sepsis, with enhanced differentiation of Th2/Th17 cells in the meanwhile. The observed Th1–Th2 shift indicated that the adaptive immunity against the polymicrobial sepsis had been impaired due to the complement depletion. This deviation toward Th2 differentiation could inhibit potentially protective cell-mediated immunity (CMI) responses and produce antibodies associated with antibody-dependent cell-mediated cytotoxicity (ADCC). The imbalanced deviation would further result in the elimination of Th cell subsets and the appearance of immunosuppression. Attenuated T-cell responses in late stage of sepsis may also be a consequence of decreased levels of C5a [20]. The persistently low level of C5a is a specific characteristic of complement exhaustion.
Fig. 6. NF-κB DNA binding activity in the spleen tissue after surgery. (A) ESMA autoradiography of NF-κB DNA binding activity (black arrow) at 24 h and 48 h post-surgery in the sham group, HuC3− group, and HuC3+ group. (B) Quantification of NF-κB DNA binding activity. Data represent as mean ± SEM; n = 5 per group; three times repeated experiment. ⁎P b .05, ⁎⁎P b .01, ⁎⁎⁎P b .001 vs. the sham group; #P b .05 vs. the HuC3− group; †††P b .001 means the HuC3− group at 24 h vs. the HuC3− group at 48 h after operation.
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The C5a receptors are extensively distributed on surface of T cells, as a specific epitope to C5a [13]. However, the role of the anaphylatoxin C5a is still not clearly understood. Besides, the enhanced expansion of CD25 +FOXP3 + Tregs has also been associated with the reduced CMI during sepsis [21]. To investigate the potential mechanism of T-cell responses in sepsis, the expression of NF-κB mRNA and its binding activity were assessed. The decreased expression of NF-κB mRNA with the attenuated T-cell responses might indicate that the negative control of NF-κB signaling is related to the Th1/Th2 shift and the expansion of Tregs, which is also consistent with the decreased phosphorylation of p65 subunit in NF-κB. Toscano et al. have demonstrated that NF-κB can control the expression of immunoregulatory galectin-1 protein on T cells [22]. T cells responding to presented antigen would express IL-10 in the presence of IL-12 [23]. Moreover, the enhanced expression of IL-10 is believed to be dependent upon the activation of NF-κB pathway based on previous study [24]. However, the concrete mechanism of this effect is still uncertain. Several limitations of current study should be noted. This study failed to investigate the role of C3 in regulation of B cells and antibody responses during the process of sepsis. Further studies should identify whether exogenous C3 administration co-stimulates B cells through the regulation of Th cells, although some studies have confirmed its role in enhancing the adaptive immunity in sepsis [25,26]. Besides, dendritic cells (DCs) also play an important role in bridging innate and adaptive immunity. We did not monitor its activity or investigate its relationship to complement exhaustion during sepsis. Moreover, several groups have shown that the expansion of natural CD4+CD25+ Tregs does not contribute to mortality in murine polymicrobial sepsis [27,28]. Therefore, Tregs seems likely to be an indirect mediator, which bridges the complement system and T-cell responses during sepsis. Some other populations, such as myeloid-derived suppressor cells (MDSC), and Natural Killer-T-cells, should be investigated in further studies. At last, we just confirmed the potential effect of NF-κB p65 activation on T cells. The other upstream signaling pathways of NF-κB, such as ERK1/2, IκBα kinase, and p-ERK1/2 should be studied with knockout mice in further steps. In conclusion, our findings provide strong evidence that complement C3 plays an important role in enhancing T cell responses to conditional bacterial infection during the polymicrobial sepsis. This potent effect is believed to work by depressing the expansion of regulatory T cells through NF-κB pathway. Exogenous C3 administration delays the complement depletion in late stage of sepsis, attenuates the Th1–Th2 shift, and enhances the differentiation of Th17 cells in the meantime. The findings have implications in rational design of effective treatment of sepsis-induced immunocompromise for humans. Acknowledgment This study was supported by grants from the National Natural Science Foundation of China (No. 30872456) and the Climbing Program in Natural Science Foundation of Jiangsu Province for Distinguished Scholars (No. BK2010017). References [1] Yalcindag A, He R, Laouini D, Alenius H, Carroll M, Oettgen HC, et al. The complement component C3 plays a critical role in both Th1 and Th2 responses to antigen. J Allergy Clin Immunol 2006;117:1455–61.
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