The present study was approved by the 309th Hospital of Chinese People's Liberation Army Medical Research Ethical Committee (Beijing, China). Male C57BL/6 mice (n=100) were purchased from Shandong University Animal Ethical Committee (Jinan, China), and were 8–10 weeks old at the time of entry into the present study.

Triton X-100 and MTT were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). RPMI-1640 medium was purchased from Hyclone; GE Healthcare Life Sciences, (Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) The CD4⁺ T cell Isolation kit was purchased from Miltenyi Biotec GmbH (Bergisch-Gladbach, Germany. Anti-CD3 and anti-CD28 monoclonal antibodies were purchased from BD Biosciences (Affymetrix, San Jose, CA, USA; cat. nos. 16-0022 and 16-0281, respectively). The enhanced chemiluminescence (ECL) plus chemiluminescence kit was purchased from GE Healthcare Life Sciences (Uppsala, Sweden). ELISA kits for interleukin IL-2 (cat. no. EM002-96), IL-4 (cat. no. EM003-96) and interferon IFN-γ (cat. no. EM007-96) were purchased from Shanghai ExCell Biology, Inc. (Shanghai, China).

Male C57BL/6 mice used in the present study (weight, 18–22 g) were provided by the Shandong University Animal Ethical Committee. All animals were housed in separate cages in a temperature-controlled room at 26°C under a 12-h light/dark cycle. All animals had free access to food and water. Polymicrobial sepsis was induced by the CLP procedure, as described by Wichterman et al (8). Briefly, anesthesia was induced through the intraperitoneal administration of thiopental (25 mg/kg), the mice were placed in a supine position and their abdomens were shaved. An abdominal midline incision of 1 cm was made to expose the cecum. According to Rittirsch et al (9), the cecum was ligated at the middle and punctured twice with a 21-gauge (0.73-mm) needle to induce sepsis of moderate severity. Sham-operated mice underwent the same laparotomy procedure with the exception of ligation and perforation.

In the present study, 100 mice were randomly divided into four groups as follows: Sham injury group (n=30), CLP group (n=30), CLP with lentivirus-RNA-TNFAIP8 group (n=20) and CLP with negative control group (n=20). Mice in all groups were sacrificed 24 h following CLP, and spleen samples were harvested for the isolation of CD4⁺ T cells.

Small RNA specific to TNFAIP8 was synthesized by GenchemBiotetchnology Company (Shanghai, China), the sequence was as follows: 5′-CCGGCATGGAGAAGTTCAAGAAGAATTCAAGAGATTCTTCTTGAACTTCTCCATGTTTTT-3′. To induce overexpression of TNFAIP8, recombinant lentiviruses [pFH-L lentiviral vector (Shanghai GeneChem Co., Ltd., Shanghai, China)], carrying TNFAIP8-RNAwere injected intraperitoneally at a multiplicity of infection (MOI) of 10⁹ TU/ml into mice 14 days prior to CLP injury. Concomitantly, the same concentration of recombinant lentiviruses that carried the negative control RNA (empty vector; Shanghai GeneChem Co., Ltd.) was injected at an MOI of 5×10⁸ TU/ml into the mice, which served as a wild type control. TNFAIP8 and negative control RNA lentivirus generation was performed according to the lentivirus vector particle manufacturer's protocol. The efficiency of overexpression was determined by western blot analysis of TNFAIP8 expression.

Spleens were obtained from the sham mice and CLP mice, and were cultured in 5 ml RPMI-1640 medium. Mononuclear cells were isolated with the use of Ficoll-Paque density gradient centrifugation at 1,500 × g for 15 min at 4°C, according to the manufacturer's protocol, and CD4⁺ T cells were isolated from them using the CD4⁺ T cell isolation kit according to the manufacturer's protocol. Mononuclear cells (10 µl/10⁷ total cells) were stained with a biotin-antibody cocktail (MiltenyiBiotec GmbH, BergischGladbach, Germany; cat. no. 130049201) and incubated for 10 min at 4°C. They were then magnetically labeled with anti-biotin MACS microbeads (20 µl/10⁷ total cells; MiltenyiBiotec GmbH), incubated for 15 min at 4°C, and harvested through a negative selection LS column (MiltenyiBiotec GmbH).

Western blotting was performed to determine the expression of TNFAIP8 in the extracted CD4⁺ T cells. Cells were lysed by radioimmunoprecipiation lysis buffer [25 mM Tris-HCL (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS] at the temperature of 0°C for 30 min. Following sonication for 5 sec, the lysed cells were then centrifuged at 12,000 × g for 30 min at 4°C. Protein levels were quantified using a bicinchoninic acid protein assay kit. Protein extracts (50 µg) were separated by 8% SDS-PAGE, and the products were electro transferred to an immobilon polyvinylidene difluoride membrane. Following blocking with 10% skim milk overnight at 4°C, the membrane was incubated for 4 h at room temperature with anti-TNFAIP8 polyclonal antibody (cat. no. ab64988; 1:500 dilution; Abcam, Cambridge, MA, USA) or control anti-β-actin antibody (cat. no. sc-130201; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The membrane was then washed three times with PBS and incubated with peroxidase-labeled mouse IgG secondary antibody (cat. no. A0548; 1:5,000; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. As aforementioned, the membrane was washed with PBS three times, and blots were then developed using the ECL plus chemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). The protein bands were detected using an ECL detection system (Pierce; Thermo Fisher Scientific, Inc.).

CD4⁺ T cells with density of 10⁶/ml in the normal group were washed with PBS three times, fixed with 4% paraformaldehyde in PBS for 20 min, then permeabilized with 0.2% Triton X-100 for 20 min both at room temperature. Sections were pre-blocked with 1% FBS in PBS for 30 min, and stained with anti-TNFAIP8 antibody (cat. no. ab64988; 1:200; Abcam) overnight at 4°C. Following washing three times in PBS, CD4⁺ T cells were stained with a fluorescein isothiocyanate-conjugated goat anti-immunoglobulin G secondary antibody (cat. no. C1309; 1:5,000; Applygen Technologies Inc., Beijing, China) for 1 h at room temperature followed by three further washes in PBS. Following washing, the nuclei were stained with 4′,6-diamidino-2-phenylindole for 5 min at room temperature. The cells were observed under a laser scanning confocal microscope.

Purified CD4⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, and were stimulated for 24 h for CD4⁺ activation with a combination of 1 µg/ml soluble anti-CD3 and 1 µg/ml soluble anti-CD28 monoclonal antibodies at 37°C. The T cells were then plated in 96-well plates at a density of 1×10⁵ cells/well, and were incubated at 37°C in 5% CO₂ for 68 h. Subsequently, modified MTT solution (5 mg/ml, 10 µl/well) (10–12) was added and the cells were incubated for a further 4 h, after which 100 µg acid isopropanol was added to dissolve the MTT crystals. The MTT crystal suspension was repeatedly mixed with a pipette, and the optical density was measured using a microplate reader at a wavelength of 540 nm.

To analyze the secretion of IL-2, IL-4 and IFN-γ into the culture medium of the cells, the supernatants obtained from CD4⁺ T cells were analyzed, and the supernatants were removed by pipetting. The levels of IL-2, IL-4 and IFN-γ were measured using commercially available ELISA kits according to the manufacturer's protocols.

All data in the present study are presented as the mean ± standard deviation of 3 independent experiments. Continuous data were examined by one-way analysis of variance followed by a post hoc Dunnett's test for multiple comparisons. SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform these statistical analyses. P<0.05 was considered to indicate a statistically significant difference.

The expression and distribution of TNFAIP8 protein in CD4⁺ T cells isolated from normal mice was investigated by means of confocal laser scanning microscopy. Green fluorescence was observed in the cytoplasm of the CD4⁺ T cells, with their nuclei stained blue (Fig. 1A-F).

In the present study, the effects of TNFAIP8 on proliferative activity of CD4⁺ T cells in a CLP mouse model were observed. TNFAIP8 was successfully upregulated following injection of mice with lentivirus-RNA-TNFAIP8 (Fig. 2). Western blot analysis revealed that the protein expression levels of TNFAIP8 were significantly upregulated in the TNFAIP8 overexpression group compared with the control groups (Fig. 2). The proliferative activity of splenic CD4⁺ T cells was significantly suppressed 24 h following CLP compared with the sham injury group (P<0.05; Fig. 3). To further clarify the involvement of TNFAIP8 in the decreased cell proliferation observed following CLP-induced sepsis, the effects of upregulated TNFAIP8 were determined in vivo 24 h following CLP. Compared with the CLP group, proliferative activity was significantly increased in the CLP with TNFAIP8 overexpression group (P<0.05; Fig. 3), indicating that TNFAIP8 may be closely associated with the immune functions of CD4⁺ T cells.

IL-2 is a potent T cell growth factor that acts upon itself in an autocrine fashion. The levels of IL-2 in culture supernatants of CD4⁺ T cells were measured by ELISA. Following 24 h of CLP, IL-2 production in the culture supernatant was significantly downregulated compared with in the sham-injured group (P<0.05; Fig. 4). Conversely, IL-2 expression levels were significantly higher following TNFAIP8 upregulation by lentivirus-RNA-TNFAIP8 infection in vivo compared with the CLP group, whereas no significant difference was observed between the negative control CLP and CLP groups (Fig. 4). It was noted that excessive TNFAIP8 expression was associated with the increased IL-2 levels secreted by CD4⁺ T cells.

It is possible for an immune response to become polarized towards either Th1 or Th2 production over time; therefore, one subtype or the other dominates. It is well known that Th1 cells produce IFN-γ and Th2 cells produce IL-4; therefore, ELISA was used in the present study to detect these T cell-produced cytokines, in order to identify the polarization of naive T cells. The results revealed that the levels of IFN-γ produced by CD4⁺ T cells were significantly reduced, and that the levels of IL-4 were significantly increased at 24 h in the CLP group compared with the sham group (P<0.01; Fig. 5). Furthermore, a significant increase in IFN-γ and a significant decrease in IL-4 were detected in CD4⁺ T cells following TNFAIP8 overexpression in the CLP-induced sepsis group (P<0.01; Fig. 5). Taken together, these results suggested that TNFAIP8 may affect T cell polarization following sepsis.