Proportion of CD14+CD16+ monocytes in peripheral blood from patients with type 2 diabetic mellitus and effect of LPS and IL-15 on expression of STAT5 in monocytes

AIM: To characterize the proportion of CD14⁺CD16⁺ monocytes in peripheral blood from type 2 diabetes (T2DM) patients and to observe the response of CD14⁺CD16⁺ monocytes to lipopolysaccharide (LPS) and interleukin-15 (IL-15) for further exploring the potential mechanism of inflammatory immune response in the pathogenesis of T2DM. METHODS: Twenty-eight patients with T2DM and 20 healthy volunteers were enrolled in the study. The peripheral blood was collected for determining the percentage of CD14⁺CD16⁺ monocytes by flow cytometry. The peripheral blood mononuclear cells (PBMC) were isolated and subject to stimulation with LPS and IL-15 for 4 h. The protein expression of STAT5 was detected by Western blotting and the phosphorylated (p)-STAT5 was determined by Western blotting and immunofluorescence. Serum levels of 25-hydroxyvitamin D₃ and IL-6, and the concentrations of IL-6 and monocyte chemoattractant protein-1(MCP-1) in the culture supernatants were assessed by ELISA. Serum level of C-reactive protein (CRP) was measured by immunoturbidimetry. RESULTS: There were positive correlations between the quantity of CD14⁺CD16⁺ monocytes and serum levels of CRP and IL-6 (r=0.394, P<0.05 and r=0.741, P<0.01), while serum 25 (OH) D₃ was negatively correlated with the quantity of CD14⁺CD16⁺ monocytes (r=-0.409, P<0.01), serum CRP(r=-0.479,P<0.01) and serum IL-6 (r=-0.774,P <0.01). After stimulated with LPS and IL-15, PBMC showed significant up-regulation of p-STAT5 protein expression, and significant increases in the supernatant levels of IL-6 and MCP-1 were observed (P<0.05). The expression of p-STAT5 existed in the nucleus.CONCLUSION: These findings suggest that the functional disturbance in monocytes occurs in T2DM, which may be related to insufficiency of vitamin D₃. The aberrant activation of STAT5 signaling pathway underlies the functional abnormalities of the monocytes in T2DM.     

Immunological mechanism of long-term stimulation by LPS in macrophages

AIM: To investigate the molecular mechanism and the immunosuppressive phenotype of macrophages under long-term exposure to lipopolysaccharide (LPS). METHODS: We used Ficoll-Hypaque density gradient centri-fugation combined with MicroBeads Separation Kits to separate peripheral blood mononuclear cells from human blood, and then induced the monocytes into macrophages. We observed the morphology of the macrophages by treating the cells with LPS for 48 h, in comparison with a negative control and IFN-γ treatment. ELISA was used to detect the levels of cytokines, such as IL-10, IL-12, IL-6 and TNF-α, and flow cytometry was used to detect the expression of the surface molecules (HLA-DR, CD14, CCR7, HLA-ABC and CD40). To observe the effect of macrophage on T cell proliferation, co-culture experiment was carried out for 6 d. Real-time PCR was used to validate the expression levels of molecules related to MyD88-independent pathway in Toll-like receptor 4 (TLR4) signal pathway. RESULTS: The antigen-presenting ability of the macrophages was reduced and the IL-10 expression level was increased after the cells were treated with LPS for 48 h. We observed a poor proliferative capacity of CD8⁺ T cells after co-culturing of LPS-induced macrophages with CD3⁺ T cells for 6 d. The results of real-time PCR indicated that TRIF, IRF3 and CIITA were down-regulated in LPS-induced macrophages.CONCLUSION: We successfully established a macrophage model in vitro and observed that LPS-induced macrophages into an immunosuppressive phenotype with poor CD8⁺ T cell proliferative capacity, in which MyD88-independent TLR4 signaling pathway was impaired.     

The characteristics of TCRVα24+ NKT cells in response to in vitro stimulation

AIM: To investigate the amount and patterns of expressing CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells, and compare with that of CD3⁺ T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin (Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-γ on TCRVα24⁺ NKT cells and CD3⁺ T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRVα24⁺ NKT cells to CD3⁺ T cells was about (1.34±0.42)%. The expression rates of CD69 on TCRVα24⁺ NKT cells and CD3⁺T cells in response to PDB+Ion for 6 h were (96.71±1.33)% and (98.60±0.47)%, respectively, while the ratio were (11.47±2.86)% and (1.07±0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-γ on TCRVα24⁺ NKT cells stimulated with PDB+Ion for 6 h were (48.62±2.44)% and (46.65±8.91)%, respectively, which were significantly higher than that of unstimulated group [(31.57±3.31)%, (13.45±6.29)%] and that of stimulated CD3⁺ T cells, though the expression rates on stimulated CD3⁺ T cells were significantly higher than that of unstimulated CD3⁺ T cells. CONCLUSION: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-γ and IL-4 on these lymphocytes are higher than CD3⁺ T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.     

Unfractionated heparin ameliorates lipopolysaccharide-induced expression of interleukin-8 in human endothelial cells through Toll-like receptor 4 signaling

AIM:To determine the effect of unfractionated heparin(UFH) on lipopolysaccharide(LPS)-induced expression of interleukin-8(IL-8) and the role of Toll-like receptor 4(TLR4) signaling. METHODS:Human pulmonary microvascular endothelial cells(HPMECs) were either exposed to LPS alone(10 mg/L) or in combination with 100 U/L or 10³ U/L UFH. UFH was added to the cells 15 min prior to stimulation with LPS. Those samples not receiving LPS or UFH received an equal volume of phosphate-buffered saline. The concentrations of IL-8 in the cell culture supernatants were detected by ELISA at 2, 6 and 12 h. The mRNA expression of IL-8, CD14 and TLR4 in HPMECs was detected by real-time fluorescence quantitative polymerase chain reaction at 2, 6 and 12 h. RESULTS:Compared with normal control group, the mRNA expression of IL-8 in LPS group was increased, and reached the peak at 6 h. The protein level of IL-8 reached the peak at 12 h. The mRNA expression of TLR4 in LPS group reached the peak at 6 h. They were down-regulated in UFH group. The mRNA expression of CD14 was not detected. CONCLUSION:The expression of IL-8 is obviously increased in LPS-treated HPMECs. UFH might exert its therapeutic effect through TLR4 signaling.     

Effect of heterogenic corneal stroma transplantation on peripheral T cells of rats

AIM: To Compare immunogenicity of three kinds of heterogenic corneal stroma. METHODS: 36 SD rats were randomized into 4 groups, each group consisting of 9 rats. Group 1 was control group. Three kinds of heterogenic corneal stroma: porcine, rabbit and chicken corneal stroma were heterotopically transplanted to subcutaneous layer of 27 (group 2-4) SD rats, respectively. The expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ on peripheral T cells was identified and analyzed by dual fluorescence flow cytometry at 7, 14, 28 days after operation. RESULTS: Compared with control group, the expression of CD4⁺, CD8⁺, CD25⁺, CD71⁺ was no significant change in porcine corneal stroma group(P＞0.05), the expression of CD4⁺ was increased in rabbit corneal stroma (P＜0.05), CD4⁺, CD4⁺ CD71⁺ markedly higher in the chicken corneal stroma (P＜0.01) at 7 days after operation. CONCLUSION: The immunogenicity of porcine stroma is the lowest in three kinds of heterogenic corneal stroma (chicken, rabbit and porcine).     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Change of antithrombin Ⅲ in patients with atherosclerotic cerebral infarction

AIM：To explore the change of antithrombin Ⅲ (AT-Ⅲ) in the patients with atherosclerotic cerebral infarction. METHODS：Chromogenic substrate assay was used to measure the activity of AT-Ⅲ in 55 patients with atherosclerotic cerebral infarction and 55 healthy controls, and the correlation analysis was applied to determine the AT-Ⅲ activity with the severity of damage in central nervous system and general biochemical parameters. The levels of TNF-α and IL-6 in the plasma were detected by ELISA. Immunocomplex in the plasma was measured by enzyme immunoassay （EIA）. The number and phenotype of the monocytes in peripheral blood were analyzed by flow cytometry. ELISA was also applied to determine the secretion of TNF-α and IL-6 from the monocytes after the stimulation of immunocomplex. The expression of AT-Ⅲ in human brain vascular endothelial cells after the stimulation of TNF-α and IL-6 was observed by Western blotting. RESULTS：The activity of AT-Ⅲ significantly decreased in the patients with atherosclerotic cerebral infarction, and negatively correlated with the damage degree of nervous system function, systolic pressure, diastolic pressure, glucose, cholesterol, triglyceride, low-density lipoprotein cholesterol and homocysteine, while positively correlated with high-density lipoprotein. In addition, the plasma levels of TNF-α and IL-6 increased significantly, accompanied with the enhancement of immunocomplex level. The numbers of CD14+ CD16+ and CD14+ CD32+ monocytes in peripheral blood were not changed, while CD14+ CD64+ monocytes increased obviously. The secretion of TNF-α and IL-6 by monocytes were significantly enhanced after stimulated with immunocomplex, while the protein expression of AT-Ⅲ in the human brain vascular endothelial cells was down-regulated after co-incubated with TNF-α or IL-6. CONCLUSION：Decreased AT-Ⅲ activity in the patients with atherosclerotic cerebral infarction is one of the risk factors of cerebral infarction, and related with the disease severity. The production of pro-inflammatory cytokines through immunocomplex from CD14+ CD64+ monocytes may be involved in the mechanism. Improvement of AT-Ⅲ activity may protect against cerebral ischemia.     

Effect of adoptive transfer of CD4+ T cells with miR-7 knockdown on acute liver injury in mice

AIM:To study the effect of adoptive transfer of CD4⁺ T cells with microRNA-7 (miR-7) knockdown (KD) on mouse acute liver injury model and to investigate its significance. METHODS:CD4⁺ CD62L⁺ T cells were purified from the spleen of normal wild-type (WT) mice and miR-7KD mice by magnetic bead sorting, and were stained with CFSE. These 2×10⁶ CFSE-labeling cells were injected into normal mice via tail vein, and then the mouse acute liver injury model was induced by intraperitoneal injection of 30 mg/kg concanavalin A. After 72 h, the appearance, weight and weight index of the liver were investigated. The pathological change of the liver tissues was observed by HE staining. Real-time PCR was used to examine the mRNA expression of Bax and P53. The expression levels of CD62L, interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the CD4⁺ T cells were analyzed by flow cytometry. RESULTS:We found that the liver tissue became lighter, and the weight (P<0.01) and weight index (P<0.05) were changed significantly in miR-7KD mice compared with control group. Moreover, HE staining showed that the liver cell damage was increased in the liver of miR-7KD mice. Meanwhile, the expression levels of Bax and P53 were significantly increased in miR-7KD group (P<0.05). The percentage of CD62L in CD4⁺ T cells was significantly decreased (P<0.01) in miR-7KD mice, with high expression of IFN-γ (P<0.05) and low expression of IL-4 (P<0.01) in CD4⁺T cells. CONCLUSION:These findings suggest that miR-7 knockdown significantly promotes the pathology of CD4⁺ T cell-mediated acute liver injury, which provides a preliminary experimental basis for further exploration on the mechanism of acute liver injury occurrence.     

Protective effect of HSF1 on mice with LPS-induced acute lung injury and screening of relevant differentially-expressed genes

AIM: To study the protective effect of heat shock factor1 (HSF1) on the mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI), and to screen the relevant differentially-expressed genes. METHODS: ALI mouse model was established by LPS intracheal instillation. The macroscopic and pathological changes of the lung tissue were observed, and the concentrations of total protein, TNF-α, IL-β, IL-6 and VEGF in the bronchoalveolar lavage fluid (BALF) were analyzed. Differentially-expressed genes in the lung tissues of HSF1^+/+ mice and HSF1^-/- mice with ALI induced by LPS were screened by gene chips. The key gene was verified by real-time qPCR. RESULTS: The macroscopic and pathological changes of the lung injury in HSF1^-/-+LPS mice were more serious than those in HSF1^+/++LPS mice. The concentrations of total protein, VEGF, TNF-α, IL-1β and IL-6 in the BALF of HSF1^-/-+LPS mice were significantly higher than those of HSF1^+/++LPS mice (P<0.05). Compared with the HSF1^+/+ mice, a total of 918 differentially-expressed genes were indentified in the HSF1^-/- mice, among which the expression levels of 65 genes had obvious diffe-rence, with 28 genes up-regulated, including Atg7, ccr1, cxcr2, Tbl1xr1, Mmp9, Pparg, Plcb2, Arrb2, Cntn1, Col4a6, etc, and 37 genes down-regulated, including Fgfr1, Fgfr2, Map4k4, Ddx58, Tfg, Stat3, Smad4, Lamc1, Sdc3, etc. The results of real-time qPCR showed that the mRNA level of CXCR2 in HSF1^-/-+ LPS mice was significantly higher than that in HSF1^+/++ LPS mice, which was consistent with the results of gene chips. CONCLUSION: HSF1 has protective effect on the mice with LPS-induced ALI. CXCR2 may be involved in the protective effect of HSF1 on this process.     

Influence of histone deacetylase inhibitor romidepsin on phenotype and function of T lymphocytes in vitro

AIM: To explore the effects of romidepsin (FK228), a novel histone deacetylase inhibitor, on the effector and regulatory T cells in vitro.METHODS: As the reactive cells, lymphocytes, CD4⁺ T cells and CD8⁺ T cells were labelled with CFSE, and stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group), or PBS (placebo group).After 72 h, the proliferation of the cells was detected in different groups. The lymphocytes were stimulated with anti-CD3 and anti-CD28 mAbs in the presence and absence of different levels of romidepsin (experimental group and positive control group),or PBS (placebo group). After 72 h, the percentage of CD4⁺ Foxp3⁺ T cells and the levels of related cytokines were detected in different groups. RESULTS: The proliferation of CFSE-labelled lymphocytes, CD4⁺ T cells and CD8⁺ T cells triggered by anti-CD3 and anti-CD28 mAbs all were inhibited when cultured with romidepsin at concentrations of 1 μmol/L, 3 μmol/L and 5 μmol/L in a dose-dependent manner (P<0.05). Compared with placebo group, in the presence of anti-CD3 and anti-CD28 mAbs, 1 μmol/L romidepsin did not increase the percentage of CD4⁺ Foxp3⁺ T cells (P>0.05). When cultured with romidepsin at concentrations of 3 μmol/L and 5 μmol/L, the percentage of CD4⁺ Foxp3⁺ T cells was enhanced markedly (P<0.05). The levels of IL-10 and TNF-α in the supernatant were markedly increased in positive control group and 3 experimental groups (P<0.05), and the levels of cytokines in different experimental groups were gradually decreased with the elevation of FK228 concentration (P<0.05). The level of TGF-β was slightly increased in positive control group with no significant difference compared with placebo group (P>0.05). With the increase in the concentration of FK228 in different experimental groups, the TGF-β level was increased in a dose-dependent manner and there were significant differences in the 3 experimental groups. Meanwhile, significant differences existed between experimental groups and placebo group and between experimental groups and positive control group (P<0.05). CONCLUSION: Romidepsin inhibits the proliferation of CD4⁺ and CD8⁺ effector T cells and increases the percentage of CD4⁺ Foxp3⁺ regulatory T cells. It may be related to the increased level of TGF-β, but independent of IL-10.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Detection and clinical significance of myeloid-derived suppressor cells in peripheral blood of patients with Parkinson disease

AIM: To detect the myeloid-derived suppressor cells (MDSCs) in peripheral blood from the patients with Parkinson disease (PD) and its clinical significance. METHODS: The patients (n=80) diagnosed PD from January 2016 to March 2017 in our hospital and 20 healthy volunteers were selected as the subjects. According to the Hoehn-Yahr staging, 80 PD patients were staged, of whom 22 were I, 24 were Ⅱ, 20 were Ⅲ, 14 were IV, and 0 was V. Peripheral blood (5 mL) samples from the patients with PD and the healthy volunteers were collected and the mononuclear cells were isolated. The levels of CD14⁺CD11b⁺ cells and CD14^-CD11b⁺ cells in the peripheral blood were detected by flow cytometry. The two populations of the cells were sorted by magnetic beads. The mRNA levels of arginase 1 (ARG1), interleukin-10 (IL-10) and cyclooxygenase 2 (COX-2) were detected by qPCR. The expression of surface membrane proteins CD14 and CD11b, and immunosuppressive factors ARG1, IL-10 and COX-2 was determined by Western blot and ELISA. RESULTS: No significant change of CD14⁺CD11b⁺ cells between the patients with PD and normal controls was observed, but the cells with CD14^-CD11b⁺ increased significantly in the patients with PD compared with the control people (P<0.05). The CD14^-CD11b⁺ cells in peripheral blood of the patients were related to the stage of Hoehn-Yahr. The CD14^-CD11b⁺ and CD14⁺CD11b⁺ cells showed high levels of IL-10 and COX-2, and the high level of ARG1 was only expressed in the CD14^-CD11b⁺ cells. The expression of ARG1 in the CD14^-CD11b⁺ population from PD patients was significantly different from that of CD14⁺CD11b⁺ population and normal subjects (P<0.05). CONCLUSION: The CD14^-CD11b⁺ cells and ARG1 expression level in peripheral blood of the PD patients can be used to evaluate the pathogenesis and staging. Immunosuppression may play an important role in the pathogenesis and development of PD.     

Effects of iNKT cells from OVA-induced asthmatic mice combined with OVA on phenotype and function of bone marrow-derived dendritic cells in vitro

AIM:To investigate the effects of invariant natural killer T-cells (iNKT cells) from ovalbumin (OVA)-induced asthmatic mice combined with OVA on the phenotypic and functional characteristics of bone marrow-derived dendritic cells (BMDCs) in vitro. METHODS:The BMDCs from wild-type (WT) BLAB/c mice were co-cultured with purified iNKT cells from WT mice immunized and challenged with OVA in the presence of 100 mg/L OVA (iNKT cells plus OVA group) or PBS (iNKT cells plus PBS group) for 20 h, and were also cultured with 50 mg/L LPS (LPS group), 100 mg/L OVA (OVA group) or PBS (PBS group) for 20 h. The expression of MHC-Ⅱ, CD40, CD86, and CD80 on the BMDCs was measured by flow cytometric analysis, and the levels of interleukin-12 (IL-12) p70, IL-6, tumor necrosis factor-α (TNF-α) and IL-10 in the culture supernatant were measured by ELISA. Splenic CD4⁺ T cells from DO11.10 transgenic mice were co-cultured for 48 h with the above mentioned BMDCs, and then the concentrations of IL-4 and interferon-γ (IFN-γ) in culture supernatants were measured by ELISA. RESULTS:The expression of MHC-Ⅱ, CD80, CD86 and CD40, and releases of proinflammatory cytokines by BMDCs in iNKT cells plus OVA group were comparable to those in the LPS group (P>0.05), but significantly higher than those in iNKT cells plus PBS group, OVA group, and PBS group (P<0.05 or P<0.01). The concentration of IL-4 in culture supernatants from BMDCs in iNKT cells plus OVA group co-cultured with DO11.10 CD4⁺ T cells was similar to that in LPS group (P>0.05), but markedly higher than that in iNKT cells plus PBS group, OVA group, and PBS group (P<0.01). CONCLUSION:Bone marrow-derived dendritic cells undergo immunogenic maturation upon interaction with iNKT cells in the presence of OVA.     

Balance of Treg/Th17 in synovium of collagen-induced arthritis rats and impact of TNF-α blockage therapy

AIM: To investigate the balance of Treg/Th17 in synovium of collagen-induced arthritis (CIA) and the impact of tumor necrosis factor α(TNF-α) blockage therapy. METHODS: Rat CIA model was established by bovine II collagen injection. The pathological score was evaluated by HE staining and toluidine blue staining. The TNF-α level in plasma was measured by ELISA. The expression of Treg/Th17 in synovium was detected by double staining immunofluorescence. RESULTS: The plasma level of TNF-α in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (P<0.01), whereas no significant difference was found between TNFR-Fc treatment group and control group (P>0.05). No significant difference between CIA group and control group in the ratio of CD4⁺Foxp3⁺Treg cells/CD4⁺ cells in synovium (23.12%±4.93% vs 24.66%±5.82%, P>0.05) was observed, whereas the ratio in TNFR-Fc treatment group was significantly increased(33.07%±5.14%). The ratio of CD4⁺RORγt⁺Th17 cells/CD4⁺ cells in CIA group was significantly higher than that in control group and TNFR-Fc treatment group (9.74%±2.23% vs 1.00%±0.59%, 5.63%±1.76%, P<0.01). CONCLUSION: Differentiation disturbance of Treg/Th17 exists in the synovium of CIA rats. TNFR-Fc may restore the balance of Treg/Th17 by inhibiting Th17 cell differentiation and inducing the production or accumulation of Treg.     

Calreticulin expression is necessary for protective immune effect of T-cell vaccine in EAE mice

AIM:To study the role of cell membrane ectopic calreticulin (CALR) expression on the protective immunie effect of T-cell vaccine (TCV) on experimental autoimmune encephalomyelitis (EAE). METHODS:EAE model was established by myelin oligodendrocyte glycoprotein 35-55 (MOG_35-55) immunization in C57BL/6 mice, and the mice were immunized with MOG_35-55-specific CALR⁺ and CALR^- T-lymphocytes. Symptomatic scores were compared at the maximum of the disease. On the 15th day after immunization, the proportion of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Treg) in the spleen, and the expression of interferon-γ (IFN-γ), interleukin-4 (IL-4), IL-10 and IL-17A in the serum were measured. RESULTS:Increased expression of CALR in activated T cells after γ-irradiation was observed. Blockade of CALR on the vaccinating T-cell surface reduced the protective effect of TCV. Furthermore, blockade of CALR reduced the number of Treg in the spleen and up-regulated pro-inflammatory cytokines. CONCLUSION:CALR expression in the T cells is necessary for the protective immunity induced by TCV in EAE mice.     

Relationship between invasion of tumor-associated macrophages and phenotype and immune efficacy of tumor-infiltrating lymphocytes in advanced ovarian carcinoma

AIM:To explore the relationship between the invasion of tumor-associated macrophages(TAM) and the phenotype and immune efficacy of tumor-infiltrating lymphocytes(TIL) in advanced ovarian carcinoma. METHODS:Immunohistochemical analysis of TAM density in 175 cases of poorly-differentiated ovarian cancer tissue biopsy was performed. The cases were divided into TAM high-density(TAM^High) group and TAM low-density(TAM^Low) group according to the median of TAM density. The control group included 32 cases of benign ovarian lesions. The changes of CD8⁺ and CD25⁺ phenotypes of TIL were detected by flow cytometry analysis. TIL in the 2 groups were cultured in vitro and the conditioned-medium was collected for detecting the expression of IL-2, IL-10, TGF-β and IFN-γ by ELISA. RESULTS:The average TAM infiltration density was 62.8/high-power field(HP, ×400) in 175 cases of poorly-differentiated ovarian carcinoma, and the median was 53.3/HP. TAM^High group was 87 cases and TAM^Low group was 88 cases. A significant difference between malignant ovarian carcinoma group and control group(10.5/HP) was observed. The mean expression of CD8⁺ TIL in TAM^High group was 24%, and CD8⁺ TIL in TAM^Low group was 52%(P<0.05). The mean expression of CD25⁺ TIL in TAM^High was 48%, and CD25⁺ TIL in TAM^Low was 25%(P<0.05). The average infiltration density of CD8⁺ and CD25⁺ TIL in control group was 7%. The average infiltration density of CD8⁺ and CD25⁺ TIL in TAM^High and TAM^Low groups was significantly higher than that in control group(P<0.05). Compared with TAM^Low group, TIL destruction cytokines IL-2 and IFN-γ were significantly decreased in TAM^High group(P<0.05), while the inhibitory cytokines IL-10 and TGF-β were significantly increased(P<0.05). CONCLUSION:In high-density TAM infiltration of ovarian cancer tissues, CD25⁺ TIL type and inhibitory cytokines IL-10 and TGF-β increase, while CD8⁺ TIL type and destruction cytokines IL-2/IFN-γ decrease, suggesting that the high-density TAM has relationship with the phenotype and immune efficacy of TIL.     

The effect and mechanism of H. polygyrus infection in T-cell-mediated inflammatory bowel disease in mice

AIM: To investigate the effect and mechanism of Heligmosomoides polygyrus (H. polygyrus) infection in mouse inflammatory bowel disease (IBD) mediated by CD4⁺ helper T-cells. METHODS: Ovalbumin (OVA) -specific CD4⁺ helper T-cells were transferred into SCID (severe combined immunodeficiency) mice to establish an IBD model. The IBD mice were infected by H. polygyrus and sacrificed 14 days later. The histological changes of the colon were observed, and the expression of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA and flow cytometry. Additionally, IL-4 monoclonal antibody was intraperitoneally injected into the H. polygyrus-infected IBD mice to block the secretion of IL-4. The IL-4-blocking IBD mice were sacrificed 9 days later and the above indexes were also determined.RESULTS: Compared with the non-infection group, the H. polygyrus-infected IBD mice had more severe colonic lesions, higher level of IL-4 and lower level of IFN-γ in mesenteric lymph nodes (all P<0.05). Compared with the non-blocking group, the H. polygyrus-infected IBD mice with IL-4 blockage had less colonic lesions, lower IL-4 level and higher IFN-γ level (all P<0.05).CONCLUSION: H. polygyrus infection in CD4⁺ T-cell-mediated IBD model promotes inflammation in the early stage probably by inducing the secretion of Th2 cytokine and inhibiting the secretion of Th1 cytokine. The finding suggests that using worms for treatment of IBD needs to be cautious.     

Influences of protein kinase C inhibitors on the expression of IL-2 and IFN-γ by human T-lymphocytes

AIM:To investigate the influences of protein kinase C(PKC) inhibitors on the expression of interleukin-2(IL-2) and interferon-γ(IFN-γ) byin vitro activated T-lymphocytes. METHODS:Double fluorescent staining together with flow cytometry was adopted to detect intracellular cytokines and to analyze the effects of H7 and gossypol on IL-2 and IFN-γ expression levels of T-lymphocytes stimulated with phorbol ester (PDB)+ionomycin(I) in the presence of monensin.RESULTS:The expression rates of IL-2 and IFN-γ of CD3⁺ T cells stimulated with PDB+I for 4 h were 16.64±2.04 and 25.81±3.53(x±s), respectively, which were significantly higher than that of control (1.06±0.22 and 3.12±0.77)(P＜0.05). Gossypol was able to inhibit the expression of IL-2 and IFN-γ significantly, with the expression rates of 2.08±0.12 and 9.01±1.90, respectively. At the presence of 50 μmol/L H7, the rates of IL-2⁺ and IFN-γ⁺ CD3⁺ T cells were 0.43±0.06 and 2.40±0.27, respectively. The effect of H7 was stronger than that of gossypol. CONCLUSION:PKC plays an important role in the expression of IL-2 and IFN-γ of CD3⁺T cells and its inhibitors H7 and gossypol exert significant inhibitory effect on the expression of these two cytokines. It is suggested that H7 and gossypol may have modulatory effect on T-cell-dependent specific immune responses by inhibiting PKC activity.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.