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.     

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.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Association between development of CD4+CD25+ regulatory T cells and thymus CD4-CD25+ cells

AIM: To explore the correlation between development of CD4⁺CD25⁺ regulatory T cells (CD4⁺CD25⁺ Tr) and thymus CD4^-CD25⁺ cells. METHODS: The ratios of CD4⁺CD25⁺ regulatory T cells to CD4+ T cells in thymus, spleen, lymph node and peripheral blood of mice from birth to mature and also the ratios of CD4^-CD25⁺ cells to CD4^- T cells in thymus were measured by flow cytometry. Purified CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells were labeled with CFDA-SE, and then stimulated with various kinds of stimulators. RESULTS: The percentages of CD4⁺CD25⁺ Tr in mouse spleen, lymph nodes and peripheral blood increased gradually, but not in thymus, from day one to week 10 of the age with rapid rising from day one to week 1. The percentages of CD4^-CD25⁺ cells in mouse thymus were quite high on day one after birth, and decreased rapidly from day one to week 1. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells showed no proliferation in response to ConA, while CD4⁺CD25⁺ Tr showed a transient enlargement of cell size. Both CD4⁺CD25⁺ Tr and CD4⁺CD25^- T cells underwent proliferation in response to PDB plus ionomycin. CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ Tr, showed a proliferative response to the stimulation of coated anti-CD3 plus soluble anti-CD28 antibody, however, CD4⁺CD25⁺ Tr showed significant proliferation and CD4⁺CD25^- T cells showed a stronger response in addition of high dose of IL-2. CONCLUSION: The thymus CD4^-CD25⁺ cells are probably the precursor of CD4⁺CD25⁺ Tr during cell development.     

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.     

Analysis of activation of T cells from draining lymph node and peripheral blood in allotransplantation mice

AIM:To study the activation of T cells from local lymph node and peripheral blood early after allotransplantation.METHODS:Transplant of myocardio-tissue into mouse forearm subcutaneously was used as a model to analyze the expression of CD69 by T subpopulations from draining lymph node and peripheral blood by flow cytometry.RESULTS:The expression rates of CD69 by both CD4⁺T cells and CD8⁺T cells from the draining lymph node were raised (P＜0.01) 72 h after allotransplantation, and it was higher on CD8⁺T cells than on CD4⁺T cells (P＜0.01). No significant difference in CD69 expression was found on CD4⁺T and CD8⁺T cells from peripheral blood among the groups, topical complete Freund's adjuvant (CFA) and systemic cyclosporin(CsA) enhanced and inhibited expression of CD69 by both CD4⁺T cells and CD8⁺T cells after allotransplantation, respectively (P＜0.05 or P＜0.01).CONCLUSION:To detect the expression of CD69 by T cells from draining lymph node can keep insight to the allorecognition early after transplantation.     

Changes of CD28, CD56 and CD57 expression on CD8+T cells in the peripheral blood of healthy elderly individuals

AIM:The expression of CD28, CD56 and CD57 on CD8⁺T cells in the peripheral blood of young (age range 20-35) and elderly(age range 60-75) healthy donors were compared to explore the change of the cellular immune function with aging.METHODS:Three-color fluorescent flow cytometry was performed to analyze the differences in percentage of CD8⁺CD28⁺, CD8⁺CD56⁺ and CD8⁺CD57⁺T cells in the peripheral blood between the young and elderly groups.RESULTS:CD8⁺CD28⁺T cells in the peripheral blood of the elderly group was significantly lower than those in the young group, with percentage of 34.07±5.28 and 49.84±7.43,respectively (P＜0.05). Conversely, CD8⁺CD56⁺T cells and CD8⁺CD57⁺T cells in the peripheral blood of the elderly group were significantly higher than those in the young group, and the percentage was 6.60±2.40 vs 2.10±0.35,41.82±6.01 vs 22.89±2.80, respectively(P＜0.05).CONCLUSION:The expression of CD28, CD56 and CD57 on the CD8⁺T cells in the peripheral blood are changed significantly with aging. The decrease in CD28 expression may play an important role in the immunosenescence, while the increase in CD56 and CD57 expression seems to be a compensatory adaptation for the immune dysfunction.     

Effect of forsythiaside A on immune function in rats with ulcerative colitis

AIM To investigate the effect of forsythiaside A (FA) on immune function in rats with ulcerative colitis and its related mechanism. METHODS Healthy SD rats were randomly divided into 5 groups: control group (no treatment, normal feeding), model group (establishment of rat ulcerative colitis model), and low, medium and high doses of FA groups (treatment of the model rats with FA at 5 mg/kg, 20 mg/kg and 80 mg/kg, respectively). The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat colon tissues were measured by colorimetry, and the serum levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2) and IL-4 were detected by ELISA. The spleen index and thymus index, the percentages of CD3⁺, CD4⁺ and CD8⁺ T-lymphocytes in peripheral blood mononuclear cells （PBMC）, the serum IgA and IgG levels, and the serum complement C3 and C4 levels were also determined. RESULTS The colon tissues of the rats in model group showed obvious inflammation and ulceration, indicating that the animal model was successfully established. Compared with model group, the colonic inflammation and ulceration were significantly attenuated in FA groups, among which the high dose had the best effect. Compared with control group, the spleen index and thymus index of the rars in model group were decreased (P<0.05), MDA content in colon tissues was increased (P<0.05), and SOD activity in colon tissues was decreased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were decreased (P<0.05), while the serum levels of IgA, IgG, TNF-α, and IL-2 were increased in model group as compared with control group. Furthermore, the spleen index and thymus index of the rats in FA groups were increased (P<0.05), the MDA content in the colon tissues was decreased (P<0.05), and the SOD activity in the colon tissues was increased (P<0.05). The levels of CD3⁺, CD4⁺, CD8⁺ and CD4⁺/CD8⁺ T-lymphocytes in PBMC, and the serum levels of C3, C4 and IL-4 were increased (P<0.05), while serum IgA, IgG, TNF-α and IL-2 levels were decreased in FA groups as compared with model group (P<0.05). CONCLUSION Forsythiaside A effectively attenuates the colonic lesions in rats with ulcerative colitis, and its mechanism may be related to reinforcement of oxygen free radical scavenging power, alleviation of inflammatory response, and enhancement of immune function.     

TL1A promotes development of intestinal fibrosis associated with chron?ic experimental colitis by regulating IL-17 and IFN-γ

AIM To explore how tumor recrosis factor ligand-related molecule 1A (TL1A) promotes the development of intestinal fibrosis associated with chronic experimental colitis by regulating interleukin-17 (IL-17) and interferon-γ (IFN-γ). METHODS Aexperimental colitis-associated wild-type (WT) and TL1A (L-Tg) transgenic intestinal fibrosis model was established by dextran sulfate sodium (DSS) induction.The severity of colitis was evaluated by detecting the disease activity index (DAI). HE staining was used to observe the histopathological changes and pathological score of the colitis.Myeloperoxidase (MPO) was measured in each group. The collagen deposition was detected by Masson’s trichrome staining and Sirius red staining. The lamina propria, spleen and mesenteric lymph nodes(MLN) mononuclear cells were isolated and counted, and the levels of IL-17 and IFN-γ weremeasured by ELISA, andthe percentages of CD4⁺IFN-γ⁺T cells and CD4⁺IL-17⁺T cells were analyzed by flow cytometry. RESULTS After drinking DSS water,the body weight of the mice in DSS/Tg group was decreased significantly as compared with WT group(P<0.05). The DAI score, histology score and MPO activity were significantly increased(P<0.05). Thelevels of IL-17 and IFN-γ, LPMC, spleen and MLN were significantly increased. The percentages of CD4⁺IFN-γ⁺T cells and CD4⁺IL-17⁺T cells were significantlyincreased.The thickness and collagen deposition of the colon were increased inTg group. CONCLUSION TL1A promotes the development of intestinal fibrosis associated with chronic experimental colitis by regulating IL-17 and IFN-γ.     

Determination of T cell cycle and the expression of bcl- 2 in asthmatic mice and its significance

AIM: To investigate the changes of T cell cycle, the expression of bcl- 2 in allergic asthmatic mice and the effects of dexamethasone on them. METHODS: An animal model with asthma was established by means of ovalbumin sensitizing-challenging. CD3 expression in spleen and lymphocytes in bronchoalveolar lavage fluid (BALF), T cell cycle and Bcl-2 expression in spleen were detected by flow cytometry. RESULTS: In BALF lymphocytes and spleen lymphocytes, CD3 expression rate in the asthmatic group was significantly higher than that of control group. In BALF lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly lower than that of the asthmatic group. However, in spleen lymphocytes, CD3 expression rate in the asthma plus dexamethasone group was significantly higher than that of the asthmatic group. In spleen lymphocytes, the cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmatic group were significantly higher than that from the control group. Cell count in S phase, G₂+M phase and apoptosis rate of T cell from the asthmaplus dexamethasone group was significantly lower than that from the asthmatic group. The Bcl-2 expression rate of T cell from the asthmatic group was significantly higher than that from the control group. CONCLUSIONS: In the allergic asthmatic mice model, T cell count, proliferation and activation of T cells, apoptosis rate of T cells in spleen lymphocytes increase, meanwhile bcl- 2 expression also increases significantly. There was no significant effect of dexamethasone on the bcl- 2 expression. The therapeutic effects of dexamethasone on asthma may be not due to the inhibition of the bcl- 2 expression in T cells.     

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.     

Elevation of blood Th17 cells and CD3+CD8-IL-21+ T cells in patients with cervical cancer

AIM: To investigate the role of Th17 cells in the patients with cervical cancer.METHODS: We measured the peripheral levels of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in 37 cervical cancer (CC) patients, 25 cervical intraepithelial neoplasia (CIN) patients and 18 healthy controls by flow cytometry. The percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells in total CD4⁺ cells were calculated.RESULTS: Compared with controls, the patients with CC or CIN had higher proportions of Th17 cells (all P<0.01) and CD3⁺CD8^-IL-21⁺ T cells (all P<0.05). Notably, in CC patients, the increased percentages of Th17 cells and CD3⁺CD8^-IL-21⁺ T cells were independently associated with the clinical stage(all P<0.05), lymph node metastasis (P<0.01,P<0.05) and vasoinvasion (all P<0.01), while the elevated percentage of CD3⁺CD8^-IL-21⁺ T cells was also associated with the tumor size(P<0.01). Furthermore, the percentage of Th17 cells was positively correlated with that of CD3⁺CD8^-IL-21⁺ T cells in healthy controls and CC patients, but not in CIN patients.CONCLUSION: Our results indicates a possible role of Th17 cells in CC patients correlated with CD3⁺CD8^-IL-21⁺T cells, and the elevated percentage of circulating Th17 cells may be involved in the development and progression of cervical cancer.     

Chinese herbal preparation Qing Yi Tang Ⅱ granule protects against experimental acute pancreatitis in mice

AIM: To investigate the protect effect of Chinese herbal preparation, Qing Yi TangⅡgranule (QYT), on acute pancreatitis (AP) mice and its mechanism. METHODS: Adult male and female C57BL/6 mice (n=24) were randomly divided into control group, AP group and AP+QYT group. Severe AP was induced by combined intra-peritoneal injection of caerulein (50 μg/kg) and lipopolysaccharide (LPS; 10 mg/kg). Drinking water or 24% QYT solution was given to the mice in AP group or AP+QYT group by oral gavage. The mice in control group were intraperitoneally injected with equivalent volume of normal saline and gavaged with water. The mice were sacrificed 3 h after the last injection. Severity of AP was assessed by biochemical markers and histology. The plasma level of IL-6 and MCP-1, and lung myeloperoxidase (MPO) levels were determined for assessing the extent of systemic inflammatory response. The intestinal microflora, T lymphocytes and T-lymphocyte subgroups were examined for assessing the function of the intestinal barrier. RESULTS: Compared with control group, the mice in AP group presented significant increases in pathological histological scores, plasma amylase activity and IL-6 and MCP-1 levels, as well as the MPO activity in the lung and pancreatic tissues. QYT attenuated these changes to some extent. Furthermore, the increased intestinal microflora was significantly reversed by QYT. No difference of the numbers of Peyer's patches in small intestine in the 3 groups was observed, but the percentage of CD3⁺ T lymphocytes decreased significantly in AP group, and increased percentage of CD4⁺ and CD4⁺/CD8⁺ ratio were found in AP group and AP+QYT group. CONCLUSION: QYT protects against cearulein and LPS-induced acute pancreatitis in mice. The mechanisms may be related to the suppression of the inflammatory response, promoting intestinal bacteria removal, and regulating the functions of T lymphocytes in the intestinal barrier.     

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.     

Relationship between Th17 cells infiltrated in lungs and pulmonary vascular reconstruction under hypobarric hypoxia

AIM: To investigate the changes of retinoid-related orphan receptor γt(RORγt) mRNA and interleukin-17(IL-17) protein in the lung tissue under hypobaric hypoxia, and the relationship between Th17 cells and hypoxic pulmonary vascular reconstruction. METHODS: Male BALB/c mice(n=50) were randomly divided into control group and 3 d, 7 d, 14 d and 28 d hypobaric hypoxia groups. The mice in hypobaric hypoxia groups were housed in a hypobaric hypoxia chamber(simulated altitude of 6 000 m) for 3 d, 7 d, 14 d or 28 d. The mice in control group were housed in normal pressure and oxygen environment. The hemodynamic data were recorded by cardiac catheterization. The hypertrophy of right ventricle was evaluated by the ratio of weight of the right ventricle to the weight of the left ventricle plus interventri-cular septum, and the right ventricular weight over body weight. The spleen was collected and the proportions of the Th17(CD4⁺IL-17⁺RORγt⁺) cells were detected by flow cytometry. The serum levels of IL-4, IL-6 and IL-17 and the change of IL-17 in the lung tissue were measured by ELISA. The mRNA expression of RORγt in the spleen and lung tissues was measured by RT-qPCR. RESULTS: Compared with control group, the mouse right ventricular systolic pressure, the hypertrophy index of right ventricle and the serum IL-17 level were significantly elevated in hypoxia groups, which was consistent with the results of flow cytometry. The mRNA expression of RORγt in the lung tissue was also significantly increased in 7 d, 14 d and 28 d hypoxia groups. The expression of IL-17 in the lung tissue was significantly increased in 14 d and 28 d hypoxia groups. CONCLUSION: Hypoxia promotes differentiation of Th0 cells to Th17 cells in the spleen. The Th17 cells infiltrated in the lung tissue under hypobarric hypoxia are involved in pulmonary vascular reconstruction.     

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.     

Effect of pachyman polysaccharides on Th17/Treg balance in systemic lupus erythematosus patients

AIM: To investigate the immunomodulatory effect of pachyman polysaccharides (PPS) on T helper 17 cell (Th17)/regulatory T cell (Treg) balance in the peripheral blood of systemic lupus erythematosus (SLE) patients. METHODS: The CD4⁺ T cells were isolated from the peripheral blood samples obtained from 45 SLE patients and 35 healthy controls enrolled in our study using magnetic bead separation method. The proportions of Th17 and Treg cells were measured by flow cytometry. The CD4⁺ T cells from SLE patients and healthy controls were treated with PPS. The cytoto-xicity of PPS was evaluated by detecting cell viability with MTT assay. The contents of interleukin-17 (IL-17), IL-6, IL-10 and transforming growth factor-β (TGF-β) were measured by ELISA. The expression of retinoid-related orphan receptor γt (RORγt) and forkhead box protein P3 (Foxp3) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. RESULTS: The Th17 cells were significantly elevated, while Treg cells were obviously decreased in the SLE patients compared with the healthy control group (P<0.05). Compare with control group, the contents of IL-17 and IL-6 were decreased, while the contents of IL-10 and TGF-β were increased (P<0.05). The expression of RORγt at mRNA and protein levels was down-regulated and the expression of Foxp3 was up-regulated (P<0.05). The ratio of Th17/Treg was decreased in 100 μg/L nontoxic PPS-treated CD4⁺ T cells isolated from the SLE patients (P<0.05). CONCLUSION: PPS treatment inhibits Th17 cells and elevates Treg cells in the CD4⁺ T cells isolated from SLE patients, which may have a therapeutic effect on SLE patients.     

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.     

Establishment of method for PD-1 immunophenotype detection in TCR Vβ repertoire of CD3+, CD4+ and CD8+ T-cell subsets

AIM:To establish the method for detecting the immunophenotype of immunosuppressive receptor programmed cell death protein 1 (PD-1) in T-cell receptor (TCR) Vβ repertoire of CD3⁺, CD4⁺ and CD8⁺ T-cell subsets, therefore to evaluate the distribution of PD-1 in T-cell repertoire from human peripheral blood (PB). METHODS:The PB samples from 10 cases of healthy individuals (HI) were collected. Using multi-colored fluorescence flow cytometry, the distribution frequency of PD-1 in TCR Vβ repertoire was detected with a wide panel of anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and 24 anti-TCR Vβ repertoire (IOTest^® Beta Mark TCR Vβ Repertoire Kit, containing 8 tubes which labeled A~H, each tube is a composite antibody of FITC and PE coupling, each cocktail contains antibodies direc-ted to 3 different Vβ subfamilies) monoclonal antibodies. RESULTS:The total number of the 24 TCR Vβ repertoire detected in CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells from 10 cases of HI was consistent with the Quick Reference Card data provided by the kit. The preliminary results showed that the frequency of Vβ usage in CD3⁺, CD4⁺ and CD8⁺ T cells was different. High usage of Vβ2, Vβ3, Vβ8, Vβ9, Vβ5.1, Vβ13.1 and Vβ13.2 was found in CD3⁺ T cells, while high usage of Vβ2, Vβ3, Vβ8, Vβ5.1, Vβ9 and Vβ13.1 in CD3⁺CD4⁺ T cells, and high usage of Vβ1, Vβ2, Vβ3, Vβ9, Vβ13.1 and Vβ13.2 in CD3⁺CD8⁺ T cells were also observed. Further analysis showed that the expression of PD-1 was detected in all 24 TCR Vβ subfamilies of CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells. The higher frequency of PD-1⁺ T cells was CD4⁺Vβ5.2⁺ T cells, whereas the higher frequency of PD1⁺ T cells in CD8⁺Vβ11⁺ and CD8⁺Vβ13.6⁺ T cells was detected. CONCLUSION:The method for detection of the immunosuppressive receptor PD-1 in TCR Vβ repertoire of T-cell subsets is successfully established, which provides a new method for further analysis of immunosuppressive characteristics of TCR Vβ repertoire in the patients with leukemia.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.