This study was aimed to construct a lentiviral vector carrying mouse RORγt and glp gene, and to detect the expression of RORγt in the 293FT cells. The RORγt fragment was amplified by RT-PCR from mouse thymus and cloned into PCR 2.1 vector. The RORγt DNA fragment was prepared by digestion and inserted into MigR1 plasmid, then the RORγt-IRES-GFP was directionally linked with lentiviral transfer plasmid pTK208 to generate a lentiviral vector pXZ9-RORγt. The recombinant lentivirus were produced by co-transfected three plasmids into 293FT packing cells using lipofectamine 2000. After transfection, the lentiviral supernatant was collected and concentrated via ultracentrifugation. The 293FT cells were infected by the concentrated lentivirus, GFP expression was examined under a fluorescent microscope and the expression of RORγt protein was detected by Western blot. The results showed that the RORγt fragment was amplified from cDNA of mouse thymus and recombinant lentiviral vector pXZ9-RORγt was constructed successfully. High titer lentivirus were prepared after one round ultracentrifugation. RORγt expression could be detected in 293FT cells after virus infection. It is concluded that the lentiviral vector pXZ9- RORγt containing mouse RORγt-IRES-GFP is successfully constructed; RORγt can express in 293FT cells via lentiviral vector transduction, which provides an optional tool for further research on the mechanism of RORγt controlling Th17 cell differentiation.
Animals ; Cell Line ; DNA, Complementary ; genetics ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; Transfection

BACKGROUND: Exposure to the ionizing radiation (IR) encountered outside the magnetic field of the Earth poses a persistent threat to the reproductive functions of astronauts. The potential effects of space IR on the circadian rhythms of male reproductive functions have not been well characterized so far. METHODS: Here, we investigated the circadian effects of IR exposure (3 Gy X-rays) on reproductive functional markers in mouse testicular tissue and epididymis at regular intervals over a 24-h day. For each animal, epididymis was tested for sperm motility, and the testis tissue was used for daily sperm production (DSP), testosterone levels, and activities of testicular enzymes (glucose-6-phosphate dehydrogenase (G6PDH), sorbitol dehydrogenase (SDH), lactic dehydrogenase (LDH), and acid phosphatase (ACP)), and the clock genes mRNA expression such as Clock, Bmal1, Ror-α, Ror-β, or Ror-γ. RESULTS: Mice exposed to IR exhibited a disruption in circadian rhythms of reproductive markers, as indicated by decreased sperm motility, increased daily sperm production (DSP), and reduced activities of testis enzymes such as G6PDH, SDH, LDH, and ACP. Moreover, IR exposure also decreased mRNA expression of five clock genes (Clock, Bmal1, Ror-α, Ror-β, or Ror-γ) in testis, with alteration in the rhythm parameters. CONCLUSION These findings suggested potential health effects of IR exposure on reproductive functions of male astronauts, in terms of both the daily overall level as well as the circadian rhythmicity.
ARNTL Transcription Factors/genetics* ; Acid Phosphatase ; Animals ; CLOCK Proteins/genetics* ; Circadian Rhythm/radiation effects* ; Epididymis/radiation effects* ; Gene Expression/radiation effects* ; Genitalia, Male/radiation effects* ; Glucosephosphate Dehydrogenase ; L-Iditol 2-Dehydrogenase ; L-Lactate Dehydrogenase ; Male ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Nuclear Receptor Subfamily 1, Group F, Member 1/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 2/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; RNA, Messenger/genetics* ; Radiation Exposure ; Radiation, Ionizing ; Reproductive Physiological Phenomena/radiation effects* ; Sperm Motility/radiation effects* ; Spermatozoa/radiation effects* ; Testis/radiation effects*

OBJECTIVETo generate engineering Th17 cells from mice CD4(+)CD25(-) naïve T cells, and to evaluate whether the phenotypes or functions of these engineering cells were similar to natural Th17 cells.

METHODSRecombinant lentivirus carrying mouse RORγt (pXZ9-RORγt) and mock control pXZ9 were generated by co-transfected three-plasmids into 293FT packing cells. CD4(+)CD25(-) naïve T cells were purified from mice spleens by magnetic activated cell sorting, and stimulated by anti-CD3ε, anti-CD28 mAb plus IL-2. The stimulated cells were further infected by pXZ9-RORγt or pXZ9 virus with or without polarization by TGF-β plus IL-6 and divided into five groups: pXZ9-RORγt (group A), pXZ9 + TGF-β + IL-6 (group B), pXZ9-RORγt + TGF-β + IL-6 (group C), pXZ9 (group D) and control (group E). Production efficiency of engineering Th17 cells was referred as the percentage of IL-17A producing cells. Cytokine production profiles of these cells were assayed by realtime RT-PCR and cells function was evaluated by susceptibility of mouse experimental autoimmune encephalomyelitis (EAE).

RESULTS(1) High-title lentivirus was prepared and was succeeded to transduce CD4(+)CD25(-) naïve T cells. Forced expression of RORγt (group A) resulted in (40.25 ± 5.46)% CD4(+)CD25(-) naïve T cells converted into engineering Th17 cells and the convert efficiency increased to (60.59 ± 8.15)% in addition of TGF-β and IL-6 (group C), or decreased to (14.36 ± 5.27)% when presence of TGF-β and IL-6 only (group B). (2) IL-17A, IL-17F and IL-21 production of pXZ9-RORγt infected cells combined with TGF-β and IL-6 were most similar to natural Th17 cells while cells over expression of RORγt alone showed deficiency in IL-21 production. (3) Both pXZ9-RORγt infected cells, TGF-β and IL-6 polarized cells and polarized of RORγt transduced cells could promote the susceptibility to mouse EAE in C57BL6 mice models.

CONCLUSIONHigh yield of engineering Th17 cells was prepared from CD4(+)CD25(-) naïve T cells by over expression RORγt plus TGF-β and IL-6 polarization. These engineering Th17 cells were similar to the natural Th17 cells in phenotypes and functional identification.

Animals ; Cells, Cultured ; Genetic Techniques ; Interleukin-17 ; pharmacology ; Interleukin-6 ; pharmacology ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; Th17 Cells ; cytology ; metabolism ; Transforming Growth Factor beta ; pharmacology

OBJECTIVETo study the effect of budesonide (BUD) on RORγt expression in the pulmonary tissue of asthmatic mice and mechanisms of BUD in the treatment of asthma.

METHODSBlab/c asthmatic mouse model was induced by ovalbumin (OVA). Thirty female mice were randomly divided into three groups: control, asthmatic and BUD-treated. IL-17 levels in bronchoalveolar lavage fluid (BALF) and serum were measured using ELISA. Total and differential cell counts in BALF were measured. Airway inflammation was evaluated by hematoxylin and eosin staining. IL-17 mRNA and RORγt mRNA expression were measured by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSRORγt mRNA and IL-l7 levels in the asthmatic group were significantly higher than in the control group (P<0.01). BUD treatment significantly decreased RORγt mRNA and IL-l7 levels compared with the asthmatic group (P<0.01). Compared with the control group, total, neutrophil and eosinophil cell count in BALF increased significantly in the asthmatic group (P<0.01). After BUD treatment, counts of total, neutrophil and eosinophil cells in BALF were significantly reduced (P<0.01) and were similar to in the control group. Inflammatory reactions in the respiratory tract were significantly alleviated in the BUD treated group.

CONCLUSIONSRORγt and IL-l7 levels in the pulmonary tissue of asthmatic mice increase and this may be associated with the pathogenesis of asthma. BUD can inhibit RORγt and IL-17 and thus reduces lung inflammation.

Animals ; Asthma ; drug therapy ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Bronchodilator Agents ; pharmacology ; Budesonide ; pharmacology ; Female ; Interleukin-17 ; analysis ; Lung ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; RNA, Messenger ; analysis

OBJECTIVETo investigate the potential correlation between the level of Th17 cells in peripheral blood and the development of human hepatocellular carcinoma (HCC).

METHODSTh17 cells in the blood samples from 61 HCC patients and 38 healthy controls were assessed by flow cytometry (FCM). The mRNA expression levels of IL-17, IL-23p19 and RORc in peripheral blood mononuclear cells (PBMC) were detected by quantitative real-time PCR. The potential correlation of increased Th17 cells in blood with the clinical characteristics of the 61 patients, including gender, age, preoperative AFP concentration, tumor diameter, portal vein tumor thrombus (PVTT), metastasis and TNM stages was analyzed. Statistical analysis was performed using the software GraphPad Prizm 5.0.

RESULTSThe number of Th17 cells in 61 HCC patients was significantly higher than that in the normal controls (4.67% ± 0.79% vs 3.25% ± 0.68%, P < 0.0001). The same tendency was also found in the mRNA levels of IL-17, IL-23p19 and RORc in PBMC (P < 0.05). The increased level of Th17 cells in HCC patients showed a positive correlation with the tumor size, PVTT, metastasis and TNM stages (P < 0.05 for each group). The level of Th17 cells in HCC patients was increased along with the increasing TNM stages I to stage IV: 4.05% ± 0.82%, 4.32% ± 0.67%, 4.94% ± 0.70%, and 5.22% ± 0.87%, respectively, where the level of Th17 cells in patients with advanced stage of HCC (III-IV) was significantly higher than that in early stage (I-II, P = 0.0008).

CONCLUSIONThe increased of level of Th17 cells in peripheral blood of HCC is significantly correlated with the tumor size, PVTT, metastasis and TNM stage, indicating that the Th17 cells might participate in promoting invasion and progression of HCC directly or indirectly by secreting characteristic cytokines.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Humans ; Interleukin-17 ; genetics ; metabolism ; Interleukin-23 Subunit p19 ; genetics ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Lymphocyte Count ; Male ; Middle Aged ; Neoplasm Metastasis ; Neoplasm Staging ; Neoplastic Cells, Circulating ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; metabolism ; Portal Vein ; pathology ; RNA, Messenger ; metabolism ; Th17 Cells ; pathology ; Tumor Burden

OBJECTIVETo study the effects of dexamethasone on intracellular expression of Th17 cytokine interleukin 17 and the mechanisms in asthmatic mice.

METHODSExperimental asthma was induced by ovalbumin (OVA) sensitization in 20 in female Balb/c mice with (dexamethasone group, n=10) or without dexamethasone treatment (model group, n=10), with another 10 serving as the control group. The levels of IL-17 in the bronchoalveolar lavage fluid (BALF) and serum of the mice were measured by enzyme-linked immunosorbent assay (ELISA), and the airway inflammation was evaluated by HE staining. The expressions of IL-17 and RORgammat mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR), and the expression of RORgammat protein was measured by immunohistochemical staining.

RESULTSThe levels of RORgammat and IL-17 mRNA and protein in the asthmatic model group were significantly higher than those in the control group (P<0.01), and the increased expressions of RORgammat and IL-17 mRNA and protein in the asthmatic mice were significantly reduced by dexamethasone treatment (P<0.05).

CONCLUSIONDexamethasone can inhibit the release of IL-17 probably by inhibiting RORgammat expression and blocking Th17 differentiation in asthmatic mice.

Animals ; Asthma ; chemically induced ; immunology ; metabolism ; Dexamethasone ; pharmacology ; Female ; Interleukin-17 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; Ovalbumin ; RNA, Messenger ; biosynthesis ; genetics ; T-Lymphocyte Subsets ; immunology ; metabolism ; T-Lymphocytes, Helper-Inducer ; immunology ; metabolism

OBJECTIVETo investigate the effects of vasoactive intestinal peptide (VIP) on the airway inflammation and its regulatory effect on Th17/Treg imbalance in asthmatic mice.

METHODSA total of 30 BALB/c mice were equally and randomly divided into three groups: control, asthma, and VIP. An acute asthmatic mouse model was established by sensitization and challenge with ovalbumin (OVA). The control group received normal saline instead of OVA. Before the challenge with OVA, the VIP group was administered VIP (20 μg/mL) by aerosol inhalation for 30 minutes. The bronchoalveolar lavage fluid (BALF) and the lung tissue were collected from mice. The pathological changes in the lung tissue were observed by hematoxylin and eosin staining. The levels of Th17/Treg-related cytokines in BALF were measured by enzyme-linked immunosorbent assay. The expression of retinoid-related orphan receptor gamma t (RORγt) and forkhead box P3 (Foxp3) were measured by real-time fluorescence quantitative PCR and immunohistochemistry.

RESULTSThe histopathological results showed that the VIP group had milder symptoms of airway inflammation than the asthma group. The level of IL-17 in BALF in the asthma group was significantly higher than that in the control group and the VIP group (P<0.01), but the level of IL-17 in the control group was significantly lower than that in the VIP group (P<0.01). The level of IL-10 in BALF in the asthma group was significantly lower than that in the control group and the VIP group (P<0.01, but the level of IL-10 in the VIP group was significantly higher than that in the control group (P<0.01). The asthma group showed significantly higher expression levels of RORγt mRNA and protein in the lung tissue and significantly lower expression levels of Foxp3 mRNA and protein than the control group (P<0.01). The VIP group had significantly lower expression levels of RORγt mRNA and protein in the lung tissue and significantly higher expression levels of Foxp3 mRNA and protein than the asthma group (P<0.05).

CONCLUSIONSThe Th17/Treg imbalance may be closely related to the airway inflammation in asthmatic mice. VIP can improve airway inflammation by regulating the Th17/Treg imbalance in asthmatic mice.

Animals ; Asthma ; drug therapy ; immunology ; Forkhead Transcription Factors ; genetics ; Interleukin-10 ; analysis ; Interleukin-17 ; analysis ; Male ; Mice ; Mice, Inbred BALB C ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology ; Vasoactive Intestinal Peptide ; pharmacology ; therapeutic use

OBJECTIVETo detect the expression levels of transcription factors and associated cytokines of Th17 and Treg cells in peripheral blood mononuclear cells (PBMC) of patients with gastric cancer, and explore the possible pathological mechanism of these cells involved in the development of gastric cancer.

METHODSThe mRNA levels of RORgammat, FoxP3 in PBMC were determined by quantitative real-time PCR (QRT-PCR) from 57 patients with gastric cancer, 31 patients with benign gastric illness and 40 healthy people. The concentration of IL-17, IL-23, TGF-beta, IL-10 in plasma were detected by enzyme linked immunosorbent assay (ELISA).

RESULTSCompared with healthy volunteers, patients with gastric cancer showed higher levels of RORgammat and FoxP3 in PBMC (P < 0.05). The ratio of FoxP3/RORgammat in gastric cancer group was higher than that in the volunteer group and benign gastric illness group (P < 0.05). The ratio of FoxP3/RORgammat was higher in advanced disease than early disease (P < 0.05). The expressions of IL-17, IL-23, TGF-beta and IL-10 were higher in patients with gastric cancer than that in healthy volunteers (P < 0.05). In addition, The expression of TGF-beta and IL-10 were significantly increased in the advanced disease group than that in the early group (P < 0.05), but IL-17 and IL-23 was not significantly changed between the two groups (P > 0.05).

CONCLUSIONThere are higher levels of Th17 and Treg cells in gastric cancer patients, and it also shows a persistent predominant tendency of Treg cells and a reduced tendency of Th17 cells in advanced disease. Detecting the expression of Th17/Treg transcription factor and related cytokines would contribute to the diagnosis and prediction of the disease development and prognosis.

Adult ; Aged ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Gastritis ; blood ; metabolism ; pathology ; Humans ; Interleukin-10 ; blood ; Interleukin-17 ; blood ; Interleukin-23 ; blood ; Male ; Middle Aged ; Neoplasm Staging ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; blood ; metabolism ; pathology ; T-Lymphocytes, Regulatory ; metabolism ; Th17 Cells ; metabolism ; Transforming Growth Factor beta ; blood

OBJECTIVETo observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats.

METHODSSixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1β, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) β were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction.

RESULTSMTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group (P<0.05 or P<0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats (P<0.05). Only middle- and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats (P<0.05). However, NCTD has no effect on vascular endothelial growth factor (VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group (P<0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group (P<0.05).

CONCLUSIONSNCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

Animals ; Arthritis, Experimental ; blood ; drug therapy ; pathology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; therapeutic use ; Cytokines ; blood ; Forkhead Transcription Factors ; metabolism ; Joints ; drug effects ; pathology ; Male ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the changes of Treg/Th17 cells related transcription factors (FOXP3, RORC) and cytokines (TGF-beta, IL-17) in the peripheral blood in patients with allergic rhinitis. METHOD: Use visual analogue scale (VAS) to assess the allergic rhinitis severity of patients. Eighteen patients with allergic rhinitis and 12 healthy control subjects were consecutively enrolled. Peripheral blood samples were obtained from all patients and healthy control subjects (after prick test). The mRNA expressions of key transcription factors (RORC, FOXP3) in PBMCs were measured by RT-PCR, and the concentrations of TGF-beta, IL-17 in plasma were measured by ELISA. RESULT: Patients with moderate/severe persistent rhinitis have a VAS of 8.5 +/- 0.8, and total patients have a VAS of 6.2 +/- 1.6. Compared with healthy control subjects, the mRNA expressions of FOXP3 and the concentrations of TGF-beta in plasma were significantly lower in patients with allergic rhinitis (P < 0.01) while the mRNA expressions of RORC and the concentrations of IL-17 in plasma were significantly higher in allergic rhinitis group (P < 0.01). CONCLUSION The imbalance of Treg/Th17 cells existed in allergic rhinitis patients, which may contribute to the proceeding of allergic rhinitis.
Adolescent ; Adult ; Case-Control Studies ; Child ; Female ; Forkhead Transcription Factors ; metabolism ; Humans ; Interleukin-17 ; metabolism ; Male ; Middle Aged ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; genetics ; Rhinitis, Allergic, Perennial ; metabolism ; T-Lymphocytes, Regulatory ; metabolism ; Th17 Cells ; metabolism ; Transforming Growth Factor beta ; metabolism ; Young Adult