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

Th17 cells are a newly discovered subsets of T cells. It can specifically secrete IL-17. The RORγt and STAT3 are specific transcription factors of Th17 cells. In recent researches, it has been found that Th17 cells and their proportion increased in a variety of autoimmune diseases. This article briefly reviews Th17 cells and its relationship with the occurrence and severity of several immune-related blood diseases, including aplastic anemia, autoimmune hemolytic anemia, immune thrombo-cytopenia and immune-related pancytopenia.
Autoimmune Diseases ; Hematologic Diseases ; immunology ; Humans ; Interleukin-17 ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; STAT3 Transcription Factor ; Th17 Cells ; immunology

OBJECTIVETo explore the role of CD4⁺ CD25⁺ Foxp3⁺ Treg/CD4⁺ IL-17A(+)Th17 cells and the related cytokines in Graves' ophthalmopathy.

METHODSBased on clinical activity scores (CAS), we divided patients with untreated Graves' ophthal- mopathy into active group (AGO group with CAS ≥ 3 (15 cases) and non-active group (NGO group) with CAS<3 (15 cases), with another 15 patients with untreated Graves' disease free of eye symptoms (GD group) and 15 normal subjects as controls. Peripheral venous blood Treg/Th17 cell ratio was determined using flow cytometry. RT-PCR was used to detect the mRNA expression levels of Treg-specific transcription factor Foxp3 and Th17-specific transcription factor RORγt. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of Th17 cell-related cytokines (IL-17A, IL-23, and IL-6) and Treg-related cytokines (TGF-β, IL-10, and IL-35).

RESULTSCompared with the normal subjects, the patients in GD, NGO, AGO groups all showed significantly increased Th17 cell count (P<0.05), which was the highest in AGO group. RT-PCR results revealed significantly increased RORγt in GD, NGO, and AGO groups, also the highest in AGO group. Serum IL-17A, IL-23, and IL-6 levels all showed significant increments in GD, NGO, and AGO groups (P<0.05), especially in AGO group. Among the Treg-related cytokines, TGF-β and IL-35 levels decreased (P<0.05) but IL-10 increased significantly (P<0.05) in GD, NGO, AGO groups.

CONCLUSIONDecreased immunosuppressive capacity of Treg cells can be an important factor in the pathogenesis of Graves' ophthalmopathy. Th17 cells may also participate in the occurrence and progression of Graves' ophthalmopathy and can serve along with related cytokines as novel indicators of the disease activity. Impaired Treg/Th17 balance may importantly contribute to the occurrence of Graves' ophthalmopathy.

Cytokines ; immunology ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Graves Ophthalmopathy ; immunology ; Humans ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology

OBJECTIVES: This study aimed to explore the changes in the expression of the characteristic transcription factor retinoid related orphan receptor γt (RORγt) and the cytokine interleukin-17 (IL-17) of T helper cell 17 (Th17) in the pressure side of the periodontal tissue of rats under different orthodontic forces. Their effects on the expression of osteoprotegerin (OPG) and the quantity of osteoclast (OC) were also explored. The role of Th17 cell in alveolar bone remodeling under different forces was preliminarily investigated. METHODS: A total of 108 rats were chosen and randomly divided into three groups. Mesial forces of 0, 50, and 100 g were loaded on the maxillary first molar in the three groups. The rats were executed at 0, 1, 3, 5, 7, and 14 days. The expression of RORγt mRNA was quantified by real-time quantitative polymerase chain reaction. The expression of IL-17 protein was quantified by enzyme linked immunosorbent assay. The expression levels of RORγt and OPG proteins were quantified, and the quantity of OC was counted via immunohistochemistry. RESULTS: The expression levels of RORγt and IL-17 and the quantity of OC increased first and then decreased in the 50 and 100 g groups, and the peak values of the two groups were on days 5 and 7, respectively. The expression levels in the 50 g group basically recovered to normal level on day 14, while that in the 100 g group remained at a high level. The expression levels in the 50 g group were higher than those in the 0 g group and lower than those in the 100 g group. The expression of OPG in the 50 g group decreased first, then increased, and finally decreased. It basically recovered to normal level on day 14. The expression of OPG in the 100 g group decreased first and then increased. It remained at a high level on day 14. The expression in the 50 g group was significantly higher than that in the 0 g group on day 7, while the expression in the 100 g group was significantly higher than that in the 0 g group on day 14. CONCLUSIONS RORγt, IL-17, and OPG were expressed regularly over time under different orthodontic forces, indicating that Th17 participated in the process of bone resorption on the pressure side of periodontal tissue by secreting IL-17.
Animals ; Bone Resorption ; Cytokines ; Interleukin-17 ; Molar ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; Osteoclasts ; Osteoprotegerin ; Rats ; Th17 Cells ; Tooth Movement Techniques

Objective To explore the phenotypic conversion of regulatory T cells (Tregs) in the lungs of mice with bronchopulmonary dysplasia (BPD)-affected mice. Methods A total of 20 newborn C57BL/6 mice were divided into air group and hyperoxia group, with 10 mice in each group. The BPD model was established by exposing the newborn mice to hyperoxia. Lung tissues from five mice in each group were collected on postnatal days 7 and 14, respectively. Histopathological changes of the lung tissues was detected by HE staining. The expression level of surfactant protein C (SP-C) in the lung tissues was examined by Western blot analysis. Flow cytometry was performed to assess the proportion of FOXP3⁺ Tregs and RORγt⁺FOXP3⁺ Tregs in CD4⁺ lymphocytes. The concentrations of interleukin-17A (IL-17A) and IL-6 in lung homogenate were measured by using ELISA. Spearman correlation analysis was used to analyze the correlation between FOXP3⁺Treg and the expression of SP-C and the correlation between RORγt⁺FOXP3⁺ Tregs and the content of IL-17A and IL-6. Results The hyperoxia group exhibited significantly decreased levels of SP-C and radical alveolar counts in comparison to the control group. The proportion of FOXP3⁺Tregs was reduced and that of RORγt⁺FOXP3⁺Tregs was increased. IL-17A and IL-6 concentrations were significantly increased. SP-C was positively correlated with the expression level of RORγt⁺FOXP3⁺ Tregs. RORγt⁺FOXP3⁺ Tregs and IL-17A and IL-6 concentrations were also positively correlated. Conclusion The number of FOXP3⁺ Tregs in lung tissue of BPD mice is decreased and converted to RORγt⁺ FOXP3⁺ Tregs, which may be involved in hyperoxy-induced lung injury.
Animals ; Mice ; Mice, Inbred C57BL ; Bronchopulmonary Dysplasia ; T-Lymphocytes, Regulatory ; Interleukin-17 ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; Hyperoxia ; Interleukin-6 ; Forkhead Transcription Factors ; Lung

This paper aimed to investigate the effects of Jinwu Jiangu recipe total extract on the IL-17/STAT3 signals in rheumatoid arthritis synovial fibroblasts(RASF). The primary RASFs were cultured by tissue piece method ， and divided into blank control group， Jinwu Jiangu recipe low dose group， Jinwu Jiangu recipe middle dose group， Jinwu Jiangu recipe high dose group， and tripterygium glycosides control group. They were then treated with corresponding serum free medium， different doses of Jinwu Jiangu recipe total extract(0.06， 0.6， 6.0 g·L⁻¹)， and tripterygium glycosides(0.03 g·L⁻¹) respectively for 24 hours. The gene expression levels of RORα， RORγt， and STAT3 mRNA were detected by polymerase chain reaction(PCR)， and the protein activity of IL-17R and pSTAT3 were measured by Western blot assay. The results showed that as compared with blank control group， the expression levels of RORα， RORγt， IL-17R and STAT3 mRNA in RASF were significantly declined(<0.01). As compared with tripterygium glycosides control group， Jinwu Jiangu recipe total extract middle dose group and high dose group can down-regulate the expression levels of RORα， RORγt， IL-17R and STAT3 mRNA(<0.05)， and the effect was more obvious in high dose group(<0.01). As compared with blank control group， the protein expression levels of IL-17R and pSTAT3 in each treatment group were obviously decreased(<0.01). As compared with tripterygium glycosides control group， Jinwu Jiangu recipe high dose group had more obvious effect in down-regulating the protein expression of pSTAT3(<0.01). Therefore， Miao medicine Jinwu Jiangu recipe total extract can down-regulate the expressions of RORα， RORγt， and STAT3 mRNA， and inhibit the protein activity of IL-17R and pSTAT3 in RASF.
Arthritis, Rheumatoid ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; Gene Expression Regulation ; Humans ; Nuclear Receptor Subfamily 1, Group F, Member 1 ; metabolism ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; Receptors, Interleukin-17 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Synovial Membrane ; Synoviocytes ; drug effects

Regulatory T (Treg) cells play an essential role in immune homeostasis by controlling the function of various immune effector cells, including RAR-related orphan receptor gammat(+) (RORγt(+)) T helper 17 (Th17) cells. Foekhead box P(3) (FoxP(3)) is the master regulator of Treg cell function, while RORγt is the key transcription factor for the induction of the interleukin (IL)-17 family of cytokines during Th17 cell differentiation. FoxP3 can directly interact with and negatively regulate the function of RORγt, to determine the balance between induced Treg (iTreg) and Th17 cell polarization. Two recent independent studies from the Pan and Chi Labs have shown how hypoxia-inducible factor 1 alpha (HIF1α) is able to tip the balance of T cell differentiation toward the Th17 lineage by responding to the local changes in metabolic shift or an increase in proinflammatory mediators in the microenvironment. By allying with HIF1α, RORγt wins the fight against FoxP3 and Treg cell commitment.
Animals ; Cell Differentiation ; Forkhead Transcription Factors ; metabolism ; Gene Expression Regulation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Immune System ; cytology ; metabolism ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; T-Lymphocytes, Regulatory ; metabolism ; physiology

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

OBJECTIVEThis study aimed to investigate the effect of trichloroethylene (TCE) intake via drinking water on Th17 cells in mice.

METHODSForty eight six weeks old female BALB/c mice were divided into blank control, vehicle control, 2.5 mg/ml TCE and 5.0 mg/ml TCE groups by random number table (12 mice each group), and exposed to TCE by drinking water. On the 14(th), 28(th), 56(th), 84(th) days, blood were collected and assayed for IL-17, IL-6, and TGF-β concentration in serum through ELISA. Animals were killed and spleen biopsies were taken sterility. The proportion of Th17 cells among CD4(+) T cells and RORγt mRNA expression level in spleen were measured by FCM and real-time PCR.

RESULTSIn 2.5 mg/ml TCE and 5.0 mg/ml TCE group mice, Th17 cells/CD4(+) T cells in spleen were (3.46 ± 0.32)% and (5.45 ± 0.45)% on day 14, (3.47 ± 0.33)% and (4.10 ± 0.39)% on day 84, which were significantly higher than those for solvent control group at the same time point ((2.15 ± 0.20)%, (2.16 ± 0.35)%, respectively) (P < 0.01). RORγt mRNA expression levels were (1.870 ± 0.084) and (1.965 ± 0.060) on 14 day, (1.998 ± 0.079) and (2.028 ± 0.073) on day 56, which were also significantly higher than those for solvent control group at the same time point (1.77 ± 0.04 and 1.75 ± 0.09, respectively) (P < 0.05). IL-17 concentrations in serum were (32.28 ± 5.38) and (34.47 ± 5.02) pg/ml on day 14, and (34.87 ± 5.48) and (41.94 ± 6.19) pg/ml on day 28, which were significantly higher than those for solvent control group at the same time point((21.57 ± 5.23), (22.11 ± 5.11) pg/ml). IL-6 concentration in serum were (43.07 ± 6.71) and (47.86 ± 8.52) pg/ml on day14, (41.32 ± 7.04) and (46.74 ± 9.33) pg/ml on day 56, which were significantly higher than solvent control group at the same time point ((7.56 ± 7.71) and (28.26 ± 7.22) pg/ml). TGF-β concentration were (17.48 ± 3.06) and (18.93 ± 3.12) pg/ml on day 14, which did not show significant difference from solvent control group ((15.25 ± 2.95) pg/ml). Correlation analysis showed that IL-6 in serum were significantly positively correlated with the proportion of Th17 cells among CD4(+) T cells and RORγt expression level in spleen (r = 0.741, 0.765, P < 0.01).

CONCLUSIONTCE might promote the differentiation of Th17 cells and increase IL-17 secretion by inducing IL-6 and up-regulating RORγt expression together with TGF-β.

Animals ; Drinking Water ; chemistry ; Female ; Interleukin-17 ; immunology ; Interleukin-6 ; immunology ; Mice ; Mice, Inbred BALB C ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; immunology ; Th17 Cells ; drug effects ; immunology ; Transforming Growth Factor beta ; immunology ; Trichloroethylene ; toxicity