1.Enhanced expression of CD40L cDNA on ovarian cancer cell line OVHM induces the secretion of Th1 cytokines from dendritic cells.
Zheng-Mao ZHANG ; Feng-Hua ZHANG ; Xi-Mei WANG ; Chao ZHANG ; Jie LIU ; Lai-Mei GU ; Quan-Hai LI ; Bao-En SHAN ; Masatoshi TAGAWA
Chinese Journal of Oncology 2008;30(3):174-178
OBJECTIVETo examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer (OVHM) cells could induce the secretion of Th1 cytokines from dendritic cells (DC).
METHODSOVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamine 2000 and then G418 resistant cells as positive cells were selected. They were examined for their expression of CD40L with flow cytometry. Bone marrow cells were firstly depleted of erythrocytes, macrophages, T and B cells with PE-conjugated magnetic beads, and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days. PKH67-labeled tumor cells were cultured with DC, and then the stained cells were analyzed for the expression of MHC-I, MHC-II, CD80, CD86, CCR7 in DC with flow cytometry. The expression of p40, p19, p35, p28, EBI3 subunits, IL-18, IFN-gamma, Mig gene in cocultured DC-tumor cells were detected by RT-PCR.
RESULTSThe CD40L cDNA was successfully transfected into OVHM cells. Bone marrow-derived DCs, when cultured with CD40L/OVHM, formed clusters with the tumors and showed an upregulated expression of MHC- I, MHC-II, CD80, CD86, CCR7. Expression of the IL-12, IL-23, IL-27, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells.
CONCLUSIONThese data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors, enhances the maturation of DCs, induces secretion of Th1 cytokines, especially for IL-12, IL-23 and IL-27, which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.
Animals ; CD40 Ligand ; genetics ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Cytokines ; secretion ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; metabolism ; Female ; Interleukin-12 ; secretion ; Interleukin-23 ; secretion ; Interleukins ; secretion ; Mice ; Ovarian Neoplasms ; metabolism ; pathology ; Th1 Cells ; secretion ; Transfection
2.Expression of T-helper 17 cells and signal transducers in patients with psoriasis vulgaris of blood-heat syndrome and blood-stasis syndrome.
Bin FAN ; Xin LI ; Kan ZE ; Rong XU ; Ruo-Fei SHI ; Lin GENG ; Fu-Lun LI ; Yi-Fei WANG ; Jie CHEN ; Bin LI
Chinese journal of integrative medicine 2015;21(1):10-16
OBJECTIVETo investigate the levels of cytokines related to T-helper (Th) 17 cells in serum and signal transducers in the psoriatic lesions of patients with psoriasis vulgaris of blood-heat syndrome (BHS) and blood-stasis syndrome (BSS).
METHODSSixty patients with psoriasis vulgaris were divided into the BHS and BSS groups according to the syndrome differentiation of Chinese medicine (CM). Ten healthy subjects were considered as the control group. Cytokine levels of interleukin (IL)-17, IL-23 and IL-6 in serum were determined by enzyme-linked immunosorbent assay. Expression levels of signal transducer and activator of transcription 3 (STAT3), p38-mitogen-activated protein kinase (MAPK) and STAT6 in the psoriatic lesions were determined using immunohistochemistry (IHC), Western blot, and real-time quantitative reverse transcription polymerase chain reaction, respectively.
RESULTSProduction of IL-17, IL-23 and IL-6 in the BHS group and BSS group were significantly increased compared with those in the control group (P<0.05). Levels of IL-17 and IL-23 in the BHS group were higher than those in the BSS group (P<0.05). Compared with the control group, IHC positive expressions and protein expressions of STAT3 and p38-MAPK, and the STAT3 mRNA expressions in the BHS and BSS groups were significantly higher (P<0.05 or P<0.01). The protein expression of STAT3 in the BHS group was significantly higher than that in the BSS group (P<0.05).
CONCLUSIONSCytokines in serum and signal transducers in the psoriatic lesions alter with various CM syndromes of psoriasis. The results provide scientific basis for the treatment based on syndrome differentiation of CM in treating psoriasis vulgaris.
Adult ; Female ; Gene Expression Regulation ; Humans ; Immunohistochemistry ; Interleukin-17 ; blood ; Interleukin-23 ; blood ; Interleukin-6 ; blood ; Male ; Psoriasis ; blood ; enzymology ; genetics ; immunology ; RNA, Messenger ; genetics ; metabolism ; STAT3 Transcription Factor ; genetics ; metabolism ; STAT6 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Syndrome ; Th17 Cells ; immunology ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism
3.Detection and significance of transcription factors and cytokines of Th17/Treg cells in peripheral blood in the gastric cancer patients.
Su-fang PENG ; Sheng-jun WANG ; Jian-guo CHEN ; Xiao-li DAI ; Yan SHI ; Ya-zhen LI ; Zhao-liang SU ; Yuan XUE ; Zhi-qiang HE ; Xin-xiang HUANG ; Hua-xi XU
Chinese Journal of Oncology 2010;32(3):185-189
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
4.Increased level of Th17 cells in peripheral blood correlates with the development of hepatocellular carcinoma.
Wei-wei WANG ; Zhen-meng WANG ; Yao-yang LIU ; Yang-hua QIN ; Qian SHEN
Chinese Journal of Oncology 2010;32(10):757-761
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
5.Myeloid differentiation primary response protein 88 blockade upregulates indoleamine 2,3-dioxygenase expression in rheumatoid synovial fibroblasts.
Mi Kyung PARK ; Hye Jwa OH ; Yang Mi HEO ; Eun Mi PARK ; Mi La CHO ; Ho Youn KIM ; Sung Hwan PARK
Experimental & Molecular Medicine 2011;43(8):446-454
Indoleamine 2,3-dioxygenase (IDO) is a key negative regulator of immune responses and has been implicated in tumor tolerance, autoimmune disease and asthma. IDO was detected in the joint synovial tissue in the inflammatory microenvironment of rheumatoid arthritis (RA), but IDO expression in joint synovial tissue is not sufficient to overcome the inflamed synovial environment. This study aimed to unravel the mechanisms involving the failure to activate tolerogenic IDO in the inflamed joint. We demonstrate that both poly (I:C) and lipopolysaccharide (LPS) induce expression of IDO in synovial fibroblasts. However, inflammatory cytokines such as IL-17, TNF-alpha, IL-12, IL-23 and IL-16 did not induce IDO expression. Poly (I:C) appeared to induce higher IDO expression than did LPS. Surprisingly, toll-like receptor (TLR)4-mediated IDO expression was upregulated after depletion of myeloid differentiation primary response protein 88 (MyD88) in synovial fibroblasts using small interfering RNA (siRNA). IDO, TLR3 and TLR4 were highly expressed in synovial tissue of RA patients compared with that of osteoarthritis patients. In addition, RA patients with severe disease activity had higher levels of expression of IDO, TLR3 and TLR4 in the synovium than patients with mild disease activity. These data suggest that upregulation of IDO expression in synovial fibroblasts involves TLR3 and TLR4 activation by microbial constituents. We showed that the mechanisms responsible for IDO regulation primarily involve MyD88 signaling in synovial fibroblasts, as demonstrated by siRNA-mediated knockdown of MyD88.
Adaptor Proteins, Vesicular Transport/genetics/metabolism
;
Arthritis, Rheumatoid/*metabolism
;
Blotting, Western
;
Cells, Cultured
;
Fibroblasts/drug effects/*metabolism
;
Humans
;
Immunohistochemistry
;
Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics/*metabolism
;
Interleukin-12/pharmacology
;
Interleukin-16/pharmacology
;
Interleukin-17/pharmacology
;
Interleukin-23/pharmacology
;
Lipopolysaccharides/pharmacology
;
Myeloid Differentiation Factor 88/genetics/*metabolism
;
Poly I-C/pharmacology
;
Polymerase Chain Reaction
;
RNA, Small Interfering/genetics/physiology
;
Synovial Membrane/*cytology
;
Toll-Like Receptor 4/genetics/metabolism
;
Tumor Necrosis Factor-alpha/pharmacology
6.Silencing IL-23 expression by a small hairpin RNA protects against asthma in mice.
Yanchun LI ; Meng SUN ; Huanji CHENG ; Shanyu LI ; Li LIU ; Hongmei QIAO ; Shucheng HUA ; Jirong LU
Experimental & Molecular Medicine 2011;43(4):197-204
To determine the impact of IL-23 knockdown by RNA interference on the development and severity of ovalbumin (OVA)-induced asthmatic inflammation, and the potential mechanisms in mice, the IL-23-specific RNAi-expressing pSRZsi-IL-23p19 plasmid was constructed and inhaled into OVA-sensitized mice before each challenge, as compared with that of control mice treated with alum or budesonide. Inhalation of the pSRZsi-IL-23p19, significantly reduced the levels of OVA-challenge induced IL-23 in the lung tissues by nearly 75%, determined by RT-PCR. In addition, knockdown of IL-23 expression dramatically reduced the numbers of eosinophils and neutrophils in BALF and mitigated inflammation in the lungs of asthmatic mice. Furthermore, knockdown of IL-23 expression significantly decreased the levels of serum IgE, IL-23, IL-17, and IL-4, but not IFNgamma, and its anti-inflammatory effects were similar to or better than that of treatment with budesonide in asthmatic mice. Our data support the notion that IL-23 and associated Th17 responses contribute to the pathogenic process of bronchial asthma. Knockdown of IL-23 by RNAi effectively inhibits asthmatic inflammation, which is associated with mitigating the production of IL-17 and IL-4 in asthmatic mice.
Animals
;
Asthma/chemically induced/genetics/metabolism/*prevention & control
;
Bronchoalveolar Lavage Fluid/cytology
;
Enzyme-Linked Immunosorbent Assay
;
Eosinophils
;
Female
;
Inflammation/metabolism
;
Interleukin-23/*genetics
;
Leukocyte Count
;
Mice
;
Mice, Inbred BALB C
;
Neutrophils
;
Ovalbumin/pharmacology
;
Plasmids/genetics
;
*RNA Interference
;
RNA, Small Interfering/*genetics
;
Reverse Transcriptase Polymerase Chain Reaction
;
Th17 Cells/immunology
7.The immune-stimulating peptide WKYMVm has therapeutic effects against ulcerative colitis.
Sang Doo KIM ; Soonil KWON ; Sung Kyun LEE ; Minsoo KOOK ; Ha Young LEE ; Ki Duk SONG ; Hak Kyo LEE ; Suk Hwan BAEK ; Chan Bae PARK ; Yoe Sik BAE
Experimental & Molecular Medicine 2013;45(9):e40-
In this study, we examined the therapeutic effects of an immune-stimulating peptide, WKYMVm, in ulcerative colitis. The administration of WKYMVm to dextran sodium sulfate (DSS)-treated mice reversed decreases in body weight, bleeding score and stool score in addition to reversing DSS-induced mucosa destruction and shortened colon. The WKYMVm-induced therapeutic effect against ulcerative colitis was strongly inhibited by a formyl peptide receptor (FPR) 2 antagonist, WRWWWW, indicating the crucial role of FPR2 in this effect. Mechanistically, WKYMVm effectively decreases intestinal permeability by stimulating colon epithelial cell proliferation. WKYMVm also strongly decreases interleukin-23 and transforming growth factor-beta production in the colon of DSS-treated mice. We suggest that the potent immune-modulating peptide WKYMVm and its receptor FPR2 may be useful in the development of efficient therapeutic agents against chronic intestinal inflammatory diseases.
Adjuvants, Immunologic/pharmacology/*therapeutic use
;
Animals
;
Caco-2 Cells
;
Cell Proliferation
;
Colitis, Ulcerative/*drug therapy/metabolism
;
Colon/pathology
;
Humans
;
Interleukin-23/genetics/metabolism
;
Intestinal Mucosa/drug effects/metabolism/pathology
;
Mice
;
Mice, Inbred C57BL
;
Oligopeptides/pharmacology/*therapeutic use
;
Permeability
;
Receptors, Formyl Peptide/antagonists & inhibitors
;
Transforming Growth Factor beta/genetics/metabolism
8.Effect of electroacupuncture on myocardial inflammatory injury and apoptosis in mice with acute myocardial ischemia based on VEGF-C/VEGFR-3 pathway.
Hai-Yan ZUO ; Sheng-Bing WU ; Xin WU ; Shuai CUI ; Lei WANG ; Xiao-Xiao WANG ; Hao-Sheng WU ; Si-Jia TONG ; Zhen-He PEI ; Mei-Qi ZHOU
Chinese Acupuncture & Moxibustion 2022;42(11):1269-1277
OBJECTIVE:
To observe the effect of electroacupuncture (EA) on vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), proinflammatory factors and apoptosis in myocardial tissue in mice with acute myocardial ischemia (AMI), and to explore the mechanism of EA for AMI.
METHODS:
Fifty male C57BL/6 mice were randomly divided into a sham operation group, a model group, an EA group, an inhibitor group and an inhibitor+EA group, 10 mice in each group. Except for the sham operation group, the mice in the remaining groups were intervented with ligation at the left anterior descending (LAD) coronary artery to establish AMI model. The mice in the sham operation group were intervented without ligation after thoracotomy. The mice in the EA group were intervented with EA at "Shenmen" (HT 7) and "Tongli" (HT 5), disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity, 30 min each time, once a day, for 3 d. The mice in the inhibitor group were treated with intraperitoneal injection of SAR 131675 (12.5 mg•kg-1•d-1, once a day for 3 d). The mice in the inhibitor+EA group were injected intraperitoneally with SAR 131675 30 min before EA. The ECG before modeling, 30 min after modeling and 3 d after intervention was detected, and the ST segment displacement was recorded; after the intervention, the ELISA method was applied to measure the contents of serum creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST) as well as tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) in myocardial tissue; the HE staining method was used to observe the morphological changes of myocardial tissue; the immunofluorescence double labeling method was applied to measure the number of co-expression positive cells of VEGF-C/VEGFR-3 in myocardial tissue; the TUNEL method was used to detect the level of cardiomyocyte apoptosis; the Western blot method was applied to measure the protein expressions of VEGF-C, VEGFR-3, b-lymphoma-2 (Bcl-2), activated caspase-3 (Cleaved Caspase-3) and activated poly adenosine diphosphate ribose polymerase-1 (Cleaved PARP-1).
RESULTS:
Compared with the sham operation group, in the model group the ST segment displacement was increased (P<0.01); the contents of CK-MB, AST, TNF-α and IL-23 were increased (P<0.01); the arrangement of myocardial fibers was disordered, and interstitial inflammatory cell infiltration was obvious; the number of co-expression positive cells of VEGF-C/VEGFR-3 was decreased (P<0.01); the number of cardiomyocyte apoptosis was increased (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were decreased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were increased (P<0.01). Compared with the model group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.01); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). There was no significant difference in all the indexes between the model group and the inhibitor group (P>0.05). Compared with the model group, the protein expression of VEGF-C was increased in the inhibitor+EA group (P<0.01). Compared with the inhibitor group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.05); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). Compared with the inhibitor+EA group, all the indexes in the EA group were improved except the protein expression of VEGF-C (P<0.01).
CONCLUSION
EA could relieve the inflammatory reaction and apoptosis in AMI mice, and its mechanism may be related to activating VEGF-C/VEGFR-3 pathway and promoting lymphangion genesis.
Mice
;
Male
;
Animals
;
Electroacupuncture
;
Vascular Endothelial Growth Factor Receptor-3
;
Caspase 3
;
Vascular Endothelial Growth Factor C
;
Tumor Necrosis Factor-alpha/genetics*
;
Vascular Endothelial Growth Factor A/genetics*
;
Poly(ADP-ribose) Polymerase Inhibitors
;
Mice, Inbred C57BL
;
Myocardial Ischemia/metabolism*
;
Apoptosis
;
Interleukin-23
;
Proto-Oncogene Proteins c-bcl-2