Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.
Mice ; Animals ; Interferon-gamma/metabolism* ; Interleukin-17/metabolism* ; Interleukin-33/metabolism* ; Cytokines/metabolism* ; Carbon Tetrachloride ; Th2 Cells ; Interleukin-4/metabolism* ; Liver Cirrhosis/pathology* ; Th1 Cells ; Th17 Cells/pathology* ; Immunity

Objective To investigate the expressions of IL-18, IL-18 binding protein isoform a (IL-18BPa) and IL-18 receptor α (IL-18Rα) in blood CD4⁺ Th2 cells of patients with allergic rhinitis (AR) and the effects of allergens on their expressions. Methods Blood samples of AR patients and healthy control subjects (HCs) were collected. Peripheral blood mononuclear cells (PBMCs) and CD4⁺ T cells sorted by immunomagnetic beads were stimulated by crude extract of Artemisia sieversiana wild allergen (ASWE), Platanus pollen (PPE) and house dust mite extract (HDME). Flow cytometry was used to detect the expression of IL-18, IL-18BPa and IL-18Rα in CD4⁺ Th2 cells, and BioPlex was used to detect the level of plasma IL-4 and analyze its correlation with the proportion of IL-18⁺ Th2 cells. Results Compared with HCs, the proportion of IL-18⁺ cells was increased in Th2 cells of AR patients; MFI of IL-18 was increased, while that of IL-18Rα was decreased. Moreover, allergens induced IL-18 and IL-18Rα expression in sorted CD4⁺ Th2 cells of HCs and induced IL-18Rα in that of AR patients. Additionally, elevated plasma IL-4 level was found in AR patients, which was moderately correlated with the percentage of IL-18⁺ Th2 cells. Conclusion Allergens may be involved in the pathogenesis of AR by inducing expression of IL-18 in peripheral blood CD4⁺ Th2 cells.
Humans ; Th2 Cells ; Interleukin-18/metabolism* ; Up-Regulation ; Leukocytes, Mononuclear/metabolism* ; Interleukin-4/metabolism* ; Rhinitis, Allergic/metabolism* ; Allergens ; Cytokines/metabolism*

OBJECTIVE: To investigate the significance of Tim-3 and Galectin-9 in Th1/Th2 imbalance in patients with multiple myeloma (MM). METHODS: 55 newly diagnosed MM patients and 20 healthy controls were included. Flow cytometry was used to detect the expression of Tim-3 on CD4⁺T cells, the proportion of Th1, Th2, Tim-3⁺Th1 and Tim-3⁺Th2 cells in peripheral blood. ELISA was used to detect the levels of cytokines IFN-γ and IL-4 in serum, and PCR was used to detect the level of Galectin-9 mRNA. Then the correlations between Galectin-9 mRNA expression and Th-cell subsets and related cytokine levels, as well as the relationship between Tim-3⁺Th1/Tim-3⁺Th2 ratio and corresponding clinical features were analyzed. RESULTS: Compared with the control group, the expression of Tim-3 on CD4⁺T cells in peripheral blood of MM patients was significantly increased (P<0.05), the proportions of Tim-3⁺Th1 cells, Tim-3⁺Th2 cells and Tim-3⁺Th1/Tim-3⁺Th2 ratio in MM patients were also increased (P<0.05), while the proportion of Th1 cells and Th1/Th2 ratio in MM patients were significantly decreased (P<0.05). The level of cytokine IFN-γ and IFN-γ/IL-4 ratio in MM patients were significantly decreased (P<0.05), while the level of cytokine IL-4 was increased (P<0.05). The mRNA levels of Galectin-9 in MM patients were significantly increased (P<0.05). The levels of Galectin-9 mRNA were positively correlated with Tim-3⁺CD4⁺T cells (r=0.663), Tim-3⁺Th2 cells (r=0.492) and IL-4 (r=0.470), while negatively correlated with IFN-γ (r=-0.593). The ratios of Tim-3⁺Th1/Tim-3⁺Th2 in MM patients were positively correlated with ISS stage (r=0.511), osteolytic damage (r=0.556) and chromosome abnormality (r=0.632). CONCLUSION These results suggest that Tim-3 and Galectin-9 are involved in Th1/Th2 imbalance in MM patients, and the high ratio of Tim-3⁺Th1/Tim-3⁺Th2 is associated with poor clinical prognosis.
Humans ; Cytokines/metabolism* ; Galectins/metabolism* ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Interleukin-4/metabolism* ; Ligands ; Multiple Myeloma/metabolism* ; RNA, Messenger/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism*

OBJECTIVE: To investigate the changes in percentage of GATA3⁺ regulatory T (Treg) cells in patients with allergic rhinitis (AR) and mouse models. METHODS: The nasal mucosa specimens were obtained from 6 AR patients and 6 control patients for detection of nasal mucosal inflammation. Peripheral blood mononuclear cells (PBMC) were collected from 12 AP patients and 12 control patients to determine the percentages of Treg cells and GATA3⁺ Treg cells. In a C57BL/6 mouse model of AR, the AR symptom score, peripheral blood OVA-sIgE level, and nasal mucosal inflammation were assessed, and the spleen of mice was collected for detecting the percentages of Treg cells and GATA3⁺ Treg cells and the expressions of Th2 cytokines. RESULTS: Compared with the control patients, AR patients showed significantly increased eosinophil infiltration and goblet cell proliferation in the nasal mucosa (P < 0.01) and decreased percentages of Treg cells and GATA3⁺ Treg cells (P < 0.05). The mouse models of AR also had more obvious allergic symptoms, significantly increased OVA-sIgE level in peripheral blood, eosinophil infiltration and goblet cell hyperplasia (P < 0.01), markedly lowered percentages of Treg cells and GATA3⁺ Treg cells in the spleen (P < 0.01), and increased expressions of IL-4, IL-6 and IL-10 (P < 0.05). CONCLUSION The percentage of GATA3⁺ Treg cells is decreased in AR patients and mouse models. GATA3⁺ Treg cells possibly participate in Th2 cell immune response, both of which are involved in the occurrence and progression of AR, suggesting the potential of GATA3⁺ Treg cells as a new therapeutic target for AR.
Animals ; Mice ; Cytokines/metabolism* ; Disease Models, Animal ; GATA3 Transcription Factor ; Inflammation ; Leukocytes, Mononuclear/metabolism* ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Nasal Mucosa/metabolism* ; Ovalbumin ; Rhinitis, Allergic/therapy* ; T-Lymphocytes, Regulatory ; Th2 Cells/metabolism* ; Humans

Objective: To clarify the chemotactic characteristics of type 2 helper T cells (Th2 cells) and type 2 follicular helper T cells (Tfh2 cells) in peripheral blood of patients with allergic rhinitis (AR), and to explore the associations between the chemokine receptors expression and the levels of antigen-specific IgE (sIgE) and the severity of the disease. Methods: The peripheral blood mononuclear cells of 41 patients with AR (20 males and 21 females, aged 35.0 (24.5, 47.0) years) and 42 healthy controls (24 males and 18 females, aged 35.0 (24.8, 46.5) years) treated in Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from April 2017 to February 2018 were isolated. The expressions of chemokine receptor (CCR)2, CCR3, CCR4, CCR5, CCR7, CCR8, chemokine C-X3-C-motif receptor 1 (CX3CR1) and C-X-C motif receptor 4 (CXCR4) in Th2 and Tfh2 cells were explored by fluorescence activated cell sorting (FACs). The relationship between the expression of these chemokine receptors in Th2 cells and Tfh2 cells and the levels of serum sIgE and the scores of visual analogue scale (VAS) was analyzed. Graphpad prism 7.0 software was used for statistical analysis. Results: The significant differences in chemotactic characteristics between Th2 cells and Tfh2 cells in the control group were found: Th2 cells highly expressed chemokine receptors CCR2, CCR3, CCR5, CCR8 and CX3CR1, while Tfh2 cells highly expressed immune cell homing chemokine receptors CCR7 and CXCR4. AR patients, compared to the control, expressed higher levels of CCR2, CCR5 and CX3CR1 on peripheral Th2 cells(all P<0.01). At the same time, the proportion of CCR2⁺and CCR5⁺Th2 cells was positively correlated with VAS score (r value was 0.58 and 0.61, respectively, both P<0.01). In AR patients, higher expression levels of CCR7 on Tfh2 cells were detected (P<0.01), and the proportion of CCR7⁺Tfh2 cells was positively correlated with the level of serum sIgE (r=0.51, P<0.01). Conclusion: The percentage of CCR2⁺ and CCR5⁺ Th2 cells in peripheral of AR patients can reflect the severity of AR to some extent, while the percentage of CCR7⁺ Tfh2 cells is positively correlated with the level of serum sIgE.
Adult ; Female ; Humans ; Leukocytes, Mononuclear ; Male ; Middle Aged ; Rhinitis, Allergic/metabolism* ; Severity of Illness Index ; Th2 Cells

OBJECTIVE: To explore the expression levels and clinical significance of helper T cell 1/helper T cell 2 (Th1/Th2) cytokine and interleukin-6 (IL-6) in patients with acute leukemia (AL) complicated by infection. METHODS: 68 patients with AL complicated by infection admitted to Wuhan Fifth Hospital from May 2017 to January 2020 were enrolled as study group, 50 AL patients without infection were enrolled as AL group, and 30 healthy volunteers checked in physical examination center were enrolled as healthy control group. The levels of serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10), and peripheral blood Th1/Th2 cells subsets were measured and compared among the three groups. The serum IL-6, IL-10, TNF-α and Th1/Th2 were compared between the patients with mild to moderate infection (n=52) and septic shock (n=16). The relationship between IL-6, IL-10, TNF-α, Th1/Th2 and AL infection was analyzed. RESULTS: The levels of IL-6, IL-10 , TNF-α, and the proportion of Th2 of the patients in study group and AL group were significantly higher than those in healthy control group (P<0.001), while the proportion of Th1 and Th1/Th2 were significantly lower than those in healthy control group (P<0.001). The levels of IL-6, IL-10 and TNF-α, and the proportion of Th2 the patients in study group were significantly higher than those in AL group (P<0.001), while the proportion of Th1 and Th1/Th2 were significantly lower than those in AL group (P<0.001). The serum IL-6, IL-10 and TNF-α level of the patients in septic shock group were significantly higher than those in mild-to-moderate infection group (P<0.001), while Th1/Th2 was lower than those in mild-to-moderate infection group (P<0.001). The results of ROC curve analysis showed that the area under the ROC curve (AUC) values of IL-6, IL-10, TNF-α and Th1/Th2 alone for the diagnosis of septic shock were 0.779, 0.761, 0.724 and 0.718, which were lower than that their combination (0.910) (P<0.05). CONCLUSION The levels of serum IL-6, IL-10 and TNF-α are high in patients with AL complicated infection and septic shock, while Th1/Th2 cell subsets is low. The combined detection of serum IL-6, IL-10, TNF-α and Th1/Th2 is a good diagnostic value for predicting the occurrence of severe septic shock.
Cytokines/metabolism* ; Humans ; Interleukin-10 ; Interleukin-6/metabolism* ; Leukemia/metabolism* ; Shock, Septic/metabolism* ; Th1 Cells/metabolism* ; Th2 Cells/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Antigens ; metabolism ; CTLA-4 Antigen ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; metabolism ; Phenotype ; Proteomics ; Receptors, Chimeric Antigen ; metabolism ; Single-Cell Analysis ; methods ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects ; Transcription, Genetic ; drug effects ; Up-Regulation ; drug effects

OBJECTIVETo study the influence of Yanyankang powder on Th1/Th2 in rats with experimental autoimmune uveitis (EAU).

METHODSThe EAU models were induced in Lewis rats by immunization with interphotoreceptor retinoid binding protein (IRBP) 1177-1191 in complete Freund's adjuvant. The rats were randomly divided into 3 groups: a model control group, a Yanyankang group, and a prednisone group, 9 rats in each group. The model control group was intervened with saline solution by gavage. The Yanyankang group was intervened with Yanyankang powder 4 g/(kg day) by gavage. The prednisone group were intervened with prednisone acetate tablets 5 mg/(kg d) by gavage. All groups were intervened after immunization once every 2 days for 18 days and monitored by slit-lamp biomicroscopy daily until day 18. The levels of gamma interferon (INF-γ) and interleukin-10 (IL-10) in the supernatants of T cells were determined by enzyme-linked immunosorbent assay. Polymerase chain reaction (PCR) technology was used for measuring Th1 and Th2 related cytokine mRNA expressions.

RESULTSSlighter intraocular inflammation was found in the Yanyankang group and the prednisone group than the control group. The levels of the IFN-γ and IL-10 in the supernatants of the spleen lymph node cells were 382.33±6.30, 155.87±4.46 μg/L in the Yanyankang group and 270.93±7.76, 265.32±11.88 μg/L in the prednisone group. Both had significant differences compared with the control group (941.53±8.59, 20.67±4.65 μg/L; =0.01). The PCR results showed the same tendency.

CONCLUSIONYanyankang powder showed favorable effects in the rats with EAU by influencing the function of Th1 and Th2 cells.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Eye ; pathology ; Female ; Immunization ; Inflammation ; pathology ; Interferon-gamma ; genetics ; metabolism ; Interleukin-10 ; genetics ; metabolism ; Lymph Nodes ; drug effects ; metabolism ; Powders ; RNA, Messenger ; genetics ; metabolism ; Rats, Inbred Lew ; Spleen ; metabolism ; Th1 Cells ; drug effects ; immunology ; Th2 Cells ; drug effects ; immunology ; Uveitis ; drug therapy ; genetics ; immunology

OBJECTIVEThe aim of the study was to investigate whether Orai1 antibody intraperitoneal injection could improve the condition of allergic rhinitis (AR) in mice.

METHODSTwenty-four BALB/C mice (SPF grade) were classified into 4 groups (AR group, Control group, Experimental group 1 and experimental group 2) according to a random number table. A mouse model of AR was established (Control group was established by phosphate buffered solution), and experimental group 1 and Experimental group 2 were established through intraperitoneal injection of 100 μg and 150 μg Orai1 antibody respectively. The number of sneezing and rubbing and eosinophilia in mice were assessed after different doses of Orai1 antibody intraperitoneal injection were applied. Then Orai1 protein and its mRNA in nasal mucosa, histomine, eosionphil cation protein (ECP), interlukin (IL)-1β, IL-4, IL-5 and IL-6 and their mRNA in nasal lavage fluid (NLF) and nasal mucosa were evaluated using enzyme linked immunosorbent assay (ELISA) and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). Furthermore, Orai1 protein and its mRNA in Th2 cells in peripheral blood, IL-4 and IL-5 in peripheral serum and their mRNAs in Th2 cells were also examined through ELISA and real-time RT-PCR. The data were analyzed by a statistical software of Graph Pad Prism 5.

RESULTSThere were significant differences in sneezing, nasal rubbing and local invading eosinophils in nasal mucosa after the treatment (t100 μg=7.88, t100 μg=9.92, t100 μg=4.30, respectively; t150 μg=16.43, t150 μg=16.31, t150 μg=9.35, respectively, all P-values<0.01). The Orai1 antibody intervention decreased contents of Orai1 in nasal mucosa, histomine, ECP, IL-1β, IL-4, IL-5 and IL-6. The contents of experimental group 1 were (0.186±0.015) μg/ml, (6.618±0.180) ng/ml, (2.555±0.031) ng/ml, (85.26±2.94) pg/ml, (55.12±1.21) pg/ml, (58.45±2.11) pg/ml and (77.12±2.13) pg/ml, respectively. The contents of experimental group 2 were (0.089±0.003) μg/ml, (4.501±0.310) ng/ml, (1.260±0.017) ng/ml, (48.49±2.12) pg/ml, (33.15±0.87) pg/ml, (38.24±0.95) pg/ml and (51.72±0.81) pg/ml, respectively. The differences were siginificant between group 1, group 2 and AR group(t value was 3.29, 10.44, 9.45, 17.53, 74.53, 87.06, 3.98; 8.54, 13.32, 23.00, 20.89, 80.73, 103.70, 13.34, all P<0.01). However, there were no significant differences in Orai1 protein and its mRNA in peripheral Th2 cells, IL-4 and IL-5 in peripheral serum and their mRNAs in Th2 cells (all P-values>0.05). In addition, the effect of 150 μg Orai1 antibody treatment was better than 100 μg one (all P-values<0.05).

CONCLUSIONOrai1 antibody intraperitoneal injection can improve the symptoms of AR mice, and alleviate the condition of allergic inflammation. Orai1 may become a novel aim in the AR study.

Animals ; Antibodies ; pharmacology ; Calcium Channels ; immunology ; metabolism ; Cytokines ; immunology ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Eosinophilia ; therapy ; Eosinophils ; immunology ; Immunotherapy ; Inflammation ; Injections, Intraperitoneal ; Mice ; Mice, Inbred BALB C ; Nasal Mucosa ; metabolism ; ORAI1 Protein ; RNA, Messenger ; Rhinitis, Allergic ; therapy ; Th2 Cells ; immunology

BACKGROUNDAtopic dermatitis (AD) is characterized by defective skin barrier and imbalance in T helper 1/T helper 2 (Th1/Th2) cytokine expression. Filaggrin (FLG) is the key protein to maintaining skin barrier function. Recent studies indicated that Th1/Th2 cytokines influence FLG expression in keratinocytes. However, the role of Th1/Th2 cytokines on FLG processing is not substantially documented. Our aim was to investigate the impact of Th1/Th2 cytokines on FLG processing.

METHODSHaCaT cells and normal human keratinocytes were cultured in low and high calcium media and stimulated by either interleukin (IL)-4, 13 or interferon-γ (IFN-γ). FLG, its major processing proteases and key protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) were measured by both real-time quantitative polymerase chain reaction and Western blotting. Their expression was also evaluated in acute and chronic AD lesions by immunohistochemistry.

RESULTSIL-4/13 significantly reduced, while IFN-γ significantly up-regulated FLG expression. IL-4/13 significantly increased, whereas IFN-γ significantly decreased the expression of kallikreins 5 and 7, matriptase and channel-activating serine protease 1. On the contrary, IL-4/13 significantly decreased, while IFN-γ increased the expression of LEKTI and caspase-14. Similar trends were observed in AD lesions.

CONCLUSIONSOur results suggested that Th1/Th2 cytokines differentially regulated the expression of major FLG processing enzymes. The imbalance between Th1 and Th2 polarized immune response seems to extend to FLG homeostasis, through the network of FLG processing enzymes.

Caspase 14 ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Dermatitis, Atopic ; metabolism ; Humans ; Immunohistochemistry ; Interferon-gamma ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; metabolism ; Intermediate Filament Proteins ; metabolism ; Keratinocytes ; enzymology ; metabolism ; Proteinase Inhibitory Proteins, Secretory ; metabolism ; Serine Peptidase Inhibitor Kazal-Type 5 ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism