Chinese medicine Yu-Ping-Feng-San attenuates allergic inflammation by regulating epithelial derived pro-allergic cytokines / 中国天然药物

This study aimed to investigate the mechanisms of Yu-Ping-Feng-San (YPFS) on attenuating allergic inflammation in the initial stage of atopic dermatitis (AD). AD mouse model was established with fluorescein isothiocyanate (FITC) sensitization and elicitation. Epithelial barrier structure was observed with transmission electron microscope. The populations of dendritic cells (DCs) and group 2 innate lymphoid cells (ILC2s) were detected by flow cytometry. Human immortalized keratinocyte (HaCaT) cells were stimulated with Poly(I:C)/TNF-α in vitro to assessthymic stromal lymphopoietin (TSLP), interleukin (IL)-33 and nuclear factor-κB (NF-κB) levels or expressions by immunofluorescence, enzyme linked immunosorbent assay (ELISA) and western blot. In the initial stage of AD, ear swelling and infiltration of inflammatory cells in ear tissues were markedly attenuated with YPFS treatments. The damaged structures of ear epithelium and the increased levels of Th2-cytokines induced by FITC were significantly rescued in YPFS-treated mice. The production of pro-allergic cytokines, TSLP and IL-33, as well as the cell populations of their target cells DCs and ILC2s were decreased in AD model, respectively. Likewise, the levels of TSLP and IL-33 in Poly(I:C)/TNF-α-stimulated HaCaT cells showed the same results. Lower levels of p-NF-κB were detected with YPFS treatment, and the expressions of TSLP and IL-33 could be further decreased with inhibiting of NF-κB. Therefore, YPFS attenuates allergic inflammation in the initial stage of AD probably through regulating NF-κB-TSLP/IL-33 pathway, which may provide a novel effective target for the prevention and treatment of allergic diseases.

Mechanism of Th2 related inflammatory factors in the pathogenesis of eczema / 药学实践杂志

Eczema is a common clinical inflammatory skin disease characterized by severe itching and recurrent attacks.Its etiology is complex, including a variety of internal and external factors.The main mechanism is related to the imbalance of Th2immune response, and related inflammatory factors play an important role.Epithelial-derived factor like thymic stromal lymphopoietin and interleukin-33could trigger Th2immune imbalance and promote the secretion of type inflammatory factors such as interleukin-4, interleukin-5, and interleukin-13.These inflammatory factors will further induce eosinophilic granulocytosis and IgE formation.In this paper, the research progress of mechanism of type 2related inflammatory factors in eczema were reviewed, which provided the great significance for treatment.

The Role of Thymic Stromal Lymphopoietin (TSLP) in Glomerulonephritis

Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine that is an important trigger and initiator of many allergic diseases. TSLP promotes a T-helper type 2 (Th2) cytokine response that can be pathological. A relationship is formed both at the induction phase of the Th2 response through polarization of dendritic cells to drive Th2 cell differentiation and at the effector phase of the response, by promoting the expansion of activated T cells and their secretion of Th2 cytokines and TSLP. In transgenic mice with TSLP overexpression, it has been reported that TSLP leads to the development of mixed cryoglobulinemic membranoproliferative glomerulonephritis. In addition, TSLP can play an important role in the pathogenesis of IgA nephropathy and systemic lupus erythematosus-related nephritis. From our knowledge of the role of TSLP in the kidney, further studies including the discovery of new therapies need to be considered based on the relationship between TSLP and glomerulonephritis.

Phloxine O, a Cosmetic Colorant, Suppresses the Expression of Thymic Stromal Lymphopoietin and Acute Dermatitis Symptoms in Mice

Cosmetics are primarily applied to the skin; therefore, the association of cosmetic dyes with skin diseases or inflammation is a topic of great interest. Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine that activates dendritic cells to promote Th2 inflammatory immune responses. TSLP is highly expressed in keratinocytes under inflammatory conditions, which suggests that it may play a critical role in the development of skin diseases, such as atopic dermatitis. Therefore, we investigated whether cosmetic dyes influenced the production of TSLP by keratinocytes. Phloxine O, also known as D&C Red No.27, is one of the most common red synthetic pigments and is widely used in colored cosmetics. Our results showed that Phloxine O downregulated phorbol 12-myristate 13-acetate-induced production of TSLP in a murine keratinocyte cell line (PAM212). Phloxine O also suppressed TSLP expression in KCMH-1 cells, which are mouse keratinocytes that constitutively produce high levels of TSLP. To investigate the in vivo effects of Phloxine O, we induced TSLP expression in mouse ear skin by topically applying MC903, a vitamin D3 analogue that is a well-known inducer of atopic dermatitis-like symptoms. Topical application of Phloxine O prevented MC903-induced TSLP production in mouse ear skin, attenuated the acute dermatitis-like symptoms and decreased serum IgE and histamine levels in mice. Suppression of TSLP expression by Phloxine O correlated with reduced expression of OX40 ligand and Th2 cytokines in mouse ear skin. Our results showed that Phloxine O may be beneficial to prevent dermatitis by suppressing the expression of TSLP and Th2 cytokines in skin.

Correlative analysis of TSLP and Th2 responses in lung tissue of young rats infected with RSV / 中国免疫学杂志

Objective:To investigate the changes of thymic lymphocyte procyanidin(TSLP)and interleukin-4(IL-4),interleukin-10(IL-10)and interleukin-13(IL-13)in the lung tissue of young rats infected with respiratory syncytial virus,and to investigate the changes of TSLP and Th2 Correlation.Methods:18 Wistar rats were randomly divided into RSV infection group and normal group,9 rats in each group.Application of RSV virus droplets nasal modeling,saline diarrhea as a control.The lung tissue of rats was taken for pathological observation;the load of syncytial virus at different time points and the expression of TSLP in lung tissue were detected by Real-time quantitative polymerase chain reaction(q-PCR);at the same time,the expression of TSLP at the protein level was detected by Western blot.The levels of cytokines IL-4,IL-10 and IL-13 in serum were measured by enzyme-linked immunosorbent assay(ELISA).And the relationship between TSLP and IL-4,IL-10 and IL-13 was analyzed by Spearman correlation.Results:Compared with the normal control group,the rats in the model group showed symptoms such as poor mental state,rough hair,decreased activity and shortness of breath after intranasal infection,and the symptoms were aggravated.Pathological observation showed that the alveolar wall was thickened in the model group,and a large number of lymphocytes,plasma cells,eosinophil infiltration.q-PCR was used to detect the syncytial virus load in model group,and reached the peak at about 5 days,then decreased gradually.RSV infection increased the secretion of TSLP in rat respiratory tract.q-PCR and Western blot showed that TSLP concentration in model group was higher than that in control group(P<0.05).The levels of IL-4,IL-10 and IL-13 in the serum of the model group were higher than those in the control group(P<0.05).Spearman correlation analysis showed that TSLP was positively correlated with IL-4 and IL-10 expression in RSV model group,and there was no correlation between TSLP and IL-13 expression.Conclusion:RSV infection in rat lung tissue can induce increased expression of TSLP,while promoting the occurrence of Th2 type of inflammatory response and the expression of related cytokines,in order to further study of RSV infection caused by bronchiolitis and asthma and other respiratory diseases,the mechanism of action and clinical treatment laid the foundation.

Affinity maturation of human scFv anti-TSLP in vitro / 中国免疫学杂志

Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.

TSLP promotes recruitment of myofibroblasts into airway in asthmatic mice / 中国免疫学杂志

Objective:To investigate whether thymic stromal lymphopoietin ( TSLP) participate in asthmatic airway remodeling partially by promoting myofibroblast accumulating in the lung. Methods:Twelve mice evenly were randomly divided into four groups:a saline group;an HDM-exposed group;an IgG isotype-treated group and an anti-TSLP-treated group. The supernatant of bronchoalveolar lavage fluid ( BALF) was used to analyze the levels of TSLP,IL-25 and IL-33 by ELISA. Fluorescence-labeled collagenⅠ( ColⅠ)/α-smooth muscle actin (α-SMA) -dual-positive myofibroblasts were examined by confocal microscopy. Results:Chronic allergen exposure induced obviously abnormal airway structural changes,which were inhibited by blocking TSLP. We detected a highly increased number of myofibroblasts in the sub-epithelial zone in mice from HDM-challenged group. However, TSLP neutralization significantly reduced myofibroblasts recruitment. Moreover,blocking TSLP not only decreased the level of TSLP,but also inhibited the expression levels of TGF-β1 and IL-33 in BAL fluid. Conclusion:The results suggest that orchestrating myofibroblasts recruiting into the lungs is one of the main pathogenesis that TSLP involves in airway remodeling in asthmatic mice.

Observation of adequate stimulus methods for key initiative factor expression in human keratinocytes of anaphylactic disease / 中国药理学通报

Aim To explore the expressed level of ini-tiative key factors TSLP and IL-33 in a human kerati-nocyte cell line,HaCaT cells were chosen to be stimu-lated by different stimulants,and develop a stable and effective in vitro model to observe allergic sensitization. Methods HaCaT cells were cultured in K-SFM with different stimulants to screen out the stimulants which could significantly improve the expressed level of TSLP and IL-33.Expressed level of TSLP and IL-33 was an-alyzed by ELISA kits and immunofluorescence.Re-sults (1 )The dose-response relationship of single stimulant indicated that both Poly(I:C)and TNF-αcould significantly improve expressed level of TSLP and IL-33 in HaCaT cells,but the rest of stimulants was not observed significant stimulation in concentration range of this experiment.(2)Dose-effect relationship of combined stimulants indicated that poly(I ∶C)1 00 mg·L -1 combined with TNF-α20 μg·L -1 was the most efficient.(3)Time-effect relationship of the a-bove-mentioned combined stimulants showed that 1 2 h was the optimal time of stimulation.Conclusions Different stimulants and different time result in various expressed levels of TSLP and IL-33 in HaCaT cells.1 2 h stimulus duration of Poly(I:C)1 00 mg·L -1 com-bined with TNF-α20 μg · L -1 is the most efficient stimulating way.This result provides an effective in vitro model to study the pathomechanism and drug effi-cacy of allergic sensitization.

Expression of TSLP and NF-κB in patients with adenomyosis / 中国免疫学杂志

Objective:To investigate the role of TSLP and NF-κB in the pathogenesis of adenomyosis.Methods:Immunohisto-chemistry was employed to detect the expression of TSLP and NF-κB p65 in the endometrial glandular cell and stromal cell of 35 adeno-myosis patients and 20 hysteromyoma patients as control , and analyze the correlation of the two proteins.The concentration of TSLP was measured by specific ELISA in the serum of healthy subjects besides of the patients with adenomyosis and hysteromyoma .Results:TSLP and NF-κB p65 were both positively expressd in the glandular cells and stromal cells of ectopic and eutopic endometrium of patients with adenomyosis , their expression was significantly higher than that of the control group ( P<0.01 ) , and the expression of TSLP and NF-κB p65 in ectopic endometrium was significantly higher than that in eutopic endometrium ( P<0.01 ).There was positive correlation between TSLP and NF-κB p65 in ectopic endometrium.The concentration of TSLP was higher in the serum of patients with adenomyosis.Conclusion:The TSLP is highly expressed in endometrial glandular and stromal cells of patients with adenomyosis , and there is higher level of TSLP in serum , and there is correlation between the expression of TSLP and NF-κB in ectopic endometrium.

Selection and identification of full human scFv against TSLP / 中国免疫学杂志

Objective:Expression of protein TSLP and selection of full human anti-TSLP single chain Fv ( scFv).Methods:The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results: The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion: The full human scFvs against for TSLP were selected suc-cessfully.

Eukaryotic expression and characterization of mouse TSLP and HIV-1 gp120 BAL V1/V2 fusion protein / 中国免疫学杂志

Objective:To express fusion protein of mouse thymic stromal lymphopoietin (TSLP) and HIV-1 gp120BAL V1/V2 subdomain in 293F cell.Methods:Full length of the V1V2 sequence from BAL isolate was fused with the C-terminus of mouse thymic stromal lymphopoietin (TSLP) and sub-cloned into pCEP-Pu vector to construct the eukaryotic expression plasmid-pCTV1V2BAL.The recombinant plasmid was confirmed by enzyme digestion and sequencing , then transfected into 293 F cells using PEI as a transfection reagent .The fusion protein was purified by metal chelate affinity chromatography and characterized by SDS -PAGE and Western blot . The epitopes of V1/V2 in fusion protein were identified by ELISA .Results:The SDS-PAGE and Western blot results showed that there were highly heterogeneous glycoprotein bands at the site between 35 kD and 55 kD, which reacted with anti-mTSLP rabbit polyclonal antibody and anti-His tag mouse monoclonal antibody .The ELISA analysis showed that antibodies to V 1/V2BAL existed in the sera of HIV-1 positive patients.Conclusion:The mTSLP-V1/V2 fusion protein was successfully expressed in 293F cells, which may be useful for HIV-1 vaccine research .

Panonychus citri Can Induce T-helper Type 2 Immune Responses via the Release of Thymic Stromal Lymphopoietin and IL-4

Panonychus citri damages the leaves of citrus trees, causing defoliation, and induces T-helper type 2 (TH2) immune responses (occupational asthma) via a hitherto unknown mechanism. This is a particular problem on Jeju Island, which is located to the south of the Korean peninsula. In this study, we show for the first time how P. citri induces TH2 immunity. Exposure to P. citri induces the production of thymic stromal lymphopoietin (TSLP) by either basophils or CD4+ T cells (it is not certain which), which results in the production of interleukin 4 (IL-4). IL-4 promotes the production of immunoglobulin E (IgE), which ultimately contributes to the process of allergic inflammation. Therefore, TSLP plays an important role in the P. citri-induced TH2 immune response.

Protease-Activated Receptor 2 Is Involved in Th2 Responses against Trichinella spiralis Infection

In order to get a better understanding of the role of protease-activated receptor 2 (PAR2) in type 2 helper T (Th2) cell responses against Trichinella spiralis infection, we analyzed Th2 responses in T. spiralis-infected PAR2 knockout (KO) mice. The levels of the Th2 cell-secreted cytokines, IL-4, IL-5, and IL-13 were markedly reduced in the PAR2 KO mice as compared to the wild type mice following infection with T. spiralis. The serum levels of parasite-specific IgE increased significantly in the wild type mice as the result of T. spiralis infection, but this level was not significantly increased in PAR2 KO mice. The expression level of thymic stromal lymphopoietin, IL-25, and eotaxin gene (the genes were recently known as Th2 response initiators) of mouse intestinal epithelial cells were increased as the result of treatment with T. spiralis excretory-secretory proteins. However, the expression of these chemokine genes was inhibited by protease inhibitor treatments. In conclusion, PAR2 might involve in Th2 responses against T. spiralis infection.

Increased Expression and Role of Thymic Stromal Lymphopoietin in Nasal Polyposis

PURPOSE: Nasal polyposis is a chronic inflammatory disease of the upper airways often associated with asthma and characterized by markedly increased numbers of eosinophils, Th2 type lymphocytes, fibroblasts, goblet cells and mast cells. Previous studies have shown elevated levels of thymic stromal lymphopoietin (TSLP) in atopic diseases like asthma, atopic dermatitis and mainly in animal models of allergic rhinitis (AR). Here, we investigated the expression of TSLP in nasal polyps from atopics and non-atopics in comparison with the nasal mucosa and its potential role in nasal polyposis. METHODS: Messenger RNA expression for TSLP, thymus and activation-regulated chemokine (TARC) and macrophage derived chemokine (MDC) in nasal polyps and nasal mucosa of atopics and non-atopics was analyzed by real time PCR. Immunoreactivity for TSLP in nasal polyps and in the nasal mucosa of patients with AR and non-allergic rhinitis (NAR) was analyzed by immunohistochemistry. Eosinophil counts was analyzed by Wright-Giemsa staining and nasal polyp tissue IgE, by ELISA. RESULTS: Messenger RNA expression for TSLP,TARC and MDC was markedly higher in nasal polyps as compared to the allergic nasal mucosa. Immunoreactivity for TSLP was detected in epithelial cells, endothelial cells, fibroblasts and inflammatory cells of the nasal mucosa and nasal polyps. The number of TSLP+ cells was significantly greater in the nasal mucosa of AR than NAR patients. The number of TSLP+ cells in nasal polyps from atopics was significantly greater than that of non-atopics and that in the allergic nasal mucosa. The number of TSLP+ cells correlated well with the number of eosinophils and the levels of IgE in nasal polyps. CONCLUSIONS: The high expression of TSLP in nasal polyps and its strong correlation to eosinophils and IgE suggest a potential role for TSLP in the pathogenesis of nasal polyps by regulating the Th2 type and eosinophilic inflammation.

TSLP promotes lung Inflammation via activating dendritic cells in OVA-induced mice asthmatic model / 中华微生物学和免疫学杂志

Objective To study the expression of thymic stromal lymphopoietin(TSLP) and the activation of DCs in OVA-induced murine asthma model, and investigate the effects and underlying mecha-nisms of TSLP on lung inflammation. Methods Thirty BALB/c mice were randomly divided into control group, OVA group and TSLP neutralizing antibody treated group. The asthma model was evaluated by airway responsiveness and histological analysis of lung tissues ; The levels of TSLP mRNA in lungs were determined by quantitative real-time PCR; The expression of TSLP in lungs were determined by immunohistochemistry and Western blot; The expression of CD40, CD80, CD86 in BALF was detected by FACS. Results Both the histological analysis of lung tissues and the airway responsiveness were all consistent with the characteris-tic of murine asthma model. The expression of TSLP and TSLP mRNA in the OVA group was significantly in-creased compared with blank group. The expression of CD40, CD80, CD86 in BALF from OVA group was increased significantly compared with the control group. Furthermore, treating mice with TSLP neutralizing antibody reduced the expression of CD40, CD80, CD86 on dendritic cells, and IL-4, IL-5, IL-13 in the OVA group. Conclusion Our study indicate that TSLP was highly expressed in the bronchial epithelia of murine asthma model, via upregulation of CD40, CD80, CD86, induce DCs to active CD4~+ T cells and pro-duce type 2 responses, so that aggravating the lung inflammation of asthma. Blocking TSLP is capable of in-hibiting the production of Th2 cytokines, thus presents a promising strategy for the treatment of asthma.

The Expression of TSLP and Its Relationship with the Number of Infiltrating Regulatory T Cells in Lung Cancer / 中国肿瘤临床

Objective: To investigate the expression of TSLP in human lung cancer tissue and the correla-tion between TSLP expression and number of regulatory T cells (Tregs). Methods: The expression of TSLP mRNA and protein was detected in different pathological lesions of the lung by Q-RT-PCR and immunohisto-chemistry. Immunohistochemistry was used to detect Foxp3+ Tregs. The correlation of TSLP with the number of Tregs was analyzed. Results: TSLP gene was expressed in tumor tissues (n=37), latero-tumor tissues (n=29) and non-tumor lung tissues (n=24), without statistical difference (P=0.148). TSLP protein was expressed in the cytoplasm and was observeed in 69.57% of tumor tissues, 13.33% of benign lesions and 30.00% of non-tumor lung tissues, with a significant difference (P<0.05). The expression of TSLP protein was correlated with tumor size (P=0.000) and lymph node metastasis (P=0.018). The number of Tregs in TSLP positive group was more than that in TSLP negative group (P<0.05). Conclusion: The expression of TSLP in lung tu-mor tissues is increased and is correlated with the number of Tregs, indicating that TSLP could induce Treg to play an important role in tumor immunotolerance.

Thymic Stromal Lymphopoietin (TSLP) is secreted by villi of early human gestation / 中国免疫学杂志

Objective:To investigate the expression of a new cytokine,thymic stromal lymphopoietin (TSLP) and its receptor TSLPR in the villi of human first trimester gestation.Methods:Villi were collected from women who had undergone an artificial abortion at 7-11 weeks of normal gestation.The trophoblast cells (Tros) were isolated and cultured.The total RNA was extracted using TRIzol reagent,from both villi and the Percoll-gradient-isolated Tros,then the DNA fragments of hTSLP and hTSLPR were amplificated by RT-PCR.Villous tissues were detected for TSLP by immunohistochemistry (IHC) and Western blot.Immunocytochemistry (ICC) was carried out on cultured trophoblast cells for TSLP/TSLPR expression.Levels of TSLP in the supernatants were detected by ELISA.Results:Normal villi and the cultured Tros transcript were found to express TSLP/TSLPR mRNA and secreted TSLP protein.In addition,TSLP receptor was also expressed on trophoblasts.Conclusion:Both TSLP and its receptor are expressed on villi and trophoblast cells,which suggests that TSLP plays an important role in maternal-fetal immuno-tolerance in human early pregnancy.