GATA-3 mRNA Levels Research Articles

Role of Th2, Th17 and Treg Cells and relevant cytokines in pathogenesis of allergic rhinitis

ObjectiveTo explore the role of different cells and molecules in the pathogenesis of allergic rhinitis (AR) with positive Artemisia allergen by detecting their expression levels.MethodsFrom January 2021 to December 2022,200 AR patients diagnosed in the Otolaryngology Clinic of Ordos Central Hospital were selected as the AR group, and 50 healthy people who underwent physical examination in the hospital during the same period were randomly selected as the healthy control (HC) group. The levels of GATA-3mRNA, RORγtmRNA and FoxP3mRNA in peripheral blood mononuclear cells were detected by real-time fluorescence quantitative PCR (qRT-PCR). The proportions of Th2, Th17 and Treg cells were detected by flow cytometry. The concentrations of IL-4, IL-5, IL-17 and IL-10 in serum were detected by enzyme-linked immunosorbent assay. The differences of transcription gene level, immune cell ratio and cytokine concentration between the two groups were analyzed.ResultsThere was no difference in age and gender between the two groups. The levels of GATA-3mRNA and RORγtmRNA transcription genes in peripheral blood mononuclear cells, the percentage of Th2, Th17 and Treg immune cells, the levels of eosinophils and basophils in peripheral blood, the concentrations of IL-4, IL-5, IL-17, IL-10 cytokines and IgE in serum of AR patients were significantly higher than those in HC group (P < 0.05). IL-4 and IL-17 were positively correlated with total IgE level.ConclusionThe secretion of immune cells and cytokines in peripheral blood of AR patients is abnormal. Th2, Th17, Treg specific transcription factors and related cells and cytokines are involved in the occurrence and development of allergic rhinitis.

Apolipoprotein A-I inhibited group II innate lymphoid cell response mediated by microRNA-155 in allergic rhinitis

BackgroundGroup 2 innate lymphoid cells (ILC2s) have been found to take part in type 2 inflammation by secreting Th2 cytokines. Apolipoprotein A-I (Apo-AI), a major structural and functional protein of high-density lipoproteins, plays anti-inflammatory effects on neutrophils, monocytes, macrophages and eosinophils. However, its effects on ILC2 are not well characterized. ObjectiveIn this study, we aimed to investigate to the effect of Apo-AI on the proliferation and function of ILC2 as well as possible mechanism. MethodsThe protein expression of Apo-AI and the percentage of ILC2 in peripheral blood between 20 AR and 20 controls were detected using enzyme-linked immunosorbnent assay (ELISA) and flow cytometry. The effect of Apo-AI and miR-155 on ILC2 proliferation and function were detected by tritiated thymidine incorporation and ELISA. Anima models were adopted to verify the effect of Apo-AI in vivo. ResultsElevated expression of Apo-AI was observed in AR patients. The Apo-AI promotes ABCA1 expression by ILC2, which can be inhibited by anti-Apo-AI. The Apo-AI decreased ILC2 proliferation and the mRNA levels of GATA3 and RORα from ILC2. The miR-155 over-expression promoted the up-regulation of GATA-3 and type II cytokines from ILC2, while the addition of Apo-AI or miR-155 inhibitor inhibited the expression of GATA-3 and type II cytokines by ILC2 significantly. Apo-AI-/- mice showed as enhanced allergen-induced airway inflammation. The miR-155 inhibitor can reverse the enhanced allergen-induced airway inflammation in Apo-AI-/- mice, while miR-155 mimics can reverse the decreased allergen-induced airway inflammation in Apo-AI treated mice. ConclusionApo-AI suppressed the proliferation and function of ILC2 through miR-155 in AR. Our data provided new insights into the mechanism of allergen-induced airway inflammation.

Xiao-qing-long-tang ameliorates OVA-induced allergic rhinitis by inhibiting ILC2s through the IL-33/ST2 and JAK/STAT pathways

BackgroundAllergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa that is mediated by immunoglobulin E (IgE). Xiao-qing-long-tang (XQLT) is a traditional Chinese medicine compound that is widely used to treat respiratory diseases such as AR. However, the underlying mechanism of the effect of XQLT on AR remains unclear. PurposeTo elucidate the effect of XQLT on ovalbumin (OVA)-induced AR and the mechanisms of action. MethodsThe therapeutic efficacy of XQLT was evaluated in a well-established OVA-induced AR mouse model. Nasal symptoms were analyzed, type 2 cytokines and OVA-sIgE levels were measured, nasal mucosa tissues were collected for histological analysis, and the changes of Group 2 innate lymphoid cells (ILC2s) and the IL-33/ST2 and JAK/STAT signaling pathways in the nasal mucosa were observed. ResultsXQLT significantly alleviated the nasal symptoms and histological damage to the nasal mucosa in AR mice, and reduced the levels of type 2 cytokines and OVA-sIgE. In addition, after XQLT treatment, the numbers of ILC2s in the nasal mucosa of AR mice were reduced, and the mRNA levels of the transcription factors GATA3 and ROR-α were decreased. Moreover, IL-33/ST2 signaling pathway was inhibited. The costimulatory cytokine associated JAK/STAT signaling pathway was also inhibited in ILC2s. ConclusionOur study demonstrated that XQLT regulated ILC2s through the IL-33/ST2 and JAK/STAT pathways to ameliorate type 2 inflammation in OVA-induced AR. These findings suggest that XQLT might be used to treat AR.

GATA3 as a Blood-Based RNA Biomarker for Idiopathic Parkinson's Disease.

Finding novel biomarkers for Parkinson's disease (PD) is crucial for early disease diagnosis, severity assessment and identifying novel disease-modifying drug targets. Our study aimed at investigating the GATA3 mRNA levels in whole blood samples of idiopathic PD (iPD) patients with different disease severities as a biomarker for iPD. The present study is a cross-sectional, case-control study, with samples obtained from the Luxembourg Parkinson's cohort (LuxPARK). iPD (N = 319) patients, along with age-matched controls without PD (non-PD; N = 319) were included in this study. Blood GATA3 mRNA expression was measured using quantitative reverse transcription PCR (RT-qPCR) assays. The capacity of GATA3 expression levels to establish the diagnosis of iPD (primary end-point) and assess disease severity (secondary end-point) was determined. The blood levels of GATA3 were significantly lower in iPD patients, compared to non-PD controls (p ≤ 0.001). Logistic regression models showed a significant association of GATA3 expression with iPD diagnosis after adjustment for the confounders (p = 0.005). Moreover, the addition of GATA3 expression to a baseline clinical model improved its iPD diagnosis capacity (p = 0.005). There was a significant association of GATA3 expression levels with the overall disease severity (p = 0.002), non-motor experiences of daily living (nm-EDL; p = 0.003) and sleep disturbances (p = 0.01). Our results suggest that GATA3 expression measured in blood may serve as a novel biomarker and may help in the diagnosis of iPD and assessment of disease severity.

Regulation of Notch Signaling Pathway to Innate Lymphoid Cells in Patients with Acute Myocardial Infarction

ABSTRACT The Notch signaling pathway is an important regulator in fate decisions and immune responses of innate lymphoid cells (ILCs). However, the function of Notch signaling in ILCs in acute coronary syndrome is still not fully elucidated. Thirty-one unstable angina pectoris (UAP) patients, 21 acute myocardial infarction (AMI) patients, and 20 controls were included in this study. Peripheral blood mononuclear cells (PBMCs) were isolated. The mRNA expression levels of Notch receptors and ligands were measured by real-time PCR, while ILC subsets were measured by flow cytometry. Lin− cells were purified and stimulated with γ-secretase inhibitor (GSI). ILC subsets, transcription factors, and secreted cytokines were assessed. Notch receptor and ligand mRNA levels were elevated in PBMCs and peripheral lin− cells from AMI patients. There was no significant difference in total lin−CD45+CD161+CD127+ ILC frequency among three groups. The CRTH2−CD117− ILC1 subset was down-regulated, while the CRTH2+ ILC2 subset was up-regulated in AMI patients. The CRTH2−CD117+ ILC3 subpopulation was comparable among the three groups. ILC1% was negatively correlated with Notch1 and Notch2 in AMI patients. Inhibition of Notch signaling pathway by GSI induced elevations in ILC1 frequency, T-bet mRNA expression, and interferon-γ secretion and reduced ILC2 frequency, GATA3 mRNA levels, and interleukin-5/interleukin-13 production by lin− cells from AMI patients. The current data indicated that activation of Notch signaling pathway might contribute to ILC1-to-ILC2 shift in peripheral blood in AMI patients.

Arsenic Induces Blood‒Brain Barrier Disruption and Regulates T Lymphocyte Subpopulation Differentiation in the Cerebral Cortex and Hippocampus Associated with the Nrf2 Pathway In Vivo.

Increasing evidence has confirmed that the nervous system shows innate and adaptive immunity, which also participates in nerve damage. This study aimed to explore the neuroimmune imbalance induced by arsenic and its possible mechanism. Mice were exposed to NaAsO2 (0, 5, 10, 25, and 50 mg/L) for 1 month by drinking water. Y-maze and Morris water maze tests revealed that arsenic impaired learning and memory. The optical density of Evans blue showed a marked dose-dependent increase in the brain, and the mRNA and protein levels of the BBB tight junctions (TJs), occludin at 25 and 50 mg/L arsenic, and claudin-5 at 50 mg/L arsenic, were markedly decreased in the cerebral cortex. Arsenic downregulated occludin and claudin-5 mRNA expression at 50 mg/L and protein expression at 25 and 50 mg/L in the hippocampus. Immunohistochemical staining showed that 50 mg/L arsenic increased corticocerebral and hippocampal CD3+ T, CD4+ T, and CD8+ T cells; CD4 and CD8 proteins were increased with 25 and 50 mg/L arsenic. Arsenic decreased the corticocerebral and hippocampal Th1, Th17, and regulatory Treg transcription factors T-bet, Rorγt, and Foxp3 and the cytokine IFN-γ, IL-17, and TGF-β mRNA levels and increased the Th2 transcription factor GATA3 and cytokine IL-4 mRNA levels. Moreover, arsenic enhanced the expression of nuclear factor E2-related factor (Nrf2) and its downstream enzymes heme oxygenase-1 (HO-1) and glutathione-S-transferase (GST). In conclusion, these results demonstrate that arsenic exposure induces BBB dysfunction and T lymphocyte infiltration and affects CD4+ T lymphocyte differentiation, which may be associated with Nrf2 activation.

RhCNB Improves Cyclophosphamide-Induced Immunodeficiency in BALB/c Mice.

Background This study aims to explore the immunomodulatory effect of rhCNB on mice with cyclophosphamide (CTX)-induced immunodeficiency through TLR4/MAPK pathway. Methods BALB/c mice were randomly divided into three groups: a negative control group, an immunosuppression model group, and a rhCNB treatment group. Tail vein injection of cyclophosphamide (40 mg/kg) was used to establish a mouse immunosuppression model. Intraperitoneal injection of rhCNB (20 mg/kg) was administered to the treatment group, whereas equal quantities of normal saline were given to the control group and model group. Perform peripheral blood routine of CD4, CD8, and CD19 lymphocyte subsets and peripheral blood Th1/Th2 cell subsets 24 hours after the last administration. RT-PCR was used to detect mRNA levels of TLR4, P38, JNK, T-bet, and GATA3, the spleen immune organ index was measured, and the histopathological status of the spleen and thymus was observed. Results The results showed that compared with the control group, WBC, PLT, LYM, NEU, immune organ index, CD4+/CD8+ and CD19+ subgroup ratio, and peripheral blood Th1/Th2 cell subgroups decreased in the model group. The mRNA levels of TLR4, P38, JNK, T-bet, and GATA3 decreased compared with the model group, while they increased in the treatment group. Conclusions rhCNB has an immunomodulatory effect by regulating the expression of Th1/Th2 cytokine balance through the TLR4/MAPK signaling pathway and promoting the differentiation and proliferation of lymphocytes, thereby improving the immune function.

Compound 511 ameliorates MRSA-induced lung injury by attenuating morphine-induced immunosuppression in mice via PI3K/AKT/mTOR pathway

BackgroundOpioids are widely used in clinical practice. However, their long-term administration causes respiratory depression, addiction, tolerance, and severe immunosuppression. Traditional Chinese medicine (TCM) can alleviate opioid-induced adverse effects. Compound 511 is particularly developed for treating opioid addiction, based on Jiumi Liangfang, an ancient Chinese drug treatment and rehabilitation monograph completed in 1833 A.D. It is an herbal formula containing eight plants, each of them contributing to the overall pharmacological effect of the product: Panax ginseng C. A. Meyer (8.8%), Astragalus membranaceus (Fisch.) (18.2%), Datura metel Linn. (10.95%), Corydalis yanhusuo W. T. Wang (14.6%), Acanthopanar gracilistμlus W. W. Smith (10.95%), Ophiopogon japonicus (Linn. f.) Ker-Gawl. (10.95%), Gynostemma pentaphyllum (Thunb.) Makino (10.95%), Polygala arvensis Willd. (14.6%). This formula effectively ameliorates opioid-induced immunosuppression. However, the underlying mechanism remains unclear. PurposeTo reveal the effects of Compound 511 on the immune response of morphine-induced immunosuppressive mice and their potential underlying molecular mechanism. This study provides information for a better clinical approach and scientific use of opioids. MethodsImmunosuppression was induced in mice by repeated morphine administration. Th1/Th2/Th17/Treg cell levels were measured using flow cytometry. Splenic transcription factors of Th1/Th2/Th17/Treg and outputs of the regulatory PI3K/AKT/mTOR signaling pathway were determined. Subsequently, methicillin-resistant Staphylococcus aureus (MRSA) was administered intranasally to morphine-induced immunosuppressive mice pretreated with Compound 511. Their lung inflammatory status was assessed using micro-computer tomography (CT), hematoxylin and eosin (H&E) staining, and enzyme-linked immunosorbent assay (ELISA). ResultsCompared to morphine, Compound 511 significantly decreased the immune organ indexes of mice, corrected the Th1/Th2 and Treg/Th17 imbalance in the immune organs and peripheral blood, reduced the mRNA levels of FOXP3 and GATA3, and increased those of STAT3 and T-bet in the spleen. It improved immune function and reduced MRSA-induced lung inflammation. ConclusionCompound 511 ameliorates opioid-induced immunosuppression by regulating the balance of Th1/Th2 and Th17/Treg via PI3K/AKT/mTOR signaling pathway. Thus, it effectively reduces susceptibility of morphine-induced immunosuppressive mice to MRSA infection.

Effects of Lactiplantibacillus plantarum 19-2 on immunomodulatory function and gut microbiota in mice.

This study aims to evaluate the effects of Lactiplantibacillus plantarum 19-2 (L. plantarum 19-2) on mice treated with the alkylating agent cyclophosphamide (CTX). Our findings show that L. plantarum 19-2 restored the spleen and thymus index and the number of white blood cells and lymphocytes% in CTX treated mice. Serum immunoglobulin levels in CTX-treated mice were increased by L. plantarum 19-2. In addition, as compared to the model group, L. plantarum 19-2 upregulated the content of SIgA, while L. plantarum 19-2 regulates the mRNA and protein expression levels of GATA-3, T-bet, IFN-γ, and IL-4 in small intestinal tissues, which adjusted mucosal barriers, structural status, and the balance of Helper T-cell 1 and Helper T-cell 2. Lactiplantibacillus plantarum 19-2 regulated the distribution of intestinal flora in mice, promoting the growth of Bacteroides and Proteobacteria. In addition, L. plantarum 19-2 inhibited the growth of several harmful bacteria, including Actinobacteria and Firmicutes.

Osteopontin mediated eosinophils activation by group II innate lymphoid cells

BackgroundOsteopontin (OPN) can regulate Th2 inflammation in allergic rhinitis (AR). A recent study suggested that group II innate lymphoid cells (ILC2s) were very important for airway inflammation. But the role of OPN in ILC2s regulation is not explored. MethodsPurified ILC2s were stimulated by human recombinant OPN. The expression of GATA3 and RORα was assayed using real-time polymerase chain reaction (PCR) and enzyme linked immunosorbent assay. MiR-181a was transfected into eosinophils to test the OPN production. The protein concentrations of interleukin (IL)-5 and IL-13 were examined using ELISA. Purified eosinophils and ILC2s were cocultured and stimulated by OPN and the activation of eosinophils was detected by ELISA. ResultsAfter OPN stimulation, the ILC2s proliferation, the mRNA levels of GATA3 and RORα, the protein of GATA3, RORα, IL-5 and IL-13 expression were up-regulated significantly in a dose dependent manner. Eosinophils cultured alone transfected with miR-181a mimics produced less OPN protein compared with eosinophils transfected with miR-control, whereas OPN production was significantly promoted when miR-181a inhibitor was transfected. In the eosinophils and ILC2s coculture system, eosinophil cationic protein (ECP) production induced by OPN or IL-33 were significantly higher than ECP production in eosinophils culture system. OPN presented similar potency with IL-33 in the activation of eosinophils. When anti-IL-5 antibody was added, the production of ECP was significantly inhibited. ConclusionsOur data for the first time provided new evidence that OPN played important roles in innate immunity of AR by regulation of ILC2s and the interaction between ILC2s and eosinophils.

Involvement of T-bet and GATA3 transcription factors in Mesenchymal stem cells and royal jelly combination treatment in brain stroke

IntroductionMesenchymal stem cells (MSC) therapy is a promising therapeutic strategy to overcome the brain stroke side effects. However, it may be associated with long-term complications, including induction of inflammation. This project was designed to examine the effects of MSC administration and its combination with royal jelly (RJ) on the differentiation of T helper subsets. Material and methodsIn this project, the mice were divided to the six groups, including control (healthy without stroke), stroke (mice model of middle cerebral artery occlusion (MCAO)), treated with mouse MSC (mMSC), royal jelly (RJ), combination of mMSC and RJ (mMSC + RJ) and MSC conditioned medium (SUP). Thereafter, sticky test, brain mRNA levels of T-bet (transcription factor for Th1 subset), GATA3 (transcription factor for Th2 subset), and ROR-γ (transcription factor for Th17 subset) and percentage of myeloperoxidase (MPO) activities were explored in the groups. ResultsAdministration of mMSC and mMSC + RJ improved the sticky test times and decreased the MPO activities. Using mMSCs and RJ was associated with increased expression of T-bet and GATA3 transcription factors. Transplantation of mMSCs in combination with RJ reduced expression of T-bet in the infarcted tissue. ConclusionUsing mMSC may be associated with Th1-related inflammation in the long term. RJ co-administration significantly reduced the risks, hence, to decrease the plausible side effects of MSCs, it can be proposed to use RJ in combination with MSC to reduce stroke complications.

Livin promotes Th2-type immune response in airway allergic diseases

ObjectivesTo investigate the effects of livin on the Th2 immune response in airway allergic diseases (AAD) and explore the interaction among livin, GATA3, IL-4 in peripheral blood CD4+ T cells of AAD patients.MethodsWT mice and livin KO mice were developed for model of AAD. Th2 cell levels in the lung tissues and spleen were assessed by flow cytometry. Also, it was assessed in the culture after exposing to livin inhibitor (Lp-15); the protein and mRNA levels of livin, GATA3 and IL-4 in peripheral blood CD4+ T cells isolated from patients with or without AAD were measured by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Finally, Co-immunoprecipitation (Co-IP) was employed to identify the interaction between livin and GATA3.ResultsCompared with WT mouse, Th2 cell frequency in lung tissues and spleen was significantly decreased in livin KO mouse; after adding Lp-15, the differentiation from Naive CD4+T cells in spleen to Th2 cells was blocked; the protein and mRNA levels of livin, GATA3 and IL-4 in AAD group were higher than that in control group. The levels of livin were positively correlated with IL-4, and GATA3 was also positively correlated with IL-4 and livin. GATA3 was detected in the protein complex co-precipitated with livin antibody, and livin was also detected in the protein complex co-precipitated by GATA3 antibody.ConclusionLivin increases the expression of IL-4 and facilitates naive CD4+ T cells to differentiate into Th2 cells, which triggers airway allergy.

Alterations in T-Cell Transcription Factors and Cytokine Gene Expression in Late-Onset Alzheimer's Disease.

Late-onset Alzheimer's disease (LOAD) is associated with many environmental and genetic factors. The effect of systemic inflammation on the pathogenesis of neurodegenerative diseases such as AD has been strongly suggested. T helper cells (Th) are one of the important components of the immune system and can easily infiltrate the brain in pathological conditions. The development of each Th-subset depends on the production of unique cytokines and their main regulator. This study aimed to compare the mRNA levels of Th-related genes derived from peripheral blood mononuclear cells of LOAD patients with control. Also, the identification of the most important Th1/Th2 genes and downstream pathways that may be involved in the pathogenesis of AD was followed by computational approaches. This study involved 30 patients with LOAD and 30 non-demented controls. The relative expression of T-cell cytokines (IFN-γ, TNF-α, IL-4, and IL-5) and transcription factors (T-bet and GATA-3) were assessed using Real-time PCR. Additionally, protein-protein interaction (PPI) was investigated by gene network construction. A significant decrease at T-bet, IFN-γ, TNF-α, and GATA-3 mRNA levels was detected in the LOAD group, compared to the controls. However, there was no significant difference in IL-4 or IL-5 mRNA levels. Network analysis revealed a list of the highly connected protein (hubs) related to mitogen-activated protein kinase (MAPK) signaling and Th17 cell differentiation pathways. The findings point to a molecular dysregulation in Th-related genes, which can promising in the early diagnosis or targeted interventions of AD. Furthermore, the PPI analysis showed that upstream off-target stimulation may involve MAPK cascade activation and Th17 axis induction.

Oral administration of Lactobacillus plantarum CQPC11 attenuated the airway inflammation in an ovalbumin (OVA)-induced Balb/c mouse model of asthma.

This study investigated the antiasthmatic and anti-inflammatory effects of Lactobacillus plantarum-CQPC11 (LP-CQPC11) on ovalbumin (OVA)-induced asthmatic Balb/c mice. Administration of different doses of LP-CQPC11 (105 , 107 , and 109 colony-forming unit [CFU]/mouse) effectively reduced airway hyperresponsiveness (AHR) and the lung W/D ratio in asthmatic mice. LP-CQPC11 treatment reduced the accumulation of inflammatory cells in the BALF and attenuated histologic edema in asthmatic mice. Administration of LP-CQPC11 decreased the serum levels of OVA-specific IgE, IgE, and OVA-specific IgG1. LP-CQPC11 treatment decreased the levels of inflammatory cytokines (TNF-α, IL-4, IL-13, IL-5, and IL-6) in the BALF of asthmatic mice. In addition, LP-CQPC11 also elevated the mRNA levels of Foxp3 and T-bet and decreased the mRNA levels of Gata3 and RORγt in asthmatic mice lungs. Administration of LP-CQPC11 also reduced OVA-induced oxidative stress by improving the activities of GSH-Px, SOD, and catalase in the lungs. Finally, LP-CQPC11 treatment also significantly decreased the activation of the NF-κB pathway to modulate the inflammatory reaction in the lungs of asthmatic mice. The results from this study clearly demonstrated that oral administration of LP-CQPC11 exhibited outstanding activity in attenuating OVA-induced asthma in a mouse model. Furthermore, LP-CQPC11 may be an effective microecologic agent in preventing allergic asthma in the future. PRACTICAL APPLICATIONS: Allergic asthma is a common chronic inflammation-associated respiratory disease. Lactic acid bacteria (LAB) are known as a health product involved in modulating immune tolerance and play important roles in disease prevention and treatment. Many studies have reported that LAB, as probiotics, exhibits great antioxidation, anticancer, and anti-inflammatory activities and have health benefits in gastrointestinal disorders. In fact, human studies have confirmed that Lactobacillus rhamnosus strains have an effective activity to reduce the risk of allergic asthma. LP-CQPC11 was isolated from Sichuan pickled cabbages (a type of LAB-fermented vegetable product, also called Sichuan paocai) and was reported to reduce d-galactose-induced aging in mice in our previous study. However, the antiasthmatic and anti-inflammatory activities of LP-CQPC11 are unclear. The current study investigated the antiasthmatic and anti-inflammatory effects of LP-CQPC11 on OVA-induced asthmatic Balb/c mice.

CRISPR/Cas9-mediated demethylation of FOXP3-TSDR toward Treg-characteristic programming of Jurkat T cells

Demethylation of FOXP3-TSDR (Treg specific demethylated region) is a hallmark of stable differentiation and suppressive function of regulatory T (Treg) cells. Previous protocols aiming at human naïve T cell differentiation failed to implement a Treg cell specific epigenetic signature. Ten-eleven translocation (TET) enzymes catalyze DNA demethylation. Plasmids towardexpression of a fusion protein encompassing nonfunctional Cas9, the catalytic domain of TET1, blue fluorescent protein, and encoding single guide RNAs (sgRNAs) targeting specific segments of the FOXP3-TSDR were engineered and transfected into Jurkat T cells. FOXP3-TSDR methylation was analyzed by deep-amplicon bisulfite sequencing while cellular Foxp3, Tbet, Gata3, and Rorgt mRNA levels were determined by real-time PCR. Overexpression of dCas9TET1 significantly decreased Jurkat cell FOXP3-TSDR methylation and increased Foxp3 mRNA expression while expressions of master transcription factor mRNAs of other major T cell lineages remained largely unaffected. dCas9-TET1 construct transfection mediated Treg programming of patients’ primary T cells might be feasible.

ILC2s induce adaptive Th2-type immunity in different stages of tuberculosis through the Notch-GATA3 pathway

The study is to investigate the roles of group 2 innate lymphoid cells (ILC2s) in different courses of tuberculosis (TB). Serum and PBMCs were respectively isolated from the TST negative, LTBI (latent TB infection), ATB (active TB) and RTB (recurrent TB) patients. Flow cytometry was used to detect Th1, Th2 and ILC2s in the peripheral blood. The mRNA and protein levels of GATA3, RORα, CRTH2, Hes1, Notch1, NF-κB, and ID2 were detected by qRT-PCR and Western blotting. ILC2 cells from ATB and RTB patients were stimulated with rJagged2 or DAPT in vitro, and co-cultured with CD4+ T cells from TST negative group. ELISA was used to detect cytokine levels. The results showed that compared with the TST negative or LTBI group, Th2 cells and serum IL-4 in ATB group increased dramatically, accompanied by an increase of Th2/Th1 ratio in ATB patients, especially in RTB group. However, ILC2s in the ATB and RTB group increased significantly, along with increased GATA3, RORα, and CRTH2 levels. After rJagged2 stimulation in vitro, the levels of Hes1, Notch1, NF-κB, RORα, GATA3 and ID2 and those of IL-4, IL-5 and IL-13 were significantly increased. These effects were abrogated by DAPT treatment. Then, ILC2s, especially those from RTB patients, induced Th2-type immune response after co-culturing with CD4+ T cells. In conclusion, our results suggest that ILC2s may promote Th2-type immune response in different stages of TB via the Notch-GATA3 pathway.

Expression of GATA3 and bcl-11b in peripheral T-cell lymphoma and their clinical significance

Objective: To study the expression of GATA3 and bcl-11b in peripheral T-cell lymphoma (PTCL) and their correlation with clinicopathological features. Methods: The Oncomine and GEO databases were used for analyzing the expression levels of GATA3 and bcl-11b mRNA in PTCL. Immunohistochemistry was used to detect the expression of GATA3 and bcl-11b proteins in 127 cases of PTCL diagnosed at Shanxi Provincial Cancer Hospital from January 2010 to June 2020, as well as 40 cases of lymph node with reactive hyperplasia. Results: The data in Oncomine and GEO databases showed that the expression of GATA3 and bcl-11b mRNA in PTCL was lower than that in normal tissues (P<0.05). Immunohistochemistry showed that the positive rates of GATA3 in PTCL and lymph nodes with reactive hyperplasia were 60.6% (77/127) and 85.0% (34/40, P<0.05), respectively. The expression rates of bcl-11b in PTCL and lymph nodes with reactive hyperplasia were 55.1% (70/127) and 75.0% (30/40, P<0.05), respectively. The expression of GATA3 was related to the pathological classification of the patients with PTCL, and was inversely related to the Ann Arbor stage of the patient, while the expression of bcl-11b was inversely correlated with the IPI score of the patient (P<0.05). The expression of GATA3 and bcl-11b was related to the patients' age, gender, LDH level, and B symptoms. Other clinicopathological characteristics were irrelevant. Spearman correlation analysis shows that the expression of GATA3 protein was associated with that of bcl-11b protein in PTCL. Conclusion: GATA3 and bcl-11b are closely related to the prognosis of PTCL, and may be important factors involved in the occurrence and development of PTCL.

Jia-Wei-Yu-Ping-Feng-San Attenuates Group 2 Innate Lymphoid Cell-Mediated Airway Inflammation in Allergic Asthma.

The incidence of asthma has increased in recent decades. Although corticosteroids and bronchodilators are used in clinical practice, the control of asthma remains a challenge. Allergic asthma is characterized airway inflammation mediated by type 2 immune response. Group 2 innate lymphoid cells (ILC2s) are an important source of type 2 cytokines IL-5 and IL-13, which contribute to the progress of asthma. Jia-Wei-Yu-Ping-Feng-San (JWYPFS), a traditional Chinese medicine, has been widely used to treat asthma in China. In this study we investigated the mechanisms of JWYPFS in the treatment of asthma, especially the effect on ILC2s important in airway inflammation. Female C57BL/6 mice were sensitized and challenged with OVA to establish a model of allergic asthma. Airway hyperresponsiveness was examined by direct airway resistance analysis. Inflammatory cell counts were determined in bronchoalveolar lavage fluid (BALF). Inflammatory cell infiltration and mucus hypersecretion in lung tissue sections was observed by HE and PAS staining, respectively. The numbers and proportions of ILC2s as well as the ILC2s-related transcription factors GATA3, IRF4, and type 2 cytokines were measured in lung tissue samples. Additionally, ILC2s were collected from mouse lung; ILC2s-related cytokines and GATA3 and IRF4 were evaluated after IL-33-induced activation of ILC2s in vitro. Elevated inflammatory cells, mucus secretion, airway hyperresponsiveness and type 2 cytokines in the OVA-treated asthma group indicated that an allergic asthma model had been established. JWYPFS treatment attenuated airway resistance and reduced inflammatory cells including eosinophils, and inhibited mucus production and type 2 cytokines in these asthmatic mice. Moreover, JWYPFS treatment dramatically decreased the numbers and proportions of ILC2s and the mRNA levels of GATA3 and IRF4. In an in vitro experiment JWYPFS significantly suppressed GATA3, IRF4 and type 2 cytokine expression, including IL-5 and IL-13 in IL-33-stimulated ILC2s. JWYPFS alleviates ILC2s-mediated airway inflammation, suggesting that JWYPFS might be an effective agent to treat allergic asthma.

ILC2 Cells Promote Th2 Cell Differentiation in AECOPD Through Activated Notch-GATA3 Signaling Pathway.

This study is to investigate the capacity of type 2 innate lymphoid cells (ILC2s) in regulating the Th2 type adaptive immune response of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). The study enrolled healthy people, stable chronic obstructive pulmonary disease (COPD) patients, and AECOPD patients. Flow cytometry was used to detect Th2 and ILC2 cells in the peripheral blood. In addition, ILC2s from the peripheral blood of AECOPD patients were stimulated with PBS, IL-33, Jagged1, DAPT, IL-33+Jagged1, IL-33+DAPT, and IL-33+Jagged-1+DAP in vitro. The levels of cytokines in the culture supernatant were detected by ELISA and the culture supernatant was used to culture CD4 + T cells. The mRNA and protein levels of Notch1, hes1, GATA3, RORα, and NF-κB of ILC2s were detected by real-time PCR and Western blot. The proportion of Th2 and ILC2s was significantly increased in the peripheral blood of AECOPD patients, alone with the increased Notch1, hes1, and GATA3 mRNA levels. In vitro results showed that the mRNA and protein levels of Notch1, hes1, GATA3 and NF-κB were significantly increased after stimulation with Notch agonist, meanwhile, the level of type 2 cytokines were increased in the supernatant of cells stimulated with Notch agonist, and significantly promoted differentiation of Th2 cells in vitro. Disruption of Notch pathway weakened GATA3 expression and cytokine production, and ultimately affected the differentiation of Th2 cells. In conclusion, our results suggest that ILC2s can promote Th2 cell differentiation in AECOPD via activated Notch-GATA3 signal pathway.

HIV‐Associated Hypertension is TNFα‐Dependent and Involves Increased Vascular Nox1 Expression in Mice

HIV is a major global public health issue which has claimed more than 37 million lives so far. While combination antiretroviral therapies (cART) have significantly increased life expectancy, 35% of all treated HIV patients are diagnosed with hypertension. While cART treatment suppresses viral replication and further progression of the disease to AIDS, viral proteins are still present at low concentrations leading to the present clinical question of whether hypertension is the result of these viral proteins, cART regimen, or a combination of the two. We hypothesize that HIV-associated hypertension involves inflammatory response to viral proteins as well as increases in TNFα production and vascular NADPH Oxidase (Nox) 1 expression. To test this hypothesis, we utilized a transgenic mouse model (Tg26) which ubiquitously expresses seven of the nine viral proteins under the viral long terminal repeat promoter. Using quantitative RT-PCR analysis, high viral mRNA concentrations were found in lymphoid organs including the spleen, intraperitoneal immune cells, and lymph nodes but absent in the vasculature, therefore providing a mouse model mimicking clinical human pathology. Blood Pressure measurements using radio telemetry revealed that viral infection elevated mean arterial pressure in both male (WT=112.3+/-1.3 vs. Tg26=121.9+/-4.0) and female (WT=110.6+/-3.01 vs. Tg26=120.3+/-6.0mmHg) mice. Viral infection also increased systolic and diastolic blood pressure and heart rate with conserved diurnal variation. Measurement of aortic reactivity using wire myography revealed impaired endothelial relaxation (male: p<0.05/female p<0.05) to acetylcholine but intact vascular response to sodium Nitroprusside (SNP) and conserved vascular constriction to KCl and phenylephrine. Remarkably, transplant of bone marrow (BM) from Tg26 mice into WT impaired endothelial function (p<0.05) while transplant of BM from WT to Tg26 mice restored endothelial function. A multiplex cytokine panel found elevated levels of the pro-inflammatory cytokine TNFα in the plasma (p<0.05) and RT-qPCR analysis of the aorta (p<0.05) and intraperitoneal immune cells (p<0.05) also yielded increased TNFα levels. Treatment with the sTNFα inhibitor, etanercept, lowered MAP and restored endothelial function in male mice. Quantification of aortic Noxes (1,2, and 4) revealed increases in Nox1 expression (p<0.05). remarkably, selective inhibition of Nox1 via GKT771 restored endothelial dependent relaxation in both Tg26 and Tg26àWT transplanted mice. Analysis of multiple immune cell markers in the aorta via RT-qPCR found increased mRNA levels of the T-cell marker GATA3 (p<0.05). Taken together, these results support the hypothesis that HIV associated hypertension at levels of low viremia and independent of cART involves bone-derived immune cells, is TNFα dependent and involves increased vascular Nox1 expression.