Piperine Ameliorates Trimellitic Anhydride-Induced Atopic Dermatitis-Like Symptoms by Suppressing Th2-Mediated Immune Responses via Inhibition of STAT6 Phosphorylation

Atopic dermatitis (AD) is a common inflammatory skin disease predominately related to Type 2 helper T (Th2) immune responses. In this study, we investigated whether piperine is able to improve AD symptoms using a trimellitic anhydride (TMA)-induced AD-like mouse model. Topical treatment with piperine reduced ear swelling (ear thickness and epidermal thickness) induced by TMA exposure. Furthermore, piperine inhibited pro-inflammatory cytokines such as TNF-α and IL-1β in mouse ears, compared with the TMA-induced AD group. In measuring allergic immune responses in draining lymph nodes (dLNs), we found that IL-4 secretion, GATA3 mRNA level, and STAT6 phosphorylation were suppressed by piperine treatment. In an ex vivo study, piperine also inhibited the phosphorylation of STAT6 on the CD4+ T cells isolated from splenocytes of BALB/c mice, and piperine suppressed IL-4-induced CCL26 mRNA expression and STAT6 phosphorylation in human keratinocytes resulting in the inhibition of infiltration of CCR3+ cells into inflammatory lesions. These results demonstrate that piperine could ameliorate AD symptoms through suppression of Th2-mediated immune responses, including the STAT6/GATA3/IL-4 signaling pathway. Therefore, we suggest that piperine is an excellent candidate as an inhibitor of STAT6 and may help to improve AD symptoms.

Polymorphic Variant in GATA3 gene Is a Hallmark of PAR1-Deleted BCP-ALL and Associates with Poor Prognosis Among Pediatric Patients Treated with the BFM Backbone Protocols

Introduction Germline variant at rs3824662 in GATA3 gene was associated with early response to treatment as well as relapse risk in childhood BCP-ALL treated with COG protocols. This effect resulted from the strong link between the GATA3 polymorphism and the presence of somatic defects in leukemic cells including IKZF1 deletions, CRLF2 rearrangements and JAK2 gene mutations, which promote aggressive course of the disease. Aims The study aimed to evaluate an association between GATA3 gene polymorphism and clinical and biological features of pediatric BCP-ALL treated with the BFM backbone protocols. Methods Between November 2010 and June 2016, 957 consecutive children with newly diagnosed BCP-ALL were enrolled into the study. 822 patients treated according to ALL-IC BFM2009 protocol (n=594) and ALL-IC BFM2002 protocol (n=228) in 15 centers of the Polish Pediatric Leukemia/Lymphoma Study Group were enrolled into the study (median age 4.5 yrs, median follow-up time 2.5 yrs). Patients with BCR-ABL1 and MLL gene rearrangements were excluded from the analysis. The rs3824662 polymorphism of the GATA3 gene was genotyped using TaqMan probes. GATA3 mRNA expression level in leukemic cell was evaluated in BCP-ALL cases using qPCR with FAM-MGB probes (n=136). Targeted copy number screening of selected 23 loci was performed using the P335-B2 SALSA MLPA kit (MRC-Holland, Netherlands) in n=807 available DNA leukemia samples. MRD at day 15 and 33 was measured by flow cytometry with EuroFlow 8-color antibody panels. Results In the study group genotypes distribution withinrs3824662 of GATA3 was as follows: AA: n=44 (5.4%); CA: n=266 (32.4%); CC: n=512 (62.3%). Median MRD15 and MRD33 were 0.31% and 0.001% respectively. IKZF1 deletion were found in 18%, PAX5 in 20%, PAR1 in 12%, CDKN2A in 24%, CDKN2B 20%, BTG1 in 6.3%, ETV6 in 21%, EBF1 in 4% and RB1 in 6% of cases. Leukemic cells harbouring AA variant withinrs3824662 showed GATA3 mRNA expression 1.6 and 2.2 times higher compared to cells with CA and CC variants respectively, with the difference close to significant (ANOVA p=0.06). The presence of AA variant was not related to any gene deletion apart from microdeletions of the pseudoautosomal region PAR1 (Xp22 and Yp11) which occurred more frequently in AA carriers as compared to CA and CC carriers (11/41 vs. 34/242 vs. 52/468, p=0.013). We did not find any association of clinical features such as initial WBC, steroid response, sex and age at diagnosis among BCP-ALL patients with GATA3 genotype. However, AA carriers had a higher risk for MRD>10% at day 15 OR (95%CI)=3.93 (1.37-11.23) as well as MRD>0.01% at day 33, OR(95%CI)=2.95 (1.12-7.73) as compared CC carriers. Cox model of survival analysis was done for variables with univariate significance of p<0.15 (risk group, sex). The results of Cox regression showedthe AA genotype remained associated with an increased risk of death regardless of risk group (assigned based on ALL-IC BFM09 protocol), HR(95%C)=2.95(1.12-7.73), P=0.028). Conclusions We showed that carriers of AA genotype in GATA3 are prone to develop PAR1 -deleted BCP-ALL. Moreover our study confirmed an association of GATA3 AA rs3824662 homozygosity with poor early response to treatment as well as risk of death among pediatric BCP-ALL patients treated with the BFM backbone protocols. Disclosures Madzio: National Science Centre: Research Funding.

Activity of Corilagin on Post-Parasiticide Liver Fibrosis in Schistosomiasis Animal Model

This study investigates the effects and possible molecular mechanisms of corilagin extraction on prevention of Schistosoma japonicum ova-induced granulomas and liver fibrosis. As a result, under a light microscope, when compared to a model group, the corilagin group showed smaller granulomas, less liver cell denaturation and less inflammatory cell infiltration, and the connective tissues were significantly decreased. By Masson staining, the liver sections from the corilagin group showed less collagen distributed around granulomas, decreased liver fibrosis in the portal tracts and less formed interlobular tissue. The expression of hydroxyproline, IL-13 in liver and GATA3 in spleen in the model group was significantly higher than that in the normal group (P<0.05 or 0.01), while the level of hydroxyproline, IL-13 and GATA3 in the corilagin group were significantly lower than that in the model group (P<0.05). In conclusion, corilagin extraction can decrease the level of Th2-associated profibrotic cytokine IL-13, and down-regulate the transcription of GATA3 mRNA in spleen cells, which alleviate the hepatic fibrosis caused by egg granuloma in Schistosoma japonicum infection.

Early Time-Dependent Dynamic Changes of TBET and GATA3 mRNA Expressions in Patients with Acute Coronary Syndrome

Background. T-box expressed in T cells (TBET) and guanine adenine thymine adenine sequence-binding protein 3 (GATA3) play important roles in the differentiation of Th1 and Th2 subsets, which contributes to the progression of acute coronary syndrome (ACS).Objective. This study aimed to investigate the temporal change of TBET/GATA3 mRNA ratio in ACS.Methods. Thirty-three patients suspected of ACS with symptom onset within 24 hours were recruited. Blood samples were taken after arrival at the emergency department and at hourly intervals until the 6th hour. The mRNA expressions of TBET and GATA3 were quantified by a real-time RT-qPCR.Results. The TBET/GATA3 mRNA ratio was elevated dramatically in patients with acute myocardial infarction (AMI) and exhibited biphasic M-shaped release kinetics with two distinct peaks. The ratio was elevated 2 hours after symptom onset, dropped to the lowest level at 10 hours, and rose to the second peak at 14 hours. A similar biphasic M-shaped curve was observed in AMI patients with blood samples taken prior to any intervention.Conclusions. The TBET/GATA3 mRNA ratio was elevated in AMI patients throughout most of the first 20 hours after symptom onset. The biphasic M-shaped release kinetics was more likely to reflect pathophysiological changes rather than treatment effects.

GATA3 Silencing Defines a Distinct Subset of ETP-ALL.

Abstract 2389 Introduction The transcription factor GATA3 plays an important role in normal T cell development. Its role in mature T cells is well understood, but its function in earlier stages of T cell development remains unclear. Whereas GATA3 levels are precisely regulated for the T cell differentiation program, aberrant expression of GATA3 has been linked to tumorigenesis. Based on these observations, we investigated the role of GATA3 in Early Thymic Progenitor Acute Lymphoblastic Leukemia (ETP-ALL), a newly defined high-risk subgroup of T-ALL, characterized by a specific gene expression profile and distinct immunophenotype. Patients and Methods Eighty-six bone marrow samples from adult patients with newly diagnosed T-ALL, including ETP-ALL (n=17) enrolled into the German Multicenter Acute Lymphoblastic Leukemia (GMALL) trials, were studied for GATA3 expression by oligonucleotide expression arrays (HG-U133 plus 2.0) within the Microarray Innovations in LEukemia study. We identified additional 71 ETP-ALL adult patients and 94 T-ALL patients enrolled on the GMALL protocol, in which GATA3 mRNA expression was measured by quantitative polymerase chain reaction (RT-PCR). Combining ETP-ALL and T-ALL cases (n=165), we defined two GATA3 expression groups GATA3null and GATA3high based on a biological gap (GATA3 expression of 0.2). DNA methylation was analyzed in both T-ALL (n=11) and ETP-ALL (n=69) samples by pyrosequencing with primers designed to include seven CpG sites of Exon 2/Intron 3 of GATA3. Samples were grouped into GATA3 high vs. low methylation according to their mean methylation being below or above 40%. Results Based on gene expression arrays we observed a high proportion of ETP-ALL (11/17) that lacked GATA3 expression, whereas only a small fraction of the remaining T-ALL cases (3/69) had no GATA3 expression. These results were validated by RT-PCR in a larger cohort: 26% of ETP-ALL (19/71) were GATA3null, but only 2% of T-ALL (2/94) were in the GATA3null expression group. To explore the regulation of this specific expression pattern, epigenetic regulation of GATA3 was analyzed by pyrosequencing. While unselected T-ALL samples were hypomethylated (< 6% methylated CpG), ETP-ALL samples had a higher GATA3 methylation status (28% methylated CpG, p<0.001). ETP-ALL cases were further categorized into high methylated (18/69) and low methylated samples (51/69) and correlated to mRNA expression. GATA3null samples showed a higher degree of GATA3 methylation (41% methylated CpGs) compared to GATA3high samples (8% methylated CpGs, p < 0.001). In an in-vitro assay of T-cell leukemia cell lines demethylating agents increased GATA3 mRNA expression by up to 5-fold. In murine hematopoetic stem cells it was shown that loss of DNMT3A induced GATA3 expression via hypomethylation. In ETP-ALL, we identified 11 DNMT3A mutations in 69 samples (16%) and correlated the DNMT3A mutation status to GATA3 methylation. Ten of 11 (91%) DNMT3A mutated samples showed low level GATA3 methylation, whereas 17 (29%) of the 58 DNMT3A wildtype cases had high methylation. Conclusion ETP-ALL is a subgroup of adult T-ALL with a distinct molecular profile. Here we show that within ETP-ALL a separate molecular entity can be defined by GATA3 silencing due to DNA methylation. In-vitro studies showed that GATA3 expression can be restored by the use of demethylating agents. As loss of function mutations in DNMT3A correlate with low GATA3 methylation in ETP-ALL, a potential role of DNMT3A in the epigenetic silencing of GATA3 is suspected. So far, the number of targeted drugs available for T-ALL is limited. Therefore, incorporating demethylating agents may resolve the T-cell differentiation block in T-ALL by increasing GATA3 expression. Future work will explore downstream effects of GATA3 in acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function

Patients with Wiskott-Aldrich syndrome (WAS) have numerous immune cell deficiencies, but it remains unclear how abnormalities in individual cell types contribute to the pathologies of WAS. In T cells, the WAS protein (WASp) regulates actin polymerization and transcription, and plays a role in the dynamics of the immunologic synapse. To examine how these events influence CD4 function, we isolated the WASp deficiency to CD4+ T cells by adoptive transfer into wild-type mice to study T-cell priming and effector function. WAS−/− CD4+ T cells mediated protective T-helper 1 (Th1) responses to Leishmania major in vivo, but were unable to support Th2 immunity to Nippostrongylus brasiliensis or L major. Mechanistically, WASp was not required for Th2 programming but was required for Th2 effector function. WAS−/− CD4+ T cells up-regulated IL-4 and GATA3 mRNA and secreted IL-4 protein during Th2 differentiation. In contrast, cytokine transcription was uncoupled from protein production in WAS−/− Th2-primed effectors. WAS−/− Th2s failed to produce IL-4 protein on restimulation despite elevated IL-4/GATA3 mRNA. Moreover, dominant-negative WASp expression in WT effector T cells blocked IL-4 production, but had no effect on IFNγ. Thus WASp plays a selective, posttranscriptional role in Th2 effector function.