Lower expression of GATA3 and T-bet correlates with downregulated IL-10 in severe falciparum malaria.
ABSTRACT: Interleukin (IL)-10, a non-redundant anti-inflammatory cytokine is produced by different cells and its production involves activation of cell-specific transcriptional regulatory machinery in response to specific pathogen. We have previously demonstrated downregulated levels of IL-10 in severe falciparum malaria. The present study investigated transcriptional regulation of IL-10 in severe malaria. Comparative expression analysis of cell-specific signalling proteins and transcription factors for IL-10 production during the stage of active infection and with resolution of parasitaemia was performed. Interestingly, T-bet and GATA3, the Th1 and Th2 transcription factors, respectively, were downregulated in severe malaria with fold change values of 0.59 and 0.86. Increase in the levels of both the factors with resolution of parasitaemia implicated a role for parasite in depressed levels of these factors. Further support for probable parasite manipulation of GATA3 was obtained from negative correlation of GATA3 with parasitaemia. In addition, a role for interferon-? in suppressing IL-10 transcription was evident from its negative correlation with GATA3 and IL-10 levels. In summary, IL-10 transcription in Th1 and Th2 is defective and appears to have major contribution to low levels in severe malaria.
Project description:Th1-type immunity is considered to be required for efficient response to BCG in bladder cancer, although Th2 predisposition of BCG responders has recently been reported. The aim was to evaluate the relationship of Th1 and Th2 components in 23 patients undergoing BCG treatment. Peripheral blood, serum and urine samples were prospectively collected at baseline, during and after BCG. Th1 (neopterin, tryptophan, kynurenine, kynurenine-to-tryptophan ratio (KTR), IL-12, IFN-?, soluble TNF-R75 and IL-2R?) and Th2 (IL-4, IL-10) biomarkers as well as CD4 expression in T helper (Th), effector and regulatory T cells were determined. Local immune cell subsets were measured on formalin-fixed, paraffin-embedded cancer tissue by immunohistochemistry to examine expression of transcription factors that control Th1 (T-bet) and Th2-type (GATA3) immunity. We confirmed a Th2 predisposition with a mean GATA3/T-bet ratio of 5.51. BCG responders showed significantly higher levels of urinary (p = 0.003) and serum neopterin (p = 0.012), kynurenine (p = 0.015), KTR (p = 0.005), IFN-? (p = 0.005) and IL-12 (p = 0.003) during therapy, whereas levels of IL-10 decreased significantly (p < 0.001) compared to non-responders. GATA3/T-bet ratio correlated positively with serum neopterin (p = 0.008), IFN-? (p = 0.013) and KTR (p = 0.018) after the first BCG instillation. We observed a significant increase in CD4 expression in the Th cell population (p < 0.05), with only a modest tendency toward higher frequency in responders compared to non-responders (p = 0.303). The combined assessment of GATA3/T-bet ratio, neopterin and KTR may be a useful biomarker in predicting BCG response. Th2-promoting factors such as GATA3 may trigger Th1-type immune responses and thus contribute to the BCG success.
Project description:The mechanisms of activation and regulation of T lymphocytes and their cytokines in malaria caused by Plasmodium vivax are complex and poorly understood. Previous data suggest that T cells balance protective immune responses with immune mediated pathology in malaria. This study investigates the lymphocytic profile of patients infected with P. vivax by identifying and quantifying the specific sub-populations of Th1, Th2, Th17 and Treg cells and observing the correlation between parasitaemia and the number of platelets.A cross-sectional study was carried out in an endemic area of the state of Acre, Brazil. In order to obtain identification and quantification of lymphocyte sub-populations through flow cytometry, blood samples were collected from 50 individuals infected with P. vivax and 20 non-infected controls. To differentiate Th1 from Th2, the presence of cytokines IL-4 and TNF was examined by enzyme-linked immunosorbent assay. Utilizing the Mann-Whitney and Spearman coefficient tests, comparison and correlation analysis were rendered to test the parasitaemia and the number of platelets relationship.The data indicate that individuals infected with P. vivax present a significant reduction in Th1, Th2 and Th17 cell sub-populations when compared to the non-infected control group. A negative correlation exists between parasitaemia and platelet counts in individuals infected with P. vivax. There is no correlation of parasitaemia or thrombocytopaenia with any sub-population of T lymphocytes analysed. Interestingly, patients with serum Th1 cytokine profile present inversely proportional parasitaemia to the increase in the number of Th1, Th2, Th17 and Treg cells while patients with serum Th2 cytokine profile present directly proportional parasitaemia to the increase in number of Th1 and Th2 cells. Regarding the number of platelets, patients with serum Th1 cytokine profile show a correlation directly proportional to the Th17 sub-population. In contrast, platelet counts are directly proportional only to Treg and activated Treg cells in patients with serum Th2 cytokine profile.During the P. vivax infection patients with serum Th1 versus Th2 cytokine profile present different biological mechanisms for activating the immune system against parasite load.
Project description:The linked IL-4 and IL-13 cytokine genes, which are activated and silenced in T helper (Th) 2 and Th1 cells, respectively, are flanked by the equivalently expressed RAD50 and KIF3A genes. A scan of DNase I hypersensitivity and DNA methylation across approximately 100 kb of the KIF3A/IL-4/IL-13/RAD50 cluster revealed differences in chromatin structure between Th1 and Th2 cells at the 3' end of the RAD50 gene, a region previously shown to contain a locus control region (LCR) regulating Th2-specific expression of IL-4 and IL-13. Naive CD4 T cells did not exhibit any DNase I hypersensitivity in this region, but stimulation under either Th1 or Th2 conditions caused rapid development of three hypersensitive sites. An additional hypersensitive site developed rapidly only under Th2 conditions, through a mechanism dependent on signal transducers and activators of transcription 6 (STAT6) but not GATA3. Our data point to a physical separation in the actions of STAT6 and its downstream effector GATA3 during Th2 differentiation: STAT6 directly remodels the RAD50 LCR, whereas GATA3 acts only in the vicinity of the IL-4 gene. We suggest that the RAD50 LCR has a complex and dual role in Th1 and Th2 differentiation, communicating early T cell antigen receptor and cytokine signals to the IL-4/IL-13 locus in both differentiating cell types.
Project description:The transcription factor GATA3 is a master regulator that modulates T helper 2 (Th2) cell differentiation and induces expression of Th2 cytokines, such as IL-4, IL-5, and IL-13. Th2 cytokines are involved in the protective immune response against foreign pathogens, such as parasites. However, excessive production of Th2 cytokines results in type-2 allergic inflammation. Therefore, the application of a GATA3 inhibitor provides a new therapeutic strategy to regulate Th2 cytokine production. Here, we established a novel high-throughput screening system for an inhibitor of a DNA-binding protein, such as a transcription factor, and identified pyrrothiogatain as a novel inhibitor of GATA3 DNA-binding activity. Pyrrothiogatain inhibited the DNA-binding activity of GATA3 and other members of the GATA family. Pyrrothiogatain also inhibited the interaction between GATA3 and SOX4, suggesting that it interacts with the DNA-binding region of GATA3. Furthermore, pyrrothiogatain significantly suppressed Th2 cell differentiation, without impairing Th1 cell differentiation, and inhibited the expression and production of Th2 cytokines. Our results suggest that pyrrothiogatain regulates the differentiation and function of Th2 cells via inhibition of GATA3 DNA binding activity, which demonstrates the efficiency of our drug screening system for the development of novel small compounds that inhibit the DNA-binding activity of transcription factors.
Project description:The transcriptional repressor Bcl6 is a critical arbiter of Th cell fate, promoting the follicular Th lineage while repressing other Th cell lineages. Bcl6-deficient (Bcl6(-/-)) mice develop a spontaneous and severe Th2-type inflammatory disease, thus warranting assessment of Bcl6 in regulatory T cell (Treg) function. Bcl6(-/-) Tregs were competent at suppressing T cell proliferation in vitro and Th1-type colitogenic T cell responses in vivo. In contrast, Bcl6(-/-) Tregs strongly exacerbated lung inflammation in a model of allergic airway disease and promoted higher Th2 responses, including systemic upregulation of microRNA-21. Further, Bcl6(-/-) Tregs were selectively impaired at controlling Th2 responses, but not Th1 and Th17 responses, in mixed chimeras of Bcl6(-/-) bone marrow with Foxp3(-/-) bone marrow. Bcl6(-/-) Tregs displayed increased levels of the Th2 transcription factor Gata3 and other Th2 and Treg genes. Bcl6 potently repressed Gata3 transcriptional transactivation, providing a mechanism for the increased expression of Th2 genes by Bcl6(-/-) Tregs. Gata3 has a critical role in regulating Foxp3 expression and functional fitness of Tregs; however, the signal that regulates Gata3 and restricts its transactivation of Th2 cytokines in Tregs has remained unexplored. Our results identify Bcl6 as an essential transcription factor regulating Gata3 activity in Tregs. Thus, Bcl6 represents a crucial regulatory layer in the Treg functional program that is required for specific suppression of Gata3 and Th2 effector responses by Tregs.
Project description:Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4(+) T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN-?/IL-4 double-positive cells. These IFN-?(+) IL-4(+) cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN-?(+) IL-4(-) (Th1) and IFN-?(-) IL-4(+) (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN-?(+) IL-4(+) cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co-existence of Th1 and Th2 characteristics.
Project description:The Src family kinase Lck is thought to facilitate Th2 differentiation; however, its role in Th1 cells has not been well explored. Using mice that lack Lck in mature T cells, we find that lck(-/-) Th1 skewed cells have normal expression of T-bet and produce IFN-? at WT levels. However, there is a 3-fold increase in IL-10 producing cells in the mutant cultures. These cells do not have elevated levels of IL-4, GATA3, IL-17 or Foxp3, indicating that they are not Th2, Th17, or Foxp3(+) T regulatory cells (Treg). Nor do these cells behave in a similar manner as the type 1 Treg. Most of the IL-10 in the lck(-/-) Th1 cultures is derived from the memory/activated subset, as the cytokine profile from Th1 cultures established from purified CD62L(+) (naïve) cells are similar to WT cells. Furthermore, this IL-10 expression appears to be dependent on IL-12 and correlates with elevated c-Maf. These data highlight a previously unappreciated role for Lck in regulating IL-10 in Th1 cells.
Project description:Malaria-enteroparasitic co-infections are known for their endemicity. Although they are prevalent, little is known about their epidemiology and effect on the immune response. This study evaluated the effect of enteroparasite co-infections with malaria caused by Plasmodium vivax in a border area between Brazil and French Guiana. The cross sectional study took place in Oiapoque, a municipality of Amapá, on the Amazon border. Malaria was diagnosed using thick blood smears, haemoglobin dosage by an automated method and coproparasitology by the Hoffman and Faust methods. The anti-PvMSP-119 IgG antibodies in the plasma were evaluated using ELISA and Th1 (IFN-?, TNF-? and IL-2), and Th2 (IL-4, IL-5 and IL-10) cytokine counts were performed by flow cytometry. The participants were grouped into those that were monoinfected with vivax malaria (M), vivax malaria-enteroparasite co-infected (CI), monoinfected with enteroparasite (E) and endemic controls (EC), who were negative for both diseases. 441 individuals were included and grouped according to their infection status: [M 6.9% (30/441)], [Cl 26.5% (117/441)], [E 32.4% (143/441)] and [EC 34.2% (151/441)]. Males prevailed among the (M) 77% (23/30) and (CI) 60% (70/117) groups. There was a difference in haemoglobin levels among the different groups under study for [EC-E], [EC-Cl], [E-M] and [Cl-M], with (p < 0.01). Anaemia was expressed as a percentage between individuals [CI-EC (p < 0.05)]. In terms of parasitaemia, there were differences for the groups [CI-M (p < 0.05)]. Anti-PvMSP-119 antibodies were detected in 51.2% (226/441) of the population. The level of cytokines evaluation revealed a large variation in TNF-? and IL-10 concentrations in the co-infected group. In this study we did not observe any influence of coinfection on the acquisition of IgG antibodies against PvMSP119, as well as on the profile of the cytokines that characterize the Th1 and Th2 patterns. However, co-infection increased TNF-? and IL-10 levels.
Project description:Picroside II isolated from Pseudolysimachion rotundum var. subintegrum has been used as traditional medicine to treat inflammatory diseases. In this study, we assessed whether picroside II has inhibitory effects on airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. In the HDM-induced asthmatic model, picroside II significantly reduced inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), the levels of total immunoglobulin (Ig) E and HDM-specific IgE and IgG1 in serum, airway inflammation, and mucus hypersecretion in the lung tissues. ELISA analysis showed that picroside II down-regulated the levels of Th2-related cytokines (including IL-4, IL-5, and IL-13) and asthma-related mediators, but it up-regulated Th1-related cytokine, IFN? in BALF. Picroside II also inhibited the expression of Th2 type cytokine genes and the transcription factor GATA3 in the lung tissues of HDM-induced mice. Finally, we demonstrated that picroside II significantly decreased the expression of GATA3 and Th2 cytokines in developing Th2 cells, consistent with in vivo results. Taken together, these results indicate that picroside II has protective effects on allergic asthma by reducing GATA3 expression and Th2 cytokine bias.
Project description:The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.