Forkhead Box P3 mRNA Levels Research Articles

Lower levels of FOXP3 are associated with prolonged inflammatory responses in kidney transplant recipients.

Immunosuppressive treatment of kidney transplant recipients is mainly aimed at pro-inflammatory T effector cells, yet they also target the immunosuppressive T regulatory cells. Here, we test the hypothesis that low levels of the master gene regulator of T regulatory cells, forkhead box P3 (FOXP3) splice variants, are associated with prolonged inflammatory responses to stimuli. From blood samples obtained the first - and 29th day post-transplant, we extracted peripheral blood mononuclear cells and measured mRNA levels of Total FOXP3, pre-mature RNA FOXP3 (pre-mRNA FOXP3), full length FOXP3 (FOXP3fl) and, FOXP3 splice variant excluding exon two (FOXP3d2). We defined the primary outcome as the number of days in which C reactive protein (CRP) was above 50 mg/L. CRP levels were gathered in two periods, the first from the second to 29 days post-transplant, and the second from 30 to 57 days post-transplant. The association was tested using adjusted negative binomial regression. From 507 included kidney transplant recipients, 382 recipients had at least one CRP measurement >50 mg/L in the first period, median duration of elevated CRP was 4 days [interquartile range (IQR) 2 to 6]. In the second period, 69 recipients had at least one CRP measurement >50 mg/L, median duration of elevated CRP was 3 days [IQR 2 to 5]. In the first period, we found a significant association between lower levels of Total FOXP3 and prolonged duration of CRP elevation, incidence rate ratio 0.61 (95% confidence interval 0.46-0.80), p<0.01. Lower levels of total FOXP3 mRNA levels in peripheral blood of kidney transplant recipients are associated with prolonged duration of inflammatory responses regardless of the underlying stimuli.

MicroRNA-155 and 194 alter expression of Th17 and T regulatory-related transcription factors in the patients with severe coronavirus disease 2019 (COVID-19)

IntroductionIt has been demonstrated that the patients with severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) suffer from severe inflammation. Due to the ethnics, the immune responses may be different. Additionally, microRNAs may alter immune responses in the patients. The current study was aimed to evaluate the expression of T helper subsets-related transcription factors, some T helper 17 (Th17) products, and two microRNAs, including miR-155 and miR-194, in the Iranian hospitalized patients. MethodsIn this study, T-box expressed in T cells (T-bet), GATA binding protein 3, The retinoid orphan receptor gamma t (RORγt), forkhead box P3 (FOXP3), interleukin (IL)-17A, IL-8, and CC ligand 20 (CCL20) mRNA levels and, miR-155 and miR-194 levels were evaluated in 70 patients suffered from severe coronavirus disease 2019 (COVID-19) and 70 healthy subjects using Real-Time qPCR technique. ResultsThe findings showed that RORγt, and FOXP3 mRNA levels were significantly increased, while IL-17A, IL-8, and CCL20 mRNA levels were significantly decreased in the hospitalized SARS-CoV-2 infected patients. Although the levels of miR-155 and miR-194 were not different between groups, miR-194 has negative and positive correlations with RORγt and IL-17A in the Iranian healthy controls. ConclusionThis study reports although RORγt was up-regulated, IL-17A, IL-8, and CCL20 mRNA levels were significantly decreased in the hospitalized SARS-CoV-2 infected patients. It may be concluded that up-regulation of FOXP3, via development of T regulatory lymphocytes suppresses Th17 functions and neutralizes Th17 activities. MiR-194 may play crucial roles in regulation of RORγt and IL-17A expression in healthy people, the phenomenon that is disrupted in the severe SARS-CoV-2 infected patients.

Association of FOXP3 and GAGE10 promoter polymorphisms and decreased FOXP3 expression in regulatory T cells with susceptibility to generalized vitiligo in Gujarat population.

Alterations in regulatory T (Treg) cells have been observed in generalized vitiligo (GV) patients and decreased Forkhead Box P3 (FOXP3) has been implicated in the disease pathogenesis. The present study examined FOXP3 rs3761547(A>G), rs3761548(C>A), rs2232365(A>G) and GAGE10 rs11798415(A>T) promoter single nucleotide polymorphisms (SNPs) in 419 GV patients and 429 controls from Gujarat population using PCR-RFLP and ARMS-PCR. Real-time PCR and flow cytometry were used for assessment of FOXP3 mRNA and protein levels respectively in 96 GV patients and 90 controls. The frequency of genotypes (p<0.001) and alleles (p=0.012 & p=0.040) for rs3761547(A>G) and rs11798415(A>T) SNPs significantly differed between GV patients and controls. FOXP3 mRNA and protein levels were significantly decreased (p<0.001) in GV Tregs compared to controls. Active vitiligo (AV) Tregs showed significantly reduced FOXP3 mRNA and protein levels compared to that of stable vitiligo (SV) (p=0.02 & p=0.039). The correlation of genotype-phenotype of FOXP3 SNPs suggested reduced FOXP3 mRNA (p=0.019, p<0.001 & p<0.001) and protein (p=0.028, p<0.001 & p=0.022) levels in patients with susceptible GG, AA and GG genotypes respectively. The GAGT, GCGT & ACGT haplotypes were prevalent in GV patients (p=0.004, p=0.004 & p=0.016); however, GAGT & GCGT were overrepresented in patients with AV (p=0.044 & p=0.024). The susceptible GAGT and GCGT haplotypes in patients exhibited reduction in FOXP3 mRNA (p=0.014 & p=0.019) and protein (p=0.024 & p=0.028). DNA-protein docking analysis revealed reduced binding for transcription factor C/EBP to the susceptible allele 'G' (rs3761547) compared to A allele. For the first time, the study suggests significant association of FOXP3 rs3761547(A>G) & GAGE10 rs11798415(A>T) SNPs with susceptibility to GV in Gujarat population. In addition, the likely role of these SNPs in altered FOXP3 expression of Tregs may possibly affect Treg suppressive function in GV.

Epigenetical Targeting of the FOXP3 Gene by S-Adenosylmethionine Diminishes the Suppressive Capacity of Regulatory T Cells Ex Vivo and Alters the Expression Profiles.

Regulatory T cells (Treg cells), a subgroup of CD4 lymphocytes, play a crucial role in serving as an immune suppressor and in maintaining peripheral tolerance. As the accumulation of Treg cells in the tumor microenvironment is significantly associated with a decreased survival time of patients, they are considered as an important therapeutic target in the immunotherapy of human cancers. These cells are either derived from the thymus, which are called (CD4CD25CD127) natural Treg cells (nTreg cells), or they are generated from CD4CD25 naive T cells by transforming growth factor-beta 1 and interleukin 2 (IL-2) in the periphery, which are called induced Treg cells (iTreg cells). Although iTreg cells are unstable, nTreg cells stably express forkhead box P3 (FOXP3) protein. Moreover, nTreg cells can be classified as memory (CD45RA) and naive (CD45RA) Treg cells, and this classification is based on the expression of CD45RA. FOXP3, which is a master regulator transcription factor, is essential for the functions of Treg cells, and it is mainly controlled by epigenetic mechanisms. The cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway is also reported to contribute to the regulatory functions of tumor-infiltrating Treg cells. As a new approach, we investigated whether S-adenosylmethionine (SAM), a substrate of DNA methyltransferase, attenuates the immune-suppressive capacity of the naive subtype of nTreg cells (CD4CD25CD127CD45RA). Moreover, we examined the effects of PGE2/COX2 pathway blockers on the suppressive capacity of Treg cells. We found that SAM diminished the suppression competency of Treg cells by decreasing the FOXP3 mRNA and protein levels in a dose-dependent manner. SAM increased the DNA methylation of FOXP3 at the first intron site. In addition, SAM decreased the mRNA and protein levels of the IL-10 cytokine, which has suppressive roles in the immune system. Moreover, mRNA levels of interferon gamma (IFNG) were found to be increased. COX2 inhibition and blockage of PGE2 receptors also reduced the protein and mRNA levels of IL-10, but they did not exhibit any significant effect on Treg cells' suppression in the coculture system. Our results show that SAM might be considered and investigated as a promising agent for immunotherapy in the future.

Association of Notch pathway down-regulation with Triple Negative/Basal-like breast carcinomas and high tumor-infiltrating FOXP3 + Tregs

T regulatory cells (Tregs) are a lineage of lymphocytes involved in immune response suppression that are characterized by the expression of the forkhead box P3 (FOXP3) transcription factor. Notch pathway regulates FOXP3 transcription in Tregs, but its role in breast cancer is unknown. We aimed at studying whether Notch pathway regulates FOXP3 expression and Tregs content in breast cancer, and its association with luminal breast carcinomas. We analyzed by quantitative Real-Time PCR the mRNA levels of FOXP3, Notch pathway genes (Notch1, Notch2, Notch4 and Jagged1) and STAT3 in a series of 152 breast carcinomas including hormone receptor-positive and -negative phenotypes (luminal and Triple Negative/Basal-like). We also studied the protein expression of Notch1, STAT3 and FOXP3 by immunohistochemistry. High FOXP3 mRNA levels correlated with larger tumor size (p=0.010), histological grade 3 (p=0.008) and positive lymph-node status (p=0.031). Also, low levels of Notch pathway genes mRNA correlated with poor prognostic factors such as larger tumor size, positive lymph-node status, tumor phenotype and infiltrating tumor Tregs. A survival analysis for the patients showed that large tumor size, histological grade 3, vascular invasion, infiltrating Tregs and low Notch1 mRNA expression were significantly associated with a decreased patients' overall survival (p≤0.05). On a multivariate analysis, high Tregs content (HR=3.00, 95% CI 1.04–8.90, p=0.042) and low Notch1 mRNA levels (HR=3.33, 95% CI 1.02–10.86, p=0.046) were independent markers for overall survival. Our results support that the Notch pathway up-regulation promotes luminal breast carcinomas, whereas down-regulation correlates with the expression of FOXP3, favors tumor Tregs infiltration and associates with Triple Negative/Basal-like tumors.

FoxP3 T Cells and the Pathophysiologic Effects of Brain Death and Warm Ischemia in Donor Kidneys

Forkhead box P3 regulatory T cells control inflammatory responses, but it remains unclear whether they inhibit brain death-initiated inflammation and tissue injury in deceased kidney donors. DESIGN, SETTING, PARTICIPANTS, MEASUREMENT: To study the actions of regulatory T cells at various stages of the donation and transplantation procedure, forkhead box P3, regulatory and inflammatory cytokine expression, and tissue injury markers were determined in time 0 kidney biopsies from deceased and living donors. Additionally, the interaction between forkhead box P3+ T cells and kidney injury molecule-1 by activated primary tubular epithelial cells was studied. After cold storage, the deceased donor kidneys expressed the higher mRNA levels of kidney injury molecule-1 and CD3ε. In these samples, the inflammatory cytokines IL-8 and IFN-γ and markers associated with regulation (forkhead box P3, TGF-β, and IL-10) were highly expressed compared with living donor kidneys. Correlations were found between mRNA expression levels of forkhead box P3 and kidney injury molecule-1 and forkhead box P3 and IFN-γ. Immunohistochemical analysis confirmed the presence of forkhead box P3+ T cells in donor kidneys. Renal function (analyzed by serum creatinine levels) at the first week posttransplantation correlated with kidney injury molecule-1 and forkhead box P3 mRNA levels. In vitro studies showed that kidney injury molecule-1 expression by primary tubular epithelial cells was 63% (mean) lower when cocultured with regulatory T cells compared with control T cells. These results show that donor forkhead box P3+ T cells infiltrate the deceased donor kidney, where they may control inflammatory and injury responses.

Elevated Interferon Regulatory Factor 4 Levels in Patients with Allergic Asthma

Objective. Allergic asthma is a common inflammatory disease regulated by the T helper (Th) cells. Interferon regulatory factor 4 (IRF4) plays an important role in the differentiation of Th cells. This study investigated whether IRF4 is involved in the systemic immune responses in allergic asthma patients. Methods. Peripheral blood mononuclear cells (PBMCs) were isolated from allergic asthmatics (n = 29) and healthy controls (n = 12). The mRNA and protein levels of IRF4 in PBMCs were measured by quantitative RT-PCR and western blotting. The frequencies of Th1, Th2, and Th17 cells were analyzed by flow cytometry. The related cytokine (interferon-γ, interleukin-4 (IL-4), and IL-17) concentrations in plasma and culture supernatants were measured by ELISA. The levels of GATA binding protein 3 (GATA3), retinoic acid-related orphan receptor γt (RORγt), and forkhead box P3 (FOXP3), the master transcription factors controlling Th2, Th17, and T regulatory cells differentiation, respectively, were examined by quantitative RT-PCR. Results. The levels of IRF4 were elevated in allergic asthmatics compared with those in healthy controls. The frequencies of Th2 and Th17 cells as well as the concentrations of Th2- and Th17-related cytokines were higher in plasma from asthma patients than those from healthy controls; similar results were observed in culture supernatants. IRF4 mRNA levels were positively correlated with GATA3, RORγt, and FOXP3 mRNA levels in allergic asthmatics but not in healthy controls. Conclusions. Our results suggested that IRF4 may play a role in the systemic inflammation of allergic asthma patients by facilitating the differentiation of Th2 and Th17 cells at least at the transcriptional level.

Th17 Transcription Factor RORC2 Is Inversely Correlated with FOXP3 Expression in the Joints of Children with Juvenile Idiopathic Arthritis

To investigate the relationship between interleukin 17 (IL-17) producing T cells (Th17) and CD4+CD25+FOXP3+ regulatory T cells (Tregs) in blood and synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA). Sixty-five children with JIA (18 males and 47 females, median age 6.2 yrs; 45 with oligoarticular and 20 with polyarticular course) and 75 age- and sex-matched healthy controls were studied. Flow cytometry was used to analyze the forkhead box P3 (FOXP3)-positive Treg cells in peripheral blood (PB) and synovial fluid mononuclear cells (SFMC). FOXP3 and retinoic-acid related orphan receptor C isoform 2 (RORC2) messenger RNA (mRNA) were assessed by real-time polymerase chain reaction analysis. Cytokines (IL-17 and Th1/Th2 related cytokines) were measured in culture supernatants of 11 paired PBMC and SFMC activated with PMA and ionomycin. FOXP3+ T cells and FOXP3 mRNA amounts were significantly lower in PB of children with JIA as compared with controls (p = 0.0002 and p = 0.001, respectively) and a higher percentage of Treg cells with concomitant higher level of FOXP3 transcript levels were observed in SF when compared with their PB counterparts (both p < 0.0001). SF CD4+FOXP3+ T cells were characterized by higher amounts of FOXP3 protein per cell when compared with peripheral CD4+FOXP3+ T cells, as revealed by the difference in FOXP3 median fluorescence intensity (median +/- SD, arbitrary units, 54 +/- 22.6 vs 19.5 +/- 4.2; p < 0.001). RORC2 transcript levels were higher in JIA joints when compared with matched PB samples (median fold increase 3.9, p < 0.0001) but negatively correlated with FOXP3 mRNA levels (r = -0.623, p = 0.04). Stimulated SFMC displayed an impaired ability to produce IL-17 when compared with PBMC and, interestingly, an inverse relationship between IL-17 levels and the percentage of CD4+CD25+FOXP3+ SF T cells (r = -0.510, p = 0.047) was seen. We demonstrated for the first time an increased synovial expression of the transcription factor of Th17, RORC2, in JIA, and its inverse relationship with FOXP3 mRNA. These results extend research on "Th17" and Tregs in JIA.