Upregulation Of Foxp3 Expression Research Articles

Epigenetic modification of CD4+ T cells into Tregs by 5-azacytidine as cellular therapeutic for atherosclerosis treatment

Recent research has explored the potential of the demethylating drug 5-azacytidine (Aza) as therapy for a range of diseases. However, the therapeutic efficacy of Aza for patients of atherosclerosis remains unclear. This study investigates the therapeutic application of Aza to atherosclerosis in order to elucidate the underlying mechanisms. We generated induced Tregs (iTregs) from CD4+ T cells by using Aza in vitro, and this was followed by the intravenous infusion of iTregs for the treatment of atherosclerosis. The adoptive transfer of Aza-iTreg significantly increased peripheral blood Treg cells, suppressed inflammation, and attenuated atherosclerosis in ApoE−/− mice. Furthermore, we observed a notable demethylation of the Forkhead box P3 (Foxp3)-regulatory T cell-specific demethylated region (TSDR) and an upregulation of Foxp3 expression in the CD4+ T cells in the spleen of the ApoE−/− mice following the transfer of Aza- iTregs. We also demonstrated that Aza converted naive CD4+ T cells into Tregs by DNA methyltransferase 1 (Dnmt1)-mediated Foxp3-TSDR demethylation and the upregulation of Foxp3 expression. Conversely, the overexpression of Dnmt1 in the CD4+ T cells attenuated the Aza-induced Foxp3-TSDR demethylation and upregulation of Foxp3 expression. Our results reveal that Aza converts naive CD4+ T cells into functional Tregs by inhibiting Dnmt1, and the transfer of Aza-iTregs suppresses atherosclerosis in mice.

Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway

BackgroundToxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii.MethodsQuantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining.To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells.ResultsIn T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site.ConclusionsT. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.Graphical

Beneficial effects of Hawthorn extract on metabolic hypertensive rats by regulating Treg/Th17 immune balance

There is an increasing global trend toward unhealthy lifestyles and dietary decisions, like the overconsumption of alcohol and high-sugar and high-fat diets (ACHSFDs). Poor eating habits can cause an imbalance of the Treg/Th17 immune axis, thereby leading to an imbalance of anti-inflammatory and pro-inflammatory factors, which damages target organs and the vascular endothelium causing MH. Hawthorn has been shown to improve gastrointestinal function, lower blood pressure, and regulate metabolic abnormalities. However, whether it acts on MH by regulating the Treg/Th17 immune axis remains unclear. In this study, the hawthorn extract reduced blood pressure[systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure (MBP)] and improved dyslipidemia in rats with ACHSFD-induced MH. In addition, the extract reduced serum AST and ALT levels and liver coefficient and improved liver fat deposition and fibrosis. On the other hand, the hawthorn extract downregulated RORγt expression, upregulated Foxp3 expression, increased IL-10 levels in the aorta, and reduced IL-17, IL-6, and TNF-α content. Moreover, the extract upregulated eNOS expression in the aorta and increased NO content in the serum. These findings indicate that hawthorn extract can inhibit aortic inflammatory injury and improve vascular endothelial function by regulating the Treg/Th17 immune imbalance.

Targeting FOXP3 Tumor-Intrinsic Effects Using Adenoviral Vectors in Experimental Breast Cancer.

The regulatory T cell master transcription factor, Forkhead box P3 (Foxp3), has been detected in cancer cells; however, its role in breast tumor pathogenesis remains controversial. Here we assessed Foxp3 tumor intrinsic effects in experimental breast cancer using a Foxp3 binder peptide (P60) that impairs Foxp3 nuclear translocation. Cisplatin upregulated Foxp3 expression in HER2+ and triple-negative breast cancer (TNBC) cells. Foxp3 inhibition with P60 enhanced chemosensitivity and reduced cell survival and migration in human and murine breast tumor cells. We also developed an adenoviral vector encoding P60 (Ad.P60) that efficiently transduced breast tumor cells, reduced cell viability and migration, and improved the cytotoxic response to cisplatin. Conditioned medium from transduced breast tumor cells contained lower levels of IL-10 and improved the activation of splenic lymphocytes. Intratumoral administration of Ad.P60 in breast-tumor-bearing mice significantly reduced tumor infiltration of Tregs, delayed tumor growth, and inhibited the development of spontaneous lung metastases. Our results suggest that Foxp3 exerts protumoral intrinsic effects in breast cancer cells and that gene-therapy-mediated blockade of Foxp3 could constitute a therapeutic strategy to improve the response of these tumors to standard treatment.

Bacillus Calmette Guerin Injection Improves Hindlimb Motor Function and Alleviates Inflammation through Upregulating Foxp3 Expression in Rats with Spinal Cord Injury

Background: Immune and inflammatory reactions are important factors leading to secondary spinal cord injury. Aim: This study aimed to investigate the effect of BCG on behavior after acute spinal cord injury in rats and its related mechanisms. Methods: Rats with spinal cord injury were generated according to modified Allen’s method. Rats were divided into the BCG group (rats with spinal cord injury intraperitoneally injecting with BCG), Model group (rats with spinal cord injury intraperitoneally injecting with normal saline), and Sham group (normal rats intraperitoneally injecting with normal saline). Basso, Beattie, and Bresnahan (BBB) scoring system was used for evaluating behavioral outcomes. Foxp3 expression in spinal cord tissues was evaluated using immunofluorescence analysis. HE staining was used for assessing the injury of spinal cord tissues. Results: BBB scores of the Model group were remarkably lower compared to that of the Sham group (P<0.05). Foxp3 expression was significantly downregulated in the Model group compared with that of the Sham group (P<0.05). BBB scores of the BCG group were significantly higher compared to that of the Model group at 3, 5, 7, and 14 days post-treatment (P<0.05). In the BCG group, there were fewer inflammatory cells, smaller cavities, and a complete structural arrangement than in the Model group. BCG treatment significantly reduced the spinal cord injury score compared to that of the Model group (P<0.05). Post BCG treatment, Foxp3 expression was predominantly enhanced compared with that of the Model group (P<0.05). Conclusion: BCG could affect inflammation after spinal cord injury through upregulating the expression of Foxp3 and further promote the recovery of hindlimb motor function in rats with spinal cord injury.

CTLA4 mRNA is downregulated by miR-155 in regulatory T cells, and reduced blood CTLA4 levels are associated with poor prognosis in metastatic melanoma patients.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint expressed in regulatory T (Treg) cells and activated T lymphocytes. Despite its potential as a treatment strategy for melanoma, CTLA-4 inhibition has limited efficacy. Using data from The Cancer Genome Atlas (TCGA) melanoma database and another dataset, we found that decreased CTLA4 mRNA was associated with a poorer prognosis in metastatic melanoma. To investigate further, we measured blood CTLA4 mRNA in 273 whole-blood samples from an Australian cohort and found that it was lower in metastatic melanoma than in healthy controls and associated with worse patient survival. We confirmed these findings using Cox proportional hazards model analysis and another cohort from the US. Fractionated blood analysis revealed that Treg cells were responsible for the downregulated CTLA4 in metastatic melanoma patients, which was confirmed by further analysis of published data showing downregulated CTLA-4 surface protein expression in Treg cells of metastatic melanoma compared to healthy donors. Mechanistically, we found that secretomes from human metastatic melanoma cells downregulate CTLA4 mRNA at the post-transcriptional level through miR-155 while upregulating FOXP3 expression in human Treg cells. Functionally, we demonstrated that CTLA4 expression inhibits the proliferation and suppressive function of human Treg cells. Finally, miR-155 was found to be upregulated in Treg cells from metastatic melanoma patients compared to healthy donors. Our study provides new insights into the underlying mechanisms of reduced CTLA4 expression observed in melanoma patients, demonstrating that post-transcriptional silencing of CTLA4 by miRNA-155 in Treg cells may play a critical role. Since CTLA-4 expression is downregulated in non-responder melanoma patients to anti-PD-1 immunotherapy, targeting miRNA-155 or other factors involved in regulating CTLA4 expression in Treg cells without affecting T cells could be a potential strategy to improve the efficacy of immunotherapy in melanoma. Further research is needed to understand the molecular mechanisms regulating CTLA4 expression in Treg cells and identify potential therapeutic targets for enhancing immune-based therapies.

Abstract 305: Dietary β-carotene Accelerates Atherosclerotic Resolution By Promoting Treg Expansion In The Atherosclerosis Lesion

Forkhead box P3 ( FoxP3 ) + regulatory T cells (Tregs) mediate atherosclerosis resolution and regression by promoting an anti-inflammatory environment in the lesion. FoxP3 is upregulated by retinoic acid, the active form of vitamin A, but whether vitamin A status influences atherosclerosis resolution remains unexplored. We hypothesized that β-carotene, the main vitamin A precursor in human diet, accelerates atherosclerosis resolution by upregulating FoxP3 expression to promote Treg expansion. We induced atherosclerosis in FoxP3 EGFP mice fed Western diet control (WD-Control) and injected with an anti-sense oligonucleotide targeting the LDLR (ASO-LDLR) for 16 weeks. After this period, we harvested a subset of mice as Baseline, while the remaining mice underwent atherosclerosis resolution upon the interruption of ASO-LDLR infusions to recover LDLR expression and normalize plasma lipids for a period of three weeks. During this time, mice either continued on the same WD-Control or were switched to a Western diet supplemented with 50 mg of β-carotene/kg of diet (WD- β-carotene). To examine the implication of Tregs, we injected a subset of WD-β-carotene with anti-CD25 (WD-β-carotene + anti-CD25), while WD-Control and a second subset of WD- β-carotene mice were injected with IgG control. We estimated atherosclerosis resolution by quantifying macrophage and collagen contents in the lesions present at the level of the aortic root. WD-β-carotene-fed mice injected with IgG showed a reduction in macrophage content accompanied by an increase in collagen in comparison to all the remaining groups, while WD-β-carotene + anti-CD25 lesions resembled those present in WD-Control mice. We examined Treg the relative number of CD25 + FoxP3 + and CD25 - FoxP3 + cells in the spleen, blood, and the lesion. CD25 + FoxP3 + numbers decreased in response to anti-CD25 infusions in all the tissues examined. Dietary β-carotene favored an increase of 269% and 653% in CD25 - FoxP3 + Tregs in comparison to the Baseline of WD-Control groups, respectively. These effects occurred independently of changes in plasma lipids or changes in body weight. Our data show that β-carotene supplementation results in Treg expansion and acceleration of atherosclerosis resolution by increasing Treg number.

The immunoregulatory effect of the TREM2-agonist Sulfavant A in human allogeneic mixed lymphocyte reaction.

Sulfavant A (SULF A) is a synthetic derivative of naturally occurring sulfolipids. The molecule triggers TREM2-related maturation of dendritic cells (DCs) and has shown promising adjuvant activity in a cancer vaccine model. the immunomodulatory activity of SULF A is tested in an allogeneic mixed lymphocyte reaction (MLR) assay based on monocyte-derived dendritic cells and naïve T lymphocytes from human donors. Flow cytometry multiparametric analyses and ELISA assays were performed to characterize the immune populations, T cell proliferation, and to quantify key cytokines. Supplementation of 10 µg/mL SULF A to the co-cultures induced DCs to expose the costimulatory molecules ICOSL and OX40L and to reduce release of the pro-inflammatory cytokine IL-12. After 7 days of SULF A treatment, T lymphocytes proliferated more and showed increased IL-4 synthesis along with downregulation of Th1 signals such as IFNγ, T-bet and CXCR3. Consistent with these findings, naïve T cells polarized toward a regulatory phenotype with up-regulation of FOXP3 expression and IL-10 synthesis. Flow cytometry analysis also supported the priming of a CD127-/CD4+/CD25+ subpopulation positive for ICOS, the inhibitory molecule CTLA-4, and the activation marker CD69. These results prove that SULF A can modulate DC-T cell synapse and stimulate lymphocyte proliferation and activation. In the hyperresponsive and uncontrolled context of the allogeneic MLR, the effect is associated to differentiation of regulatory T cell subsets and dampening of inflammatory signals.

An Engineered Probiotic Produces a Type III Interferon IFNL1 and Reduces Inflammations in in vitro Inflammatory Bowel Disease Models.

The etiology of inflammatory bowel diseases (IBDs) frequently results in the uncontrolled inflammation of intestinal epithelial linings and the local environment. Here, we hypothesized that interferon-driven immunomodulation could promote anti-inflammatory effects. To test this hypothesis, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce and secrete a type III interferon, interferon lambda 1 (IFNL1), in response to nitric oxide (NO), a well-known colorectal inflammation marker. We then validated the anti-inflammatory effects of the engineered EcN strains in two in vitro models: a Caco-2/Jurkat T cell coculture model and a scaffold-based 3D coculture IBD model that comprises intestinal epithelial cells, myofibroblasts, and T cells. The IFNL1-expressing EcN strains upregulated Foxp3 expression in T cells and thereafter reduced the production of pro-inflammatory cytokines such as IL-13 and -33, significantly ameliorating inflammation. The engineered strains also rescued the integrity of the inflamed epithelial cell monolayer, protecting epithelial barrier integrity even under inflammation. In the 3D coculture model, IFNL1-expressing EcN treatment enhanced the population of regulatory T cells and increased anti-inflammatory cytokine IL-10. Taken together, our study showed the anti-inflammatory effects of IFNL1-expressing probiotics in two in vitro IBD models, demonstrating their potential as live biotherapeutics for IBD immunotherapy.

Forkhead box M1 recruits FoxP3+ Treg cells to induce immune escape in hilar cholangiocarcinoma.

ObjectiveHilar cholangiocarcinoma (HCCA) is a malignancy related to chronic biliary tract inflammation. Tumor immune escape is a necessary process of tumorigenesis. Forkhead box M1 (FoxM1) could affect the progression of various carcinomas. This study attempted to elaborate on the mechanism of FoxM1 in HCCA immune escape.MethodsHCCA cell lines were collected to measure the expression of FoxM1 and FoxP3. CD8+ T cells were extracted to establish the co‐culture system with HCCA cells and Treg cells. pcDNA3.1‐FoxM1 or si‐FoxP3 was transfected into HCCA cells in the co‐culture system. HCCA cell viability, mobility, and invasiveness as well as levels of transforming growth factor (TGF)‐β and interleukin (IL)‐6 were evaluated. The binding relation between FoxM1 and FoxP3 promoter was verified. HCCA cells with pcDNA3.1‐FoxM1 were subcutaneously injected into mice to establish the xenograft mouse models.ResultsFoxM1 and FoxP3 were overexpressed in HCCA cells. The co‐culture of CD8+ T and HCCA cells inhibited HCCA cell activity and Treg cells limited CD8+ T killing. FoxM1 overexpression strengthened the inhibiting role of Treg cells in CD8+ T killing, upregulated TGF‐β and IL‐6 levels, and encouraged HCCA immune escape. FoxM1 bound to the FoxP3 promoter region to promote FoxP3 transcription. Silencing of FoxP3 neutralized the promoting role of FoxM1 overexpression in Treg cell immunosuppression and HCCA cell immune escape. FoxM1 aggravated tumor development, upregulated FoxP3 expression, increased Treg cells, and reduced CD8+ T cells.ConclusionFoxM1 bound to the FoxP3 promoter region to promote FoxP3 transcription and recruited FoxP3+ Treg cells, thereby inducing HCCA immune escape.

Lactate induces metabolic and epigenetic reprogramming of pro-inflammatory Th17 cells.

Increased lactate levels in the tissue microenvironment are a well-known feature of chronic inflammation. However, the role of lactate in regulating T cell function remains controversial. Here, we demonstrate that extracellular lactate predominantly induces deregulation of the Th17-specific gene expression program by modulating the metabolic and epigenetic status of Th17 cells. Following lactate treatment, Th17 cells significantly reduced their IL-17A production and upregulated Foxp3 expression through ROS-driven IL-2 secretion. Moreover, we observed increased levels of genome-wide histone H3K18 lactylation, a recently described marker for active chromatin in macrophages, in lactate-treated Th17 cells. In addition, we show that high lactate concentrations suppress Th17 pathogenicity during intestinal inflammation in mice. These results indicate that lactate is capable of reprogramming pro-inflammatory T cell phenotypes into regulatory T cells.

Mesenchymal stem cells augment regulatory T cell function via CD80-mediated interactions and promote allograft survival

Mesenchymal stem cells (MSCs) and regulatory T cells (Tregs) both have been shown to modulate the alloimmune response and promote transplant survival. Mounting evidence suggests that MSCs augment Treg function, but the mechanisms underlying this phenomenon have not been fully deciphered. Here, we identified that MSCs express substantial levels of CD80 and evaluated its immunoregulatory function using in vivo and in vitro experiments. Our in vitro culture assays demonstrated that MSCs induce expression of FoxP3 in Tregs in a contact-dependent manner, and the blockade of CD80 abrogates this FoxP3 induction and Treg-mediated suppression of T cell proliferation. Moreover, supplementation of soluble CD80significantly upregulated FoxP3 expression. Using a well-characterized murine model of corneal transplantation, we show that silencing CD80 in MSCs diminishes the capacity of MSCs to promote selective graft infiltration of Tregs, promote FoxP3 expression and upregulate suppressive function of Tregs. Consequently, MSCs, following CD80knockdown, failed to promote corneal allograft survival.

PRDM1 Drives Human Primary T Cell Hyporesponsiveness by Altering the T Cell Transcriptome and Epigenome.

T cell hyporesponsiveness is crucial for the functional immune system and prevents the damage induced by alloreactive T cells in autoimmune pathology and transplantation. Here, we found low expression of PRDM1 in T cells from donor and recipients both related to the occurrence of acute graft-versus-host disease (aGVHD). Our systematic multiomics analysis found that the transcription factor PRDM1 acts as a master regulator during inducing human primary T cell hyporesponsiveness. PRDM1-overexpression in primary T cells expanded Treg cell subset and increased the expression level of FOXP3, while decreased expression had the opposite effects. Moreover, the binding motifs of key T cell function regulators, such as FOS, JUN and AP-1, were enriched in PRDM1 binding sites and that PRDM1 altered the chromatin accessibility of these regions. Multiomics analysis showed that PRDM1 directly upregulated T cell inhibitory genes such as KLF2 and KLRD1 and downregulated the T cell activation gene IL2, indicating that PRDM1 could promote a tolerant transcriptional profile. Further analysis showed that PRDM1 upregulated FOXP3 expression level directly by binding to FOXP3 upstream enhancer region and indirectly by upregulating KLF2. These results indicated that PRDM1 is sufficient for inducing human primary T cell hyporesponsiveness by transcriptomic and epigenetic manners.

Effects of dandelion tea on type II collagen induced arthritis in mice by regulation of ROR-γt/Foxp3 signaling

In this study, type II collagen was used to induce arthritis in mice to test the effect and mechanism of dandelion tea. The visual manifestations of arthritis, histopathological conditions of spleen, serum inflammatory factors and gene expression of synovium of foot and ankle joints in mice were detected by the methods of arthritis index (AI), spleen index, enzyme linked immunosorbent assay (ELISA), hematoxylin-eosin (H&E) staining and quantitative polymerase chain reaction (qPCR). The compound components of dandelion tea were determined by high performance liquid chromatography (HPLC). The experimental results show that dandelion tea can reduce the AI value, spleen index and paw swelling degree of arthritis mice, reduce the pathological damage of spleen, and increase the serum IL-10 and TGF-β levels, and can up-regulate Foxp3 expression and down regulate ROR-γt expression in the synovium of ankle joint. At the same time, the results of component analysis showed that the efficacy of dandelion tea came from the contents of gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, p-coumaric acid, rutin, myricetin, isoquercitrin, isochlorogenic acid and luteolin. It can be seen that dandelion tea is a health drink with good intervention effect on arthritis.

Notch2 suppresses the development of allergic rhinitis by promoting FOXP3 expression and Treg cell differentiation

AimsNotch signaling is closely related to a variety of diseases, but the role of Notch2 in allergic rhinitis (AR) remain unclear. This study was performed to investigate the effects of Notch2 on the differentiation of Treg cells and on the inflammatory response of AR. Materials and methodsPeripheral blood (including 101 AR patients and 66 Controls) and nasal mucosa (including 19 AR patients and 17 Controls) were collected to detect the expression levels of Notch2, NICD2 and FOXP3. CD4+ T cells of human origin were selected to detect the effects of Notch2 on the differentiation of Treg cells and FOXP3. An AR mouse model was established, and lentiviruses overexpressing Notch2 were administered. Then, allergic symptoms, OVA-sIgE titers, nasal mucosal inflammation, Th1/Th2/Th17 cytokines and splenic Treg cells were assessed. Key findingsCompared with that in the Control group, the expression of Notch2 in the AR group was decreased, and Notch2 expression was negatively correlated with the degree of allergy (P < 0.01). The expression levels of Notch2, NICD2 and FOXP3 were decreased in the nasal mucosa of AR patients. Notch2 can promote the differentiation of human Treg cells in vitro (P < 0.05), and Notch2 can directly promote FOXP3 transcription. Animal experiments showed after the upregulation of Notch2 expression, the allergic inflammatory of mice with AR was reduced, the differentiation of Treg cells was increased, and the imbalance of T cells was reversed (P < 0.05). SignificanceNotch2 promotes the differentiation of Treg cells by upregulating FOXP3 expression, thus significantly inhibiting the inflammatory response of AR.

Lessons from humanized mice: Is Foxp3 a bona fide marker of human regulatory T cells?

Abstract Regulatory T cells (Tregs) suppress conventional T cells (Tconv) and are critical for homeostasis of the immune system. Tregs are identified by the transcription factor Foxp3 and, to a large extent, by the transcription factor Helios (expressed by 80% of human Tregs). One confounding factor in the study of human Tregs is that Tconv cells upregulate Foxp3 expression upon activation in vitro. However, it remains unclear whether this is the case in vivo. To investigate differences in phenotype between Tregs and activated Tconv cells, peripheral blood mononuclear cells (PBMCs) from healthy human donors were activated in vitro and in vivo and analyzed for expression of activation markers and cytokines using flow cytometry. For in vitro activation, PBMCs were stimulated in xenogeneic reactions with mouse splenocytes or with anti-CD3. For in vivo activation, human PBMC-engrafted NOD-scid IL2Rgnull (NSG) mice were used. NSG mice develop xenogeneic graft-versus-host disease (GVHD) as human cells begin to attack mouse tissues. During in vitro activation, there was an increase in Foxp3 expression by IL-2 producing, Helios− Tconv cells. On the contrary, during the course of GVHD in vivo, activated Tconv cells, identified as CD25+ and Foxp3−, produced IL-2 but remained Foxp3− and Helios−. Foxp3+ Tregs did not produce IL-2 and remained predominantly Helios+. In conclusion, while expression of Foxp3 is not a reliable marker of activated human Tregs in vitro, it appears to be a specific marker of activated human Tregs in vivo as observed in this humanized mouse model. Using Foxp3 to distinguish human Tregs can improve diagnosis and treatment for autoimmune disorders and cancer.

Suppression of Th1 and Th17 Proinflammatory Cytokines and Upregulation of FOXP3 Expression by a Humanized Anti-DNAM-1 Monoclonal Antibody.

DNAM-1 is an activating immunoreceptor expressed on hematopoietic cells, including both CD4+ and CD8+ T cells, natural killer cells, and platelets. Since DNAM-1 is involved in the pathogenesis of various inflammatory diseases and cancers in humans as well as mouse models, it is a potential target for immunotherapy for these diseases. In this study, we generated a humanized neutralizing antihuman DNAM-1 monoclonal antibody (mAb), named TNAX101A, which contains an engineered Fc portion of human IgG1 to reduce Fc-mediated effector functions. We show that TNAX101A efficiently interfered the binding of DNAM-1 to its ligand CD155 and showed unique functions; it decreased production of the inflammatory cytokines such as interferon-gamma, tumor necrosis factor alpha, interleukin (IL)-6, IL-17A, and IL-17F by anti-CD3 antibody-stimulated or alloantigen-stimulated T cells and increased FOXP3 expression in anti-CD3-stimulated regulatory T (Treg) cells. These dual functions of TNAX101A may be advantageous for the treatment of T cell-mediated inflammatory diseases through both downregulation of effector T cell function and upregulation of Treg cell function.

Daifan San intervenes in forkhead box P3 and the interleukin (IL)-23/IL-17A signaling pathway to help prevent and treat primary biliary cirrhosis.

To investigate the mechanism by which Daifan San (DFS) prevents and treats primary biliary cirrhosis (PBC) via the forkhead box P3 (FoxP3) and interleukin (IL)-23/IL-17A signaling pathways. Ninety C57BL/6 mice were randomly divided into the control, model, DFS low-dose, DFS middle-dose, DFS high-dose and ursodeoxycholic acid (UDCA) groups (n = 15 per group). A mouse model of PBC was induced using polyinosinic polycytidylic acids (poly I:C). Lymphocyte subset expression in the peripheral blood was analyzed via flow cytometry. The inflammatory cytokines and antimitochondrial autoantibody (AMA) levels were detected via enzyme-linked immunosorbent assays. The expressions and location of type I collagen, type III collagen, cytokeratin 19 and FoxP3 in the liver tissue were evaluated via immunohistochemistry. FoxP3, IL-23 and IL-17 expressions in the peripheral blood and liver tissue were evaluated via real-time polymerase chain reaction and western blotting. IL-17, IL-23, IL-8, IL-33, TNF-a, and AMA expressions were significantly increased in the model group and decreased in the DFS and UDCA groups. Conversely, Treg cell and FoxP3 expressions were significantly decreased in the model group and increased in the DFS and UDCA groups. The IL-23/IL-17A signaling pathway was closely correlated with chronic inflammation of the bile duct in PBC and functional deletion of Treg cells, leading to reduced FoxP3 levels and mediating the loss of tolerance in PBC. DFS may delay the occurrence and relieve the symptoms of PBC by downregulating IL-23/IL-17A signaling pathway expression and upregulating FoxP3 expression.

Compound C induces autophagy and apoptosis in parental and hydroquinone-selected malignant leukemia cells through the ROS/p38 MAPK/AMPK/TET2/FOXP3 axis.

Hydroquinone (HQ), a major metabolic product of benzene, causes acute myeloid leukemia (AML) elicited by benzene exposure. Past studies found that continuous exposure of human AML U937 cells to HQ selectively produces malignant U937/HQ cells in which FOXP3 upregulation modulates malignant progression. Other studies revealed that AMPK promotes TET2 activity on DNA demethylation and that TET2 activity is crucial for upregulating FOXP3 expression. This study was conducted to elucidate whether compound C, an AMPK inhibitor, blocked the AMPK-TET2-FOXP3 axis in AML and in HQ-selected malignant cells. We found higher levels of AMPKα, TET2, and FOXP3 expression in U937/HQ cells compared to U937 cells. Treatment of parental Original Article and HQ-selected malignant U937 cells with compound C induced ROS-mediated p38 MAPK activation, leading to a suppression of AMPKα, TET2, and FOXP3 expression. Moreover, compound C induced apoptosis and mTOR-independent autophagy. The suppression of the autophagic flux inhibited the apoptosis of compound C-treated U937 and U937/HQ cells, whereas co-treatment with rapamycin, a mTOR inhibitor, sensitized the two cell lines to compound C cytotoxicity. Overexpression of AMPKα1 or pretreatment with autophagic inhibitors abrogated compound C-induced autophagy and suppression of TET2 and FOXP3 expression. Restoration of AMPKα1 or FOXP3 expression increased cell survival after treatment with compound C. In conclusion, our results show that compound C suppresses AMPK/TET2 axis-mediated FOXP3 expression and induces autophagy-dependent apoptosis in parental and HQ-selected malignant U937 cells, suggesting that the AMPK/TET2/FOXP3 axis is a promising target for improving AML therapy and attenuating benzene exposure-induced AML progression.

Resveratrol Attenuates Allergic Asthma and Associated Inflammation in the Lungs Through Regulation of miRNA-34a That Targets FoxP3 in Mice.

Asthma is a chronic inflammatory disease of airways mediated by T-helper 2 (Th2) cells involving complex signaling pathways. Although resveratrol has previously been shown to attenuate allergic asthma, the role of miRNA in this process has not been studied. We investigated the effect of resveratrol on ovalbumin-induced experimental allergic asthma in mice. To that end, BALB/c mice were immunized with ovalbumin (OVA) intraperitoneally followed by oral gavage of vehicle (OVA-veh) or resveratrol (100 mg/kg body) (OVA-res). On day 7, the experimental groups received intranasal challenge of OVA followed by 7 days of additional oral gavage of vehicle or resveratrol. At day 15, all mice were euthanized and bronchioalveolar fluid (BALF), serum and lung infiltrating cells were collected and analyzed. The data showed that resveratrol significantly reduced IL-5, IL-13, and TGF-β in the serum and BALF in mice with OVA-induced asthma. Also, we saw a decrease in CD3+CD4+, CD3+CD8+, and CD4+IL-4+ cells with increase in CD4+CD25+FOXP3+ cells in pulmonary inflammatory cell infiltrate in OVA-res group when compared to OVA-veh. miRNA expression arrays using lung infiltrating cells showed that resveratrol caused significant alterations in miRNA expression, specifically downregulating the expression of miR-34a. Additionally, miR-34a was found to target FOXP3, as evidenced by enhanced expression of FOXP3 in the lung tissue. Also, transfection studies showed that miR-34a inhibitor upregulated FOXP3 expression while miR-34a-mimic downregulated FOXP3 expression. The current study suggests that resveratrol attenuates allergic asthma by downregulating miR-34a that induces increased expression of FOXP3, a master regulator of Treg development and functions.