FOXP3 (in)stability and cancer immunotherapy.

Dysregulation of regulatory T cells (Tregs) is described in the context of inflammatory and autoimmune diseases, and cancer. Forkhead box P3 (FOXP3) is a transcription factor that its activity is an indicator of Treg identity. FOXP3 induces metabolic versatility in intra-tumoral Tregs, so that its deficiency mediates Treg instability or even gives rise to the acquisition of effector T cell phenotype. FOXP3 dysregulation and defectiveness occurs upon ubiquitination, methylation and presumably acetylation. Stimulators of PTEN, mammalian target of rapamycin complex 2 (mTORC2), and nucleus accumbens-associated protein-1 (NAC1), and inhibitors of B lymphocyte-induced maturation protein-1 (Blimp-1), Deltex1 (DTX1) and ubiquitin-specific peptidase 22 (USP22) are suggested to hamper FOXP3 stability, and to promote its downregulation and further Treg depletion. A point is that Treg subsets reveal different reliance on FOXP3, which indicates that not all Tregs are strictly dependent on FOXP3, and presumably Tregs with different origin rely on diverse regulators of FOXP3 stability. The focus of this review is over the current understanding toward FOXP3, its activity in Tregs and influence from different regulators within tumor microenvironment (TME). Implication of FOXP3 targeting in cancer immunotherapy is another focus of this paper.

ß-carotene accelerates the resolution of atherosclerosis in mice.

ß-carotene oxygenase 1 (BCO1) catalyzes the cleavage of ß-carotene to form vitamin A. Besides its role in vision, vitamin A regulates the expression of genes involved in lipid metabolism and immune cell differentiation. BCO1 activity is associated with the reduction of plasma cholesterol in humans and mice, while dietary ß-carotene reduces hepatic lipid secretion and delays atherosclerosis progression in various experimental models. Here we show that ß-carotene also accelerates atherosclerosis resolution in two independent murine models, independently of changes in body weight gain or plasma lipid profile. Experiments in Bco1-/- mice implicate vitamin A production in the effects of ß-carotene on atherosclerosis resolution. To explore the direct implication of dietary ß-carotene on regulatory T cells (Tregs) differentiation, we utilized anti-CD25 monoclonal antibody infusions. Our data show that ß-carotene favors Treg expansion in the plaque, and that the partial inhibition of Tregs mitigates the effect of ß-carotene on atherosclerosis resolution. Our data highlight the potential of ß-carotene and BCO1 activity in the resolution of atherosclerotic cardiovascular disease.

Immunoregulatory Effect of Jiedu Tongluo Shengjin Prescription on Sjögren's Syndrome Mice Based on Stability of Treg Cells / 中国实验方剂学杂志

ObjectiveTo investigate the effect of different doses of Jiedu Tongluo Shengjin prescription （JTSP） on serum interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α） and forkhead box P3 （FoxP3） in submandibular gland of NOD/Ltj mice with Sjögren's syndrome， and to explore the mechanism of JTSP on immune regulation in NOD/Ltj mice. MethodThirty NOD/Ltj mice （eight weeks old） were randomly divided into model group， JTSP low-dose group， JTSP medium-dose group， JTSP high-dose group and hydroxychloroquine group， and were administrated with normal saline， JTSP 9， 18， and 36 g·kg-1， and hydroxychloroquine 60 mg·kg-1 daily， respectively from the age of 12 weeks. Six ICR mice were given an equal amount of normal saline by gavage as the control group. During the experiment， daily water consumption and saliva secretion of mice at the age of 9， 12， 16 weeks were recorded. After 4 weeks of administration， submandibular gland and spleen tissues were dissected to calculate corresponding indexes. The pathological morphology of submandibular gland was observed by hematoxylin-eosin （HE） staining. Meso Scale Discovery （MSD） and immunohistochemistry were employed to detect the serum levels of IL-6， TNF-α and IL-10， and the expression and distribution of FoxP3 in submandibular gland， respectively. The protein expression of FoxP3 in mouse submandibular gland was determined by Western blot， and the mRNA expressions of FoxP3 and TNF-α were determined by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the control group， the model group presented increased daily water consumption， decreased saliva secretion， lowered submandibular gland index， elevated pathological score of submandibular gland， up-regulated serum IL-6 and TNF-α and mRNA expression of TNF-α while down-regulated serum IL-10 and protein and mRNA expressions of FoxP3 in submandibular gland （P<0.05）. Compared with the conditions in model group， daily water consumption in JTSP groups was reduced while saliva secretion was increased， especially in medium-dose and high-dose groups （P<0.05）， and there was an increase in the submandibular gland index of JTSP medium-dose group （P<0.05） while a decrease in the spleen index of JTSP high-dose group （P<0.05）. Additionally， JTSP groups had lower pathological score of submandibular gland than the model group （P<0.05）， especially high-dose group， as well as lower serum IL-6 and TNF-α and mRNA expression of TNF-α while higher serum IL-10 （P<0.05）. JTSP at medium and high doses up-regulated the protein and mRNA expressions of FoxP3 in submandibular gland （P<0.05）. ConclusionJTSP may inhibit the secretion of inflammatory cytokines by regulating the stability of FoxP3+ regulatory T （Treg） cells， thus alleviating the systemic immune inflammation in Sjögren's syndrome.

Fine-tuning of regulatory T cells is indispensable for the metabolic steatosis-related hepatocellular carcinoma: A review.

The majority of chronic hepatic diseases are caused by nutritional imbalance. These nutritional inequities include excessive intake of alcohol and fat, which causes alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), respectively. The pathogenesis of hepatic diseases is mainly dependent on oxidative stress, autophagy, DNA damage, and gut microbiota and their metabolites. These factors influence the normal physiology of the liver and impact the hepatic microenvironment. The hepatic microenvironment contains several immune cells and inflammatory cytokines which interact with each other and contribute to the progression of chronic hepatic diseases. Among these immune cells, Foxp3+ CD4+ regulatory T cells (Tregs) are the crucial subset of CD4+ T cells that create an immunosuppressive environment. This review emphasizes the function of Tregs in the pathogenesis of ALD and NAFLD and their role in the progression of NAFLD-associated hepatocellular carcinoma (HCC). Briefly, Tregs establish an immunosuppressive landscape in the liver by interacting with the innate immune cells and gut microbiota and their metabolites. Meanwhile, with the advancement of steatosis, these Tregs inhibit the proliferation, activation and functions of other cytotoxic T cells and support the progression of simple steatosis to HCC. Briefly, it can be suggested that targeting Tregs can act as a favourable prognostic indicator by modulating steatosis and insulin resistance during the pathogenesis of hepatic steatosis and NAFLD-associated HCC.

Chemotherapy Shifts the Balance in Favor of CD8+ TNFR2+ TILs in Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is primarily treated via chemotherapy; in parallel, efforts are made to introduce immunotherapies into TNBC treatment. CD4+ TNFR2+ lymphocytes were reported as Tregs that contribute to tumor progression. However, our published study indicated that TNFR2+ tumor-infiltrating lymphocytes (TNFR2+ TILs) were associated with improved survival in TNBC patient tumors. Based on our analyses of the contents of CD4+ and CD8+ TILs in TNBC patient tumors, in the current study, we determined the impact of chemotherapy on CD4+ and CD8+ TIL subsets in TNBC mouse tumors. We found that chemotherapy led to (1) a reduction in CD4+ TNFR2+ FOXP3+ TILs, indicating that chemotherapy decreased the content of CD4+ TNFR2+ Tregs, and (2) an elevation in CD8+ TNFR2+ and CD8+ TNFR2+ PD-1+ TILs; high levels of these two subsets were significantly associated with reduced tumor growth. In spleens of tumor-bearing mice, chemotherapy down-regulated CD4+ TNFR2+ FOXP3+ cells but the subset of CD8+ TNFR2+ PD-1+ was not present prior to chemotherapy and was not increased by the treatment. Thus, our data suggest that chemotherapy promotes the proportion of protective CD8+ TNFR2+ TILs and that, unlike other cancer types, therapeutic strategies directed against TNFR2 may be detrimental in TNBC.

Relative expression and prognostic significance of forkhead box P3 in childhood B-cell acute lymphoblastic leukemia.

BACKGROUND: Despite the favorable survival rates of childhood B-cell acute lymphoblastic leukemia (B-ALL), a significant number of patients present a dismal prognosis. Forkhead box P3 (FOXP3), a marker of regulatory T cells, functions as a transcription factor involved in immune cell regulation, and its expression correlates with prognosis in many malignancies. Therefore, this study aimed to assess the relative gene expression level of FOXP3 in childhood B-ALL and to detect its prognostic utility. METHODS: The study included 139 bone marrow samples obtained from 112 patients at diagnosis and 27 healthy children. Following extraction, RNA was reverse transcribed and the relative expression level of FOXP3 was quantified by quantitative PCR. Cytogenetics, immunophenotype, and minimal residual disease were analyzed according to international guidelines. RESULTS: A highly significant overexpression of FOXP3 was detected in childhood B-ALL patients at diagnosis, which was associated with a stronger risk for disease relapse and patients' worse survival. Moreover, multivariate regression models highlighted the independent prognostic value of FOXP3 for childhood B-ALL. Finally, the combination of FOXP3 relative expression with clinically used disease markers clearly enhanced the prediction of treatment stratification. CONCLUSIONS: High FOXP3 relative expression was associated with inferior outcome suggesting its potentiality as a molecular prognostic marker to predict childhood B-ALL patients' outcomes.

Thymic origins of autoimmunity-lessons from inborn errors of immunity.

During their intrathymic development, nascent T cells are empowered to protect against pathogens and to be operative for a life-long acceptance of self. While autoreactive effector T (Teff) cell progenitors are eliminated by clonal deletion, the intrathymic mechanisms by which thymic regulatory T cell (tTreg) progenitors maintain specificity for self-antigens but escape deletion to exert their regulatory functions are less well understood. Both tTreg and Teff development and selection result from finely coordinated interactions between their clonotypic T cell receptors (TCR) and peptide/MHC complexes expressed by antigen-presenting cells, such as thymic epithelial cells and thymic dendritic cells. tTreg function is dependent on expression of the FOXP3 transcription factor, and induction of FOXP3 gene expression by tTreg occurs during their thymic development, particularly within the thymic medulla. While initial expression of FOXP3 is downstream of TCR activation, constitutive expression is fixed by interactions with various transcription factors that are regulated by other extracellular signals like TCR and cytokines, leading to epigenetic modification of the FOXP3 gene. Most of the understanding of the molecular events underlying tTreg generation is based on studies of murine models, whereas gaining similar insight in the human system has been very challenging. In this review, we will elucidate how inborn errors of immunity illuminate the critical non-redundant roles of certain molecules during tTreg development, shedding light on how their abnormal development and function cause well-defined diseases that manifest with autoimmunity alone or are associated with states of immune deficiency and autoinflammation.

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.

Tumor Necrosis Factor α Reduces SNAP29 Dependent Autolysosome Formation to Increase Prion Protein Level and Promote Tumor Cell Migration.

Tumor Necrosis Factor α (TNFα) is best known as a mediator of inflammation and immunity, and also plays important roles in tumor biology. However, the role of TNFα in tumor biology is complex and not completely understood. In a human melanoma cell line, M2, and a lung carcinoma cell line, A549, TNFα up-regulates prion protein (PrP) level, and promotes tumor cell migration in a PrP dependent manner. Silencing PRNP abrogates TNFα induced tumor cell migration; this phenotype is reversed when PRNP is re-introduced. Treatment with TNFα activates nuclear factor kappa B (NF-κB) signaling, which then mitigates autophagy by reducing the expression of Forkhead Box P3 (FOXP3). Down regulation of FOXP3 reduces the transcription of synaptosome associated protein 29 (SNAP29), which is essential in the fusion of autophagosome and lysosome creating autolysosome. FOXP3 being a bona fide transcription factor for SNAP29 is confirmed in a promoter binding assay. Accordingly, silencing SNAP29 in these cell lines also up-regulates PrP, and promotes tumor cell migration without TNFα treatment. But, when SNAP29 or FOXP3 is silenced in these cells, they are no longer respond to TNFα. Thus, a reduction in autophagy is the underlying mechanism by which expression of PrP is up-regulated, and tumor cell migration is enhanced upon TNFα treatment. Disrupting the TNFα-NF-κB-FOXP3-SNAP29 signaling axis may provide a therapeutic approach to mitigate tumor cell migration.

Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

Mouse Double Minute 2 Homolog-Mediated Ubiquitination Facilitates Forkhead Box P3 Stability and Positively Modulates Human Regulatory T Cell Function.

Regulatory T cells (Treg cells) are essential for maintaining immune tolerance, and the dysfunction of Treg cells may cause autoimmune diseases and tumors. Forkhead box P3 (FOXP3) is the key transcription factor controlling Treg cell development and suppressive function. Mouse double minute 2 homolog (MDM2), an E3 ubiquitin ligase, has been identified as an oncoprotein that mediates the ubiquitination and degradation of tumor suppressor p53; however, whether it has functions in Treg cells remains unknown. Here, we demonstrate that MDM2 positively regulates human Treg cell suppressive function via its mediated ubiquitination and stabilization of FOXP3. Knockdown of MDM2 with shRNA in human primary Treg cells leads to the impaired ability of FOXP3 to regulate the expression levels of downstream genes and the attenuated suppressive capacity of Treg cells, due to FOXP3 instability. Consistently, MDM2 overexpression in human Treg cells enhances FOXP3 stability and Treg cell suppressive capacity. Mechanistically, MDM2 interacts with FOXP3, and mainly mediates monoubiquitination and polyubiquitination of FOXP3, thus stabilizing the protein level of FOXP3. We have also found lysine residues in FOXP3 required for MDM2-mediated ubiquitination. In addition, TCR/CD28 signaling upregulates the expression level of MDM2 and its mediated FOXP3 ubiquitination in human Treg cells. Therefore, our findings reveal that MDM2 in Treg cells could be a potential therapeutic target for treating autoimmune diseases and tumors.

Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics.

Excessive activation of the proinflammatory cytokine tumor necrosis factor-α (TNFα) is a major cause of autoimmune diseases, including rheumatoid arthritis. TNFα induces immune responses via TNF receptor 1 (TNFR1) and TNFR2. Signaling via TNFR1 induces proinflammatory responses, whereas TNFR2 signaling is suggested to suppress the pathophysiology of inflammatory diseases. Therefore, selective inhibition of TNFR1 signaling and preservation of TNFR2 signaling activities may be beneficial for managing autoimmune diseases. To this end, we developed a TNFR1-selective, antagonistic TNFα mutant (R1antTNF). Here, we developed an R1antTNF derivative, scR1antTNF-Fc, which represents a single-chain form of trimeric R1antTNF with a human IgG-Fc domain. scR1antTNF-Fc had properties similar to those of R1antTNF, including TNFR1-selective binding avidity, TNFR1 antagonistic activity, and thermal stability, and had a significantly extended plasma t1/2in vivo In a murine rheumatoid arthritis model, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF (a previously reported PEGylated form) delayed the onset of collagen-induced arthritis, suppressed arthritis progression in mice, and required a reduced frequency of administration. Interestingly, with these biologic treatments, we observed an increased ratio of regulatory T cells to conventional T cells in lymph nodes compared with etanercept, a commonly used TNF inhibitor. Therefore, scR1antTNF-Fc and 40-kDa PEG-scR1antTNF indirectly induced immunosuppression. These results suggest that selective TNFR1 inhibition benefits the management of autoimmune diseases and that R1antTNF derivatives hold promise as new-modality TNF-regulating biologics.

Abnormal Expression of Indoleamine 2, 3-Dioxygenase in Human Recurrent Miscarriage.

Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that mediates the conversion of tryptophan to kynurenine, was shown to play a key role in placental development during normal pregnancy. However, little is known about the pattern of IDO expression in the endometrium and its attendant functional significance in pregnancies complicated with recurrent miscarriage (RM). Immunohistochemical studies of IDO, Foxp3, CD56, and CD163 expression were performed in endometrial samples from women with RM and healthy fertile controls. Our study found that IDO was localized in glandular epithelial cells, surface epithelial cells, and a small number of cells within the stromal compartment (including stromal cells and leukocytes) in endometrium. Indoleamine 2, 3-dioxygenase expression in the RM group was significantly lower than control group. The Foxp3 and CD56 expression were significantly increased with the elevated IDO expression in controls but not in RM. The percentage of Foxp3 + Tregs was significantly correlated with the level of IDO expression in the control group. Comparatively, no correlation was found between the percentage of CD56 + cells, CD163 + cells, and the level of IDO expression, no matter in controls and RM patients. This study demonstrated that the downregulation of IDO expression and noncoordinated association between IDO and other endometrial immune cells were associated with RM. Our findings provide insights into the contribution of IDO in immune regulation to maintain normal pregnancy, which could be used to develop potential therapeutic methods for RM.

The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

A positive feedback loop formed by NGFR and FOXP3 contributes to the resistance of non-small cell lung cancer to icotinib.

BACKGROUND: The study was aimed to investigate the mechanisms causing acquired chemoresistance to icotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), in non-small cell lung cancer (NSCLC). METHODS: Three wildtype NSCLC cell lines were used to produce icotinib-resistant (IR) cell lines. Real-time PCR and western blot assays were used to detect the mRNA and protein levels of nerve growth factor receptor (NGFR) and forkhead box P3 (FOXP3). MTT assay was used to detect the viability of cells. Luciferase activity and chromatin immunoprecipitation (ChIP) assays were used to detect the transactivation activity of FOXP3. RESULTS: NGFR and FOXP3 were dramatically increased in three IR NSCLC cell lines, and both proteins were required for induction of icotinib resistance. NGFR-induced icotinib resistance was partially related to activation of AKT, a well-known chemoresistance inducer in many tumor types. Activated AKT could induce the expression of FOXP3 which further induce icotinib through transactivating NGFR expression by binding to its promoter. In addition, the inducing of FOXP3 could also induce icotinib resistance solely. CONCLUSIONS: NGFR, AKT and FOXP3 form a positive feedback loop, by which the abilities of NGFR and FOXP3 on inducing icotinib resistance are further enhanced. We believe that NGFR and FOXP3 might be novel therapeutic targets in NSCLC.

Effect of Warming and Heat-clearing Method on Expressions of FoxP3 and ROR-γt in Colon Tissue of Rats with Intestinal Endotoxemia Caused by Acute-on-chronic Liver Failure / 中国实验方剂学杂志

Objective::To study the effect of warming and heat-clearing method (Wenyang Jiedu Huayu decoction) on the expressions of Forkhead box P3 (FoxP3), Retinoic acid-related orphan receptor gamma t (ROR-γt) in colon tissue of mice with acute-on-chronic liver failure (ACLF), in order to explore the possible regulatory mechanism on intestinal endotoxemia (IETM) in liver failure mice. Method::The 130 SD rats were randomly divided into normal group (10 rats) and model group (120 rats). The ACLF mice model was established through the subcutaneous injection with bovine serum albumin and the intraperitoneal injection with D-galactosamine(D-Gal) and lipopolysaccharide (LPS). The model mice were randomly divided into model group, heat-clearing group (Yinchenhao decoction, 6.68 g·kg-1), warming group (Yinchen Zhufu decoction, 7.09 g·kg-1) and warming and heat-clearing group (Wenyang Jiedu Huayu decoction, 19.53 g·kg-1). The normal group and the model group were given distilled water by gastric lavage, while the other groups were given equal volume of corresponding Chinese herbal medicines for a week. The value of each index at 1, 12 and 24 h was measured. The ratio of Treg/Th17 cell in peripheral blood were detected and calculated by flow cytometry. Real-time fluorescence quantitative PCR (Real-time PCR) was used to detect the expressions of FoxP3 and ROR-γt in colon tissues of mice at different time points. In situ hybridization and immunohistochemistry were used to observe the expressions of FoxP3 and ROR-γt genes and proteins. Result::Compared with normal group, the ratio of Treg/Th17 in the model group decreased significantly at each time point (P<0.01). Compared with the model group, the Treg/Th17 ratio increased only in the warming and heat-clearing method group (P<0.05). Compared with normal group, the expression of ROR-γt in the model group was significantly higher (P<0.01), and the expression of ROR-γt in the model group was higher than FoxP3.Compared with the model group, the expressions of FoxP3 and ROR-γt mRNA in the heat-clearing group and the warming group decreased at each time point (P<0.05), and the expressions of FoxP3 and ROR-γt in the warming and heat-clearing method group decreased significantly (P<0.01). The expressions of FoxP3 and ROR-γt mRNA in warming and heat-clearing group decreased compared with those in the warming group and heat-clearing group (P<0.05). Conclusion::The mechanism of the warming and heat-clearing method on IETM in liver failure may be related to the regulation of FoxP3 and ROR-γt expressions.

NLRP3 negatively regulates Treg differentiation through Kpna2-mediated nuclear translocation.

Naïve CD4+ T cells in the periphery differentiate into regulatory T cells (Tregs) in which Foxp3 is expressed for their suppressive function. NLRP3, a pro-inflammatory molecule, is known to be involved in inflammasome activation associated with several diseases. Recently, the expression of NLRP3 in CD4+ T cells, as well as in myeloid cells, has been described; however, a role of T cell-intrinsic NLRP3 in Treg differentiation remains unknown. Here, we report that NLRP3 impeded the expression of Foxp3 independent of inflammasome activation in Tregs. NLRP3-deficient mice elevate Treg generation in various organs in the de novo pathway. NLRP3 deficiency increased the amount and suppressive activity of Treg populations, whereas NLRP3 overexpression reduced Foxp3 expression and Treg abundance. Importantly, NLRP3 interacted with Kpna2 and translocated to the nucleus from the cytoplasm under Treg-polarizing conditions. Taken together, our results identify a novel role for NLRP3 as a new negative regulator of Treg differentiation, mediated via its interaction with Kpna2 for nuclear translocation.

Abnormal Expression of Indoleamine 2, 3-Dioxygenase in Human Recurrent Miscarriage.

Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that mediates the conversion of tryptophan to kynurenine, was shown to play a key role in placental development during normal pregnancy. However, little is known about the pattern of IDO expression in the endometrium and its attendant functional significance in pregnancies complicated with recurrent miscarriage (RM). Immunohistochemical studies of IDO, Foxp3, CD56, and CD163 expression were performed in endometrial samples from women with RM and healthy fertile controls. Our study found that IDO was localized in glandular epithelial cells, surface epithelial cells, and a small number of cells within the stromal compartment (including stromal cells and leukocytes) in endometrium. Indoleamine 2, 3-dioxygenase expression in the RM group was significantly lower than control group. The Foxp3 and CD56 expression were significantly increased with the elevated IDO expression in controls but not in RM. The percentage of Foxp3+ Tregs was significantly correlated with the level of IDO expression in the control group. Comparatively, no correlation was found between the percentage of CD56+ cells, CD163+ cells, and the level of IDO expression, no matter in controls and RM patients. This study demonstrated that the downregulation of IDO expression and noncoordinated association between IDO and other endometrial immune cells were associated with RM. Our findings provide insights into the contribution of IDO in immune regulation to maintain normal pregnancy, which could be used to develop potential therapeutic methods for RM.

Vitamin D alleviates acute graft-versus-host disease through promoting the generation of Foxp3+ T cells.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is a medical complication which may result in significant morbidity and mortality after transplantation. The aim of this study investigated the therapeutic effect and underlying mechanism of 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in the treatment of aGVHD. METHOD: An aGVHD model was built by transferring splenocytes of B6 mice into B6D2F1 mice. 1α,25(OH)2D3 was added to evaluate the protective function to aGVHD; the phenotype and cytokine expression profile of spleen cells from the aGVHD model were determined using flow cytometry 2 weeks after the model is established. RESULT: Administration of 1α,25(OH)2D3 significantly slowed aGVHD progression and improved survival of B6D2F1 recipients of grafted B6 splenocytes. 1α,25(OH)2D3 treatment also resulted in an increased number of CD4+Foxp3+ regulatory T cells (Tregs) but decreased the number of CD4+IL-4+ cells. In vitro analysis demonstrated that 1α,25(OH)2D3 directly increased forkhead box P3 (Foxp3) and IL-10 expression and enhanced the function of induced Tregs (iTregs). CONCLUSIONS: This analysis indicated that the effect of 1α,25(OH)2D3 is mediated in part by improving the number of Tregs. 1α,25(OH)2D3 administration thus represents a viable approach for treating aGVHD.

Ring finger protein 31-mediated atypical ubiquitination stabilizes forkhead box P3 and thereby stimulates regulatory T-cell function.

The CD4+CD25+FOXP3+ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.