Foxp3 Protein Expression Research Articles

Arctigenin ameliorates high-fat diet-induced metabolic disorders by reshaping gut microbiota and modulating GPR/HDAC3 and TLR4/NF-κB pathways

BackgroundArctigenin (AG), a phenylpropanoid lignan from the medicinal and food homologous plant Arctium lappa l., is known for its anti-cancer, anti-inflammatory and antioxidant properties. However, the pharmacological effects of AG on metabolic disorders remain limited, and specific mechanisms based on gut microbiota have not been reported. PurposeThis study aimed to evaluate the regulation of glycolipid metabolism by AG in obese mice and investigate the potential mechanisms associated with gut microbes. MethodsThe anti-obesity efficacy of AG was evaluated in high-fat diet (HFD)-fed mice. 16S rRNA gene sequencing and GC–MS were used to detect changes in gut microbes and metabolite levels. Immunohistochemistry, immunofluorescence, and polymerase chain reaction were used to validate the molecular mechanisms of gut microbe-derived metabolites involved in the improvement of intestinal homeostasis and hepatic metabolism by AG. ResultsWe found that AG significantly ameliorated HFD-induced glucolipid metabolism disorders, liver degeneration and the imbalance of macrophage M1/M2 polarization. In addition, AG attenuated intestinal barrier damage, inflammation and imbalance of Th17/Treg immune in HFD mice. Importantly, AG promoted short-chain fatty acid (SCFA)-producing bacteria and SCFA levels, which regulated the G protein-coupled receptor (GPR)41/43 and HDAC3 pathways to induce FOXP3 protein expression and consequently maintained intestinal Th17/Treg immunity. AG also inhibited lipopolysaccharide (LPS) production leading to attenuation of TLR4/NF-κB-mediated intestinal inflammation. Furthermore, AG upregulated intestinal MCT1 protein levels to promote absorption of SCFA and activated the hepatic GPR41/43/109a-AMPK pathway to regulate lipid metabolism, and thus reduced lipid accumulation. ConclusionThis study first demonstrated that AG could modulate the gut microbiota and derived metabolites to repair intestinal damage and regulate hepatic metabolic pathways, thereby ameliorating metabolic disorders induced by HFD. These findings support the great potential of AG as a novel prebiotic to fight obesity and chronic metabolic diseases by targeting the gut microbiota.

Correlation of Th17/Treg associated transcription factors with clinicopathological features of colorectal cancer and their prognostic significance.

To analyze the correlation of Th17/Treg associated transcription factors (TFs) with clinicopathological features of colorectal cancer (CRC) and their prognostic significance. This research enrolled 56 CRC patients (experimental group, EG) and 50 healthy controls (control group, CG), who presented to Deqing People's Hospital between June 2017 and January 2019. The levels of Th17, Treg and their TFs [forkhead box protein P3 (Foxp3), retinoid acid receptor-related orphan receptor gamma t (RORγt)] and secreted inflammatory factors (IFs) [interleukin-17 (IL-17), interleukin-22 (IL-22)] were detected in the peripheral blood (PB) of both groups, and the TFs' phosphorylated protein expression was observed by Western blot. Further, the correlation of TFs with patients' pathological features was analyzed. Finally, a 3-year prognostic follow-up was performed on CRC patients. Receiver operating characteristic (ROC) determined the predictive value of Th17/Treg on the prognostic mortality of patients. Peripheral blood Th17 and Treg showed higher levels in the EG than in the CG, demonstrating excellent diagnostic effects on CRC (P<0.05). The EG also exhibited reduced Foxp3 and p-Foxp3 protein expression, and elevated RORγt and p-RORγt levels compared with the CG (all P<0.0001). In addition, the EG exhibited statistically higher IL-17 and IL-22 levels than the CG (all P<0.05). Further, the analysis of pathological features revealed close correlations of Th17/Treg, RORγt and Foxp3 with tumor size, TNM staging, degree of differentiation, and lymph node metastasis (LNM) of CRC patients (all P<0.001). Finally, the prognostic follow-up results identified that TNM staging, degree of differentiation, LNM, RORγt, Th17 and Treg were independent risk factors for prognostic mortality of CRC patients, while Foxp3 was an independent protective factor (all P<0.001). Th17/Treg associated TFs are of great significance for the prognosis evaluation of CRC, the imbalance of which can cause aggravation of the inflammatory reaction and promote malignancy of CRC.

Abstract B038: Targeting a Treg-specific deubiquitinase module for antitumor immune therapy

Abstract Checkpoint immunotherapies have transformed the treatment landscape for multiple human malignancies, but still with very limited success in treatment of many human cancers. A major hurdle is mediated by regulatory T (Treg) cells, which suppress the function of antitumor effector T cells. However, current approaches in targeting Treg to activate antitumor immune responses have shown only transient efficacy and are highly unspecific. Importantly, our recent studies, through unbiased CRISPR screening, identified the death from cancer cell signature gene USP22 required for optimal FoxP3 expression. USP22 genetic inhibition resulted in an approximately 15-25% reduction of FoxP3 expression in Tregs and partially diminishes Treg suppressive functions, which consequently results in elevated antitumor immunity against a broad spectrum of cancer types in mice. Therefore, a 20% reduction in FoxP3 protein expression, in this case, by USP22 inhibition, creates an ideal therapeutic window to boost antitumor immunity without triggering extensive autoimmune inflammatory responses (Nature, 2020). More recently, we further demonstrated that tumor microenvironment factors, including TGF-β, hypoxia, and tumor metabolites, induce Treg fitness/adaptation through selectively upregulating a deubiquitinase module, including USP21 and USP22 (Science Advances, 2022). Our ongoing study has identified USP22-specific small molecule inhibitors that largely diminish Treg fitness and dramatically inhibit tumor growth at a clinically relevant setting. In addition, our recent unpublished single-cell RNA-seq characterization revealed a novel function of the Treg-specific deubiquitination module in Treg terminal differentiation into a unique immune suppressive subpopulation, specifically in the tumor microenvironment. Collectively, our study defines a novel Treg deubiquitination module in tumor immunosurveillance and provides a rationale for the first Treg-specific targeting in antitumor immune therapy. Citation Format: Deyu Fang. Targeting a Treg-specific deubiquitinase module for antitumor immune therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B038.

Low vitamin D during pregnancy is associated with infantile eczema by up-regulation of PI3K/AKT/mTOR signaling pathway and affecting FOXP3 expression: A bidirectional cohort study

Vitamin D has received increasing attention because of its association with atopic disease development. Limited studies that have been done on the impact of maternal vitamin D levels during pregnancy on infantile eczema are still debatable. We wanted to discover the effect of maternal vitamin D on infantile eczema and explore whether regulatory T cells (Treg) play a role in this process. 219 pairs of mothers and children were enrolled. Maternal fasting venous blood was collected in pregnancy's second and third trimesters to determine vitamin D levels. Cord blood and placenta samples were collected during childbirth for detecting levels of genes, proteins and cytokines. Pediatricians followed up the prevalence of eczema in infants within 1 year. The reported rate of vitamin D deficiency and insufficiency was 35.6% and 28.3%. Lower maternal 25(OH)D3 levels were related to a higher risk of infantile eczema. Foxp3 gene expression is lower in cord blood of infants with eczema compared to infants without eczema. There was a positive correlation between maternal 25(OH)D3 levels and the expression of FOXP3 gene in cord blood. Compared to vitamin D sufficiency women, vitamin D deficiency women's placental FOXP3 protein expression was decreased and PI3K/AKT/mTOR protein was up-regulated. Our study demonstrates that low prenatal maternal vitamin D levels increased the risk of infantile eczema aged 0–1 year, which might be related to the downregulating of the FOXP3 gene expression in cord blood and decreased placental FOXP3 protein expression. Low placental FOXP3 protein was related with activating PI3K/AKT/mTOR signaling pathway.

Yin Yang 1 impacts upon preeclampsia by regulating Treg/TH17 cells and PI3K/AKT pathway

Preeclampsia (PE) is a common obstetric syndrome with an unclear etiology and pathogenesis. The study here aimed to investigate the role of Yin Yang 1 (YY1) in PE, and to reveal any YY1-regulated mechanisms in PE. Peripheral blood, placenta, and endometrial tissues of PE patients, healthy volunteers, and patients who had undergone an elective Cesarean section and had a scarred uterus (control group) were collected for analyses. Rat PE models were established by lipopolysaccharide induction. Subsets of these rats were then made to over-express YY1. At 18 d after the PE was established, urine, blood, and placental tissues from all rats were collected. Levels of regulatory-T (Treg) and helper T-type 17 (TH17) cells in both human and rat blood were measured by flow cytometry. ELISA kits were used to evaluate blood levels of inflammatory factors (i.e. IL-6, IL-10, and IL-17) as well. RT-qPCR and Western blot assays were performed to quantify levels of forkhead box P3 (Foxp3), retinoic acid-related orphan receptor C (RORc), and YY1 in the human and rat placenta and endometrial tissues. Expressions of PI3K/AKT pathway-related proteins were also evaluated by Western blots. The results indicated that the PE patients, relative to levels in control group and the healthy control subjects, had decreased circulating levels of Treg cells/increased TH17 cells; tissues from these patients also had relatively-decreased FoxP3 mRNA and protein expressions and elevated RORc mRNA and protein expressions. YY1 was expressed only at low levels in the PE patient placenta and endometrial tissues. In rats, PE rats treated with over-expressed YY1 had (relative to in PE rats without over-induced YY1) increased circulating levels of Treg cells/decreased TH17 cells; tissues from these rats had elevated FoxP3 mRNA and protein expressions and reduced mRNA and protein RORc expressions, as well as indications of alleviated inflammation. In the rat placenta samples, YY1 was also determined to activate the PI3K/AKT pathway. In summary, YY1 regulates the balance among Treg/TH17 cells and so affect the PE process in part through activation of the PI3K/AKT pathway.

CD4+ regulatory T cells lacking Helios and Eos

The transcriptional regulators that drive regulatory T (Treg) cell development and function remain partially understood. Helios (Ikzf2) and Eos (Ikzf4) are closely-related members of the Ikaros family of transcription factors. They are highly expressed in CD4+ Treg cells and functionally important for Treg cell biology, as mice deficient for either Helios or Eos are susceptible to autoimmune diseases. However, it remains unknown if these factors exhibit specific or partially redundant functions in Treg cells. Here we show that mice with germline deletions of both Ikzf2 and Ikzf4 are not very different from animals with single Ikzf2 or Ikzf4 deletions. Double knockout Treg cells differentiate normally, and efficiently suppress effector T cell proliferation in vitro. Both Helios and Eos are required for optimal Foxp3 protein expression. Surprisingly, Helios and Eos regulate different, largely non-overlapping, sets of genes. Only Helios is required for proper Treg cell aging, as Helios deficiency results in reduced Treg cell frequencies in the spleen of older animals. These results indicate that Helios and Eos are required for distinct aspects of Treg cell function.

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment.

This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis. An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions. After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05). In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

S3I-201, a selective stat3 inhibitor, ameliorates clinical symptoms in a mouse model of experimental autoimmune encephalomyelitis through the regulation of multiple intracellular signalling in Th1, Th17, and treg cells.

CD4+T cells, specifically Th cells (Th1 and Th17) and regulatory T cells (Tregs), play a pivotal role in the pathogenesis of multiple sclerosis (MS), a demyelinating autoimmune disease of the CNS. STAT3 inhibitors are potential therapeutic targets for several immune disorders. In this study, we investigated the role of a well-known STAT3 inhibitor, S3I-201, in experimental autoimmune encephalomyelitis (EAE), a model of MS. Following induction of EAE, mice were intraperitoneally administered S3I-201 (10 mg/kg) each day, beginning on day 14 and continuing till day 35 and were evaluated for clinical signs. Flow cytometry was used to investigate further the effect of S3I-201 on Th1 (IFN-γ, STAT1, pSTAT1, and T-bet), Th17 (IL-17A, STAT3, pSTAT3, and RORγt), and regulatory T cells (Treg, IL-10, TGF-β1, and FoxP3) expressed in splenic CD4+ T cells. Moreover, we analyzed the effects of S3I-201 on mRNA and protein expression of IFN-γ, T-bet, IL-17A, STAT1, STAT3, pSTAT1, pSTAT3, RORγ, IL-10, TGF-β1, and FoxP3 in the brains of EAE mice. The severity of clinical scores decreased in S3I-201-treated EAE mice compared to vehicle-treated EAE mice. S3I-201 treatment significantly decreased CD4+IFN-γ+, CD4+STAT1+, CD4+pSTAT1+, CD4+T-bet+, CD4+IL-17A+, CD4+STAT3+, CD4+pSTAT3+, and CD4+RORγt+ and increased CD4+IL-10+, CD4+TGF-β1+, and CD4+FoxP3+ in the spleens of EAE mice. Additionally, S3I-201 administration in EAE mice significantly decreased the mRNA and protein expression of Th1 and Th17 and increased those of Treg. These results suggest that S3I-201 may have novel therapeutic potential against MS.

Abstract 4638: Integrative spatial analysis of paired IHC and H&amp;E images identifies Foxp3 enriched tumor-infiltrating lymphocytes associated with disease-free survival in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma

Abstract Background: Recent studies support that high levels of tumor-infiltrating lymphocytes (TILs) were associated with better prognosis in HPV(+) oropharyngeal squamous cell carcinoma (OPSCC). While these studies provide a potential role for TILs as a prognostic biomarker, the analyses were relied on manual quantification performed by pathologists, resulting in inter-observer variability. Deep Learning (DL)-based whole slide hematoxylin and eosin (H&amp;E) image analyses may overcome these challenges. However, this analysis only provides the presence of cell types without consideration for the functional roles of each cell within the tumor immune microenvironment (TME). In this work, we develop a computational pipeline to integrate paired H&amp;E and immunohistochemistry (IHC) images to functionally characterize TILs and investigate their prognostic utility. Methods: We analyzed 88 patients: 42 stage I, 39 stage II, and 7 stage III. Our data contain both H&amp;E and IHCs examining FoxP3, CD3, PD-L1, CD20, etc. on serial sections of the tissue. In-house DL-based H&amp;E analysis used to identify TILs, tumors, and stroma in each tumor, then performed registration between adjacent H&amp;E and IHC images from the same tissue. The patients were then classified into three basic immune phenotypes: immune inflamed (IN; high TILs in the tumor region), immune excluded (EX; TILs are mostly localized in stroma), and immune desert (ID; few/no TILs) based on TIL enrichment in the TME. To functionally characterize TILs, we quantified protein expression from the adjacent IHCs. For example, we further classified each patient into different subtypes based on enriched protein expression (e.g., FoxP3 high IN, FoxP3 low IN). We used the Kaplan-Meier method and multivariate Cox analysis to evaluate the prognostic value of different subtypes enriched with different proteins to predict disease-free survival (DFS). Results: The IN group with 43 patients was significantly associated with good prognosis. Interestingly, further stratification of the IN subgroup based on Foxp3 quantification on TIL regions (i.e., high FoxP3 IN and low FoxP3 IN) showed that high protein expression of FoxP3 in TILs in the IN subgroup is significantly associated with a better prognosis compared to other immune subgroups (HR, 0.16; p-value, 0.003). Multivariate analysis, including other clinical covariates showed that the immune subtypes associated with high FoxP3 are independently associated with DFS. These results demonstrate that DL-based integrative IHC and H&amp;E image analysis could be used to identify subgroups with distinct clinical outcomes. Furthermore, our results reveal unknown roles for Foxp3 expression in the TILs in HPV(+) OPSCC as a prognostic biomarker, a finding which should be evaluated in a larger cohort. Citation Format: Sumanth Reddy Nakkireddy, Inyeop Jang, Minji Kim, Linda X. Yin, Michael Rivera, Joaquin J. Garcia MD, Kathleen R. Bartemes, David M. Routman, Eric J. Moore, Daniel J. Ma, Kathryn M. Van Abel, Tae Hyun Hwang. Integrative spatial analysis of paired IHC and H&amp;E images identifies Foxp3 enriched tumor-infiltrating lymphocytes associated with disease-free survival in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4638.

Compound small peptide of Chinese medicine alleviates cyclophosphamide induced immunosuppression in mice by Th17/Treg and jejunum intestinal flora.

The aim of this study was to explore the efficacy of Compound small peptide of Chinese medicine (CSPCM) on cyclophosphamide (CTX) induced immunosuppression in mice. The 100 male Kunming mice were divided into 5 groups: group A (control group), group B (model group), group C (100 mg/kg.bw CSPCM), group D (200 mg/kg.bw CSPCM) and group E (400 mg/kg.bw CSPCM). At 1-3 days, mice of group B, C, D and E were intraperitoneally injected with 80 mg/kg.bw CTX. The results showed that compared with group A, the immune organ index, body weight change, RORγ T gene expression, RORγ T protein expression, CD3+ cell number, Th17 number and Alpha index, white blood cell count, lymphocyte count and monocyte count were significantly decreased in group B (p < 0.05), while Foxp3 gene expression, Foxp3 protein expression and Treg cell number were significantly increased (p < 0.05), CSPCM has a good therapeutic effect on the above abnormalities caused by CTX. CTX caused the decrease of intestinal flora richness and the abnormal structure of intestinal flora, and CSPCM could change the intestinal flora destroyed by CTX to the direction of intestinal flora of healthy mice. On the whole, CSPCM has a good therapeutic effect on CTX-induced immunosuppression in mice, which is reflected in the index of immune organs, the number of T lymphocytes and Th17 cells increased, the number of Treg cells decreased and the structure of intestinal flora was reconstructed.

Differential Spatial Gene and Protein Expression Associated with Recurrence Following Chemoradiation for Localized Anal Squamous Cell Cancer.

The identification of transcriptomic and protein biomarkers prognosticating recurrence risk after chemoradiation of localized squamous cell carcinoma of the anus (SCCA) has been limited by a lack of available fresh tissue at initial presentation. We analyzed archival FFPE SCCA specimens from pretreatment biopsies prior to chemoradiation for protein and RNA biomarkers from patients with localized SCCA who recurred (N = 23) and who did not recur (N = 25). Tumor cells and the tumor microenvironment (TME) were analyzed separately to identify biomarkers with significantly different expression between the recurrent and non-recurrent groups. Recurrent patients had higher mean protein expression of FoxP3, MAPK-activation markers (BRAF, p38-MAPK) and PI3K/Akt activation (phospho-Akt) within the tumor regions. The TME was characterized by the higher protein expression of immune checkpoint biomarkers such as PD-1, OX40L and LAG3. For patients with recurrent SCCA, the higher mean protein expression of fibronectin was observed in the tumor and TME compartments. No significant differences in RNA expression were observed. The higher baseline expression of immune checkpoint biomarkers, together with markers of MAPK and PI3K/Akt signaling, are associated with recurrence following chemoradiation for patients with localized SCCA. These data provide a rationale towards the application of immune-based therapeutic strategies to improve curative-intent outcomes beyond conventional therapies for patients with SCCA.

Myofibroblast stroma differentiation in infiltrative basal cell carcinoma is accompanied by regulatory T-cells.

The implications of infiltrative compared to non-infiltrative growth of cutaneous basal cell carcinoma (BCC) on the tumor stroma and immune cell landscape are unknown. This is of clinical importance, because infiltrative BCCs, in contrast to other BCC subtypes, are more likely to relapse after surgery and radiotherapy. This descriptive cross-sectional study analyzed 38 BCCs collected from 2018 to 2021. In the first cohort (n=28), immune cells were characterized by immunohistochemistry and multiplex immunofluorescence staining for CD3, CD8, CD68, Foxp3, and α-SMA protein expression. In the second cohort (n=10) with matched characteristics (age, sex, location, and BCC subtype), inflammatory parameters, including TGF-β1, TGF-β2, ACTA2, IL-10, IL-12A, and Foxp3, were quantified via RT-qPCR after isolating mRNA from BCC tissue samples and perilesional skin. Infiltrative BCCs showed significantly increased levels of α-SMA expression in fibroblasts (p=0.0001) and higher levels of Foxp3+ (p=0.0023) and CD3+ (p=0.0443) T-cells compared to non-infiltrative BCCs. CD3+ (p=0.0171) and regulatory T-cells (p=0.0026) were significantly increased in α-SMA-positive tumor stroma, whereas CD8+ T-cells (p=0.1329) and CD68+ myeloid cells (p=0.2337) were not affected. TGF-β1 and TGF-β2 correlated significantly with ACTA2/α-SMA mRNA expression (p=0.020, p=0.005). Infiltrative growth of BCCs shows a myofibroblastic stroma differentiation and is accompanied by an immunosuppressive tumor microenvironment.

Effects of lactate on metabolism and differentiation of CD4+T cells

BackgroundLactate accumulation caused by abnormal tumor metabolism can induce the formation of an inhibitory immune microenvironment through a variety of pathways, which is characterized by regulatory T cells (Treg) infiltration and effector T cells (Teff) depletion. Studies have found that the key reason why Treg cells can survive in harsh environments lies in their flexible metabolic mode, which can use lactate in tumor microenvironment (TME) as an alternative energy substance to maintain their inhibitory activity. In addition, lactate could also promote the differentiation of CD4+T cells into Treg, but the mechanism was not completely clear. The purpose of this study was to investigate the possible mechanism by which lactate is utilized by CD4+T cells to influence Th17/Treg ratio. MethodsBasal cytokines (anti-CD3, anti-CD28, TGF-β) and 10 mM lactate was added into Naïve CD4+T cells basal medium for 3 days. After TCR stimulation, Naïve CD4+T converted to CD4+T. Flow cytometry was used to detect the proportion of Treg cells; ELISA was used to detect the activity of LDHA, LDHB and NADH and the amount of α -Ketoglutaric Acid (α-KG) and 2-Hydroxyglutaric Acid (2HG) after lactate entered the cells; Western Blot and RT-PCR were used to detect the protein and gene expression of Foxp3, RORγt, LDHA and LDHB. In the validation experiment, lactate uptake inhibitor AZD3965, LDHA inhibitor GSK2837808A and NADH conversion inhibitor Rotenone were added respectively to observe the differentiation ratio of Treg cells and confirm the key points of metabolism; the degradation of Treg cell transcription factor Foxp3 was interfered with ubiquitination inhibitors to observe whether it co-ubiquitinated with HIF-1α; the expression and activity of LDHA, LDHB and NADH in mitochondria and cytoplasm were detected to confirm cell localization. ResultsWhen basal cytokines (anti-CD3, anti-CD28, TGF-β) stimulated, lactate was added to the culture medium, and CD4+T cells absorbed a large amount of lactate not only through MCT1 (monocarboxylic acid transporter), but also increased the expression of lactate dehydrogenase and accelerated the intracellular metabolism of lactate. LDHB in cytoplasm mainly catalyzed the dehydrogenation of lactate to pyruvate, accompanied by the transformation reaction between NAD+ and NADH. The latter further entered the mitochondria and participates in the tricarboxylic acid cycle metabolism. In addition, lactate could significantly increase the level of LDHA in mitochondria and promote the transformation of α-KG to 2HG, accompanied by the transformation of NADH to NAD+. These metabolic changes eventually led to an increase in the intracellular 2HG/α-KG ratio. Abnormal 2HG increased the proportion of Treg by inhibiting ATP5B-mediated phosphorylation of mTOR and the synthesis of HIF-1α, causing it not be enough to ubiquitinate and degrade with Foxp3. ConclusionsLactate plays an important role in regulating the differentiation of Treg cells, inducing the expression and function of LDHA and promoting the transformation of α-KG to 2HG may be an important mechanism.

Expression of Foxp3 in Fibromas and Fibrosarcomas of Skin and Subcutaneous Tissue in Dogs.

Foxp3 is a transcription factor responsible for the formation of T regulatory lymphocytes. Foxp3 expression can be associated with either neoplastic progression or regression. The aim of the study was to evaluate Foxp3 expression in soft tissue tumours (fibromas and fibrosarcomas) of skin and subcutaneous tissue in dogs and to describe its relationship with tumour malignancy grade. The study was conducted on 71 skin and subcutaneous tumours including 31 fibromas and 40 fibrosarcomas. The samples underwent histological and immunohistochemical evaluation using anti-Foxp3, anti-Ki, and vimentin antibodies. Cytoplasmic expression of Foxp3 protein in the cutaneous and subcutaneous fibrosarcomas in dogs was confirmed. Moreover, a positive relationship between the expression of Foxp3 and tumour malignancy grade and between Foxp3 and Ki-67 expression was noted. A positive correlation between the Foxp3 expression intensity and malignancy grade suggests a significant role of Foxp3 in the carcinogenesis of skin and subcutaneous fibrosarcomas in dogs. Increased expression of Foxp3 may have a positive effect on the progression of cancer.

Gancao Fuzi decoction regulates the Th17/Treg cell imbalance in rheumatoid arthritis by targeting Foxp3 via miR-34a

Ethnopharmacological relevanceDuring the Eastern Han Dynasty, Zhang Zhongjing first recorded the Gancao Fuzi decoction (GCFZD) formula in the “Synopsis of the Golden Chamber”, which is reportedly an effective and safe treatment for rheumatoid arthritis (RA). However, the mechanism underlying the observed improvement in the T helper 17 (Th17)/regulatory T (Treg) cell imbalance in RA obtained with GCFZD has not been reported. Aim of the studyThis study aimed to demonstrate whether GCFZD ameliorated RA by modulating the Th17/Treg imbalance in RA mice. Materials and methodsCollagen was used to induce a model of collagen-induced arthritis (CIA) in mice. GCFZD was administered by gavage, and the arthritis index score, imaging and histopathological changes of the ankle joints, and the levels of the immunoglobulin G (IgG) class antibodies and proinflammatory factors in serum were determined. In addition, the frequencies of Th17 and Treg cells, the levels of relevant transcription factors and functional factors and the miR-34a gene in the spleen and the levels of interleukin-17A (IL-17A) and IL-10 in serum were determined. ResultsGCFZD significantly reduced the arthritis score, improved joint swelling and bone damage, reduced the pathological score, and decreased the serum levels of IgG class antibody (IgG and IgG2a) and proinflammatory factor [tumour necrosis factor-alpha (TNF-α), IL-1β and IL-6]. Moreover, the Th17-cell proportion, the expression level of the Th17-specific transcription factor retinoic acid-related orphan receptor γt (RORγt) and functional factor IL-17A in the spleen, and the serum IL-17A level were decreased, whereas the Treg cell proportion, expression levels of the Treg-specific transcription factor forkhead box P3 (Foxp3) and functional factor IL-10 in the spleen, and the serum IL-10 level were increased. Furthermore, GCFZD inhibited miR-34a gene expression while promoting Foxp3 protein expression. ConclusionsThe findings of this study demonstrate the therapeutic effect of GCFZD on mice with CIA, and the mechanism is related to an improvement in the Th17/Treg cell imbalance by targeting Foxp3 via miR-34a.

Glycyrrhiza uralensis Fisch. alleviates dextran sulfate sodium-induced colitis in mice through inhibiting of NF-κB signaling pathways and modulating intestinal microbiota

Ethnopharmacological relevanceLicorice is widely used in traditional Chinese Medicine (TCM) for compound compatibility, which could reduce toxicity and increase efficacy of certain herbal medicine, and its active components prominently effects of inhibit of inflammation and regulate of immunity. Aim of the studyThe study probed into the mechanism of the anti-inflammatory and immunomodulatory effects of licorice based on the domination of the T helper type 17/regulatory T cells (Th17/Treg) differentiation balance and the composition and structure of the intestinal flora through the nuclear factor kappa B (NF-κB) signaling pathway. Materials and methodsBALB/c mice were inoculated with dextran sulfate sodium (DSS) to establish animal models of ulcerative colitis (UC). For the pharmacodynamic study, UC mice were observed for the anti-inflammatory effect of licorice water extraction (LWE) in vivo, including clinical observation and measurement of colon length. Hematoxylin-eosin (HE) staining was used to evaluate pathological conditions. Immunohistochemistry (IHC) and transmission electron microscopy (TEM) were performed to observe the intestinal barrier of the colons. Inflammatory cytokine levels were measured using with enzyme-linked immunosorbent assay (ELISA) kits. The proportions of T helper (Th) cells in the colons was assessed using flow cytometry. Gut microbiota diversity was detected using 16S ribosomal (r)DNA sequencing. In addition, Western blot (WB) assays were used to verify ROR-γt, Foxp3, TLR4, MyD88 and NF-κB expression according to a standard protocol. ResultsLWE exerted a pharmacological anti-inflammatory effect by attenuating inflammation in the colonic tissues through affecting the protein expression of TLR4/MyD88/NF-κB, and increasing the expression of tight junction (TJ) protein in the colons, improving the integrity of the intestinal mucosal barrier in vivo. Moreover, LWE reversed the imbalance in Th17/Treg cells differentiation and influenced the protein expression of ROR-γt and Foxp3 in UC mouse colons. In particular, LWE significantly affected the diversity of the gut microbiota in UC mice, ameliorated the composition of dominant species, and significantly increased the type and quantity of probiotics. ConclusionLicorice tends to reduce inflammation and enhance the protective action of the intestinal mucosal barrier via the TLR4/MyD88/NF-κB signal transduction pathway and alter the imbalance of Th-cell differentiation. Notably, licorice may affect the diversity of intestinal microbiota and the content of beneficial bacteria in the colon, which is a potential mechanism for understanding anti-inflammatory and immunomodulatory effects in UC mice in vivo.

Bifidobacterium infantis Promotes Foxp3 Expression in Colon Cells via PD-L1-Mediated Inhibition of the PI3K-Akt-mTOR Signaling Pathway.

AimOur objective was to investigate whether Bifidobacterium infantis inhibits PI3K-Akt-mTOR signaling and upregulates Foxp3 expression through PD-L1 and to explore the possible mechanism of action of B. infantis in cellular immunosuppression. MethodThe effects of B. infantis supernatant on PD-L1, PD-1, Foxp3, and the PI3K-Akt-mTOR signaling pathway were observed by culturing HCT-116 cells. Simultaneously, the effects of blocking PD-L1 on PD-1, on Foxp3 protein and mRNA, and on the PI3K-Akt-mTOR signaling pathway protein were observed.ResultsB. infantis supernatant was able to upregulate the protein and mRNA expression of PD-L1 and Foxp3 and downregulate the phosphorylated protein expression of PI3K, Akt, and mTOR (P < 0.05); however, for PI3K, Akt, and mTOR, there was no change in the total protein expression. After the blocking of PD-L1, the stimulatory effect of B. infantis supernatant on Foxp3 and the inhibitory effect on the phosphorylated protein expression of PI3K, Akt, and mTOR were weakened.ConclusionB. infantis may inhibit the PI3K-Akt-mTOR signaling pathway and promote the expression of Foxp3 through PD-L1, which may be a target via which B. infantis exerts its immunosuppressive effect.

Wheatgerm Supplementation Reduces Gut Inflammation and Epithelial Barrier Dysfunction in IL-10 KO Mice Fed Atherogenic Diet

Abstract Objectives Wheat germ (WG) contains many bioactive compounds with the potential to maintain an anti-inflammatory gut environment. This study investigated the effects of WG supplementation on gut inflammation and integrity in high-fat fed interleukin (IL)-10 KO mice. Methods Eight-wk-old female B6.129P2-Il10tm1Cgn/J (IL-10KO) and C57BL/6 (WT) mice (n = 10/group) were randomly assigned to diets: WT fed a control diet (WTCO; AIN93-M) and IL-10 KO mice fed control (KOCO), high-fat with high-cholesterol (HFHC; 45% fat kcal, 1% cholesterol), or HFHC + 10% WG (HFWG) for 3 m. Disease activity indices (fecal blood, ruffled fur, stool softness, and rectal prolapse) were monitored twice a week. Fecal indole and short chain fatty acids (SCFAs) concentration were assessed at the beginning and end of study. Proinflammatory cytokines were assessed in the serum and ileum. Ileal and colonic protein expression of transcription factors (STAT3, p-STAT3, PPARg, FoxP3, and AhR), tight junction proteins (ZO-1, occludin), and tryptophan catabolizing enzyme (IDO-1) were assessed by immunoblotting. Relative ileal and colonic gene expression of IL-22 and antimicrobial peptides (Reg3b and Reg3g) were assessed using qRT-PCR. P &amp;lt; 0.05 was considered statistically significant. Results WG increased (P = 0.003) colon length compared to the HFHC group. Weight loss (12.2% in HFHC vs WTCO) was not prevented by WG, but disease activity indices were significantly reduced in the WG vs HFHC group. WG also increased fecal indole, total SCFAs and acetate accompanied by an increase in colonic protein expression of PPARg (P &amp;lt; 0.0001) and FoxP3 (P = 0.001). Ileal STAT3 phosphorylation was reduced (P = 0.0076) due to WG supplementation. An increased colon and ileal protein expression of IDO-1 in the HFHS group was reduced by WG, while also increasing the expression of AhR, ZO-1, and occludin. The relative gene expression of the antimicrobial peptides (Reg3b and Reg3g) was increased (P &amp;lt; 0.05) while serum and ileal tissue concentration of the proinflammatory cytokine, IL-17 was reduced (P = 0.0165 and p = 0.0248 respectively) by WG. Conclusions WG modulated changes that are associated with HF-feeding in IL-10 KO mice, and might be a promising regimen for ameliorating the effects of gut inflammation. Funding Sources Oklahoma Agriculture Experiment Station, Jim and Lynn Williams Professorship, and Barbara K. Pass Grant.

A novel immune cell signature predicts pathological complete response to neoadjuvant chemotherapy in triple negative breast cancer patients in the Q-CROC3 trial.

e12614 Background: Tumor infiltrating lymphocytes (TILs) have been associated with good prognosis and response to neoadjuvant chemotherapy. Several reports have shown that the heterogeneity of tumor infiltrating immune cells affects the response to chemotherapy, with for example low levels of FOXP3 expressing T cells associated with good prognosis and pathological complete response (pCR) to chemotherapy. Methods: We examined different immune cell markers on 52 pre-chemotherapy biopsy specimens obtained from triple negative breast cancer patients undergoing neo-adjuvant chemotherapy from the Q-CROC-03 trial. Slides were stained for CD8, CD3,PD-1, PDL-1, FOXP-3 and Granzyme B using multi-colour immunohistochemistry and automated cell counting of stroma and epithelial counts was conducted using the Vectra/inForm image analysis platform. We had total of 39 variables for analysis and we performed Penalized logistic regression for variable selection. Results: Nine variables were found statistically significant to predict response to chemotherapy, PD1+ stroma counts being the one with the highest probability of association with response. A tree algorithm was then used on all 9 variables to identify the best variable and threshold combination to identify patients who respond to chemotherapy. We separated our cohort in test (25% of samples n = 13) and training (75% of samples n = 39) sets for this analysis. Restricting the tree depth to 2 variables for clinical interpretability identified the combination of average counts of stromal PD1+ and average density of stromal FOXP3+ as predictors of chemo response (accuracy 0.82). Both stromal average PD1+ counts and average stromal FOXP3+ density positively correlated with the levels of TILS. Conclusions: Combining FOXP3 and PD1 protein expression in the stroma of pre-treatment biopsies of triple negative breast cancers receiving neoadjuvant chemotherapy is highly predictive of pCR.

LncRNA JPX contributes to Treg/Th17 imbalance in allergic rhinitis via targeting the miR-378g/CCL5 axis

Aim: T-regulatory (Treg)/T-helper (Th) 17 imbalance contributes to the pathogenesis of allergic rhinitis (AR). Long non-coding RNAs (lncRNAs) participate in the progression of AR. Herein, the effect of lncRNA JP X on Treg/Th17 balance in AR was explored. Methods: CD4+ T cells were isolated from patients with AR and healthy control. The percentage of Treg and Th17 cells were examined by flow cytometry. The levels of JP X, miR-378g, CCL5, T GF-β, and IL-17A were tested using qRT-P CR. The protein expression of Foxp3 and RORγt was measured by western blot. Results: The data showed that an imbalance of Treg/Th17 was associated with AR. Upregulation of JP X was found in AR, and knockdown of which improved the imbalance of Treg/Th17. Furthermore, JP X functioned as a sponge of miR-378g to upregulate CCL5. Inhibition of miR-378g reversed the effects on Treg/Th17 induced by silencing of JP X. Moreover, overexpression of CCL5 reversed miR-378g-induced effects. Conclusion: In conclusion, depletion of JP X promoted Treg/Th17 balance in AR via regulating the miR-378g/CCL5 axis. The findings provided a novel therapeutic insight for AR.