Effect of Dietary Supplementation with Omega-3 Fatty Acid on the Generation of Regulatory T Lymphocytes and on Antioxidant Parameters and Markers of Oxidative Stress in the Liver Tissue of IL-10 Knockout Mice.

INTRODUCTION: chronic low-grade inflammation, or inflammaging, emerges as a crucial element in the aging process and is associated with cardiovascular and neurological diseases, sarcopenia, and malnutrition. Evidence suggests that omega-3 fatty acids present a potential therapeutic agent in the prevention and treatment of inflammatory diseases, mitigating oxidative stress, and improving muscle mass, attributes that are particularly relevant in the context of aging. The objective of the present study was to evaluate the effectiveness of supplementation with omega-3 fish oil in improving the immune response and oxidative stress in knockout mice for interleukin IL-10 (IL-10-/-). MATERIAL AND METHODS: female C57BL/6 wild-type (WT) and interleukin IL-10 knockout (IL-10-/-) mice were fed during 90 days with a standard diet (control groups), or they were fed/supplemented with 10% of the omega-3 polyunsaturated fatty acid diet (omega-3 groups). Muscle, liver, intestinal, and mesenteric lymph node tissue were collected for analysis. RESULTS: the IL-10-/-+O3 group showed greater weight gain compared to the WT+O3 (p = 0.001) group. The IL-10-/-+O3 group exhibited a higher frequency of regulatory T cells than the IL-10-/- group (p = 0.001). It was found that animals in the IL-10-/-+O3 group had lower levels of steatosis when compared to the IL-10-/- group (p = 0.017). There was even greater vitamin E activity in the WT group compared to the IL-10-/-+O3 group (p = 0.001) and WT+O3 compared to IL-10-/-+O3 (p = 0.002), and when analyzing the marker of oxidative stress, MDA, an increase in lipid peroxidation was found in the IL-10-/-+O3 group when compared to the IL-10-/- group (p = 0.03). Muscle tissue histology showed decreased muscle fibers in the IL-10-/-+O3, IL-10-/-, and WT+O3 groups. CONCLUSION: the findings show a decrease in inflammation, an increase in oxidative stress markers, and a decrease in antioxidant markers in the IL-10-/-+O3 group, suggesting that supplementation with omega-3 fish oil might be a potential intervention for inflammaging that characterizes the aging process and age-related diseases.

Calcitriol restrains microglial M1 polarization and alleviates dopaminergic degeneration in hemiparkinsonian mice by boosting regulatory T-cell expansion.

OBJECTIVE: Vitamin D deficiency is a risk factor for Parkinson's disease (PD) and vitamin D supplementation robustly alleviates neurodegeneration in PD models. However, the mechanisms underlying this effect require further clarification. Current evidence suggests that harnessing regulatory T cells (Treg) may mitigate neuronal degeneration. In this study, we investigated the therapeutic effects of vitamin D receptor activation by calcitriol on PD, specifically focusing on its role in Treg. METHODS: Hemiparkinsonian mice model was established through the injection of 6-OHDA into the striatum. Mice were pretreated with calcitriol before 6-OHDA injection. The motor performance, dopaminergic neuronal survival, contents of dopamine, and dopamine metabolites were evaluated. The pro-inflammatory cytokines levels, T-cell infiltration, mRNA expression of indicated microglial M1/M2 phenotypic markers, and microglial marker in the midbrain were detected. Populations of Treg in the splenic tissues were assessed using a flow cytometry assay. PC61 monoclonal antibody was applied to deplete Treg in vivo. RESULTS: We show that calcitriol supplementation notably improved motor performance and reduced dopaminergic degeneration in the 6-OHDA-induced PD model. Mechanistically, calcitriol promoted anti-inflammatory/neuroprotective Treg and inhibited pro-inflammatory/neurodestructive effector T-cell generation in this model. This process significantly inhibited T-cell infiltration in the midbrain, restrained microglial activation, microglial M1 polarization, and decreased pro-inflammatory cytokines release. This more favorable inflammatory microenvironment rescued dopaminergic degeneration. To further verify that the anti-inflammatory effects of calcitriol are associated with Treg expansion, we applied an antibody-mediated Treg depletion assay. As predicted, the anti-inflammatory effects of calcitriol in the PD model were diminished following Treg depletion. CONCLUSION: These findings suggest that calcitriol's anti-inflammatory and neuroprotective effects in PD are associated with its potential to boost Treg expansion.

Huangqi-Guizhi-Wuwu decoction regulates differentiation of CD4+ T cell and prevents against experimental autoimmune encephalomyelitis progression in mice.

BACKGROUND: Multiple sclerosis (MS) is a demyelination disorder caused by an overactive immune response. Its pathological characteristics include CNS inflammation, white matter demyelination, glial cell proliferation, and so on. Huangqi-Guizhi-Wuwu Decoction (HGWD), which is recorded in the Synopsis of the Golden Chamber, is used clinically for the therapy of MS, but its mechanism is still elusive. PURPOSE: This study was aimed to investigate the impact of HGWD on the classical animal model for MS, experimental autoimmune encephalomyelitis (EAE), and explore the underlying action mechanism. RESULTS: HGWD ameliorated the pathogenesis of EAE mice, and improved their neurobehavior and pathological tissue damage. Network pharmacology predictions revealed the action mechanism of HGWD in EAE mice might be related to its effect on the immune system of mice. HGWD effectively suppressed the inflammatory infiltration in CNS, while also preventing the elevation of CD4+T cells of mice with EAE. HGWD could increase the ratio of Treg cells, up-regulate the secretion of IL-10 and Foxp3 mRNA expression, inhibit the ratio of Th1 and Th17 cells, down-regulate the IFN-γ and IL-17 protein expression, as well as the RORγT and T-bet gene expression in EAE mice. In addition, HGWD-containing serum modulated Th1/Th17/Treg cell differentiation in vitro. Moreover, HGWD inhibited the p-JAK1, p-JAK2, p-STAT1, p-STAT3 and p-STAT4 proteins and elevated the p-STAT5 protein in lymphoid tissues of EAE mice. CONCLUSION: HGWD improved the progress of EAE by regulating the proportion of CD4+T cell subtype differentiation, which might be exerted through JAK/STAT signaling pathway, providing a pharmacological basis for the clinical treatment of MS.

A novel mechanism for regulating lung immune homeostasis: Zukamu granules alleviated acute lung injury in mice by inhibiting NLRP3 inflammasome activation and regulating Th17/Treg cytokine balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a severely acute lung inflammation with high morbidity and mortality. Zukamu granules (ZKMG) is one of the Uygur patent drugs commonly used in clinic, which is included in the National Essential Drugs List (2018 edition). Clinical studies have shown that ZKMG has a significant effect on acute upper respiratory tract infection, and has better anti-inflammatory and antipyretic effects. However, the immunomodulatory mechanism of ZKMG on ALI is still not clear. AIM OF THE STUDY: The aim of this study is to investigate the lung protective effect and immunomodulatory mechanism of ZKMG on lipopolysaccharide (LPS) -induced ALI mice, and to provide an important basis for the treatment strategy and theoretical basis of ALI. MATERIALS AND METHODS: First, network pharmacology was used to predict the potential signaling pathways and biological processes of ZKMG related to immunology. Molecular docking technique was used to predict the possibility between the core components of ZKMG acting on NLRP3 protein. In addition, protein levels of F4/80 in lung tissues were assessed by Immunohistochemistry (IHC). The contents of IL-1ß, IL-18, IL-17A and IL-10 in the lung tissue and serum, MPO in the lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR analysis (RT-qPCR) was used to detect NLRP3 mRNA in lung tissue. Protein levels of NLRP3, Caspase-1, Cleaved caspase-1 p20, ASC, and GSDMD were detected by Western blot (WB). RESULTS: The results of network pharmacology showed that the immune pathways of ZKMG were mainly Th17 signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, etc. Molecular docking results showed that the core components of ZKMG had good binding ability to NLRP3 protein. The verification experiments showed that ZKMG can reduce the degree of lung injury, and reduce the level of inflammatory infiltration of neutrophils and macrophages by reducing the content of MPO and F4/80. In addition, ZKMG can reduce NLRP3 mRNA, inhibit the expression of NLRP3/Caspase-1/GSDMD and other related pathway proteins, and reduce inflammatory factors such as IL-1ß and IL-18. It can also reduce the content of pro-inflammatory cytokine IL-17A, increase the content of anti-inflammatory cytokine IL-10 in lung tissue. CONCLUSION: ZKMG can reduce the degree of lung tissue injury in ALI by inhibiting NLRP3/Caspase-1/GSDMD signaling pathway and restoring the IL-17A/IL-10 cytokine balance, and its protective mechanism may be related to the regulation of lung immune homeostasis. It will provide a new strategy for studying the regulation of lung immune homeostasis.

Phellinus linteus activates Treg cells via FAK to promote M2 macrophage polarization in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent malignant tumor worldwide. Within HCC's tumor microenvironment, focal adhesion kinase (FAK) plays a critical role. Regulatory T cells (Treg) modulate the polarization of tumor-associated macrophages , but the relationship between FAK, Treg cells, and macrophages remains underexplored. Phellinus linteus (PL) shows promise as a treatment for HCC due to its pharmacological effects. This study aimed to explore the relationship between FAK and Treg-macrophages and to assess whether PL could exert a protective effect through the FAK process in HCC. Initially, C57BL/6-FAK-/- tumor-bearing mice were utilized to demonstrate that FAK stimulates HCC tumor development. High dosages (200 µM) of FAK and the FAK activator ZINC40099027 led to an increase in Treg (CD4+CD25+) cells, a decrease in M1 macrophages (F4/80+CD16/32+, IL-12, IL-2, iNOS), and an increase in M2 macrophages (F4/80+CD206+, IL-4, IL-10, Arg1, TGF-ß1). Additionally, FAK was found to encourage cell proliferation, migration, invasion, and epithelial-mesenchymal transition while inhibiting apoptosis in HepG2 and SMMC7721 cells. These effects were mediated by the PI3K/AKT1/Janus Kinase (JAK)/ signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase (p38 MAPK)/Jun N-terminal Kinase (JNK) signaling pathways. Furthermore, PL exhibited a potent antitumor effect in vivo in a dose-dependent manner, reducing FAK, Treg cells, and M2 macrophages, while increasing M1 macrophages. This effect was achieved through the inhibition of the PI3K/AKT/JAK/STAT3, and p38/JNK pathways. Overall, our findings suggest that FAK promotes HCC via Treg cells that polarize macrophages toward the M2 type through specific signaling pathways. PL, acting through FAK, could be a protective therapy against HCC.

Pien Tze Huang alleviates Concanavalin A-induced autoimmune hepatitis by regulating intestinal microbiota and memory regulatory T cells.

BACKGROUND: Traditional Chinese medicine has used the drug Pien Tze Huang (PTH), a classic prescription, to treat autoimmune hepatitis (AIH). However, the precise mode of action is still unknown. AIM: To investigate the mechanism of PTH in an AIH mouse model by determining the changes in gut microbiota structure and memory regulatory T (mTreg) cells functional levels. METHODS: Following induction of the AIH mouse model induced by Concanavalin A (Con A), prophylactic administration of PTH was given for 10 d. The levels of mTreg cells were measured by flow cytometry, and intestinal microbiota was analyzed by 16S rRNA analysis, while western blotting was used to identify activation of the toll-like receptor (TLR)2, TLR4/nuclear factor-κB (NF-κB), and CXCL16/CXCR6 signaling pathways. RESULTS: In the liver of mice with AIH, PTH relieved the pathological damage and reduced the numbers of T helper type 17 cells and interferon-γ, tumor necrosis factor-alpha, interleukin (IL)-1ß, IL-2, IL-6, and IL-21 expression. Simultaneously, PTH stimulated the abundance of helpful bacteria, promoted activation of the TLR2 signal, which may enhance Treg/mTreg cells quantity to produce IL-10, and suppressed activation of the TLR4/NF-κB and CXCL16/CXCR6 signaling pathways. CONCLUSION: PTH regulates intestinal microbiota balance and restores mTreg cells to alleviate experimental AIH, which is closely related to the TLR/CXCL16/CXCR6/NF-κB signaling pathway.

A Study of the Predictive Value of Treg and Th1/Th2 Cytokines on Pregnancy Outcome in Patients with Recurrent Pregnancy Loss.

Objectives: To investigate the influence of helper T cell type 1 (Th1)/Th2 cytokines and Treg cells on the pregnancy outcome of patients with recurrent pregnancy loss (RPL) and to reveal the predictive value of their combination on the pregnancy outcome. Methods: The 165 RPL patients admitted to the Reproductive Medicine Department of the Second Hospital of Lanzhou University from September 2019 to June 2021 served as the research subjects for the study. The subjects comprised a live birth group (102 patients) or a non-live birth group (63 patients) based on their pregnancy outcomes. All patients were tracked through the end of pregnancy. Flow cytometry was applied to determine Treg and Th1/Th2 cytokine levels in the peripheral blood of patients without pregnancy. Results: The levels of interleukin-6 (IL-6), IL-2, IL-10, and Treg in the RPL live birth group were significantly higher than those in the non-live birth group. The ratio of TNF - α/ IL-6, NF-α/ IL-10, IFN- γ/ IL-6, IFN- γ/ IL-10 in the non-live birth group increased significantly. The area under the curve (AUC) of Th1/Th2 cytokines combined with Treg cells was 0.786 (95% CI: 0.712-0.860), the specificity was 76.2%, and the sensitivity was 74.5%. Conclusion: Treg and Th1/Th2 cytokines showed a predictive ability for the pregnancy outcome of patients with recurrent pregnancy loss and their higher combined predictive efficacy. Meanwhile, in the Th1/Th2 immune response, its ratio was more important than the expression of a single cytokine.

Association of Nonpuerperal Mastitis with Cytokines Related to Helper T Cells TH1/TH2 and TH17/Treg.

Objective: To investigate the association of nonpuerperal mastitis with cytokines related to the helper T cells TH1/TH2 and TH17/Treg and associated immune balance. Methods: From 2016 to 2021, we included 40 patients with non-puerperal mastitis who underwent surgery at China-Japan Friendship Hospital and compared them with 40 control patients with benign non-infectious breast disease. Hematoxylin-eosin staining detects inflammatory infiltrates of breast tissue. The expression of interferon γ and interleukin 4 in breast tissue was detected by immunofluorescence imaging, and the relative protein expression of TH1/TH2 and TH17/Treg cell-associated cytokines in CD4+ T cells was detected by western blotting. CD4+ T cells were isolated by fluorescence-activated cell sorting for detection of the relative protein expression of interferon γ and interleukin 4 in CD4+ T cells. Results: Hematoxylin-eosin staining showed that the nonpuerperal mastitis group had significantly greater inflammatory infiltration than the control group. Immunofluorescence images showed the relative fluorescence intensity of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative fluorescence intensity of interleukin 4 did not significantly differ between the 2 groups (P = .0686). Western blotting revealed that the relative protein expression of interferon γ, interleukin 2, and interleukin 17 was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 (P = .0512), interleukin 10 (P = .3088), and transforming growth factor ß (P = .0653) did not significantly differ between the 2 groups. Flow cytometry of isolated CD4+ T cells showed the relative protein expression of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 did not significantly differ between the 2 groups (P = .0680). Conclusion: The expression of the TH1 cytokines interferon γ and interleukin 2 and the TH17 cytokine interleukin 17 was significantly higher in patients with nonpuerperal mastitis, while the TH2 cytokine interleukin 4 and the Treg cytokines interleukin 10 and transforming growth factor ß were expressed at lower levels. This study provides new research ideas for the treatment of mastitis.

[The features of regulatory T-blood cells subpopulation and the level of CD25 expression in patients with Graves' disease in dynamics after radioactive iodine therapy].

BACKGROUND: The content of regulatory T cells (Treg) at different stages in formation of effector subpopulations and the level of CD25 expression on the membrane of their various fractions in Graves' disease can determine the long-term autoimmune process persistence and be the target of immunotropic therapy of the disease. AIM: To study the features of regulatory T-blood cells subpopulation and the level of CD25 expression in patients with Graves' disease in dynamics after radioactive iodine therapy (RIT) to identify the specific Treg subpopulations for potential immunotropic therapy targets of the disease. MATERIALS AND METHODS: A single-center, prospective, cohort, open, controlled study was conducted with the participation of women with laboratory-confirmed Graves' disease. The features of regulatory T-blood cells subpopulation and the level of expression (MFI) CD25 surface receptor were studied by flow cytometry using direct immunofluorescence using monoclonal antibodies. RESULTS: The study included 36 women with recurrent Graves' disease, middle age 46.34±14.32 years. In patients with Graves' disease before and during the entire period after RIT a low percentage of naive (CD45R0-CD62L+) and terminally differentiated (CD45R0-CD62L-) Treg was established relative to the control, and on 3 and 6 months after RIT a significant decrease of cells with this phenotype was revealed relative to the values detected in patients before and 1 month after RIT (p<0.001). Against the background of compensated hypothyroidism the most significant changes of expression CD25 receptor in patients with Graves' disease were found on 3 and 6 months after RIT: reduced levels of MFI CD25 on surface of naive and terminally differentiated Treg. CONCLUSION: A decrease in the level of naive Treg was found (apparently due to a violation of differentiation processes in thymus) and terminally differentiated Tregs (due to maturation and survival processes), which are supplemented by a reduced expression of the CD25 receptor on the surface of these cells and do not depend on hyperthyroidism compensation, the titer of TSH receptor antibodies, previous conservative therapy with thiamazole and RIT. The obtained new data reveal the role of naive and terminally differentiated Treg subpopulations in immunopathogenesis and help to outline further ways to develop approaches for immunotropic therapy.

Indole Propionic Acid Increases T Regulatory Cells and Decreases T Helper 17 Cells and Blood Pressure in Mice with Salt-Sensitive Hypertension.

Hypertension affects over a billion adults worldwide and is a major risk factor for cardiovascular disease. Studies have reported that the microbiota and its metabolites regulate hypertension pathophysiology. Recently, tryptophan metabolites have been identified to contribute to and inhibit the progression of metabolic disorders and cardiovascular diseases, including hypertension. Indole propionic acid (IPA) is a tryptophan metabolite with reported protective effects in neurodegenerative and cardiovascular diseases; however, its involvement in renal immunomodulation and sodium handling in hypertension is unknown. In the current study, targeted metabolomic analysis revealed decreased serum and fecal IPA levels in mice with L-arginine methyl ester hydrochloride (L-NAME)/high salt diet-induced hypertension (LSHTN) compared to normotensive control mice. Additionally, kidneys from LSHTN mice had increased T helper 17 (Th17) cells and decreased T regulatory (Treg) cells. Dietary IPA supplementation in LSHTN mice for 3 weeks resulted in decreased systolic blood pressure, along with increased total 24 h and fractional sodium excretion. Kidney immunophenotyping demonstrated decreased Th17 cells and a trend toward increased Treg cells in IPA-supplemented LSHTN mice. In vitro, naïve T cells from control mice were skewed into Th17 or Treg cells. The presence of IPA decreased Th17 cells and increased Treg cells after 3 days. These results identify a direct role for IPA in attenuating renal Th17 cells and increasing Treg cells, leading to improved sodium handling and decreased blood pressure. IPA may be a potential metabolite-based therapeutic option for hypertension.

Immune-related microRNAs in breast milk and their relation to regulatory T cells in breastfed children.

BACKGROUND: The immunomodulatory capacity of breast milk may partially be mediated by microRNAs (miRNA), small RNA molecules that regulate gene expression on a post-transcriptional level and are hypothesized to be involved in modulation of immunological pathways. Here, we evaluate the expression of immune-related miRNAs in breast milk after pre- and postnatal supplementation with Limosilactobacillus reuteri and omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), and the association to infant regulatory T cell (Treg) frequencies. METHODS: One-hundred and twenty women included in a double-blind, randomized, placebo-controlled allergy intervention trial received L. reuteri and/or ω-3 PUFAs daily from gestational week 20. Using Taqman qPCR, 24 miRNAs were analyzed from breast milk obtained at birth (colostrum) and after 3 months (mature milk) of lactation. The proportion of activated and resting Treg cells were analyzed in infant blood using flow cytometry at 6, 12, and 24 months. RESULTS: Relative expression changed significantly over the lactation period for most of the miRNAs; however, the expression was not significantly influenced by any of the supplements. Colostrum miR-181a-3p correlated with resting Treg cell frequencies at 6 months. Colostrum miR-148a-3p and let-7d-3p correlated with the frequencies of activated Treg cells at 24 months, as did mature milk miR-181a-3p and miR-181c-3p. CONCLUSION: Maternal supplementation with L. reuteri and ω-3 PUFAs did not significantly affect the relative miRNA expression in breast milk. Interestingly, some of the miRNAs correlate with Treg subpopulations in the breastfed children, supporting the hypothesis that breast milk miRNAs could be important in infant immune regulation. TRIAL REGISTRATION: ClinicalTrials.gov-ID: NCT01542970.

[Effect of electroacupuncture on Treg/Th17 balance in spleen and serum inflammatory factors in obese mice].

OBJECTIVE: To observe the therapeutic effect of electroacupuncture(EA) on obese mice, and to explore the underlying mechanism of EA in treating obesity by focusing on the balance of regulatory T cells (Treg) and T helper 17 cells (Th17) and related inflammatory factors. METHODS: C57BL/6J male mice were randomly divided into normal group, model group and EA group, with 10 mice in each group. The obesity model was established by feeding the mice with high-fat diet. Mice in the EA group was treated with EA at "Zhongwan"(CV12), "Guanyuan"(CV4), "Zusanli"(ST36) and "Fenglong"(ST40) for 20 min every time, 3 times every week, for a total of 8 weeks. The food intake and body weight of mice were observed and recorded, and Lee's index was calculated; the contents of interleukin 2(IL-2), IL-4, IL-6, IL-10, IL-17A, gamma interferon (IFN-γ) and tumor necrosis factor(TNF)-α in serum were detected by multiplex liquid chip quantitative technique; the levels of Treg and Th17 cells in mice spleen tissues were detected by flow cytometry; and the expression levels of foxhead box p3(Foxp3) and retinoic acid related orphan receptor γt(ROR-γt) mRNA in spleen were detected by real-time quantitative PCR. RESULTS: Compared with the normal group, the food intake, body weight, Lee's index, the contents of IL-2, IL-6, IL-17A, IFN-γ and TNF-α in the serum, and the percentage of Th17 and expression of ROR-γt mRNA in the spleen tissues were significantly increased (P<0.01, P<0.001), while the contents of IL-4 and IL-10 in the serum, the percentage of Treg and expression of Foxp3 mRNA in the spleen tissues were significantly decreased (P<0.001, P<0.01) in the model group. Compared with the model group, the food intake, body weight, Lee's index, the contents of IL-2, IL-6, IL-17A, IFN-γ, and TNF-α in the serum, the percentage of Th17 and expression of ROR-γt mRNA in the spleen tissues were significantly decreased (P<0.01), while the contents of IL-4 and IL-10 in serum, the percentage of Treg and expression of Foxp3 mRNA in the spleen tissues were significantly increased(P<0.01, P<0.05) in the EA group. CONCLUSION: EA may improve the obese state of mice by regulating the balance of Treg/Th17 in spleen and the expression of inflammatory factors in serum.

Prognostic Impact of Aberrantly Expressed Protein-coding Gene Associated With Gastric Cancer's Regulatory T Cells, Based on Online Databases.

Context: Gastric cancer (GC) remains one of the most prevalent malignancies worldwide, and no effective cure exists for advanced GC. Clinicians believe that molecularly targeted therapy through PCGs may replace surgery, radiotherapy, and other treatments as a breakthrough in curing malignancies. Objective: The study intended to examine the impact of aberrant expression of the protein-coding genes (PCGs) associated with regulatory T cells on the prognosis of patients with gastric cancer (GC). Design: The research team performed a genetic study through research of genetic data in online databases. Setting: The study took place at Zhongda Hospital. Outcome Measures: The research team selected a publicly available dataset, genetic suppressor element 109476 (GSE109476), from the Gene Expression Omnibus (GEO) database for differential gene analysis, gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to screen for PCGs associated with regulatory T cells as well as the Gene Expression Profiling Interactive Analysis (GEPIA) database with the Kaplan-Meier Plotter database to analyze the expression of the above PCGs in GC and the prognostic impact on GC. Results: The GEO2R analysis found 315 differentially expressed PCGs in GSE109476, among which nine PCGs were associated with regulatory T cells: (1) chemokine (C-C motif) ligand 19 (CCL19), (2) CCL21, (3) C-C chemokine receptor type 7 (CCR7), (4) cluster of differentiation 70 (CD70), (5) ephrin B3 (EFNB3), (6) early growth response 3 (EGR3), (7) interleukin-7 receptor (IL7R), (8) galectin-1 (LGALS1), and (9) tumor necrosis factor (TNF) receptor superfamily member 13C (TNFRSF13C). The GEPIA database indicated that no significant differences existed between the expression of CCL19, CCL21, CD70, EFNB3, EGR3, IL7R, and TNFRSF13C in stomach adenocarcinoma (STAD) tissues and that in normal tissues (P > .05), while expressions of CCR7 and LGALS1 were significantly elevated in STAD tissues compared to the normal tissues (P < .05). The Kaplan-Meier Plotter database analysis, on the other hand, showed a significant relationship between all of the above-mentioned PCGs, except CCL19, and the prognosis of GC. Conclusions: CCL19, CCL21, CCR7, CD70, EFNB3, EGR3, IL7R, LGALS1, and TNFRSF13C are PCGs are differentially expressed in GC and closely associated with regulatory T cells. They may affect the occurrence and development of GC through a variety of pathways, including regulation of immune infiltration and inflammation, and are of great potential research value.

Electro-acupuncture treatment inhibits the inflammatory response by regulating γδ T and Treg cells in ischemic stroke.

BACKGROUND: Electro-acupuncture (EA) is an effective and safe treatment for ischemic stroke. It is not only capable of reducing cerebral damage but also alleviating intestinal inflammation. However, its mechanism has not been fully elucidated. METHODS: All rats were randomly divided into three experimental groups: the SHAM group, the MCAO group, and the MEA (MCAO+EA) group. Ischemic-reperfusion (I/R) injury was induced by MCAO surgery. Rats in the MEA group were treated with EA stimulation in the "Baihui" acupoint (1 mA, 2/15 Hz, 20 min for each time). The Real-time (RT)-qPCR was used to evaluate the mRNA expression of inflammation factors in the ischemic brain and the small intestine after I/R injury. In addition, our research evaluated the effects of EA on regulatory T cells (Tregs) and γδ T cells in the small intestine and brain via Flow cytometry analysis. Finally, we applied CM-Dil and CFSE injection and explored the potential connections of T cells between the ischemic hemisphere and the small intestine. RESULTS: Our results suggested that EA treatment could significantly reduce the inflammation response in the ischemic brain and small intestine 3 days after I/R injury in rats. To be specific, EA increased the percentage of Tregs in the brain and the small intestine and decreased intestinal and cerebral γδ T cells. Concomitantly, after EA treatment, the percentage of cerebral CD3+TCRγδ+CFSE+ cells dropped from 12.06% to 6.52% compared with the MCAO group. CONCLUSIONS: These findings revealed that EA could regulate the Tregs and γδ T cells in the ischemic brain and the small intestine, which indicated its effect on inhibiting inflammation. And, EA could inhibit the mobilization of intestinal T cells, which may contribute to the protection of EA after ischemic stroke.

Electro-acupuncture treatment ameliorates intestinal inflammatory injury in cerebral ischemia-reperfusion rats via regulating the balance of Treg / γδ T cells.

Electro-acupuncture (EA) has an anti-inflammatory role in ischemic stroke, but whether the protective effect of EA involves the regulation of the intestine barrier and Treg/ γδ T cells is unclear. Cerebral ischemia-reperfusion (I/R) injury was induced by middle cerebral artery occlusion(MCAO) for 2 h followed by reperfusion for 24 h. The rats have treated with EA at the "Baihui" acupoint(GV20). Triphenyl tetrazolium chloride (TTC) staining and Longa neurologic score were performed to evaluate the outcomes after ischemic stroke. Inflammatory factor expression levels in the serum, ischemic hemisphere brain, and small intestine were detected by ELISA or RT-qPCR. Additionally, the morphology change of the small intestine was evaluated by analyzing villus height and smooth muscle thickness. Meanwhile, the expression of tight-junction proteins, including Zonula Occludens-1 (ZO-1), Occludin, and Claudin-1, were detected to evaluate the impact of EA on mucosal permeability in the small intestine. The percentages of regulatory T cells (Tregs) (CD45+CD4+Foxp3+) and γδ T cells (CD45+CD4-γδ T+) were measured to assess the effect of EA on intestinal T cells. EA decreased the brain infarction volume and intestine barrier injury in ischemic stroke rats. At the same time, it effectively suppressed the post-stroke inflammation in the brain and small intestine. More importantly, EA treatment increased the percentage of Tregs in the small intestine while reducing the rate of γδ T cells, and ultimately increased the ratio of Treg/ γδ T cells. These results demonstrated that EA ameliorated intestinal inflammation damage by regulating the Treg/ γδ T cell polarity shift and improving the intestine barrier integrity in rats with I/R injury. This may be one of the mechanisms underlying the anti-ischemic injury effects of acupuncture on stroke.

Salidroside alleviates ulcerative colitis via inhibiting macrophage pyroptosis and repairing the dysbacteriosis-associated Th17/Treg imbalance.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by flora disequilibrium and mucosal immunity disorder. Here, we report that salidroside effectively restricts experimental colitis from two aspects of intestinal macrophage pyroptosis and dysbacteriosis-derived colonic Th17/Treg imbalance. In innate immunity, the upregulated TREM1 and pyroptosis-related proteins in inflamed colons were inhibited by salidroside administration and further experiments in vitro showed that salidroside suppressed LPS/ATP-induced bone marrow-derived macrophages (BMDMs) pyroptosis evident by the decline of LDH and IL-1ß release as well as the protein level of NLRP3, caspase-1, and GSDMD p30. Moreover, the TREM1 inhibitor weakened the effect of salidroside on BMDMs pyroptosis, whereas salidroside still could downregulate TREM1 when NLRP3 was inhibited. In adaptive immunity, salidroside improved the gut microflora diversity and Th17/Treg ratio in DSS-induced mice, especially promoting the abundance of Firmicutes. Clearance of the gut flora blocked the benefit of salidroside on colonic inflammation and Th17/Treg adaptive immunity, but transplanting salidroside-treated foecal bacterium into flora-depleted wild mice reproduced the resistance of salidroside to gut inflammation. Taken together, our data demonstrated that salidroside protected experimental colitis via skewing macrophage pyroptosis and Th17/Treg balance, indicating its potential effect on UC and other immune disorders.

Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-ß in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-ß. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Manual acupuncture at ST36 attenuates rheumatoid arthritis by inhibiting M1 macrophage polarization and enhancing Treg cell populations in adjuvant-induced arthritic rats.

OBJECTIVES: Acupuncture has been found to be effective at relieving many inflammatory pain conditions, including rheumatoid arthritis (RA). We aimed to assess the anti-inflammatory potential of manual acupuncture (MA) treatment of RA using adjuvant-induced arthritic (AIA) rats and to explore the underlying mechanisms. METHODS: The anti-inflammatory and analgesic actions of MA at ST36 (Zusanli) in AIA rats were assessed using paw withdrawal latency and swelling, histological examination and cytokine detection by enzyme-linked immunoassay (ELISA). The cell-cell communication (CCC) network was analyzed with a multiplex immunoassay of 24 immune factors expressed in the inflamed joints, and the macrophage and Treg populations and associated cytokines regulated by MA were investigated using reverse-transcription quantitative polymerase chain reaction (RT-qPCR), ELISA and flow cytometry. RESULTS: MA markedly decreased heat hyperalgesia and paw swelling in AIA rats. MA-treated rats also exhibited decreased levels of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß) coupled with increased anti-inflammatory cytokines (IL-10, transforming growth factor (TGF)-ß1) in the ankle joints at protein and mRNA levels. CCC network analysis confirmed that macrophages are of critical importance and are potential therapeutic targets in RA. Repeated treatment with MA triggered a macrophage phenotypic switch in the paws, with fewer M1 macrophages. Prominent increases in the Treg cell population and TGF-ß1 in the popliteal lymph nodes demonstrated the immunomodulatory effects of MA. Furthermore, a selective TGF-ß1-receptor inhibitor, SB431542, attenuated the anti-inflammatory effects of MA and MA-induced suppression of the levels of M1-released cytokines. CONCLUSION: These findings provide novel evidence that the anti-inflammatory and analgesic effects of MA on RA act through phenotypic modulation involving the inhibition of M1 macrophage polarization and an increase in the Treg cell population, highlighting the potential therapeutic advantages of acupuncture in controlling pain and ameliorating inflammatory conditions.

Regulating Th17/Treg Balance Contributes to the Therapeutic Effect of Ziyuglycoside I on Collagen-Induced Arthritis.

To investigate the therapeutic effect and primary pharmacological mechanism of Ziyuglycoside I (Ziyu I) on collagen-induced arthritis (CIA) mice. CIA mice were treated with 5, 10, or 20 mg/kg of Ziyu I or 2 mg/kg of methotrexate (MTX), and clinical manifestations, as well as pathological changes, were observed. T cell viability and subset type were determined, and serum levels of transforming growth factor-beta (TGF-ß) and interleukin-17 (IL-17) were detected. The mRNA expression of retinoid-related orphan receptor-γt (RORγt) and transcription factor forkhead box protein 3 (Foxp3) in mouse spleen lymphocytes was ascertained by the real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Molecular docking was used to detect whether there was a molecular interaction between Ziyu I and protein kinase B (Akt). The activation of mechanistic target of rapamycin (mTOR) in T cells was verified by Western blotting or immunofluorescence. Ziyu I treatment effectively alleviated arthritis symptoms of CIA mice, including body weight, global score, arthritis index, and a number of swollen joints. Similarly, pathological changes of joints and spleens in arthritic mice were improved. The thymic index, T cell activity, and RORγt production of Ziyu I-treated mice were significantly reduced. Notably, through molecular docking, western blotting, and immunofluorescence data analysis, it was found that Ziyu I could interact directly with Akt to reduce downstream mTOR activation and inhibit helper T cell 17 (Th17) differentiation, thereby regulating Th17/regulatory T cell (Treg) balance and improving arthritis symptoms. Ziyu I effectively improves arthritic symptoms in CIA mice by inhibiting mTOR activation, thereby affecting Th17 differentiation and regulating Th17/Treg balance.

The Role of FOXP3 on Tumor Metastasis and Its Interaction with Traditional Chinese Medicine.

Forkhead box protein 3 (FOXP3) is an important transcription factor for regulatory T cells (Tregs) and plays an important role in their immunosuppressive function. In recent years, studies have found that FOXP3 is expressed in many kinds of tumors and plays different roles in tumors' biological behaviors, including tumor proliferation, metastasis, drug resistance, and prognosis. However, the effects of FOXP3 on tumor metastasis and its interaction with traditional Chinese medicine (TCM) remain unclear. Therefore, in this review, we focus on the effects of FOXP3 on tumor metastasis and its relationship with TCM, which can provide evidence for further research and therapy in clinical settings.