Dihydroartemisinin alleviates erosive bone destruction by modifying local Treg cells in inflamed joints: A novel role in the treatment of rheumatoid arthritis.

Treg cell-based therapy has exhibited promising efficacy in combatting rheumatoid arthritis (RA). Dihydroartemisinin (DHA) exerts broad immunomodulatory effects across various diseases, with its recent spotlight on T-cell regulation in autoimmune conditions. The modulation of DHA on Treg cells and its therapeutic role in RA has yet to be fully elucidated. This study seeks to unveil the influence of DHA on Treg cells in RA and furnish innovative substantiation for the potential of DHA to ameliorate RA. To this end, we initially scrutinized the impact of DHA-modulated Treg cells on osteoclast (OC) formation in vitro using Treg cell-bone marrow-derived monocyte (BMM) coculture systems. Subsequently, employing the collagen-induced arthritis (CIA) rat model, we validated the efficacy of DHA and probed its influence on Treg cells in the spleen and popliteal lymph nodes (PLN). Finally, leveraging deep proteomic analysis with data-independent acquisition (DIA) and parallel accumulation-serial fragmentation (PASEF) technology, we found the alterations in the Treg cell proteome in PLN by proteomic analysis. Our findings indicate that DHA augmented suppressive Treg cells, thereby impeding OC formation in vitro. Consistently, DHA mitigated erosive joint destruction and osteoclastogenesis by replenishing splenic and joint-draining lymph node Treg cells in CIA rats. Notably, DHA induced alterations in the Treg cell proteome in PLN, manifesting distinct upregulation of alloantigen Col2a1 (Type II collagen alfa 1 chain) and CD8a (T-cell surface glycoprotein CD8 alpha chain) in Treg cells, signifying DHA's targeted modulation of Treg cells, rendering them more adept at sustaining immune tolerance and impeding bone erosion. These results unveil a novel facet of DHA in the treatment of RA.

Hyperoside Suppresses Renal Inflammation by Regulating Macrophage Polarization in Mice With Type 2 Diabetes Mellitus.

Accumulating evidence reveals that both inflammation and lymphocyte dysfunction play a vital role in the development of diabetic nephropathy (DN). Hyperoside (HPS) or quercetin-3-O-galactoside is an active flavonoid glycoside mainly found in the Chinese herbal medicine Tu-Si-Zi. Although HPS has a variety of pharmacological effects, including anti-oxidative and anti-apoptotic activities as well as podocyte-protective effects, its underlying anti-inflammatory mechanisms remain unclear. Herein, we investigated the therapeutic effects of HPS on murine DN and the potential mechanisms responsible for its efficacy. We used C57BLKS/6J Lepdb/db mice and a high glucose (HG)-induced bone marrow-derived macrophage (BMDM) polarization system to investigate the potentially protective effects of HPS on DN. Our results showed that HPS markedly reduced diabetes-induced albuminuria and glomerular mesangial matrix expansion, accompanied with a significant improvement of fasting blood glucose level, hyperlipidaemia and body weight. Mechanistically, pretreatment with HPS effectively regulated macrophage polarization by shifting proinflammatory M1 macrophages (F4/80+CD11b+CD86+) to anti-inflammatory M2 ones (F4/80+CD11b+CD206+) in vivo and in bone marrow-derived macrophages (BMDMs) in vitro, resulting in the inhibition of renal proinflammatory macrophage infiltration and the reduction in expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) while increasing expression of anti-inflammatory cytokine Arg-1 and CD163/CD206 surface molecules. Unexpectedly, pretreatment with HPS suppressed CD4+ T cell proliferation in a coculture model of IL-4-induced M2 macrophages and splenic CD4+ T cells while promoting their differentiation into CD4+IL-4+ Th2 and CD4+Foxp3+ Treg cells. Taken together, we demonstrate that HPS ameliorates murine DN via promoting macrophage polarization from an M1 to M2 phenotype and CD4+ T cell differentiation into Th2 and Treg populations. Our findings may be implicated for the treatment of DN in clinic.

Huangbai Liniment Ameliorates Skin Inflammation in Atopic Dermatitis.

Atopic dermatitis (AD), also known as atopic eczema, is one of the most common skin diseases and is characterized by allergic skin inflammation, redness, and itchiness and is associated with a hyperactivated type 2 immune response. The leading causes of AD include an imbalance in the immune system, genetic predisposition, or environmental factors, making the development of effective pharmacotherapies complex. Steroids are widely used to treat AD; however, they provide limited efficacy in the long term and can lead to adverse effects. Thus, novel treatments that offer durable efficacy and fewer side effects are urgently needed. Here, we investigated the therapeutic potential of Huangbai Liniment (HB), a traditional Chinese medicine, using an experimental AD mouse model, following our clinical observations of AD patients. In both AD patient and the mouse disease model, HB significantly improved the disease condition. Specifically, patients who received HB treatment on local skin lesions (3-4 times/day) showed improved resolution of inflammation. Using the 1-Chloro-2,4-dinitrobenzene (DNCB)-induced AD model in BALB/c mice, we observed that HB profoundly alleviated severe skin inflammation and relieved the itching. The dermatopathological results showed markedly reversed skin inflammation with decreased epidermal thickness and overall cellularity. Correspondingly, HB treatment largely decreased the mRNA expression of proinflammatory cytokines, including IL-1ß, TNF-α, IL-17, IL-4, and IL-13, associated with declined gene expression of IL-33, ST2, and GATA3, which are connected to the type 2 immune response. In addition, HB restored immune tolerance by promoting regulatory T (TREG) cells and inhibiting the generation of TH1, TH2, and TH17 cells in vitro and in the DNCB-induced AD mouse model. For the first time, we demonstrate that HB markedly mitigates skin inflammation in AD patients and the DNCB-induced AD mouse model by reinvigorating the T cell immune balance, shedding light on the future development and application of novel HB-based therapeutics for AD.

The effects of oxygen-ozone therapy on regulatory T-cell responses in multiple sclerosis patients.

Multiple sclerosis (MS) is a common degenerative disorder of the central nervous system. The decreased frequency and dysfunction of Treg cells cause inflammation and disease progression. Ozone autohemotherapy can be used as a potential therapeutic approach to regulate the immune system responses and inflammation in MS. For this purpose, 20 relapsing-remitting multiple sclerosis patients were under treatment with ozone twice weekly for 6 months. The frequency of Treg cell, the expression levels of the Treg cell-related factors (FoxP3, IL-10, TGF-ß, miR-17, miR-27, and miR-146A), and the secretion levels of IL-10 and TGF-ß were assessed. We found a significant increase in the number of Treg cells, expression levels of FoxP3, miRNAs (miR-17 and miR-27), IL-10, and TGF-ß factors in patients after oxygen-ozone (O2 -O3 ) therapy compared to before treatment. In contrast, oxygen-ozone therapy notably decreased the expression level of miR-146a in treated patients. Interestingly, the secretion levels of both IL-10 and TGF-ß cytokines were considerably increased in both serum and supernatant of cultured peripheral blood mononuclear cells in posttreatment condition compared to pretreatment condition. According to results, oxygen-ozone therapy raised the frequency of Treg cell and its relevant factors in treated MS patients. Oxygen-ozone therapy would contribute to improving the MS patients by elevating the Treg cell responses.

High-Dose Neonatal Vitamin A Supplementation to Bangladeshi Infants Increases the Percentage of CCR9-Positive Treg Cells in Infants with Lower Birthweight in Early Infancy, and Decreases Plasma sCD14 Concentration and the Prevalence of Vitamin A Deficiency at Two Years of Age.

BACKGROUND: Vitamin A (VA) stores are low in early infancy and may impair development of the immune system. OBJECTIVE: This study determined if neonatal VA supplementation (VAS) affects the following: 1) development of regulatory T (Treg) cells; 2) chemokine receptor 9 (CCR9) expression, which directs mucosal targeting of immune cells; and 3) systemic endotoxin exposure as indicated by changed plasma concentrations of soluble CD14 (sCD14). Secondarily, VA status, growth, and systemic inflammation were investigated. METHODS: In total, 306 Bangladeshi infants were randomly assigned to receive 50,000 IU VA or placebo (PL) within 48 h of birth, and immune function was assessed at 6 wk, 15 wk, and 2 y. Primary outcomes included the following: 1) peripheral blood Treg cells; 2) percentage of Treg, T, and B cells expressing CCR9; and 3) plasma sCD14. Secondary outcomes included the following: 4) VA status measured using the modified relative dose-response (MRDR) test and plasma retinol; 5) infant growth; and 6) plasma C-reactive protein (CRP). Statistical analysis identified group differences and interactions with sex and birthweight. RESULTS: VAS increased (P = 0.004) the percentage of CCR9+ Treg cells (13.2 ± 1.37%) relative to PL (9.17 ± 1.15%) in children below the median birthweight but had the opposite effect (P = 0.04) in those with higher birthweight (VA, 9.13 ± 0.89; PL, 12.1 ± 1.31%) at 6 and 15 wk (values are combined mean ± SE). VAS decreased (P = 0.003) plasma sCD14 (1.56 ± 0.025 mg/L) relative to PL (1.67 ± 0.032 mg/L) and decreased (P = 0.034) the prevalence of VA deficiency (2.3%) relative to PL (9.2%) at 2 y. CONCLUSIONS: Neonatal VAS enhanced mucosal targeting of Treg cells in low-birthweight infants. The decreased systemic exposure to endotoxin and improved VA status at 2 y may have been due to VA-mediated improvements in gut development resulting in improved barrier function and nutrient absorption. This trial was registered at clinicaltrials.gov as NCT01583972 and NCT02027610.

Bufei Yishen Formula Restores Th17/Treg Balance and Attenuates Chronic Obstructive Pulmonary Disease via Activation of the Adenosine 2a Receptor.

Bufei Yishen formula (BYF) is a Traditional Chinese Medicine (TCM) reported to ameliorate chronic obstructive pulmonary disease (COPD) by regulating the balance between T helper (Th) 17 and regulatory T (Treg) cells. However, its mechanism remains unknown. Therefore, this study aimed to explore the underlying mechanisms of BYF. Naïve CD4+ T cells were exposed to anti-CD3, anti-CD28, transforming growth factor (TGF)-ß, and/or interleukin (IL)-6 to promote their differentiation into Th17 or Treg cells. A rat model of cigarette smoke- and bacterial infection-induced COPD was established and orally treated with BYF and/or an adenosine 2a receptor (A2aR) antagonist. Then, the rats were sacrificed, their lung tissues were removed for histological analysis, and their spleens were collected to evaluate Th17 and Treg cells. The results showed that BYF significantly suppressed Th17 cell differentiation and its related cytokines and enhanced Treg cell differentiation and its related cytokines. In addition, BYF activated the A2aR, increased the levels of p-signal transducer and activator of transcription (STAT)5, and decreased the level of p-STAT3 in Treg and Th17 cells. The A2aR antagonist suppressed the changes induced by BYF treatment in Th17 and Treg cells. Furthermore, the A2aR antagonist diminished the therapeutic effect of BYF on COPD, as indicated by the lung injury scores, bronchiole wall thickness, small pulmonary vessels wall thickness, bronchiole stenosis, alveolar diameters, decrease in inflammatory cytokines, increase in alveolar number, and lung functions. Similarly, the A2aR antagonist reversed the effects of BYF on the proportion of Th17 and Treg cells in the spleen. Additionally, BYF increased the protein and mRNA levels of A2aR and regulated the phosphorylation of STAT3 and STAT5 in spleen and lung tissues, which were inhibited by cotreatment with the A2aR antagonist. In conclusion, this study suggested that BYF exhibited its anti-COPD efficacy by restoring the Th17/Treg balance via activating A2aR, which may provide evidence for the clinical application of BYF in the treatment of COPD.

Rotating magnetic field ameliorates experimental autoimmune encephalomyelitis by promoting T cell peripheral accumulation and regulating the balance of Treg and Th1/Th17.

Multiple sclerosis (MS) is an autoimmune disease characterized by T cell infiltration and demyelination of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a classical preclinical animal model of MS. In this study, we found that rotating magnetic field (RMF) treatment exerts potential preventive effects on the discovery of EAE, including reducing the severity of the disease and delaying the onset of the disease. The results indicated that RMF (0.2 T, 4 Hz) treatment increases the accumulation of CD4+ cells in the spleen and lymph nodes by downregulating the expression of CCL-2, CCL-3 and CCL-5, but has no significant effect on myelin oligodendrocyte glycoprotein (MOG) specific T cell responses. Simultaneously, RMF treatment adjusted the imbalance between regulatory T (Treg) cell and T helper 1 (Th1) cells or T helper 17 (Th17) cells by increasing the proportion of Treg cells and inhibiting the ratio of Th1 and Th17 cell subsets. These findings suggest that exposure to RMF may improve EAE disease by promoting CD4+ cell accumulation into peripheral lymphoid tissue, improving the imbalance between Treg and Th1/Th17 cells. Therefore, as a mild physical therapy approach, RMF, is likely to be a potential way to alter the development of EAE.

Baitouweng Decoction Ameliorates Ulcerative Colitis in Mice Partially Attributed to Regulating Th17/Treg Balance and Restoring Intestinal Epithelial Barrier.

Ulcerative colitis (UC) is a chronic intestinal disease with unclear pathogenesis. With an increasing global prevalence over the past two decades, UC poses a serious threat to public health. Baitouweng decoction (BTW), a traditional Chinese medicine, has been shown to have good clinical efficacy for treating intestinal inflammation. Yet, the efficacy of BTW in UC and the underlying mechanism remain unclear. The current study aimed to determine whether BTW suppressed intestinal inflammation in mice and the potential mechanism. We used a dextran sulfate sodium (DSS)-induced murine colitis model to test the anti-inflammatory efficacy of BTW. Clinical symptoms were scored by the disease activity index (DAI), and the colon length and pathological changes in colon tissue were also used to further evaluate the efficacy of BTW. Precisely how BTW affected immune function and the intestinal barrier of UC mice was also examined. BTW significantly reduced DAI score and colonic pathological damage. BTW regulated the balance between T helper (Th)17 and regulatory T (Treg) cells, decreased interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α, and increased IL-10 levels. BTW reduced intestinal permeability of UC mice, increased expression of tight junction proteins (occludin and zonula occludens-1), and decreased expression of phospho-nuclear factor (p-NF)-κB and phospho-extracellular signal-regulated kinase (p-ERK) in the colon. BTW inhibited the ERK/p-NF-κB signaling pathway and suppressed expression of cyclo-oxygenase-2 and inducible NO synthase in lipopolysaccharide-activated RAW 264.7 cells. BTW significantly promoted the synthesis of short-chain fatty acids in the gut, particularly acetate, propionate, isobutyric acid, and isovalerate. The results suggest that BTW can protect against DSS-induced UC. The mechanism may be partially attributed to regulating the balance of Th17/Treg cells and restoring the intestinal epithelial barrier.

Mangiferin suppresses allergic asthma symptoms by decreased Th9 and Th17 responses and increased Treg response.

Mangiferin is the major bioactive ingredient in the leaves of Mangifera indica L., Aqueous extract of such leaves have been traditionally used as an indigenous remedy for respiratory diseases including cough and asthma in Traditional Chinese Medicine. Mangiferin was shown to exert its anti-asthmatic effect by modulating Th1/Th2 cytokines imbalance via STAT6 signaling pathway. However, compelling evidence indicated that subtypes of T helpers and regulatory T cells other than Th1/Th2 were also involved in the pathogenesis of asthma. In current study, we investigated the effects of mangiferin on the differentiation and function of Th9, Th17 and Treg cells in a chicken egg ovalbumin (OVA)-induced asthmatic mouse model. Mangiferin significantly attenuated the symptoms of asthma attacks, reduced the total number of leukocytes, EOS and goblet cells infiltration in lung. Simultaneously, treatment with mangiferin remarkably decreased the proportion of Th9 and Th17 cells; reduced the levels of IL-9, IL-17A; inhibited the expression of PU.1 and RORγt in lung. However, the proportion of Treg cells, the expression of IL-10, TGF-ß1 and Foxp3 were increased by mangiferin. Our data suggest that mangiferin exerted anti-asthmatic effect through decreasing Th9 and Th17 responses and increasing Treg response in OVA-induced asthmatic mouse model.

Effect of compound sophorae decoction on dextran sodium sulfate (DSS)-induced colitis in mice by regulating Th17/Treg cell balance.

OBJECTIVE: Compound sophorae decoction, a Chinese medicinal formulae composed of six Chinese herbs, is effective for the clinical treatment of ulcerative colitis (UC). Some of its effective monomers had been proven to have suppressive effect on UC models. The aim of this study is to further explore the mechanism whether compound sophorae decoction ameliorates dextran sodium sulfate (DSS)-induced mice colitis by regulating the balance between T helper (Th) 17 and regulatory T (Treg) cells. METHODS: Experimental model of UC, established by drinking water with DSS, was treated with compound sophorae decoction and mesalazine. The stool, activity, body weight of the mice, colon length and colon histopathology were observed to evaluate severity of colitis. The concentration of cytokines in colonic tissues were detected by ELISA. The expression of phosphorylated nuclear factor-kappaB (NF-κB) p65, STAT3 and phosphorylated STAT3 in colonic tissues were determined by western blotting and immunohistochemistry. The percentage of Th17 and Treg cells in spleen and mesenteric lymph nodes (MLNs) were detected by flow cytometry. The levels of transcription factor ROR-γt and FOXP3 in colon tissues were detected by qRT-PCR and immunohistochemistry. RESULTS: The aqueous extract of compound sophorae decoction was able to improve the symptoms and pathological damage of mice. The body weight of mice were increased and DAI were significantly decreased; ulcers were slighter than DSS group. The administration of compound sophorae decoction reduced the level of inflammatory factors interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and phospho-NF-κB p65, and also decreased the proportions of Th17 cells in spleen and MLNs and the expression of ROR-γt, IL-17A, STAT3, IL-6 in colonic tissues; while the percentage of Treg cells in spleen and MLNs and the expression of FOXP3, transforming growth factor (TGF)-ß1, IL-10 in colonic tissues were upregulated. CONCLUSION: Overall, this study suggested that compound sophorae decoction significantly improves the symptoms and the pathological damage of mice with colitis and influences the immune function by regulating Th17/Treg cell balance in DSS-induced colitis in mice.

Clinical Efficacy of Shensuyin in Treating Adult Viral Myocarditis with Syndrome of Insufficiency of Lung-Qi / 中国实验方剂学杂志

Objective: To investigate the efficacy of Shensuyin in treating viralmyocarditis (VMC) with syndrome of insufficiency of lung-qi and the effect on levels of Th17 and Treg cells and relevant factors. Method: One hundred-four VMC cases were regaded as object of study and randomly divided into control group and observation group, with 52 cases in each group. Control group was treated with routine therapy by reference to ‘Chinese Guidelines for Diagnosis and Treatment of Heart Failure 2014’. In addition to the therapy of control group, observation group on the basis of treatment in the control group with Shensuyin, 1 dose/d, bid. One course of treatment was 8 weeks for both groups. Scores of Shensuyin, serum levels of Troponin I (cTnI) and cardiac free fatty acid binding protein (H-FABP), heart function and total efficacy were compared for both groups. Flow cytometry was used to detect peripheral blood levels of Th17 and Treg cells for the two groups. Serum levels of interleukin (IL) -17, IL-21, IL-10 were detected in both groups. Result: After treatment, scores of syndrome of Yin and Yang deficiency (shortness of breath, fatigue, dull chest pain, bad breath, cough) of observation group were obviously lower than those of control group (PPPPPConclusion: In addition to the routine therapy of VMC, the efficacy of Shensuyin has a significant effect in treating VMC with syndrome of lung qi deficiency, and the regulatory effect on levels of Th17 and Treg cells and relevant factors may be one of the effective ways.

Combination of NIR therapy and regulatory T cell modulation using layer-by-layer hybrid nanoparticles for effective cancer photoimmunotherapy.

The efficacy of combined near-infrared (NIR) and immune therapies for inhibiting tumor growth and recurrence has gained increasing research attention. Regulatory T cells in the tumor microenvironment constitute a major obstacle in achieving robust CD8+ T cell antitumor immunotherapy. In the present study, we designed a photoimmunotherapy-based strategy involving a combination of photothermal and photodynamic therapies, followed by Treg cell suppression, for eliciting an immune response with IR-780- and imatinib-loaded layer-by-layer hybrid nanoparticles. Methods: The layer-by-layer hybrid nanoparticles were prepared through electrostatic interactions. Their photothermal effect, photodynamic effect as well as their effect on inhibiting Treg cells' suppressive function were investigated in vitro and in vivo. Their antitumor effect was evaluated using B16/BL6 and MC-38 tumor-bearing mice. Results: The layer-by-layer hybrid nanoparticles, which were pH-sensitive, enabled the release of IR-780 dye for NIR-induced photothermal and photodynamic effects, and the release of imatinib-loaded glucocorticoid-induced TNF receptor family-related protein/poly(lactic-co-glycolic acid) (GITR-PLGA) nanoparticles to initiate antitumor immunotherapy. The photothermal and photodynamic effects caused by IR-780 under NIR exposure resulted in direct tumor apoptosis/necrosis and the production of tumor-associated antigen, promoted dendritic cell maturation, and enhanced the presentation of tumor-associated antigen to T cells, while the imatinib-loaded GITR-PLGA cores reduced the suppressive function of Treg cells, and consequently activated effective CD8+ T cells towards tumors. Conclusion: With the significant photothermal, photodynamic and immunotherapies, the system successfully eradicated tumor growth, diminished tumor recurrence, and improved survival in vivo. The proposed nanoparticles provide a novel and versatile approach to boost antitumor photoimmunotherapy.

Zhenbao Pill reduces Treg cell proportion in acute spinal cord injury rats by regulating TUG1/miR-214/HSP27 axis.

Background: Acute spinal cord injury (SCI) is one of the weakest pathologies that seriously affect the quality of life of patients. Objective: To study the mechanism of how Zhenbao Pill reduces Treg cell proportion and improves acute SCI. Methods: A rat SCI model was established. Flow cytometry analysis was performed to determine the Treg cell proportion. RNA immunoprecipitation (RIP) and RNA pull-down were applied in confirming taurine up-regulated gene 1 (TUG1) and miR-214 binding. Intrathecal injection of TUG1 siRNA was also conducted to determine the effect of TUG1 in vivoResults: Zhenbao Pill promoted the expression of TUG1 and heat shock protein 27 (HSP27) protein, and reduced the expression of miR-214 and forkhead box protein p3 (Foxp3) as well as Treg cell proportion in a concentration-dependent manner in SCI rats or in vitro cultured CD4+ T cells. Knockdown of TUG1 reversed the high protein expression of HSP27 and the inhibition of Treg cell proportion as well as Foxp3 protein induced by Zhenbao Pill, and miR-214 inhibitor canceled the TUG1 knockdown effect. Further, miR-214 mimic reversed the inhibition of Treg cell proportion and Foxp3 protein expression by Zhenbao Pill, which was abolished by the overexpression of HSP27. The mechanism was validated in animal experiments. Conclusion: Zhenbao Pill regulated TUG1/miR-214/HSP27 signaling pathway to reduce Treg cell proportion and thus relieve acute SCI.

Xihuang pill promotes apoptosis of Treg cells in the tumor microenvironment in 4T1 mouse breast cancer by upregulating MEKK1/SEK1/JNK1/AP-1 pathway.

OBJECTIVE: To determine the role of the MEKK1/SEK1/JNK1/AP-1 pathway in the action of Xihuang pill (XHP) in reducing regulatory T (Treg) cell numbers in the tumor microenvironment in a 4T1 mouse breast cancer model, and to clarify the anti-tumor mechanism of XHP in breast cancer. METHODS: We established a mouse 4T1 breast cancer model. Model mice were administered XHP for 2 weeks, and tumor tissues were then removed, weighed, sliced, and homogenized. Treg cells in the tumor microenvironment were isolated by magnetic cell sorting and analyzed by immunohistochemistry and flow cytometry. Treg cell apoptosis was detected by TdT-mediated dUTP nick end labeling. mRNA expression levels of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment were detected by quantitative real-time PCR and their protein expression levels were detected by immunofluorescence staining and western blot. RESULTS: Tumor weights were significantly lower in the XHP groups compared with the untreated control group. The overall number of Treg cells in the tumor microenvironment decreased while the number of apoptotic Treg cells increased with increasing doses of XHP. mRNA and protein expression levels of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment increased with increasing doses of XHP. CONCLUSION: XHP might promote Treg cell apoptosis in the tumor microenvironment and further inhibit the tumor growth of 4T1 mouse breast cancer. The mechanism of XHP may be related to upregulation of gene and protein expression of MEKK1, SEK1, JNK1, and AP-1 in Treg cells in the tumor microenvironment.

Lactobacillus acidophilus Improves Intestinal Inflammation in an Acute Colitis Mouse Model by Regulation of Th17 and Treg Cell Balance and Fibrosis Development.

Disruption of the balance among the microbiota, epithelial cells, and resident immune cells in the intestine is involved in the pathogenesis of inflammatory bowel disease (IBD). Probiotics exert protective effects against IBD, and probiotic commensal Lactobacillus species are common inhabitants of the natural microbiota, especially in the gut. To investigate the effects of Lactobacillus acidophilus on the development of IBD, L. acidophilus was administered orally in mice with dextran sodium sulfate (DSS)-induced colitis. DSS-induced damage and the therapeutic effect of L. acidophilus were investigated. Treatment with L. acidophilus attenuated the severity of DSS-induced colitis. Specifically, it suppressed proinflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor-α, IL-1ß, and IL-17 in the colon tissues, which are produced by T helper (Th) 17 cells. Moreover, in vitro L. acidophilus treatment directly induced T regulatory (Treg) cells and the production of IL-10, whereas the production of IL-17 was suppressed in splenocytes. In addition, we found that L. acidophilus treatment decreased the levels of α-smooth muscle actin, a marker of activated myofibroblasts, and type I collagen compared with control mice. These results suggest that L. acidophilus may be a novel treatment for IBD by modulating the balance between Th17 and Treg cells, as well as fibrosis development.

Sea Cucumber Lipid-Soluble Extra Fraction Prevents Ovalbumin-Induced Allergic Airway Inflammation.

In a previous study, our research group demonstrated that sea cucumber (Apostichopus japonicus) extracts ameliorated allergic airway inflammation through CD4+CD25+Foxp3+ T (regulatory T; Treg) cell activation and recruitment to the lung. In this study, we aimed to determine which components of sea cucumber contribute to the amelioration of airway inflammation. We used n-hexane fractionation to separate sea cucumber into three phases (n-hexane, alcohol, and solid) and evaluated the ability of each phase to elevate Il10 expression in splenocytes and ameliorate symptoms in mice with ovalbumin (OVA)/alum-induced asthma. Splenocytes treated with the n-hexane phase showed a significant increase in Il10 expression. In the n-hexane phase, 47 fatty acids were identified. Individual fatty acids that comprised at least 5% of the total fatty acids were 16:0, 16:1n-7, 18:0, 18:1n-7, 20:4n-6, and 20:5n-3 (eicosapentaenoic acid). After administering the n-hexane phase to mice with OVA/alum-induced asthma, their asthma symptoms were ameliorated. Several immunomodulatory effects were observed in the n-hexane phase-pretreated group, compared with a vehicle control group. First, eosinophil infiltration and goblet cell hyperplasia were significantly reduced around the airways. Second, the concentrations of Th2-related cytokines (IL-4, IL-5, and IL-13) and Th17-related cytokines (IL-17) were significantly decreased in the spleen and bronchoalveolar lavage fluid (BALF). Finally, the concentrations of TGF-ß and IL-10, which are associated with Treg cells, were significantly increased in the BALF and splenocyte culture medium. In conclusion, a fatty acid-rich fraction (n-hexane phase) of sea cucumber extract ameliorated allergic airway inflammation in a mouse model.

Mangiferin corrects the imbalance of Th17/Treg cells in mice with TNBS-induced colitis.

In the previous study, 80% ethanol extract of the rhizome mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC) and its main constituent mangiferin improved TNBS-induced colitis in mice by inhibiting macrophage activation related to the innate immunity. In the preliminary study, we found that AC could inhibit Th17 cell differentiation in mice with TNBS-induced colitis. Therefore, we investigated whether AC and it main constituent mangiferin are capable of inhibiting inflammation by regulating T cell differentiation related to the adaptive immunity in vitro and in vivo. AC and mangiferin potently suppressed colon shortening and myeloperoxidase activity in mice with TNBS-induced colitis. They also suppressed TNBS-induced Th17 cell differentiation and IL-17 expression, but increased TNBS-suppressed Treg cell differentiation and IL-10 expression. Moreover, AC and mangiferin strongly inhibited the expression of TNF-α and IL-17, as well as the activation of NF-κB. Furthermore, mangiferin potently inhibited the differentiation of splenocytes into Th7 cells and increased the differentiation into Treg cells in vitro. Mangiferin also inhibited RORγt and IL-17 expression and STAT3 activation in splenocytes and induced Foxp3 and IL-10 expression and STAT5 activation. Based on these findings, mangiferin may ameliorate colitis by the restoration of disturbed Th17/Treg cells and inhibition of macrophage activation.

Oleanolic acid ameliorates dextran sodium sulfate-induced colitis in mice by restoring the balance of Th17/Treg cells and inhibiting NF-κB signaling pathway.

In a preliminary experiment, it was found that oleanolic acid (OA), which is widely distributed in food and medicinal plants, inhibited interleukin (IL)-6/tumor growth factor beta-induced differentiation of splenic T cells into Th17 cells. Moreover, OA induced the differentiation of splenic T cells into Treg cells. Therefore, we examined the anti-inflammatory effect of OA in mice with dextran sodium sulfate (DSS)-induced colitis. Oral administration of OA significantly inhibited DSS-induced colon shortening, macroscopic score, and myeloperoxidase activity. Treatment with OA inhibited DSS-induced differentiation to Th17 cells and downregulated the expression of RORγt and IL-17 in the lamina propria of colon and Treg cell differentiation and Foxp3 and IL-10 expression were increased. OA treatment increased the DSS-suppressed expression of tight junction proteins such as ZO-1, occludin, and claudin-1 in the colon. Moreover, OA treatment inhibited DSS-induced expression of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-17, the activation of NF-κB and mitogen-activated protein kinases, and increased IL-10 expression. OA also inhibited the activation of NF-κB and expression of proinflammatory cytokines in LPS-stimulated peritoneal macrophages. These findings suggest that OA may ameliorate inflammatory diseases such as colitis by inhibiting Th17 cell differentiation and increasing Treg cell differentiation.

Baicalin and geniposide attenuate atherosclerosis involving lipids regulation and immunoregulation in ApoE-/- mice.

Baicalin and geniposide, which are respectively isolated from Scutellariae radix and Gardenia jasminoides, have been known to exhibit a number of pharmacological effects, including anti-inflammatory and anti-oxidant. Here, we primarily aimed to observe the protective effects of these two Chinese herbs on inhibiting the development of atherosclerosis in apolipoprotein E knockout mice via lipids regulation and immunoregulation. After the ApoE-/- mice with high-cholesterol diet had received 12-weeks׳ oral administration of either baicalin or geniposide (100 mg/kg), atherosclerotic plaque areas in aorta were measured and exhibited a prominent decrease in the treated mice. We then assayed serum lipids levels, serum Treg-cell-associated cytokines (TGF-ß1 and IL-10) and the frequency of splenic Treg cells. We found that geniposide notably decreased serum TC and LDL-c. Both baicalin and geniposide treated mice showed much more splenic Treg cells and the correlated cytokines (TGF-ß1 and IL-10). Foxp3, as the marker of Treg cell, was detected in atherosclerotic lesions, and we found that Foxp3 expression at both mRNA and protein levels was up-regulated in addition to increased Foxp3 positive Treg cells detected by immunohistochemistry in baicalin or geniposide treated mice. In conclusion, baicalin and geniposide up-regulated the expression of foxp3, promoted the number and function of Treg cells and ameliorated the atherosclerotic lesions progression partly through lipids regulation and immunoregulation.

Clematichinenoside AR induces immunosuppression involving Treg cells in Peyer׳s patches of rats with adjuvant induced arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematichinenoside AR (AR) has been defined as a major active ingredient of triterpenoid saponins extracted from Clematidis Radix et Rhizoma, which is a traditional Chinese herbal medicine that has long been used in the treatment of rheumatoid arthritis (RA). To further explore the mechanism of AR in the treatment of RA, we investigated whether its immunomodulatory effects are related to Treg-mediated suppression derived from Peyer׳s patches (PPs) in adjuvant induced arthritis (AIA) rat model. MATERIALS AND METHODS: AR (8, 16, 32 mg/kg) was orally administered daily from Day 18 to Day 31 after immunization. The effect of AR on AIA rats was evaluated by hind paw swelling and histopathological examination. Percentages of CD4(+)CD25(+)Foxp3(+) T regulatory cells were determined by flow cytometry. Levels of IL-10, TGF-ß1, IL-17A and TNF-α were measured by ELISA. Expressions of Foxp3 and RORγ in synovium were detected using immunohistochemical analysis. RESULTS: AR treatment significantly reduced paw swelling of AIA rats, and histopathological analysis confirmed it could suppress severity of established arthritis. AR treatment upregulated the percentages of CD4(+)CD25(+)Foxp3(+) Treg cells among CD4+ T cells in PPs lymphocytes, and increased the levels of IL-10 and TGF-ß1 secreted from ConA-activated PPs lymphocytes, whereas decreased the levels of IL-17 A and TNF-α. Similar tendency of circulating CD4(+)CD25(+)Foxp3(+) Treg cells percentages and serum cytokine levels were observed. Moreover, AR decreased the expression levels of Foxp3 and RORγ in joint synovial membrane. CONCLUSIONS: In conclusion, these results suggested AR has a potent protective effect on the progression of AIA, probably by augmenting CD4(+)CD25(+)Foxp3(+) Treg cells in PPs to induce immunosuppression, and modulating the balance between Treg cells and Th17 cells systemically. These findings may help to develop AR as a potent immunosuppressive agent for the treatment of RA.