Promoted Treg Differentiation Research Articles

Fermented Gracilaria lemaneiformis polysaccharides alleviate food allergy by regulating Treg cells and gut microbiota

Food allergy has a significant impact on the health and well-being of individuals, affecting both their physical and mental states. Research on natural bioactive compounds, such as polysaccharides extracted from seaweeds, holds great promise in the treatment of food allergies. In this study, fermented Gracilaria lemaneiformis polysaccharides (F-GLSP) were prepared using probiotic fermentation. Probiotic fermentation of Gracilaria lemaneiformis reduces the particle size of polysaccharides. To compare the anti-allergic activity of F-GLSP with unfermented Gracilaria lemaneiformis polysaccharides (UF-GLSP), an OVA-induced mouse food allergy model was established. F-GLSP exhibited a significant reduction in OVA-specific IgE and mMCP levels in allergic mice. Moreover, it significantly inhibited Th2 differentiation and IL-4 production and significantly promoted Treg differentiation and IL-10 production in allergic mice. In contrast, UF-GLSP only reduced OVA-specific IgE and mMCP in the serum of allergic mice. Furthermore, F-GLSP demonstrated a more pronounced regulation of intestinal flora abundance compared to UF-GLSP, significantly influencing the populations of Firmicutes, Bacteroidetes, Lactobacillus, and Clostridiales in the intestines of mice with food allergy. These findings suggest that F-GLSP may regulate food allergies in mice through multiple pathways. In summary, this study has promoted further development of functional foods with anti-allergic properties based on red algae polysaccharides.

Active targeting microemulsion-based thermosensitive hydrogel against periodontitis by reconstructing Th17/Treg homeostasis via regulating ROS-macrophages polarization cascade

Periodontitis is a multifactorial inflammatory disease characterized by severe alveolar bone damage and attachment loss. The imbalance of T help 17 (Th17) / regulatory T cells (Treg) induces excessive interleukin (IL)-17, which leads to alveolar bone damage and aggravates the development of periodontitis. Therefore, we proposed a therapeutic strategy to restore Th17/Treg homeostasis by interfering reactive oxygen species (ROS)-macrophage polarization cascade using active targeting microemulsions-based thermosensitive hydrogel. Folic acid-modified quercetin-loaded microemulsions (FA-Qu-MEs) were dispersed in poloxamer 407 and poly(N-isopropylacrylamide) matrix of hydrogel (FA-Qu-MEs@Gel). FA-Qu-MEs@Gel could be locally injected into the periodontal pocket and sustainedly release drugs. FA-Qu-MEs exhibited excellent ROS scavenging potency by targeting macrophages, resulting M1 phenotype macrophage from to M2 phenotype macrophage. Subsequently, the phenotypic changes of macrophages lead to decreased expression of IL-6 and tumor necrosis factor-α, which inhibited activated Th17, while IL-10 secreted by M2 macrophages promoted Treg differentiation. Finally, the restored Th17/Treg homeostasis reduced the level of IL-17 to accelerate alveolar bone regeneration. This study deigns a novel system that promote alveolar bone regeneration by remodeling Th17/Treg homeostasis via regulating ROS-macrophages polarization cascade for periodontitis treatment.

Translational kinetic-pharmacodynamics of mRNA-6231, an investigational mRNA therapeutic encoding mutein interleukin-2.

Regulatory T cells (Tregs) are essential for maintaining immune homeostasis by serving as negative regulators of adaptive immune system effector cell responses. Reduced production or function of Tregs has been implicated in several human autoimmune diseases. The cytokine interleukin 2 plays a central role in promoting Treg differentiation, survival, and function invivo and may therefore have therapeutic benefits for autoimmune diseases. mRNA-6231 is an investigational, lipid nanoparticle-encapsulated, mRNA-based therapy that encodes a modified human interleukin 2 mutein fused to human serum albumin (HSA-IL2m). Herein, we report the development of a semi-mechanistic kinetic-pharmacodynamic model to quantify the relationship between subcutaneous dose(s) of mRNA-6231, HSA-IL2m protein expression, and Treg expansion in nonhuman primates. The nonclinical kinetic-pharmacodynamic model was extrapolated to humans using allometric scaling principles and the physiological basis of pharmacological mechanisms to predict the clinical response to therapy a priori. Model-based simulations were used to inform the dose selection and design of the first-in-human clinical study (NCT04916431). The modeling approach used to predict human responses was validated when data became available from the phase I clinical study. This validation indicates that the approach is valuable in informing clinical decision-making.

Gut microbiota-derived butyrate restores impaired regulatory T cells in patients with AChR myasthenia gravis via mTOR-mediated autophagy

More than 80% of patients with myasthenia gravis (MG) are positive for anti-acetylcholine receptor (AChR) antibodies. Regulatory T cells (Tregs) suppress overproduction of these antibodies, and patients with AChR antibody-positive MG (AChR MG) exhibit impaired Treg function and reduced Treg numbers. The gut microbiota and their metabolites play a crucial role in maintaining Treg differentiation and function. However, whether impaired Tregs correlate with gut microbiota activity in patients with AChR MG remains unknown. Here, we demonstrate that butyric acid-producing gut bacteria and serum butyric acid level are reduced in patients with AChR MG. Butyrate supplementation effectively enhanced Treg differentiation and their suppressive function of AChR MG. Mechanistically, butyrate activates autophagy of Treg cells by inhibiting the mammalian target of rapamycin. Activation of autophagy increased oxidative phosphorylation and surface expression of cytotoxic T-lymphocyte-associated protein 4 on Treg cells, thereby promoting Treg differentiation and their suppressive function in AChR MG. This observed effect of butyrate was blocked using chloroquine, an autophagy inhibitor, suggesting the vital role of butyrate-activated autophagy in Tregs of patients with AChR MG. We propose that gut bacteria derived butyrate has potential therapeutic efficacy against AChR MG by restoring impaired Tregs.

INHBA(+) cancer-associated fibroblasts generate an immunosuppressive tumor microenvironment in ovarian cancer

Effective targeting of cancer-associated fibroblasts (CAFs) is hindered by the lack of specific biomarkers and a poor understanding of the mechanisms by which different populations of CAFs contribute to cancer progression. While the role of TGFβ in CAFs is well-studied, less attention has been focused on a structurally and functionally similar protein, Activin A (encoded by INHBA). Here, we identified INHBA(+) CAFs as key players in tumor promotion and immunosuppression. Spatiotemporal analyses of patient-matched primary, metastatic, and recurrent ovarian carcinomas revealed that aggressive metastatic tumors enriched in INHBA(+) CAFs were also enriched in regulatory T cells (Tregs). In ovarian cancer mouse models, intraperitoneal injection of the Activin A neutralizing antibody attenuated tumor progression and infiltration with pro-tumorigenic subsets of myofibroblasts and macrophages. Downregulation of INHBA in human ovarian CAFs inhibited pro-tumorigenic CAF functions. Co-culture of human ovarian CAFs and T cells revealed the dependence of Treg differentiation on direct contact with INHBA(+) CAFs. Mechanistically, INHBA/recombinant Activin A in CAFs induced the autocrine expression of PD-L1 through SMAD2-dependent signaling, which promoted Treg differentiation. Collectively, our study identified an INHBA(+) subset of immunomodulatory pro-tumoral CAFs as a potential therapeutic target in advanced ovarian cancers which typically show a poor response to immunotherapy.

Adipose-derived stem cell exosomes promote tumor characterization and immunosuppressive microenvironment in breast cancer

Adipose-derived stem cells (ASC) or autologous fat transplantation could be used to ameliorate breast cancer postoperative deformities. This study aims to explore the action of ASC and ASC-exosomes (ASC-exos) in breast cancer characterization and tumor microenvironment immunity, which provided a new method into the application of ASC-exos. ASC were extracted from human adipose tissue for the isolation and verification of ASC-exos. ASC-exos were co-cultured with CD4+T cells, CD14+ monocytes and MCF-7 cells, respectively. The tumor formation of nude mice was also constructed. Cell characterization was determined by CCK8, scratch assay, and Transwell. Hematoxylin–eosin (HE), immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to observe the histopathology and protein expression. CD4+T cell and CD14+ monocytes differentiation was detected by flow cytometry. Western blot, qRT-PCR and RNAseq were used to detect the action of ASC-exos on gene and protein expression. CD4+T cells could take up ASC-exos. ASC-exos inhibited Th1 and Th17 differentiation and promoted Treg differentiation of CD4+T cells. ASC-exos inhibited M1 differentiation and promoted M2 differentiation of CD14+ monocytes. ASC-exos promoted the migration, proliferation, and invasion, while inhibited apoptosis of MCF-7 cells. ASC-exos promoted the tumor formation of breast cancer. The effect of ASC-exos on tumor microenvironment immunity was in accordance with the above in vitro results. TOX, CD4 and LYZ1 genes were upregulated, while Mettl7b and Serpinb2 genes were downregulated in ASC-exos group. Human T-cell leukemia virus 1 infection pathway was significantly enriched in ASC-exos. Thus, ASC-exos promoted breast cancer characterization and tumor microenvironment immunosuppression by regulating macrophage and T cell differentiation.

P1223 Lactobacillus rhamnosus GG/p40 encapsuled cationic host defense peptide play protective roles on immune-related colitis by inducing differentiation and immune suppression of regulatory T cells

Abstract Background The immune-related colitis (IRC) is the most prominent in immune-related adverse events (irAE). The association between gut microbiota and IRC development has been revealed. Our study aimed to explore the protective effect and mechanism of the probiotic Lactobacillus rhamnosus GG (LGG) and its secretory protein p40 on IRC. The self-assembled cationic host defense peptide (CHDP) hydrogel was applied to protect oral p40 protein from digestive damage and target enriched the site of colitis. Methods The IRC model mice were intervened with LGG by gavage (i.g.), p40 by enema (en.), or p40 encapsuled by CHDP (i.g.). The T cell phenotype of colonic lamina propria mononuclear cells (LPMC) was measured by flow cytometry. The indocyanine green (ICG) encapsuled by CHDP was gavage in IRC model mice, the distribution of CHDP was monitored by fluorescence signals imaging via IVIS, to assess the adhesive effect of CHDP in mice colitis. In vitro, the proportion of regulatory T cells (Treg) differentiated was measured by LGG supernatant co-cultured with CD4+ naïve T cells, and the division index of CD4+T cells was measured by LGG supernatant co-cultured with CD4+T cells and Treg (CD4+T cells: Treg=1:1) by carboxyfluorescein succinimidyl ester (CFSE) staining test. Results Compared with DSS+anti-PD-1+anti-CTLA-4 IRC model group, LGG (i.g.) group had lower weight loss, DAI score, histopathological score, relative IL-6 and TNF-α mRNA level, IL-6 and TNF-α concentration and higher colon length (P<0.05) (Fig.1A-L). P40 (en.) group had lower weight loss, DAI score, histopathological score and higher colon length (P<0.05) (Fig.1M-T). CHDP+p40 (i.g.) group had lower weight loss, DAI score, histopathological score, relative IL-6 and TNF-α mRNA level, IL-6 concentration and higher colon length (P<0.05) (Fig.1M-W). The percentages of CD25+FoxP3+Treg/CD4+T cells of colonic LPMC in LGG, p40 (en.), and CHDP+p40 (i.g.) group were significantly higher than IRC model group (P<0.05) (Fig.1X-Z, α). The average 28hr-residual rate of fluorescence imaging radiance in IRC-CHDP+ICG group was higher than both NC-CHDP+ICG and IRC-ICG group (Fig.2β-δ), and the average radiance of colon in IRC-CHDP+ICG group was also the highest (P<0.05) (Fig.2ε-ζ). In vitro, LGG 107 CFU/mL, 108 CFU/mL and 5 × 108 CFU/mL (P<0.05) could significantly promote Treg differentiation with concentration dependent manner (Fig.2η-ι), and the suppression of Treg on CD4+T cells with lower division index compared with control group (Fig.2κ). Conclusion LGG/p40 played protective role by promoting the differentiation and immune suppression of Treg cells in IRC. CHDP hydrogel could protect p40 protein from the digestive damage, and target enriched the inflamed lesion to improve the efficacy.

Tracking the role of Aire in immune tolerance to the eye with a TCR transgenic mouse model

Roughly one-half of mice with partial defects in two immune tolerance pathways (AireGW/+Lyn-/- mice) spontaneously develop severe damage to their retinas due to T cell reactivity to Aire-regulated interphotoreceptor retinoid-binding protein (IRBP). Single-cell T cell receptor (TCR) sequencing of CD4+ T cells specific for a predominate epitope of IRBP showed a remarkable diversity of autoantigen-specific TCRs with greater clonal expansions in mice with disease. TCR transgenic mice made with an expanded IRBP-specific TCR (P2.U2) of intermediate affinity exhibited strong but incomplete negative selection of thymocytes. This negative selection was absent in IRBP-/- mice and greatly defective in AireGW/+ mice. Most P2.U2+/- mice and all P2.U.2+/-AireGW/+ mice rapidly developed inflammation of the retina and adjacent uvea (uveitis). Aire-dependent IRBP expression in the thymus also promoted Treg differentiation, but the niche for this fate determination was small, suggesting differences in antigen presentation leading to negative selection vs. thymic Treg differentiation and a stronger role for negative selection in preventing autoimmune disease in the retina.

Lactobacillus acidophilus and its metabolite ursodeoxycholic acid ameliorate ulcerative colitis by promoting Treg differentiation and inhibiting M1 macrophage polarization.

Lactobacillus acidophilus (LA) is a common clinical probiotic that improves ulcerative colitis (UC) by restoring intestinal immune balance. However, the interaction of LA with the gut microbiota and its metabolites in the treatment of UC remains unknown. Therefore, this study seeks to elucidate whether the gut microbiota and its metabolites act as pivotal effectors in LA's therapeutic mechanisms and how precisely they modulate intestinal immunity. In this study, we verified that LA can obviously ameliorate the disease severity, and regulate intestinal immune disorders in UC mice. Subsequently, antibiotic (ABX)-mediated depletion of the gut microflora demonstrated that the therapeutic efficiency of LA was closely associated with gut microbiota. In addition, the results of metabolomics revealed that ursodeoxycholic acid (UDCA), a metabolite of intestinal flora, may be a potential effector molecule mediating therapeutic effects of LA. Indeed, we found that UDCA can improve the macro pathological characteristics of UC mice, and through a comprehensive set of in vivo and in vitro experiments, we discovered that UDCA exerts dual effects on immune regulation. Firstly, it promotes the differentiation of Treg cells, resulting in increased secretion of anti-inflammatory cytokines. Secondly, UDCA inhibits the polarization of M1 macrophages, effectively reducing the secretion of pro-inflammatory cytokines. Moreover, we found that UDCA regulation of immune response is directly related to the RapGap/PI3K-AKT/NF-κB signaling pathway. In conclusion, LA and its metabolite, UDCA, may treat UC by activating the RapGap/PI3K-AKT/NF-κB signaling pathway and modulating Treg cells and M1 macrophages. All in all, our findings highlight the potential of microbial metabolites in enhancing probiotic for UC treatment.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Integrin αvβ8-Mediated Activation of TGF-β1 Is Involved in Regulating the Sustained Response after TPO-Ras Withdrawal in ITP Patients

Background: Thrombopoietin receptor agonists（TPO-RAs）are effective and safe in immune thrombocytopenia (ITP), and are currently the most important second-line drugs. The effect of TPO-RAs is transient, platelet counts usually return to baseline within 2 weeks unless treatment is continued. Only one-third of patients can maintain after drug discontinuation, but the mechanism is unknown and worthy of further exploration. Method and Results：In this study we observed 66 patients treated with TPO-RAs and followed-up to three months after drug withdrawal. Patients were divided into three groups: non-response group (NR), sustained group (Group A), and non- sustained group (Group B) (Fig1-A). The blood samples were collected at the drug discontinuation, and we found that compared with total TGF-β1 levels，plasma activated TGF-β1 levels were extremely lower in NR patients and were also reduced in group B (Fig1-B,C). At the same time, regulatory T cells have the same trend. By using the qPCR and Western blot we preliminary found that integrin αvβ8 regulate the activation of TGF-β1 to promote the formation of Tregs, which affects the sustained response to TPO-RAs withdrawal. Furthermore, lower levels of activated TGF-β1 in the bone marrow were associated with higher numbers of megakaryocytes, fewer plate-producing megakaryocytes, and decreased proportion of hyperploid megakaryocytes (≥16N). Through in vitro cell culture, we found that recombinant human integrin avb8 protein can promote Treg differentiation and inhibit T cell activation in ITP patients' PBMCs, but this phenomenon can be reversed by anti-β8 antibody. Then, we constructed PF4-TGF-β1 conditional knockout mice (Cre+ mice) and constructed of an ITP passive model by injecting CD41 antibody and found that the Cre+ mice have higher platelets at baseline, while the platelets dropped rapidly than Cre- mice after CD41 antibody injection (Fig2-A). Using rhTPO to treat the ITP mice, we found that Cre+ mice were less effective than Cre- mice, and the sustained response time was shorter after drug withdrawal. When sacrificed the mice, decreased Treg cells and elevated M1 macrophages were detected in Cre+ mice. Meanwhile, HE staining of bone marrow found a greater number of megakaryocytes, but the cell diameter decreased significantly (Fig2-B, D). Next, we treated the mice with rhTPO in combined with recombinant mice integrin avb8 (50ng/kg or 250ng/kg), the therapeutic efficacy and sustained response time in the high-dose avb8 group were significantly improved (Fig2-C) We also constructed a more representative ITP active model of autoimmune diseases, by immunizing CD61KO mice to produce autoantibodies and infusing spleen cells back into SCID mice. Simultaneously through transfection siRNA or shRNA to activate or silence mouse T cell integrin β8 and reinfuse it into SCID mice. We found that activating integrin β8 in mice had a more significant therapeutic effect and maintained a longer response time. Finally, through single-cell sequencing analysis, the smads signaling pathways，integrin binding pathways and AP-1 family signaling pathways were significantly enriched in group A patients. Further study confirmed that TPO-RAs can regulate the AP-1 family, which upregulated the β8 subunit, resulting in enhanced activation of TGF-β1 (Fig3-A, B). Conclusion: Integrin αvβ8 activates TGF-β1 and upregulates Tregs to restore immune intolerance in ITP patients, meanwhile, activated TGF-β1 also promotes hyperploidy megakaryocyte differentiation in bone marrow, which co-regulates the sustained response after TPO-RAs discontinuation.

Transcriptome analysis of T cells from Ldlr−/− mice and effects of in vitro vitamin D treatment

Vitamin D is known for its immunosuppressive effects on T cells, suppressing Th1 and Th17 and promoting Treg differentiation. Th1 cells contribute to inflammatory responses such as inflammatory cytokine production and macrophage activation, which accelerate the progression of atherosclerosis. However, the mechanisms underlying the modulation of T cell functions by vitamin D in atherosclerosis have not been investigated. This study analyzed the gene expression profiles of T cells, using RNA-seq transcriptome analysis, to investigate the effects of in vitro vitamin D treatment on T cell differentiation and signal transduction pathways in Ldlr knock-out (Ldlr−/−) mice. C57BL/6 mice were randomly assigned to two groups and fed a control diet (CON) or a Western diet (WD) for 16 weeks, while Ldlr−/- mice (LDLR−/−) were fed a Western diet. Splenic T cells were isolated and stimulated with anti-CD3e and anti-CD28 mAb for 48 h with or without 10 nM 1,25(OH)2D3. RNA sequencing was performed, followed by KEGG and GO enrichment analyses. Populations of T cell subsets and cytokine production were measured to assess T cell lineage differentiation. The JAK-STAT, HIF-1, and calcium signaling pathways of Ldlr−/− mice significantly differed from those of control mice, and 1,25(OH)2D3 treatment affected MAPKKK binding molecular function of Ldlr−/− mice. Percentages of Th1 cells and IL-17 production were significantly reduced by 1,25(OH)2D3 treatment in all three mouse groups. These results suggest that 1,25(OH)2D3 has anti-inflammatory effects in atherosclerosis and is involved in cell signaling pathways that could prevent disease progression by regulating T cell differentiation and effector functions.

Mechanistic insights into the role of probiotics in modulating immune cells in ulcerative colitis.

Ulcerative colitis (UC) is a persistent inflammatory disorder that affects the gastrointestinal tract, mainly the colon, which is defined by inflammatory responses and the formation of ulcers. Probiotics have been shown to directly impact various immune cells, including dendritic cells (DCs), macrophages, natural killer (NK) cells, and T and B cells. By interacting with cell surface receptors, they regulate immune cell activity, produce metabolites that influence immune responses, and control the release of cytokines and chemokines. This article is a comprehensive review wherein we conducted an exhaustive search across published literature, utilizing reputable databases like PubMed and Web of Science. Our focus centered on pertinent keywords, such as "UC," 'DSS," "TNBS," "immune cells," and "inflammatory cytokines," to compile the most current insights regarding the therapeutic potential of probiotics in managing UC. This overview aims to provide readers with a comprehensive understanding of the effects of probiotics on immune cells in relation to UC. Probiotics have a crucial role in promoting the proliferation of regulatory T cells (Tregs), which are necessary for preserving immunological homeostasis and regulating inflammatory responses. They also decrease the activation of pro-inflammatory cells like T helper 1 (Th1) and Th17 cells, contributing to UC development. Thus, probiotics significantly impact both direct and indirect pathways of immune cell regulation in UC, promoting Treg differentiation, inhibiting pro-inflammatory cell activation, and regulating cytokine and chemokine release. Probiotics demonstrate significant potential in modulating the immune reactions in UC. Their capacity to modulate different immune cells and inflammation-related processes makes them a promising therapeutic approach for managing UC. However, further studies are warranted to optimize their use and fully elucidate the molecular mechanisms underlying their beneficial effects in UC treatment.

Ginkgo biloba extract promotes Treg differentiation to ameliorate ischemic stroke via inhibition of HIF-1α/HK2 pathway.

The ischemic brain can dialogue with peripheral tissues through the immune system. Ginkgo biloba extract (EGb) was used to regulate various neurological disorders; however, the impact of EGb on ischemic stroke is still unclear. Here, we aimed to investigate whether immunomodulation has participated in the beneficial effects of EGb on ischemia/reperfusion (I/R) brain injury. Mice were orally administered with EGb once daily for 7 days before the induction of I/R. Neurobehavioral scores, infarct volume, and brain inflammation were determined. The proportion of CD4+ T cells was detected by flow cytometry. EGb significantly lowered neurobehavioral scores, infarct volume, and the level of inflammatory cytokines in I/R mice. Interestingly, EGb altered the proportion of CD4+ T cells, particularly increasing the proportion of Treg cells. Depletion of Treg cells weakened the neuroprotective effects of EGb on ischemic stroke; furthermore, EGb decreased the expression of HIF-1α and HK2 and promoted the differentiation of Treg cells in vitro. EGb suppressed the HIF-1α/HK2 signaling pathway to promote the differentiation of Treg cells and ameliorate ischemic stroke in mice. The expansion effect of EGb on Treg cells could be exploited as part of future stroke therapy.

Sphingosine 1‐phosphate combining with S1PR4 promotes regulatory T cell differentiation related to FAO through Nrf2/PPARα

AbstractMetabolism and metabolic processes have long been considered to shape the tumour immunosuppressive microenvironment. Recent research has demonstrated that T regulatory cells (Tregs) display high rates of fatty acid oxidation (FAO) and a relatively low rate of glycolysis. Sphingosine 1‐phosphate (S1P), which is a G protein signalling activator involved in immune regulation and FAO modulation, has been implicated in Treg differentiation. However, the precise relation between Treg differentiation and S1P remains unclear. In this study, we isolated naïve CD4+ T cells from the spleens of 6–8‐week‐old BALB/c mice using magnetic bead sorting, which was used in our study for Treg differentiation. S1P stimulation was performed during Treg differentiation. We examined the oxygen consumption and palmitic acid metabolism of the differentiated Tregs and evaluated the expression levels of various proteins, including Nrf2, CPT1A, Glut1, ACC1 and PPARα, through Western blotting. Our results demonstrate that S1P promotes Treg differentiation and enhances FAO, and that the expression of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and peroxisome proliferator‐activated receptor α (PPARα) is upregulated. Furthermore, Nrf2 or PPARα knockdown dampened the Treg differentiation and FAO that were promoted by S1P, confirming that S1P can bind with S1PR4 to promote Treg differentiation through the Nrf2/PPARα signalling pathway, which may be related to FAO facilitation.

Content of myeloid-derived suppressor cells in autoimmune diseases in children

Myeloid-derived suppressor cells (MDSCs) play an important role in regulation of immune response. An increase in their number in adult patients with autoimmune diseases has been reported. G-MDSCs, M-MDSCs, and MDSCs(M-G-) at different stages of autoimmune disease may both activate T cell proliferation, leading to disease progression, or inhibit it, thus promoting Treg differentiation. Arginase-1 (Arg- 1) is an enzyme in MDSCs that reduces the concentration of arginine required for T lymphocyte proliferation. Our aim was to evaluate the content of MDSCs populations and functional activity of MDSCs in children with autoimmune diseases. 75 children with inflammatory bowel diseases (IBD), 60 children with multiple sclerosis (MS), 69 children with psoriasis (PS), 62 healthy age-matched children were included into the study. The content of MDSCs ((CD3, CD19, CD56, HLA-DR)-, CD11b+ and CD33+), subpopulations of MDSCs (M-MDSCs, G-MDSCs expressing CD14 and CD15), assessment of Arg-1 activity were performed by flow cytometry techniques. The content of MDSCs in patients with IBD, MS and PS was significantly higher than in the comparison group and depended on the state of exacerbation/remission. In exacerbation and remission of IBD, MS and PS, a significant increase of MDSCs was revealed when compared with healthy children; the highest values were found in children in exacerbation of MS (Me-3.5 (2.5-5.6) % MNC against Me-1.6 (0.9-2.5) % MNC, p 0.001). In patients with MS, the content of G-MDSC, M-MDSC was significantly higher, and MDSC(M-G-) was lower than in healthy children. An increase in absolute amounts of G-MDSCs was shown in MS exacerbation compared to the disease remission state (p = 0.022). For patients with IBD, a significant increase in percentage of MDSCs and M-MDSCs (p = 0.014 and p = 0.045, respectively) was obtained in exacerbation of the disease relative to remission state. In patients with IBD, MS, and PS, a significant increase in Arg-1 activity in MDSCs was found, with a decreased number of MDSCs in patients in remission compared to exacerbation phase of the disease. In children with autoimmune diseases, an increase in the MDSC populations was found. The activity of arginase-1 in MDSCs is increased in remission, along with a decrease in their numbers.

Kuijieling decoction regulates the Treg/Th17 cell balance in ulcerative colitis through the RA/RARα signaling pathway

Ethnopharmacological relevanceUlcerative colitis (UC) is an idiopathic intestinal disease characterized by chronic inflammation with unknown etiology. Kuijieling decoction is a traditional Chinese Medicine with unique therapeutic efficacy for UC. Aim of the studyTo validate the effects of Kuijieling decoction on vitamin A metabolism and retinoic acid (RA) production, along with evaluation of its immunomodulatory activity, and further clarify the upstream mechanisms underlying Treg/Th17 regulation that contribute to therapeutic effects against UC. Materials and methodsNetwork pharmacology and molecular docking analyses were employed to predict the potential anti-UC targets of Kuijieling and associated pathways. A rat model of UC was generated by treatment with trinitrobenzene sulfonic acid (TNBS) to induce inflammation. T lymphocytes were induced to differentiate into Th17 via combined stimulation with the cytokines TGF-β1, IL-6 and IL-23. Expression levels of RA/RARα-related factors (RARα, CRABPII, Smad3, IL-6R and IL-23R) and Treg/Th17 cell-related factors (Foxp3, RORγt) were measured via western blot (WB), quantitative real-time PCR (RT-qPCR), and immunohistochemistry analyses. Components of the vitamin A metabolic pathway (vitamin A, retinol, retinoic acid) and Treg/Th17 cell-related cytokines (IL-10, IL-17, IL-21) were evaluated using ELISA. Flow cytometry was performed to determine the percentages of Treg and Th17 cells. ResultsThe action targets of Kuijieling were significantly associated with T cell activation, Th17 cell differentiation and the immune response. IL-2, IL-6, STAT3 and RARα displayed strong binding affinities with the main components of Kuijieling, suggesting an important role in its therapeutic efficacy. Kuijieling promoted vitamin A metabolism and RA/RARα signaling in UC rats and T lymphocytes. Moreover, Kuijieling effectively regulated the Treg/Th17 cell balance in UC rats and T lymphocytes and relieved inflammation. The protective effect of Kuijieling was weakened after inhibition of the RA/RARα signaling pathway. ConclusionsKuijieling promotes vitamin A metabolism and RA synthesis, enhances interactions between RA, intracellular binding protein CRABPII and nuclear receptor RARα, and upregulates Smad3 and Foxp3, thus promoting Treg differentiation. Simultaneously, Kuijieling inhibited expression of IL-6R and IL-23R genes and production of RORγt, leading to suppression of Th17 differentiation, and ultimately, reduction of the Th17/Treg cell ratio.

Characterization of the alternative splicing landscape in lung adenocarcinoma reveals novel prognosis signature associated with B cells.

Lung cancer is the second most commonly diagnosed cancer and the leading cause of cancer-related death. Malignant pleural effusion (MPE) is a special microenvironment for lung cancer metastasis. Alternative splicing, which is regulated by splicing factors, affects the expression of most genes and influences carcinogenesis and metastasis. mRNA-seq data and alternative splicing events in lung adenocarcinoma (LUAD) were obtained from The Cancer Genome Atlas (TCGA). A risk model was generated by Cox regression analyses and LASSO regression. Cell isolation and flow cytometry were used to identify B cells. We systematically analyzed the splicing factors, alternative splicing events, clinical characteristics, and immunologic features of LUAD in the TCGA cohort. A risk signature based on 23 alternative splicing events was established and identified as an independent prognosis factor in LUAD. Among all patients, the risk signature showed a better prognostic value in metastatic patients. By single-sample gene set enrichment analysis, we found that among tumor-infiltrating lymphocytes, B cells were most significantly correlated to the risk score. Furthermore, we investigated the classification and function of B cells in MPE, a metastatic microenvironment of LUAD, and found that regulatory B cells might participate in the regulation of the immune microenvironment of MPE through antigen presentation and promotion of regulatory T cell differentiation. We evaluated the prognostic value of alternative splicing events in LUAD and metastatic LUAD. We found that regulatory B cells had the function of antigen presentation, inhibited naïve T cells from differentiating into Th1 cells, and promoted Treg differentiation in LUAD patients with MPE.

Periosteum-derived mesenchymal stem cell alleviates renal fibrosis through mTOR-mediated Treg differentiation

Background Mesenchymal stem cells (MSCs) are the hotspots of cellular therapy due to their low immunogenicity, potent immunoregulation, and unique renoprotection. The present study aimed to investigate the effects of periosteum-derived MSCs (PMSCs) in ischemia–reperfusion (IR)-mediated renal fibrosis. Methods Using cell proliferation assay, flow cytometry, immunofluorescence, and histologic analysis, the differences in cell characteristics, immunoregulation, and renoprotection of PMSCs were compared to the bone marrow-derived MSCs (BMSCs), the most frequently studied stem cells in cellular therapy. In addition, the mechanism of PMSC renoprotection was investigated by 5′ end of the RNA transcript sequencing (SMART-seq) and mTOR knockout mice. Results The proliferation and differentiation capabilities of PMSCs were stronger than those of BMSCs. Compared with BMSCs, the PMSCs exerted a better effect on alleviating renal fibrosis. Meanwhile, the PMSCs more effectively promote Treg differentiation. Treg exhaustion experiment indicated that Tregs exerted an important effect on inhibiting renal inflammation and acted as a critical mediator in PMSC renoprotection. Additionally, SMART-seq results implied that the PMSCs promoted Treg differentiation, possibly via the mTOR pathway. In vivo and in vitro experiments showed that PMSC inhibited mTOR phosphorylation of Treg. After mTOR knockout, the PMSCs failed to promote Treg differentiation. Conclusions Compared with BMSCs, the PMSCs exerted stronger immunoregulation and renoprotection that was mainly attributed to PMSC promotion for Treg differentiation by inhibiting the mTOR pathway.

Immunomodulatory functions of the gut protist Pentatrichomonas hominis in health and disease

Abstract Protists have been identified as part of the mammalian gut microbiome and are known to impact gut immunity and complex intestinal diseases such as inflammatory bowel disease (IBD). In humans, studies mainly describe host interactions with disease-causing species, while little is known about how commensal protists impact the immune system. Protists from the Parabasalid family, including Pentatrichomonas hominis (P. hominis), have been identified in about 30% of healthy individuals; however, the role of these non-pathogenic commensal Parabasalids remains unstudied. Our data shows that P. hominis promotes Treg differentiation in vitro. Interestingly, this ability was found to be contact-independent, as culture supernatant was sufficient to induce Tregs. Moreover, by doing de-novo genome and transcriptome assembly and high-resolution mass spectrometry we have identified immunomodulatory candidate pathways and metabolites that might be mediating this effect. Re-establishment of tolerance is a major therapeutic strategy for treating intestinal diseases. Hence, we were interested in studying P. hominis impact on the gut immune landscape. To that aim, we successfully and stably colonized mice with P. hominis in gnotobiotic housing conditions and under antibiotic regimen and we are testing P. hominis role under steady state and IBD mouse models. Altogether, our research will address a present gap in knowledge, potentiating the study of protists in the microbiome and their association with intestinal disorders.