Splenic Treg Research Articles

Zingerone improves the immune responses in an animal model of breast cancer.

The potent anti-tumorigenic effects were attributed to ginger and there are some reports regarding the anti-cancer and immunomodulatory properties ginger-derived components. This study aimed to investigate the effects of zingerone on some immune-related parameters in an animal model of breast cancer. The breast cancer was established in female BALB/c mice using a carcinogenic 4T1 cell line. At day 10 after cancer induction, tumor-bearing mice were divided into five groups and treated intraperitoneal (daily from days 11-30) with saline or zingerone (at doses 10, 20, 50 and 100mg/kg/day). The mice were sacrificed on day 31 and the number of splenic Th1- and Treg cells, the expression of IFN-γ and TGF-β in the blood mononuclear cells, the antibody production against sheep red blood cell (SRBC) were determined using flow cytometry, real time-PCR and a standard hemagglutination assay, respectively. Zingerone at doses 50 and 100mg/kg enhanced the number of splenic Th1 cells (p<0.03 and 0.007, respectively); at doses 10, 20, 50 and 100mg/kg reduced the number of splenic Treg cells (p<0.02, 0.01, and 0.01, respectively), at doses 50 and 100mg/kg enhanced the expression of IFN-γ (p<0.03), at doses 50 and 100mg/kg reduced the expression of TGF-β, at doses 50mg/kg reduced the titer of anti-SRBC antibody (p<0.05). Zingerone improve the T cell-mediated and antibody responses in a mouse model of breast cancer. Theimmunotherapeutic potentials of zingerone in cancers need more considerations.

The effect of fingolimod on regulatory T cells in a mouse model of brain ischaemia

BackgroundThe role of the immune system in stroke is well-recognised. Fingolimod, an immunomodulatory agent licensed for the management of relapsing-remitting multiple sclerosis, has been shown to provide benefit in rodent models of stroke. Its mechanism of action, however, remains unclear. We hypothesised fingolimod increases the number and/or function of regulatory T cells (Treg), a lymphocyte population which promotes stroke recovery. The primary aim of this study was to rigorously investigate the effect of fingolimod on Tregs in a mouse model of brain ischaemia. The effect of fingolimod in mice with common stroke-related comorbidities (ageing and hypercholesteremia) was also investigated.MethodsYoung (15–17 weeks), aged C57BL/6 mice (72–73 weeks), and ApoE−/− mice fed a high-fat diet (20–21 weeks) underwent permanent electrocoagulation of the left middle cerebral artery. Mice received either saline or fingolimod (0.5 mg/kg or 1 mg/kg) at 2, 24, and 48 h post-ischaemia via intraperitoneal injection. Another cohort of young mice (8–9, 17–19 weeks) received short-term (5 days) or long-term (10 days) fingolimod (0.5 mg/kg) treatment. Flow cytometry was used to quantify Tregs in blood, spleen, and lymph nodes. Immunohistochemistry was used to quantify FoxP3+ cell infiltration into the ischaemic brain.ResultsFingolimod significantly increased the frequency of Tregs within the CD4+ T cell population in blood and spleen post-ischaemia in all three mouse cohorts compared to untreated ischemic mice. The highest splenic Treg frequency in fingolimod-treated mice was observed in ApoE−/− mice (9.32 ± 1.73% vs. 7.8 ± 3.01% in young, 6.09 ± 1.64% in aged mice). The highest circulating Treg frequency was also noted in ApoE−/− mice (8.39 ± 3.26% vs. 5.43 ± 2.74% in young, 4.56 ± 1.60% in aged mice). Fingolimod significantly increased the number of FoxP3+ cells in the infarct core of all mice. The most pronounced effects were seen when mice were treated for 10 days post-ischaemia.ConclusionsFingolimod increases Treg frequency in spleen and blood post-ischaemia and enhances the number of FoxP3+ cells in the ischaemic brain. The effect of fingolimod on this regulatory cell population may underlie its neuroprotective activity and could be exploited as part of future stroke therapy.

Exposure of pregnant rats to staphylococcal enterotoxin B increases offspring splenic Treg number and function via decreasing FoxP3 methylation

Staphylococcus aureus is an infectious pathogen that is relatively common, but that can cause severe disease in pregnant women and their fetus. We previously demonstrated that exposing pregnant rats to staphylococcal enterotoxin B (SEB) altered splenic CD4/CD8 T cell frequencies in their offspring. Whether prenatal SEB exposure impacts Tregs in these offspring, however, remains to be determined. As such, in this study, we intravenously injected pregnant rats with 15 μg of SEB on gestational day 16. Splenic tissue was then harvested from 1-, 3-, and 5-day-old neonatal rats and analyzed via flow cytometry to assess Treg numbers. In addition, FoxP3 expression levels were assessed via qPCR and western blotting, while FoxP3 methylation status was evaluated via methyl-DNA immunoprecipitation qPCR. Immunosuppression assays were additionally used to gauge Treg suppressive functionality. We found that exposing pregnant rats to SEB resulted in a significant increase in Treg numbers, FoxP3 expression, and Treg suppressive capacity in the spleens of both neonatal and adult offspring. In addition, total T cell, CD4+T cell, and non-Treg CD4+ T cell numbers were elevated in the spleens of offspring following prenatal SEB exposure. We additionally determined that SEB exposure resulted in a significant reduction in FoxP3 DNA methylation. Together, our results indicate that prenatal SEB exposure can markedly enhance offspring splenic Treg numbers and functionality at least in part by decreasing FoxP3 methylation.

Irreversible electroporation induces CD8+ T cell immune response against post-ablation hepatocellular carcinoma growth.

Ablative treatment evokes antitumor immunity, but knowledge on the emerging irreversible electroporation (IRE)-induced immunity in hepatocellular carcinoma (HCC) is limited. To investigate the immune effects induced by IRE and its role in preventing post-ablation HCC progression, a C57BL/6J mouse model bearing subcutaneous H22 hepatoma was employed. IRE treatment significantly suppresses HCC growth, and treated mice are tumor-free after secondary tumor injection and show increased splenic interferon-gamma (IFN-γ)+CD8+ T cells. Additionally, more CD8+ T and dendritic cells, but not CD4+ T, B or NK cells, infiltrate into peri-ablation zones after IRE at day 7. Depletion of CD8+ T cells induces local tumor regrowth and distant metastasis after IRE. Vaccination using IRE-processed H22 lysates prevents tumorigenesis in mice, suggesting a protective immune response. IRE also alleviates immunosuppression by reducing local and splenic Treg and PD-1+ T cells. Regarding mechanism, IRE induces cell necrosis and significant release of danger-associated molecular patterns including ATP, high mobility group box 1 and calreticulin that are pivotal to CD8+ T cell immunity. Together, IRE is a promising approach to evoke CD8+ T cell immunity, which help prevent post-ablation HCC progression.

Rutin prevents inflammation-associated colon damage via inhibiting the p38/MAPKAPK2 and PI3K/Akt/GSK3β/NF-κB signalling axes and enhancing splenic Tregs in DSS-induced murine chronic colitis.

A large body of emerging evidence has revealed the role of p38/MK2 and PI3K/Akt/GSK3β cascades in the orchestrating process of colitis. Rutin, a bioflavonoid present in many fruits and vegetables, has been recognized to offer therapeutic attributes in acute colitis. However, its role in chronic colitic condition has not yet been delineated in reference to p38/MK2 and PI3K/Akt/GSK3β signalling. The present investigation assessed the efficacy and underlying molecular mechanism of rutin in alleviating DSS-induced chronic colitis. The analysis of signalling pathways demonstrated the robust activation of PI3K/Akt/GSK3β/MAPKs/NF-κB and p38/MK2 in DSS-induced colitis in animals, which was efficiently alleviated following the rutin treatment. In silico studies indicated its target specificity with these pathways. Rutin administration markedly improved the disease activity score, colon length, goblet cell loss and compromised colon epithelial integrity in colitic mice. Decreased expression of oxi-inflammatory markers such as IgM, IgE, iNOS, ICAM-1, HO-1 and Th1/IL-10 cytokines ratios after treatment suggests its efficacy in regulating effector, regulatory and B cell homeostasis. Additionally, rutin demonstrated its role in restoring epithelial integrity by modulating the transcript levels of tight junction proteins, mucus-secreting proteins, epithelial cell proliferation and apoptosis. Treg expansion revealed that rutin supplementation also exhibits an immune regulatory potential and suppresses inflammatory aggravation mediated by adaptive immune responses. Overall, results indicate that the modulation of p38/MK2 and PI3K/Akt/GSK3β/NF-κB pathways by rutin represents a novel therapeutic approach in chronic colitis that help to curb dysregulated intestinal integrity, cytokine ratio and splenic Tregs.

The characteristics of CD4+T-helper cell subset differentiation in experimental Clonorchis sinensis-infected FVB mice.

Objective(s):Immune responses are tightly regulated in the development of clonorchiasis. However, the adaptive immune regulatory pathway contributing to the pathological processes of clonorchis sinensis (C. sinensis) infection is still not clear. In this study, we assessed the dynamic changes of CD4+T cell subsets and the related cytokines as well as transcription factors during C. sinensis infection. Materials and Methods:We used female FVB mice to establish the infection model. H&E and Masson’s stain were performed in 2 or 8 weeks post-infection (PI) liver of C. sinensis. The percentages of splenic Th1, Th2, and Treg in CD4+T cells were detected by flow cytometry. The transcription factors T-bet, GATA3, Foxp3, and RORγt gene expression were detected by qPCR. The protein expression of IL-10, IL-17, IL-4, IL-2, and Tumor Necrosis Factor-α (TNF-α) were examined using ELISA.Results:The percentages of splenic Th1, Th2, and Treg in CD4+T cells were significantly increased in both 2 and 8 weeks PI of C. sinensis, while the ratio of Treg/Th17 as well as the percentage of Treg in serum was gradually increased during the development of infection. The expressions of T-bet, GATA3, Foxp3, and RORγt were increased in 8 weeks PI. Serum levels of IL-10, IL-17, IL-4, and IL-2 were profoundly increased in infected mice, while the concentrations of TNF-α increased to peak two weeks PI. Conclusion:Our results suggested that the imbalance of CD4+T cell subsets may regulate and contribute to an appropriate compromise between pathology, tissue repair, and elimination in a susceptible murine host.

Tumor-Infiltrating Regulatory T-cell Accumulation in the Tumor Microenvironment Is Mediated by IL33/ST2 Signaling.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression-related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3- conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

Ubiquitination of MHC Class II Is Required for Development of Regulatory but Not Conventional CD4+ T Cells.

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI /KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March8 -/- mice were largely unaltered. In MLRs, March8 -/-, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- and allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March8 -/- mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development.

Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10–6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

Induction of systemic immune responses and reversion of immunosuppression in the tumor microenvironment by a therapeutic vaccine for cervical cancer.

Cervical cancer is the most common malignant tumor of the genital tract in females worldwide. Persistent human papillomavirus (HPV) infection is closely associated with the occurrence of cervical cancer. No licensed therapeutic HPV vaccines for cervical cancer are currently available. In our previous study, we demonstrated that the vaccine containing the HPV16 E7 43-77 peptide and the adjuvant unmethylated cytosine-phosphate-guanosine oligodeoxynucleotide elicited significant prophylactic and therapeutic effects on cervical cancer. In the current study, we comprehensively evaluated the effect of the vaccine on systemic immune responses and the tumor microenvironment (TME) in a mouse model of cervical cancer. The results showed that the administration of the vaccine induced a significant increase in splenic IFN-γ-producing CD4 and CD8 T cells as well as tumor infiltrating CD4 and CD8 T cells. Moreover, marked decreases in splenic MDSCs and Tregs as well as intratumoral MDSCs, Tregs and type 2-polarized tumor-associated macrophages were observed in the vaccine group. The profile of cytokines, chemokines and matrix metalloproteinases (MMPs) in the TME revealed significantly increased expression of IL-2, IL-12, TNF-α, IFN-γ, CCL-20, CXCL-9, CXCL-10 and CXCL-14 and decreased expression of IL-6, IL-10, TGF-β, CCL-2, CCL-3, CCL-5, CXCL-8, MMP-2, MMP-9 and VEGF in the vaccine group. The expression of the cell proliferation indicator Ki67, apoptosis regulatory protein p53 and angiogenesis marker CD31 was significantly decreased in the vaccine group. In conclusion, the vaccine reversed tolerogenic systemic and local TME immunosuppression and induced robust antitumor immune responses, which resulted in the inhibition of established implanted tumors.

Limited efficacy of rapamycin monotherapy in vascularized composite allotransplantation

BackgroundVascularized composite allotransplantation (VCA) is a novel and life-enhancing procedure to restore a patient's function and/or appearance. Current immunosuppression in VCA recipients is based on calcineurin inhibitor (CNI) therapy that can lead to severe complications, such that inducing immune tolerance is a major goal of VCA research. In contrast to CNI, rapamycin (RPM) is thought to be beneficial to the development of immune tolerance by suppressing T-effector cells (Teffs) and expanding T-regulatory (Treg) cells. However, we found high dose RPM monotherapy prolonged VCA survival by only a few days, leading us to explore the mechanisms responsible. MethodsA mouse orthotopic forelimb transplantation model (BALB/c- > C57BL/6) was established using WT mice, as well as C57BL/6 recipients with conditional deletion of T-bet within their Treg cells. Events in untreated VCA recipients or those receiving RPM or FK506 therapy were analyzed by flow-cytometry, histopathology and real-time qPCR. Results. Therapy with RPM (2 mg/kg/d, p < .005) or FK506 (2 mg/kg/d, p < .005) each prolonged VCA survival. In contrast to FK506, RPM increased the ratio of splenic Treg to Teff cells (p < .05) by suppressing Teff and expanding Treg cells. While the proportion of activated splenic CD4 + Foxp3- T cells expressing IFN-γ were similar in control and RPM-treated groups, RPM decreased the proportions ICOS+ and CD8+ IFN-γ + splenic T cells. However, RPM also downregulated CXCR3+ expression by Tregs, and forelimb allografts had reduced infiltration by CXCR3+ Treg cells. In addition, allograft recipients whose Tregs lacked T-bet underwent accelerated rejection compared to WT mice despite RPM therapy. ConclusionsWe demonstrate that while RPM increased the ratio of Treg to Teff cells and suppressed CD8+ T cell allo-activation, it failed to prevent CD4 Teff cell activation and impaired CXCR3-dependent Treg graft homing, thereby limiting the efficacy of RPM in VCA recipients.

Research Highlights

Research Highlights

Prunella vulgaris L. attenuates experimental autoimmune thyroiditis by inducing indoleamine 2,3-dioxygenase 1 expression and regulatory T cell expansion

BackgroundPrunella vulgaris L. (P. vulgaris) has traditionally been used to treat swelling and inflammation of the thyroid gland. This study aimed to evaluate the effects of P. vulgaris on experimental autoimmune thyroiditis (EAT) and explore the roles of indoleamine 2,3-dioxygenase 1 (IDO1) and regulatory T cells (Tregs) in these P. vulgaris-mediated effects. MethodsThe main bioactive compounds in P. vulgaris were analysed by high-performance liquid chromatography. An EAT model was established by immunization of Lewis rats with thyroglobulin via subcutaneous injection. Thyroid volume was assessed by ultrasound, and lymphatic infiltration in the thyroid was evaluated by haematoxylin and eosin staining. The serum levels of thyroglobulin antibody (TgAb) and cytokines were measured by indirect enzyme-linked immunosorbent assay. The percentage of CD4+CD25+Foxp3+ Tregs was detected by flow cytometry. The mRNA and protein levels of IDO1 were measured by qRT-PCR and Western blotting, respectively. The levels of tryptophan (Trp) and kynurenine (Kyn) in serum and faecal samples were assessed with a fluorometric kit and spectrophotometry. ResultsThe main bioactive compound in P. vulgaris was rosmarinic acid. The TgAb level and thyroid volume in EAT rats were significantly decreased after administration of P. vulgaris (P < 0.01). The inflammation score in EAT rats that were administered P. vulgaris was significantly lower than that in the EAT controls (P < 0.01). In addition, P. vulgaris promoted the expansion of splenic Tregs and increased the production of IL-10 and TGF-β (P < 0.01) in EAT rats. Moreover, P. vulgaris induced IDO1 mRNA and protein expression in the spleen and intestine in P. vulgaris-treated EAT rats (P < 0.01). Finally, Trp levels were reduced and Kyn levels and the Kyn/Trp ratio were increased in the serum of P. vulgaris-treated EAT rats. ConclusionWe were the first to demonstrate the role of IDO1-induced Treg expansion in P. vulgaris-mediated attenuation of EAT. Our study provides insight into the immunopathogenesis of autoimmune thyroiditis and shows the potential therapeutic value of P. vulgaris.

Promoting antitumor immunity and increased T-cell function by depletion and blockade of the immunosuppressive cell compartment in murine cancer

Abstract Tumor growth occurs due to a failure of the immune system to recognize and destroy malignant cells. The cause of breakdown in immune surveillance is at the systemic level and at in the tumor microenvironment. These changes can even occur in the tumor cells themselves to make them less detectable by anticancer immune cells such as downregulation of tumor-associated antigens or decreased expression of antigen presenting molecules such as MHCI. In addition, many cancer types will upregulate immune checkpoint molecules, such as PD-L1 which binds to PD-1 on cytotoxic T-cells to suppress antitumor immune cell function. Immune surveillance can also be curtailed by the presence of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and T-regulatory cells (T-regs) which both serve to dampen antitumor immunity. Our lab utilizes a metronomic low-dose therapeutic approach to examine the effects of anticancer drugs against murine breast cancer and the effects on the immunosuppressive cell compartments. Recent data has shown a reduction in T-regs following treatment with cyclophosphamide, a common chemotherapy. We have also shown that guadecitabine, a second-generation DNA methyl-transferase inhibitor, depletes MDSCs. Here we show that combining low-dose metronomic treatments of guadecitabine and cyclophosphamide leads to a depletion in splenic and tumor MDSCs and T-regs, increased CD8+ T-cell function in the tumor, as well as a reduction in tumor size in FoxP3 reporter mice bearing triple-negative 4T1 tumors. In addition, preliminary studies suggest this antitumor effect is amplified by combining guadecitabine and cyclophosphamide with checkpoint inhibitors such as anti-PD-1, in vivo.

Heat shock protein gp96 controls regulatory T cell lineage stability via IL-2/pSTAT5 signaling

Abstract Gp96 is an essential endoplasmic reticulum chaperone for various proteins including GARP, TLRs, integrins, Wnt co-receptor LRP6 and the platelet glycoprotein Ib/IX/V complex. Our laboratory has previously shown that loss of gp96 in murine Foxp3+ regulatory T cells (Tregs) results in their instability as determined by FOXP3 downregulation, impaired immunosuppressive function and production of effector cytokines (Zhang, et al., J Clin Invest., 2015, 125: 859–869). However, the mechanism underlying Treg dysfunction upon gp96 deletion remains unknown. Using tamoxifen-inducible Treg-specific gp96 gene (encoded by Hsp90b1) knockout (KO) mice (Foxp3-ERT2creHsp90b1f/f), we first phenotypically characterized gp96-deleted Tregs in mouse spleen and peripheral lymph nodes (pLNs). Gp96 deficient Tregs showed decreased frequency of TCF1−CD62L− effector population, but were enriched with short-lived KLRG1+ and Helios+ subsets, in both spleen and pLNs. We also confirmed that the expression of Foxp3 was decreased in splenic gp96-deleted Tregs, which correlated with a reduction of cell surface CD25. Intriguingly, both Foxp3 and surface CD25 levels stayed high in gp96-null Tregs in pLNs. In addition, gp96-null Tregs in mouse spleen, but not in pLNs, demonstrated compromised suppressive function. We further found that IL-2/CD25 signaling, as assessed by STAT5 phosphorylation, was more activated in gp96-null Tregs in pLNs, compared to that in spleen. In vitro IL-2 treatment was able to rescue the attenuated Foxp3 expression in gp96-null Tregs. Our work indicates that IL-2/pSTAT-5 signaling may be a key factor in maintaining Treg Foxp3 expression and its lineage stability upon gp96 deletion.

Oral therapy with colonization factor antigen I prevents development of type 1 diabetes in Non-obese Diabetic mice

Antigen (Ag)-specific tolerization prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but proved less effective in humans. Several auto-Ags are fundamental to disease development, suggesting T1D etiology is heterogeneous and may limit the effectiveness of Ag-specific therapies to distinct disease endotypes. Colonization factor antigen I (CFA/I) fimbriae from Escherichia coli can inhibit autoimmune diseases in murine models by inducing bystander tolerance. To test if Ag-independent stimulation of regulatory T cells (Tregs) can prevent T1D onset, groups of NOD mice were orally treated with Lactococcus lactis (LL) expressing CFA/I. LL-CFA/I treatment beginning at 6 weeks of age reduced disease incidence by 50% (p < 0.05) and increased splenic Tregs producing both IL-10 and IFN-γ 8-fold (p < 0.005) compared to LL-vehicle treated controls. To further describe the role of these Tregs in preventing T1D, protective phenotypes were examined at different time-points. LL-CFA/I treatment suppressed splenic TNF-α+CD8+ T cells 6-fold at 11 weeks (p < 0.005) and promoted a distinct microbiome. At 17 weeks, IFN-γ+CD4+ T cells were suppressed 10-fold (p < 0.005), and at 30 weeks, pancreatic Tbet+CD4+ T cells were suppressed (p < 0.05). These results show oral delivery of modified commensal organisms, such as LL-CFA/I, may be harnessed to restrict Th1 cell-mediated immunity and protect against T1D.

Resolvin D1 Protects Against Ischemia/Reperfusion-Induced Acute Kidney Injury by Increasing Treg Percentages via the ALX/FPR2 Pathway.

AimsTo evaluate whether Resolvin D1 attenuates ischemia/reperfusion-induced (IRI) acute kidney injury (AKI) via affecting Tregs.Materials and MethodsThe IRI-AKI mouse model was established, and RvD1 was injected into the mouse tail vein. Further, the renal function, histological changes, injury markers and serum cytokines were detected at 24 and 72 h after IRI. Flow cytometry was used to categorize regulatory T cells (Tregs) in the spleen and kidney. Treg cells were stripped with the anti-CD25 antibody blocker PC61 to assess its role in the protective effect of RvD1 on IRI mice. CD4+ T cells were obtained from spleen monocytes by magnetic bead sorting and differentiated into induced Treg (iTreg) cells. The effect of RvD1 on iTreg cell differentiation was observed in vitro. In addition, neutralizing antibodies against the orphan receptor G-protein-coupled receptor 32 (anti-GPR32) and LXA4 receptor (anti-ALX/FPR2), both RvD1 receptor blockers, were used to evaluate the effect of RvD1 on iTreg cell differentiation. Boc-1, an ALX/FPR2 receptor inhibitor, was administered via the tail vein to observe its effects on the ameliorative efficacy of RvD1 in IRI-AKI mice in vivo.ResultsIn vivo, RvD1 increased Treg percentages, alleviated renal tubular injury and reduced the serum levels of IFN-γ, TNF-α and IL-6 in IRI-AKI mice, while PC61 depleted the number of Tregs and reversed the protective effects of RvD1. In vitro, RvD1 induced the generation of iTregs. Importantly, preincubation with anti-ALX/FPR2 neutralizing antibodies but not with anti-GPR32 neutralizing antibodies, abrogated the enhancement activity of RvD1 on iTregs. In addition, in vivo blockade of the receptor ALX/FPR2 by Boc-1 reversed the beneficial effects of RvD1 on the splenic and kidney Treg percentages, renal tubular injury and serum IFN-γ, TNF-α, and IL-6 levels.ConclusionOur study demonstrates that RvD1 protects against IRI-AKI by increasing the percentages of Tregs via the ALX/FPR2 pathway.

A Diet High in Saturated Fat Results in Greater Increases in Splenic Tregs in Female vs Male Sprague Dawley Rats

BackgroundDiets high in saturated fats promote inflammation and are linked to the development of many cardiovascular diseases (CVD). Young females are typically protected from CVD vs males. However, epidemiological and basic science studies suggest that the cardiovascular protection in females is compromised by a high fat diet (HFD). Little is known regarding the impact of a HFD on T cells in males vs. females. Therefore, the current study tested the hypothesis that a HFD results in a more pro‐inflammatory T cell profile in females vs. males.Methods26‐day‐old male and female Sprague Dawley rats (n=3–5 per group) were randomized to a control (Ctrl; 7.2% calories from fat) or HFD (36% of calories from fat) for 10 weeks. 24 hour food consumption was measured every 3 weeks. Body weights were measured weekly. At the end of the 10 week treatment, body fat composition was measured via NMR. Rats were euthanized and spleen were isolated for flow cytometric analysis of Pan T cells (CD3+), CD4+ T cells (CD3+CD4+), and T regulatory cells (Tregs; CD3+CD4+FOXP3+).ResultsThroughout the treatment, rats on HFD consumed more kcal from fat vs Ctrls (P&lt;0.05). There were no differences in body weight between Ctrl and HFD rats of the same sex (P&gt;0.05 at each time point), although males were heavier than females at all time points (P&lt;0.05), Table 1. Following 10 weeks of treatment, HFD increased body fat percentage in both sexes vs Ctrl, (PDiet=0.01). Increases in body fat were similarly in both sexes (PSex=0.33; Pinteraction=0.37). At end of treatment, all groups had similar percentages of splenic CD3+ cells (Pdiet=0.27; Psex=0.14; Pinteraction=0.41). However, rats on a HFD had more CD4+ T cells vs Ctrl (PDiet=0.006). Moreover, females had more CD4+ T cells vs males (PSex=0.002; Pinteraction=0.09). HF females had the most splenic Tregs vs all groups (PDiet=0.28, PSex=0.09, PInteraction=0.007.DiscussionThis dietary model allows for investigation of the effects of a HFD on body composition and T cell count independent of obesity. Evidence in the literature suggests high anti‐inflammatory Tregs in female rats contributes to cardiovascular protection and decrease in renal Tregs compromises this protection. Thus, the increase in splenic Tregs in females on a HFD may show a compensatory increase to counteract the decreased renal Tregs observed in the literature. Future studies will test this hypothesis. Data following 0 and 10 weeks of dietary treatment expressed as means ± SEM. Assay Weeks of treatment Population Ctrl F HF‐F Ctrl‐M HF‐M Body weight (g) 0 73.25 ± 0.48 70 ± 1.05 83.2 ± 2.13 80.2 ± 1.07 10 254 ± 8.57 242.4 ± 11.05 411 ± 11.79 410.8 ± 13.72 Body fat percentage 10 7.94 ± 0.49 11.35 ± 1.23 7.84 ± 1.69 10.18 ± 0.82 Splenic T cells 10 CD3 21.5 ± 1.55 20.96 ± 2.02 25.9 ± 2.3 22.28 ± 0.65 CD4 73.48 ± 1.38 77.36 ± 2.85 58.8 ± 2.91 72.23 ± 2.44 Tregs 6.05 ± 0.31 8.47 ± 0.79 7.55 ± 0.92 5.63 ± 0.32

IL-2/IL-2R, TL1A/TNFRSF25 or Their Combined Stimulation Results in Distinct CD4+FoxP3+ Regulatory T Cell Phenotype and Suppressive Function

CD4+Foxp3+ regulatory T cells (Tregs) are non-redundant mediators of immunity and tolerance. Transfer of Tregs has emerged as a promising therapy for graft-versus-host disease (GVHD) following allogeneic HSCT (aHSCT). Our prior work demonstrated that a 2-pathway in vivo strategy targeting TNFRSF25 (with TL1A-Ig) and IL-2 receptor (with low-dose IL-2, IL-2LD) can elicit a strong increase in Treg numbers and function. In fact, very low numbers of these in vivo expanded donor Tregs suppressed GVHD post-aHSCT. Based on these findings and the success of IL-2 in pre-clinical and clinical studies, we asked: are there phenotypic/functional differences between Tregs expanded via IL-2 LD, TL1A-Ig or their combined application? Mice were administered IL-2LD, TL1A-Ig or TL1A-Ig+IL-2LD over 6 days. Splenic and lymph node (LN) Treg phenotype was determined by flow cytometry. Treg functionality was assessed with sorted populations using an in vivo MHC-mismatched aHSCT. Treatment of B6-FoxP3RFP mice with TL1A-Ig+IL-2LD vs. IL-2LD only resulted in significantly higher levels of Treg activation/differentiation and functional markers, i.e. KLRG1, CD103, Nrp1, ICOS (Fig. 1A). Ki67 expression was higher in 2-pathway vs. IL-2LD stimulation alone (Fig.1B). Notably, TL1A-Ig treatment alone induced the above phenotype. CD25 (IL-2Rα) expression was reduced after TL1A-Ig vs. IL-2-mediated expansion further emphasizing the difference between the TNFRSF25 and IL-2 receptor stimulation (Fig. 1A). Results were corroborated using a second independent mouse strain, BALB/c, following use of these protocols. To test if the observed phenotypic differences could be related to more potent Treg function in vivo, an MHC-mismatched aHSCT (B6→BALB/c) was performed using 200,000 sorted Tregs (>98% purity by CD4+FoxP3+ selection from C57BL/6-FoxP3RFP reporter mice) from donor unexpanded, IL-2LD, or TL1A-Ig+IL-2LD treated mice + T cells. As anticipated, transfer 200,000 TL1A-Ig+IL-2LD stimulated Tregs ameliorated acute GVHD (Fig. 1C). Interestingly, lower GVHD clinical scores were obtained using the same number of IL-2LD only vs.TL1A-Ig+IL-2LD stimulated Tregs (Fig. 1C). Moreover, early post-transplant, higher LN and splenic CD4/CD8 ratios were detected in aHSCT recipients treated with IL-2LD expanded Tregs vs. TL1A-Ig+IL-2LD (Fig. 1D). Overall, TL1A-Ig+IL-2LD promotes a more activated/differentiated and proliferative Treg phenotype accompanied by less effective long-term GVHD inhibition vs. IL-2LD only stimulated Tregs. We posit that these different 1- and/or 2-pathway driven Tregs may promote development of new strategies utilizing one or both populations dependent on the desire for GVHD prophylaxis or therapeutic treatment following aHSCT.

Chronic ethanol consumption and HBV induce abnormal lipid metabolism through HBx/SWELL1/arachidonic acid signaling and activate Tregs in HBV-Tg mice.

Rationale: Chronic ethanol consumption as a public health problem worldwide boosts the development of chronic liver diseases in hepatitis B virus (HBV)-infected patients. Arachidonic acid metabolite prostaglandin E2 (PGE2) activates regulatory T cells (Tregs) function. Here, we aim to investigate the underlying mechanism by which chronic ethanol consumption enriches the HBV-induced abnormal lipid metabolism and Tregs.Methods: The si-RNAs were used to weaken the expression of SWELL1 in HepG2, HepG2.2.15 and K180 cancer cell lines, followed by RNA sequencing from HepG2 cells. Arachidonic acid metabolite PGE2 and LTD4 were measured by ELISA assay in vivo and in vitro. Western blot analysis and RT-qPCR were used to examine HBx and SWELL1 and transcriptional factor Sp1 in clinical HCC samples and cell lines. The effect of chronic ethanol consumption on Tregs was tested by flow cytometry in HBV-Tg mice. The splenic Tregs were collected and analyzed by RNA sequencing.Results: The cooperative effect of ethanol and HBV in abnormal lipid metabolism was observed in vivo and in vitro. The depression of SWELL1 (or HBx) resulted in the reduction of lipid content and arachidonic acid metabolite, correlating with suppression of relative gene atlas. Ethanol and SWELL1 elevated the levels of PGE2 or LTD4 in the liver of mice and cell lines. Interestingly, the ethanol modulated abnormal lipid metabolism through activating HBx/Sp1/SWELL1/arachidonic acid signaling. Chronic ethanol consumption remarkably increased the population of PBL Tregs and splenic Tregs in HBV-Tg mice, consistently with the enhanced expression of PD-L1 in vivo and in vitro. Mechanically, RNA-seq data showed that multiple genes were altered in the transcriptomic atlas of Tregs sorting from ethanol-fed mice or HBV-Tg mice.Conclusion: The chronic ethanol intake enriches the HBV-enhanced abnormal lipid metabolism through HBx/SWELL1/arachidonic acid signaling and activates Tregs in mice.