Treg Differentiation Research Articles

In human CD4+ T-Cells, omeprazole suppresses proliferation, downregulates V-ATPase, and promotes differentiation toward an autoimmunity-favoring phenotype

BackgroundProton pump inhibitors (PPIs) represent a commonly prescribed class of medications. Triggered by findings indicating a correlation between PPI usage and susceptibility to infectious or autoimmune diseases, we studied the impact of a pharmacological concentration of omeprazole on human CD4+ T-cells. MethodsIn mixed lymphocyte reactions (MLRs), we analyzed the proliferation index and measured the concentration of key cytokines representative of distinct CD4+ T-cell subsets. In CD4+ T-cells isolated from the MLRs, we evaluated proliferation markers and pathways, the expression of signature transcription factors of CD4+ T-cell subsets, vacuolar H+- ATPase (V-ATPase) levels, and the activation status of AMP-activated kinase (AMPK) and mammalian target of rapamycin complex-1 (mTORC1). ResultsOmeprazole reduced proliferation index in MLRs, and in isolated CD4+ T-cells, it downregulated the proliferation marker Ki-67, possibly mediated by the p53- p21 pathway. Analysis of cytokines and signature transcription factors of CD4+ T-cell subsets indicated that omeprazole decreased T helper 1 (Th1) differentiation, had negligible impact on Th2 differentiation, increased Th17 differentiation, and reduced regulatory T-cell (Treg) differentiation. Omeprazole also decreased V-ATPase, a known target of PPIs and a site for AMPK and mTORC1 activation. Consequently, this led to diminished activation of these kinases, potentially elucidating the mechanism by which omeprazole influences CD4+ T-cell differentiation. ConclusionOmeprazole downregulates V-ATPase and inhibits activation of AMPK and mTORC1. As a result, omeprazole suppresses CD4+ T-cell clonal expansion, potentially contributing to the observed association between PPIs and susceptibility to infections. Additionally, it modulates CD4+ T-cell differentiation in a manner that favors autoimmunity.

Overcoming GABA-induced Treg suppression of immunity by ABAT to augment CD8+T cell anti-tumor immune response in liver cancer

Background: ABAT, a key enzyme in GABA catabolism, modulates anti-tumor immune activity across various cancers. However, the molecular mechanisms by which the ABAT/GABA axis exerts immune regulation in the liver cancer microenvironment remain unclear. Methods: ABAT expression in liver cancer tissues was scrutinized via the TCGA-LIHC database, and the 5-year survival rates of liver cancer patients were appraised through Kaplan-Meier survival analyses. The mRNA levels of ABAT in liver cancer cell lines were quantified by qRT-PCR. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), colony formation, and Transwell assays were employed to gauge the influence of ABAT overexpression on liver cancer cell growth, proliferation, migration, and invasion, respectively. Western blot evaluated EMT-related protein expression. ELISA quantified GABA, IL-10, TGF-β1, Granzyme B, and IFN-γ in the culture medium. Flow cytometry was used to measure the frequency of CD25+FOXP3+ cells and the expression of CD25, CD69, and PD-1 on CD8+T cells in co-culture. Carboxifluorescein diacetate succinimidyl ester (CFSE) dilution assays were performed to assess the proliferative activity of CD8+T cells. Results: ABAT was found to be underexpressed in liver cancer tissues and cells, and such underexpression is indicative of a poorer prognosis for patients, while its overexpression was shown to curb the malignancy of liver cancer cells. Upon overexpression of ABAT, there is a decrease in GABA levels in the cell supernatant, coupled with an increase in IL-10 and TGF-β1 cell number, and an upsurge in the CD25+FOXP3+ cell ratios, all of which were restored by the addition of exogenous GABA. Furthermore, the depletion of ABAT led to a reduction in the proliferation and tumor-killing ability of CD8+T cells, effects that were reversed by the application of GABAA receptor inhibitors. Conclusion: ABAT functions to inhibit Treg differentiation in liver cancer through downregulation of GABA, thus promoting the antitumor activity of CD8+T cells.

Cancer Cell-Derived Exosomal miR-500a-3p Modulates Hepatic Stellate Cell Activation and the Immunosuppressive Microenvironment.

Hepatocellular carcinoma (HCC) mainly depends on liver fibrosis/cirrhosis, which is regulated by tumor cells and the tumor microenvironment (TME), and is a crucial factor in tumor progression. This study aimed to identify abnormally expressed miR-500a-3p in the hepatitis-cirrhosis-HCC pathway and explored the roles of miR-500a-3p in HCC progression. A clinical cohort of patients with HCC is studied retrospectively. Subsequently, the role of miR-500a-3p transported by HCC exosomes in hepatic stellate cell (HSC) activation, hepatoma growth and invasion, and immune cell differentiation is determined by in vitro and in vivo experiments. In clinical tissues, miR-500a-3p is significantly enriched in HCC and cirrhosis tissues, and co-expression of the immune marker CD4 or PD-L1 significantly correlates with low survival rates in patients. Extracellular miR-500a-3p is taken up by HSC and PBMC, which promotes the secretion of the cytokines TGF-β1 and IL-10, increases PD-L1 expression in HSC, and stabilizes PD-1 expression in PBMC to affect the TME. Moreover, miR-500a-3p is associated with CD4+ T-cell exhaustion and Treg differentiation and is significantly associated with increased tumorigenicity in in situ mouse HCC models. Mechanistically, HCC-derived exosomal miR-500a-3p directly influences SOCS2 to regulate the JAK3/STAT5A/STAT5B signaling pathway. MiR-500a-3p promotes the growth and migration of HCC through the SOCS2/JAK3/STAT5A/STAT5B axis.

CD38 modulates cytokine secretion by NK cells through the Sirt1/NF-κB pathway, suppressing immune surveillance in colorectal cancer.

Tregs and M2-type macrophages are essential for immune surveillance. CD38 + NK cells are involved in immunoregulation by modulating cytokine secretion. This study investigated how CD38 + NKs affect Tregs and macrophages in colorectal cancer (CRC). Higher proportions of CD38 + NKs and Tregs were detected in bloods and tumor tissues of CRC patients than that in the samples from healthy controls (HCs). Compared with CD38 + NKs from HCs, the NK cells from CRC promoted the differentiation of Tregs from CD4 + T cells, and secreted increased levels of IL-10, TGF-β and TNF-α and decreased levels of IFN-γ. CD38 + NKs from CRC expressed higher levels of CD38, NF-κB and acetyl-NF-κB and lower levels of Sirt1. When CRC CD38 + NK cells were treated with anti-CD38 monoclonal antibody, the above trends were reversed. CRC CD38 + NKs with treatment of NF-κB inhibitor also showed opposite effects on cytokine secretion and CD4 + T-cell differentiation. After treatment with a Sirt1 activator, NF-κB signaling was inhibited in these CD38 + NKs, whereas treatment with a Sirt1 inhibitor activated NF-κB signaling. The supernatants of CRC CD38 + NK culture promoted M0 macrophage polarization to M2-type. We suggest that CD38 modulates cytokine secretion by NK cells through Sirt1/NF-κB signaling pathway, thereby suppressing immune surveillance in tumorigenesis.

β-catenin disruption decreases macrophage exosomal α-SNAP and impedes Treg differentiation in acute liver injury.

Hepatic macrophages and regulatory T cells (Tregs) play an important role in the maintenance of liver immune homeostasis, but the mechanism by which hepatic macrophages regulate Tregs in acute liver injury remains largely unknown. Here, we found that the hepatic Treg proportion and β-catenin expression in hepatic macrophages were associated with acetaminophen (APAP) and D-galactosamine (D-GalN)/ lipopolysaccharide (LPS)-induced acute liver injury. Interestingly, β-catenin was markedly upregulated only in infiltrating macrophages, but not in resident Kupffer cells. Myeloid-specific β-catenin knockout mice showed an increased inflammatory cell infiltration and hepatocyte apoptosis. Moreover, myeloid β-catenin deficiency decreased the hepatic Treg proportion in the injured liver. Mechanistically, in vitro co-culture experiments revealed that macrophage β-catenin modulated its exosome composition, and influenced Treg differentiation. Using mass spectrometry-based proteomics, we identified that macrophage β-catenin activation increased the level of exosomal α-SNAP, which in turn promoted Treg differentiation. Overall, our findings demonstrated a molecular mechanism that macrophage β-catenin regulated the Treg proportion in the liver by enhancing the expression of exosomal α-SNAP, providing insights into the pathophysiology of acute liver injury.

Abstract PR002: Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis

Abstract The majority of cancer-associated deaths result from distant organ metastasis, yet the mechanisms that enable this process remain poorly understood. Colonization of locoregional or distant lymph nodes (LNs) typically precedes the formation of distant organ metastases, yet it has been unclear whether LN metastasis plays a functional role in disease progression. LNs are major sites of anti-tumor lymphocyte education, including in the context of immunotherapy, yet LN metastasis frequently correlates with further disease progression. By leveraging our mouse models of LN metastasis, we find that LN colonization represents a critical step in tumor progression by inducing tumor-specific immune tolerance in a manner that promotes further dissemination of tumors to distant organs. This promotion of distant seeding by LN metastases is reliant upon adaptive immunity and is tumor specific. Using flow cytometry and single-cell sequencing to perform comprehensive immune profiling, we identify multiple cellular mediators of tolerance. LN metastases evade NK cells, induce shifts in DC and B cell populations, and promote CD8 T cell exhaustion, but of particular importance, we find that regulatory T cells (Tregs) are enriched in the LNs of mice and patients with LN metastases. LN metastatic cells exhibit an enhanced capacity to induce Treg differentiation from naïve CD4 T cells, and ablation of Tregs using FoxP3-DTR mice eliminates the ability of LN metastases to promote distant metastasis. Furthermore, adoptive transfer of Tregs from the LNs of mice bearing LN metastases to naïve mice facilitates lung metastasis in a manner that Tregs from mice without LN metastases cannot. These Tregs are induced in a tumor antigen-specific manner and TCR recognition of tumors is required for the metastasis-promoting tolerance. Using photoconvertible mice, we track the trafficking dynamics of Tregs from tumor involved LNs and demonstrate their elevated trafficking to sites of distant metastasis prior to the formation of detectable metastases in those sites. Through bulk and single-cell TCR sequencing of the Treg repertoire we reveal how LN colonization alters the clonal architecture of the Treg compartment and demonstrate how the evolution of metastatic traits coincides with shifts in the Treg repertoire. Parallel analyses in patients with melanoma and head and neck cancer reveal similar CD4 T cell dynamics in the context of LN involvement. Together, these findings demonstrate a critical role for LN metastasis in promoting tumor-specific immune tolerance and metastatic progression. Citation Format: Cort B. Breuer, Norma A. Gutierrez, Zhewen Xiong, Amanda R. Kirane, John B. Sunwoo, Nathan E. Reticker-Flynn. Lymph node colonization reprograms lymphocyte responses to generate systemic tolerance and promote distant metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR002.

Analysis of the possibility and progress of FoxP3 in tumor treatment based on its effect on Tregs

Abstract. Treg can inhibit immunity in cancer and a variety of autoimmune diseases. And foxp3 is a kind of spectrum specificity markers. Its also the main regulatory factor that can ensure the generation, maintenance and the immunosuppressive function of the Treg cells. At the same time, foxp3 can also promote the differentiation and function of Treg. In this paper, the interaction between foxp3 and various transcription factors (TFs) as well as their joint action are analyzed. These effects can have an impact on the suppressive effect of Treg cells, which in turn affects the status of the tumor. The post-translational modifications of foxp3 itself are also analyzed. foxp3 has been modified by methylation, acetylation, phosphorylation, ubiquitination, glycosylation and lactatation. Its own activity has been affected, and its binding ability to target genes has been changed. From these analyses, a conclusion can be drawn that the effect of CAR-T therapy can be enhanced by targeting multiple TFs and signaling pathways. At the same time, targeted inhibitors can be explored for various enzymes involved in post-translational modification. And these inhibitors can reduce the ability of foxp3. However, the research on TFs and post-translational modifications still has some limitations. Future studies can focus on these two aspects.

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

Neutrophil Extracellular Traps Participate in the Pathogenesis of Lupus Through S100A10-Mediated Regulatory T-Cell Differentiation and Functional Abnormalities.

In systemic lupus erythematosus (SLE), neutrophil dysregulation and neutrophil extracellular traps (NETs) formation contribute to disease pathogenesis, potentially worsening the autoimmune response. Although research indicates NETs' involvement in various autoimmune conditions, their relationship with regulatory T cells (Tregs) in SLE remains elusive. In this study, in vivo experiments were involved in administering NET injections to C57BL/6 and MRL/Ipr mice. In vitro, a co-culture system facilitated interaction between Tregs and NETs. Proteomic analysis elucidated NET composition, while RNA sequencing delineated their impact on Treg differentiation. We demonstrated that increased NET levels correlate inversely with Treg abundance in SLE patients, influencing both their proportion and functionality. NET administration reduced Treg levels and induced lupus-like symptoms in C57BL/6 mice, exacerbating symptoms in MRL/Ipr mice. DNase I treatment mitigated NET effects, restoring Treg levels and alleviating symptoms. RNA sequencing revealed altered gene expression in naïve CD4+ T cells exposed to NETs. Additionally, proteomic analysis showed S100A10 protein changes between SLE patients and healthy controls, hindering Treg differentiation. NETs influence TLR-4 of naïve CD4+ T cells via S100A10, thereby modulating Treg proportion and functionality. These findings highlight the critical role of NETs in Treg differentiation in SLE, suggesting that targeting NETs may provide a novel therapeutic approach.

Impairment of regulatory T cell stability in axial spondyloarthritis: role of EZH2 and pSTAT5.

Axial spondyloarthritis (axSpA) is a chronic inflammatory disease involving the spine, peripheral joints, and entheses. Functional impairment of regulatory T cells (Treg) is linked to inflammatory diseases, but limited data is available regarding Treg involvement in axSpA. Treg stability refers to their ability to maintain their functions and characteristics in pro-inflammatory environments. EZH2 and phosphorylated STAT5 (pSTAT5) play a critical role in maintaining Treg stability. We aimed to characterize Treg stability in patients with axSpA. Peripheral blood mononuclear cells (PBMCs) from axSpA patients, either naïve from targeted therapy or treated by TNF inhibitors (TNFi), and from healthy donors (HD), were freshly isolated. Expression of stability (EZH2, pSTAT5) and suppressive (TNFR2 and CD39) markers by Treg was analyzed by flow cytometry. EZH2 expression by Treg was decreased in axSpA patients as compared to HD (p<0.01). Mechanistic study showed that inhibition of EZH2 attenuated Treg differentiation and suppressive phenotype in vitro. EZH2 was predominantly expressed by highly suppressive TNFR2+ and CD39+ Treg. Additionally, axSpA patients also exhibited a reduced frequency of pSTAT5+ Treg compared to HD (p<0.05), and pSTAT5+ Treg frequency increased at 3 months of TNFi treatment compared to baseline (p<0.05). This last result suggested a restoration of Treg stability upon TNFi treatment. By highlighting a deficient expression of EZH2 and pSTAT5 by Treg, we revealed an impaired Treg stability in axSpA. Deciphering the pathways influenced by these molecules is necessary to assess the potential therapeutic benefits of restoring Treg stability in axSpA.

Engineering M2 macrophage-derived exosomes modulate activated T cell cuproptosis to promote immune tolerance in rheumatoid arthritis

Nanomedicines for immune modulation have made advancements in the treatment of rheumatoid arthritis (RA). However, due to aberrations in patients' immune systems, inducing antigen-specific immune tolerance while halting disease progression remains a significant challenge. Here, we develop a highly targeted multifunctional nanocomplex, termed M2Exo@CuS-CitP-Rapa (M2CPR), with the aim of selectively inhibiting inflammatory immune reactions while promoting immune tolerance towards specific antigens. M2CPR specifically targets inflammatory tissues in RA, delivering CuS NPs, CitP, Rapa, and endogenous anti-inflammatory factors, thereby ameliorating the inflammatory joint microenvironment. CuS NPs induce Cuproptosis of activated T cells, whose fragments are engulfed by resident or recruited macrophages, resulting in abundant production of TGF-β. TGF-β acts synergistically with Rapa to induce the iDCs into tDCs. tDCs present CitP to Naive T cells, promoting Tregs differentiation. Tregs, in turn, produce more TGF-β, inducing tDCs differentiation, thereby establishing a cycle of immune tolerance. Through in vitro and in vivo experiments, we validate that M2CPR can induce robust and durable antigen-specific immune tolerance, offering a new paradigm for RA therapy.

Pro-inflammatory NK-like T cells are expanded in the blood and inflamed intestine in Crohn’s disease

Altered intestinal immune homeostasis leads to chronic inflammation in Crohn’s disease (CD). To address disease- and tissue-specific alterations, we performed a T cell-centric mass cytometry analysis of peripheral and intestinal lymphocytes from patients with CD and healthy donors’ PBMCs. Chronic intestinal inflammation enforced activation, exhaustion, and terminal differentiation of CD4+ and CD8+ T cells and a relative enrichment of CD4+ regulatory T (Treg) cells. Moreover, enigmatic rare Treg subsets appeared upon inflammation, e.g. CD4+FOXP3+HLA-DR+TIGIT– and CD4+FOXP3+CD56+, expressing pro-inflammatory IFN-γ upon in vitro stimulation. Some conventional T (Tcon) cells acquired NK-like features. In CD patients’ blood, not well studied CD16+CCR6+CD127+ T cells appeared, being CD4+ or CD8+, a phenotype inducible on healthy T cells by CD blood plasma. Upon CD16-mediated antibody binding, they could attain effector function. These findings suggest an uncommon pro-inflammatory innate-like differentiation of Treg and Tcon cells with acquisition of non-specific cytotoxicity. Most likely, this is both cause and consequence of intestinal inflammation during CD.

Oxymatrine attenuates chronic allograft rejection by modulating immune responses and inhibiting fibrosis

BackgroundChronic rejection (CR) is a significant obstacle to long-term allograft survival. Oxymatrine (OMT) is a prominent bioactive compound widely utilized in traditional Chinese medicine for the management of inflammatory disorders and it has considerable potential as a therapeutic candidate for the treatment of CR. MethodsWell-established major histocompatibility complex (MHC) class II mismatched B6 mice. C-H-2bm12-to-C57BL/6 mouse transplantation was used as a CR model. Hematoxylin and eosin (H&E) staining, immunohistochemistry, and Masson's trichrome staining were used to assess pathological changes in the grafts, and the percentages of immune cells were determined by flow cytometry. The effects of OMT on the regulation of CD4+ T cell differentiation and cytokine secretion were verified in vitro. ResultsOMT effectively alleviated pathological graft damage, characterized by chronic changes in intimal lesions, vasculopathy, and fibrosis and significantly prolonged cardiac allograft survival. OMT exerted its immunomodulatory effects by inhibiting T helper 1 (Th1) and T helper 17 (Th17) cell differentiation while promoting Treg differentiation both in vivo and in vitro. Further studies revealed that OMT inhibited the phosphorylation of mammalian target of rapamycin (mTOR), which is a potential mechanism underlying its immunosuppressive effects. OMT also inhibited the activation of B cells and the production of donor-specific antibody (DSA). In addition, OMT effectively alleviated chronic changes in fibrosis in cardiac allografts, and these changes may be related to the inhibition of the transforming growth factor-β (TGF-β)-Smad 2/3 pathway. ConclusionsOMT attenuated CR by modulating the immune response and inhibiting graft fibrosis. Further in-depth investigations of OMT may provide valuable insights into the development of novel therapeutic strategies for CR inhibition.

Multistability and predominant hybrid phenotypes in a four node mutually repressive network of Th1/Th2/Th17/Treg differentiation

Elucidating the emergent dynamics of cellular differentiation networks is crucial to understanding cell-fate decisions. Toggle switch – a network of mutually repressive lineage-specific transcription factors A and B – enables two phenotypes from a common progenitor: (high A, low B) and (low A, high B). However, the dynamics of networks enabling differentiation of more than two phenotypes from a progenitor cell has not been well-studied. Here, we investigate the dynamics of a four-node network A, B, C, and D inhibiting each other, forming a toggle tetrahedron. Our simulations show that this network is multistable and predominantly allows for the co-existence of six hybrid phenotypes where two of the nodes are expressed relatively high as compared to the remaining two, for instance (high A, high B, low C, low D). Finally, we apply our results to understand naïve CD4+ T cell differentiation into Th1, Th2, Th17 and Treg subsets, suggesting Th1/Th2/Th17/Treg decision-making to be a two-step process.

Nicotinamide enhances Treg differentiation by promoting Foxp3 acetylation in immune thrombocytopenia.

Imbalanced nicotinamide adenine dinucleotide (NAD+) homeostasis has been reported in multiple autoimmune diseases and supplementation with NAD+ precursors has consistently demonstrated positive therapeutic benefits for these conditions. Immune thrombocytopenia (ITP) is an acquired autoimmune disease, in which the decreased number and impaired function of regulatory T cells (Tregs) contribute to the main pathogenesis. Here we found NAD+ level was decreased in the plasma and CD4+ T cells of ITP patients. Supplementation with NAD+ precursor nicotinamide (NAM), but not nicotinamide mononucleotide (NMN), increased Treg frequency and ameliorated thrombocytopenia in an ITP murine model. Moreover, whilst both NAM and NMN restored cytosolic NAD+ level in the CD4+ T cells from ITP patients, only NAM promoted Treg differentiation. Mechanistically, Sirtuin1 (Sirt1), a major consumer of NAD+, was highly expressed in the CD4+ T cells of ITP patients, potentially contributing to the low level of NAD+. NAM, which could act as Sirt1 inhibitor, promoted Foxp3 acetylation and stability in induced Tregs derived from naïve CD4+ T cells of ITP patients. These findings suggest that NAM holds promise as a novel therapeutic strategy for restoring immune balance in ITP.

CircDDX21 alleviates trophoblast dysfunction and Treg differentiation in recurrent spontaneous abortion via miR-520a-5p/ FOXP3/PD-L1 axis.

Recurrent spontaneous abortion (RSA) is a common complication during pregnancy, which is a burden to patients both physically and mentally. Circular RNAs (circRNAs) play important roles in RSA. However, the roles of circDDX21 in RSA development remain unknown. Decidual samples were harvested from healthy pregnant women and RSA patients. In HTR-8/SVneo and Bewo trophoblast cells, proliferation and migration were analyzed by cell counting kit-8 (CCK-8)/5-ethynyl-2'-deoxyuridine (EdU) staining and transwell/wound healing assays, respectively. CD4+ T cells from peripheral blood mononuclear cells of patients were incubated with trophoblast-conditioned medium. Regulatory T cells (Treg) proliferation was detected by carboxyfluorescein succinimidyl ester (CFSE) assay. Treg proportion, Treg/T helper 17 cells (Th17) ratio, and cytokines were measured using flow cytometry. The association among genes was validated using dual-luciferase assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP). CircDDX21 and Forkhead box P3 (FOXP3) decreased, while miR-520a-5p increased in the decidual tissues of RSA patients. CircDDX21 overexpression promoted trophoblast proliferation and migration, and facilitated CD4+ T cell differentiation into Treg. CircDDX21 targeted miR-520a-5p to elevate FOXP3. MiR-520a-5p overexpression reversed the promoted trophoblast cell function of circDDX21 overexpression in HTR-8/SVneo cells. FOXP3 overexpression reversed the repressed trophoblast cell function elicited by miR-520a-5p overexpression in HTR-8/SVneo cells. FOXP3 promoted Treg differentiation by transcriptionally upregulating programmed cell death ligand 1 (PD-L1). CircDDX21 ameliorated trophoblast dysfunction and Treg differentiation in RSA via miR-520a-5p/FOXP3/PD-L1 axis.

CCL21 Induces Plasmacytoid Dendritic Cell Migration and Activation in a Mouse Model of Glioblastoma

Dendritic cells (DCs) are professional antigen-presenting cells that are traditionally divided into two distinct subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). pDCs are known for their ability to secrete large amounts of cytokine type I interferons (IFN- α). In our previous work, we have demonstrated that pDC infiltration promotes glioblastoma (GBM) tumor immunosuppression through decreased IFN-α secretion via TLR-9 signaling and increased suppressive function of regulatory T cells (Tregs) via increased IL-10 secretion, resulting in poor overall outcomes in mouse models of GBM. Further dissecting the overall mechanism of pDC-mediated GBM immunosuppression, in this study, we identified CCL21 as highly upregulated by multiple GBM cell lines, which recruit pDCs to tumor sites via CCL21-CCR7 signaling. Furthermore, pDCs are activated by CCL21 in the GBM microenvironment through intracellular signaling of β-arrestin and CIITA. Finally, we found that CCL21-treated pDCs directly suppress CD8+ T cell proliferation without affecting regulatory T cells (Tregs) differentiation, which is considered the canonical pathway of immunotolerant regulation. Taken together, our results show that pDCs play a multifaced role in GBM immunosuppression, and CCL21 could be a novel therapeutic target in GBM to overcome pDC-mediated immunosuppression.

Interleukin-2 improves insulin sensitivity through hypothalamic sympathetic activation in obese mice

BackgroundIL-2 regulates T cell differentiation: low-dose IL-2 induces immunoregulatory Treg differentiation, while high-dose IL-2 acts as a potent activator of cytotoxic T cells and NK cells. Therefore, high-dose IL-2 has been studied for use in cancer immunotherapy. We aimed to utilize low-dose IL-2 to treat inflammatory diseases such as obesity and insulin resistance, which involve low-grade chronic inflammation.Main bodySystemic administration of low-dose IL-2 increased Treg cells and decreased inflammation in gonadal white adipose tissue (gWAT), leading to improved insulin sensitivity in high-fat diet-fed obese mice. Additionally, central administration of IL-2 significantly enhanced insulin sensitivity through the activation of the sympathetic nervous system. The sympathetic signaling induced by central IL-2 administration not only decreased interferon γ (IFNγ) + Th1 cells and the expression of pro-inflammatory cytokines, including Il-1β, Il-6, and Il-8, but also increased CD4 + CD25 + FoxP3 + Treg cells and Tgfβ expression in the gWAT of obese mice. These phenomena were accompanied by hypothalamic microgliosis and activation of pro-opiomelanocortin neurons. Furthermore, sympathetic denervation in gWAT reversed the enhanced insulin sensitivity and immune cell polarization induced by central IL-2 administration.ConclusionOverall, we demonstrated that IL-2 improves insulin sensitivity through two mechanisms: direct action on CD4 + T cells and via the neuro-immune axis triggered by hypothalamic microgliosis.

Macrophage Notch1 signaling modulates regulatory T cells via the TGFB axis in early MASLD

Background & aimsHepatic immune imbalance is critical in driving metabolic dysfunction-associated steatotic liver disease (MASLD) progression. The role of hepatic regulatory T cells (Tregs) in MASLD initiation and the mechanisms responsible for their change are not completely understood. MethodsA mouse model subjected to a short-term high-fat diet (HFD) to mimic early steatosis, along with liver biopsy samples from patients with simple steatosis and macrophage-specific Notch1 knockout mice (Notch1M-KO)， was used to investigate the role of Tregs in early MASLD and the effect of hepatic macrophage Notch1 signaling on Treg frequency. Using Exos-miRNA-sequencing, we analyzed the miRNA correlated with Treg differentiation. ResultsHere we showed that a decrease in Tregs contributed to HFD-induced hepatic steatosis and insulin resistance (n=5/group/time point, p<0.001). Remarkably, the frequency of Tregs exhibited a negative correlation with Notch1 activation in hepatic macrophages during hepatic steatosis (n=38/group, r=-0.735, p<0.001). Furthermore, we found that Notch1 deficiency attenuated hepatic lipid deposition and reversed Treg levels (n=5 per group, p<0.01, p<0.05). Moreover, Treg depletion in Notch1M-KO mice greatly diminished the ameliorative effect of macrophagic Notch1 deletion on hepatic steatosis. Mechanistically, macrophage Notch1 activation increased its exosomal miR-142a-3p level (1-2-fold), impairing Treg differentiation by targeting TGFBR1 on T cells. Consistently, HFD-fed Notch1M-KO mice exhibited reduced miR-142a-3p levels, elevated TGFBR1 expression on T cells, and increased Tregs frequency in the liver. ConclusionsThese findings highlight the critical role of hepatic Tregs in the early stage of MASLD and add a novel, non-negligible pathway for macrophage involvement in hepatic steatosis. We identify a previously unrecognized molecular mechanism of macrophage Notch1/exosomal miR-142a-3p/TGFBR1 pathway in regulating Treg differentiation, providing the rationale for refined therapeutic strategies for MASLD. Impact and implicationsThe immune mechanisms driving MASLD progression, particularly in the early stages, are not fully understood, which limits the development of effective interventions. This study elucidated a novel mechanism by which hepatic macrophage Notch1 signaling modulated Tregs through the exosomal miR-142a-3p/TGFBR1 axis, contributing to the progression of MASLD. These findings provide a rationale for a potential immunological approach to treat MASLD in the future.

Bone marrow mesenchymal stem cells-derived exosomal miR-381 alleviates lung ischemia-reperfusion injury by activating Treg differentiation through inhibiting YTHDF1 expression

AimOur study aimed to investigate whether BMSCs-derived exosomal miR-381 promotes Treg cell differentiation in lung ischemia-reperfusion injury (LIRI), and the underlying mechanism. MethodsThe in vitro and in vivo models of LIRI were established by hypoxia/reoxygenation (H/R) treatment and lung ischemia/reperfusion (I/R) surgery, respectively. BMSCs-derived exosomes were isolated and identified by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Cell viability, proliferation, and apoptosis were assessed by CCK-8, EdU, and flow cytometry assay, respectively. IL-18 secretion level in lung microvascular endothelial cells (LMECs) and lung tissue homogenate was examined by ELISA. Treg cell differentiation was determined using flow cytometry. The relationships between miR-381, YTHDF1, and IL-18 were investigated using dual-luciferase reporter gene, RIP, and/or RNA pull-down assays. MeRIP assay was employed to determine m6A modification of IL-18 mRNA in LMECs. The ubiquitination level of Foxp3 protein in CD4+ T cells was analyzed by Co-IP assay. ResultsBMSCs-derived exosomes reduced LMECs injury and increased Treg cell differentiation in LIRI, whereas miR-381 inhibition in BMSCs weakened these impacts. Mechanistically, miR-381 inhibited IL-18 translation in LMECs by inhibiting YTHDF1 expression via binding to its 3’-UTR. As expected, YTHDF1 overexpression in LMECs abolished the effects of miR-381-overexpressed exosomes on LMECs injury and Treg cell differentiation. Moreover, LMECs-secreted IL-18 inhibited Treg cell differentiation by promoting the ubiquitination degradation of Foxp3 protein. ConclusionBMSCs-derived exosomal miR-381 suppressed IL-18 translation in LMECs through binding to YTHDF1 3’-UTR, thus suppressing the ubiquitination degradation of Foxp3 in CD4+ T cells, which promoted Treg cell differentiation and mitigated LIRI development.