Jian Gu

Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions

Significance Natural regulatory T cells (nTregs) play important roles in preventing autoimmune diseases, but they may be unstable in the presence of inflammation. Here we report that all-trans RA (atRA) but not rapamycin prevents human nTregs from converting to Th1/Th17 cells and sustains their suppressive function in inflammatory environments. Adoptive transfer of nTregs pretreated with atRA enhances their suppressive effects on xenograft-vs.-host diseases. Moreover, we show that atRA suppresses IL-1 receptor upregulation, accelerates IL-6 receptor downregulation, and affects the epigenetic modifications in Foxp3 locus in nTregs following inflammatory stimulation. We suggest that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated diseases. Recent studies have demonstrated that thymus-derived naturally occurring CD4+Foxp3+ regulatory T cells (Tregs) in human and mouse may be unstable and dysfunctional in the presence of proinflammatory cytokines. All-trans RA (atRA), the active derivative of vitamin A, has been shown to regulate Treg and T effector cell differentiation. We hypothesize atRA stabilizes human natural Tregs (nTregs) under inflammatory conditions. atRA prevents human nTregs from converting to Th1 and/or Th17 cells and sustains their Foxp3 expression and suppressive function in vitro or in vivo following encounters with IL-1 and IL-6. Interestingly, adoptive transfer of human nTregs pretreated with atRA significantly enhanced their suppressive effects on xenograft-vs.-host diseases (xGVHDs), and atRA- but not rapamycin-pretreated nTregs sustained the functional activity against xGVHD after stimulation with IL-1/IL-6. atRA suppresses IL-1 receptor (IL-1R) up-regulation, accelerates IL-6R down-regulation, and diminishes their signaling events as well as prevents the up-regulation of STIP1 homology and U-Box containing protein 1 on Foxp3+ cells following IL-1/IL-6 stimulation. atRA also increases histone acetylation on Foxp3 gene promoter and CpG demethylation in the region of Foxp3 locus (i.e., Treg-specific demethylated region). These results strongly implicate that nTregs primed with atRA may represent a novel treatment strategy to control established chronic immune-mediated autoimmune and inflammatory diseases.

Differential role of all-trans retinoic acid in promoting the development of CD4 and CD8 regulatory T cells

It is known that ATRA promotes the development of TGF‐β‐induced CD4+Foxp3+ iTregs, which play a vital role in the prevention of autoimmune diseases; however, the role of ATRA in facilitating the differentiation and function of CD8+Foxp3+ iTregs remains elusive. Using a head‐to‐head comparison, we found that ATRA promoted expression of Foxp3 and development of CD4+ iTregs, but it did not promote Foxp3 expression on CD8+ cells. Using a standard in vitro assay, we demonstrated that CD8+ iTregs induced by TGF‐β and ATRA were not superior to CD8+ iTregs induced by TGF‐β alone. In cGVHD, in a typical lupus syndrome model where DBA2 spleen cells were transferred to DBA2xC57BL/6 F1 mice, we observed that both CD8+ iTregs induced by TGF‐β and ATRA and those induced by TGF‐β alone had similar therapeutic effects. ATRA did not boost but, conversely, impaired the differentiation and function of human CD8+ iTregs. CD8+ cells expressed the ATRA receptor RAR and responded to ATRA, similar to CD4+ cells. We have identified the differential role of ATRA in promoting Foxp3+ Tregs in CD4+ and CD8+ cell populations. These results will help to determine a protocol for developing different Treg cell populations and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

Human CD39hi regulatory T cells present stronger stability and function under inflammatory conditions

Autoimmune diseases are characterized by an imbalance between regulatory T cells and effector T-cell subsets, such as Th1 and Th17 cells. Studies have confirmed that natural CD4+Foxp3+ Tregs were unstable and dysfunctional in the presence of pro-inflammatory cytokines. In the current study, human CD39hi Tregs and CD39low Tregs were sorted from Tregs in vitro after 7 days of expansion. The functions of both Treg subsets were investigated under inflammatory conditions in vitro and in vivo. In the presence of IL-1β and IL-6, cultured CD4+CD39hi Tregs maintained stable forkhead box protein 3 expression, whereas CD4+CD39low Tregs lost Foxp3 expression and trans-differentiated into Th1 or Th17 cells. Decreased IL-1βR and IL-6R expression on the CD39hi Tregs was the primary mechanism responsible for Treg stability. In addition, reduced activation of downstream molecules, such as STAT1 and STAT3, through the modulation of CpG demethylation played an important role. Finally, human CD4+CD39hi Tregs but not CD4+CD39low Tregs protected against xenograft versus host disease in model mice. These results strongly implied the physiological importance of CD39 expression and suggested that manipulation of CD39hi Tregs might represent a novel strategy for the treatment of autoimmune diseases.

LncRNA HULC affects the differentiation of Treg in HBV-related liver cirrhosis.

BACKGROUND AND AIMS Recently, a couple of the long noncoding RNAs (lncRNAs) have been proved to participate in hepatocellular carcinoma development and progression. However, their associations with liver cirrhosis have not been reported. In this study, we aimed to identify the affection of HULC on regulatory T cells (Tregs) differentiation in HBV-related liver cirrhosis. METHODS Seven lncRNAs were chosen as candidate lncRNAs based on the association with liver disease. The candidate lncRNAs were validated by RT-qPCR. Additional flow cytometry of Tregs was performed in 34 HBV-related liver cirrhosis patients and 34 healthy volunteers. To investigate the function of HULC, HULC expression was modified by gene overexpression via lentivirus vector. RIP assay was performed further to validate the association between HULC and p18. RESULTS Circulation Tregs and HULC were significantly up-regulated in plasma samples of HBV-related cirrhosis patients. In addition, overexpression of HULC by lentivirus vector elevated Treg frequency in vitro. Furthermore, RIP assay showed that HULC down-regulated the level of p18 directly. CONCLUSIONS We confirmed the effects of HULC on Tregs differentiation in HBV-related liver cirrhosis. In addition, it was proved that HULC regulates the function of Tregs through down-regulated the level of p18 directly.

miR-146b antagomir-treated human Tregs acquire increased GVHD inhibitory potency.

CD4(+)CD25(+)FoxP3(+) thymic-derived regulatory T cells (tTregs) are indispensable for maintaining immune system equilibrium. Adoptive transfer of tTregs is an effective means of suppressing graft-versus-host disease (GVHD) in murine models and in early human clinical trials. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an ubiquitin-conjugating enzyme that mediates nuclear factor κB (NF-κB) activation, plays an essential role in modulating regulatory T cell survival and function. MicroRNAs (miRNAs) are noncoding RNAs, which mediate RNA silencing and posttranscriptional gene repression. By performing comprehensive TaqMan Low Density Array miRNA assays, we identified 10 miRNAs differentially regulated in human tTreg compared with control T cells. One candidate, miR-146b, is preferentially and highly expressed in human naive tTregs compared with naive CD4 T cells. miRNA prediction software revealed that TRAF6 was the one of the top 10 scored mRNAs involved tTreg function with the highest probability as a potential miR-146b target. Antagomir-mediated knockdown of miRNA-146b, but not another miRNA-146 family member (miRNA-146a), enhanced TRAF6 expression. TRAF6, in turn, increases NF-κB activation, which is essential for tTreg function as well as Foxp3 protein and antiapoptotic gene expression, and downregulates proapoptotic gene expression. miR-146b knockdown increased the nuclear localization and expression of genes regulated by NF-κB, which was associated with enhanced tTreg survival, proliferation, and suppressive function measured in vitro and in vivo. TRAF6 inhibition had the opposite effects. We conclude that an miR-146b-TRAF6-NF-κB-FoxP3 signaling pathway restrains regulatory T cell survival, proliferation, and suppressor function. In vitro exposure of human tTregs to miR-146b antagomirs can be exploited to improve the clinical efficacy of human adoptive tTreg transfer in a GVHD setting.

Human Gingiva-Derived Mesenchymal Stem Cells Inhibit Xeno-Graft-versus-Host Disease via CD39–CD73–Adenosine and IDO Signals

Mesenchymal stem cells have the capacity to maintain immune homeostasis and prevent autoimmunity. We recently reported that human-derived gingival mesenchymal stem cells (GMSCs) have strong capacity to suppress immune responses and T cell-mediated collagen-induced arthritis in animals. However, it is unclear whether these cells can suppress human T cell-mediated diseases. Here, we used a xenogenic GVHD model in the NOD/SCID mouse, which is a useful preclinical construct for evaluating the therapeutic and translational potential of this approach for applications in human disease. We found that GMSCs potently suppressed the proliferation of PBMC and T cells in vitro. Co-transfer of GMSC with human PBMC significantly suppressed human cell engraftment and markedly prolonged the mouse survival. Moreover, we demonstrated that GMSCs inhibited human PBMC-initiated xenogenic responses via CD39/CD73/adenosine and IDO signals. These findings suggest the potential for GMSCs to suppress human immune responses in immune system-mediated diseases, offering a potential clinical option to be used for modulating GVHD and autoimmune diseases.

Rapamycin Regulates iTreg Function through CD39 and Runx1 Pathways

It has been shown that rapamycin is able to significantly increase the expression of FoxP3 and suppress activity in induced Treg (iTreg) cells in vivo and in vitro. CD39 is a newly determined Treg marker that relates to cell suppression. Runx1, a regulator of FoxP3, controls the expression of adenosine deaminase (ADA) gene, which is found recently in the downstream of CD39 pathway in trophoblast cells. Whether rapamycin would influence CD39 pathway and regulate the expression of Runx1 remains to be determined. The addition of rapamycin to human CD4+ naïve cells in the presence of IL-2, TGF-β promotes the expression of FoxP3. In this paper, we found that CD39 positively correlated with the FoxP3 expression in iTreg cells. Rapamycin induced iTreg cells showed a stronger CD39/Runx1 expression with the enhanced suppressive function. These data suggested that CD39 expression was involved in iTreg generation and the enhanced suppressive ability of rapamycin induced Treg was partly due to Runx1 pathway. We conclude that rapamycin favors CD39/Runx1 expression in human iTreg and provides a novel insight into the mechanisms of iTreg generation enhanced by rapamycin.

Anti-IL-17 Antibody Improves Hepatic Steatosis by Suppressing Interleukin-17-Related Fatty Acid Synthesis and Metabolism

To investigate the relationship between interleukin-17 and proteins involved in fatty acid metabolism with respect to alcoholic liver disease, male ICR mice were randomized into five groups: control, alcoholic liver disease (ALD) at 4 weeks, 8 weeks, and 12 weeks, and anti-IL-17 antibody treated ALD. A proteomic approach was adopted to investigate changes in liver proteins between control and ALD groups. The proteomic analysis was performed by two-dimensional difference gel electrophoresis. Spots of interest were subsequently subjected to nanospray ionization tandem mass spectrometry (MS/MS) for protein identification. Additionally, expression levels of selected proteins were confirmed by western blot. Transcriptional levels of some selected proteins were determined by RT-PCR. Expression levels of 95 protein spots changed significantly (ratio >1.5, P < 0.05) during the development of ALD. Sterol regulatory element-binding protein-lc (SREBP-1c), carbohydrate response element binding protein (ChREBP), enoyl-coenzyme A hydratase (ECHS1), and peroxisome proliferator-activated receptor alpha (PPAR-α) were identified by MS/MS among the proteins shown to vary the most; increased IL-17 elevated the transcription of SREBP-1c and ChREBP but suppressed ECHS1 and PPAR-α. The interleukin-17 signaling pathway is involved in ALD development; anti-IL-17 antibody improved hepatic steatosis by suppressing interleukin-17-related fatty acid metabolism.

iTreg induced from CD39+ naive T cells demonstrate enhanced proliferate and suppressive ability

CD4(+)CD25(+)FoxP3(+) regulatory T (Treg) cells which consist of naturally occurring Treg (nTreg) and induced Treg (iTreg) cells are associated with the maintenance of immune homeostasis. Previous studies were focused on their potential to ameliorate graft-versus-host disease (GVHD) in human and mice. CD39 is a surface marker both expressed on CD4(+)CD25(-) T cells and Treg cells. CD39(+) Treg cells demonstrate stronger suppressive ability compared to conventional Treg cells. However, whether the potential of CD39(+) naïve T cells induced Treg cells is different from conventional naïve T cells induced Treg cells in vivo and vitro remains to be inconclusive. Here we demonstrate that CD39(+) iTreg cells show enhanced proliferation and suppressive ability as well as lower inflammatory cytokines compared to CD39(-) iTreg cells. To conclude, our findings demonstrate that CD39(+) iTreg cells acquire high suppressive capacity in vitro and vivo, and this may provide a new insight into Treg cell therapy in GVHD clinical trials.

miR-142-3p regulates autophagy by targeting ATG16L1 in thymic-derived regulatory T cell (tTreg)

Thymic-derived regulatory T cell (tTreg) clinical trials show therapeutic promise in the prevention of acute graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation patients. However, strategies are needed to improve tTreg proliferative ability and survival as a means to improve tTreg therapy and reduce the requirement for producing large numbers of Treg cells for adoptive tTreg transfer. Autophagy is a self-degradative process for cytosolic components, which is involved in cells death, differentiation, lymphocyte homeostasis, and tTreg function. Studies have shown that mice with tTreg cells that have a disrupted autophagy process have defective tTreg cell generation and function, resulting in autoimmune disease and failed GVHD prevention by adoptively transferred tTreg cells. We found the attenuated autophagy status during ex vivo expansion, which leads us to determine whether tTreg cell survival could be augmented by miR-142-3p, the miRNA which is highly expressed in tTreg cells and potentially targets autophagy-related protein (ATG)-1, ATG16L1. We demonstrate that miR-142-3p downregulates ATG16L1 mRNA and production of ATG16L1, that has been linked to autoimmune diseases. Conversely, miR-142-3p knock-down improved tTreg cell expansion, survival and function in vitro and vivo. In aggregate, these studies provide a new approach that uses miR-142-3p knockdown to increase tTreg cell efficacy by increasing ATG16L1 mRNA and protein and the autophagy process.