Murine Th2 Cells Research Articles

Transcriptional signature of human pro-inflammatory TH17 cells identifies reduced IL10 gene expression in multiple sclerosis

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ−IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases.

Lumican Inhibits the pathogenicity of murine encephalitic TH17 cells

Abstract TH17 cells play an essential role in the development of both human multiple sclerosis and animal experimental autoimmune encephalomyelitis (EAE). Nevertheless, it is not well understood how the pathogenicity of TH17 cells is controlled in the autoimmune neuro-inflammation. In vitro, we found Lumican (Lum), an extracellular matrix (ECM) protein, is selectively expressed by TH17 cells among tested murine TH subsets. Lum-deficiency leads to earlier onset and enhanced severity of experimental autoimmune encephalomyelitis. This enhanced disease in Lum-deficient mice is associated with increased production of IL-17 and IL-21 and decreased TH17 cell apoptosis. Dysregulation in cytokine production appears to be specific to TH17 cells as TH1 and TH2 cell polarization and/or cytokine production were unaltered. Furthermore, adoptive transfer of MOG-specific TH17 cells derived from Lum-deficient mice led to earlier onset and increased severity of disease compared to controls highlighting a TH17 cell-intrinsic effect of Lum. Taken together, our results suggest that Lum negatively regulates encephalitic TH17 cells, implicating a potential therapeutic pathway in TH17 cell-mediated autoimmune and inflammatory diseases.

Murine Th17 cells utilize IL-2 receptor gamma chain cytokines but are resistant to cytokine withdrawal-induced apoptosis.

Adoptive cellular therapy (ACT) with the Th17 subset of CD4+ T cells can cure established melanoma in preclinical models and holds promise for treating human cancer. However, little is known about the growth factors necessary for optimal engraftment and anti-tumor activity of Th17 cells. Due to the central role of IL-2 receptor gamma chain (IL2Rγ-chain) cytokines (IL-2, IL-7, and IL-15) in the activity and persistence of many T cell subsets after adoptive transfer, we hypothesized that these cytokines are important for Th17 cells. We found that Th17 cells proliferated in response to IL-2, IL-7, and IL-15 in vitro. However, in contrast to many other T cell subsets, including conventionally activated CD8+ T cells, we found that Th17 cells were resistant to apoptosis in the absence of IL2Rγ-chain cytokines. To determine whether Th17 cells utilize IL2Rγ-chain cytokines in vivo, we tracked Th17 cell engraftment after adoptive transfer with or without cytokine depletion. Depletion of IL-7 and/or IL-2 decreased initial engraftment, while depletion of IL-15 did not. Supplementation of IL-2 increased initial Th17 engraftment. To assess the clinical relevance of these findings, we treated melanoma-bearing mice with Th17 cell adoptive transfer and concurrent cytokine depletion or supplementation. We found that simultaneous depletion of IL-2 and IL-7 decreased therapeutic efficacy, depletion of IL-15 had no effect, and IL-2 supplementation increased therapeutic efficacy. Our results show that Th17 cells are responsive to IL2Rγ-chain cytokines, and provide insight into the application of these cytokines for Th17-based therapeutic strategies.

Lumican negatively controls the pathogenicity of murine encephalitic TH17 cells.

TH17 cells play an essential role in the development of both human multiple sclerosis and animal experimental autoimmune encephalomyelitis (EAE). Nevertheless, it is not well understood how the pathogenicity of TH17 cells is controlled in the autoimmune neuroinflammation. In vitro, we found Lumican (Lum), an extracellular matrix (ECM) protein, is selectively expressed by TH17 cells among tested murine TH subsets. Lum deficiency leads to earlier onset and enhanced severity of EAE. This enhanced disease in Lum-deficient mice is associated with increased production of IL-17 and IL-21 and decreased TH17 cell apoptosis. Dysregulation in cytokine production appears to be specific to TH17 cells as TH1 and TH2 cell polarization and/or cytokine production were unaltered. Furthermore, adoptive transfer of myelin oligodendrocyte glycoprotein specific TH17 cells derived from Lum-deficient mice led to earlier onset and increased severity of disease compared to controls highlighting a TH17-cell-intrinsic effect of Lum. Taken together, our results suggest that Lum negatively regulates encephalitic TH17 cells, implicating a potential therapeutic pathway in TH17 cell mediated autoimmune and inflammatory diseases.

Tiam1/Rac1 complex controls Il17a transcription and autoimmunity.

RORγt is a master transcription factor of Th17 cells and considered as a promising drug target for the treatment of autoimmune diseases. Here, we show the guanine nucleotide exchange factor, Tiam1, and its cognate Rho-family G protein, Rac1, regulate interleukin (IL)17A transcription and autoimmunity. Whereas Tiam1 genetic deficiency weakens IL-17A expression partially and inhibits the development of experimental autoimmune encephalomyelitis (EAE), deletion of Rac1 in T cells exhibits more robust effects on Th17 cells and EAE. We demonstrate Tiam1 and Rac1 form a complex with RORγt in the nuclear compartment of Th17 cells, and together bind and activate the Il17 promoter. The clinical relevance of these findings is emphasized by pharmacological targeting of Rac1 that suppresses both murine and human Th17 cells as well as EAE. Thus, our findings highlight a regulatory pathway of Tiam1/Rac1 in Th17 cells and suggest that it may be a therapeutic target in multiple sclerosis.

ERβ-Dependent Direct Suppression of Human and Murine Th17 Cells and Treatment of Established Central Nervous System Autoimmunity by a Neurosteroid.

Multiple sclerosis (MS), an autoimmune disease of the CNS, is mediated by autoreactive Th cells. A previous study showed that the neurosteroid dehydroepiandrosterone (DHEA), when administered preclinically, could suppress progression of relapsing-remitting experimental autoimmune encephalomyelitis (EAE). However, the effects of DHEA on human or murine pathogenic immune cells, such as Th17, were unknown. In addition, effects of this neurosteroid on symptomatic disease, as well as the receptors involved, had not been investigated. In this study, we show that DHEA suppressed peripheral responses from patients with MS and reversed established paralysis and CNS inflammation in four different EAE models, including the 2D2 TCR-transgenic mouse model. DHEA directly inhibited human and murine Th17 cells, inducing IL-10-producing regulatory T cells. Administration of DHEA in symptomatic mice induced regulatory CD4(+) T cells that were suppressive in an IL-10-dependent manner. Expression of the estrogen receptor β by CD4(+) T cells was necessary for DHEA-mediated EAE amelioration, as well as for direct downregulation of Th17 responses. TGF-β1 as well as aryl hydrocarbon receptor activation was necessary for the expansion of IL-10-producing T cells by DHEA. Thus, our studies demonstrate that compounds that inhibit pathogenic Th17 responses and expand functional regulatory cells could serve as therapeutic agents for autoimmune diseases, such as MS.

Inherent low Erk and p38 activity reduce Fas Ligand expression and degranulation in T helper 17 cells leading to activation induced cell death resistance.

Activation Induced Cell Death of T helper cells is central to maintaining immune homeostasis and a perturbation often manifests in aberrant T helper cells that is associated with immunopathologies. Significant presence of T cells positive for IL-17A (Th17) and dual positive for IFN-γ/IL-17A (Th1/Th17) in both effector (CD45RA+RO+) and memory (CD45RA−RO+) compartments with differential FasL protein in RA peripheral blood suggested their differential TCR AICD sensitivity. Lowered active caspase-3 in Th17 and Th1/Th17 over Th1 cells confirmed their capability to resist AICD and pointed to early upstream events. Differential MAPK activities, FasL protein and downstream caspase-3 activities in murine Th1 and Th17 cells established distinct TCR mediated signaling pathways and suggested low Erk and p38 activity as pivotal for AICD sensitivity. We extrapolated our mouse and human data and report that Fas-FasL is the preferred death pathway for both Th1 and Th17 and that inherently low Erk2 activity protected Th17 cells from TCR AICD. The presence of significantly higher numbers of aberrant T helper cells in RA also suggest an inflammatory cytokine milieu and AICD insensitive T cell link to sustained inflammation. Re sensitization to apoptosis by targeting MAPK activity especially Erk2 in RA might be of therapeutic value.

Abstract 565: RORγ agonists regulate immune checkpoint receptors to enhance anti-tumor immunity

Abstract As the master transcription factor for Type 17 T cells, RORγt activates a program of gene expression associated with enhancing effector function and overcoming immune suppression. Lycera is developing synthetic, small molecule RORγ agonists for immunotherapy of cancer. We have previously reported that RORγγ agonists increase type 17 cytokine and chemokine production, enhance cell survival and have single agent anti-tumor activity in syngeneic tumor models. Interestingly, transcriptional profiling and cytometry studies revealed that treatment of murine and human Th17 or Tc17 cells with RORγ agonists increases the expression of costimulatory receptors such as CD137 and decreased expression of coinhibitory receptors like PD-1. Given the importance of PD-1 in anti-tumor immunity, we further characterized the effects of RORγ agonist on this pathway. In vitro treatment with RORγ agonists significantly decreases mean fluorescent intensity and percent PD-1+ cells after resting and repetitive restimulation of Type 17 T cells. RORγ agonists do not modulate PD-1 expression in RORγ-/- T cells. In co-cultures of wild type and RORγ-/- T cells in the presence of the agonist, RORγ-/- T cells have reduced PD-1 expression suggesting that RORγ agonists induce a transmissible effect on RORγ-/- T cells. However, ChIP-seq data indicates that RORγt does not directly bind to the promoter or enhancer element of PD-1. Transcriptional and epigenetic profiling experiments have identified several pathways modulated by RORγ agonists that may regulate PD-1 expression. Reduced PD-1 expression following RORγ agonist treatment has a functional impact as treated cells also resist PD-L1-mediated inhibition of cytokine production and proliferation. Importantly, the decreased PD-1 expression observed after in vitro treatment with RORγ agonists is maintained following adoptive transfer of tumor specific T cells. These cells are highly effective at controlling tumor growth without further agonist treatment in vivo, suggesting that in vitro RORγ agonist treatment results in durable epigenetic changes. In summary, RORγ agonists have been shown to decrease checkpoint receptor expression while enhancing cytokine production and promoting long term survival and self-renewal of T cells. These results provide rationales for combining an RORγ agonist with checkpoint inhibitor such as anti-CTLA4 or anti-PD-1. By integrating effects on different effector pathways, RORγ agonists represent a promising immunotherapy approach for the treatment of cancer. Citation Format: Xiao Hu, Xikui Liu, Jacques Moisan, Chrystal Paulos, Yahong Wang, Chauncey Spooner, Charles Lesch, Rodney Morgan, David Mertz, Dick Bousley, Clarke Taylor, Chad Van Huis, Don Skalitzky, Thomas Aicher, Peter Toogood, Laura Carter. RORγ agonists regulate immune checkpoint receptors to enhance anti-tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 565.

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis.

The small adaptor protein growth factor receptor-bound protein 2 (Grb2) modulates and integrates signals from receptors on cellular surfaces in inner signaling pathways. In murine T cells, Grb2 is crucial for amplification of TCR signaling. T cell-specific Grb2(fl/fl) Lckcre(tg) Grb2-deficient mice show reduced T cell numbers due to impaired negative and positive selection. In this study, we found that T cell numbers in Grb2(fl/fl) CD4cre(tg) mice were normal in the thymus and were only slightly affected in the periphery. Ex vivo analysis of CD4(+) Th cell populations revealed an increased amount of Th1 cells within the CD4(+) population of Grb2(fl/fl) CD4cre(tg) mice. Additionally, Grb2-deficient T cells showed a greater potential to differentiate into Th17 cells in vitro. To test whether these changes in Th cell differentiation potential rendered Grb2(fl/fl) CD4cre(tg) mice more prone to inflammatory diseases, we used the murine Th1 cell- and Th17 cell-driven model of experimental autoimmune encephalomyelitis (EAE). In contrast to our expectations, Grb2(fl/fl) CD4cre(tg) mice developed a milder form of EAE. The impaired EAE disease can be explained by the reduced proliferation rate of Grb2-deficient CD4(+) T cells upon stimulation with IL-2 or upon activation by allogeneic dendritic cells, because the activation of T cells by dendritic cells and the subsequent T cell proliferation are known to be crucial factors for the induction of EAE. In summary, Grb2-deficient T cells show defects in T cell development, increased Th1 and Th17 cell differentiation capacities, and impaired proliferation after activation by dendritic cells, which likely reduce the clinical symptoms of EAE.

Regulatory T-cell–intrinsic amphiregulin is dispensable for suppressive function

Regulatory T-cell–intrinsic amphiregulin is dispensable for suppressive function

BCL-2 protects human and mouse Th17 cells from glucocorticoid-induced apoptosis

Glucocorticoid resistance has been associated with Th17-driven inflammation, the mechanisms of which are not clear. We determined whether human and mouse Th17 cells are resistant to glucocorticoid-induced apoptosis. Freshly isolated human blood Th17 cells and in vitro differentiated Th17 cells from IL-17F red fluorescent protein reporter mice were treated with dexamethasone, a potent glucocorticoid. Apoptosis was measured using annexin V and DAPI staining. Screening of apoptosis genes was performed using the apoptosis PCR array. Levels of molecules involved in apoptosis were measured using quantitative RT-PCR, flow cytometry, and Western blotting. Knockdown of BCL-2 in murine Th17 cells was performed via retroviral transduction. Cytokines were measured using ELISA. A murine Th17-driven severe asthma model was examined for Th17 glucocorticoid sensitivity in vivo. Human and mouse Th17 cells and mouse Th2 cells were resistant to glucocorticoid-induced apoptosis. Th17 cells had glucocorticoid receptors levels comparable to those in other T effectors cells. Th17 cells had high levels of BCL-2, knockdown of which sensitized Th17 cells to dexamethasone-induced apoptosis. Production of IL-22, but not IL-17A and IL-17F, was suppressed by glucocorticoids. STAT3 phosphorylation in Th17 cells was insensitive to glucocorticoid inhibition. Lung Th17 cells in the murine severe asthma model were enhanced, rather than suppressed, by glucocorticoids. Th17 cells are resistant to glucocorticoid-induced apoptosis and cytokine suppression, at least in part due to high levels of BCL-2. These findings support a role of Th17 cells in glucocorticoid-resistant inflammatory conditions such as certain endotypes of asthma.

B7-H1 Selectively Controls TH17 Differentiation and Central Nervous System Autoimmunity via a Novel Non-PD-1-Mediated Pathway.

It is currently acknowledged that TH17 cells are critically involved in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). In this article, we demonstrate that signals delivered by the coinhibitory molecule B7-homologue 1 (B7-H1) via a B7-homologue 1 mouse-IgG2aFc (B7-H1-Ig) fusion protein nearly abolish TH17, but not TH1 and TH2, differentiation via direct interaction with the T cell. These effects were equally pronounced in the absence of programmed death-1 or B7.1 and B7.2 on the T cell side, thus providing clear evidence that B7-H1 modulates T cell differentiation via a novel receptor. Mechanistically, B7-H1 interfered with early TCR-mediated signaling and cytokine-mediated induction of the TH17-determining transcription factors retinoic acid-related orphan receptor γ t and IFN regulator factor-4 in a programmed death-1 and B7-independent fashion. In an animal model of MS, active myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, B7-H1-Ig exhibited a significant and long-lasting effect on disease severity upon administration during the first 5 d of the priming phase, which was accompanied by reduced TH17 responses in the periphery and within the CNS. Importantly, B7-H1-Ig was even capable of interfering with T cell encephalitogenicity when interaction with the T cells occurred after priming using an adoptive transfer experimental autoimmune encephalomyelitis model. In line with this, both naive human CD4(+) T cells and differentiated TH17 effector cells from MS patients were highly sensitive toward B7-H1-Ig-mediated TH17 suppression. Together, we propose the existence of a novel B7-H1-mediated immune-regulatory pathway in T cells, which selectively limits murine and human TH17 cell responses and might be therapeutically exploited to control TH17-mediated autoimmunity.

Th Cell Diversity in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis.

Multiple sclerosis (MS) is believed to be initiated by myelin-reactive CD4(+) Th cells. IL-12-polarized Th1 cells, IL-23-polarized Th17 cells, and Th17 cells that acquire Th1 characteristics were each implicated in autoimmune pathogenesis. It is debated whether Th cells that can drive the development of demyelinating lesions are phenotypically diverse or arise from a single lineage. In the current study, we assessed the requirement of IL-12 or IL-23 stimulation, as well as Th plasticity, for the differentiation of T cells capable of inducing CNS axon damage. We found that stable murine Th1 and Th17 cells independently transfer experimental autoimmune encephalomyelitis (widely used as an animal model of MS) in the absence of IL-23 and IL-12, respectively. Plastic Th17 cells are particularly potent mediators of demyelination and axonopathy. In parallel studies, we identified MS patients who consistently mount either IFN-γ- or IL-17-skewed responses to myelin basic protein over the course of a year. Brain magnetic resonance imaging revealed that patients with mixed IFN-γ and IL-17 responses have relatively high T1 lesion burden, a measure of permanent axon damage. Our data challenge the dogma that IL-23 and Th17 plasticity are universally required for the development of experimental autoimmune encephalomyelitis. This study definitively demonstrates that autoimmune demyelinating disease can be driven by distinct Th-polarizing factors and effector subsets, underscoring the importance of a customized approach to the pharmaceutical management of MS.

Dual Inhibition of Interleukin-23 and Interleukin-17 Offers Superior Efficacy in Mouse Models of Autoimmunity.

Therapies targeting either interleukin (IL)-23 or IL-17 have shown promise in treating T helper 17 (Th17)-driven autoimmune diseases. Although IL-23 is a critical driver of IL-17, recognition of nonredundant and independent functions of IL-23 and IL-17 has prompted the notion that dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating autoimmune diseases relative to targeting either cytokine alone. To test this hypothesis, we generated selective inhibitors of IL-23 and IL-17 and tested the effect of either treatment alone compared with their combination in vitro and in vivo. In vitro, using a novel culture system of murine Th17 cells and NIH/3T3 fibroblasts, we showed that inhibition of both IL-23 and IL-17 completely suppressed IL-23-dependent IL-22 production from Th17 cells and cooperatively blocked IL-17-dependent IL-6 secretion from the NIH/3T3 cells to levels below either inhibitor alone. In vivo, in the imiquimod induced skin inflammation model, and in the myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis model, we demonstrated that dual inhibition of IL-17 and IL-23 was more efficacious in reducing disease than targeting either cytokine alone. Together, these data support the hypothesis that neutralization of both IL-23 and IL-17 may provide enhanced benefit against Th17 mediated autoimmunity and provide a basis for a therapeutic strategy aimed at dual targeting IL-23 and IL-17.

Distinct glucocorticoid sensitivity of Th17 cytokines in murine T hybridomas and primary cells (IRC11P.428)

Abstract Cytokine suppression contributes to the anti-inflammatory actions of glucocorticoids (GCs). However, IL-17 has been reported to be resistant to GC regulation in asthma, nasal polyps, and Crohn’s disease. In contrast, a negative GC-response element has been identified in the promoter region of the IL-17 gene and IL-17 was sensitive to GC suppression in a murine asthma model. To clarify whether IL-17 and other Th17 cytokines are sensitive to GC regulation, we generated Th17 hybridoma cells by fusing in vitro differentiated murine (bl/6) Th17 cells with BW5147 thymoma. Two clones selectively expressing IL-17A, IL-17F, IL-22 and GM-CSF were stable for over 30 generations. Additional clones selectively expressing IFN-γ or IL-4, but not Th17 cytokines and BW5147 cells that produce little or no cytokines were used as controls. Dexamethasone, a potent GC, significantly inhibited anti-CD3 and anti-CD28-stimulated IFN-γ, IL-4, as well as IL-17A, IL-17F, and IL-22 in various clones. Interestingly, IL-17A and IL-17F, but not IL-22, were resistant to GC suppression in in vitro differentiated Th17 cells. Furthermore, hybridomas, but not in vitro differentiated Th17 cells underwent GC-induced apoptosis. IL-6 partially rescues hybridomas from GC-induced apoptosis, however it did not modulate IL-17 suppression by GCs. Our findings support that IL-17A and IL-17F are capable of being downregulated by GCs and Th17 cell microenvironment and cellular milleu engender GC resistance.

Interferon-β suppresses murine Th1 cell function in the absence of antigen-presenting cells.

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals.

IκBNS regulates murine Th17 differentiation during gut inflammation and infection.

IL-17-producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A-producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS (-/-) Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS (-/-) mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections.

Glycogen synthase kinase-3 controls IL-10 expression in CD4(+) effector T-cell subsets through epigenetic modification of the IL-10 promoter.

The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4+ T helper cells. Treatment of naive murine CD4+ T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.

MiR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim.

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

Gene network inference using continuous time Bayesian networks: a comparative study and application to Th17 cell differentiation.

BackgroundDynamic aspects of gene regulatory networks are typically investigated by measuring system variables at multiple time points. Current state-of-the-art computational approaches for reconstructing gene networks directly build on such data, making a strong assumption that the system evolves in a synchronous fashion at fixed points in time. However, nowadays omics data are being generated with increasing time course granularity. Thus, modellers now have the possibility to represent the system as evolving in continuous time and to improve the models’ expressiveness.ResultsContinuous time Bayesian networks are proposed as a new approach for gene network reconstruction from time course expression data. Their performance was compared to two state-of-the-art methods: dynamic Bayesian networks and Granger causality analysis. On simulated data, the methods comparison was carried out for networks of increasing size, for measurements taken at different time granularity densities and for measurements unevenly spaced over time. Continuous time Bayesian networks outperformed the other methods in terms of the accuracy of regulatory interactions learnt from data for all network sizes. Furthermore, their performance degraded smoothly as the size of the network increased. Continuous time Bayesian networks were significantly better than dynamic Bayesian networks for all time granularities tested and better than Granger causality for dense time series. Both continuous time Bayesian networks and Granger causality performed robustly for unevenly spaced time series, with no significant loss of performance compared to the evenly spaced case, while the same did not hold true for dynamic Bayesian networks. The comparison included the IRMA experimental datasets which confirmed the effectiveness of the proposed method. Continuous time Bayesian networks were then applied to elucidate the regulatory mechanisms controlling murine T helper 17 (Th17) cell differentiation and were found to be effective in discovering well-known regulatory mechanisms, as well as new plausible biological insights.ConclusionsContinuous time Bayesian networks were effective on networks of both small and large size and were particularly feasible when the measurements were not evenly distributed over time. Reconstruction of the murine Th17 cell differentiation network using continuous time Bayesian networks revealed several autocrine loops, suggesting that Th17 cells may be auto regulating their own differentiation process.