Author Correction: Requirement for the basic helix-loop-helix transcription factor Dec2 in initial TH2 lineage commitment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The signaling suppressor CIS controls proallergic T cell development and allergic airway inflammation.

Transcription factors of the STAT family are critical in the cytokine-mediated functional differentiation of CD4(+) helper T cells. Signaling inhibitors of the SOCS family negatively regulate the activation of STAT proteins; however, their roles in the differentiation and function of helper T cells are not well understood. Here we found that the SOCS protein CIS, which was substantially induced by interleukin 4 (IL-4), negatively regulated the activation of STAT3, STAT5 and STAT6 in T cells. CIS-deficient mice spontaneously developed airway inflammation, and CIS deficiency in T cells led to greater susceptibility to experimental allergic asthma. CIS-deficient T cells showed enhanced differentiation into the TH2 and TH9 subsets of helper T cells. STAT5 and STAT6 regulated IL-9 expression by directly binding to the Il9 promoter. Our data thus demonstrate a critical role for CIS in controlling the proallergic generation of helper T cells.

Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Airway Neutrophilia.

Heightened unfolded protein responses (UPRs) are associated with the risk for asthma, including severe asthma. Treatment-refractory severe asthma manifests a neutrophilic phenotype with TH17 responses. However, how UPRs participate in the deregulation of TH17 cells leading to neutrophilic asthma remains elusive. This study found that the UPR sensor IRE1 is induced in the murine lung with fungal asthma and is highly expressed in TH17 cells relative to naïve CD4+ T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by both human and mouse TH17 cells. Ern1 (encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation of Ern1 leads to alleviated TH17 responses and airway neutrophilia in a fungal airway inflammation model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPR-mediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.

Requirement for the basic helix-loop-helix transcription factor Dec2 in initial TH2 lineage commitment.

How naive CD4(+) T cells commit to the T helper type 2 (T(H)2) lineage is poorly understood. Here we show that the basic helix-loop-helix transcription factor Dec2 was selectively expressed in T(H)2 cells. CD4(+) T cells from Dec2-deficient mice showed defective T(H)2 differentiation in vitro and in vivo in an asthma model and in response to challenge with a parasite antigen. Dec2 promoted expression of interleukin 4 (IL-4), IL-5 and IL-13 during early T(H)2 differentiation and directly bound to and activated transcription of genes encoding the transcription factors JunB and GATA-3. As GATA-3 induces Dec2 expression, our findings also indicate a feed-forward regulatory circuit during T(H)2 differentiation.

Transcription of Il17 and Il17f is controlled by conserved noncoding sequence 2.

T helper 17 (Th17) cells specifically transcribe the Il17 and Il17f genes, which are localized in the same chromosome region, but the underlying mechanism is unclear. Here, we report a cis element that we previously named conserved noncoding sequence 2 (CNS2) physically interacted with both Il17 and Il17f gene promoters and was sufficient for regulating their selective transcription in Th17 cells. Targeted deletion of CNS2 resulted in impaired retinoic acid-related orphan receptor gammat (RORγt)-driven IL-17 expression in vitro. CNS2-deficient T cells also produced substantially decreased amounts of IL-17F. These cytokine defects were associated with defective chromatin remodeling in the Il17-Il17f gene locus, possibly because of effects on CNS2-mediated recruitment of histone-modifying enzymes p300 and JmjC domain-containing protein 3 (JMJD3). CNS2-deficient animals were also shown to be resistant to experimental autoimmune encephalomyelitis (EAE). Our results thus suggest that CNS2 is sufficient and necessary for Il17 and optimal Il17f gene transcription in Th17 cells.

Toll-like receptor 2 signaling in CD4(+) T lymphocytes promotes T helper 17 responses and regulates the pathogenesis of autoimmune disease.

Toll-like receptors (TLRs) have previously been shown to play critical roles in the activation of innate immunity. Here, we describe that T cell expression of TLR2 regulates T helper 17 (Th17) cell responses. Stimulation with TLR2 agonists promoted Th17 differentiation in vitro and led to more robust proliferation and Th17 cytokine production. Using the experimental autoimmune encephalomyelitis (EAE) model, we found that TLR2 regulated Th17 cell-mediated autoimmunity in vivo and that loss of TLR2 in CD4(+) T cells dramatically ameliorated EAE. This study thus reveals a critical role of a TLR in the direct regulation of adaptive immune response and pathogenesis of autoimmune diseases.

Effects of spinal non-viral interleukin-10 gene therapy formulated with d-mannose in neuropathic interleukin-10 deficient mice: Behavioral characterization, mRNA and protein analysis in pain relevant tissues.

Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-ß1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.

Critical regulation of early Th17 cell differentiation by interleukin-1 signaling.

T helper (Th) 17 cells have been recently discovered in both mouse and human. Here we show that interleukin-1 (IL-1) signaling on T cells is critically required for the early programming of Th17 cell lineage and Th17 cell-mediated autoimmunity. IL-1 receptor1 expression in T cells, which was induced by IL-6, was necessary for the induction of experimental autoimmune encephalomyelitis and for early Th17 cell differentiation in vivo. Moreover, IL-1 signaling in T cells was required in dendritic cell-mediated Th17 cell differentiation from naive or regulatory precursors and IL-1 synergized with IL-6 and IL-23 to regulate Th17 cell differentiation and maintain cytokine expression in effector Th17 cells. Importantly, IL-1 regulated the expression of the transcription factors IRF4 and RORgammat during Th17 cell differentiation; overexpression of these two factors resulted in IL-1-independent Th17 cell polarization. Our data thus indicate a critical role of IL-1 in Th17 cell differentiation and this pathway may serve as a unique target for Th17 cell-mediated immunopathology.

T helper 17 cells promote cytotoxic T cell activation in tumor immunity.

Although T helper 17 (Th17) cells have been found in tumor tissues, their function in cancer immunity is unclear. We found that interleukin-17A (IL-17A)-deficient mice were more susceptible to developing lung melanoma. Conversely, adoptive T cell therapy with tumor-specific Th17 cells prevented tumor development. Importantly, the Th17 cells retained their cytokine signature and exhibited stronger therapeutic efficacy than Th1 cells. Unexpectedly, therapy using Th17 cells elicited a remarkable activation of tumor-specific CD8(+) T cells, which were necessary for the antitumor effect. Th17 cells promoted dendritic cell recruitment into the tumor tissues and in draining lymph nodes increased CD8 alpha(+) dendritic cells containing tumor material. Moreover, Th17 cells promoted CCL20 chemokine production by tumor tissues, and tumor-bearing CCR6-deficient mice did not respond to Th17 cell therapy. Thus, Th17 cells elicited a protective inflammation that promotes the activation of tumor-specific CD8(+) T cells. These findings have important implications in antitumor immunotherapies.

Leptin Enhances TH2 and ILC2 Responses in Allergic Airway Disease.

Allergic asthma and obesity are the leading health problems in the world. Many studies have shown that obesity is a risk factor of development of asthma. However, the underlying mechanism has not been well established. In this study, we demonstrate that leptin, an adipokine elevated in obese individuals, promoted proliferation and survival of pro-allergic type 2 helper T cells and group 2 innate lymphoid cells and production of type 2 cytokines, which together contribute to allergic responses. Leptin activates mTORC1, MAPK, and STAT3 pathways in TH2 cells. The effects of leptin on TH2 cell proliferation, survival, and cytokine production are dependent on the mTORC1 and MAPK pathways as revealed by specific inhibitors. In vivo, leptin-deficiency led to attenuated experimental allergic airway inflammation. Our results thus support that obesity-associated elevation of leptin contributes to the increased susceptibility of asthma via modulation of pro-allergic lymphocyte responses.

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.

Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages.

After activation, CD4(+) helper T (Th) cells differentiate into distinct effector subsets. Although chemokine (C-X-C motif) receptor 5-expressing T follicular helper (Tfh) cells are important in humoral immunity, their developmental regulation is unclear. Here we show that Tfh cells had a distinct gene expression profile and developed in vivo independently of the Th1 or Th2 cell lineages. Tfh cell generation was regulated by ICOS ligand (ICOSL) expressed on B cells and was dependent on interleukin-21 (IL-21), IL-6, and signal transducer and activator of transcription 3 (STAT3). However, unlike Th17 cells, differentiation of Tfh cells did not require transforming growth factor beta (TGF-beta) or Th17-specific orphan nuclear receptors RORalpha and RORgamma in vivo. Finally, naive T cells activated in vitro in the presence of IL-21 but not TGF-beta signaling preferentially acquired Tfh gene expression and promoted germinal-center reactions in vivo. This study thus demonstrates that Tfh is a distinct Th cell lineage.

Molecular antagonism and plasticity of regulatory and inflammatory T cell programs.

Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.

Depletion of JunB increases adipocyte thermogenic capacity and ameliorates diet-induced insulin resistance.

The coexistence of brown adipocytes with low and high thermogenic activity is a fundamental feature of brown adipose tissue heterogeneity and plasticity. However, the mechanisms that govern thermogenic adipocyte heterogeneity and its significance in obesity and metabolic disease remain poorly understood. Here we show that in male mice, a population of transcription factor jun-B (JunB)-enriched (JunB+) adipocytes within the brown adipose tissue exhibits lower thermogenic capacity compared to high-thermogenic adipocytes. The JunB+ adipocyte population expands in obesity. Depletion of JunB in adipocytes increases the fraction of adipocytes exhibiting high thermogenic capacity, leading to enhanced basal and cold-induced energy expenditure and protection against diet-induced obesity and insulin resistance. Mechanistically, JunB antagonizes the stimulatory effects of PPARγ coactivator-1α on high-thermogenic adipocyte formation by directly binding to the promoter of oestrogen-related receptor alpha, a PPARγ coactivator-1α downstream effector. Taken together, our study uncovers that JunB shapes thermogenic adipocyte heterogeneity, serving a critical role in maintaining systemic metabolic health.

Essential autocrine regulation by IL-21 in the generation of inflammatory T cells.

After activation, CD4+ helper T (T(H)) cells differentiate into distinct effector subsets that are characterized by their unique cytokine expression and immunoregulatory function. During this differentiation, T(H)1 and T(H)2 cells produce interferon-gamma and interleukin (IL)-4, respectively, as autocrine factors necessary for selective lineage commitment. A distinct T(H) subset, termed T(HIL-17), T(H)17 or inflammatory T(H) (T(H)i), has been recently identified as a distinct T(H) lineage mediating tissue inflammation. T(H)17 differentiation is initiated by transforming growth factor-beta and IL-6 (refs 5-7) and reinforced by IL-23 (ref. 8), in which signal transduction and activators of transcription (STAT)3 and retinoic acid receptor-related orphan receptor (ROR)-gamma mediate the lineage specification. T(H)17 cells produce IL-17, IL-17F and IL-22, all of which regulate inflammatory responses by tissue cells but have no importance in T(H)17 differentiation. Here we show that IL-21 is another cytokine highly expressed by mouse T(H)17 cells. IL-21 is induced by IL-6 in activated T cells, a process that is dependent on STAT3 but not ROR-gamma. IL-21 potently induces T(H)17 differentiation and suppresses Foxp3 expression, which requires STAT3 and ROR-gamma, which is encoded by Rorc. IL-21 deficiency impairs the generation of T(H)17 cells and results in protection against experimental autoimmune encephalomyelitis. IL-21 is therefore an autocrine cytokine that is sufficient and necessary for T(H)17 differentiation, and serves as a target for treating inflammatory diseases.

Unfolded protein response factor ATF6 augments T helper cell responses and promotes mixed granulocytic airway inflammation.

The unfolded protein response (UPR) is associated with the risk of asthma, including treatment-refractory severe asthma. Recent studies demonstrated a pathogenic role of activating transcription factor 6a (ATF6a or ATF6), an essential UPR sensor, in airway structural cells. However, its role in T helper (TH) cells has not been well examined. In this study, we found that ATF6 was selectively induced by signal transducer and activator of transcription6 (STAT6) and STAT3 in TH2 and TH17 cells, respectively. ATF6 upregulated UPR genes and promoted the differentiation and cytokine secretion of TH2 and TH17 cells. T cell-specific Atf6-deficiency impaired TH2 and TH17 responses in vitro and in vivo and attenuated mixed granulocytic experimental asthma. ATF6 inhibitor Ceapin A7 suppressed the expression of ATF6 downstream genes and TH cell cytokines by both murine and human memory clusters of differentiation 4 (CD4)+ T cells. At the chronic stage of asthma, administration of Ceapin A7 lessened TH2 and TH17 responses, leading to alleviation of both airway neutrophilia and eosinophilia. Thus, our results demonstrate a critical role of ATF6 in TH2 and TH17 cell-driven mixed granulocytic airway disease, suggesting a novel option to combat steroid-resistant mixed and even T2-low endotypes of asthma by targeting ATF6.

Unconventional Activation of IRE1 Enhances TH17 Responses and Promotes Neutrophilic Airway Inflammation.

Treatment-refractory severe asthma manifests a neutrophilic phenotype associated with TH17 responses. Heightened unfolded protein responses (UPRs) are associated with the risk of asthma, including severe asthma. However, how UPRs participate in the deregulation of TH17 cells leading to this type of asthma remains elusive. In this study, we investigated the role of the UPR sensor IRE1 in TH17 cell function and neutrophilic airway inflammation. We found that IRE1 is induced in fungal asthma and is highly expressed in TH17 cells relative to naïve CD4+ T cells. Cytokine (e.g. IL-23) signals induce the IRE1-XBP1s axis in a JAK2-dependent manner. This noncanonical activation of the IRE1-XBP1s pathway promotes UPRs and cytokine secretion by TH17 cells. Ern1 (encoding IRE1)-deficiency decreases the expression of ER stress factors and impairs the differentiation and cytokine secretion of TH17 cells. Genetic ablation of Ern1 leads to alleviated TH17 responses and airway neutrophilia in a Candida albicans asthma model. Consistently, IL-23 activates the JAK2-IRE1-XBP1s pathway in vivo and enhances TH17 responses and neutrophilic infiltration into the airway. Taken together, our data indicate that IRE1, noncanonically activated by cytokine signals, promotes neutrophilic airway inflammation through the UPRmediated secretory function of TH17 cells. The findings provide a novel insight into the fundamental understanding of IRE1 in TH17-biased TH2-low asthma.

COX-2 Deficiency Promotes White Adipogenesis via PGE2-Mediated Paracrine Mechanism and Exacerbates Diet-Induced Obesity.

Cyclooxygenase-2 (COX-2) plays a critical role in regulating innate immunity and metabolism by producing prostaglandins (PGs) and other lipid mediators. However, the implication of adipose COX-2 in obesity remains largely unknown. Using adipocyte-specific COX-2 knockout (KO) mice, we showed that depleting COX-2 in adipocytes promoted white adipose tissue development accompanied with increased size and number of adipocytes and predisposed diet-induced adiposity, obesity, and insulin resistance. The increased size and number of adipocytes by COX-2 KO were reversed by the treatment of prostaglandin E2 (PGE2) but not PGI2 and PGD2 during adipocyte differentiation. PGE2 suppresses PPARγ expression through the PKA pathway at the early phase of adipogenesis, and treatment of PGE2 or PKA activator isoproterenol diminished the increased lipid droplets in size and number in COX-2 KO primary adipocytes. Administration of PGE2 attenuated increased fat mass and fat percentage in COX-2 deficient mice. Taken together, our study demonstrated the suppressing effect of adipocyte COX-2 on adipogenesis and reveals that COX-2 restrains adipose tissue expansion via the PGE2-mediated paracrine mechanism and prevents the development of obesity and related metabolic disorders.

Adipocyte-derived PGE2 is required for intermittent fasting-induced Treg proliferation and improvement of insulin sensitivity.

The intermittent fasting (IF) diet has profound benefits for diabetes prevention. However, the precise mechanisms underlying IF's beneficial effects remain poorly defined. Here, we show that the expression levels of cyclooxygenase-2 (COX-2), an enzyme that produces prostaglandins, are suppressed in white adipose tissue (WAT) of obese humans. In addition, the expression of COX-2 in WAT is markedly upregulated by IF in obese mice. Adipocyte-specific depletion of COX-2 led to reduced fractions of CD4+Foxp3+ Tregs and a substantial decrease in the frequency of CD206+ macrophages, an increase in the abundance of γδT cells in WAT under normal chow diet conditions, and attenuation of IF-induced antiinflammatory and insulin-sensitizing effects, despite a similar antiobesity effect in obese mice. Mechanistically, adipocyte-derived prostaglandin E2 (PGE2) promoted Treg proliferation through the CaMKII pathway in vitro and rescued Treg populations in adipose tissue in COX-2-deficient mice. Ultimately, inactivation of Tregs by neutralizing anti-CD25 diminished IF-elicited antiinflammatory and insulin-sensitizing effects, and PGE2 restored the beneficial effects of IF in COX-2-KO mice. Collectively, our study reveals that adipocyte COX-2 is a key regulator of Treg proliferation and that adipocyte-derived PGE2 is essential for IF-elicited type 2 immune response and metabolic benefits.

Cutting edge: in vitro generated Th17 cells maintain their cytokine expression program in normal but not lymphopenic hosts.

Upon activation, naive CD4(+) T cells differentiate into effector Th cell subsets. The stability and plasticity of effector Th cells have not been well understood. In this study we used an IL-17F-red fluorescent protein reporter mouse to analyze the plasticity of Th17 cells in vitro and in vivo. We found that in vitro generated Th17 cells poorly maintained their differentiation program in vitro and could be reprogrammed into other T cell lineages. Moreover, upon transfer into lymphopenic hosts, Th17 cells rapidly lost their IL-17 expression and were converted into Th1 cells independently of IL-7 signaling. However, Th17 cells maintained their phenotypes well in normal animals, even in the absence of Ag and inflammation. These results, although suggesting the plasticity of Th17 cells, also indicate active maintenance of their program in vivo.