Huan Ning

Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation

TH17 cells are recognized as a unique subset of T helper cells that have critical roles in the pathogenesis of autoimmunity and tissue inflammation. Although RORγt is necessary for the generation of TH17 cells, the molecular mechanisms underlying the functional diversity of TH17 cells are not fully understood. Here we show that a member of interferon regulatory factor (IRF) family of transcription factors, IRF8, has a critical role in silencing TH17-cell differentiation. Mice with a conventional knockout, as well as a T cell-specific deletion, of the Irf8 gene exhibited more efficient TH17 cells. Indeed, studies of an experimental model of colitis showed that IRF8 deficiency resulted in more severe inflammation with an enhanced TH17 phenotype. IRF8 was induced steadily and inhibited TH17-cell differentiation during TH17 lineage commitment at least in part through its physical interaction with RORγt. These findings define IRF8 as a novel intrinsic transcriptional inhibitor of TH17-cell differentiation.

Activation of IL-27 p28 Gene Transcription by Interferon Regulatory Factor 8 in Cooperation with Interferon Regulatory Factor 1

Interferon regulatory factor (IRF) family members, especially interferon regulatory factor-1 (IRF-1) and interferon regulatory factor-8 (IRF-8 or ICSBP), play important roles in interferon signaling in a wide range of host responses to infection and tumor growth. Interleukin-27 (IL-27), as a member of the IL-12 cytokine family, not only acts as a proinflammatory cytokine that regulates the differentiation of naive T helper cells but also possesses anti-inflammatory properties. IL-27 consists of EBI3 (Epstein-Barr virus-induced gene 3) and p28 subunits. Our previous work has shown that IRF-1 regulates IL-27 p28 gene transcription by specifically binding to the IRF-1 response element in the p28 promoter. In this study, we found that IRF-8-deficient macrophages were highly defective in the production of IL-27 p28 at both mRNA and protein levels. Circulating IL-27 p28 in serum was also decreased in IRF-8−/− mice in a septic shock model. Lipopolysaccharide, as a potent inducer of IL-27 p28 expression, could activate IRF-8 expression in a MyD88-dependent pathway, which in turn induced p28 gene transcription through NF-κB and/or IRF-8. Transcriptional analyses revealed that IRF-8 activated p28 gene transcription through binding to a site located at −57 to −48 in the p28 promoter overlapping the IRF-1 binding site. Consistent with this observation, overexpression of both IRF-8 and IRF-1 additively activated IL-27 p28 promoter. This study provides further mechanistic information regarding how signals initiated during innate and adaptive immune responses synergize to yield greater IL-27 production and sustained cellular immunity.

Posttranscriptional Regulation of IL-23 Expression by IFN-γ through Tristetraprolin

IL-23 plays an essential role in maintenance of IL-17–producing Th17 cells that are involved in the pathogenesis of several autoimmune diseases. Regulation of Th17 cells is tightly controlled by multiple factors such as IL-27 and IFN-γ. However, the detailed mechanisms responsible for IFN-γ–mediated Th17 cell inhibition are still largely unknown. In this study, we demonstrate that IFN-γ differentially regulates IL-12 and IL-23 production in both dendritic cells and macrophages. IFN-γ suppresses IL-23 expression by selectively targeting p19 mRNA stability through its 3′-untranslated region (3′UTR). Furthermore, IFN-γ enhances LPS-induced tristetraprolin (TTP) mRNA expression and protein production. Overexpression of TTP suppresses IL-23 p19 mRNA expression and p19 3′UTR-dependent luciferase activity. Additionally, deletion of TTP completely abolishes IFN-γ–mediated p19 mRNA degradation. We further demonstrate that IFN-γ suppresses LPS-induced p38 phosphorylation, and blockade of p38 MAPK signaling pathway with SB203580 inhibits IFN-γ– and LPS-induced p19 mRNA expression, whereas overexpression of p38 increases p19 mRNA expression via reducing TTP binding to the p19 3′UTR. Finally, inhibition of p38 phosphorylation by IFN-γ leads to TTP dephosphorylation that could result in stronger binding of the TTP to the adenosine/uridine-rich elements in the p19 3′UTR and p19 mRNA degradation. In summary, our results reveal a direct link among TTP, IFN-γ, and IL-23, indicating that IFN-γ–mediated Th17 cell suppression might act through TTP by increasing p19 mRNA degradation and therefore IL-23 inhibition.

Transcriptional Suppression of IL-27 Production by Mycobacterium tuberculosis-Activated p38 MAPK via Inhibition of AP-1 Binding

Mycobacterium tuberculosis remains a major global challenge to human health care, and the mechanisms of how M. tuberculosis evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that bacillus Calmette–Guérin (BCG) and viable M. tuberculosis as well as M. tuberculosis lysates could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and M. tuberculosis lysate stimulation of macrophages induced activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. M. tuberculosis lysate-induced p28 transcription was dependent on MyD88 signaling pathway. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after M. tuberculosis lysate stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to M. tuberculosis lysate stimulation. Furthermore, blockade of p38 by SB203580 enhanced M. tuberculosis-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhanced the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective tuberculosis immunity.

Suppression of IL-12 production by tristetraprolin through blocking NF-kcyB nuclear translocation.

Tristetraprolin (TTP), an mRNA-binding protein, plays a significant role in regulating the expression of adenylate-uridylate–rich elements containing mRNAs. Mice deficient of TTP (TTP−/−) develop a systemic autoimmune inflammatory syndrome characterized by cachexia, conjunctivitis, and dermatitis. IL-12 plays a crucial role in immune defense against infectious and malignant diseases. In this study, we found increased production of IL-12 during endotoxic shock and enhanced Th1 cells in TTP knockout mice. The levels of IL-12 p70 and p40 protein as well as p40 and p35 mRNA were also increased in activated macrophages deficient of TTP. In line with these findings, overexpression of TTP suppressed IL-12 p35 and p40 expression at the mRNA and promoter level, whereas it surprisingly had little effects on their mRNA stability. Our data showed that the inhibitory effects of TTP on p35 gene transcription were completely rescued by overexpression of NF-кB p65 and c-Rel but not by the p50 in activated macrophages. Our data further indicated that TTP acquired its inhibition on IL-12 expression through blocking nuclear translocation of NF-кB p65 and c-Rel while enhancing p50 upon stimulation. In summary, our study reveals a novel pathway through which TTP suppresses IL-12 production in macrophages, resulting in suppression of Th1 cell differentiation. This study may provide us with therapeutic targets for treatment of inflammatory and autoimmune disorders.

Tristetraprolin induces cell cycle arrest in breast tumor cells through targeting AP-1/c-Jun and NF-κB pathway

The main characteristic of cancers, including breast cancer, is the ability of cancer cells to proliferate uncontrollably. However, the underlying mechanisms of cancer cell proliferation, especially those regulated by the RNA binding protein tristetraprolin (TTP), are not completely understood. In this study, we found that TTP inhibits cell proliferation in vitro and suppresses tumor growth in vivo through inducing cell cycle arrest at the S phase. Our studies demonstrate that TTP inhibits c-Jun expression through the C-terminal Zn finger and therefore increases Wee1 expression, a regulatory molecule which controls cell cycle transition from the S to the G2 phase. In contrast to the well-known function of TTP in regulating mRNA stability, TTP inhibits c-Jun expression at the level of transcription by selectively blocking NF-κB p65 nuclear translocation. Reconstitution of NF-κB p65 completely abolishes the inhibition of c-Jun transcription by TTP. Moreover, reconstitution of c-Jun in TTP-expressing breast tumor cells diminishes Wee1 overexpression and promotes cell proliferation. Our results indicate that TTP suppresses c-Jun expression that results in Wee1 induction which causes cell cycle arrest at the S phase and inhibition of cell proliferation. Our study provides a new pathway for TTP function as a tumor suppressor which could be targeted in tumor treatment.

Monocyte chemotactic protein–induced protein 1 controls allergic airway inflammation by suppressing IL-5–producing TH2 cells through the Notch/Gata3 pathway

Background: Asthmatic and allergic inflammation is mediated by TH2 cytokines (IL‐4, IL‐5, and IL‐13). Although we have learned much about how TH2 cells are differentiated, the TH2 checkpoint mechanisms remain elusive. Objectives: In this study we investigate how monocyte chemotactic protein–induced protein 1 (MCPIP1; encoded by the Zc3h12a gene) regulates IL‐5–producing TH2 cell differentiation and TH2‐mediated inflammation. Methods: The functions of Zc3h12a−/− CD4 T cells were evaluated by checking the expression of TH2 cytokines and transcription factors in vivo and in vitro. Allergic airway inflammation of Zc3h12a−/− mice was examined with murine asthma models. In addition, antigen‐specific CD4 T cells deficient in MCPIP1 were transferred to wild‐type recipient mice, challenged with ovalbumin (OVA) or house dust mite (HDM), and accessed for TH2 inflammation. Results: Zc3h12a−/− mice have spontaneous severe lung inflammation, with an increase in mainly IL‐5– and IL‐13–producing but not IL‐4–producing TH2 cells in the lung. Mechanistically, differentiation of IL‐5–producing Zc3h12a−/− TH2 cells is mediated through Notch signaling and Gata3 independent of IL‐4. Gata3 mRNA is stabilized in Zc3h12a−/− TH2 cells. MCPIP1 promotes Gata3 mRNA decay through the RNase domain. Furthermore, deletion of MCPIP1 in OVA‐ or HDM‐specific T cells leads to significantly increased TH2‐mediated airway inflammation in OVA or HDM murine models of asthma. Conclusions: Our study reveals that MCPIP1 regulates the development and function of IL‐5–producing TH2 cells through the Notch/Gata3 pathway. MCPIP1 represents a new and promising target for the treatment of asthma and other TH2‐mediated diseases. Graphical abstract: Figure. No caption available.

Tristetraprolin inhibits macrophage IL-27-induced activation of antitumour cytotoxic T cell responses

IFN-γ-producing cytotoxic T lymphocytes are essential for host defense against viral infection and cancer. Here we show that the RNA-binding tristetraprolin, encoded by Zfp36, is needed for CD8+ T-cell production of IFN-γ in vivo. When activated in vitro, however, IFN-γ production by naive wild type and tristetraprolin-deficient CD8+ T-cells is comparable. IL-27 is overproduced by tristetraprolin-deficient macrophages and increased systemically in tristetraprolin-deficient mice. Tristetraprolin suppresses IL-27 production by promoting p28 mRNA degradation. Importantly, deletion of IL-27 receptor WSX-1 in tristetraprolin-deficient mice (WSX-1/tristetraprolin double knockout) leads to a reduction in cytotoxic T lymphocyte numbers. Moreover, tumor growth is accelerated, not only in tristetraprolin-deficient mice after cytotoxic T lymphocyte depletion, but also in WSX-1/tristetraprolin double knockout mice, with substantial reduction in the number of tumor cytotoxic T lymphocytes. This study describes a regulatory pathway for IL-27 expression and cytotoxic T lymphocyte function mediated by tristetraprolin, contributing to regulation of antitumour immunity.IL-27 is one of a number of cytokines that can induce antitumour CD8+ T cell responses. Here the authors show that TTP, encoded by Zfp36, degrades p28 to inhibit IL-27 production by macrophages and is thereby a negative regulator of the antitumour response.

Abstract LB-042: MCPIP1 suppresses breast tumor progression by targeting anti-apoptosis pathway

The ability to evade apoptosis is a hallmark of cancer and also plays a significant role in cancer resistance to conventional therapy. While recent progress has broadened our understanding of the apoptosis-evasion mechanisms by cancer cells, how apoptosis resistance develops in cancer cells through posttranscriptional mechanisms, especially by the newly discovered RNA-binding protein monocyte chemotactic protein induced protein 1 (MCPIP1), remains unknown. Here, we report that MCPIP1 expression is impaired in breast tumors and breast tumor cell lines, with the severest impairment in highly metastatic tumors. Ectopic expression of MCPIP1 causes apoptosis of breast tumor cells through selectively suppression of anti-apoptotic gene transcripts, including Bcl2L1, Bcl2A1, RelB, Birc3 and Bcl3. This suppression is medicated through a physical interaction between the PIN domain of MCPIP1 and the 3′UTRs of anti-apoptotic transcripts, resulting in mRNA degradation and tumor cell apoptosis. In difference from the RNA-binding protein tristetraprolin which binds to the ARE in the 3′UTR of target genes for mRNA decay, MCPIP1 specially recognizes a stem-loop structure in the 3′UTR of anti-apoptotic genes for binding and mRNA decay. Furthermore, induction of MCPIP not only ameliorates breast tumor formation but also completely shrinks the established tumors within six days. Lung metastasis is also significantly reduced by MCPIP1 induction. The tumor suppressive effect of MCPIP1 acts through activation of apoptosis. Importantly, by analysis of the excised breast tumors of 251 patients, we found that low MCPIP1 levels in tumors are strongly associated with poor survival of patients over 13 years of follow up. Taken together, we demonstrate that MCPIP1 is a novel potent tumor suppressor which induces tumor apoptosis through tipping the balance between pro-apoptotic and anti-apoptotic genes via selectively targeting the mRNAs of anti-apoptotic genes for degradation. MCPIP1 can serve as a new therapeutic target for treatment of breast cancer and other cancers as well. Citation Format: Jianguo Liu, Wenbao Lu, Huan Ning, Hui Peng, Qinghong Wang, Rong Hou. MCPIP1 suppresses breast tumor progression by targeting anti-apoptosis pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-042. doi:10.1158/1538-7445.AM2015-LB-042