Xuetao Cao

MicroRNA-146a Feedback Inhibits RIG-I-Dependent Type I IFN Production in Macrophages by Targeting TRAF6, IRAK1, and IRAK2

Upon recognition of viral components by pattern recognition receptors, including TLRs and retinoic acid-inducible gene I (RIG-I)- like helicases, cells are activated to produce type I IFN and proinflammatory cytokines. These pathways are tightly regulated by host to prevent inappropriate cellular response, but viruses can down-regulate these pathways for their survival. Recently, identification of negative regulators for cytoplasmic RNA-mediated antiviral signaling, especially the RIG-I pathway, attract much attention. However, there is no report about negative regulation of RIG-I antiviral pathway by microRNAs (miRNA) to date. We found that vesicular stomatitis virus (VSV) infection up-regulated miR-146a expression in mouse macrophages in TLR-myeloid differentiation factor 88-independent but RIG-I-NF-κB-dependent manner. In turn, miR-146a negatively regulated VSV-triggered type I IFN production, thus promoting VSV replication in macrophages. In addition to two known miR-146a targets, TRAF6 and IRAK1, we proved that IRAK2 was another target of miR-146a, which also participated in VSV-induced type I IFN production. Furthermore, IRAK1 and IRAK2 participated in VSV-induced type I IFN production by associating with Fas-associated death domain protein, an important adaptor in RIG-I signaling, in a VSV infection-inducible manner. Therefore, we demonstrate that miR-146a, up-regulated during viral infection, is a negative regulator of the RIG-I-dependent antiviral pathway by targeting TRAF6, IRAK1, and IRAK2.

Identification of miRNomes in Human Liver and Hepatocellular Carcinoma Reveals miR-199a/b-3p as Therapeutic Target for Hepatocellular Carcinoma

The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ∼88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.

Cancer-Expanded Myeloid-Derived Suppressor Cells Induce Anergy of NK Cells through Membrane-Bound TGF-β1

NK cells, the important effector of innate immunity, play critical roles in the antitumor immunity. Myeloid-derived suppressor cells (MDSC), a population of CD11b+Gr-1+ myeloid cells expanded dramatically during tumor progression, can inhibit T cells and dendritic cells, contributing to tumor immune escaper. However, regulation of NK cell innate function by MDSC in tumor-bearing host needs to be investigated. In this study, we found that the function of NK cells from liver and spleen was impaired significantly in all tumor-bearing models, indicating the impairment of hepatic NK cell function by tumor is a universal phenomenon. Then we prepared the orthotopic liver cancer-bearing mice as tumor model to investigate how hepatic NK cells are impaired. We show that down-regulation of NK cell function is inversely correlated with the marked increase of MDSC in liver and spleen. MDSC inhibit cytotoxicity, NKG2D expression, and IFN-γ production of NK cells both in vitro and in vivo. After incubation with MDSC, NK cells could not be activated to produce IFN-γ. Furthermore, membrane-bound TGF-β1 on MDSC is responsible for MDSC-mediated suppression of NK cells. The impaired function of hepatic NK cells in orthotopic liver cancer-bearing mice could be restored by depletion of MDSC, but not regulatory T cells. Therefore, cancer-expanded MDSC can induce anergy of NK cells via membrane-bound TGF-β1. MDSC, but not regulatory T cells, are main negative regulator of hepatic NK cell function in tumor-bearing host. Our study provides new mechanistic explanations for tumor immune escape.

The STAT3-Binding Long Noncoding RNA lnc-DC Controls Human Dendritic Cell Differentiation

L[i]nc to Dendritic Cell Activation Long noncoding RNAs (lncRNAs) are important regulators of gene expression, but whether they are important regulators of the immune system is poorly understood. Wang et al. (p. 310) identify a lncRNA expressed exclusively in human dendritic cells (DC), called lnc-DC, that is required for optimal DC differentiation from human monocytes and that regulates DC activation of T cells. Lnc-DC interacts with the transcription factor STAT3, which is also required for DC development and function, to prevent interaction with and to block dephosphorylation by tyrosine phosphatase SHP1. A long noncoding RNA regulates dendritic cell differentiation and function. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes; however, few have been identified that regulate immune cell differentiation and function. Here, we identified lnc-DC, which was exclusively expressed in human conventional dendritic cells (DCs). Knockdown of lnc-DC impaired DC differentiation from human monocytes in vitro and from mouse bone marrow cells in vivo and reduced capacity of DCs to stimulate T cell activation. lnc-DC mediated these effects by activating the transcription factor STAT3 (signal transducer and activator of transcription 3). lnc-DC bound directly to STAT3 in the cytoplasm, which promoted STAT3 phosphorylation on tyrosine-705 by preventing STAT3 binding to and dephosphorylation by SHP1. Our work identifies a lncRNA that regulates DC differentiation and also broadens the known mechanisms of lncRNA action.

Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells

The fates of dendritic cells (DCs) after antigen presentation have been studied extensively, but the influence of lymphoid microenvironments on DCs is mostly unknown. Here, using splenic stromal cells to mimic the immune microenvironment, we show that contact with stromal cells promoted mature DCs to proliferate in a fibronectin-dependent way and that both stromal cell contact and stromal cell–derived transforming growth factor-β induced their differentiation into a new regulatory DC subset. We have identified an in vivo counterpart in the spleen with similar phenotype and functions. These differentiated DCs secreted nitric oxide, which mediated the suppression of T cell proliferation in response to antigen presentation by mature DCs. Thus, our findings identify an important mechanism by which the microenvironment regulates immune responses.

The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenin-dependent pathway.

Intracellular nucleic acid sensors detect microbial RNA and DNA and trigger the production of type I interferon. However, the cytosolic nucleic acid–sensing system remains to be fully identified. Here we show that the cytosolic nucleic acid–binding protein LRRFIP1 contributed to the production of interferon-β (IFN-β) induced by vesicular stomatitis virus (VSV) and Listeria monocytogenes in macrophages. LRRFIP1 bound exogenous nucleic acids and increased the expression of IFN-β induced by both double-stranded RNA and double-stranded DNA. LRRFIP1 interacted with β-catenin and promoted the activation of β-catenin, which increased IFN-β expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-β enhanceosome via IRF3. Therefore, LRRFIP1 and its downstream partner β-catenin constitute another coactivator pathway for IRF3-mediated production of type I interferon.

Inducible microRNA-155 Feedback Promotes Type I IFN Signaling in Antiviral Innate Immunity by Targeting Suppressor of Cytokine Signaling 1

Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Our previous study showed that a panel of microRNAs, including miR-155, was markedly upregulated in macrophages upon vesicular stomatitis virus infection; however, the biological function of miR-155 during viral infection remains unknown. In this paper, we show that RNA virus infection induces miR-155 expression in macrophages via TLR/MyD88-independent but retinoic acid-inducible gene I/JNK/NF-κB–dependent pathway. And the inducible miR-155 feedback promotes type I IFN signaling, thus suppressing viral replication. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a canonical negative regulator of type I IFN signaling, is targeted by miR-155 in macrophages, and SOCS1 knockdown mediates the enhancing effect of miR-155 on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible miR-155 feedback positively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting SOCS1.

Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b

Integrins are critical for the migration and function of leukocytes in inflammation. However, the interaction between integrin αM (CD11b), which has high expression in monocytes and macrophages, and Toll-like receptor (TLR)-triggered innate immunity remains unclear. Here we report that CD11b deficiency enhanced TLR-mediated responses in macrophages, rendering mice more susceptible to endotoxin shock and Escherichia coli–caused sepsis. CD11b was activated by TLR-triggered phosphatidylinositol 3-OH kinase (PI(3)K) and the effector RapL and fed back to inhibit TLR signaling by activating the tyrosine kinases Src and Syk. Syk interacted with and induced tyrosine phosphorylation of MyD88 and TRIF, which led to degradation of these adaptor molecules by the E3 ubiquitin ligase Cbl-b. Thus, TLR-triggered, active CD11b integrin engages in crosstalk with the MyD88 and TRIF pathways and subsequently inhibits TLR signaling in innate immune responses.

Involvement of ERK, p38 and NF‐κB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll‐like receptors (TLR) are sentinel receptors capable of recognizing pathogen‐associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG‐containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram‐positive and Gram‐negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up‐regulation of co‐stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up‐regulated following LPS stimulation. The up‐regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor‐α induced by LPS plus CpG ODN. While inhibition of extracellular signal‐related kinase and NF‐κB activation suppressed the up‐regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up‐regulation of TLR2 and TLR4 mRNA expression but enhanced the up‐regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up‐regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.

Phosphatase SHP-1 promotes TLR- and RIG-I-activated production of type I interferon by inhibiting the kinase IRAK1

Unbalanced production of proinflammatory cytokines and type I interferons in immune responses may lead to immunopathology; thus, the mechanisms that ensure the beneficial production of proinflammatory cytokines and type I interferons are of particular importance. Here we demonstrate that the phosphatase SHP-1 negatively regulated Toll-like receptor–mediated production of proinflammatory cytokines by inhibiting activation of the transcription factor NF-κB and mitogen-activated protein kinase. Simultaneously, SHP-1 increased the production of type I interferon mediated by Toll-like receptors and the helicase RIG-I by directly binding to and inhibiting activation of the kinase IRAK1. Our data demonstrate that SHP-1 contributes to immune homeostasis by balancing the production of proinflammatory cytokines and type I interferons in the innate immune response.