Xingguang Liu

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.

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.

MicroRNA-466l Upregulates IL-10 Expression in TLR-Triggered Macrophages by Antagonizing RNA-Binding Protein Tristetraprolin-Mediated IL-10 mRNA Degradation

MicroRNAs (miRNAs) are generally recognized as regulating gene expression posttranscriptionally by inhibiting translation or inducing target mRNA degradation. New mechanisms for miRNAs to regulate gene expression also still attract much attention. More and more novel miRNAs are discovered by the advanced sequencing technology, but yet their biological functions are largely unknown. Up to now, the function of miR-466l, a miRNA discovered in mouse embryonic stem cells, remains unclear. In this study, we report that miR-466l can upregulate both mRNA and protein expression of IL-10 in TLR-triggered macrophages. Furthermore, we show that miR-466l can competitively bind to the IL-10 3′ untranslated region AU-rich elements, which is a typical binding site for RNA-binding protein (RBP). Tristetraprolin is a well-known RBP, and mediates rapid degradation of IL-10 mRNA. miRNA always mediates target mRNA degradation or translation repression modestly; thus, the net effect of miR-466l’s binding to IL-10 AU-rich elements is to prevent IL-10 mRNA degradation mediated by tristetraprolin, resulting in extended t1/2 of IL-10 mRNA and elevated IL-10 expression. Thus, competitive binding with RBP to the same target mRNA and subsequent stabilization of target mRNA is an alternative mechanism for gene regulation by miRNAs. Also, a mechanism for regulation of IL-10 by miRNAs is outlined.

Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk

The molecular mechanisms involved in the full activation of innate immunity achieved through Toll-like receptors (TLRs) remain to be fully elucidated. In addition to their classical antigen-presenting function, major histocompatibility complex (MHC) class II molecules might mediate reverse signaling. Here we report that deficiency in MHC class II attenuated the TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and dendritic cells, which protected mice from endotoxin shock. Intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD40 and maintained Btk activation, but cell surface MHC class II molecules did not. Then, Btk interacted with the adaptor molecules MyD88 and TRIF and thereby promoted TLR signaling. Therefore, intracellular MHC class II molecules can act as adaptors, promoting full activation of TLR-triggered innate immune responses.

MicroRNA-148/152 Impair Innate Response and Antigen Presentation of TLR-Triggered Dendritic Cells by Targeting CaMKIIα

MicroRNAs (miRNAs) are involved in the regulation of immunity, including the lymphocyte development and differentiation, and inflammatory cytokine production. Dendritic cells (DCs) play important roles in linking innate and adaptive immune responses. However, few miRNAs have been found to regulate the innate response and APC function of DCs to date. Calcium/calmodulin-dependent protein kinase II (CaMKII), a major downstream effector of calcium (Ca2+), has been shown to be an important regulator of the maturation and function of DCs. Our previous study showed that CaMKIIα could promote TLR-triggered production of proinflammatory cytokines and type I IFN. Inspired by the observations that dicer mutant Drosophila display defect in endogenous miRNA generation and higher CaMKII expression, we wondered whether miRNAs can regulate the innate response and APC function of DCs by targeting CaMKIIα. By predicting with software and confirming with functional experiments, we demonstrate that three members of the miRNA (miR)-148 family, miR-148a, miR-148b, and miR-152, are negative regulators of the innate response and Ag-presenting capacity of DCs. miR-148/152 expression was upregulated, whereas CaMKIIα expression was downregulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. We showed that miR-148/152 in turn inhibited the production of cytokines including IL-12, IL-6, TNF-α, and IFN-β upregulation of MHC class II expression and DC-initiated Ag-specific T cell proliferation by targeting CaMKIIα. Therefore, miRNA-148/152 can act as fine-tuner in regulating the innate response and Ag-presenting capacity of DCs, which may contribute to the immune homeostasis and immune regulation.

CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages

Calcium and its major downstream effector, calcium/calmodulin-dependent protein kinase II (CaMKII), are found to be important for the functions of immune cells. Lipopolysaccharide (LPS) has been shown to induce intracellular calcium release in macrophages; however, whether and how CaMKII is required for Toll-like receptor (TLR) signaling remain unknown. Here we demonstrate that TLR 4, 9, and 3 ligands markedly induce intracellular calcium fluxes and activate CaMKII-alpha in macrophages. Selective inhibition or RNA interference of CaMKII significantly suppresses TLR4, 9, 3-triggered production of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferon-alpha/beta (IFN-alpha/beta) in macrophages. Coincidently, overexpression of constitutively active CaMKII-alpha significantly enhances production of the above cytokines. In addition to the activation of mitogen-activated protein kinase and nuclear factor kappaB pathways, CaMKII-alpha can directly bind and phosphorylate transforming growth factor beta-activated kinase 1 (TAK1) and IFN regulatory factor 3 (IRF3; serine on 386) via the N-terminal part of its regulatory domain. Therefore, CaMKII can be activated by TLR ligands, and in turn promotes both myeloid differentiating factor 88 and Toll/IL-1 receptor domain-containing adaptor protein-inducing IFN-beta-dependent inflammatory responses by directly activating TAK1 and IRF3. The cross-talk with the calcium/CaMKII pathway is needed for full activation of TLR signaling in macrophages.

Constitutive MHC class I molecules negatively regulate TLR-triggered inflammatory responses via the Fps–SHP-2 pathway

The molecular mechanisms that fine-tune Toll-like receptor (TLR)-triggered innate inflammatory responses remain to be fully elucidated. Major histocompatibility complex (MHC) molecules can mediate reverse signaling and have nonclassical functions. Here we found that constitutively expressed membrane MHC class I molecules attenuated TLR-triggered innate inflammatory responses via reverse signaling, which protected mice from sepsis. The intracellular domain of MHC class I molecules was phosphorylated by the kinase Src after TLR activation, then the tyrosine kinase Fps was recruited via its Src homology 2 domain to phosphorylated MHC class I molecules. This led to enhanced Fps activity and recruitment of the phosphatase SHP-2, which interfered with TLR signaling mediated by the signaling molecule TRAF6. Thus, constitutive MHC class I molecules engage in crosstalk with TLR signaling via the Fps-SHP-2 pathway and control TLR-triggered innate inflammatory responses.

Notch signal suppresses Toll-like receptor-triggered inflammatory responses in macrophages by inhibiting extracellular signal-regulated kinase 1/2-mediated nuclear factor κB activation.

Background: Notch signaling has been implicated in the development and function of the immune system, whereas its role in TLRs-triggered innate inflammatory responses remains unclear. Results: Notch signaling suppresses TLR-triggered inflammatory responses by inhibiting ERK1/2-mediated NF-κB activity in macrophages. Conclusion: Cross-regulation exists between Notch and TLR signaling. Significance: Understanding Notch signaling in the context of the TLR-triggered macrophage inflammatory response may provide insights into the mechanisms for inflammation regulation. Multiple signaling pathways are involved in the tight regulation of Toll-like receptor (TLR) signaling, which is important for the tailoring of inflammatory response to pathogens in macrophages. It is widely accepted that TLR signaling can activate Notch pathway; however, whether full activation of Notch signaling can feedback modulate TLR signaling pathway so as to control inflammation response remains unclear. Here, we demonstrated that stimulation with TLR ligands up-regulated Notch1 and Notch2 expression in macrophages. The expression of Notch target genes including Hes1 and Hes5 was also induced in macrophages by LPS, suggesting that TLR4 signaling enhances the activation of Notch pathway. Importantly, overexpression of constituted active form of Notch1 (NICD1) and Notch2 (NICD2) suppressed production of TLR4-triggered proinflammatory cytokines such as TNF-α and IL-6 but promoted production of antiinflammatory cytokine IL-10, which is dependent on the PEST domain of NICD. In addition, NICD1 and NICD2 suppressed TLR-triggered ERK phosphorylation, which is indispensable for Notch-mediated inhibition of TLR4-triggered proinflammatory cytokine production. Furthermore, activation of Notch signaling inhibited NF-κB transcription activity by MyD88/TRAF6 and TRIF pathways, which was dependent on ERK activity. Therefore, our results showed that Notch signaling negatively regulates TLR-triggered inflammation responses, revealing a new mechanism for negative regulation of TLR signaling via Notch pathway.

Immune Responsive Gene 1 (IRG1) Promotes Endotoxin Tolerance by Increasing A20 Expression in Macrophages through Reactive Oxygen Species

Background: The molecular mechanisms of endotoxin tolerance remain not well elucidated. Results: IRG1, up-regulated by LPS and during sepsis, can feedback suppress the Toll-like receptor-triggered inflammatory response by increasing A20 expression via reactive oxygen species (ROS) in LPS-tolerized macrophages. Conclusion: Inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS. Significance: Providing new molecular mechanisms regulating hypoinflammation of sepsis and endotoxin tolerance. Sepsis-associated immunosuppression (SAIS) is regarded as one of main causes for the death of septic patients at the late stage because of the decreased innate immunity with a more opportunistic infection. LPS-tolerized macrophages, which are re-challenged by LPS after prior exposure to LPS, are regarded as the common model of hypo-responsiveness for SAIS. However, the molecular mechanisms of endotoxin tolerance and SAIS remain to be fully elucidated. In addition, negative regulation of the Toll-like receptor (TLR)-triggered innate inflammatory response needs further investigation. Here we show that expression of immune responsive gene 1 (IRG1) was highly up-regulated in the peripheral blood mononuclear cells of septic patients and in LPS-tolerized mouse macrophages. IRG1 significantly suppressed TLR-triggered production of proinflammatory cytokines TNF-α, IL-6, and IFN-β in LPS-tolerized macrophages, with the elevated expression of reactive oxygen species (ROS) and A20. Moreover, ROS enhanced A20 expression by increasing the H3K4me3 modification of histone on the A20 promoter domain, and supplement of the ROS abrogated the IRG1 knockdown function in breaking endotoxin tolerance by increasing A20 expression. Our results demonstrate that inducible IRG1 promotes endotoxin tolerance by increasing A20 expression through ROS, indicating a new molecular mechanism regulating hypoinflammation of sepsis and endotoxin tolerance.