SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway,and its C-terminal region is critical for the antagonism

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein which can inhibit the synthesis of type I interferon (IFN), but the molecular mechanism of this antagonism remains to be identified. In this study, we demonstrated that the N protein of SARS-CoV could inhibit IFN-beta (IFN-β) induced by poly(I:C) or Sendai virus. However, we found that N protein could not inhibit IFN-β production induced by overexpression of downstream signaling molecules of two important IFN-β induction pathways, toll-like receptor 3 (TLR3)- and RIG-I-like receptors (RLR)-dependent pathways. These results indicate that SARS-CoV N protein targets the initial step, probably the cellular PRRs (pattern recognition receptors)-RNAs-recognition step in the innate immune pathways, to suppress IFN expression responses. In addition, co-immunoprecipitation assays revealed that N protein did not interact with RIG-I or MDA5. Further, an assay using truncated mutants revealed that the C-terminal domain of N protein was critical for its antagonism of IFN induction, and the N deletion mutant impaired for RNA-binding almost completely lost the IFN-β antagonist activity. These results contribute to our further understanding of the pathogenesis of SARS-CoV.     

Pellino3 ligase negatively regulates influenza B dependent RIG-I signalling through downregulation of TRAF3-mediated induction of the transcription factor IRF3 and IFNβ production

Viral infection activates the innate immune system, which recognizes viral components by a variety of pattern recognition receptors and initiates signalling cascades leading to the production of pro-inflammatory cytokines. To date, signalling cascades triggered after virus recognition are not fully characterized and are investigated by many research groups. The critical role of the E3 ubiquitin ligase Pellino3 in antibacterial and antiviral response is now widely accepted, but the precise mechanism remains elusive. In this study, we sought to explore Pellino3 role in the retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this work, the molecular mechanisms of the innate immune response, regulated by Pellino3, were investigated in lung epithelial cells during influenza B virus infection. We used wild-type and Pellino3-deficient A549 cells as model cell lines to examine the role of Pellino3 ligase in the type I interferon (IFN) signalling pathway. Our results indicate that Pellino3 is involved in direct ubiquitination and degradation of the TRAF3, suppressing interferon regulatory factor 3 (IRF3) activation and interferon beta (IFNβ) production.     

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1β-stimulated human alveolar epithelial cells

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1β. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.     

Antibacterial immunity in respiratory tract: what role for the myeloid differentiation primary response protein 88 (MyD88) adaptor?

As an essential interface between the external environment and the organism, the respiratory tract is particularly exposed to the potential injuries due to microorganisms, such as bacteria. Against these aggressions, the immune system has a key role, involving detection of germs followed by the establishment of innate and adaptive immune processes. During these responses, the adaptor protein MyD88 has a central place in the development of immune response: i) it contributes to innate immune response through the production of cytokines and chemokines, allowing recruitment and activation of effector cells; ii) it is involved secondarily in the development and orientation of antibacterial adaptive response, iii) and participates in sustaining immune homeostasis. Its importance is reflected in human pathology (MyD88 hereditary deficiency) by increasing susceptibility to few pathogenic bacteria responsible for respiratory infections which mainly occur in the upper respiratory tract. Since it has a key role, MyD88 is an essential factor for antibacterial immunity and more broadly for anti-infectious immunity. This protein might be therefore a possible therapeutic target.     

The induction of innate and adaptive immunity by biodegradable poly(γ-glutamic acid) nanoparticles via a TLR4 and MyD88 signaling pathway

The induction of adaptive immunity through the activation of innate immunity is indispensable for vaccine development. Although strategies for particulate antigen delivery are widely investigated, their immunological mechanisms are unclear. We describe in this study that biodegradable nanoparticles (NPs) elaborated with poly(γ-glutamic acid) (γ-PGA) are able to induce potent innate and adaptive immune responses through Toll-like receptor 4 (TLR4) and MyD88 signaling pathways. The production of inflammatory cytokines from macrophages and the maturation of dendritic cells were impaired in MyD88-knockout and TLR4-deficient mice compared with their wild-types, when the cells were stimulated with γ-PGA NPs. The immunization of these mice with antigen-carrying γ-PGA NPs also resulted in diminished induction of antigen-specific cellular immune responses. These results suggest that γ-PGA NPs have not only an antigen-carrying capacity but also a potent adjuvant function of eliciting adaptive immune responses to the carrying antigen through recognition of the first-line host-sensor system.     

Interferon-α/β receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006

Background  

Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported.     

Activation of cardiac human ether-a-go-go related gene potassium currents is regulated by α1A-adrenoceptors

Patients with cardiac disease typically develop life-threatening ventricular arrhythmias during physical or emotional stress, suggesting a link between adrenergic stimulation and regulation of the cardiac action potential. Human ether-a-go-go related gene (hERG) potassium channels conduct the rapid component of the repolarizing delayed rectifier potassium current, I_Kr. Previous studies have revealed that hERG channel activation is modulated by activation of the -adrenergic system. In contrast, the influence of the -adrenergic signal transduction cascade on hERG currents is less well understood. The present study examined the regulation of hERG currents by _1A-adrenoceptors. hERG channels and human _1A-adrenoceptors were heterologously coexpressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Stimulation of _1A-receptors by applying 20 µM phenylephrine caused hERG current reduction due to a 9.6-mV shift of the activation curve towards more positive potentials. Simultaneous application of the ₁-adrenoceptor antagonist prazosin (20 µM) prevented the activation shift. Inhibition of PKC (3 µM Ro-32-0432) or PKA (2.5 µM KT 5720) abolished the -adrenergic activation shift, suggesting that PKC and PKA are required within the regulatory mechanism. The effect was still present when the PKA- and PKC-dependent phosphorylation sites in hERG were deleted by mutagenesis. In summary, cardiac repolarizing hERG/I_Kr potassium currents are modulated by _1A-adrenoceptors via PKC and PKA independently of direct channel phosphorylation. This novel regulatory pathway of ₁-adrenergic hERG current regulation provides a link between stress and ventricular arrhythmias, in particular in patients with heart disease.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Transcription factor Smad-independent T helper 17 cell induction by transforming-growth factor-β is mediated by suppression of eomesodermin

1. Download : Download high-res image (67KB)
2. Download : Download full-size image

Highlights? Suppression of Eomes by TGF-β is an important mechanism for Th17 cell differentiation ? Eomes expression is suppressed by TGF-β via a Smad-independent mechanism ? The JNK-c-Jun pathway suppresses Eomes expression and promotes Th17 cell differentiation ? Eomes directly inhibits RORγt and IL-17A expression through the T-box domain     

GDF-5 is suppressed by IL-1β and enhances TGF-β3-mediated chondrogenic differentiation in human rheumatoid fibroblast-like synoviocytes

Objective

To investigate the expression of growth differentiation factor-5 (GDF-5) in chondrocytes (HC) and fibroblast-like synoviocytes (FLS) from humans with rheumatoid arthritis (RA) when stimulated with proinflammatory cytokines and to explore whether GDF-5 plays a role in regulating the differentiation of FLS-RA into chondrocytes.

Methods

Expression of GDF-5 in synovium and cartilage in RA and osteoarthritis (OA) was assessed by immunohistochemistry. GDF-5 production in FLS-RA and HC-RA was examined through real-time quantitative RT-PCR (Q-PCR) and western blotting. Expressions of GDF-associated receptors on FLS-RA were determined by semiquantitative-PCR, and MTT assay was used to study the effects on FLS-RA proliferation. Effect of GDF-5 and TGF-β3 on in vitro chondrogenic ability of FLS-RA was investigated using pellet-culture system, Q-PCR and histological analysis.

Results

Immunohistochemical analysis demonstrated that GDF-5 expression in the synovium and cartilage from joints of RA patients was much lower than that of OA patients. Addition of IL-1β or TNF-α appeared to downregulate the expression of GDF-5 in HC-RA and FLS-RA. Inhibition of GDF-5 expression by IL-1β in RA-FLS was attenuated by pretreatment with MEK1/2 inhibitor. GDF-5-associated receptors were expressed in FLS-RA, but GDF-5 had no effect on FLS-RA proliferation. GDF-5 had a strong chondrogenic-promoting effect on TGF-β3-induced chondrocyte differentiation in FLS-RA.

Conclusions

GDF-5 is expressed in FLS-RA and HC-RA, and its expression is strongly downregulated by proinflammatory cytokines. MEK-ERK pathway is a negative regulator of GDF-5 expression in FLS-RA. In FLS-RA, synergy between GDF-5 and TGF-β3 enhances chondrogenesis. Anti-inflammatory drugs combined with GDF-5 might be a new therapeutic treatment for RA.     

Lipopolysaccharide and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in human gingival fibroblasts

Both lipopolysaccharide (LPS) and interleukin (IL)-1β activate the MyD88-dependent signaling pathways to stimulate proinflammatory cytokine expression. However, it remains unknown how LPS and IL-1β interact with each other to coordinate the stimulation. In this study, we sought to investigate the interaction between LPS and IL-1β on MyD88-dependent signaling pathways in human gingival fibroblasts (HGFs). Results showed that LPS derived from Porphyromonas gingivalis (Pg LPS) and IL-1β cooperatively stimulated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling pathways, and subsequent expression of proinflammatory cytokine expression. Furthermore, our results showed that Pg LPS and IL-1β exerted a synergy on MyD88 expression and knockdown of MyD88 expression by small interfering RNA diminished the synergistic effect of Pg LPS and IL-1β on IL-6 expression, suggesting that upregulation of MyD88 is involved in the coordinated stimulation by Pg LPS and IL-1β of proinflammatory cytokine expression. Finally, our results showed that pharmacological inhibitors for MAPK and NFκB significantly reduced IL-6 secretion stimulated by Pg LPS and IL-1β, indicating that the MyD88-dependent MAPK and NFκB signaling pathways are essential for the upregulation of proinflammatory cytokine expression by Pg LPS and IL-1β. Taken together, this study showed that LPS and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs.     

β1–4-galactosyltransferase gene expression is regulated during entry into the cell cycle and during the cell cycle

Mammalian glycosyltransferases have been implicated in a wide variety of functions besidesN-linked glycosylation, including developmental processes. For this reason, we studied the effects of cell cycle and entry into the cell cycle on 1–4-galactosyltransferase gene expression. In this study we report that 1–4-galactosyltransferase (GalTase) gene expression is, indeed, regulated during the normal cell cycle, peaking during late G₁-, S, and early G₂ phase of the cell cycle. In addition, GalTase gene expression is regulated in a manner that resembles other early response genes such asjun andfos upon reentry into the cell cycle from quiescence. Finally, we show that the GalTase gene is differentially expressed during murine embryogenesis and in terminally differentiated adult tissues. It is most abundant in testis, followed by skeletal muscle and spleen. The reasons for this pattern of differential expression in adult tissues are unknown. These studies should provide important new information regarding GalTase gene expression, its regulation, and its potential link to other developmental functions.     

Amyloid-β oligomer synaptotoxicity is mimicked by oligomers of the model protein HypF-N

Protein misfolded oligomers are thought to be the primary pathogenic species in many protein deposition diseases. Oligomers by the amyloid-β peptide play a central role in Alzheimer's disease pathogenesis, being implicated in synaptic dysfunction. Here we show that the oligomers formed by a protein that has no link with human disease, namely the N-terminal domain of HypF from Escherichia coli (HypF-N), are also synaptotoxic. HypF-N oligomers were found to (i) colocalize with post-synaptic densities in primary rat hippocampal neurons; (ii) induce impairment of long-term potentiation in rat hippocampal slices; and (iii) impair spatial learning of rats in the Morris Water Maze test. By contrast, the native protein and control nontoxic oligomers had none of such effects. These results raise the importance of using HypF-N oligomers as a valid tool to investigate the pathogenesis of Alzheimer's disease, with advantages over other systems for their stability, reproducibility, and costs. The results also suggest that, in the context of a compromised protein homeostasis resulting from aggregation of the amyloid β peptide, a number of oligomeric species sharing common synaptotoxic activity can arise and cooperate in the pathogenesis of the disease.