Molecular cloning, characterization and expression analysis of the tumor necrosis factor (TNF) superfamily gene, TNF receptor superfamily gene and lipopolysaccharide-induced TNF-α factor (LITAF) gene from Litopenaeus vannamei

In vertebrates, the tumor necrosis factor (TNF)-receptor (TNFR) system participates in diverse physiological and pathological events, such as inflammation and protective immune responses to microbial infections. There are few reports about the role of the invertebrate TNF-TNFR system in immune responses. Here, we isolated and characterized the TNF superfamily (LvTNFSF) gene, TNFR superfamily (LvTNFRSF) gene and lipopolysaccharide-induced TNF-α factor (LvLITAF) gene from Litopenaeus vannamei. LvTNFSF consists of 472 amino acids with a conserved C-terminal TNF domain and has 89.8% identity with the Marsupenaeus japonicus TNF superfamily gene. LvTNFRSF consists of 296 amino acids with a conserved TNFR domain and has 18.0% identity with Chlamys farreri TNFR, 14.6% identity with Drosophila melanogaster Wengen and 14.6% identity with Homo sapiens TNFR1. LvLITAF consists of 124 amino acids with the LITAF domain and shows 62.6% identity with D. melanogaster LITAF and 32.3% identity with H. sapiens LITAF. The promoter region of LvTNFSF was cloned and used to construct a luciferase reporter. In Drosophila S2 cells, the promoter of LvTNFSF can be activated by LvLITAF, L. vannamei NF-κB family proteins (LvRelish and LvDorsal) and LvSTAT. Unlike its mammalian counterparts, LvTNFRSF could not activate the NF-κB pathway in Drosophila S2 cells. Using real-time quantitative PCR, we obtained expression profiles of LvTNFSF, LvTNFRSF and LvLITAF in the gill, intestine and hepatopancreas of L. vannamei after challenge with Gram-negative Vibrio alginolyticus, Gram-positive Staphylococcus aureus, the fungus Candida albicans and white spot syndrome virus (WSSV). Taken together, our results reveal that LvTNFSF, LvTNFRSF and LvLITAF may be involved in shrimp immune responses to pathogenic infections.     

Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions

A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)-associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR-superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T-cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3-mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post-translational modifications involving ubiquitin that are important in various signaling pathways.     

Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development,function, and homeostasis of the immune system

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein–protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factor-β receptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs.     

TRAF6 knockdown promotes survival and inhibits inflammatory response to lipopolysaccharides in rat primary renal proximal tubule cells

Aim: TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). Methods: PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-α, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. Results: We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-α and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. Conclusion: Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells.     

TRAF4 promotes the growth and invasion of colon cancer through the Wnt/β-catenin pathway

The tumor necrosis factor receptor-associated factor 4 (TRAF4) has been linked to carcinogenesis. However, the role of TRAF4 in colon cancer is still unclear. Therefore, we investigated the role of TRAF4 in colon cancer and the underlying mechanism. In the present study, we found that TRAF4 was overexpressed in colon cancer tissues and cells, and small interfering RNA (siRNA)-mediated gene knockdown of TRAF4 significantly inhibited cell proliferation, invasion and tumorigenesis, both in vitro and in vivo, but induced apoptosis in colon cancer cells. Furthermore, siRNA-TRAF4 significantly inhibited the expression levels of β-catenin, cyclinD1, and c-myc proteins in colon cancer cells. Taken together, these results suggest that TRAF4 promoted colon cancer cell growth and invasion by potentiating the Wnt/β-catenin pathway, suggesting that TRAF4 may be a potential molecular target for colon cancer prevention and therapy.     

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4⁺ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.     

Minimal structure of IRAK-1 to induce degradation of TRAF6

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1–102), a serine/proline/threonine-rich ProST domain (amino acids 103–197), a central kinase domain with an activation loop (amino acids 198–522), and the C-terminal C1 and C2 domains (amino acids 523–712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585–591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants–including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.     

Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spätzle-like Toll ligands (LvSpz1-3) from Litopenaeus vannamei

Toll-like receptor-mediated NF-κB pathways are essential for inducing immune related-gene expression in the defense against bacterial, fungal and viral infections in insects and mammals. Although a Toll receptor (LvToll1) was cloned in Litopenaeus vannamei, relatively little is known about other types of Toll-like receptors and their endogenous cytokine-like ligand, Spätzle. Here, we report two novel Toll-like receptors (LvToll2 and LvToll3) and three Spätzle-like proteins (LvSpz1-3) from L. vannamei. LvToll2 has 1009 residues with an extracellular domain containing 18 leucine-rich repeats (LRRs) and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain of 139 residues. LvToll3 is 1244 residues long with an extracellular domain containing 23 LRRs and a cytoplasmic TIR domain of 138 residues. The Spätzle-like proteins LvSpz1, LvSpz2 and LvSpz3 are 237, 245 and 275 residues in length, respectively, and all of them have a putative C-terminal cystine-knot domain. In Drosophila Schneider 2 (S2) cells, LvToll1 and LvToll3 were localized to the membrane and cytoplasm, and LvToll2 was confined to the cytoplasm. In Drosophila S2 cells, LvToll2 could significantly activate the promoters of NF-κB-pathway-controlled antimicrobial peptide genes, whereas LvToll1 and LvToll3 had no effect on them. LvSpz1 exerted some degree of inhibition on the promoter activities of Drosophila Attacin A and L. vannamei Penaeidin4. LvSpz3 also inhibited the Drosophila Attacin A promoter, but LvSpz2 could only slightly activate it. LvToll1, LvToll2 and LvToll3 were constitutive expressed in various tissues, while LvSpz1, LvSpz2 and LvSpz3 exhibited tissue-specific expression in the epithelium, eyestalk, intestine, gill and muscle. In the gill, after Vibrio alginolyticus challenge, LvToll1 was upregulated, but LvToll2 and LvToll3 showed no obvious changes. LvSpz1 and LvSpz3 were also strongly induced by V. alginolyticus challenge, but LvSpz2 only showed a slight downregulation. In the gill, after white spot syndrome virus (WSSV) challenge, LvToll1, LvToll2, LvToll3, LvSpz1 and LvSpz3 were upregulated, but LvSpz2 showed no obvious change, except for a slight downregulation at 12 h post-injection of WSSV. These findings might be valuable in understanding the innate immune signal pathways of shrimp and enabling future studies on the host-pathogen interactions in V. alginolyticus and WSSV infections.     

Pellino protein from pacific white shrimp Litopenaeus vannamei positively regulates NF-κB activation

Pellino, named after its property that binds Pelle (the Drosophila melanogaster homolog of IRAK1), is a highly conserved E3 class ubiquitin ligase in both vertebrates and invertebrates. Pellino interacts with phosphorylated IRAK1, causing polyubiquitination of IRAK1, and plays a critical upstream role in the toll-like receptor (TLR) pathway. In this study, we firstly cloned and identified a crustacean Pellino from pacific white shrimp Litopenaeus vannamei (LvPellino). LvPellino contains a putative N-terminal forkhead-associated (FHA) domain and a C-terminal ring finger (RING) domain with a potential E3 ubiquitin-protein ligase activity, and shows a high similarity with D. melanogaster Pellino. LvPellino could interact with L. vannamei Pelle (LvPelle) and over-expression of LvPellino could increase the activity of LvDorsal (a L. vannamei homolog of NF-κB) on promoters containing NF-κB binding motifs and enhance the expression of arthropod antimicrobial peptides (AMPs). The LvPellino protein was located in the cytoplasm and nucleus and LvPellino mRNA was detected in all the tissues examined and could be up-regulated after lipopolysaccharides, white spot syndrome virus (WSSV), Vibrio parahaemolyticus, and Staphylococcus aureus challenges, suggesting a stimulation response of LvPellino to bacterial and immune stimulant challenges. Knockdown of LvPellino in vivo could significantly decrease the expression of AMPs and increase the mortality of shrimps caused by V. parahaemolyticus challenge. However, suppression of the LvPellino expression could not change the mortality caused by WSSV infection, and dual-luciferase reporter assays demonstrated that over-expression of LvPellino could enhance the promoters of WSSV genes wsv069 (ie1), wsv303, and wsv371, indicating a complex role of LvPellino in WSSV pathogenesis and shrimp antiviral mechanisms.     

Multiple roles of TRAF3 signaling in lymphocyte function

Members of the tumor necrosis factor receptor (TNFR) superfamily employ cytoplasmic adapter proteins called TNF-R associated factors (TRAF) to initiate and regulate signaling pathways. Although many of these receptors associate with TRAF3, it has been unclear how this TRAF functions in immune responses. New information appearing through the use of novel experimental models reveals that TRAF3 can mediate both activating and inhibitory signals, and can participate in regulation of multiple members of the TNFR superfamily. TRAF3 is also important for signaling via innate immune receptors, as well as an oncogenic mimic of a normal receptor that is implicated in promoting both malignancies and autoimmunity.     

Characterization of an immune deficiency homolog (IMD) in shrimp (Fenneropenaeus chinensis) and crayfish (Procambarus clarkii)

The immune deficiency (IMD) signal pathway mediates immunity against Gram-negative bacteria in Drosophila. Recent studies show that the IMD pathway also involves in antiviral innate immune responses. The functions of the pathway in crustacean immunity are largely unknown. In this paper, two IMDs (FcIMD and PcIMD), one of the key elements of the IMD pathway, were identified from Chinese white shrimp Fenneropenaeus chinensis and red swamp crayfish Procambarus clarkii. Both proteins have a death domain located at the C-terminal. FcIMD was mainly expressed in the gills and stomach and PcIMD was mainly detected in the heart, hepatopancreas, and stomach. FcIMD peaked in hemocytes at 12 h after white spot syndrome virus (WSSV) challenge and it peaked in the gills at 6 h after WSSV challenge, but it was decreased at 2 h and kept the low level to 24 h in hemocytes and no obviously change in gill after Vibrio anguillarum challenge. PcIMD first decreased in hemocytes at 2 h and peaked at 12 h in hemocytes after V. anguillarum challenge. It was also upregulated in gill after bacterial challenge, peaked at 2 h, and decreased at 6 h, and then gradually increased at 12–24 h. PcIMD has no significant change in hemocytes and gill after WSSV challenge. Western blot analysis detected FcIMD protein in all tissues, and immunocytochemical analysis localized FcIMD in the cytoplasm of hemocytes. RNA interference analysis showed that the IMD pathway was involved in regulating the expression of three kinds AMP genes, including crustins, anti-lipopolysaccharide factors and lysozymes, in shrimp and crayfish. They are Cru 1, Cru 2, ALF 1, ALF 2 and Lys 1 in crayfish, and Cru1, Cru 3, ALF 6, ALF 8, and Lys2 in shrimp. These results suggest that although IMD distribution and expression patterns have some differences, the IMD pathway may have conserved function for AMP regulation in shrimp and crayfish immunity against Gram-negative bacteria.     

Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei

Dicer (Dcr) is the key protein of the RNA interference (RNAi) pathway. To investigate the role of the RNAi pathway in shrimp anti-viral immunity, Litopenaeus vannamei Dcr2 (designated as LvDcr2) was identified and characterized. The full-length cDNA of LvDcr2 was 5513 bp long, with an open reading frame encoding a putative protein of 1502 amino acids. In addition, five proteins homologous to the single von Willebrand factor type C (VWC) domain protein (SVC) were also identified in L. vannamei and named LvSVC1-5. These LvSVCs were between 102 and 190 amino acids in length and all contained a motif similar to Drosophila melanogaster SVC proteins (DmSVCs). By co-immunoprecipitation assays and pull-down assays, we demonstrated that LvDcr2, L. vannamei Argonaute 2 (LvAgo2), and L. vannamei transactivating response RNA-binding protein isoform 1 (LvTRBP1) interacted with each other. A luciferase reporter assay indicated that the promoters of LvSVC1, LvSVC4, LvSVC5, and DmSVC Vago (DmVago) were activated by LvDcr2 as well as by Drosophila Dcr2 (DmDcr2). Real-time RT-PCR showed that LvDcr2 and LvSVCs were up-regulated in immune responses against Poly(C-G) or WSSV challenge. These results suggested that LvDcr2 formed complexes with LvAgo2 and LvTRBP1 to act as the cores of shrimp small interfering RNA (siRNA)-induced silencing complex (siRISC)/siRISC-loading complex (siRLC), role in shrimp siRNA pathway. Furthermore, these results also suggested that LvDcr2 may engage in non-specific activation of anti-viral immunity.