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PubMed2007

Immunopathogenesis in hepatitis C virus cirrhosis.

HCV (hepatitis C virus) has a high propensity to persist and to cause chronic hepatitis C, eventually leading to cirrhosis. Since HCV itself is not cytopathic, liver damage in chronic hepatitis C is commonly attributed to immune-mediated mechanisms. HCV proteins interact with several pathways in the host's immune response and disrupt pathogen-associated pattern recognition pathways, interfere with cellular immunoregulation via CD81 binding and subvert the activity of NK (natural killer) cells as well as CD4(+) and CD8(+) T-cells. Finally, HCV-specific T-cells become increasingly unresponsive and apparently disappear, owing to several possible mechanisms, such as escape mutations in critical viral epitopes, lack of sufficient help, clonal anergy or expansion of regulatory T-cells. The role of neutralizing antibodies remains uncertain, although it is still possible that humoral immunity contributes to bystander damage of virally coated cells via antibody-dependent cellular cytotoxicity. Cytotoxic lymphocytes kill HCV-infected cells via the perforin/granzyme pathway, but also release Fas ligand and inflammatory cytokines such as IFNgamma (interferon gamma). Release of soluble effector molecules helps to control HCV infection, but may also destroy uninfected liver cells and can attract further lymphocytes without HCV specificity to invade the liver. Bystander damage of these non-specific inflammatory cells will expand the tissue damage triggered by HCV infection and ultimately activate fibrogenesis. A clear understanding of these processes will eventually help to develop novel treatment strategies for HCV liver disease, independent from direct inhibition of HCV replication.

Antibodies, Viral↗
PubMed2006

The IFN-independent response to virus particle entry provides a first line of antiviral defense that is independent of TLRs and retinoic acid-inducible gene I.

The innate immune system responds to pathogen infection by eliciting a nonspecific immune response following the recognition of various pathogen-associated molecular patterns. TLRs and the RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 recognize foreign nucleic acid within endosomal and cytoplasmic compartments, respectively, initiating a signaling cascade that involves the induction of type I IFN through the transcription factors IFN regulatory factor (IRF) 3 and NF-kappaB. However, a recent paradigm has emerged in which bacterial DNA and double-stranded B-form DNA trigger type I IFN production through an uncharacterized TLR- and RIG-I-independent pathway. We have previously described a response in primary fibroblasts wherein the entry of diverse RNA- and DNA-enveloped virus particles is sufficient to induce a subset of IFN-stimulated genes and a complete antiviral response in an IRF3-dependent, IFN-independent manner. In this study, we show that the innate immune response to virus particle entry is independent of both TLR and RIG-I pathways, confirming the existence of novel innate immune mechanisms that result in the activation of IRF3. Furthermore, we propose a model of innate antiviral immunity in which exposure to increasing numbers of virus particles elevates the complexity of the cellular response from an intracellular, IFN-independent response to one involving secretion of cytokines and activation of infiltrating immune cells.

DEAD Box Protein 58↗
PubMed2004

Involvement of MEK1 MAPKK, NTF6 MAPK, WRKY/MYB transcription factors, COI1 and CTR1 in N-mediated resistance to tobacco mosaic virus.

The tobacco N gene, a member of the Toll-interleukin 1 homology region/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class of resistance (R) genes, confers resistance to tobacco mosaic virus (TMV). We used a candidate gene approach to identify known defense genes that were also involved in N signaling. The requirement for these genes was determined by downregulating their expression using the well-established tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS). Silencing of genes encoding a mitogen-activated protein kinase (MAPK) NTF6/NRK1, or an MAPK kinase (MAPKK) MEK1/NQK1, attenuated N-mediated resistance to TMV. We also found that N resistance is compromised in plants in which expression of WRKY1-WRKY3 and MYB1 transcription factors were downregulated. In addition, suppression of jasmonic acid (JA) signaling component COI1 ortholog affected N function. However, downregulation of expression of CTR1 ortholog leads to more rapid hypersensitive response (HR). The involvement of these genes in N- and other R-gene-mediated defense provides further evidence for the convergence of downstream signaling pathways of different R genes.

Gene Silencing↗
PubMed2004

Molecular mechanisms of endotoxin tolerance.

The phenomenon of endotoxin tolerance has been widely investigated, but to date, the molecular mechanisms of endotoxin tolerance remain to be resolved clearly. The discovery of the Toll-like receptor (TLR) family as the major receptors for lipopolysaccharide (LPS) and other bacterial products has prompted a resurgence of interest in endotoxin tolerance mechanisms. Changes of cell surface molecules, signaling proteins, pro-inflammatory and anti-inflammatory cytokines and other mediators have been examined. During tolerance expression of LPS-binding protein (LBP), CD14, myeloid differentiation protein-2 (MD-2) and TLR2 are unchanged or up-regulated, whereas TLR4 is transiently suppressed or unchanged. Proximal post-receptor signaling proteins that are altered in tolerance include augmented degradation of interleukin-1 receptor-associated kinase (IRAK), and decreased TLR4-myeloid differentiation factor 88 (MyD88) and IRAK-MyD88 association. Tolerance has also been shown to be associated with decreased Gi protein content and activity, decreased protein kinase C (PKC) activity, reduction in mitogen-activated protein kinase (MAP kinase) activity, and reduced activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB) induced gene transactivation. However, not all signaling proteins and pathways are suppressed in tolerance and induction of specific anti-inflammatory proteins and signaling pathways may serve important counter inflammatory functions. The latter include induction of IRAK-M and suppressor of cytokine-signaling-1 (SOCS-1), phosphoinositide-3-kinase (PI3K) signaling, and increased or maintained expression of inhibitor-kappaB (IkappaB) isoforms. Also at the nuclear level, increase in the NF-kappaB subunit p50 homodimer expression and increased activation of peroxisome-proliferator-activated receptors-gamma (PPARgamma) have been linked to tolerance phenotype. Although there are species and cellular variations in manifestation of the LPS tolerant phenotype, it is clear that the tolerance phenomena have evolved as a complex orchestrated counter regulatory response to inflammation.

Animals↗
PubMed2006

MyD88-dependent signaling affects the development of meningococcal sepsis by nonlipooligosaccharide ligands.

The Toll-like receptors (TLRs) and the adaptor myeloid differentiation factor 88 (MyD88) are important in the innate immune defenses of the host to microbial infections. Meningococcal ligands signaling via TLRs control inflammatory responses, and stimulation can result in fulminant meningococcal sepsis. In this study, we show that the responses to nonlipooligosaccharide (non-LOS) ligands of meningococci are MyD88 dependent. An isogenic LOS-deficient mutant of the serogroup C meningococcal strain FAM20 caused fatal disease in wild type C57BL/6 mice that was not observed in MyD88-/- mice. Fatality correlated with high proinflammatory cytokine and C5a levels in serum, high neutrophil numbers in blood, and increased bacteremia at 24 h postinfection in the wild-type mice. Infection with the parent strain FAM20 resulted in fatality in 100% of the wild-type mice and 50% of the MyD88-/- mice. We conclude that both LOS and another neisserial ligand cause meningococcal sepsis in an in vivo mouse model and confirm that meningococcal LOS can act via both the MyD88- dependent and -independent pathways, while the non-LOS meningococcal ligand(s) acts only via the MyD88-dependent pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2005

TAK1, but not TAB1 or TAB2, plays an essential role in multiple signaling pathways in vivo.

TGF-beta-activated kinase 1 (TAK1), a member of the MAPKKK family, is thought to be a key modulator of the inducible transcription factors NF-kappaB and AP-1 and, therefore, plays a crucial role in regulating the genes that mediate inflammation. Although in vitro biochemical studies have revealed the existence of a TAK1 complex, which includes TAK1 and the adapter proteins TAB1 and TAB2, it remains unclear which members of this complex are essential for signaling. To analyze the function of TAK1 in vivo, we have deleted the Tak1 gene in mice, with the resulting phenotype being early embryonic lethality. Using embryonic fibroblasts lacking TAK1, TAB1, or TAB2, we have found that TNFR1, IL-1R, TLR3, and TLR4-mediated NF-kappaB and AP-1 activation are severely impaired in Tak1(m/m) cells, but they are normal in Tab1(-/-) and Tab2(-/-) cells. In addition, Tak1(m/m) cells are highly sensitive to TNF-induced apoptosis. TAK1 mediates IKK activation in TNF-alpha and IL-1 signaling pathways, where it functions downstream of RIP1-TRAF2 and MyD88-IRAK1-TRAF6, respectively. However, TAK1 is not required for NF-kappaB activation through the alternative pathway following LT-beta signaling. In the TGF-beta signaling pathway, TAK1 deletion leads to impaired NF-kappaB and c-Jun N-terminal kinase (JNK) activation without impacting Smad2 activation or TGF-beta-induced gene expression. Therefore, our studies suggests that TAK1 acts as an upstream activating kinase for IKKbeta and JNK, but not IKKalpha, revealing an unexpectedly specific role of TAK1 in inflammatory signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Differential role for TLR3 in respiratory syncytial virus-induced chemokine expression.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young infants worldwide. Previous studies have reported that the induction of interleukin-8/CXCL8 and RANTES/CCL5 correlates with disease severity in humans. The production of these chemokines is elicited by viral replication and is NF-kappaB dependent. RSV, a negative-sense single-stranded RNA virus, requires full-length positive-sense RNA for synthesis of new viral RNA. The aim of our studies was to investigate whether active viral replication by RSV could evoke chemokine production through TLR3-mediated signaling pathways. In TLR3-transfected HEK 293 cells, live RSV preferentially activated chemokines in both a time- and dose-dependent manner compared to vector controls. RSV was also shown to upregulate TLR3 in human lung fibroblasts and epithelial cells (MRC-5 and A549). Targeting the expression of TLR3 with small interfering RNA decreased synthesis of IP-10/CXCL10 and CCL5 but did not significantly reduce levels of CXCL8. Blocking the expression of the adapter protein MyD88 established a role for MyD88 in CXCL8 production, whereas CCL5 synthesis was found to be MyD88 independent. Production of CCL5 by RSV was induced directly through TLR3 signaling pathways and did not require interferon (IFN) signaling through the IFN-alpha/beta receptor. TLR3 did not affect viral replication, since equivalent viral loads were recovered from RSV-infected cells despite altered TLR3 expression. Taken together, our studies indicate that TLR3 mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells.

Adaptor Proteins, Signal Transducing↗
PubMed2002

An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus.

In Arabidopsis, EDS1 is essential for disease resistance conferred by a structural subset of resistance (R) proteins containing a nucleotide-binding site, leucine-rich-repeats and amino-terminal similarity to animal Toll and Interleukin-1 (so-called TIR-NBS-LRR proteins). EDS1 is not required by NBS-LRR proteins that possess an amino-terminal coiled-coil motif (CC-NBS-LRR proteins). Using virus-induced gene silencing (VIGS) of a Nicotiana benthaminana EDS1 orthologue, we investigated the role of EDS1 in resistance specified by structurally distinct R genes in transgenic N. benthamiana. Resistance against tobacco mosaic virus mediated by tobacco N, a TIR-NBS-LRR protein, was EDS1-dependent. Two other R proteins, Pto (a protein kinase), and Rx (a CC-NBS-LRR protein) recognizing, respectively, a bacterial and viral pathogen did not require EDS1. These data, together with the finding that expression of N. benthamiana and Arabidopsis EDS1 mRNAs are similarly regulated, lead us to conclude that recruitment of EDS1 by TIR-NBS-LRR proteins is evolutionarily conserved between dicotyledenous plant species in resistance against bacterial, oomycete and viral pathogens. We further demonstrate that VIGS is a useful approach to dissect resistance signaling pathways in a genetically intractable plant species.

Amino Acid Sequence↗
PubMed2006

Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages.

Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IkappaB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.

Animals↗
PubMed2002

IRF3 mediates a TLR3/TLR4-specific antiviral gene program.

We have identified a subset of genes that is specifically induced by stimulation of TLR3 or TLR4 but not by TLR2 or TLR9. Further gene expression analyses established that upregulation of several primary response genes was dependent on NF-kappaB, commonly activated by several TLRs, and interferon regulatory factor 3 (IRF3), which was found to confer TLR3/TLR4 specificity. Also identified was a group of secondary response genes which are part of an autocrine/paracrine loop activated by the primary response gene product, interferon beta (IFNbeta). Selective activation of the TLR3/TLR4-IRF3 pathway potently inhibited viral replication. These results suggest that TLR3 and TLR4 have evolutionarily diverged from other TLRs to activate IRF3, which mediates a specific gene program responsible for innate antiviral responses.

3T3 Cells↗
PubMed2005

Specific inhibition of MyD88-independent signaling pathways of TLR3 and TLR4 by resveratrol: molecular targets are TBK1 and RIP1 in TRIF complex.

TLRs can activate two distinct branches of downstream signaling pathways. MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) pathways lead to the expression of proinflammatory cytokines and type I IFN genes, respectively. Numerous reports have demonstrated that resveratrol, a phytoalexin with anti-inflammatory effects, inhibits NF-kappaB activation and other downstream signaling pathways leading to the suppression of target gene expression. However, the direct targets of resveratrol have not been identified. In this study, we attempted to identify the molecular target for resveratrol in TLR-mediated signaling pathways. Resveratrol suppressed NF-kappaB activation and cyclooxygenase-2 expression in RAW264.7 cells following TLR3 and TLR4 stimulation, but not TLR2 or TLR9. Further, resveratrol inhibited NF-kappaB activation induced by TRIF, but not by MyD88. The activation of IFN regulatory factor 3 and the expression of IFN-beta induced by LPS, poly(I:C), or TRIF were also suppressed by resveratrol. The suppressive effect of resveratrol on LPS-induced NF-kappaB activation was abolished in TRIF-deficient mouse embryonic fibroblasts, whereas LPS-induced degradation of IkappaBalpha and expression of cyclooxygenase-2 and inducible NO synthase were still inhibited in MyD88-deficient macrophages. Furthermore, resveratrol inhibited the kinase activity of TANK-binding kinase 1 and the NF-kappaB activation induced by RIP1 in RAW264.7 cells. Together, these results demonstrate that resveratrol specifically inhibits TRIF signaling in the TLR3 and TLR4 pathway by targeting TANK-binding kinase 1 and RIP1 in TRIF complex. The results raise the possibility that certain dietary phytochemicals can modulate TLR-derived signaling and inflammatory target gene expression and can alter susceptibility to microbial infection and chronic inflammatory diseases.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Stimulation of Toll-like receptor 4 by lipopolysaccharide during cellular invasion by live Salmonella typhimurium is a critical but not exclusive event leading to macrophage responses.

Invasion of macrophages by salmonellae induces cellular responses, with the bacterial inducers likely to include a number of pathogen-associated molecular patterns. LPS is one of the prime candidates, but its precise role in the process, especially when presented as a component of live infecting bacteria, is unclear. We thus investigated this question using the lipid A antagonist E5531, the macrophage-like cell line RAW 264.7, and primary macrophage cultures from C3H/HeJ and Toll-like receptor 4(-/-) (TLR-4(-/-)) mice. We show that LPS presented on live salmonellae provides an essential signal, via functional TLR-4, for macrophages to produce NO and TNF-alpha. Furthermore, the mitogen-activated protein kinase c-Jun N-terminal kinase and p38 are activated, and the transcription factor NF-kappa B is translocated to the nucleus when RAW 264.7 cells are presented with purified LPS or live salmonellae. Purified LPS stimulates rapid, transitory mitogen-activated protein kinase activation that is inhibited by E5531, whereas bacterial invasion stimulates delayed, prolonged activation, unaffected by E5531. Both purified LPS and bacterial invasion caused translocation of NF-kappa B, but whereas E5531 always inhibited activation by purified LPS, activation by bacterial invasion was only inhibited at later time points. In conclusion, we show for the first time that production of NO and TNF-alpha is critically dependent on activation of TLR-4 by LPS during invasion of macrophages by salmonellae, but that different patterns of activation of intracellular signaling pathways are induced by purified LPS vs live salmonellae.

Animals↗
PubMed2004

Structural analysis of CYP2C9 and CYP2C5 and an evaluation of commonly used molecular modeling techniques.

This work had two separate aims: to evaluate different modeling techniques and to make a detailed structural characterization of CYP2C9. To achieve these goals, the consensus principal component analysis (CPCA) technique and distance measurements were used to explore available crystal structures, newly built homology models, and repeated molecular dynamics simulations. The CPCA was based on molecular interaction fields focused on the active site regions of the proteins and include detailed amino acid analysis. The comparison of the CYP2C9 and CYP2C5 crystal structures revealed differences in the flexible regions such as the B-C and F-G loop and the N and C termini. Cross homology models of CYP2C9 and CYP2C5, using their respective crystal structures as templates, indicated that such models were more similar to their templates than to their target proteins. Inclusion of multiple templates slightly improved the similarity to the crystal target in some cases and could be recommended even though it requires a careful manual alignment process. The application of molecular dynamics simulations to highly flexible proteins such as cytochromes P450 is also explored and the information is extracted by the CPCA. Advantages and drawbacks are presented for the different modeling techniques. Despite the varying modeling success, the models give insight and understanding by the mutual forming and discarding of hypotheses. This is a dynamic process since the crystal structures are improving with time and, therefore, the answers to the models are also changing accordingly.

Aryl Hydrocarbon Hydroxylases↗
PubMed2005

Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimulated macrophages via inhibition of Egr-1 and a serum response element/MEK-ERK1/2 pathway.

Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5'-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.

Animals↗
PubMed2025

Trichomonas vaginalis extracellular vesicles activate the NLRP3 inflammasome and TLR3-mediated inflammatory cascades in host cells.

Trichomonas vaginalis (TV) is a flagellated parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection (STI), with over 275 million cases annually. TV has been shown to secrete extracellular vesicles (TV-EVs) to regulate intercellular communication between parasites and host immune response; however, the mechanisms by innate immunity against TV-EVs are largely unknown. Herein, we aim to investigate the molecular mechanisms of inflammation induced by TV-EVs and identify novel proteins modulating the immune response in host cells. Firstly, the morphological characteristics of TV-EVs have been analyzed by transmission electron microscope (TEM) and nanoparticle tracking analysis, revealing that the vesicles are round-shaped bilayer membrane structures with size mostly about 100-120 nm. Additionally, the internalization of TV-EVs by host cells has been validated through immunofluorescence and TEM analysis. The multiplex immunoassay identified that TV-EVs induce the secretion of inflammatory cytokines, including CXCL1, IL-6, IL-8 and MIP-1β in THP-1 macrophages and ectocervical cells (Ect). Mechanistically, TV-EVs induce TLR3 overexpression to activate the NF-κB/NLRP3 pathway in THP-1 macrophages. Additionally, TV-EVs activate the PI3K-mediated NF-κB, p38 MAPK and ERK pathways in Ect. Moreover, TV-EV-induced TLR3 overexpression positively regulates the PI3K and NF-κB pathways, while simultaneously suppressing the p38 MAPK and ERK pathways in Ect. Proteomic analysis identified that TV-EVs upregulate MICB and TRAF3IP2, which are also positively regulated by TLR3 and involved in TV-EV-induced inflammatory cascade. Altogether, this study significantly advances our understanding of the immunomodulatory roles of TV-EVs in host cells, paving the way for future treatment of trichomoniasis and TV-associated STIs.

Humans↗
PubMed2004

CpG oligodeoxynucleotides stimulate IFN-gamma-inducible protein-10 production in human B cells.

Several classes of CpG oligodeoxynucleotides (ODNs) with different immune stimulatory profiles were recently identified: the A-, B- and C-classes. In this study, we investigated the CpG-dependent stimulation of IFN-gamma-inducible protein 10 (IP-10 or CXCL10) in different human immune cell types. CpG ODNs induced IP-10 in monocytes, pDCs and in B cells. Purified B cells as well as RPMI 8226 cells responded to CpG stimulation by IP-10 production. Treatment with exogenous IFN-alpha2b sensitized PBMCs, purified B cells as well as RPMI 8226 cells to respond more efficiently to stimulation with CpG ODNs by IP-10 production. IP-10 signaling could be directly stimulated via TLR9 in CpG-unresponsive HEK293 cells transfected with human TLR9 and an IP-10 reporter construct. Therefore, CpG-mediated IP-10 production is stimulated through IFN-alpha in cells that express the IFN-alpha receptor, a second pathway for IP-10 induction exists in TLR9-expressing B cells and pDCs where IP-10 is stimulated directly upon CpG-mediated TLR9 signaling. Our data provide a better understanding of the mechanisms through which CpG ODNs induce efficient Th1 responses.

Adjuvants, Immunologic↗
PubMed2000

Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes.

The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well.

Amino Acid Sequence↗
PubMed2004

Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice.

The Mycobacterium bovis bacillus Calmette-Guérin cell-wall skeleton (BCG-CWS) activates Toll-like receptor (TLR) 2 and TLR4, but unlike the typical TLR4 agonist bacterial lipopolysaccharide barely induces type 1 IFN. BCG-CWS has been used for adjuvant immunotherapy for patients with cancer. We investigated the adjuvant potential of BCG-CWS for induction of CTLs subsequent to TLR-mediated dendritic cell (DC) maturation, using a syngeneic mouse tumor model (B16 melanoma in C57BL/6). We evaluated the retardation of tumor growth and cytotoxic response in wild-type and MyD88-/- mice immunized with tumor debris and/or BCG-CWS. Delays in tumor growth and cytotoxic response were induced by immunization with a mixture of BCG-CWS emulsion and the tumor. BCG-CWS was capable of activating DCs ex vivo by the criteria of CD80/CD86 up-regulation and cytokine (interleukin-12, tumor necrosis factor-alpha) induction. Efficient tumor suppression and ex vivo cytokine induction did not occur in MyD88-deficient mice and cells, suggesting that the MyD88 adapter is crucial for induction of tumor cytotoxicity. Because TLR4 is involved in both MyD88-dependent and -independent pathways and the latter affects DC maturation, our findings indicate that both pathways cooperate to induce CTL-based tumor immunity.

Adaptor Proteins, Signal Transducing↗