SARM: a novel Toll-like receptor adaptor, is functionally conserved from arthropod to human

Sterile-alpha and Armadillo motif containing protein (SARM) was recently identified as the fifth member of the Toll-like receptor (TLR) adaptor family. Whilst the Caenorhabditis elegans SARM homologue, TIR-1, is crucial for efficient immune responses against bacterial infections, human SARM was demonstrated to function as a specific inhibitor of TRIF-dependent TLR signaling. The opposing role of SARM in C. elegans and human is intriguing, prompting us to seek clarification on the enigmatic function of SARM in an ancient species which relies solely on innate immunity for survival. Here, we report the discovery of a primitive but functional SARM (CrSARM) in the immune defense of a "living fossil", the horseshoe crab, Carcinoscorpius rotundicauda. CrSARM shares numerous signature motifs and displays significant homology with vertebrate and invertebrate SARM homologues. CrSARM downregulates TRIF-dependent TLR signaling suggesting the conservation of SARM function from horseshoe crab to human. During infection by Pseudomonas aeruginosa, CrSARM is rapidly upregulated within 3h and strongly repressed at 6h, coinciding with the timing of bacterial clearance, thus demonstrating its dynamic role in innate immunity. Furthermore, yeast-two-hybrid screening revealed several potential interaction partners of CrSARM implying the role of SARM in downregulating TLR signaling events. Altogether, our study shows that, although C. elegans SARM upregulates immune signaling, its disparate role as a suppressor of TLR signaling, specifically via TRIF and not MyD88, is well-conserved from horseshoe crab to human.     

Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation

Toll-like receptor (TLR) signals that initiate innate immune responses to pathogens must be tightly regulated to prevent excessive inflammatory damage to the host. The adaptor protein Mal is specifically involved in signaling via TLR2 and TLR4. We demonstrate here that after TLR2 and TLR4 stimulation Mal becomes phosphorylated by Bruton's tyrosine kinase (Btk) and then interacts with SOCS-1, which results in Mal polyubiquitination and subsequent degradation. Removal of SOCS-1 regulation potentiates Mal-dependent p65 phosphorylation and transactivation of NF-kappaB, leading to amplified inflammatory responses. These data identify a target of SOCS-1 that regulates TLR signaling via a mechanism distinct from an autocrine cytokine response. The transient activation of Mal and subsequent SOCS-1-mediated degradation is a rapid and selective means of limiting primary innate immune response.     

TLR接头蛋白研究进展

Toll样受体(TLR)是近年来备受关注的一类病原体识别受体,能选择性地识别侵入机体的病原微生物所携带的病原相关分子模式(PAMPs),继而激活信号级联放大,产生免疫应答.目前研究发现,TLR识别相应配体后,由一类包含TLR结构域的接头蛋白MYD88、MAL、TRIF、TRAM和SARM通过MYD88依赖途径或MYD8...     

The function of TRADD in signaling through tumor necrosis factor receptor 1 and TRIF-dependent Toll-like receptors

The physiological function of the adaptor protein TRADD remains unclear because of the unavailability of a TRADD-deficient animal model. By generating TRADD-deficient mice, we found here that TRADD serves an important function in tumor necrosis factor receptor 1 (TNFR1) signaling by orchestrating the formation of TNFR1 signaling complexes. TRADD was essential for TNFR1 signaling in mouse embryonic fibroblasts but was partially dispensable in macrophages; abundant expression of the adaptor RIP in macrophages may have allowed some transmission of TNFR1 signals in the absence of TRADD. Although morphologically normal, TRADD-deficient mice were resistant to toxicity induced by TNF, lipopolysaccharide and polyinosinic-polycytidylic acid. TRADD was also required for TRIF-dependent Toll-like receptor signaling in mouse embryonic fibroblasts but not macrophages. Our findings definitively establish the biological function of TRADD in TNF signaling.     

Enhanced Toll-like receptor responses in the absence of signaling adaptor DAP12

DAP12 is a signaling adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) that pairs with receptors on myeloid cells and natural killer cells. We examine here the responses of mice lacking DAP12 to stimulation through Toll-like receptors (TLRs). Unexpectedly, DAP12-deficient macrophages produced higher concentrations of inflammatory cytokines in response to a variety of pathogenic stimuli. Additionally, macrophages deficient in spleen tyrosine kinase (Syk), which signals downstream of DAP12, showed a phenotype identical to that of DAP12-deficient macrophages. DAP12-deficient mice were more susceptible to endotoxic shock and had enhanced resistance to infection by the intracellular bacterium Listeria monocytogenes. These data suggest that one or more DAP12-pairing receptors negatively regulate signaling through TLRs.     

TLR接头蛋白SARM研究进展

SARM是最后一个发现的TLR接头蛋白,也是唯一具有抑制作用的接头蛋白,SARM的功能在不同物种和不同研究体系中结果差异较大,本文综述了人和小鼠中SARM的基因定位、基因结构和表达情况,并综述了线虫、鲎、文昌鱼、斑马鱼、小鼠和人等不同物种中SARM功能研究的最新进展。     

TNF receptor-associated factor-1 (TRAF1) negatively regulates Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-mediated signaling

Toll-like receptor 3 (TLR3) plays an important role in antiviral responses through recognizing viral double-stranded RNA produced during viral infection and mediating induction of type I IFN. TRIF is a Toll/IL-1 receptor (TIR) domain-containing adaptor protein that is associated with TLR3 and critically involved in TLR3-mediated signaling. In yeast two-hybrid screens, we identified TNF receptor-associated factor (TRAF)1 as a TRIF-interacting protein. The TRAF-C domain of TRAF1 and the TIR domain of TRIF were responsible for their interaction. Overexpression of TRAF1 inhibited TRIF- and TLR3-mediated activation of NF-kappaB, IFN-stimulated response element and the IFN-beta promoter. Overexpression of TRIF caused caspase-dependent cleavage of TRAF1. The cleaved N-terminal but not C-terminal fragment of TRAF1 was responsible for inhibiting TRIF signaling. Mutation of the caspase cleavage site of TRAF1 or addition of the caspase inhibitor crmA inhibited TRAF1 cleavage and abolished the ability of TRAF1 to inhibit TRIF signaling, suggesting that TRIF-induced cleavage of TRAF1 is required for its inhibition of TRIF signaling. Our findings provide a novel mechanism for negative regulation of TRIF-mediated signaling.     

Toll-like receptor function and signaling

Mammals sense pathogen invasion through pattern-recognition receptors. A group of transmembrane proteins, Toll-like receptors (TLRs), play critical roles as pattern-recognition receptors. They are mainly expressed on antigen-presenting cells, such as macrophages or dendritic cells, and their signaling activates antigen-presenting cells to provoke innate immunity and to establish adaptive immunity. Each TLR has common effects, such as inflammatory cytokine induction or upregulation of costimulatory molecule expression, but also has its specific function, exemplified by type I IFN-inducing ability. These immunoadjuvant effects are not only critical in antimicrobial immunity but are also involved in manifestations of autoimmunity. Furthermore, some TLR agonists are now promising therapeutic tools for various immune disorders, including allergy. Therefore understanding molecular mechanisms on TLRs should be quite useful in the development of therapeutic maneuvers against allergy and autoimmune diseases.     

Inherited disorders of human Toll-like receptor signaling: immunological implications

Summary: In vitro nine of 10 known human Toll‐like receptors (TLRs) are engaged by well‐defined chemical agonists that mimic microbial compounds, raising the possibility that human TLRs play a critical role in protective immunity in vivo. We thus review here the recently described human primary immunodeficiencies caused by germline mutations in genes encoding molecules involved in cell signaling downstream from TLRs. Subjects with anhidrotic ectodermal dysplasia with immunodeficiency (EDA‐ID) carry either X‐linked recessive hypomorphic mutations in NEMO or autosomal dominant hypermorphic mutations in IKBA. Their cells show a broad defect in nuclear factor‐κB (NF‐κB) activation, with an impaired, but not abolished response to a large variety of stimuli including TLR agonists. EDA‐ID patients show developmental anomalies of skin appendages and a broad spectrum of infectious diseases. Patients with autosomal recessive amorphic mutations in IRAK4 present a purely immunological syndrome and more restricted defects, with specific impairment of the Toll and interleukin‐1 receptor (TIR)–interleukin‐1 receptor‐associated kinase (IRAK) signaling pathway. In these subjects, the NF‐κB‐ and mitogen‐activated protein kinase‐mediated induction of inflammatory cytokines in response to TIR agonists is impaired. The patients present a narrow range of pyogenic bacterial infections that become increasingly rare with age. Altogether, these data suggest that human TLRs play a critical role in host defense. However, they do not provide compelling evidence, as even the infectious phenotype of patients with mutations in IRAK4 may result from impaired signaling via receptors other than TLRs. Paradoxically, these experiments of nature raise the possibility that the entire set of human TLRs is largely redundant in protective immunity in vivo.     

Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells

Toll-like receptor 4 (TLR4) is the signal-transducing component of the LPS recognition complex and is essential for LPS-induced septic shock. Here we demonstrate that TLR1 has the capacity to abrogate TLR4 signaling. Human microvascular endothelial cells express TLR4 but not TLR1 and respond to LPS through TLR4. The ability of these cells to respond to LPS was lost, however, when they were transfected with TLR1. Inhibition was specific for TLR1 because TL5 failed to block TLR4 function. Moreover, TLR1 had no effect upon TNF-alpha signaling, indicating that TLR1 operated at a step upstream of the convergence between the two pathways. Inhibition of TLR4 signaling was mediated by the extracellular, but not cytoplasmic domain of TLR1. In addition, TLR1 physically associated with TLR4 in co-precipitation experiments. These findings suggest that TLR1 might restrain potentially dangerous innate response to LPS by binding to TLR4 and preventing the formation of active signaling complexes.     

Toll-like receptor 9 mediates CpG-DNA signaling

Among the bacterial products known to activate the innate immune '1system is bacterial DNA. This activity resides within the nonmethylated CpG motifs of the DNA and is recapitulated using appropriate synthetic CpG containing oligodeoxynucleotides (CpG-ODN). TLR9-deficient mice were shown to exhibit a nonresponsive phenotype-to-bacterial DNA and CpG-ODN. Here, we describe a model system to further characterize CpG-ODN and TLR9 interactions using ectopically expressed TLR9 in HEK293 cells. Expression of TLR9 confers cellular responsiveness to CpG-ODN but not to the other bacterial products. Previous studies identified species-specific CpG-containing sequences; here, we show that expression of murine TLR9 favors responses to CpG-ODN motifs specific to mouse cells, and expression of human TLR9 favors CpG-ODN known to preferentially activate human cells. Response patterns to various CpG-ODN motifs were parallel when cells containing an ectopically expressed TLR9 and endogenous receptor were compared. Here, we also show that TLR9 acts at the cell surface and engages an intracellular signaling pathway that includes MyD88, IRAK, and TRAF6.     

NOX enzymes and Toll-like receptor signaling

Invading microorganisms are recognized by the host innate immune system through pattern recognition receptors. Among these receptors, Toll-like receptors (TLRs) are able to sense the molecular signatures of microbial pathogens, protozoa, fungi, and virus and activate proinflammatory signaling cascades. In addition to their role in bacterial killing by phagocytes, reactive oxygen species generated by NADPH oxidase (NOX) homologues also play key roles in signaling and host defense in a variety of cell types. Recent studies have demonstrated a link between TLR activation and NOX homologues following microbial recognition highlighting their important role in the innate immune response and host defense.     

SPOP negatively regulates Toll-like receptor-induced inflammation by disrupting MyD88 self-association

Toll-like receptor (TLR) signaling pathways need to be tightly controlled to avoid excessive inflammation and unwanted damage to the host. Myeloid differentiation primary response gene 88 (MyD88) is a critical adaptor of TLR signaling. Here, we identified the speckle-type POZ protein (SPOP) as a MyD88-associated protein. SPOP was recruited to MyD88 following TLR4 activation. TLR4 activation also caused the translocation of SPOP from the nucleus to the cytoplasm. SPOP depletion promoted the aggregation of MyD88 and recruitment of the downstream signaling kinases IRAK4, IRAK1 and IRAK2. Consistently, overexpression of SPOP inhibited the TLR4-mediated activation of NF-κB and production of inflammatory cytokines, whereas SPOP depletion had the opposite effects. Furthermore, knockdown of SPOP increased MyD88 aggregation and inflammatory cytokine production upon TLR2, TLR7 and TLR9 activation. Our findings reveal a mechanism by which MyD88 is regulated and highlight a role for SPOP in limiting inflammatory responses.     

Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses

HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-gamma1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3tau (a phosphorylated serine-binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3tau. Our results indicate that HPK1 negatively regulates TCR signaling and T cell-mediated immune responses.     

Toll-like receptor 4-mediated signaling regulates IL-7-driven proliferation and differentiation of B-cell precursors

Lipopolysaccharide （LPS） is known to be a potent activator of mature B cells by signaling through Toll-like receptor 4 （TLR4）. Its impact on early B-cell development, however, is not well defined. When comparing to C3H/HeN mice, TLR4-mutant C3H/HeJ mice showed an increase in the number of pro-B and pre-B cells in the bone marrow. When cultured in the presence of IL-7, the proliferation of pro-B and large pre-B cells was significantly inhibited by LPS, possibly due to reduced IL-7 receptor-a （IL-7Ra） expression. Meanwhile, the generation of IgM＋/IgD＋ B cells was greatly enhanced in IL-7 cultures of pro-B and pre-B cells. Consistent with these results, treatment with LPS facilitated the progression of adoptively transferred B220＋IgM-IgD- precursors into IgD＋ cells. Overall, these data suggest that LPS has a profound influence on early B-cell development, which may contribute to the deregulated B-cell development under physiological and pathological conditions such as bacterial infections.     

The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity

The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro . Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca²⁺, although the difference was not observed without Ca²⁺. A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.