TLR4信号转导通路在肿瘤中的作用

慢性炎症与恶性肿瘤密切相关,Toll样受体4（TLR4）在肿瘤中的广泛表达提示其在慢性炎症致瘤机制中发挥重要作用。活化肿瘤细胞TLR4不仅促进肿瘤的生成和转移,而且参与肿瘤的免疫逃逸。另一方面,免疫佐剂又通过激活免疫细胞的TLR4信号产生抗肿瘤免疫。因此,TLR4在肿瘤中起着双刃剑的作用。     

TLR9与先天性免疫反应

TLR9（Toll-likereceptor9）是一种微生物病原相关分子结构模式识别受体,TLR9能够识别CpG—ODN（胞嘧啶磷酸鸟甘-寡聚脱氧核苷酸）,使病原相关受体在先天性免疫细胞上表达,并激活下游炎性通路。研究表明,TLR9在先天性免疫反应中产生了重要作用,如脓毒血症、自身免疫性疾病、刀豆体球蛋白A介导肝炎性肝脏损伤、炎性泡沫细胞形成、缺血再灌注损伤等,并且与多种致病因子相关联,如肝x受体、甲酰多肽受体、线粒体DNA等。     

Identification of Toll-Like Receptor 9 as Parapoxvirus Ovis-Sensing Receptor in Plasmacytoid Dendritic Cells

Parapoxvirus ovis (PPVO) is known for its immunostimulatory capacities and has been successfully used to generate vector vaccines effective especially in non-permissive host species. Murine conventional and plasmacytoid dendritic cells (cDC and pDC) are able to recognize PPVO. The PPVO-sensing receptor on pDC is hitherto unknown. In this study we aimed to define the pattern recognition receptor responsible for the activation of murine pDC by inactivated and replication-competent PPVO. We show that PPVO-induced expression of type I and type III interferons, pro-inflammatory cytokines, and co-stimulatory CD86 by bone marrow-derived pDC but not cDC is blocked by chloroquine, an inhibitor of endosomal maturation. The activation of pDC is independent of viral replication and depends mainly on TLR9. Moreover, the use of phosphatidylinositol 3-kinase inhibitor wortmannin or C-Jun-N-terminal kinase inhibitor SP600125 results in significant reduction of PPVO-induced pDC activation. Taken together, our data identify endosomal TLR9 as PPVO-sensing receptor in pDC.     

Impaired Innate COPD Alveolar Macrophage Responses and Toll-Like Receptor-9 Polymorphisms

Background

Dysfunctional innate responses of alveolar macrophages to nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae contribute to morbidity in chronic obstructive pulmonary disease (COPD). Our earlier studies discovered impaired COPD alveolar macrophage responses to Toll-like receptor (TLR) ligands of nontypeable H. influenzae and provide rationale for further evaluation of TLR signaling. While the role of TLR single nucleotide polymorphisms is increasingly recognized in inflammatory diseases, TLR single nucleotide polymorphisms in COPD have only recently been explored. We hypothesized that specific TLR polymorphisms are associated with dysfunctional innate immune COPD alveolar macrophage responses and investigated polymorphisms of TLR2(Arg753Gln), TLR4(Thr399Ile; Asp299Gly), and TLR9(T1486C; T1237C).

Methods

DNA was purified from cells of 1) healthy nonsmokers (n = 20); 2) COPD ex-smokers (n = 83); 3) COPD active smokers (n = 93). DNA amplifications (polymerase chain reaction) were performed for each SNP. Alveolar macrophages from each group were incubated with nontypeable H. influenzae, M. catarrhalis and S. pneumoniae. Cytokine induction of macrophage supernatants was measured and the association with TLR single nucleotide polymorphism expression was determined.

Results

No significant inter-group differences in frequency of any TLR SNP existed. However both TLR9 single nucleotide polymorphisms were expressed in high frequency. Among COPD ex-smokers, diminished IL-8 responsiveness to nontypeable H. influenzae, M. catarrhalis and S. pneumoniae was strongly associated with carriage of TLR9(T1237C) (p = 0.02; p = 0.008; p = 0.02), but not TLR9(T1486C). Carriage of TLR9(T1237C), but not TLR9(T1486C), correlated with diminished FEV₁%predicted (p = 0.037).

Conclusion

Our results demonstrate a notable association of TLR9(T1237C) expression with dysfunctional innate alveolar macrophage responses to respiratory pathogens and with severity of COPD.     

Role of Toll-Like Receptor 4 in Colorectal Carcinogenesis: A Meta-Analysis

Objective

This meta-analysis was performed to evaluate the role of toll-like receptor 4 (TLR-4) in colorectal carcinogenesis.

Methods

The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library, and CBM databases were searched from inception through November 1st, 2013 without language restrictions. Odds ratios (ORs) or standardized mean differences (SMD) with their 95% confidence intervals (CI) were calculated.

Results

Fourteen case-control studies met the inclusion criteria for this meta-analysis. A total of 1,209 colorectal cancer (CRC) cases and 1,218 healthy controls were involved in this meta-analysis. Two common polymorphisms (299 A>G and 399 C>T) in the TLR-4 gene, TLR-4 mRNA and protein expression were assessed. Our meta-analysis results revealed that the TLR-4 399 C>T polymorphism might increase the risk of CRC (allele model: OR = 1.77, 95%CI = 1.32∼2.36, P<0.001; dominant model: OR = 1.83, 95%CI = 1.32∼2.52, P<0.001; respectively). However, we found no correlation between the TLR-4 299 A>G polymorphism and CRC risk (all P>0.05). A subgroup analysis by ethnicity suggested that TLR-4 genetic polymorphisms were associated with an increased risk of CRC among Asians (allele model: OR = 1.50, 95%CI = 1.19∼1.88, P = 0.001; dominant model: OR = 1.49, 95%CI = 1.16∼1.92, P = 0.002; respectively), but not among Caucasians and Africans (all P>0.05). Furthermore, our results showed that TLR-4 mRNA and protein levels in CRC patients were higher than those in healthy controls (TLR-4 mRNA: SMD  = 2.51, 95%CI  = 0.98∼4.05, P = 0.001; TLR-4 protein: OR  = 4.75, 95%CI  = 1.16∼19.36, P = 0.030; respectively).

Conclusion

Our findings provide empirical evidence that TLR-4 may play an important role in colorectal carcinogenesis. Thus, TLR-4 is a promising potential biomarker for the early diagnosis of CRC.     

Toll-Like Receptor 9 in Plasmacytoid Dendritic Cells Fails To Detect Parvoviruses

Toll-like receptor 9 (TLR9) recognizes genomes of double-stranded DNA (dsDNA) viruses in the endosome to stimulate plasmacytoid dendritic cells (pDCs). However, how and if viruses with single-stranded DNA (ssDNA) genomes are detected by pDCs remain unclear. Here we have shown that despite the ability of purified genomic DNA to stimulate TLR9 and despite the ability to enter TLR9 endosomes, ssDNA viruses of the Parvoviridae family failed to elicit an interferon (IFN) response in pDCs.     

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.     

The T Antigen Locus of Merkel Cell Polyomavirus Downregulates Human Toll-Like Receptor 9 Expression

Establishment of a chronic infection is a key event in virus-mediated carcinogenesis. Several cancer-associated, double-stranded DNA (dsDNA) viruses act via their oncoproteins to downregulate Toll-like receptor 9 (TLR9), a key receptor in the host innate immune response that senses viral or bacterial dsDNA. A novel oncogenic virus, Merkel cell polyomavirus (MCPyV), has been recently identified that causes up to 80% of Merkel cell carcinomas (MCCs). However, it is not yet known whether this oncogenic virus also disrupts immune-related pathways. We find that MCPyV large T antigen (LT) expression downregulates TLR9 expression in epithelial and MCC-derived cells. Accordingly, silencing of LT expression results in upregulation of mRNA TLR9 levels. In addition, small T antigen (sT) also appears to inhibit TLR9 expression, since inhibition of its expression also resulted in an increase of TLR9 mRNA levels. LT inhibits TLR9 expression by decreasing the mRNA levels of the C/EBPβ transactivator, a positive regulator of the TLR9 promoter. Chromatin immunoprecipitation reveals that C/EBPβ binding at a C/EBPβ response element (RE) in the TLR9 promoter is strongly inhibited by expression of MCPyV early genes and that mutation of the C/EBP RE prevents MCPyV downregulation of TLR9. A survey of BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI polyomavirus (KIPyV), MCPyV, simian virus 40 (SV40), and WU polyomavirus (WUPyV) early genes revealed that only BKPyV and MCPyV are potent inhibitors of TLR9 gene expression. MCPyV LT targeting of C/EBP transactivators is likely to play an important role in viral persistence and potentially inhibit host cell immune responses during MCPyV tumorigenesis.     

Toll-Like Receptor 9-Activation during Onset of Myocardial Ischemia Does Not Influence Infarct Extension

Aim

Myocardial infarction (MI) remains a major cause of death and disability worldwide, despite available reperfusion therapies. Inflammatory signaling is considered nodal in defining final infarct size. Activation of the innate immune receptor toll-like receptors (TLR) 9 prior to ischemia and reperfusion (I/R) reduces infarct size, but the consequence of TLR9 activation timed to the onset of ischemia is not known.

Methods and Results

The TLR9-agonist; CpG B was injected i.p. in C57BL/6 mice immediately after induction of ischemia (30 minutes). Final infarct size, as well as area-at-risk, was measured after 24 hours of reperfusion. CpG B injection resulted in a significant increase in circulating granulocytes and monocytes both in sham and I/R mice. Paradoxically, clear evidence of reduced cardiac infiltration of both monocytes and granulocytes could be demonstrated in I/R mice treated with CpG B (immunocytochemistry, myeloperoxidase activity and mRNA expression patterns). In addition, systemic TLR9 activation elicited significant alterations of cardiac inflammatory genes. Despite these biochemical and cellular changes, there was no difference in infarct size between vehicle and CpG B treated I/R mice.

Conclusion

Systemic TLR9-stimulation upon onset of ischemia and subsequent reperfusion does not alter final infarct size despite causing clear alterations of both systemic and cardiac inflammatory parameters. Our results question the clinical usefulness of TLR9 activation during cardiac I/R.     

House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4

House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.     

The Toll-Like Receptor Agonist Imiquimod Is Active against Prions

Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI ⁺] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.     

Stress-Induced Sphingolipid Signaling: Role of Type-2 Neutral Sphingomyelinase in Murine Cell Apoptosis and Proliferation

Background

Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg²⁺-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations.

Methodology and Principal Findings

For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation.

Conclusion and Significance

nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.     

Administration of a Toll-Like Receptor 9 Agonist Decreases the Proviral Reservoir in Virologically Suppressed HIV-Infected Patients

Toll-like receptor (TLR) agonists can reactivate HIV from latently infected cells in vitro. We aimed to investigate the TLR-9 agonist, CPG 7909''s in vivo effect on the proviral HIV reservoir and HIV-specific immunity. This was a post-hoc analysis of a double-blind randomized controlled vaccine trial. HIV-infected adults were randomized 1∶1 to receive pneumococcal vaccines with or without 1 mg CPG 7909 as adjuvant at 0, 3 and 9 months. In patients on suppressive antiretroviral therapy we quantified proviral DNA at 0, 3, 4, 9, and 10 months (31 subjects in the CPG group and 37 in the placebo-adjuvant group). Furthermore, we measured HIV-specific antibodies, characterized T cell phenotypes and HIV-specific T cell immunity. We observed a mean reduction in proviral DNA in the CPG group of 12.6% (95% CI: −23.6–0.0) following each immunization whereas proviral DNA in the placebo-adjuvant group remained largely unchanged (6.7% increase; 95% CI: −4.2–19.0 after each immunization, p = 0.02). Among participants with additional cryo-preserved PBMCs, HIV-specific CD8+ T cell immunity as indicated by increased expression of degranulation marker CD107a and macrophage inflammatory protein 1β (MIP1β) tended to be up-regulated following immunization with CPG 7909 compared with placebo as adjuvant. Further, increasing proportion of HIV-specific CD107a and MIP1β-expressing CD8+ T cells were strongly correlated with decreasing proviral load. No changes were observed in T cell phenotype distribution, HIV-specific CD4+ T cell immunity, or HIV-specific antibodies. TLR9-adjuvanted pneumococcal vaccination decreased proviral load. Reductions in proviral load correlated with increasing levels of HIV specific CD8+ T cells. Further investigation into the potential effect of TLR9 agonists on HIV latency is warranted.     

Macrophage Receptors for Influenza A Virus: Role of the Macrophage Galactose-Type Lectin and Mannose Receptor in Viral Entry

Although sialic acid has long been recognized as the primary receptor determinant for attachment of influenza virus to host cells, the specific receptor molecules that mediate viral entry are not known for any cell type. For the infection of murine macrophages by influenza virus, our earlier study indicated involvement of a C-type lectin, the macrophage mannose receptor (MMR), in this process. Here, we have used direct binding techniques to confirm and characterize the interaction of influenza virus with the MMR and to seek additional macrophage surface molecules that may have potential as receptors for viral entry. We identified the macrophage galactose-type lectin (MGL) as a second macrophage membrane C-type lectin that binds influenza virus and is known to be endocytic. Binding of influenza virus to MMR and MGL occurred independently of sialic acid through Ca²⁺-dependent recognition of viral glycans by the carbohydrate recognition domains of the two lectins; influenza virus also bound to the sialic acid on the MMR. Multivalent ligands of the MMR and MGL inhibited influenza virus infection of macrophages in a manner that correlated with expression of these receptors on different macrophage populations. Influenza virus strain A/PR/8/34, which is poorly glycosylated and infects macrophages poorly, was not recognized by the C-type lectin activity of either the MMR or the MGL. We conclude that lectin-mediated interactions of influenza virus with the MMR or the MGL are required for the endocytic uptake of the virus into macrophages, and these lectins can thus be considered secondary or coreceptors with sialic acid for infection of this cell type.Infection of host cells by influenza virus is initiated by attachment of virus to sialic acid residues on the host cell surface through the receptor-binding site at the distal tip of the viral hemagglutinin (HA) (43). After attachment, the virus is internalized by endocytosis, and acidification of the endosome triggers a conformational change in viral HA that results in fusion of the viral envelope and host cell membrane (34). At the cell surface, sialic acid residues are commonly found at the termini of oligosaccharide chains that are attached in O or N linkage to cell surface proteins; they are also an essential component of acidic glycosphingolipids (gangliosides) that are present in all mammalian cell membranes. Although the abundance of sialic acid on mammalian cells provides influenza virus with multiple potential receptors, virus attachment does not always lead to virus entry (5, 8, 46). Furthermore, sialic acid-independent infection of Madin-Darby canine kidney (MDCK) cells by influenza virus has been reported (35). The specific host cell molecules that serve as functional receptors (or coreceptors) for the infectious entry of influenza virus have yet to be defined.We have studied the infectious entry of influenza virus into macrophages (Mφ), which represents an early event in recognition of the virus by the innate immune system (23, 44). After intranasal infection of mice, influenza virus replicates productively in cells of the respiratory epithelium. Mφ are also infected and viral proteins are produced, but replication is abortive and no live progeny are released (32); infection of Mφ is thus a dead-end for the virus leading to a reduction in viral load. In addition, influenza virus infection of Mφ stimulates production and release of proinflammatory cytokines and alpha/beta interferon (28), which may assist in further limiting viral replication and spread within the respiratory tract. Depletion of airway Mφ from mice prior to intranasal influenza virus infection leads to increased virus titers in the lung, attesting to the important role of Mφ in early host defense against the virus (38, 44).We observed in a previous study (30) that influenza A virus strains differed in their ability to infect murine Mφ, strains carrying a more highly glycosylated hemagglutinin (HA) molecule being more efficient at infecting Mφ than less glycosylated strains, although binding of viruses to the Mφ cell surface was equivalent. Our investigation of this phenomenon indicated involvement of the Mφ mannose receptor MMR (CD206), a C-type lectin, in infectious viral entry (29, 30). The involvement of other receptors was not excluded, and our subsequent observation that influenza virus can infect the RAW 264.7 Mφ cell line, which does not express the MMR, indeed points to the existence of other routes of infectious entry of the virus into Mφ.In the present study we used direct binding methods to confirm and characterize the interaction of influenza virus with the MMR and to seek additional Mφ surface molecules that may have potential as receptors for viral entry. We identify the Mφ galactose-type lectin (MGL) as a second Mφ membrane C-type lectin that binds influenza virus and investigate its involvement in the infectious process.     

Toll-Like Receptor (TLR2 and TLR4) Polymorphisms and Chronic Obstructive Pulmonary Disease

Toll-like receptors (TLRs) participate in the defence against bacterial infections that are common in patients with Chronic Obstructive Pulmonary Disease (COPD). We studied all tagging SNPs in TLR2 and TLR4 and their associations with the level and change over time of both FEV(1) and sputum inflammatory cells in moderate-to-severe COPD. Nine TLR2 SNPs and 17 TLR4 SNPs were genotyped in 110 COPD patients. Associations of SNPs with lung function and inflammatory cells in induced sputum were analyzed cross-sectionally with linear regression and longitudinally with linear mixed-effect models. Two SNPs in TLR2 (rs1898830 and rs11938228) were associated with a lower level of FEV(1) and accelerated decline of FEV(1) and higher numbers of sputum inflammatory cells. None of the TLR4 SNPs was associated with FEV(1) level. Eleven out of 17 SNPs were associated with FEV(1) decline, including rs12377632 and rs10759931, which were additionally associated with higher numbers of sputum inflammatory cells at baseline and with increase over time. This is the first longitudinal study showing that tagging SNPs in TLR2 and TLR4 are associated with the level and decline of lung function as well as with inflammatory cell numbers in induced sputum in COPD patients, suggesting a role in the severity and progression of COPD.     

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

Background

Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number {"type":"entrez-protein","attrs":{"text":"Q8BGW0","term_id":"81896053","term_text":"Q8BGW0"}}Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 ({"type":"entrez-protein","attrs":{"text":"Q91YX0","term_id":"73920022","term_text":"Q91YX0"}}Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.

Methodology/Principal Findings

Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase ({"type":"entrez-protein","attrs":{"text":"P25911","term_id":"2507208","term_text":"P25911"}}P25911), the Rho guanine nucleotide exchange factor, Vav ({"type":"entrez-protein","attrs":{"text":"P27870","term_id":"137483","term_text":"P27870"}}P27870), and the adaptor protein Grb2 ({"type":"entrez-protein","attrs":{"text":"Q60631","term_id":"2498425","term_text":"Q60631"}}Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.

Conclusions/Significance

We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.     

Conservation of Toll-Like Receptor Signaling Pathways in Teleost Fish

In mammals, toll-like receptors (TLR) recognize ligands, including pathogen-associated molecular patterns (PAMPs), and respond with ligand-specific induction of genes. In this study, we establish evolutionary conservation in teleost fish of key components of the TLR-signaling pathway that act as switches for differential gene induction, including MYD88, TIRAP, TRIF, TRAF6, IRF3, and IRF7. We further explore this conservation with a molecular phylogenetic analysis of MYD88. To the extent that current genomic analysis can establish, each vertebrate has one ortholog to each of these genes. For molecular tree construction and phylogeny inference, we demonstrate a methodology for including genes with only partial primary sequences without disrupting the topology provided by the high-confidence full-length sequences. Conservation of the TLR-signaling molecules suggests that the basic program of gene regulation by the TLR-signaling pathway is conserved across vertebrates. To test this hypothesis, leukocytes from a model fish, rainbow trout (Oncorhynchus mykiss), were stimulated with known mammalian TLR agonists including: diacylated and triacylated forms of lipoprotein, flagellin, two forms of LPS, synthetic double-stranded RNA, and two imidazoquinoline compounds (loxoribine and R848). Trout leukocytes responded in vitro to a number of these agonists with distinct patterns of cytokine expression that correspond to mammalian responses. Our results support the key prediction from our phylogenetic analyses that strong selective pressure of pathogenic microbes has preserved both TLR recognition and signaling functions during vertebrate evolution.     

Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

The early inflammatory response to influenza virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which neutrophils gain entry to the respiratory tract and their role during pathogenesis remain unclear. Here, we report that neutrophils significantly contributed to morbidity in a pathological mouse model of influenza virus infection. Using extensive immunohistochemistry, bone marrow transfers, and depletion studies, we identified neutrophils as the predominant pulmonary cellular source of the gelatinase matrix metalloprotease (MMP) 9, which is capable of digesting the extracellular matrix. Furthermore, infection of MMP9-deficient mice showed that MMP9 was functionally required for neutrophil migration and control of viral replication in the respiratory tract. Although MMP9 release was toll-like receptor (TLR) signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil migration. These results were extended using multiplex analyses of inflammatory mediators to show that neutrophil chemotactic factor, CCL3, and TNFα were reduced in the Myd88 ^−/− airways. Furthermore, TNFα induced MMP9 secretion by neutrophils and blocking TNFα in vivo reduced neutrophil recruitment after infection. Innate recognition of influenza virus therefore provides the mechanisms to induce recruitment of neutrophils through chemokines and to enable their motility within the tissue via MMP9-mediated cleavage of the basement membrane. Our results demonstrate a previously unknown contribution of MMP9 to influenza virus pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral replication that could be exploited for therapeutic purposes.