TRIF Pathway Research Articles

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1–IKKϵ–IRF3 axis activation

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NF-κB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway.

Crucial Role of Linear Ubiquitin Chain Assembly Complex-Mediated Inhibition of Programmed Cell Death in TLR4-Mediated B Cell Responses and B1b Cell Development.

Linear ubiquitin chain assembly complex (LUBAC)-mediated linear polyubiquitin plays crucial roles in thymus-dependent and -independent type II Ab responses and B1 cell development. In this study, we analyzed the role of LUBAC in TLR-mediated B cell responses. A mouse strain in which LUBAC activity was ablated specifically in B cells (B-HOIPΔlinear mice) showed defective Ab responses to a type I thymus-independent Ag, NP-LPS. B cells from B-HOIPΔlinear mice (HOIPΔlinear B cells) underwent massive cell death in response to stimulation of TLR4, but not TLR9. TLR4 stimulation induced caspase-8 activation in HOIPΔlinear B cells; this phenomenon, as well as TLR4-induced cell death, was suppressed by ablation of TRIF, a signal inducer specific for TLR4. In addition, LPS-induced survival, proliferation, and differentiation into Ab-producing cells of HOIPΔlinear B cells were substantially restored by inhibition of caspases together with RIP3 deletion, but not by RIP3 deletion alone, suggesting that LPS stimulation kills HOIPΔlinear B cells by apoptosis elicited via the TRIF pathway. Further examination of the roles of cell death pathways in B-HOIPΔlinear mice revealed that deletion of RIP3 increased the number of B1 cells, particularly B1b cells, in B-HOIPΔlinear mice, indicating that B1b cell homeostasis is controlled via LUBAC-mediated suppression of necroptosis. Taken together, the data show that LUBAC regulates TLR4-mediated B cell responses and B1b cell development and/or maintenance by inhibiting programmed cell death.

Elucidating synergistic and antagonistic effects of B cell receptor and toll-like receptors 3, 4, and 9 in B cell activation

Abstract B cells are key components of the humoral immune response. Two forms of B cell activation come from engagement of the B cell receptor (BCR) with foreign antigens and from pathogen-associated molecular patters (PAMPs) binding to toll-like receptors (TLRs). For TLRs, MyD88 and TRIF are the major adaptors that lead to NFkB and IRF3 activation respectively. Among various TLRs that are present in B cells, TLR9 is only able to use MyD88 and TLR3 only signals through TRIF, while TLR4 uses both pathways. Therefore, these TLRs provide excellent tools to investigate the roles of TRIF and MyD88 pathways in B cell function and the interplay between them. Here, using stimulation through these three different TLRs either individually or in combination with each other and with/or without anti-IgM, we observed that there is synergy and antagonism between these pathways in terms of activation induced metabolic boost, cytokine production, IgM secretion, expression of various cell surface markers, and antigen presentation. We identified the TRIF pathway as an inducer of Type-I interferon secretion by B cells, which eventually favors the expression of costimulatory molecules such as CD86. On the other hand, MyD88 stimulation antagonized the expression of costimulatory molecules and dramatically induced rapid proliferation and production of IgM as well as proinflammatory cytokines such as IL-6 and TNF. Using B cells obtained from MyD88 or TRIF KO mice, we showed that the interplay between pathways is of limited scale; however, for TLR4 signaling MyD88 has a largely permissive effect while TRIF determines the magnitude of response. These results further our understanding of the complex TLR signaling machinery in B lymphocytes.

Human Binge Alcohol Intake Inhibits TLR4-MyD88 and TLR4-TRIF Responses but Not the TLR3-TRIF Pathway: HspA1A and PP1 Play Selective Regulatory Roles.

Binge/moderate alcohol suppresses TLR4-MyD88 proinflammatory cytokines; however, alcohol's effects on TLR-TRIF signaling, especially after in vivo exposure in humans, are unclear. We performed a comparative analysis of the TLR4-MyD88, TLR4-TRIF, and TLR3-TRIF pathways in human monocytes following binge alcohol exposure. Mechanistic regulation of TLR-TRIF signaling by binge alcohol was evaluated by analyzing IRF3 and TBK1, upstream regulator protein phosphatase 1 (PP1), and immunoregulatory stress proteins HspA1A and XBP-1 in alcohol-treated human and mouse monocytes/macrophages. Two approaches for alcohol exposure were used: in vivo exposure of primary monocytes in binge alcohol-consuming human volunteers or in vitro exposure of human monocytes/murine macrophages to physiological alcohol concentrations (25-50 mM ethanol), followed by LPS (TLR4) or polyinosinic-polycytidylic acid (TLR3) stimulation ex vivo. In vivo and in vitro binge alcohol exposure significantly inhibited the TLR4-MyD88 cytokines TNF-α and IL-6, as well as the TLR4-TRIF cytokines/chemokines IFN-β, IP-10, and RANTES, in human monocytes, but not TLR3-TRIF-induced cytokines/chemokines, as detected by quantitative PCR and ELISA. Mechanistic analyses revealed TBK-1-independent inhibition of the TLR4-TRIF effector IRF3 in alcohol-treated macrophages. Although stress protein XBP-1, which is known to regulate IRF3-mediated IFN-β induction, was not affected by alcohol, HspA1A was induced by in vivo alcohol in human monocytes. Alcohol-induced HspA1A was required for inhibition of TLR4-MyD88 signaling but not TLR4-TRIF cytokines in macrophages. In contrast, inhibition of PP1 prevented alcohol-mediated TLR4-TRIF tolerance in macrophages. Collectively, our results demonstrate that in vivo and in vitro binge alcohol exposure in humans suppresses TLR4-MyD88 and TLR4-TRIF, but not TLR3-TRIF, responses. Whereas alcohol-mediated effects on the PP1-IRF3 axis inhibit the TLR4-TRIF pathway, HspA1A selectively suppresses the TLR4-MyD88 pathway in monocytes/macrophages.

Effects of gut-derived endotoxin on anxiety-like and repetitive behaviors in male and female mice

BackgroundGut dysbiosis is observed in several neuropsychiatric disorders exhibiting increases in anxiety behavior, and recent work suggests links between gut inflammation and such disorders. One source of this inflammation may be lipopolysaccharide (LPS), a toxic component of gram-negative bacteria. Here, we (1) determine whether oral gavage of LPS, as a model of gut-derived endotoxemia, affects anxiety-like and/or repetitive behaviors; (2) test whether these changes depend on TLR4 signaling; and (3) test the extent to which gut-derived endotoxin and TLR4 antagonism affects males and females differently.MethodsIn experiment 1, male wild-type (WT) and Tlr4−/− mice were tested for locomotor, anxiety-like, and repetitive behaviors in an automated open field test apparatus, 2 h after oral gavage of LPS or saline. In experiment 2, male and female WT mice received an oral gavage of LPS and an injection of one or two TLR4 antagonists that target different TLR4 signaling pathways ((+)-naloxone and LPS derived from R. sphaeroides (LPS-RS)). Univariate and multivariate analyses were used to identify effects of treatment, sex, and genotype and their interaction.ResultsIn experiment 1, oral gavage of LPS increased anxiety-like behavior in male WT mice but not in Tlr4−/− mice. In experiment 2, oral gavage of LPS increased anxiety-like and decreased repetitive behaviors in WT mice of both sexes. Neither antagonist directly blocked the effects of orally administered LPS. However, treatment with (+)-naloxone, which blocks the TRIF pathway of TLR4, had opposing behavioral effects in males and females (independent of LPS treatment). We also identified sex differences in the expression of interleukin-6, a pro-inflammatory cytokine, in the gut both in basal conditions and in response to LPS.ConclusionIn spite of the ubiquitous nature of LPS in the gut lumen, this is the first study to demonstrate that intestinally derived LPS can initiate behavioral aspects of the sickness response. While an increased enteric load of LPS increases anxiety-like behavior in both sexes, it likely does so via sex-specific mechanisms. Similarly, TLR4 signaling may promote baseline expression of repetitive behavior differently in males and females. This study lays the groundwork for future interrogations into connections between gut-derived endotoxin and behavioral pathology in males and females.

Teleost-Specific TLR19 Localizes to Endosome, Recognizes dsRNA, Recruits TRIF, Triggers both IFN and NF-κB Pathways, and Protects Cells from Grass Carp Reovirus Infection.

TLRs are pivotal pattern recognition receptors in initiating innate immunity and triggering adaptive immunity. TLR pathways have been comprehensively investigated in mammals. However, the teleost-specific TLR19 pathway remains largely unknown. In this study, we identified TLR19 from grass carp (Ctenopharyngodon idella), and explored the ligand, adaptor, and signaling pathways. Pathogen-associated molecular pattern binding and luciferase activity assays indicate that TLR19 recognizes and responds to dsRNA analog (polyinosinic:polycytidylic acid). Confocal fluorescence microscopy demonstrates that TLR19 is synthesized in ribosomes not binding on endoplasmic reticulum, then transfers to early endosome post-polyinosinic:polycytidylic acid stimulation. Fluorescence colocalization and immunoprecipitation experiments confirm TLR19 interacts with adaptor TRIF, not MyD88, TIRAP, or SARM1. TLR19 facilitates protein and phosphorylation levels of IRF3, inhibits phosphorylation of IRF7. TLR19 enhances the promoter activities and mRNA expressions of major IFNs and NF-κBs; in contrast, grass carp TLR3 just significantly motivates IFN1 expression post-grass carp reovirus (GCRV) infection. Further investigations reveal that TLR19 inhibits GCRV replication by overexpression, knockdown, Western blotting techniques and virus titer assays, and protects cells from GCRV infection by flow cytometry and MTT method. Collectively, these results demonstrate that teleost-specific TLR19 recognizes dsRNA, recruits adaptor molecule TRIF, enhances IRF3 protein and phosphorylation levels, triggers both IFN and NF-κB pathways, and prevents viral proliferation. This is the first attempt to systematically clarify the TLR19 signaling pathway, which is the third TLR member recognizing dsRNA. The results will serve the antiviral immune mechanisms in teleost and evolutionary immunology.

The Family of LPS Signal Transducers Increases: the Arrival of Chanzymes

After LPS recognition, the MyD88-dependent and the TRIF-dependent pathways are consecutively activated in macrophages. Schappe etal. (2018) show that the chanzyme TRPM7 is required for an efficient LPS receptor complex endosomal relocation and the activation of the TRIF pathway.

A Novel Agonist of the TRIF Pathway Induces a Cellular State Refractory to Replication of Zika, Chikungunya, and Dengue Viruses.

ABSTRACTThe ongoing concurrent outbreaks of Zika, Chikungunya, and dengue viruses in Latin America and the Caribbean highlight the need for development of broad-spectrum antiviral treatments. The type I interferon (IFN) system has evolved in vertebrates to generate tissue responses that actively block replication of multiple known and potentially zoonotic viruses. As such, its control and activation through pharmacological agents may represent a novel therapeutic strategy for simultaneously impairing growth of multiple virus types and rendering host populations resistant to virus spread. In light of this strategy’s potential, we undertook a screen to identify novel interferon-activating small molecules. Here, we describe 1-(2-fluorophenyl)-2-(5-isopropyl-1,3,4-thiadiazol-2-yl)-1,2-dihydrochromeno[2,3-c]pyrrole-3,9-dione, which we termed AV-C. Treatment of human cells with AV-C activates innate and interferon-associated responses that strongly inhibit replication of Zika, Chikungunya, and dengue viruses. By utilizing genome editing, we investigated the host proteins essential to AV-C-induced cellular states. This showed that the compound requires a TRIF-dependent signaling cascade that culminates in IFN regulatory factor 3 (IRF3)-dependent expression and secretion of type I interferon to elicit antiviral responses. The other canonical IRF3-terminal adaptor proteins STING and IPS-1/MAVS were dispensable for AV-C-induced phenotypes. However, our work revealed an important inhibitory role for IPS-1/MAVS, but not TRIF, in flavivirus replication, implying that TRIF-directed viral evasion may not occur. Additionally, we show that in response to AV-C, primary human peripheral blood mononuclear cells secrete proinflammatory cytokines that are linked with establishment of adaptive immunity to viral pathogens. Ultimately, synthetic innate immune activators such as AV-C may serve multiple therapeutic purposes, including direct antimicrobial responses and facilitation of pathogen-directed adaptive immunity.

Asunaprevir Evokes Hepatocytes Innate Immunity to Restrict the Replication of Hepatitis C and Dengue Virus.

Type I Interferon-mediated innate immunity against Flaviviridae, such as Hepatitis C virus (HCV) and Dengue virus (DENV), involves TLR3, RIG-I-like receptor (RLR) and JAK-STAT signal pathways. Asunaprevir is a newly developed HCV protease inhibitor for HCV treatment. Whether, asunaprevir activates innate immunity to restrict viral infection is unclear. Thus, this study investigates the effect of asunaprevir on innate immunity and its influence on HCV and DENV infection. Huh 7.5.1, Hep-G2 cells, JFH-1 infection model, and DENV-2 infection were used for the analysis. The activity of asunaprevir-regulated innate immunity signal pathway was assessed with IFN-β promoter or IFN-stimulated responsive element (ISRE) reporter assays and immunoblotting of key signal proteins. siRNA-mediated MAVS and TRIF knockdown of cells was performed to assess the effect of asunaprevir-regulated innate immunity against HCV and DENV. Asunaprevir treatment activated ISRE and IFN-β promoter-luciferase activities and signaling proteins in the JAK-STAT, MAVS, and TRIF pathways in Huh 7.5.1 cells. Asunaprevir-mediated signaling activation was decreased in MAVS-knockdown cells. Importantly, both RNA and protein levels of DENV-2 NS3 were decreased in asunaprevir-treated Huh 7.5.1 and HepG2 cells. In MAVS-knockdown cells, the restrictive effect of asunaprevir on HCV and DENV was attenuated. Our findings reveal an unexpected activity of asunaprevir, the activation of MAVS dependent innate immunity to restrict HCV and DENV infection.

Toll like receptor 4: an important molecule in recognition and induction of appropriate immune responses against Chlamydia infection.

Chlamydia species are obligate intracellular pathogens causing different infectious diseases particularly asymptomatic genital infections and are also responsible for a wide range of complications. Previous studies showed that there are different immune responses to Chlamydia species and their infections are limited to some cases. Moreover, Chlamydia species are able to alter immune responses through modulating the expression of some immune system related molecules including cytokines. Toll like receptors (TLRs) belonge to pathogen recognition receptors (PRRs) and play vital roles in recognition of microbes and stimulation of appropriate immune responses. Therefore, it appears that TLRs may be considered as important sensors for recognition of Chlamydia and promotion of immune responses against these bacterial infections. Accordingly, TLR4 detects several microbial PAMPs such as bacterial lipopolysacharide (LPS) and subsequently activates transcription from pro-inflammatory cytokines in both MYD88 and TRIF pathways dependent manner. The purpose of this review is to provide the recent data about the status and major roles played by TLR4 in Chlamydia species recognition and promotion of immune responses against these infections and also the relationship between TLR4 activities and pathogenesis of Chlamydia infections.

The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a typeI interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

TRIF and MAVS pathway is essential to induce Rab27A and melanosome transportation by TLR3 agonist Poly(I:C) in human epidermal melanocytes

TRIF and MAVS pathway is essential to induce Rab27A and melanosome transportation by TLR3 agonist Poly(I:C) in human epidermal melanocytes

P2008 Lipopolysaccharide-induced gene expression of CD14 in TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

Cluster of differentiation 14 (CD14) is the pattern recognition receptor (PRR) involved in the recognition of bacterial component lipopolysaccharide (LPS) through the MyD88-dependent and TRIF pathway of innate immunity. It may be a modulator to prevent and mitigate the LPS-induced lung inflammation in pigs. However, reports on CD14 activation induced by LPS in TRIF pathway are controversial. Furthermore, the gene expression regulation of CD14 by the epigenetic factor sulforaphane (SFN) is still poorly understood. To identify the epigenetic changes of CD14 mediated with SFN in LPS-induced TRIF pathway, the PAMs model in vitro was investigated. For this, the mRNA expression of CD14 and downstream genes of TRIF pathway were quantified using qPCR. The cytokine levels of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA). The gene expression of the epigenetic enzymes DNA methyltransferase-1 (DNMT1) and DNMT3a were quantified. The protein level of NF-κB was analyzed by western blot. Furthermore, the DNA methylation alterations of CD14 at promotor and gene-body (CDS region) were analyzed using bisulfite sequencing in SFN- and LPS-treated PAMs. It was shown that CD14 gene expression was induced by 5 µg/ml LPS in time-dependent manner. At time point 12 h, the gene expression of CD14 and downstream genes in TRIF pathway, including TRIF, TRAF6, NFγB, TRAF3, IRF7, and cytokines, such as TNF-α, IL-1β, IL-6, and IFN-β, were significantly induced by LPS. The LPS-induced gene expression was suppressed by SFN in a dose-dependent manner. The LPS-induced cytokine levels, including TNFα, IL-1β, and NF-κB levels, were also inhibited by SFN. Similarly, the DNMT3a mRNA expression was increased by LPS and downregulated by SFN at a dose of 5 µM. Furthermore, the bisulfite sequencing results showed that gene body methylation of CD14 was positively associated with gene expression of LPS-treated PAMs and this methylation status was inhibited by SFN in a dose-dependent manner. This in vitro study suggests that CD14 is involved in TRIF pathway, including TRIF-TRAF6 and TRIF-TRAF3 pathway, by LPS induction. Furthermore, this LPS-CD14 activation was suppressed by SFN via the epigenetic regulation of CD14 gene body methylation associated with DNMT3a. This study provided novel insights into SFN-mediated epigenetic downregulation of CD14 gene in LPS-induced TRIF pathway inflammation and may open new avenues for approaches to prevent and mitigate LPS-induced inflammation in pigs.

A distinct brain pathway links viral RNA exposure to sickness behavior.

Sickness behaviors and metabolic responses to invading pathogens are common to nearly all types of infection. These responses evolved to provide short-term benefit to the host to ward off infection, but impact on quality of life, and when prolonged lead to neurodegeneration, depression, and cachexia. Among the major infectious agents, viruses most frequently enter the brain, resulting in profound neuroinflammation. We sought to define the unique features of the inflammatory response in the brain to these infections. We demonstrate that the molecular pathway defining the central response to dsRNA is distinct from that found in the periphery. The behavioral and physical response to the dsRNA mimetic poly I:C is dependent on signaling via MyD88 when it is delivered centrally, whereas this response is mediated via the TRIF pathway when delivered peripherally. We also define the likely cellular candidates for this MyD88-dependent step. These findings suggest that symptom management is possible without ameliorating protective antiviral immune responses.

HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes.

In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) βII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10. In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC-βII, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it may be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.

Novel Findings for the Role of Microenvironment and Heat Shock Proteins (HSP) in B -Cell Recptor (BCR) Activation, Disease Progression and Transformation of Follicular Non-Hodgkin Lymphoma (FL): Gene Expression Profiling (GEP) and Immuno-Histochemical (IH) Studies Reveal New Pathways for Potential Targeted Therapies

Introduction: Tumour infiltrating lymphocytes (TIL), tissue macrophages/monocytes (TMM) and follicular dendritic cells (FDC) have an important role in the biology of FL. Previous GEP studies used stored material, non-trizol freezing, negative T cell selection and CD3 stimulation and culture which could change signatures. None looked at peripheral blood (PB) T-cell (PBT) or MM (PBMM) profiles concurrently or specifically at fresh MM signatures though numbers are implicated in the disease's biology.Aims and methods: In the first stage we looked at positively selected TIL and TMM gene expression profile signatures in fresh diagnostic samples of histologically confirmed grades 1-3a FL in 14 patients and compared the profiles with similarly selected cells from controls and PB samples taken at the same time. We positively selected for CD2, CD14 and CD19 in that order with microbead technology. CD2 selection with no expansion in culture were used to minimise T-cell receptor stimulation. Cells were liquidised in Trizol prior to freezing. RNA was extracted and analysed using Affymetrix Microarray Chips. Statistical analysis used 3 parameters to define significant change in expression: fold change of >1.5, p value of > 1x10-3 and FDR of <0.05. Samples were analysed as paired and pooled with a fold (over or under expression) cut off threshold of 10. Results were validated with q-PCR for 10 of the most under or overexpressed genes. In the second stage we performed immunohistochemistry on paraffin embedded tissue from the same sample population looking at expression of HSP70, TLR2 & 4, CD21, CD22, CD200, PAX 5, p-AKT, and NF-κB p50 and p65. Co-expression by double staining was tested in 4 samples for CD3/HSP70, CD68/200, CD21/CD200 and CD68/PAX5.Results:GEP: T-cells paired samples (10) showed 97 over (41) or under (56) expressed genes in TIL compared to PBT whilst pooled samples (14) showed 778 over (380) or under (398) expressed genes. Several HSP genes are over expressed relating to HSP70 pathway. MM: Only pooled analysis was performed as the cell numbers were small. More (3239) genes were over (1494) or under (1745) expressed in LN compared to PBMM. The top over expressed genes include those that stimulate B cell receptor (BCR) growth and transformation and HSP70. Immunohistochemistry: FL cells lack nuclear HSP70 expression though TIL, TMM and FDC universally express it. Cytoplasmic NF-κB p50 and p65 and p-AKT are uniformly expressed in FL whilst TIL and TMM are weak or negative. FL cells strongly express TLR4 and to lesser extent TLR2 and are much weaker in TIL and TMM. CD200 is strongly expressed by FDC which show co-localisation with HSP70 and PAX5 double staining. TMM have granular cytoplasmic CD22 expression.Discussion: HSP suppress the apoptotic signalling at the premitochondrial stage through regulation of the pro-survival signalling and can be actively released extracellularly from inflammatory cells through the Golgi complex and passively from necrotic cells. HSP70 as a TLR4 and 2 ligand has a potent cytokine activity rapidly activates the MyD88/IRAK/NF-κB and TRIF pathways through MAPK/AP-1 and AKT increasing cell proliferation, adhesion and migration and resistance to apoptosis. Entry of the activated NF-κB into nuclei is the final step of signalling cascades and contributes to enhanced BCL-2 expression in FL.Our immunohistochemical and GEP findings are complimentary and show for the first time that in FL, HSP70 expressed and probably released by TIL and TMM for self-preservation could act as a ligand for TLR2 & 4, activating the NF-κB and p-AKT pathways leading to tumour growth and metastasis. We also show novel data that TMM and FDC appear to play a major role in disease proliferation and transformation through expression and secretion of BCR activating ligands. Targeting the HSP70/TLR pathway could induce apoptosis of both FL and TIL cells inhibiting tumour growth and spread whilst targeting HSP70, TMM and FDC could in addition abrogate histological transformation. DisclosuresNo relevant conflicts of interest to declare.

A Single Bacterial Immune Evasion Strategy Dismantles Both MyD88 and TRIF Signaling Pathways Downstream of TLR4.

During bacterial infections, Toll-like receptor 4 (TLR4) signals through the MyD88- and TRIF-dependent pathways to promote pro-inflammatory and interferon (IFN) responses, respectively. Bacteria can inhibit the MyD88 pathway, but if the TRIF pathway is also targeted is unclear. We demonstrate that, in addition to MyD88, Yersinia pseudotuberculosis inhibits TRIF signaling through the type III secretion system effector YopJ. Suppression of TRIF signaling occurs during dendritic cell (DC) and macrophage infection and prevents expression of type I IFN and pro-inflammatory cytokines. YopJ-mediated inhibition of TRIF prevents DCs from inducing natural killer (NK) cell production of antibacterial IFNγ. During infection of DCs, YopJ potently inhibits MAPK pathways but does not prevent activation of IKK- or TBK1-dependent pathways. This singular YopJ activity efficiently inhibits TLR4 transcription-inducing activities, thus illustrating a simple means by which pathogens impede innate immunity.

Abstract 19953: Interactions of MyD88 and TRIF-Pathways of Innate Immune Responses Regulate Angiotensin II Hypertension

We have reported that angiotensin II (Ang II) induces toll-like receptors (TLRs) in the innate immune system. Here we tested which of the two major adaptor proteins of TLRs, the myeloid differentiation protein 88 (MyD88) or the TIR-domain containing adaptor inducing interferon β (TRIF) mediates the Ang II-induced responses. Infusion of Ang II (3000 ng/kg/min) subcutaneously for 3 weeks in WT mice (C57BL/6J) increased systolic blood pressure to a peak value of 147 ± 4 mm Hg and resulted in a 40% increase in heart weight to body weight ratio (HW/BW). Surprisingly, in MyD88-/- mice the pressor response was enhanced significantly to 163 ± 6 mm Hg (P&lt;0.05) and the HW/BW was increased by 60%. Cardiac and renal RNA expression of TNF-α, NOX4 and Type IA -Ang II receptor were also significantly more elevated in MyD88-/- than in WT. In contrast, in a mouse strain with nonfunctional TRIF gene, Trifmut (C57BL/6J-Ticam1Lps2/J), all the Ang II responses were either uniformly decreased or abrogated. We tested whether the enhanced response in MyD88-/- mice represented a phenomenon of ‘Signaling Flux Redistribution” whereby blockade of one signaling pathway (i.e., MyD88) results in a flux of signaling substrates through the alternative pathway (TRIF). We found that expression of the RNA for chemokine CXCL10 which is dependent on TRIF pathway, was significantly enhanced in MyD88-/- hearts. We also found that expression of transcripts for TLR3, TLR4 and the TRIF adaptor protein were all enhanced in MyD88-/- and must have contributed to the exaggerated Ang II response. Conversely, Trifmut had enhanced MyD88 expression. These results suggest that: (1) Ang II induced hypertension, hypertrophy and inflammatory gene expression are primarily mediated by the TRIF pathway and (2) the MyD88 pathway is simultaneously activated by Ang II and exerts a negative regulatory influence on the pressor and inflammatory responses. We conclude that dual activation of two innate immune pathways defines the net response in WT mice with a significant translational potential. Accordingly, impairment of MyD88 may contribute to pathogenesis of hypertension. Conversely, targeting the TRIF pathway may be therapeutic by blocking the inflammatory response and enhancing the negative regulatory effects of MyD88.

Abstract 072: TRIF-Pathway of Innate Immune Responses Mediates Angiotensin II Hypertension

We tested the role of two distinct adaptors of toll-like receptor (TLR) signaling on Ang II-induced hypertension and cardiac hypertrophy. These TLR adaptors, myeloid differentiation protein 88 (MyD88) and TIR domain-containing adaptor inducing interferon β (TRIF) facilitate distinct inflammatory signaling pathways. In an earlier study, we reported that MyD88-/- mice are protected from cardiac hypertrophy and pro-inflammatory gene expression after myocardial infarction. Our current results with 3 weeks infusion of Ang II (3000 ng/kg/min) vs. saline indicate that in MyD88-/- mice, the pressor response to Ang II and cardiac hypertrophy were increased more than in wild type (WT) mice. In Ang II-infused WT, systolic blood pressure (SBP) peaked at 147 ± 4 mmHg whereas in Ang II-infused MyD88-/- mice SBP reached a peak value of 163 ± 6 mmHg. However, in mice with non-functional TRIF adaptor mutant (Trifmut), SBP did not increase during Ang II infusion and remained similar to the SBP in saline-infused mice (115 ± 3 mmHg). Baseline SBP was not different among WT, MyD88-/- and Trifmut mice. The increase in heart weight to body weight ratio (HW/BW) between saline and Ang II-infused mice was greater in MyD88-/- mice than WT mice (60% increase in MyD88-/- vs. 40% increase in WT), whereas it was less in Trifmut mice (22% increase). Accordingly, expression of several inflammatory genes (Tnfa, Nox4 and Agtr1a) was significantly greater (P&lt; 0.05) in the heart and kidney of Ang II-infused MyD88-/- mice compared with Ang II-infused WT mice, whereas expression of these genes in Trifmut mice was either unchanged or reduced. We conclude that- (1) Ang II-induced hypertension, cardiac hypertrophy and inflammatory gene expression are mediated by activation of a TRIF-dependent pathway, but not by the MyD88-dependent pathways, and (2) Enhanced Ang II effects on SBP and hypertrophy in MyD88-/- mice suggest that MyD88 may serve as a negative regulator of the TRIF pathway in Ang II-induced hypertension. Selective targeting of these adaptor proteins may have significant therapeutic implications.