Scaffolding role of TcpB in disrupting TLR4-Mal interactions: Three to tango

Toll/interleukin-1 like receptors (TLRs) are membrane-spanning proteins crucially involved in innate immunity. On activation, the cytoplasmic toll/interleukin-1 receptor (TIR) domains of these receptors undergo homo- or heterodimerization. Brucella sp. are bacterial pathogens that affect the immune system by suppressing the TLR signaling pathway. They enact this by encoding a TIR domain–containing protein, TcpB, which suppresses NF-κB activation and proinflammatory cytokine secretion mediated by TLR4 receptors. TcpB has been shown to target the Mal-mediated pathway to suppress TLR signaling. The recent identification of its mechanism of interference with TLR4 signaling involving Mal prompted us to further study the structural aspects of TcpB binding with TLR4 and Mal. Our triprotein model displays the overall scaffolding role of TcpB in anchoring TLR4 and Mal thereby inhibiting their interaction leading to the attenuation of the TLR4 pathway.     

Crystal Structures of the Toll/Interleukin-1 Receptor (TIR) Domains from the Brucella Protein TcpB and Host Adaptor TIRAP Reveal Mechanisms of Molecular Mimicry

The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys⁸⁹ and Cys¹³⁴. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.     

Modulation of microtubule dynamics by a TIR domain protein from the intracellular pathogen Brucella melitensis

TIR (Toll/interleukin-1 receptor) domain-containing proteins play a crucial role in innate immunity in eukaryotes. Brucella is a highly infectious intracellular bacterium that encodes a TIR domain protein (TcpB) to subvert host innate immune responses to establish a beneficial niche for pathogenesis. TcpB inhibits NF-κB (nuclear factor κB) activation and pro-inflammatory cytokine secretions mediated by TLR (Toll-like receptor) 2 and TLR4. In the present study, we have demonstrated that TcpB modulates microtubule dynamics by acting as a stabilization factor. TcpB increased the rate of nucleation as well as the polymerization phases of microtubule formation in a similar manner to paclitaxel. TcpB could efficiently inhibit nocodazole- or cold-induced microtubule disassembly. Microtubule stabilization by TcpB is attributed to the BB-loop region of the TIR domain, and a point mutation affected the microtubule stabilization as well as the TLR-suppression properties of TcpB.     

Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.     

Discovery of substituted 4-aminoquinazolines as selective Toll-like receptor 4 ligands

The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure–activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.     

Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system

Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING and MAVS. Herein, we review the advances in B. abortus DNA sensing by host innate immune receptors and the progress in this field.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk regulates nucleic acid-induced endosomal TOLL-like receptor-7 and -9 activation,cellular signaling and pro-inflammatory responses

The precise mechanism(s) by which intracellular TOLL-like receptors (TLRs) become activated by their ligands remains unclear. Here, we report a molecular organizational G-protein coupled receptor (GPCR) signaling platform to potentiate a novel mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B GPCR, all of which form a tripartite complex with TLR-7 and -9. siRNA silencing Neu1, MMP-9 and neuromedin-B GPCR in RAW-blue macrophage cells significantly reduced TLR7 imiquimod- and TLR9 ODN1826-induced NF-κB (NF-κB-pSer⁵³⁶) activity. Tamiflu, specific MMP-9 inhibitor, neuromedin B receptor specific antagonist BIM23127, and the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 significantly block nucleic acid-induced TLR-7 and -9 MyD88 recruitment, NF-κB activation and proinflammatory TNFα and MCP-1 cytokine responses. For the first time, Neu1 clearly plays a central role in mediating nucleic acid-induced intracellular TLR activation, and the interactions involving NMBR–MMP9–Neu1 cross-talk constitute a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death

Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-κB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.     

Innate immune recognition of,and response to,Clostridium sordellii

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-κB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFα and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFα following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated.     

miR-146a suppresses the sensitivity to interferon-α in hepatocellular carcinoma cells

Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-κB) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to IκB degradation and activation of NF-κB. NF-κB activation was confirmed by nuclear localization of NF-κB p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-κB signaling attenuated LPS-induced TNFα plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-κB signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-κB does not sensitize GCTs to TRAIL or cisplatin.