Toll-Like Receptor Signaling Pathways

Toll-like receptors (TLRs) play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. TLRs signal through the recruitment of specific adaptor molecules, leading to activation of the transcription factors NF-kB and IRFs, which dictate the outcome of innate immune responses. During the past decade, the precise mechanisms underlying TLR signaling have been clarified by various approaches involving genetic, biochemical, structural, cell biological, and bioinformatics studies. TLR signaling appears to be divergent and to play important roles in many aspects of the innate immune responses to given pathogens. The main players in innate immunity are phagocytes such as neutrophils, macrophages, and dendritic cells. These cells can discriminate between pathogens and self by utilizing signals from the Toll-like receptors (TLRs)1. TLRs recognize conserved motifs predominantly found in microorganisms but not in vertebrates. Stimulation of TLRs causes an immediate defensive response, including the production of an array of antimicrobial peptides and cytokines. Accumulating evidence has shown that individual TLRs can activate overlapping as well as distinct signaling pathways, ultimately giving rise to distinct biological effects.

Toll-like receptor (TLR) and interferon-gamma (IFN-gamma) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-gamma and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-alpha (TNF-alpha). Comparable synergism was observed between IFN-gamma and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-gamma enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-kappaB activation. Interestingly, we found that IRF-8-/- macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-gamma plus LPS. Retroviral transduction of IRF-8 into IRF-8-/- macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-gamma and TLR signal pathways.

Toll-like receptor (TLR) signaling is thought to be one of the most important pathways initiating periodontitis onset. We have previously reported that the TLR signaling pathway is upregulated in periodontitis-affected gingival tissues by microarray pathway frequency analysis. The aim of the present study was to quantitatively analyze specific upregulated genes in the TLR signaling pathway, as compared to healthy controls. Healthy and periodontitis-affected gingival tissues were taken from distinct sites of 3 patients with severe chronic periodontitis. Total RNAs from 6 gingival tissue samples were used for microarray. Samples were taken from 14 chronic periodontitis patients and 14 healthy individuals for quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) analysis. Data-mining analyses, such as pathway analyses, were performed and significant biological pathways in periodontitis were identified. In addition, qRT-PCR analysis was performed for 5 genes—cluster of differentiation 14 (CD14), lymphocyte antigen 96 (MD-2), interleukin-1 beta (IL-1β), interleukin 8 (IL-8), and chemokine ligand 9 (CXCL-9), which are associated with TLR signaling, in order to confirm the results of pathway analysis. qRT-PCR verified that the transcripts for 5 genes in the TLR signaling pathway were significantly upregulated (MD-2 p = 0.0082, CD14 p = 0.0322, IL-1β p = 0.0126, IL-8 p = 0.0438, CXCL-9 p = 0.0325), which was consistent with pathway analyses. We confirmed upregulated MD-2 gene expression levels and associated TLR pathway gene expression, including CD14, IL-1β, IL-8 and CXCL-9, in periodontitis-affected gingival tissues, as compared with healthy controls.

Toll-like receptors (TLRs) are proteins involved in recognition of foreign pathogen-associated molecular patterns and activation of processes leading to innate immune recognition. We show that stimulation of fibroblasts with a TLR5 ligand, flagellin, can induce proliferation of serum-starved cells or prevent cell cycle exit upon serum withdrawal independently of autologous growth factor secretion. Other TLR ligands, such as poly(I:C) and lipopolysaccharide, can have a similar effect only if the action of type I interferons is blocked. Flagellin stimulation can prevent cell cycle arrest induced by overexpression of exogenous cyclin-dependent kinase inhibitor p27. Stimulation of TLR5 and overexpression of MyD88, but not TRIF, TIRAP, or TRAM, result in p27 degradation, which can be suppressed by dominant negative Akt and mutation of the p27 C-terminal Thr(187) site. These data provide evidence for a nonimmune and cell autonomous role of TLR signaling, whereby TLR stimulation provides a positive signal for cell division.

(734) - Hyperglycemia Exacerbates Ischemia-Reperfusion Injury of the Lung By Activating TLR4 Signaling Pathway

The innate immune system provides a first line of defense against invading pathogens, in which the pattern recognition receptors (PRR) recognize pathogen-associated molecular patterns (PAMP) and initiate the downstream signaling pathways to eliminate the encountered pathogens. There are two main classes of such signaling pathways: NOD-like receptor (NLR) signaling pathway and Toll-like receptor (TLR) signaling pathway. The microbial pathogens under selective pressure have evolved numerous mechanisms to avoid and/or manipulate the NLR and TLR signal transduction for survival and replication. To evade the NLR signaling pathway, pathogens interfere and/or inhibit inflammasome activation in innate immune cells by producing virulence factors or reducing PAMPs expression. The mechanisms for pathogens to evade TLR signaling pathway include: inhibition of mitogen activated protein kinases (MAPKs) cascade reaction, inhibition of NF-КB activation, and interference of down-stream signal transduction by producing Toll/interleukin-1 receptor (TIR)-containing proteins which bind directly with TLRs or adaptor proteins in the signaling pathway.

Introduction: The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is clinically active in lymphoproliferative diseases driven by B-cell receptor (BCR) and Toll-like receptor (TLR) signaling, including chronic lymphocytic leukemia (CLL) and Waldenstrom macroglobulinemia (WM), respectively. However, deep remissions are uncommon and resistance to single agent has been described. The hallmark of WM is an activating mutation in MYD88 in the TLR signaling pathway. While mutations in MYD88 are uncommon in CLL, our previous study identified gene signatures indicative of active BCR and TLR signaling in CLL cells residing in lymphoid tissues (Herishanu, Blood 2011). Further, TLR9 activating CpG oligonucleotides induce proliferation and extend CLL cell survival in vitro. These observations suggest that BCR and TLR signaling may cooperate to activate CLL cells in the tissue microenvironment. Here, we tested the hypothesis that targeting both BCR and TLR signaling could improve therapy for CLL. Methods: CLL PBMCs were treated with ibrutinib and/or an IRAK1/4 inhibitor (Calbiochem) for 1h and then stimulated with soluble αIgM, CpG, or both. We quantified changes in phosphorylation of BTK, PLCγ2 and ERK (BCR pathway) and STAT3 and STAT1, as well as total IRAK1 (TLR pathway). Results: As expected, ibrutinib inhibited phosphorylation of BTK, PLCγ2 and ERK (P<.05) and decreased the survival of CLL cells stimulated with αIgM (P=.001). CpG stimulated TLR signaling degrades IRAK1 and the stimulates cytokine secretion that can, in autocrine fashion, activate STAT phopsphorylation. The IRAK1/4 inhibitor effectively inhibited TLR signaling resulting in stabilization of IRAK1 (P=.002), decreased phosphorylation of STAT1/3 (P=.04) and decreased viability compared to CpG stimulated but not IRAK inhibitor treated cells. Ibrutinib had no effect on CpG-induced IRAK1 degradation, but was comparable to IRAK1/4 inhibition in reducing STAT phosphorylation, suggesting that inhibition of BTK can antagonize downstream effects of TLR activation but not upstream IRAK dependent steps. In contrast, IRAK1/4 inhibition had no effect on αIgM-induced BCR activation. Next we evaluated the effect of dual BCR and TLR activation, modelling co-operative activation of both pathways in the tumor microenvironment. Under these in vitro conditions, ibrutinib prevented BCR activation and partial TLR activation, while IRAK1/4 affected only the TLR pathway. When ibrutinib and the IRAK1/4 inhibitor were combined, activation of both BCR and TLR signaling was prevented resulting in a significant reduction in CLL cell viability (P=.01) compared to co-activation of both pathways. Conclusion: While ibrutinib partially inhibited TLR signaling, an IRAK1/4 inhibitor was required for full inhibition of the pathway. The combination of BTK and IRAK1/4 inhibition for the treatment of lymphoproliferative diseases warrants further investigation. Citation Format: Eman L. Dadashian, Sarah Herman, Adrian Wiestner. Dual inhibition of BCR and TLR signaling has therapeutic potential in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2686. doi:10.1158/1538-7445.AM2017-2686

The interleukin-1 receptor-associated kinases: Critical regulators of innate immune signalling

BackgroundThe Toll-like receptor (TLR) signaling pathway constitutes an essential component of the innate immune system. Highly conserved proteins, indicative of their critical roles in host survival, characterize this pathway. Selective constraints could vary depending on the gene’s position within the pathway as TLR signaling is a sequential process and that genes downstream of the TLRs may be more selectively constrained to ensure efficient immune responses given the important role of downstream genes in the signaling process. Thus, we investigated whether gene position influenced protein evolution in the TLR signaling pathway of the Suidae. The members of the Suidae examined included the European Sus scrofa (wild boar), Asian Sus scrofa (wild boar), Sus verrucosus, Sus celebensis, Sus scebifrons, Sus barbatus, Babyrousa babyrussa, Potamochoerus larvatus, Potamochoerus porcus and Phacochoerus africanus.ResultsA total of 33 TLR signaling pathway genes in the Suidae were retrieved from resequencing data. The evolutionary parameter ω (dn/ds) had an overall mean of 0.1668 across genes, indicating high functional conservation within the TLR signaling pathway. A significant relationship was inferred for the network parameters gene position, number of protein-protein interactions, protein length and the evolutionary parameter dn (nonsynonymous substitutions) such that downstream genes had lower nonsynonymous substitution rates, more interactors and shorter protein length than upstream genes. Gene position was significantly correlated with the number of protein-protein interactions and protein length. Thus, the polarity in the selective constraint along the TLR signaling pathway was due to the number of molecules a protein interacted with and the protein’s length.ConclusionResults indicate that the level of selective constraints on genes within the TLR signaling pathway of the Suidae is dependent on the gene’s position and network parameters. In particular, downstream genes evolve more slowly as a result of being highly connected and having shorter protein lengths. These findings highlight the critical role of gene network parameters in gene evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0602-7) contains supplementary material, which is available to authorized users.

The Toll receptor family.

Toll-like Receptor 9 Triggers an Innate Immune Response to Helper-dependent Adenoviral Vectors

Lung cancer, particularly non-small-cell lung cancer (NSCLC), remains a leading cause of cancer-related mortality worldwide. Recent studies have implicated pyrroline-5-carboxylate reductase 1 (PYCR1), a key enzyme in proline biosynthesis, in cancer progression, yet its specific role in lung cancer remains unclear. Here we demonstrate that PYCR1 plays a critical role in NSCLC progression through its functional association with the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR) signaling pathways. An analysis of patient datasets revealed that PYCR1 is upregulated in NSCLC tissues, with the enrichment of cancer-associated pathways in PYCR1-upregulated patients. Functional studies in PYCR1-knockout (PYCR1-KO) lung cancer cells generated via CRISPR–Cas9 showed reduced cell proliferation, migration, colony formation and tumor spheroid growth both in vitro and in vivo. Mechanistically, PYCR1 stabilizes EGFR by forming a complex with EGFR and USP11, thereby enhancing EGFR deubiquitination and stability. In addition, PYCR1 promotes TLR signaling by interacting with key downstream molecules, including TRAF6, TAK1, ECSIT and TAB2, facilitating their ubiquitination and NF-κB activation. The loss of PYCR1 attenuates EGFR- and TLR-induced signaling cascades, resulting in reduced activation of AKT, TAK1 and NF-κB. Importantly, treatment with PYCR1-IN-1, a selective PYCR1 inhibitor, significantly suppressed EGFR- and TLR-induced tumor spheroid growth in multiple lung cancer cell lines, underscoring PYCR1’s potential as a therapeutic target. Collectively, our findings establish PYCR1 as a critical regulator of EGFR and TLR signaling pathways, driving lung cancer progression. Targeting PYCR1 with pharmacological inhibitors such as PYCR1-IN-1 offers a promising strategy for combating EGFR- and TLR-driven NSCLC progression.

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

Ibrutinib Inhibits Both B-Cell Receptor and Toll-like Receptor Signaling in Chronic Lymphocytic Leukemia

Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factor 6 (traf6) like gene involved in antibacterial innate immune of fresh water crayfish, Procambarus clarkii