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Bacterial endotoxin-induced sepsis causes 30–40% of the deaths in the intensive care unit (ICU) globally, for which there is no pharmacotherapy. Lipopolysaccharide (LPS), a bacterial endotoxin, stimulates the Toll-like receptor (TLR)-4 signalling pathways to upregulate the expression of various inflammatory mediators. Here, we show that the TIRAP and c-Jun protein signalling complex forms in macrophages in response to LPS stimulation, which increases the AP1 transcriptional activity, thereby amplifying the expression of inflammatory mediators. Using a computer-aided molecular docking platform, we identified gefitinib as a putative inhibitor of the TIRAP-c-Jun signalling complex. Further, we demonstrated the ability of gefitinib to inhibit the interaction of TIRAP-c-Jun with in vitro experiments and with a mouse model of sepsis. Importantly, pre-treatment with gefitinib increased the survival of the mice that received a lethal dose of LPS compared to that of the controls. These findings verify the ability of gefitinib to directly disrupt the interaction of TIRAP and c-Jun, thereby inhibiting a major inflammatory response that is often observed in patients experiencing sepsis. • LPS stimulates AP1/NFkB transcriptional activity through the TLR-4 receptor during inflammation. • The TIRAP and c-Jun complex regulates AP1/NFkB transcriptional activity. • Targeting the TIRAP and c-Jun complex is a promising strategy to inhibit the inflammatory response. • Gefitinib is a putative inhibitor of inflammation via the targeting of the TIRAP-c-Jun complex. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important lipid regulator of membrane signaling and remodeling processes. Accumulating evidence indicates a link between PIP2 metabolism and Toll-like receptor (TLR) signaling, a key transducer of immune responses such as inflammation, phagocytosis, and autophagy. Microglia are immune effector cells that serve as macrophages in the brain. Here, we examined the potential role of phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα), a PIP2-producing enzyme, in TLR2 signaling in microglial cells. Treatment of BV2 microglial cells with lipoteichoic acid (LTA), a TLR2 agonist, increased PIP5Kα expression in BV2 and primary microglial cells, but not in primary cultures from TLR2-deficient mice. PIP5Kα knockdown of BV2 cells with shRNA significantly suppressed LTA-induced activation of TLR2 downstream signaling, including the production of proinflammatory cytokines and phosphorylation of NF-κB, JNK, and p38 MAP kinase. Such suppression was reversed by complementation of PIP5Kα. PIP5Kα knockdown lowered PIP2 levels and impaired LTA-induced plasma membrane targeting of TIRAP, a PIP2-dependent adaptor required for TLR2 activation. Besides, PIP5Kα knockdown inhibited phagocytic uptake of E. coli particles and autophagy-related vesicle formation triggered by LTA. Taken together, these results support that PIP5Kα can positively mediate TLR2-associated immune responses through PIP2 production in microglial cells. [ABSTRACT FROM AUTHOR]

Toll-interleukin-1 receptor domain containing adapter protein (TIRAP) plays a crucial role in TLR2 and TLR4 signaling pathways. Glycosylphospatidylinositol (GPI), considered a toxin molecule of Plasmodium falciparum, interacts with TLR2 and 4 to induce an immune inflammatory response. A single nucleotide polymorphism at coding region of TIRAP (S180L) has been reported to influence TLRs signaling. In the present study, we investigated the association of TIRAP (S180L) polymorphism with susceptibility/resistance to severe P. falciparum malaria in a cohort of adult patients from India. TIRAP S180L polymorphism was typed in 347 cases of severe malaria (SM), 232 uncomplicated malaria and 150 healthy controls. Plasma levels of TNF-α was quantified by ELISA. Heterozygous mutation (S/L) conferred significant protection against MOD (multi organ dysfunction), NCSM (non-cerebral severe malaria) as well as mortality. Interestingly, homozygous mutants (L/L) had 16 fold higher susceptibility to death. TIRAP mutants (S/L and L/L) were associated with significantly higher plasma TNF-α levels compared to wild type (S/S). The results of the present study demonstrate that TIRAP S180L heterozygous mutation may protect patients against severe malaria and mortality. [ABSTRACT FROM AUTHOR]

Background Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), an important adaptor protein downstream of the Toll-like receptor (TLR) 2 and 4 pathways, is highly involved in the activation and coordination of the anti-mycobacterial immune response. We performed a meta-analysis to assess the association between TIRAP C539T polymorphism and tuberculosis (TB) risk. Methods A systematic literature search for relevant studies up to February 27, 2014 was conducted in PUBMED, EMBASE, Web of science, CNKI, VIP, and Wanfang database. The association between gene and disease was assessed using odds ratios (ORs) with 95% confidence intervals (95%CIs) based on five genetic models. Results A total of 16 qualified studies were enrolled in this meta-analysis. The results of pooling all studies detected statistically resistance of TIRAP C539T mutants to TB risk (T vs. C: OR 0.80, 95%CI 0.65–0.97; TC vs. CC: OR 0.71, 95%CI 0.55–0.92; TT + TC vs. CC: OR 0.74, 95% CI 0.58–0.94). Further subgroup analyses by ethnicity also demonstrated reduced risk of TB in European population (T vs. C: OR 0.71, 95%CI 0.52–0.95; TC vs. CC: OR 0.56, 95%CI 0.35–0.91; TT + TC vs. CC: OR 0.61, 95%CI 0.40–0.92), whereas no such effects were observed in other ethnicities. Conclusion This present meta-analysis suggests TIRAP C539T polymorphism is significantly correlated with reduced risk of TB infection, with stronger effect in European. Additional well-designed, larger-scale epidemiological studies among different ethnicities are needed. [ABSTRACT FROM AUTHOR]

A number of diseases including sepsis, rheumatoid arthritis, diabetes, cardiovascular diseases and hyperinflammatory immune disorders have been associated with Toll like receptor (TLR) 2 and TLR4. Endogenous adaptor protein known as MyD88 adapter-like protein (MAL) bind exclusively to the cytosolic portions of TLR2 and TLR4 to initiate downstream signalling. Brutons tyrosine kinase (BTK) and protein kinase C delta (PKCδ) have been implicated to phosphorylate MAL and activate it to initiate downstream signalling. BTK has been associated with phosphorylation at positions Tyr86 and Tyr106, necessary for the activation of MAL but definite residual target of PKCδ in MAL is still to be explored. To produce a better understanding of the functional domains involved in the formation of MAL-kinase complexes, computer-aided studies were used to characterize the protein-protein interactions (PPIs) of phosphorylated BTK and PKCδ with MAL. Docking and physicochemical studies indicated that BTK was involved in close contact with Tyr86 and Tyr106 of MAL whereas PKCδ may phosphorylate Tyr106 only. Moreover, the electrostatics charge distribution of binding interfaces of BTK and PKCδ were distinct but compatible with respective regions of MAL. Our results implicate that position of Tyr86 is specifically phosphorylated by BTK whereas Tyr106 can be phosphorylated by competitive action of both BTK and PKCδ. Additionally, the residues of MAL which are necessary for interaction with TLR2, TLR4, MyD88 and SOCS-1 also play their roles in maintaining interaction with kinases and can be targeted in future to reduce TLR2 and TLR4 induced pathological responses. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor (TLR) signaling is initiated by the binding of various adaptor proteins through ligand-induced oligomerization of the Toll/interleukin-1 receptor (TIR) domains of the TLRs. TLR2, which recognizes peptidoglycans, lipoproteins or lipopeptides derived from Gram-positive bacteria, is known to use the TIR domain-containing adaptor proteins myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal). Molecular analyses of the binding specificity of MyD88, Mal, and TLR2 are important for understanding the initial defenses mounted against Gram-positive bacterial infections such as Streptococcus pneumoniae. However, the detailed molecular mechanisms involved in the multiple interactions of these TIR domains remain unclear. Our study demonstrates that the TIR domain proteins MyD88, Mal, TLR1, and TLR2 directly bind to each other in vitro. We have also identified two binding interfaces of the MyD88 TIR domain for the TLR2 TIR domain. A residue at these interfaces has recently been found to be mutated in innate immune deficiency patients. These novel insights into the binding mode of TIR proteins will contribute to elucidation of the mechanisms underlying innate immune deficiency diseases, and to future structural studies of hetero-oligomeric TIR–TIR complexes. [Copyright &y& Elsevier]

Abstract: Toll-like receptor adaptor molecule 1/2 (TICAM-1/2) and Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP) play key roles in the Toll-like receptor (TLR) signaling pathways, which respond to viral and bacterial infections. These genes have been identified and studied in several vertebrates. However, our understanding of their evolutionary history and their roles in immune responses is far from complete. In this study, comparative and evolutionary analyses were performed for TICAM-1, TICAM-2 and TIRAP within the range of 25 representative species. Our data show that the origin of the TICAM-like and TIRAP-like genes may coincide with the origin of chordates (amphioxus). Several putative TICAMs and TIRAPs were identified for different chordate species. Shark is the only non-mammalian species whose genome contains a TICAM-2 gene. Structural modeling and comparison of TIR domains of these adaptors support their potential functional motifs and residues. Together with analyses of other genes involved in the TLR signaling pathways, we speculate that TICAM-1, TICAM-2 and TIRAP might have co-evolved with the TLR3/22 antivirus signaling, the LPS-specific TLR4 signaling and the Gram-positive bacteria-induced TLR2 signaling pathways, respectively. Our results are valuable contributions to the understanding of TICAM/TIRAP evolutional functions and may provide targets for therapeutic intervention in TLR-mediated vertebrate diseases. [Copyright &y& Elsevier]

Summary: The missense variant S180L in TIRAP (Toll-interleukin-1 receptor domain-containing adaptor protein) gene is implicated in attenuating TLRs signal transaction and may affect individual response to Mycobacterium tuberculosis infection. Several studies investigated the association between TIRAP S180L and risk of tuberculosis (TB), but the results were controversial. In this study, we quantitatively synthesized nine studies relevant to the association between TIRAP S180L polymorphism and TB risk with total 6584 TB cases and 7294 controls using meta-analysis. We found that the variant allele Leu180 and heterozygous genotype Ser/Leu were not significantly associated with risk of TB (allelic OR = 0.99, 95%CI: 0.88–1.11; Ser/Leu vs Ser/Ser: OR = 0.99, 95%CI: 0.87–1.13) with heterogeneity P values > 0.05. In subgroup analysis, none of the significant associations were observed for S180L and TB risk in Africans (allelic OR = 0.58, 95%CI: 0.29–1.61; heterozygous OR = 0.65, 95%CI: 0.32–1.32) or Asians (allelic OR = 1.30, 95%CI: 0.97–1.74; heterozygous OR = 1.17, 95%CI: 0.84–1.65) or risk of pulmonary tuberculosis (PTB) (allelic OR = 0.92, 95%CI: 0.69–1.22; heterozygous OR = 0.98, 95%CI: 0.86–1.12). This meta-analysis indicates that TIRAP S180L polymorphism is unlikely to substantially contribute to TB susceptibility. [ABSTRACT FROM AUTHOR]

Summary: Toll-like receptors (TLRs) are pattern recognition receptors and play an important role in innate immunity. Changes in TLRs and signaling molecules that result from polymorphisms are often associated with susceptibility to various infectious diseases. In the present study, we investigated whether variants in the TLR-1 1805T/G (Ile602Ser), TLR-2 2258G/A (Arg753Gln), TLR-4 896A/G (Asp299Gly), TLR-4 1196C/T (Thr399Ile), TLR-6 745C/T (Ser249Pro), TIRAP 975C/T (Ser180Leu) genes and TLR-9 promoter region polymorphisms at positions −1237C/T and −1486C/T are associated with susceptibility or resistance to pulmonary tuberculosis (PTB). Genotyping of TLR and TIRAP gene polymorphisms was performed by polymerase chain reaction followed by restriction fragment length polymorphism method in 212 healthy control subjects (HCs) and 206 PTB patients. The allele and genotype frequencies of various TLR genes were not different between the HCs and PTB patients. However, the study is underpowered to detect minor associations. The frequency of T allele of TIRAP 975C/T (Ser180Leu) polymorphism was significantly increased among PTB patients as compared to HCs [p = 0.026; Odds ratio (OR) 1.49, 95% Confidence interval (CI) 1.049–2.22]. A trend towards an increased frequency of TT genotype of TIRAP 975C/T was also observed in PTB patients [p = 0.078, OR 3.10 95% CI (0.96–10.05)]. The present study suggests that T allele of TIRAP 975C/T polymorphism may be associated with susceptibility to pulmonary TB in south Indian population. Further study on the regulatory role of this polymorphism may be helpful to understand the innate immunity in TB. [ABSTRACT FROM AUTHOR]

[Display omitted] • Phycocyanin diminishes the viability of A549 cells. • Phycocyanin significantly promotes expressions of miR-3150a-3p, miR-6883-3p and miR-627-5p. • TIRAP is a shared directed target of miR-3150a-3p, miR-6883-3p and miR-627-5p. • Knockdown TIRAP expression resulted in similar cell phenotype as miRNAs overexpression. • Overexpression TIRAP restores the diminished A549 viability caused by miRNAs and phycocyanin. As a type of functional food additives, phycocyanin is well-known for its extraordinary antineoplastic activity in non-small cell lung cancer (NSCLC). Previously, a high-throughput miRNA sequencing was employed to excavate miRNAs altered by phycocyanin in A549 cells, while the in-depth regulatory mechanism was inadequate. In this work, three significant up-regulated miRNAs in phycocyanin-treated cells are identified for the first time, including miR-3150a-3p, miR-6883-3p and miR-627-5p. Overexpression of miRNAs obviously diminished the cell growth and colony formation capacity, respectively. Dual-luciferase reporter assay verified that toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) was a direct shared target of three miRNAs. Knockdown the expression of TIRAP resulted in similar cell growth inhibition phenotype as miRNAs overexpression. Strikingly, overexpression of TIRAP could significantly restore the diminished cell viability caused by miRNAs up-regulation, as well as phycocyanin treatment. Consequently, this study proposes that phycocyanin plays an inhibitory effect of NSCLC cells via a miR-3150a-3p/miR-6883-3p/miR-627-5p-TIRAP axis. [ABSTRACT FROM AUTHOR]

Abstract: Background and aim: The involvement of Toll-like receptor (TLR)-mediated pathways in infectious and autoimmunity has been suggested. The MyD88 adaptor-like (Mal) protein, also known as the TIR domain-containing adaptor protein (TIRAP), is implicated in the TLR2- and TLR4-mediated MyD88-dependent signaling pathway. The aim of this study was to investigate the influence of the functional TIRAP (MAL) S180L polymorphism on tuberculosis (TB) and four autoimmune diseases namely: rheumatoid arthritis (RA), primary Sjögren''s syndrome (pSS), systemic lupus erythematosus (SLE) and type 1 diabetes mellitus (T1D). Methods: This was a case–control and family based association study in which 1325 individuals from a well-defined Colombian population were involved. TIRAP (MAL) S180L genotyping was done by using a polymerase chain reaction-restriction fragment length polymorphism technique and by direct sequencing. Results: Leu180 allele was found to be a protective factor against developing TB (odd ratio (OR): 0.53, 95% confidence interval (CI): 0.29–0.97) and SLE (OR: 0.29, 95% CI: 0.14–0.61) while no significant influence on RA, pSS and T1D was observed. Conclusion: These results support the influence of TIRAP (MAL) S180L polymorphism on TB and indicate that TB and SLE might share a common immunogenetic pathway in the innate immune response. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish. In this study, we isolated the cDNA sequence of Nile tilapia TLR1 (On TLR1). The deduced OnTLR1 protein contains a signal peptide, 7 leucine-rich repeats (LRRs), a C-terminal LRR (LRR-CT), a transmembrane region and a highly conserved TIR domain. In healthy Nile tilapia, the On TLR1 transcript was broadly expressed in all examined tissues, with the highest expression levels in the spleen. After infection with Streptococcus agalactiae , the On TLR1 transcripts were upregulated in the gill and kidney. After stimulation with polyinosinic-polycytidylic acid (poly(I:C)), the expression levels of On TLR1 were significantly downregulated in the intestine, whereas On TLR1 transcripts were significantly upregulated in the kidney. After challenge with lipopolysaccharide (LPS), the expression levels of On TLR1 were significantly upregulated in the spleen and kidney. The subcellular localization showed that On TLR1 was expressed in the cytoplasm. TLR1 significantly increased MyD88-dependent NF-κB activity. However, the results of a pull-down assay showed that On TLR1 did not interact with MyD88 or TIRAP. Binding assays revealed the specificity of OnTLR1 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae , Aeromonas hydrophila and poly(I:C) and LPS. Taken together, these findings suggest that OnTLR1, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion. • The cDNA sequence of TLR1 was identified from tilapia (Oreochromis niloticus). • The change of the TLR1 gene expression was observed following the stimulation of S. agalactiae , LPS and polyI:C. • The overexpression of TLR1 induced significantly the NF-kappaB activity in cell line. • The TLR1 was not interactive with MyD88 nor TIRAP adaptors. [ABSTRACT FROM AUTHOR]

Toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) and toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF) are crucial adaptors of signal transduction for the signaling pathways of toll-like receptors (TLRs). TIRAP and TRIF perform an essential function in an antimicrobial immune response; however, their function in Nile tilapia remains unknown. Herein, TIRAP and TRIF from Nile tilapia were identified and functionally characterized. Phylogenetic analysis showed that OnTIRAP and OnTRIF clustered with corresponding homologs from other fish species, with comparable gene structures to those of select vertebrate TIRAP and TRIF genes, respectively. The expression profiles of OnTIRAP and OnTRIF were broadly distributed in the ten tissues investigated, with high transcript levels noticed in immune organs. The transcription levels of OnTIRAP and OnTRIF were upregulated in response to bacterial and poly (I:C) challenges. GFP signals were only detected in the cytoplasmic region of fish cells transfected with OnTIRAP-GFP and OnTRIF-GFP expression plasmids. Moreover, overexpression of OnTIRAP and OnTRIF activated interferon-β (IFN-β) and activator protein 1 (AP1) reporters in HEK 293 cells. Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter was only observed in OnTRIF-overexpressing HEK 293 cells. Furthermore, the results of the co-immunoprecipitation analysis showed that OnTRIF, but not OnTIRAP, was recruited as an adaptor protein by OnTLR25. This study provides the first evidence on the functions of OnTIRAP and OnTRIF in the immune system of Nile tilapia against pathogens and may serve as the basis for further investigations on TLR signaling in fish. • Nile tilapia TIRAP and TRIF orthologs were cloned. • OnTIRAP and OnTRIF increase significantly after bacterial and poly (I:C) injection. • OnTIRAP activates IFN-β, AP1, and fails to induce the NF-κB reporter in HEK 293 cells. • OnTRIF can activate the NF-κB, IFN-β, and AP1 reporters in HEK 293 cells. • OnTLR25 recruits OnTRIF as its adaptor protein. [ABSTRACT FROM AUTHOR]

Though several genetic variants have been recognized to be associated with susceptibility to Tuberculosis (TB) infection and disease, a recent observation on the association of TIRAP C975T (S180L) variants with TB disease severity in mice model prompted us to assess their relevance in humans. In addition, TIRAP variants have also been reported to be associated with varied circulating Interferon-gamma induced protein (IP-10) levels. We investigated the association of TIRAP variants with severity of TB disease and IP-10 production in humans, which may be useful in predicting poor clinical outcome. Culture positive symptomatic adult pulmonary TB (PTB) patients enrolled between August 2014 and October 2017 were included in this investigation. Allelic discrimination PCR and conventional IP-10 quantification methods were employed for genotyping and IP-10 measurement followed by statistical investigations to analyse patients' variables. Among 211 participants, C/C allele was identified in 70% (n = 147); 26% (n = 55) and 4% (n = 9) had C/T and T/T alleles respectively. There was no significant association between TIRAP variants and smear grade, chest-X-ray score, symptom severity score and circulating IP-10 levels. However, significant association was observed between i) circulating IP-10 levels and time to Mycobacterium Growth Indicator Tube (MGIT) culture conversion (p =0.032); ii) smear grade among active TB patients and circulating IP-10 levels (p =0.032). Although mice experiments showed promising results with more severe disease in C/C and T/T individuals, we did not observe any such association in humans. • Association between TIRAP S180 and correla • Although mice experiments showed a significant association between TIRAP S180 an • Circulating IP-10 levels showed a strong association with smear grade, indicating its role in assessing disease burden and possibly treatment response among active TB cases. [ABSTRACT FROM AUTHOR]