SUMMARY “Triple-defective” (3d) mice carrying a mutation in UNC93B1, a chaperone for the endosomal nucleic-acid sensing (NAS) Toll-Like Receptors TLR3, TLR7 and TLR9, are highly susceptible to Toxoplasma gondii infection. However, none of the single or even the triple NAS-TLR deficient animals recapitulated the 3d susceptible phenotype to Toxoplasma infection. Investigating this further, we find that while parasite RNA and DNA activate innate immune responses via the NAS-TLRs 7 and 9, TLR11 and TLR12 working as heterodimers are required for sensing and responding to Toxoplasma profilin. Consequently, the triple TLR7/TLR9/TLR11 deficient mice are highly susceptible to T. gondii infection, recapitulating the phenotype of 3d mice. Humans lack functional TLR11 and TLR12 genes. Consistently, human cells produce high levels of pro-inflammatory cytokines in response to parasite derived RNA and DNA, but not to Toxoplasma profilin supporting a more critical role for NAS-TLRs in human toxoplasmosis.

Toll/interleukin-1 (TIR)receptor-containing adapters are critical in orchestrating the different signal transduction pathways following Toll-like receptor (TLR) activation. MyD88 adapter-like (Mal), also termed TIRAP, is involved in bridging MyD88 to the receptor complex for TLR-2 and TLR4 signaling in response to bacterial infection. We have previously reported an interaction between Mal and tumor necrosis factor receptor-associated factor 6 (TRAF6) via a TRAF6-binding motif, the disruption of which inhibited TLR-mediated NF-B-luciferase reporter activity. Given the recent report of intracellular TRAM localization promoting sequential signaling in TLR4 responses, we further characterized Mal interaction with TRAF6, the cellular localization, and the outcomes of disrupting this association on TLR inflammatory responses. We found that Mal and TRAF6 directly interact in response to TLR2 and TLR4 stimulation, although membrane localization is not necessary to facilitate interaction. Critically, reconstitution of murine Mal-deficient macrophages with MalE190A, containing a mutation within the TRAF6-binding motif, fails to reconstitute the proinflammatory response to TLR2 and TLR4 ligands compared with wild type Mal. Furthermore, Mal interaction with TRAF6 mediates Ser phosphorylation of the p65 subunit of NF-B and thus controls transcriptional activation but not nuclear translocation of NF-B. This study characterizes the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2-and TLR4-induced transactivation of NF-B and regulation of the subsequent pro-inflammatory response.

TLRs are critical pattern recognition receptors that recognize bacterial and viral pathogen-associated molecular patterns leading to innate and adaptive immune responses. TLRs signal via homotypic interactions between their cytoplasmic Toll/IL-1R (TIR) domains and TIR domain-containing adaptor proteins. Over the course of evolution, viruses have developed various immune evasion strategies, one of which involves inhibiting TLR signaling pathways to avoid immune detection. Thus, vaccinia virus encodes the A46 protein, which binds to multiple TIR-domain containing proteins, ultimately preventing TLRs from signaling. We have identified an 11-aa–long peptide from A46 (termed viral inhibitor peptide of TLR4, or VIPER), which, when fused to a cell-penetrating delivery sequence, potently inhibits TLR4-mediated responses. VIPER was TLR4 specific, being inert toward other TLR pathways, and was active in murine and human cells and in vivo, where it inhibited LPS-induced IL-12p40 secretion. VIPER also prevented TLR4-mediated MAPK and transcription factor activation, suggesting it acted close to the TLR4 complex. Indeed, VIPER directly interacted with the TLR4 adaptor proteins MyD88 adaptor-like (Mal) and TRIF-related adaptor molecule (TRAM). Viral proteins target host proteins using evolutionary optimized binding surfaces. Thus, VIPER possibly represents a surface domain of A46 that specifically inhibits TLR4 by masking critical binding sites on Mal and TRAM. Apart from its potential therapeutic and experimental use in suppressing TLR4 function, identification of VIPER’s specific binding sites on TRAM and Mal may reveal novel therapeutic target sites. Overall, we demonstrate for the first time disruption of a specific TLR signaling pathway by a short virally derived peptide.

DNA hypomethylation is a characteristic feature of systemic lupus erythematosus (SLE) immune cells. Numerous reports have implicated the involvement of the MEK/ERK pathway in the reduction of DNA methyltransferase (DNMT) expression, hence inducing the transcription of methylation-sensitive genes in SLE patients. However, the molecular mechanisms involved remain unclear. Here, we investigated whether the catalytic subunit of protein phosphatase 2A (PP2Ac), which is overexpressed in SLE T-cells, contributes to reduced DNA methylation. We show that both chemical suppression and siRNA silencing of PP2Ac in T-cells resulted in sustained phosphorylation of MEK and ERK following stimulation with phorbol 12-myristate 13-acetate and ionomycin. Furthermore, PP2Ac suppression resulted in increased DNMT enzyme activity, DNA hypermethylation, and decreased expression of methylation-sensitive genes. Similarly, in SLE T-cells, suppression of PP2Ac resulted in increased MEK/ERK phosphorylation, enhanced DNMT1 expression and suppressed expression of the methylation-sensitive CD70 gene. Our results demonstrate that PP2A regulates DNA methylation by influencing the phosphorylation of MEK/ERK. We propose that enhanced PP2Ac in SLE T-cells may dephosphorylate and activate the signaling pathway upstream of DNMT1, thus disturbing the tight control of methylation-sensitive genes, which are involved in SLE pathogenesis.

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain-containing adaptor-inducing IFN-b (TRIF)-dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor-like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN-b production. Co-immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand-induced IFN-b induction. Interestingly, Mal does not affect the induction of IL-6 and TNF-a upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN-b induction through the positive regulatory domains I-III enhancer element of the IFN-b gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)-induced IRF7 activation and concomitant IFN-b production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN-b production.

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The adapter protein MyD88 adapter-like (Mal), encoded by TIR-domain containing adapter protein (Tirap) (MIM 606252), is the most polymorphic of the five adapter proteins involved in Toll-like receptor signaling, harboring eight non-synonymous single nucleotide polymorphisms in its coding region. We screened reported mutations of Mal for activity in reporter assays to test the hypothesis that variants of Mal existed with altered signaling potential. A TIR domain variant, Mal D96N (rs8177400), was found to be inactive. In reconstituted cell lines, Mal D96N acted as a hypomorphic mutation, with impaired cytokine production and NF-B activation upon lipopolysaccharide or PAM 2 CSK 4 stimulation. Moreover, co-immunoprecipitation studies revealed that Mal D96N is unable to interact with MyD88, a prerequisite for downstream signaling to occur. Computer modeling data suggested that residue 96 resides in the MyD88 binding site, further supporting these findings. Genotyping of Mal D96N in three different cohorts suggested that it is a rare mutation. We, thus, describe a rare variant in Mal that exerts its effect via its inability to bind MyD88.

Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. Unlike wildtype MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. We observed that IRAK4 co-localizes with MyD88 in these aggregates, and thus these foci appear to be "Myddosomes." The MyD88 S34Y lossof-function mutant demonstrates how proper cellular localization of MyD88 to the Myddosome is a feature required for MyD88 function.Toll-like receptors (TLRs) 4 are a part of the innate immune system and play a critical role in host defense against a variety of pathogens. Ten functional TLRs in humans and 12 in mice have been identified to date (1). Each TLR recognizes different categories of evolutionarily conserved patterns called pathogen-associated molecular patterns in microbes. For example, TLR4 recognizes lipopolysaccharide (LPS) from the cell walls of bacteria, whereas CpG-rich DNA from bacteria and viruses acts as the ligand for TLR9.Much of the diversity of TLR pathways can be attributed to differential use of the TIR domain-containing adapter proteins. There are four adapter proteins: MyD88, MyD88 adapter-like (Mal), also called TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter-inducing interferon-␤ (TRIF), and TRIF-related adaptor molecule (TRAM).MyD88 is a key adapter protein involved in signaling from all of the TLRs except TLR3 and the TRIF-mediated arm of TLR4 signaling. Thus, depending on its use, TLR pathways can be divided into MyD88-dependent and MyD88-independent pathways. The engagement of a particular TLR by its ligand triggers a signaling cascade, which culminates in the secretion of various proinflammatory cytokines and interferons.MyD88 is a 296-amino acid protein with a modular domain structure. In addition to the N-terminal TIR domain, it has a death domain at its C terminus. The crystal structure of the MyD88 death domain has recently been resolved, and it reveals that MyD88 exists in oligomeric form in solution (2). These oligomers of MyD88 can recruit the death domains of interleukin-1 receptor-associated kinase (IRAK)4 and IRAK2 to form a ternary complex called the Myddosome.The importance of single nucleotide polymorphisms (SNPs) in TLR-related proteins has recently been brought to the forefront, with many studies implicating SNPs in disease susceptibility and outcome (3-7). Mal and MyD88 are the most polymorphic of the four adapter proteins, and Mal has received the most scrutiny in this regard (8 -10). However, other than a study by Von Bernuth et al., where children with MyD88 ...