0 5 5Recognition of pathogen-associated molecules in microbes by TLRs leads to activation of transcription factors such as NF-κB that promote increased transcription of proinflammatory cytokines and interferons 1 . All mammalian TLRs, with the exception of TLR3, use the adaptor MyD88 as the receptor-proximal signaling molecule to trigger downstream activation of NF-κB 2 . The association of MyD88 with TLRs facilitates recruitment of members of the IRAK family of kinases that in turn activate the E3 ubiquitin ligase TRAF6 (refs. 3-5). The formation of polyubiquitin chains by TRAF6 serves to bring TAK1 into close proximity with its substrates, including IκB kinases (IKKs). The TAK1-induced phosphorylation and activation of IKKα and IKKβ promotes IKK-induced phosphorylation of IκB proteins 6 that normally sequester NF-κB in an inactive form in the cytoplasm. Phosphorylated forms of IκB are subject to polyubiquitination and subsequently proteasome-dependent degradation, thus liberating NF-κB to translocate to the nucleus and transcriptionally upregulate the expression of a plethora of genes 7 . Most TLRs use this MyD88-dependent pathway to activate NF-κB, but TLR4 can additionally deploy another adaptor protein, TRIF, to trigger a MyD88-independent pathway that also activates NF-κB 8 . Among TLRs, TLR3 uses TRIF as its exclusive receptor-proximal adaptor protein. TRIF interacts with RIP1 kinase to trigger downstream IKK-mediated activation of NF-κB 9,10 . TRAF6 has been reported to associate with TRIF and mediate activation of NF-κB 11-13 , but other studies had concluded that TRAF6 is dispensable for TLR3 signaling 14,15 . Such discrepancies in relation to the role of TRAF6 in TRIF signaling may be due to cell-specific roles for TRAF6 and/or functional redundancy of TRAF6 with other members of the TRAF family 11 . In addition to activation of NF-κB, TRIF can also trigger activation of interferon-regulatory factor (IRF) transcription factors. Thus, TRIF forms a complex with the kinases TBK1 and IKKi (also known as IKKε) and both kinases can catalyze phosphorylation and activation of IRF3 and IRF7, leading to their nuclear translocation and induction of type I interferons 1,16 . The latter are key antiviral molecules that block viral replication 17,18 .It is clear from the above that ubiquitination is important in TLR signal transduction. Additionally, there is an emerging appreciation of the roles of the E3 ubiquitin ligase family of Pellino proteins in TLR signaling. The mammalian Pellino family consists of four members: Pellino1, Pellino2 and splice variants of Pellino3 termed Pellino3 long (Pellino3L; also known as Pellino3a) and Pellino3 short (Pellino3S; also known as Pellino3b) 19,20 . Each Pellino family member contains an N-terminal forkhead-associated (FHA) domain that recognizes phosphothreonine residues and mediates association with IRAKs 21 , and a C-terminal RING-like domain that confers E3 ubiquitin ligase activity and an ability to catalyze lysine 63 (Lys63)-linked polyubiquitination of IRAKs [22][23]...

Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in “danger” recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-β. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-β induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-α induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-β and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKKε-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-β induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-β. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKKε-mediated induction of IRF3 and concomitant IFN-β and RANTES production.

The pathogenesis and complications of type 2 diabetes (T2DM) are closely linked with defective glucose metabolism, obesity, cardiovascular disease and an inability to mount an effective immune response to certain pathogenic organisms. Perturbations in key innate immune receptors known as Toll-like receptors (TLRs) and inflammatory mediators such as IL-6, TNFα and IL-1β have been linked with T2DM. Herein, we sought to establish whether patients with T2DM and underlying complications exhibit perturbations in cytokine and TLR expression. Serum cytokine and mRNA levels of cytokines/TLRs in monocytes (M) and neutrophils (N) were measured in a cohort of 112 diabetic patients: good glycaemic control without complications (GC), good glycaemic control with complications (GCC), poor glycaemic control without complications (PC) and poor glycaemic control with complications (PCC) and compared them with 34 non-diabetic volunteers (NGT). Serum cytokine levels were normal in all study participants. In the GC group, cytokine and TLR gene expression were enhanced compared to NGT. In contrast, suppressed cytokine and TLR gene expression were evident in PC, GCC & PCC groups when compared to the GC. In conclusion, whereas serum pro-inflammatory cytokine levels are unaltered in T2DM patients, differences in inflammatory gene profiles exist among the T2DM patient groups.

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.

Tumour necrosis factor-a (TNF) can activate NF-kB to induce pro-inflammatory genes but can also stimulate the caspase cascade to promote apoptosis. Here we show that deficiency of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis without inhibiting the NF-kB pathway. Suppressed expression of Pellino3 leads to enhanced formation of the death-induced signalling complex, complex II, in response to TNF. We show that Pellino3 targets RIP1, in a TNF-dependent manner, to inhibit TNF-induced complex II formation and caspase 8-mediated cleavage of RIP1 in response to TNF/cycloheximide co-stimulation. Pellino3-deficient mice also show increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and an important determining factor in dictating whether TNF induces cell survival or death.

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Background:The expression of IFN-␤ is under tight regulatory control. Results: NF-B induces YY1 that negatively regulates TLR3-mediated expression of IFN-␤. Conclusion: NF-B, via YY1, causes post-induction repression of IFN-␤ expression in response to TLR3. Significance: This is a novel regulatory mechanism for controlling IFN-␤ expression.

We report here a "green" approach for the synthesis of gold nanoparticles (GNPs) in which the Mentha piperita extract was applied for the bioreduction of chloroauric acid and the stabilization of the formed nanostructures. The obtained GNPs were characterized by UV-Vis absorption spectroscopy and transmission electron microscopy (TEM). The reduction of gold ions with the plant extract leads to the production of nanoparticles with various shapes (spherical, triangular and hexagonal) and sizes (from 10 to 300 nm). The kinetics of the reaction was monitored and various conditions of the synthesis were investigated. As a result, we established protocols optimized towards the synthesis of nanospheres and nanoprisms of gold. The cytotoxic effect of the obtained gold nanoparticles was studied by performing MTT assay, which showed lower cytotoxicity of the biosynthesized GNPs compared to gold nanorods synthesized using the usual seed-mediated growth. The results suggest that the synthesis using plant extracts may be a useful method to produce gold nanostructures for various biological and medical applications.