Publications

Role of adapters in Toll-like receptor signalling

Toll-like receptors (TLRs) play a critical role in the detection of invading pathogens within the body and the subsequent immune response. Individual TLRs recognize distinct microbial components. The TLRs are a type 1 transmembrane receptor that possess an extracellular leucine-rich repeat domain and cytoplasmic domain homologous with that of the interleukin 1 receptor (IL-1R) family. Upon stimulation, TLR recruits the IL-1R-associated kinase (IRAK) via the adapter MyD88, ultimately leading to the activation of nuclear factor-κB. Cytokine production in response to all TLR ligands is completely abolished in MyD88-deficient cells, indicating that MyD88 is an essential signalling molecule shared among members of the IL-1R/Toll family. However, several novel adaptor molecules have recently been identified. Evidence is now accumulating showing that differential utilization of these adaptors may activate overlapping as well as distinct signalling pathways, and ultimately give rise to distinct biological effects exerted by individual TLR family members.

Lack of Poly(ADP-Ribose) Polymerase-1 Gene Product Enhances Cellular Sensitivity to Arsenite

Arsenite (As3+) has long been known to induce cancer and other degenerative diseases. Arsenite exerts its toxicity in part by generating reactive oxygen species. Identification of genetic factors that contribute to arsenic mutagenicity and carcinogenicity is critical for the treatment and prevention of arsenic exposure in human population. As poly(ADP-ribose) polymerase (PARP) is critical for genomic DNA stability, role of PARP-1 was evaluated in arsenic-induced cytotoxic and genotoxic effects. Our study revealed that telomere attrition, probably owing to arsenite-induced oxidative stress, was much more pronounced in PARP-1−/− mouse embryonic fibroblasts (MEF; 40%) compared with PARP-1+/+ MEFs (10-20%). Correlation observed between telomere reduction and apoptotic death in PARP-1 null cells strongly indicates that the telomere attrition might be a trigger for enhanced apoptotic death after arsenite treatment. Elevated DNA damage detected by alkaline comet assay points to an impaired repair ability of arsenite-induced DNA lesions in PARP-1−/− MEFs. Consistent with elevated DNA damage, increased micronuclei induction reflecting gross genomic instability was also observed in arsenite-treated PARP-1−/− MEFs. Microarray analysis has revealed that arsenite treatment altered the expression of about 311 genes majority of which have known functions in cellular responses to stress/external stimulus and cell growth and/or maintenance. Our results suggest an important role for PARP-1 gene product in the maintenance of chromosome-genome stability in response to arsenite-induced DNA damage.

The chemotherapeutic agent DMXAA potently and specifically activates the TBK1–IRF-3 signaling axis

Vascular disrupting agents (VDAs) represent a novel approach to the treatment of cancer, resulting in the collapse of tumor vasculature and tumor death. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is a VDA currently in advanced phase II clinical trials, yet its precise mechanism of action is unknown despite extensive preclinical and clinical investigations. Our data demonstrate that DMXAA is a novel and specific activator of the TANK-binding kinase 1 (TBK1)–interferon (IFN) regulatory factor 3 (IRF-3) signaling pathway. DMXAA treatment of primary mouse macrophages resulted in robust IRF-3 activation and ∼750-fold increase in IFN-β mRNA, and in contrast to the potent Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS), signaling was independent of mitogen-activated protein kinase (MAPK) activation and elicited minimal nuclear factor κB–dependent gene expression. DMXAA-induced signaling was critically dependent on the IRF-3 kinase, TBK1, and IRF-3 but was myeloid differentiation factor 88–, Toll–interleukin 1 receptor domain–containing adaptor inducing IFN-β–, IFN promoter-stimulator 1–, and inhibitor of κB kinase–independent, thus excluding all known TLRs and cytosolic helicase receptors. DMXAA pretreatment of mouse macrophages induced a state of tolerance to LPS and vice versa. In contrast to LPS stimulation, DMXAA-induced IRF-3 dimerization and IFN-β expression were inhibited by salicylic acid. These findings detail a novel pathway for TBK1-mediated IRF-3 activation and provide new insights into the mechanism of this new class of chemotherapeutic drugs.

A case with Mcleod syndrome associated with a novel mutation in XK gene

No abstract is provided for this article.

Involvement of Toll-Like Receptor 2 in Experimental Invasive Pulmonary Aspergillosis

Aspergillus fumigatus, an opportunistic fungal pathogen, causes severe and usually fatal invasive pulmonary aspergillosis in immunocompromised hosts. Interestingly, Drosophila cells lacking the Toll protein are prone to A. fumigatus infection. In the current study, we looked for the involvement of Toll-like receptor 2 (TLR2) in the recognition of A. fumigatus by analyzing the in vivo and ex vivo responses of immunocompromised TLR2(-/-) and TLR2(+/+) mice to this fungus. Upon intratracheal administration of conidia, survival and tumor necrosis factor alpha (TNF-alpha), interleukin-12, and macrophage inhibitory protein-2 alpha concentrations in the airspaces of TLR2(-/-) mice were significantly lower than those of TLR2(+/+) animals. In vitro analysis of TNF-alpha production by conidia-challenged alveolar macrophages from TLR2(-/-) revealed a significant deficiency in comparison with macrophages from TLR2(+/+) mice. Infected TLR2(-/-) mice also have a higher respiratory distress and a higher pathogen burden than TLR2(+/+) mice. These data demonstrate that TLR2 plays a significant role in the defense of the host against A. fumigatus infection.

Toll-Like Receptor 4 Mediates the Antitumor Host Response Induced by a 55-Kilodalton Protein Isolated from <i>Aeginetia indica</i> L., a Parasitic Plant

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-κB-dependent reporter plasmid, AILb-A induced NF-κB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-κB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Oestrogen at the neonatal stage is critical for the reproductive ability of male mice as revealed by supplementation with 17beta-oestradiol to aromatase gene (Cyp19) knockout mice

Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to oestrogens. We generated CYP19 knockout (ArKO) mice by targeted disruption of Cyp19 and studied the role of oestrogens in male reproductive ability. Approximately 85% of ArKO males were unable to sire offspring. However, no obvious difference was found in testicular and epididymal weights, numbers of sperm in the epididymis or the ability of sperm to fertilize eggs in vitro between wild-type and ArKO males. An examination of mating behaviour demonstrated that ArKO males showed an impairment in mounting behaviour against sexually mature females. The inability of more than 90% of ArKO males to sire offspring was reversed by repeated subcutaneous injections of 17beta-oestradiol when initiated on the day of birth. The effects of 17beta-oestradiol on reproduction were concentration dependent and evident when supplementation was initiated on day 7, but not on day 15 after birth. These findings suggest that oestrogens acting during neonatal life are required for normal mating behaviour in adulthood.

Deletion of Tribbles Pseudokinase-3 Ameliorates Retinal Function in CEP290RD16 Mice

Pleiotropic function of Toll-like receptors

A group of type I transmembrane proteins, Toll-like receptors (TLRs) discriminate various microorganism-associated molecular structures that can function as immune adjuvants. Each TLR signaling has an overlapping but distinct function, which largely depends on intracellular adaptor molecules. Clarifying the functions and signaling of TLRs should provide us with critical information for manipulating the host defense mechanism.

NF-IL6 and NF-kappa B in cytokine gene regulation.

No abstract is provided for this article.

Self-DNA release mediates the adjuvant effects of aluminum salts

No abstract is provided for this article.

Glucocorticoid augments lipopolysaccharide-induced activation of the I B -dependent genes encoding the anti-microbial glycoproteins lipocalin 2 and pentraxin 3

Bacterial lipopolysaccharide (LPS), one of the most potent inducers of inflammation, activates the transcription factor NF-κB to induce expression of both proinflammatory mediators and anti-microbial glycoproteins such as lipocalin 2 (Lcn2) and pentraxin 3 (PTX3) in macrophages. Glucocorticoids are known to inhibit LPS-induced expression of proinflammatory cytokines via glucocorticoid receptor (GR)-mediated transrepression of NF-κB, whereas their effect on induction of anti-microbial effectors has remained to be elucidated. Here we show that the synthetic glucocorticoid dexamethasone (Dex) strongly enhances LPS-induced transcription of Lcn2 and Ptx3, although Dex by itself fails to trigger their transcription. In macrophages deficient in IκBζ (an inducible coactivator of NF-κB), Lcn2 and Ptx3 are not activated by LPS either alone or in combination with Dex. Association of GR as well as Brg1 (a subunit of the chromatin remodelling Swi/Snf complex) with a functional glucocorticoid response element in Lcn2 requires both the costimulation with LPS and the presence of IκBζ. Although Ptx3 does not contain the element, LPS induces recruitment of Dex-liganded GR to NF-κB-binding sites in regulatory regions of Ptx3, an event that does not occur in IκBζ-deficient macrophages. Thus glucocorticoids likely regulate infection-induced inflammation by increasing anti-microbial effectors in an IκBζ-dependent manner, while repressing proinflammatory genes.

Molecular and cellular mechanisms of DNA vaccines

Although DNA vaccines are already in use for treatment of some animal diseases, they suffer from lower immunogenicity in humans which limits their effectiveness. Thus, recent studies have been focused on strategies to improve the immunogenicity of DNA vaccines. However, there is little known about the molecular and immunological mechanisms by which DNA vaccines work. It has long been the central dogma that DNA vaccine immunogenicity can be attributed to its immunostimulatory CpG motifs acting as 'a built-in adjuvant', which is recognized by Toll-like receptor (TLR) 9, the sole receptor for CpG motifs. Recent research, however, has provided evidence for a new mechanism of action for DNA vaccines. It was reported that the adjuvant effect of plasmid DNA is mediated by its double-stranded structure, which activates TBK1-dependent innate immune signaling pathways in the absence of TLRs. Moreover, TBK1-signaling may delineate direct or indirect (cross) antigen presentation through distinct types of cells in vivo, critical for the induction of antigen-specific CD4+ or CD8+ T cells, respectively. This additional information about the mechanism of action of DNA vaccines will lead to improvements in their efficacy and safety.

Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus

Raised serum levels of interferon (IFN)-α have been observed in systemic lupus erythematosus (SLE) patients, and these levels are correlated with both disease activity and severity. The origin of this IFN-α is still unclear, but increasing evidence suggests the critical involvement of activated plasmacytoid predendritic cells (PDCs). In SLE patients, DNA and RNA viruses, as well as immune complexes (ICs), that consist of autoantibodies specific to self-DNA and RNA protein particles can stimulate production of IFN-α. We have developed three series of oligonucleotide (ODN)-based inhibitors of Toll-like receptor (TLR) signaling. These ODNs include inhibitors of TLR9, inhibitors of TLR7 but not TLR9, and sequences that inhibit both TLR7 and TLR9. Specificity of these inhibitors is confirmed by inhibition of IFN-α production by PDCs in response to DNA or RNA viruses. We show that mammalian DNA and RNA, in the form of ICs, are potent self-antigens for TLR9 and TLR7, respectively, and induce IFN-α production by PDCs. This work suggests that TLRs may have a critical role in the promotion of lupus through the induction of IFN-α by PDCs. These inhibitors of TLR signaling thus represent novel therapeutic agents with potential for the treatment of lupus.

Phosphorylation-dependent Regnase-1 release from endoplasmic reticulum is critical in IL-17 response

Regnase-1 (also known as Zc3h12a or MCPIP-1) is an endoribonuclease involved in mRNA degradation of inflammation-associated genes. Regnase-1 is inactivated in response to external stimuli through post-translational modifications including phosphorylation, yet the precise role of phosphorylation remains unknown. Here, we demonstrate that interleukin (IL)-17 induces phosphorylation of Regnase-1 in an Act1-TBK1/IKKi–dependent manner, especially in nonhematopoietic cells. Phosphorylated Regnase-1 is released from the endoplasmic reticulum (ER) into the cytosol, thereby losing its mRNA degradation function, which leads to expression of IL-17 target genes. By using CRISPR/Cas-9 technology, we generated Regnase-1 mutant mice, in which IL-17–induced Regnase-1 phosphorylation is completely blocked. Mutant mice (Regnase-1AA/AA and Regnase-1ΔCTD/ΔCTD) were resistant to the IL-17–mediated inflammation caused by T helper 17 (Th17) cells in vivo. Thus, Regnase-1 plays a critical role in the development of IL-17–mediated inflammatory diseases via the Act1-TBK1-IKKi axis, and blockade of Regnase-1 phosphorylation sites may be promising for treatment of Th17-associated diseases.

<scp>TDAG51</scp> promotes transcription factor <scp>FoxO1</scp> activity during <scp>LPS</scp>‐induced inflammatory responses

No abstract is provided for this article.

Downstream Signals for MyD88-Mediated Phagocytosis of <i>Borrelia burgdorferi</i> Can Be Initiated by TRIF and Are Dependent on PI3K

We previously have shown that MyD88 is important for uptake of Borrelia burgdorferi by bone marrow derived macrophages (BMDMs). The mechanism by which MyD88 is involved in uptake of B. burgdorferi is currently is not well characterized. Here, we report that MyD88-mediated defect in the phagocytosis of B. burgdorferi can be complemented by TLR3/Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) activation in BMDMs from MyD88(-/-) mice. This effect of TLR3/TRIF activation was not due to its induction of type I IFNs, suggesting instead a convergence of signaling pathways downstream of MyD88 and TRIF. To characterize signaling pathways involved in MyD88-mediated phagocytosis of B. burgdorferi, BMDMs were treated with specific inhibitors of MAPK, protein kinase C, JAK/STAT, or PI3K. Only inhibition of PI3K resulted in a significant decrease of B. burgdorferi uptake. Consistent with this, B. burgdorferi activation of MyD88 or TLR3/TRIF signaling resulted in increased activity of PI3K. Additionally, association of B. burgdorferi with actin-related protein (Arp2/3) complexes, which facilitate actin rearrangements during phagocytosis, was similarly reduced in MyD88(-/-) BMDMs and in BMDMs treated with a PI3K inhibitor. Taken together, these findings define an essential pathway whereby downstream signals from MyD88 or TRIF converge on PI3K, which triggers actin polymerization to initiate the phagocytosis of B. burgdorferi.

TLR9 signalling in microglia attenuates seizure-induced aberrant neurogenesis in the adult hippocampus

Pathological conditions such as epilepsy cause misregulation of adult neural stem/progenitor populations in the adult hippocampus in mice, and the resulting abnormal neurogenesis leads to impairment in learning and memory. However, how animals cope with abnormal neurogenesis remains unknown. Here we show that microglia in the mouse hippocampus attenuate convulsive seizure-mediated aberrant neurogenesis through the activation of Toll-like receptor 9 (TLR9), an innate immune sensor known to recognize microbial DNA and trigger inflammatory responses. We found that microglia sense self-DNA from degenerating neurons following seizure, and secrete tumour necrosis factor-α, resulting in attenuation of aberrant neurogenesis. Furthermore, TLR9 deficiency exacerbated seizure-induced cognitive decline and recurrent seizure severity. Our findings thus suggest the existence of bidirectional communication between the innate immune and nervous systems for the maintenance of adult brain integrity.