Publications

Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

Toll-like receptor (TLR) can activate dendritic cells (DC) through common signaling pathways requiring a cytoplasmic adapter, MyD88. However, the signaling is differentially regulated among TLR family members. TLR4 can activate MyD88-deficient bone marrow-derived DC (BMDC), and lead to induction of IFN-inducible genes and up-regulation of co-stimulatory molecules such as CD40, implying that the MyD88-independent signaling pathway functions downstream of TLR4. Because these effects can also be induced by type I IFN, we have analyzed whether type I IFN is involved in TLR4-induced responses. In response to lipopolysaccharide (LPS), IFN-beta gene expression was augmented in both wild-type and MyD88-deficient BMDC. Expression of all IFN-inducible genes except immune-responsive gene 1 (IRG1) was abolished and CD40 up-regulation was decreased in LPS-stimulated BMDC lacking either IFN-alpha/beta receptor (IFN-alpha/betaR) or signal transducer and activator of transcription 1 (STAT-1). Similar to the LPS response, TLR9 signaling can also induce expression of IFN-beta and IFN-inducible genes, and up-regulation of CD40. However, all these effects were MyD88 dependent. Thus, in TLR4 signaling, IFN-beta expression can be induced either by the MyD88-dependent or -independent pathway, whereas, in TLR9 signaling, it is dependent on MyD88. In CpG DNA-stimulated DC, expression of IFN-inducible genes except IRG1 was dependent on type I IFN signaling as in LPS-stimulated DC. However, in contrast to TLR4 signaling, TLR9 signaling requires type I IFN signaling for CD40 up-regulation. Taken together, this study demonstrates differential involvement of type I IFN in TLR4- and TLR9-induced effects on DC.

Recurrent seizures may cause neuronal damage in the hippocampus. As neurons form intimate interactions with astrocytes via glutamate, this neuron–glia circuit may play a pivotal role in neuronal excitotoxicity following such seizures. On the other hand, astrocytes contact vascular endothelia with their endfeet. Recently, we found kainic acid (KA) administration induced microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin E2 (PGE2) receptor EP3 in venous endothelia and on astrocytes, respectively. In addition, mice deficient in mPGES-1 exhibited an improvement in KA-induced neuronal loss, suggesting that endothelial PGE2 might modulate neuronal damage via astrocytes. In this study, we therefore investigated whether the functional associations between endothelia and astrocytes via endothelial mPGES-1 lead to neuronal injury using primary cultures of hippocampal slices. We first confirmed the delayed induction of endothelial mPGES-1 in the wild-type (WT) slices after KA-treatment. Next, we examined the effects of endothelial mPGES-1 on Ca2+ levels in astrocytes, subsequent glutamate release and neuronal injury using cultured slices prepared from WT and mPGES-1 knockout mice. Moreover, we investigated which EP receptor on astrocytes was activated by PGE2. We found that endothelial mPGES-1 produced PGE2 that enhanced astrocytic Ca2+ levels via EP3 receptors and increased Ca2+-dependent glutamate release, aggravating neuronal injury. This novel endothelium–astrocyte–neuron signaling pathway may be crucial for neuronal damage after repetitive seizures, and hence could be a new target for drug development.

No abstract is provided for this article.

Oral Diseases (2012) Objectives: This investigation aimed to conduct a case–control study of mandibular morphology and dental anomalies to propose a relationship between mandibular/dental phenotypes and deficiency of CCAAT/enhancer‐binding protein beta (CEBPB). Materials and methods: Skulls of CEBPB −/− , CEBPB +/− and CEBPB +/+ mice were inspected with micro‐computed tomography. Mandibular morphology was assessed with a method of Euclidean distance matrix analysis. Results: Elongation of the coronoid process was identified in CEBPB +/− ( P ≤ 0.046) and CEBPB −/− 12‐month‐olds ( P ≤ 0.028) but not in 14‐day‐olds ( P ≥ 0.217) and 0‐day‐olds ( P ≥ 0.189) of either genotype. Formation of supernumerary teeth in CEBPB −/− adult mice was demonstrated (χ 2 = 6.00, df = 1, P = 0.014). Conclusions: CEBPB deficiency was related to elongation of the coronoid process and formation of supernumerary teeth. The mandibular and dental phenotypes of CEBPB deficiency were unseen by the 14th day after birth. Future investigations into the influence of CEBPB on mandibular and dental development are needed.

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by the loss of nigrostriatal dopaminergic neurons. Although increased production of prostaglandin E2 (PGE2) has been implicated in tissue damage in several pathological settings, the role of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE2 synthesis, in dopaminergic neurodegeneration remains unclear. Here we show that mPGES-1 is up-regulated in the dopaminergic neurons of the substantia nigra of postmortem brain tissue from PD patients and in neurotoxin 6-hydroxydopamine (6-OHDA)-induced PD mice. The expression of mPGES-1 was also up-regulated in cultured dopaminergic neurons stimulated with 6-OHDA. The genetic deletion of mPGES-1 not only abolished 6-OHDA-induced PGE2 production but also inhibited 6-OHDA-induced dopaminergic neurodegeneration both in vitro and in vivo. Nigrostriatal projections, striatal dopamine content, and neurological functions were significantly impaired by 6-OHDA administration in wild-type (WT) mice, but not in mPGES-1 knockout (KO) mice. Furthermore, in cultured primary mesencephalic neurons, addition of PGE2 to compensate for the deficiency of 6-OHDA-induced PGE2 production in mPGES-1 KO neurons recovered 6-OHDA toxicity to almost the same extent as that seen in WT neurons. These results suggest that induction of mPGES-1 enhances 6-OHDA-induced dopaminergic neuronal death through excessive PGE2 production. Thus, mPGES-1 may be a valuable therapeutic target for treatment of PD.

Significance Chronic obstructive pulmonary disease (COPD/emphysema) is a life-threatening disorder with high morbidity and mortality that is prevalent worldwide. It is characterized by destruction of the alveolar wall and a decline in lung function. However, few palliative therapies are currently available. We show that FCH and double SH3 domains 1 ( Fchsd1 ) knockout mice are protected against airspace enlargement induced by elastase. FCHSD1 deficiency enhanced nuclear translocation of NRF2 and attenuated elastase-induced reduction in SIRT1 levels, which reduced inflammation and apoptosis of lung cells. These data indicate that FCHSD1 promotes the initiation phase of emphysema development and indicate potential therapeutic targets for COPD.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in the early innate immune response to microbial challenge. To clarify the functional roles of TLRs 2 and 4 in mast cells, we examined bone marrow–derived mast cells (BMMCs) from TLR2 or TLR4 gene-targeted mice. Peptidoglycan (PGN) from Staphylococcus aureus stimulated mast cells in a TLR2-dependent manner to produce TNF-α, IL-4, IL-5, IL-6, and IL-13, but not IL-1β. In contrast, LPS from Escherichia coli stimulated mast cells in a TLR4-dependent manner to produce TNF-α, IL-1β, IL-6, and IL-13, but not IL-4 nor IL-5. Furthermore, TLR2- but not TLR4-dependent mast cell stimulation resulted in mast cell degranulation and Ca2+ mobilization. In a mast cell–dependent model of acute sepsis, TLR4 deficiency of BMMCs in mice resulted in significantly higher mortality because of defective neutrophil recruitment and production of proinflammatory cytokines in the peritoneal cavity. Intradermal injection of PGN led to increased vasodilatation and inflammation through TLR2-dependent activation of mast cells in the skin. Taken together, these results suggest that direct activation of mast cells via TLR2 or TLR4 by respective microligands contributes to innate and allergic immune responses.

A lipopolysaccharide from Pantoea agglomerans (LPSpa) has been applied to various fields for human use as a Toll-like receptor 4 ligand and its safety has been confirmed. Here, we showed for the first time the application of LPSpa as an effective mucosal adjuvant for activating vaccine-induced antigen specific immune responses. Mice sublingually immunized with influenza vaccine (HA split vaccine) with LPSpa induced both HA-specific IgG (systemic) and IgA (mucosal) antibody responses, which led to a significant increase in survival rate against lethal influenza virus challenge compared with subcutaneous vaccination. After sublingual administration of ovalbumin with LPSpa, ovalbumin-specific mucosal IgA responses were induced at both mucosal surfaces close to the immunized site and at remote mucosal surfaces. Sublingual administration of LPSpa evoked local antigen-uptake by dendritic cells in cervical lymph nodes. LPSpa induced cytokine production and the maturation and proliferation of innate immune cells via Toll-like receptor 4 in dendritic cells. Collectively, these results suggest that LPSpa can be used as an effective mucosal adjuvant to stimulate and activate local innate immune cells to improve and enhance mucosal vaccine potency against various pathogens.

MyD88 is an adaptor protein that plays a major role in TLR/IL‐1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo , MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis . Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild‐type (WT) mice ( P <0.01). The pulmonary tissue levels of mRNA for iNOS and IL‐18 were slightly lower, but levels of mRNA for IL‐1β, IL‐2, IL‐4, IL‐6, IL‐10, IFN‐γ, and TGF‐β were higher in MyD88 KO mice. IFN‐γ, TNF‐α, IL‐1β, and IL‐12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF‐α, IL‐12, and ICAM‐1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF‐κB activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2‐specific, MyD88‐independent IL‐1 receptor/TLR‐mediated pathway to activate NF‐κB in the host defense against mycobacterial infection.

Rheumatology has pioneered in the study of autoantibodies by showing that they are not only involved in pathogenesis but are also highly useful as diagnostic biomarkers.The diagnostic biomarker aspect of autoimmunity has gained increasing importance in cancer and many of the insights gained in Rheumatology have contributed to understanding the significance of autoantibodies in cancer.Features of autoantibodies in rheumatic disorders: In rheumatic diseases no individual autoantibody-antigen system has sufficient combination of sensitivity and specificity to serve as a useful diagnostic biomarker.Instead, several antigen-antibody systems constructed as profiles of biomarkers are highly effective in distinguishing one disorder from another.In lupus, anti-double strand DNA and anti-Sm distinguishes it from scleroderma, where the profile is anti-DNA topoisomerase 1 and anti-centromere proteins.The autoantigensare cell components involved in universal and basic gene expression pathways, such as Sm in precursor mRNA splicing and DNA topoisomerase 1 in DNA replication and transcription [1].Features of autoantibodies in cancer: Autoantibodies in cancer target intracellular molecules referred to as TAAs (tumor-associated antigens).As in rheumatic disorders, no individual autoantibody-antigen system has sensitivity and specificity to serve as a stand-alone diagnostic marker [2].Most tumors show multiple antibody specificities and with panels of TAAanti-TAAs (analogous toprofiles) the cumulative sensitivity and specificity reaches diagnostic significance.Different tumorigenesis pathways are

Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation.

A complex mechanism may be operational for dendritic cell (DC) maturation, wherein Toll-like receptor and other signaling pathways may be coordinated differently depending on the nature of the pathogens, in order for DC maturation to be most effective to a given threat. Here, we show that IFN-α/β signaling is selectively required for the maturation of DCs induced by double-stranded RNA or viral infection in vitro . Interestingly, the maturation is still observed in the absence of either of the two target genes of IFN-α/β, TLR3 and PKR (double-stranded-RNA-dependent protein kinase R), indicating the complexity of the IFN-α/β-induced transcriptional program in DCs. We also show that the DCs stimulated in vivo by these agents can migrate into the T cell zone of the spleen but fail to mature without the IFN signal. The immune system may have acquired the selective utilization of this cytokine system, which is essential for innate antiviral immunity, to effectively couple with the induction of adaptive immunity.

The T-box containing Tbr2 gene encodes for a transcription factor essential for the specification of the intermediate neural progenitors (INPs) originating the excitatory neurons of the cerebral cortex. However, its overall mechanism of action, direct target genes and cofactors remain unknown. Herein, we carried out global gene expression profiling combined with genome-wide binding site identification to determine the molecular pathways regulated by TBR2 in INPs. This analysis led to the identification of novel protein–protein interactions that control multiple features of INPs including cell-type identity, morphology, proliferation and migration dynamics. In particular, NEUROG2 and JMJD3 were found to associate with TBR2 revealing unexplored TBR2-dependent mechanisms. These interactions can explain, at least in part, the role of this transcription factor in the implementation of the molecular program controlling developmental milestones during corticogenesis. These data identify TBR2 as a major determinant of the INP-specific traits by regulating both genetic and epigenetic pathways.

MyD88 is a key adaptor molecule for signalling via Toll‐like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut‐associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88 –/– , TLR2 –/– and TLR4 –/– mice. In MyD88 –/– mice, the organogenesis of PPs was not disturbed. However, PPs in 2‐week‐old MyD88 –/– mice were significantly smaller than those in MyD88 +/– mice. Also, in 2‐week‐old TLR4 –/– , but not TLR2 –/– mice, PPs did not develop rapidly. The development of PPs in MyD88 –/– and TLR4 –/– mice was completely recovered in 10 weeks. PP cells from MyD88 –/– mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10‐week‐old MyD88 –/– mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88‐independent signalling is critically involved in the development of GALT in adult mice.

Multiple lung pathogens such as chemical agents, H5N1 avian flu, or SARS cause high lethality due to acute respiratory distress syndrome. Here we report that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced acute lung injury (ALI). We show that TLR4-TRIF-TRAF6 signaling is a key disease pathway that controls the severity of ALI. The oxidized phospholipid (OxPL) OxPAPC was identified to induce lung injury and cytokine production by lung macrophages via TLR4-TRIF. We observed OxPL production in the lungs of humans and animals infected with SARS, Anthrax, or H5N1. Pulmonary challenge with an inactivated H5N1 avian influenza virus rapidly induces ALI and OxPL formation in mice. Loss of TLR4 or TRIF expression protects mice from H5N1-induced ALI. Moreover, deletion of ncf1, which controls ROS production, improves the severity of H5N1-mediated ALI. Our data identify oxidative stress and innate immunity as key lung injury pathways that control the severity of ALI.