Publications

An analog of 1α,25-dihydroxyvitamin D 3 , 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components. Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects with Salmonella lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from Staphylococcus aureus , both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti-CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed with MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed with LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic cells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.

No abstract is provided for this article.

Central control of energy balance depends on the ability of proopiomelanocortin (POMC) or agouti-related protein (Agrp) hypothalamic neurons to sense and respond to changes in peripheral energy stores. Leptin and insulin have been implicated as circulating indicators of adiposity, but it is not clear how changes in their levels are perceived or integrated by individual neuronal subtypes. We developed mice in which a fluorescent reporter for PI3K activity is targeted to either Agrp or POMC neurons and used 2-photon microscopy to measure dynamic regulation of PI3K by insulin and leptin in brain slices. We show that leptin and insulin act in parallel to stimulate PI3K in POMC neurons but in opposite ways on Agrp neurons. These results suggest a new view of hypothalamic circuitry, in which the effects of leptin and insulin are integrated by anorexigenic but not by orexigenic neurons.

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-α and IL-6. Administration of anti-TNF-α Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-α as the molecular mediator of bacterial clearance and septic shock.

IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1β and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1β, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1β and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1β production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection.

Hypoxia-ischaemia (HI) is a major cause of neonatal brain injury. Although a number of biochemical cascades have been implicated, the downstream targets, at the level of transcriptional regulation still remain unclear. The signal transduction and activator of transcription factor 3 (STAT3) is strongly upregulated following peripheral and central trauma and thus a possible candidate. In the current study, we investigated the regulation and functional role of STAT3 in the neonatal HI brain injury in the Rice-Vannucci model in postnatal day 7 mice, using 30 (mild) or 60 min (severe) exposure to 8% Oxygen and assessment of outcome based on size of infarct (Nissl), extent of cell death (TUNEL density) and microglial activation (alphaM&X levels). On immunohistochemical level, HI insult resulted in transient upregulation of phosphorylated STAT3 (Y705) in cortical, hippocampal and thalamic neurons, with a peak at 2–4 h after insult. Moreover, cell-type specific deletion of STAT3 in neurons using Synapsin:Cre in homozygous STAT3-flox mutant mice (n=5) resulted in a significant and strong reduction of tissue damage (−80%, p<5% t-test) in hippocampus, cortex, striatum and thalamus following the severe, 60 min insult, compared with their STAT3+ littermate controls (n=5). A more moderate effect was also observed in the subcortical white matter. We are currently exploring the results in the milder, 30 min HI, to obtain data on severity-specific sensitivity to STAT3 deletion. Since Y705-phosphorylation plays an important role in STAT3 function, the use of direct kinase inhibitors could serve as a candidate target for therapeutic intervention in neonatal brain damage.

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.

No abstract is provided for this article.

We previously demonstrated that ocular surface epithelium expressed TLR3 and that its ligand, polyI:C, stimulation induced the secretion of inflammatory cytokines and type I IFN. It was recently reported that RIG-I and MDA5 also recognize viral dsRNA mimicking polyI:C. In this study, we investigated whether RIG-I and/or MDA5 contribute to polyI:C-inducible responses in conjunctival epithelium. The expression of RIG-I, MDA5, and TLR3 in human conjunctival epithelium was examined by RT-PCR and their up-regulation after polyI:C stimulation by quantitative RT-PCR and immunoblot analysis. Human conjunctival epithelial cells also expressed RIG-I, MDA5 and TLR3 mRNA and protein. The expression of RIG-I and MDA5, but not of TLR3, was markedly up-regulated upon polyI:C stimulation. We also examined the function of IPS-1 (an adaptor molecule common to RIG-I and/or MDA5) and TLR3 in conjunctival epithelium using IPS-1 KO and TLR3 KO mice. To analyze in vivo murine conjunctival epithelial cells, 10μl of a 100μg/ml polyI:C solution were delivered subconjunctivally and as eye drops, then conjunctival epithelial cells were subjected to gene expression analysis. We focused on 10 transcripts up-regulated in murine conjunctival epithelium upon polyI:C stimulation. Cxcl10, Mx1, Ifi44, Ifi203, Iigp2 and Rtp4 were dominantly regulated by IPS-1, Ccl5 by TLR3, and Rsad2, Mx2 and Cmpk2 were regulated by TLR3 and IPS-1. Our results showed that conjunctival epithelial cells express RIG-I and MDA5, and IPS-1, an adaptor molecule common to RIG-I and MDA5, contributes to polyI:C-inducible cytokine production in conjunctival epithelial cells.

Candida albicans infection can cause skin, vulvar, or oral pain. Despite the obvious algesic activity of C. albicans, the molecular mechanisms of fungal nociception remain largely unknown. Here we show that the C. albicans-specific signaling pathway led to severe mechanical allodynia. We discovered that C. albicans-derived β-glucan stimulated nociceptors depending on Dectin-1, and two pathways in inflammatory pain. The major pathway operates via the Dectin-1-mediated ATP-P2X3/P2X2/3 axis through intercellular relationships between keratinocytes and primary sensory neurons, which depends on the ATP transporter vesicular nucleotide transporter (VNUT). The other pathway operates via the Dectin-1-mediated PLC-TRPV1/TRPA1 axis in primary sensory neurons. Intriguingly, C. albicans-derived β-glucan has the ability to enhance histamine-independent pruritus, and VNUT inhibitor clodronate can be used to treat unpleasant feelings induced by β-glucan. Collectively, this is the first report to indicate that Dectin-1 and VNUT mediated innate sensory mechanisms that detect fungal infection.

Lipocalin 2 (Lcn2) plays an important role in defense against bacterial infection by interfering with bacterial iron acquisition. Although Lcn2 is expressed in a number of aseptic inflammatory conditions, its role in these conditions remains unclear. We examined the expression and role of Lcn2 after spinal cord injury (SCI) in adult mice by using a contusion injury model. Lcn2 expression at the protein level is rapidly increased 12-fold at 1 d after SCI and decreases gradually thereafter, being three times as high as control levels at 21 d after injury. Lcn2 expression is strongly induced after contusion injury in astrocytes, neurons, and neutrophils. The Lcn2 receptor (Lcn2R), which has been shown to influence cell survival, is also expressed after SCI in the same cell types. Lcn2-deficient ( Lcn2 −/− ) mice showed significantly better locomotor recovery after spinal cord contusion injury than wild-type ( Lcn2 +/+ ) mice. Histological assessments indicate improved neuronal and tissue survival and greater sparing of myelin in Lcn2 −/− mice after contusion injury. Flow cytometry showed a decrease in neutrophil influx and a small increase in the monocyte population in Lcn2 −/− injured spinal cords. This change was accompanied by a reduction in the expression of several pro-inflammatory chemokines and cytokines as well as inducible nitric oxide synthase early after SCI in Lcn2 −/− mice compared with wild-type animals. Our results, therefore, suggest a role for Lcn2 in regulating inflammation in the injured spinal cord and that lack of Lcn2 reduces secondary damage and improves locomotor recovery after spinal cord contusion injury.

Autographa californica nuclear polyhedrosis virus (AcNPV) is a double-stranded-DNA virus that is pathogenic to insects. AcNPV was shown to induce an innate immune response in mammalian immune cells and to confer protection of mice from lethal viral infection. In this study, we have shown that production of type I interferon (IFN) by AcNPV in murine plasmacytoid dendritic cells (pDCs) and non-pDCs, such as peritoneal macrophages and splenic CD11c+ DCs, was mediated by Toll-like receptor (TLR)-dependent and -independent pathways, respectively. IFN regulatory factor 7 (IRF7) was shown to play a crucial role in the production of type I IFN by AcNPV not only in immune cells in vitro but also in vivo. In mouse embryonic fibroblasts (MEFs), AcNPV produced IFN-beta and IFN-inducible chemokines through TLR-independent and IRF3-dependent pathways, in contrast to the TLR-dependent and IRF3/IRF7-independent production of proinflammatory cytokines. Although production of IFN-beta and IFN-inducible chemokines was severely impaired in IFN promoter-stimulator 1 (IPS-1)-deficient MEFs upon infection with vesicular stomatitis virus, AcNPV produced substantial amounts of the cytokines in IPS-1-deficient MEFs. These results suggest that a novel signaling pathway(s) other than TLR- and IPS-1-dependent pathways participates in the production of type I IFN in response to AcNPV infection.