Tetsuya Matsuguchi

Gene Expressions of Toll-Like Receptor 2, But Not Toll-Like Receptor 4, Is Induced by LPS and Inflammatory Cytokines in Mouse Macrophages

Toll-like receptors (TLRs) are a family of mammalian homologues of Drosophila Toll and play important roles in host defense. Two of the TLRs, TLR2 and TLR4, mediate the responsiveness to LPS. Here the gene expression of TLR2 and TLR4 was analyzed in mouse macrophages. Mouse splenic macrophages responded to an intraperitoneal injection or in vitro treatment of LPS by increased gene expression of TLR2, but not TLR4. Treatment of a mouse macrophage cell line with LPS, synthetic lipid A, IL-2, IL-15, IL-1β, IFN-γ, or TNF-α significantly increased TLR2 mRNA expression, whereas TLR4 mRNA expression remained constant. TLR2 mRNA increase in response to synthetic lipid A was severely impaired in splenic macrophages isolated from TLR4-mutated C3H/HeJ mice, suggesting that TLR4 plays an essential role in the process. Specific inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase and p38 kinase did not significantly inhibit TLR2 mRNA up-regulation by LPS. In contrast, LPS-mediated TLR2 mRNA induction was abrogated by pretreatment with a high concentration of curcumin, suggesting that NF-κB activation may be essential for the process. Taken together, our results indicate that TLR2, in contrast to TLR4, can be induced in macrophages in response to bacterial infections and may accelerate the innate immunity against pathogens.

Roles of Toll-Like Receptors in C-C Chemokine Production by Renal Tubular Epithelial Cells

Pyelonephritis, in which renal tubular epithelial cells are directly exposed to bacterial component, is a major predisposing cause of renal insufficiency. Although previous studies have suggested C-C chemokines are involved in the pathogenesis, the exact source and mechanisms of the chemokine secretion remain ambiguous. In this study, we evaluated the involvement of Toll-like receptors (TLRs) in C-C chemokine production by mouse primary renal tubular epithelial cells (MTECs). MTECs constitutively expressed mRNA for TLR1, 2, 3, 4, and 6, but not for TLR5 or 9. MTECs also expressed MD-2, CD14, myeloid differentiation factor 88, and Toll receptor-IL-1R domain-containing adapter protein/myeloid differentiation factor 88-adapter-like. Synthetic lipid A and lipoprotein induced monocyte chemoattractant protein 1 (MCP-1) and RANTES production in MTECs, which strictly depend on TLR4 and TLR2, respectively. In contrast, MTECs were refractory to CpG-oligodeoxynucleotide in chemokine production, consistently with the absence of TLR9. LPS-mediated MCP-1 and RANTES production in MTECs was abolished by NF-κB inhibition, but unaffected by extracellular signal-regulated kinase inhibition. In LPS-stimulated MTECs, inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase significantly decreased RANTES, but did not affect MCP-1 mRNA induction. Thus, MTECs have a distinct expression pattern of TLR and secrete C-C chemokines in response to direct stimulation with a set of bacterial components.

Cutting Edge: Naturally Occurring Soluble Form of Mouse Toll-Like Receptor 4 Inhibits Lipopolysaccharide Signaling

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-α production and NF-κB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.

Gene Expression of Osteoclast Differentiation Factor Is Induced by Lipopolysaccharide in Mouse Osteoblasts Via Toll-Like Receptors

Osteoclast differentiation factor (ODF), a recently identified cytokine of the TNF family, is expressed as a membrane-associated protein in osteoblasts and stromal cells. ODF stimulates the differentiation of osteoclast precursors into osteoclasts in the presence of M-CSF. Here we investigated the effects of LPS on the gene expression of ODF in mouse osteoblasts and an osteoblast cell line and found that LPS increased the ODF mRNA level. A specific inhibitor of extracellular signal-regulated kinase or protein kinase C inhibited this up-regulation, indicating that extracellular signal-regulated kinase and protein kinase C activation was involved. A protein synthesis inhibitor, cycloheximide, rather enhanced the LPS-mediated increase of ODF mRNA, and both a neutralizing Ab of TNF-α and a specific inhibitor of PGE synthesis failed to block the ODF mRNA increase by native LPS. Thus, LPS directly induced ODF mRNA. Mouse osteoblasts and an osteoblast cell line constitutively expressed Toll-like receptor (TLR) 2 and 4, which are known as putative LPS receptors. ODF mRNA increases in response to synthetic lipid A were defective in primary osteoblasts from C3H/HeJ mice that contain a nonfunctional mutation in the TLR4 gene, suggesting that TLR4 plays an essential role in the process. Altogether, our results indicate that ODF gene expression is directly increased in osteoblasts by LPS treatment via TLR, and this pathway may play an important role in the pathogenesis of LPS-mediated bone disorders, such as periodontitis.

Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2

ABSTRACT Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-α) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-κB activation and TNF-α production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall ≫ polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-α secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2−/− mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-κB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.

Th2 Cytokine Production from Mast Cells Is Directly Induced by Lipopolysaccharide and Distinctly Regulated by c-Jun N-Terminal Kinase and p38 Pathways

Mast cells secrete multiple cytokines and play an important role in allergic inflammation. Although it is widely accepted that bacteria infection occasionally worsens allergic airway inflammation, the mechanism has not been defined. In this study, we show that LPS induced Th2-associated cytokine production such as IL-5, IL-10, and IL-13 from mast cells and also synergistically enhanced production of these cytokines induced by IgE cross-linking. LPS-mediated Th2-type cytokine production was abolished in mouse bone marrow-derived mast cells derived from C3H/HeJ mice, suggesting that Toll-like receptor 4 is essential for the cytokine production. Furthermore, we found that mitogen-activated protein kinases including extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 kinase were activated by LPS stimulation in bone marrow-derived mast cells. Inhibition of extracellular signal-regulated kinase activation has little effect on LPS-mediated cytokine production. In contrast, inhibition of c-Jun N-terminal kinase activation significantly suppressed both IL-10 and IL-13 expression at both mRNA and protein levels. Interestingly, although inhibition of p38 did not down-regulate the mRNA induction, it moderately decreased all three cytokine productions by LPS. These results indicate that LPS-mediated production of IL-5, IL-10, and IL-13 was distinctly regulated by mitogen-activated protein kinases. Our findings may indicate a clue to understanding the mechanisms of how bacteria infection worsens the clinical features of asthma.

Expression of Toll-Like Receptor 2 on γδ T Cells Bearing Invariant Vγ6/Vδ1 Induced by Escherichia coli Infection in Mice

We recently reported that the number of γδ T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of γδ T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified γδ T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by Vγ6-Jγ1/Vδ1-Dδ2-Jδ2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive γδ T cells bearing canonical Vγ6/Vδ1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the γδ T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the γδ T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical Vγ6/Vδ1 T cells by native E. coli lipid A.

NF-κB and STAT5 Play Important Roles in the Regulation of Mouse Toll-Like Receptor 2 Gene Expression

Toll-like receptor 2 (TLR2) is involved in the innate immunity by recognizing various bacterial components. We have previously reported that TLR2 gene expression is rapidly induced by LPS or inflammatory cytokines in macrophages, and by TCR engagement or IL-2/IL-15 stimulation in T cells. Here, to investigate the mechanisms governing TLR2 transcription, we cloned the 5′ upstream region of the mouse TLR2 (mTLR2) gene and mapped its transcriptional start site. The 5′ upstream region of the mTLR2 gene contains two NF-κB, two CCAAT/enhancer binding protein, one cAMP response element-binding protein, and one STAT consensus sequences. In mouse macrophage cell lines, deletion of both NF-κB sites caused the complete loss of mTLR2 promoter responsiveness to TNF-α. NF-κB sites were also important but not absolutely necessary for LPS-mediated mTLR2 promoter activation. In T cell lines, mTLR2 responsiveness to IL-15 was abrogated by the 3′ NF-κB mutation, whereas 5′ NF-κB showed no functional significance. The STAT binding site also seemed to contribute, as the deletion of this sequence significantly reduced the IL-15-mediated mTLR2 promoter activation. EMSAs confirmed nuclear protein binding to both NF-κB sites in macrophages following LPS and TNF-α stimulation and to the 3′ NF-κB site in T cells after IL-15 treatment. Thus, NF-κB activation is important but differently involved in the regulation of mTLR2 gene expression in macrophages and T cells following LPS or cytokine stimulation.

Glucocorticoids and Tumor Necrosis Factor Alpha Cooperatively Regulate Toll-Like Receptor 2 Gene Expression

ABSTRACT Tumor necrosis factor alpha (TNF-α) and glucocorticoids are widely recognized as mutually antagonistic regulators of adaptive immunity and inflammation. Surprisingly, we show here that they cooperatively regulate components of innate immunity. The Toll-like receptor 2 (TLR2) gene encodes a transmembrane receptor critical for triggering innate immunity. Although TLR2 mRNA and protein are induced by inflammatory molecules such as TNF-α, we show that TLR2 is also induced by the anti-inflammatory glucocorticoids in cells where they also regulate MKP-1 mRNA and protein levels. TNF-α and glucocorticoids cooperate to regulate the TLR2 promoter, through the involvement of a 3′ NF-κB site, a STAT-binding element, and a 3′ glucocorticoid response element (GRE). Molecular studies show that the IκBα superrepressor or a STAT dominant negative element prevented TNF-α and dexamethasone stimulation of TLR2 promoter. Similarly, an AF-1 deletion mutant of glucocorticoid receptor or ablation of a putative GRE notably reduced the cooperative regulation of TLR2. Using chromatin immunoprecipitation assays, we demonstrate that all three transcription factors interact with both endogenous and transfected TLR2 promoters after stimulation by TNF-α and dexamethasone. Together, these studies define novel signaling mechanism for these three transcription factors, with a profound impact on discrimination of innate and adaptive immune responses.

JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation.

Lipopolysaccharide (LPS) is recognized by Toll‐like receptor (TLR) 4 and activates NF‐κB and a set of MAP kinases. Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two‐hybrid screening and identified JNK‐interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4‐associated protein. In mammalian cells, JIP3, through its N‐terminal region, constitutively associates with TLR4. The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co‐expression of JIP3 significantly increased the complex formation of TLR4–JNK and LPS‐mediated JNK activation. In contrast, expression of C‐terminally truncated forms of JIP3 impaired LPS‐induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibited LPS‐mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK‐1, but not with TAK‐1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs.