Naotake Tsuboi

Metformin Inhibits Proinflammatory Responses and Nuclear Factor-κB in Human Vascular Wall Cells

Objective—Metformin may benefit the macrovascular complications of diabetes independently of its conventional hypoglycemic effects. Accumulating evidence suggests that inflammatory processes participate in type 2 diabetes and its atherothrombotic manifestations. Therefore, this study examined the potential action of metformin as an inhibitor of pro-inflammatory responses in human vascular smooth muscle cells (SMCs), macrophages (M&phgr;s), and endothelial cells (ECs). Methods and Results—Metformin dose-dependently inhibited IL-1&bgr;–induced release of the pro-inflammatory cytokines IL-6 and IL-8 in ECs, SMCs, and M&phgr;s. Investigation of potential signaling pathways demonstrated that metformin diminished IL-1&bgr;–induced activation and nuclear translocation of nuclear factor-kappa B (NF-&kgr;B) in SMCs. Furthermore, metformin suppressed IL-1&bgr;–induced activation of the pro-inflammatory phosphokinases Akt, p38, and Erk, but did not affect PI3 kinase (PI3K) activity. To address the significance of the anti-inflammatory effects of a therapeutically relevant plasma concentration of metformin (20 &mgr;mol/L), we conducted experiments in ECs treated with high glucose. Pretreatment with metformin also decreased phosphorylation of Akt and protein kinase C (PKC) in ECs under these conditions. Conclusions—These data suggest that metformin can exert a direct vascular anti-inflammatory effect by inhibiting NF-&kgr;B through blockade of the PI3K–Akt pathway. The novel anti-inflammatory actions of metformin may explain in part the apparent clinical reduction by metformin of cardiovascular events not fully attributable to its hypoglycemic action.

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.

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.

Differences in Expression of Toll-Like Receptors and Their Reactivities in Dendritic Cells in BALB/c and C57BL/6 Mice

ABSTRACT We have previously reported that differences in early production of interleukin 12 (IL-12) by dendritic cells (DC) underlies the difference between the susceptibilities to Listeria monocytogenes of C57BL/6 and BALB/c mice. To elucidate mechanisms for the different abilities of DC to produce cytokine in C57BL/6 and BALB/c mice, we examined Toll-like receptor (TLR) expression by DC and their responses in vitro to known microbial ligands for TLRs. We found that DC isolated from the spleens of naive C57BL/6 mice preferentially expressed TLR9 mRNA, whereas DC from naive BALB/c mice strongly expressed TLR2, -4, -5, and -6 mRNAs. C57BL/6 DC produced a higher level of IL-12p40 in response to the ligands for TLR4 (lipopolysaccharide), TLR2 (lipoprotein), and TLR9 (CpG), whereas BALB/c DC responded to these ligands by producing a larger amount of monocyte chemoattractant protein 1. C57BL/6 DC expressed higher levels of CD40 and Stat4 than BALB/c DC did, suggesting that naive C57BL/6 mice contained more-mature subsets of DC than naive BALB/c mice. Differences in reactivities of DC to microbial molecules through TLRs may be associated with susceptibility and resistance to Listeria infection in BALB/c and C57BL/6 mice.

Human Neutrophil Fcγ Receptors Initiate and Play Specialized Nonredundant Roles in Antibody-Mediated Inflammatory Diseases

Inflammation mediated by antibody-antigen complexes contributes to autoimmune diseases. Mice deficient in the common Fcgamma-chain are protected from IgG-mediated glomerulonephritis and the reverse passive Arthus (RPA) reaction and FcR-bearing macrophages, and mast cells have been assigned primary roles in these processes. Here we demonstrate that neutrophil-selective transgenic expression of the two uniquely human neutrophil Fc gamma receptors (FcgammaRs), FcgammaRIIA and FcgammaRIIIB, in Fcgamma-chain-deficient mice restored susceptibility to progressive glomerulonephritis and the cutaneous RPA reaction. FcgammaRIIIB and FcgammaRIIA mediated neutrophil accumulation, whereas FcgammaRIIA alone promoted organ injury. In a model of soluble immune complexes deposited within the vasculature, FcgammaRIIIB was responsible for neutrophil slow rolling and adhesion whereas in the cremaster RPA, induced by both vascular and tissue soluble immune complexes, FcgammaRIIA predominated. Thus, human FcgammaRs on neutrophils serve as molecular links between antibody and immunological disease, with FcgammaRIIA promoting tissue injury and FcgammaRIIIB and FcgammaRIIA displaying specialized context-dependent functions in neutrophil recruitment.

Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo

Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human FcγRIIA and glycophosphatidylinositol (GPI)-linked FcγRIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil FcγRs to demonstrate that FcγRIIIB alone, in the absence of its known signaling partners FcγRIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. FcγRIIA also uses this pathway. As shown by intravital microscopy, FcγRIIA but not FcγRIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, FcγRIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for FcγRIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas FcγRIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.

A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA–induced IL-12 production and Th cell differentiation

A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal-regulated kinase (ERK) activation and production of TNF-alpha and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2-independent manner. Peritoneal macrophages and bone marrow-derived DCs from Cot/Tpl2-/- mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2-/- macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter-binding protein (GA-12-binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2-/- mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA-guided vaccination.

Mac-1 (CD11b/CD18) Links Inflammation and Thrombosis After Glomerular Injury

Background— Inflammation and thrombosis coexist in several disorders. Although it is recognized that leukocytes may induce a procoagulant state at sites of inflammation, the critical molecular determinants of this process remain largely unknown. Methods and Results— To examine mechanisms of inflammation-induced thrombosis, we developed a murine model of thrombotic glomerulonephritis (TGN), a known cause of acute renal failure in patients. This model, induced by lipopolysaccharide and antibody to the glomerular basement membrane, led to rapid glomerular neutrophil recruitment, thrombotic glomerular lesions with endothelial cell injury, and renal dysfunction. In mice immunodepleted of neutrophils or lacking the leukocyte-specific integrin Mac-1, neutrophil recruitment, endothelial injury, glomerular thrombosis, and acute renal failure were markedly attenuated despite the robust generation of renal cytokines. Neutrophil elastase is a likely effector of Mac-1 because its activity was reduced in Mac-1–deficient mice and the phenotype in mice deficient in Mac-1 or neutrophil elastase was similar. Platelets accumulated in glomerular capillaries within 4 hours of TGN before evidence of thrombosis. Platelet immunodepletion before TGN markedly exacerbated hematuria (hemorrhage), inflammation, and injury, whereas thrombocytopenic Mac-1–deficient mice remained resistant to disease, indicating that initial glomerular platelet deposition protects the vessel wall from neutrophil-mediated sequelae. The subsequent thrombosis relied on the interaction of Mac-1 on recruited neutrophils with glycoprotein Ibα on platelets as antibody-mediated disruption of this interaction attenuated TGN without affecting renal neutrophil accumulation. Conclusions— These observations establish Mac-1 on neutrophils as a critical molecular link between inflammation and thrombosis and suggest it as an attractive target for antithrombotic therapy.

Regulation of human neutrophil Fcγ receptor IIa by C5a receptor promotes inflammatory arthritis in mice

OBJECTIVE Rheumatoid arthritis culminates in joint destruction that, in mouse models of disease, is supported by innate immune molecules, including Fcγ receptors (FcγR) and complement. However, these findings may not be predictive of the outcome in humans, given the structural differences between murine and human activating FcγR on neutrophils, a prominent component of joint exudates. The aim of this study was to examine the role of human neutrophil FcγRIIa in the development of arthritis and probe the underlying mechanism by which FcγRIIa initiates disease. METHODS K/BxN mouse serum transfer-induced arthritis was examined in mice expressing human FcγRIIa on neutrophils but lacking their own activating FcγR (γ-chain-deficient mice). The role of mast cells, complement (C3 and C5a), and CD18 integrins in FcγRIIa-initiated disease was examined using cell reconstitution approaches, inhibitors, and functional blocking antibodies, respectively. Crosstalk between the complement receptor C5aR and FcγRIIa on neutrophils was evaluated in vitro. RESULTS The expression of human FcγRIIa on neutrophils was sufficient to restore susceptibility to K/BxN serum-induced neutrophil recruitment, synovitis, and bone destruction in γ-chain-deficient mice. Joint inflammation was robust and proceeded even in the absence of mast cells and vascular permeability, features shown to contribute to disease in wild-type mice. Neutrophil recruitment was dependent on the presence of a CD18 integrin, lymphocyte function-associated antigen 1, and C5aR. In addition, C5aR significantly enhanced FcγRIIa-mediated phagocytosis and oxidative burst in vitro. CONCLUSION Human and murine activating FcγR on neutrophils are not functionally equivalent, and in humans, they may play a primary role in arthritis. Crosstalk between neutrophil C5aR and FcγRIIa is essential for disease progression, thus highlighting a new aspect of complement during the effector phase of inflammatory arthritis.

Programmed Death 1 Ligand (PD-L) 1 and PD-L2 Limit Autoimmune Kidney Disease: Distinct Roles

The programmed death 1/programmed death 1 ligand (PD-L) pathway is instrumental in peripheral tolerance. Blocking this pathway exacerbates experimental autoimmune diseases, but its role in autoimmune kidney disease has not been explored. Therefore, we tested the hypothesis that the programmed death 1 ligands (PD-L1 and PD-L2), provide a protective barrier during T cell- and macrophage (Mφ)-dependent autoimmune kidney disease. For this purpose, we compared nephrotoxic serum nephritis (NSN) in mice lacking PD-L1 (PD-L1−/−), PD-L2 (PD-L2−/−), or both (PD-L1/L2−/−) to wild-type (WT) C57BL/6 mice. Kidney pathology, loss of renal function, and intrarenal leukocyte infiltrates were increased in each PD-L−/− strain as compared with WT mice. Although the magnitude of renal pathology was similar in PD-L1−/− and PD-L2−/− mice, our findings suggest that kidney disease in each strain is regulated by distinct mechanisms. Specifically, we detected increased CD68+ cells along with elevated circulating IgG and IgG deposits in glomeruli in PD-L2−/− mice, but not PD-L1−/− mice. In contrast, we detected a rise in activated CD8+ T cells in PD-L1−/− mice, but not PD-L2−/− mice. Furthermore, since PD-L1 is expressed by parenchymal and hemopoietic cells in WT kidneys, we explored the differential impact of PD-L1 expression on these cell types by inducing NSN in bone marrow chimeric mice. Our results indicate that PD-L1 expression on hemopoietic cells, and not parenchymal cells, is primarily responsible for limiting leukocyte infiltration during NSN. Taken together, our findings indicate that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints poised to suppress autoimmune renal disease.