Tomoh Matsumiya

Critical Role of an Antiviral Stress Granule Containing RIG-I and PKR in Viral Detection and Innate Immunity

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

Interferon-γ stimulates the expression of galectin-9 in cultured human endothelial cells

Galectin‐9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin‐9 expression in endothelial cells. Galectin‐9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon‐γ (IFN‐γ). IFN‐γ also enhanced the adhesion of human eosinophilic leukemia‐1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin‐4, which induces eotaxin expression, did not affect the expression of galectin‐9. The in situ endothelium from patients with inflammatory diseases was found to express galectin‐9. IFN‐γ‐induced production of galectin‐9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Expression of Tumor Necrosis Factor-α in Cultured Human Endothelial Cells Stimulated With Lipopolysaccharide or Interleukin-1α

Abstract —Tumor-necrosis factor-α (TNF-α) is a proinflammatory cytokine with a wide variety of biological effects. The most important source of this cytokine is monocytes/macrophages. It is a potent agonist in the activation of endothelial cells; however, the precise role of endothelial cells as a source of TNF-α is not known. In the present study, we addressed the possibility that TNF-α is produced by cultured human umbilical vein endothelial cells (HUVEC) stimulated with factors such as lipopolysaccharide (LPS) or interleukin-1α (IL-1α). LPS and IL-1α induced expression of TNF-α mRNA in HUVEC. IL-1α induced expression and secretion of TNF-α protein, but LPS did not induce production of TNF-α protein. Most of the TNF-α protein in cell lysate was found in the membrane fraction. The mRNA for TNF-α–converting enzyme (TACE) was expressed in unstimulated HUVEC, and its level was not altered by treatment with LPS or IL-1α. Transfection of HUVEC with full-length cDNA encoding the precursor TNF-α enhanced secretion of TNF-α protein by these cells, and treatment of the cells with a TACE inhibitor reduced the secretion. These results suggest that HUVEC produce TNF-α and have TACE activity. Secreted TNF-α may be involved in autocrine activation of endothelial cells, and TNF-α retained in cell membrane may serve as a juxtacrine system to activate target cells on the endothelial surface.

Synergistic stimulation, by tumor necrosis factor-α and interferon-γ, of fractalkine expression in human astrocytes

Fractalkine is a CX3C chemonkine that appears to be a neuron-to-microglia signal molecule in the central nervous system. We studied the expression of fractalkine in normal human astrocytes in culture, by using semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that tumor-necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically enhance the expression of fractalkine. The expression of both fractalkine mRNA and protein was increased in time- and concentration-dependent manners in the cells co-stimulated with TNF-alpha and IFN-gamma. Cycloheximide, an inhibitor of protein synthesis, and dexamethasone had no effect on the synergy of the stimulation of fractalkine expression. We conclude that normal human astrocytes produce fractalkine by co-stimulation with pro-inflammatory cytokines and it may serve as a potential signal for immune and inflammatory responses in the central nervous system.

Interleukin-1beta stimulates galectin-9 expression in human astrocytes.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1β (IL-1β) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 ×g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1β, and this may contribute to inflammatory reactions in the CNS.

Emerging functional cross-talk between the Keap1-Nrf2 system and mitochondria

Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) was originally identified as a positive regulator of drug detoxifying enzyme gene expression during exposure to environmental electrophiles. Currently, Nrf2 is known to regulate the expression of hundreds of cytoprotective genes to counteract endogenously or exogenously generated oxidative stress. Furthermore, when activated in human tumors by somatic mutations, Nrf2 confers growth advantages and chemoresistance by regulating genes involved in various processes such as the pentose phosphate pathway and nucleotide synthesis in addition to antioxidant proteins. Interestingly, increasing evidence shows that Nrf2 is associated with mitochondrial biogenesis during environmental stresses in certain tissues such as the heart. Furthermore, SKN-1, a functional homolog of Nrf2 in C. elegans, is activated by mitochondrial reactive oxygen species and extends life span by promoting mitochondrial homeostasis (i.e., mitohormesis). Similarly, Nrf2 activation was recently observed in the heart of surfeit locus protein 1 (Surf1) -/- mice in which cellular respiration was decreased due to cytochrome c oxidase defects. In this review, we critically examine the relationship between Nrf2 and mitochondria and argue that the Nrf2 stress pathway intimately communicates with mitochondria to maintain cellular homeostasis during oxidative stress.

Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a human macrophage cell line

Prostaglandins (PGs) play regulatory roles in a variety of physiological and pathological processes, including the immune response, cytoprotection and inflammation. Desferrioxamine (DFX), an iron chelator, is known to reduce free radical-mediated cell injury and to upregulate certain inflammatory mediators. We investigated the effects of DFX on the production of PGs and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGs, using a human macrophage cell line, U937. Our results showed that COX-2 expression and PGE(2) production are upregulated by DFX treatment and that this upregulation is dependent on both COX-2 promoter activity and alteration of mRNA stability. COX-2 promoter activity may be, at least in part, mediated by activation of the extracellular signal-regulated kinase pathway. These findings suggest that iron metabolism may regulate inflammatory processes by modulating PGs as well as other inflammatory mediators.

Characterization of Synergistic Induction of CX3CL1/Fractalkine by TNF-α and IFN-γ in Vascular Endothelial Cells: An Essential Role for TNF-α in Post-Transcriptional Regulation of CX3CL1

CX3CL1/Fractalkine, a chemokine specific to monocytes and NK cells, is induced synergistically by TNF-α and IFN-γ in vascular endothelial cells. However, the mechanism for this synergism remains unclear. This study explored the hypothesis that the CX3CL1 expression is regulated at a posttranscriptional level, which may responsible for the synergism between TNF-α and IFN-γ. Brief exposure of HUVECs to TNF-α led to a robust increase in IFN-γ–induced CX3CL1 production. We found that TNF-α stabilized CX3CL1 mRNA in HUVECs stimulated with IFN-γ. Cloning of 3′untranslated region (UTR) of CX3CL1 mRNA revealed the presence of a single copy of nonametric AU-rich element in its 3′UTR, and a luciferase reporter assay showed that a single AU-rich element is a crucial cis-element in the posttranscriptional regulation of CX3CL1. TNF-α treatment resulted in the phosphorylation of p38 MAPK and its downstream target, MAPK-activated protein kinase-2, but IFN-γ did not affect the levels of MAPK and MAPK-activated protein kinase-2 phosphorylation induced by TNF-α. Treatment of the cells with an inhibitor of p38 MAPK accelerated the decay of CX3CL1 mRNA induced by TNF-α or the combination of TNF-α and IFN-γ. Immunoprecipitation assay revealed that mRNA stabilizer HuR directly binds to 3′UTR of CX3CL1 mRNA. CX3CL1 expression is under control of posttranscriptional regulation, which is involved in the synergistic induction of CX3CL1 in response to the combined stimulation with TNF-α and IFN-γ.

Lipopolysaccharide induces the expression of cellular inhibitor of apoptosis protein-2 in human macrophages

Apoptosis is an important process in normal animal development as well as in diseases, and inhibitor of apoptosis protein (IAP) is one of the important factors that regulate apoptotic cell death. We found that lipopolysaccharide (LPS) enhances the expression of mRNA and protein of cellular IAP-2 (cIAP2) in human monoblastic U937 cells differentiated by phorbol ester pretreatment. cIAP2 mRNA was not detected in undifferentiated U937 cells. mRNAs of cIAP1 and X-chromosome-linked IAP (XIAP) were expressed constitutively and not affected by LPS in both undifferentiated and differentiated cells. LPS stimulated the expression of cIAP2 mRNA and protein in time- and concentration-dependent manners. LPS enhanced the expression of cIAP2 mRNA and protein in human monocyte-derived macrophages, which was associated with the inhibition of the caspase-3 activation, i.e., decrease in active p17 fragment of caspase-3 with simultaneous accumulation of precursor p20 fragment. We conclude that LPS may inhibit apoptosis of macrophages, at least in part, through the induction of cIAP2.

Retinoic acid-inducible gene-I is induced by double-stranded RNA and regulates the expression of CC chemokine ligand (CCL) 5 in human mesangial cells

BACKGROUND Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase involved in immune reactions against RNA viruses and various inflammatory and autoimmune diseases. The purpose of the present study was to investigate the role of RIG-I in glomerular diseases. METHODS We treated human mesangial cells in culture with polyinosinic-polycytidylic acid (poly IC), which is an authentic double-stranded RNA, and analysed the expression of RIG-I, CC chemokine ligand 5 (CCL5) and interferon (IFN)-β by western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assay (ELISA). To elucidate the poly IC-signalling pathway, we subjected the cells to RNA interference (RNAi) against RIG-I, IFN-β or Toll-like receptor (TLR) 3. Furthermore, we studied the effects of IFN-β receptor blocking and IFN-β overexpression. RESULTS Poly IC induced the expression of RIG-I and CCL5 in human mesangial cells, and RNAi against RIG-I inhibited this poly IC-induced CCL5 expression. Poly IC-induced RIG-I expression was also inhibited by RNAi against IFN-β and by an antibody against the IFN-β receptor. IFN-β overexpression induced the expression of both RIG-I and CCL5. The knockdown of TLR3 abolished poly IC-induced RIG-I expression. CONCLUSIONS The TLR3/IFN-β/RIG-I/CCL5 signalling pathway may mediate immune and inflammatory responses against viral infection in mesangial cells, suggesting the role of this pathway in the aggravation of glomerulonephritis due to viral infection.