Resiquimod Induces C-C Motif Chemokine Ligand 2 Via Nuclear Factor-Kappa B in SH-SY5Y Human Neuroblastoma Cells.

Toll-like receptor (TLR) 7 plays an important role in recognizing virus-derived nucleic acids. TLR7 signaling in astrocytes and microglia is critical for activating immune responses against neurotrophic viruses. Neurons express TLR7, similar to glial cells; however, the role of neuronal TLR7 has not yet been fully elucidated. This study sought to determine whether resiquimod, the TLR7/8 agonist, induces the expression of inflammatory chemokines in SH-SY5Y human neuroblastoma cells. Immunofluorescence microscopy revealed that TLR7 was constitutively expressed in SH-SY5Y cells. Stimulation with resiquimod induced C-C motif chemokine ligand 2 (CCL2) expression, accompanied by the activation of nuclear factor-kappa B (NF-κB) in SH-SY5Y cells. Resiquimod increased mRNA levels of C-X-C motif chemokine ligand 8 (CXCL8) and CXCL10, while the increase was slight at the protein level. Knockdown of NF-κB p65 eliminated resiquimod-induced CCL2 production. This study provides novel evidence that resiquimod has promising therapeutic potential against central nervous system viral infections through its immunostimulatory effects on neurons.

TMEM2 suppresses TLR3-mediated IFN-ß/ISG56/CXCL10 expression in BEAS-2B bronchial epithelial cells.

BACKGROUND: Bronchial epithelial cells are at the front line of viral infections. Toll-like receptor 3 (TLR3) cascade causes the expression of interferon (IFN)-ß and IFN-stimulated genes (ISGs), which in turn induce an antiviral response. Members of the transmembrane protein (TMEM) family are expressed in various cell types. Although the prognostic value of TMEM2 in various cancers has been reported, its association with infectious diseases remains unknown. In this study, we investigated the effects of TMEM2 on antiviral immunity in BEAS-2B bronchial epithelial cells. METHODS AND RESULTS: TMEM2 protein was found in the cytoplasm of normal human bronchial epithelial cells and differed between organs using immunohistochemistry. Cultured BEAS-2B cells were transfected with TMEM2 siRNA, followed by administration of TLR3 ligand polyinosinic-polycytidylic acid (poly IC) or recombinant human (r(h)) IFN-ß. The expression of TMEM2, IFN-ß, ISG56, C-X-C motif chemokine ligand 10 (CXCL10) and hyaluronan were evaluated appropriately by western blotting, quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. TMEM2 expression was not altered by poly IC stimulation. Knockdown of TMEM2 increased poly IC-induced expression of IFN-ß, CXCL10, and ISG56, while IFN-ß-induced expression of ISG56 and CXCL10 were not changed by TMEM2 knockdown. The hyaluronan concentration in the medium was decreased by either TMEM2 knockdown or poly IC, but additive or synergistic effects were not observed. CONCLUSIONS: TMEM2 knockdown enhanced TLR3-mediated IFN-ß, CXCL10, and ISG56 expression in BEAS-2B cells. This implies that TMEM2 suppresses antiviral immune responses and prevents tissue injury in bronchial epithelial cells.

Polygonum tinctorium leaf extract ameliorates high-fat diet-induced intestinal epithelial damage in mice.

Dietary fat strongly influences the intestinal mucosal barrier, which protects against invading pathogenic bacteria. A high-fat diet (HFD) compromises the integrity of epithelial tight junctions (TJs) and reduces mucin production, leading to intestinal barrier disruption and metabolic endotoxemia. It has been shown that the active constituents of indigo plants can protect against intestinal inflammation; however, their protective role in HFD-induced intestinal epithelial damage remains unknown. The present study aimed to investigate the effects of Polygonum tinctorium leaf extract (indigo Ex) on HFD-induced intestinal damage in mice. Male C57BL6/J mice were fed a HFD and injected intraperitoneally with either indigo Ex or phosphate-buffered saline (PBS) for 4 weeks. The expression levels of TJ proteins, zonula occludens-1 and Claudin-1, were analyzed by immunofluorescence staining and western blotting. The colon mRNA expression levels of tumor necrosis factor-α, interleukin (IL)-12p40, IL-10 and IL-22 were measured by reverse transcription-quantitative PCR. The results revealed that indigo Ex administration attenuated the HFD-induced shortening of the colon. Colon crypt length was shown to be significantly greater in the indigo Ex-treated group mice compared with that in the PBS-treated group mice. Moreover, indigo Ex administration increased the number of goblet cells, and ameliorated the redistribution of TJ proteins. Notably, indigo Ex significantly increased the colon mRNA expression levels of IL-10. Indigo Ex displayed little effect on the gut microbial composition of HFD-fed mice. Taken together, these results suggested that indigo Ex may protect against HFD-induced epithelial damage. The leaves of indigo plants contain promising natural therapeutic compounds that could be used to treat obesity-associated intestinal damage and metabolic inflammation.

The double-stranded RNA-dependent protein kinase PKR negatively regulates the protein expression of IFN-ß induced by RIG-I signaling.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α (eIF2α) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-ß expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-ß mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-ß, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-ß expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-ß protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-ß in RIG-I-mediated antiviral immune responses.

ER export signals mediate plasma membrane localization of transmembrane protein TMEM72.

Transmembrane protein 72 (TMEM72) is involved in normal kidney development and tumorigenesis in renal cell carcinoma. However, the function of TMEM72 has not been experimentally examined; therefore, the role of TMEM72 is incompletely understood. In this study, we initially demonstrated that TMEM72 has four transmembrane domains (TMDs) and a long C-terminal tail. Immunofluorescence analysis showed that TMEM72 is localized on the plasma membrane but not on the outer mitochondrial membrane. Experiments performed with a series of TMEM72 deletion mutants and an evaluation of the unfolded protein response indicated that these TMDs are needed for proper protein folding or assembly. In contrast, domain-specific replacement analysis indicated the essential role of the C-terminal region of TMEM72 in protein transport. Spatial colocalization and immunoprecipitation assays showed that the proximal C-terminal region is responsible for anterograde protein transport. An amino acid sequence analysis and an immunocytochemical evaluation revealed that KRKKRKAAPEVLA, which corresponds to amino acid positions 132-144 in TMEM72, participates in efficient cellular transport. The motifs 132KRKKRK137 and 139APEVLA144 are associated with COPII and are considered to cooperate with membrane trafficking. Because efficient membrane trafficking is crucial for cells to maintain normal function, our data may contribute to elucidating the pathogenesis of membrane trafficking-associated diseases, particularly renal carcinoma and chronic kidney disease.

Tryptanthrin attenuates TLR3-mediated STAT1 activation in THP-1 cells.

Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)-mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)-differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC-induced mRNA expression of interferon (IFN)-stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-ß mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.

Hepatic Macrophages Express Melanoma Differentiation-Associated Gene 5 in Nonalcoholic Steatohepatitis.

The activation of innate immune system is essential for the pathogenesis of nonalcoholic steatohepatitis (NASH). Among pattern recognition receptors, it is well-characterized that toll-like receptors (TLRs) are deeply involved in the development of NASH to reflect exposure of the liver to gut-driven endotoxins. In contrast, it has not been elucidated whether retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) are similarly implicated in the disease progression. In the present study, we examined the expression of melanoma differentiation-associated antigen 5 (MDA5), known to be a member of RLRs, in a diet-induced murine model of NASH. The liver tissues were collected from C57BL/6 J mice at 1, 3, and 6 weeks after choline-deficient L-amino acid-defined high-fat diet (CDAHFD), and the expression of MDA5 was analyzed by western blotting, immunofluorescence (IF), and real-time quantitative PCR (qPCR). The results of western blotting showed that hepatic expression of MDA5 was increased at 3 and 6 weeks. In IF, MDA5-positive cells co-expressed F4/80 and CD11b, indicating they were activated macrophages, and these cells began to appear at 1 week after CDAHFD. The mRNA expression of MDA5 was significantly upregulated at 1 week. Additionally, we performed IF using liver biopsy specimens collected from 11 patients with nonalcoholic fatty liver diseases (NAFLD), and found that MDA5-positive macrophages were detected in eight out of eleven patients. In an in vitro study, MDA5 was induced upon stimulation with lipopolysaccharide in murine bone marrow-derived macrophages and THP-1 cells. Our findings suggest that MDA5 may be involved in the inflammation of NASH.

Porphyromonas gingivalis lipopolysaccharide induces interleukin-6 and c-c motif chemokine ligand 2 expression in cultured hCMEC/D3 human brain microvascular endothelial cells.

OBJECTIVE: This paper describes the effect of Porphyromonas gingivalis (P gingivalis) lipopolysaccharide (LPS) on the expression of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in cultured hCMEC/D3 human brain microvascular endothelial cells. BACKGROUND: P gingivalis is one of the important pathogens in periodontitis, and periodontitis is a risk factor for brain disorders including cerebrovascular diseases and Alzheimer's disease. However, the mechanisms underlying the pathogenesis of P gingivalis-mediated brain diseases are incompletely understood. Effects of P gingivalis LPS on brain endothelial cells are not known well. METHODS: The hCMEC/D3 human brain microvascular endothelial cells were cultured and treated with P gingivalis LPS. The expression of IL-6 and CCL2 mRNA and protein was examined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Effect of inhibitors of Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was also investigated. Phosphorylation of NF-κB p65, p38 MAPK and JNK was examined using Western blotting. RESULTS: P gingivalis LPS-induced mRNA and protein expression of IL-6 and CCL2 in hCMEC/D3 cells in a concentration-dependent manner at the concentration of 0.5-50 µg/mL. Maximal mRNA expression of IL-6 and CCL2 was found 2 and 4 hours after stimulation, respectively. Induction of IL-6 and CCL2 by P gingivalis LPS was almost completely inhibited by pretreatment of cells with TLR4 inhibitor but not by TLR2 inhibitor. Treatment of cells with P gingivalis LPS for up to 2 hours induced phosphorylation of NF-κB p65, p38 MAPK and JNK. IL-6 induction was decreased by pretreatment of cells with NF-κB inhibitor SN50 or p38 MAPK inhibitor SB203580, while CCL2 induction was reduced by SN50 or JNK inhibitor SP600125. CONCLUSIONS: IL-6 and CCL2 produced upon P gingivalis LPS stimulation may contribute to the inflammatory reactions in brain endothelial cells and subsequent neurological disorders such as cerebrovascular and Alzheimer's diseases.

Expression of Zinc-Finger Antiviral Protein in hCMEC/D3 Human Cerebral Microvascular Endothelial Cells: Effect of a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Invasion of viruses into the brain causes viral encephalitis, which can be fatal and causes permanent brain damage. The blood-brain barrier (BBB) protects the brain by excluding harmful substances and microbes. Brain microvascular endothelial cells are important components of the BBB; however, the mechanisms of antiviral reactions in these cells have not been fully elucidated. Zinc-finger antiviral protein (ZAP) is a molecule that restricts the infection of various viruses, and there are 2 major isoforms: ZAPL and ZAPS. Toll-like receptor 3 (TLR3), a pattern-recognition receptor against viral double-stranded RNA, is implicated in antiviral innate immune reactions. The aim of this study was to investigate the expression of ZAP in cultured hCMEC/D3 human brain microvascular endothelial cells treated with an authentic TLR3 agonist polyinosinic-polycytidylic acid (poly IC). METHODS: hCMEC/D3 cells were cultured and treated with poly IC. Expression of ZAPL and ZAPS mRNA was investigated using quantitative reverse transcription-polymerase chain reaction, and protein expression of these molecules was examined using western blotting. The role of nuclear factor-κB (NF-κB) was examined using the NF-κB inhibitor, SN50. The roles of interferon (IFN)-ß, IFN regulatory factor 3 (IRF3), tripartite motif protein 25 (TRIM25), and retinoic acid-inducible gene-I (RIG-I) in poly IC-induced ZAPS expression were examined using RNA interference. Propagation of Japanese encephalitis virus (JEV) was examined using a focus-forming assay. RESULTS: ZAPS mRNA and protein expression was upregulated by poly IC, whereas the change of ZAPL mRNA and protein levels was minimal. Knockdown of IRF3 or TRIM25 decreased the poly IC-induced upregulation of ZAPS, whereas knockdown of IFN-ß or RIG-I did not affect ZAPS upregulation. SN50 did not affect ZAPS expression. Knockdown of ZAP enhanced JEV propagation. CONCLUSION: ZAPL and ZAPS were expressed in hCMEC/D3 cells, and ZAPS expression was upregulated by poly IC. IRF3 and TRIM25 are involved in poly IC-induced upregulation of ZAPS. ZAP may contribute to antiviral reactions in brain microvascular endothelial cells and protect the brain from invading viruses such as JEV.

Interleukin-6 via Toll-Like Receptor 3 Signaling Attenuates the Expression of Proinflammatory Chemokines in Human Podocytes.

BACKGROUND: Although toll-like receptor 3 (TLR3) signaling is involved in the development of certain chronic kidney diseases, the specific molecular mechanisms underlying inflammatory reactions via activation of TLR3 signaling in human podocytes remain unclear. Interleukin (IL)-6 is a pleiotropic cytokine associated with innate and adaptive immune responses; however, little is known about the implication of IL-6 via the activation of regional TLR3 signaling in the inflammatory reactions in human podocytes. METHODS: We treated immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and assessed the expression of IL-6, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against IFN-ß and IL-6. RESULTS: We found that the activation of TLR3 induced expression of IL-6, MCP-1, CCL5, and IFN-ß in human podocytes. RNA interference experiments revealed that IFN-ß was involved in the poly IC-induced expression of IL-6, MCP-1, and CCL5. Interestingly, IL-6 knockdown markedly increased the poly IC-induced expression of MCP-1 and CCL5. Further, treatment of cells with IL-6 attenuated the expression of CCL5 and MCP-1 mRNA and proteins. CONCLUSION: IL-6 induced by TLR3 signaling negatively regulates the expression of representative TLR3 signaling-dependent proinflammatory chemokines in human podocytes.

Inhibitory effect of anti-malarial agents on the expression of proinflammatory chemokines via Toll-like receptor 3 signaling in human glomerular endothelial cells.

OBJECTIVE: Although anti-malarial agents, chloroquine (CQ) and hydroxychloroquine (HCQ) are currently used for the treatment of systemic lupus erythematosus, their efficacy for lupus nephritis (LN) remains unclear. Given that upregulation of glomerular Toll-like receptor 3 (TLR3) signaling plays a pivotal role in the pathogenesis of LN, we examined whether CQ and HCQ affect the expression of the TLR3 signaling-induced representative proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) in cultured human glomerular endothelial cells (GECs). METHODS: We examined the effect of polyinosinic-polycytidylic acid (poly IC), an agonist of TLR3, on MCP-1, CCL5 and interferon (IFN)-ß expression in GECs. We then analyzed whether pretreatment with CQ, HCQ, or dexamethasone (DEX) inhibits poly IC-induced expression of these chemokines using real-time quantitative reverse transcriptase PCR and ELISA. Phosphorylation of signal transducers and activator of transcription protein 1 (STAT1) was examined using western blotting. RESULTS: Poly IC increased MCP-1 and CCL5 expression in a time- and concentration-dependent manner in GECs. Pretreating cells with CQ, but not DEX, attenuated poly IC-induced MCP-1 and CCL5 expression; however, HCQ pretreatment attenuated poly IC-induced CCL5, but not MCP-1. HCQ did not affect the expression of IFN-ß and phosphorylation of STAT-1. CONCLUSION: Considering that TLR3 signaling is implicated, at least in part, in LN pathogenesis, our results suggest that anti-malarial agents exert a protective effect against the development of inflammation in GECs, as postulated in LN. Interestingly, CQ is a rather powerful inhibitor compared with HCQ on TLR3 signaling-induced chemokine expression in GECs. In turn, these findings may further support the theory that the use of HCQ is safer than CQ in a clinical setting. However, further detailed studies are needed to confirm our preliminary findings.

Daily Brazilian green propolis intake elevates blood artepillin C levels in humans.

BACKGROUND: Propolis is a natural product collected by worker bees from a variety of plant species. As a type of propolis, Brazilian green propolis contains a large amount of artepillin C. Artepillin C is a cinnamic acid derivative and has been shown to have a wide variety of biological functions, including anti-inflammatory, antiviral and antitumor activities, in both cell culture and animal models. However, how propolis is digested and absorbed remains to be elucidated. Moreover, blood artepillin C levels after propolis intake have not been shown in human studies. RESULTS: A randomized, single-blind placebo-controlled study on the effect of Brazilian green propolis on serum artepillin C levels was conducted with healthy volunteers. The participants (n = 133) were randomly allocated in an approximately 2:1 ratio to two groups: propolis (n = 91) and placebo (n = 42). The participants took daily propolis or placebo, and blood tests were performed on day 0 (before propolis intake) and days 1, 3 and 7. Artepillin C was detected in serum in almost all individuals in the propolis groups. No serum artepillin C was detected in the placebo group. Serum artepillin C levels in the female group tended to be higher than those in the male group. In the female group, menstrual status was unrelated to serum artepillin C levels. CONCLUSION: These results suggested that propolis intake might be more effective for females than for males. © 2021 Society of Chemical Industry.

Human aortic valve interstitial cells obtained from patients with aortic valve stenosis are vascular endothelial growth factor receptor 2 positive and contribute to ectopic calcification.

Since aortic valve stenosis (AVS) is the most frequent and serious valvular heart disease in the elderly, and is accompanied by irreversible valve calcification, medicinal prevention of AVS is important. Although we recently demonstrated that human aortic valve interstitial cells (HAVICs) obtained from patients with AVS were highly sensitive to ectopic calcification stimulation, the cell types contributing to calcification are unknown. We aimed to immunocytochemically characterize HAVICs and identify their contribution to valve calcification. HAVICs were isolated from patients with AVS and cultured on non-coated dishes. Immunocytochemical features and HAVIC differentiation were analyzed in passage 1 (P1). The immunohistochemical features of the calcified aortic valve were analyzed. Most cultured P1 HAVICs were CD73-, CD90-, and CD105-positive, and CD45-and CD34-negative. HAVICs were vascular endothelial growth factor receptor 2 (VEGFR2)-positive; however, approximately half were α-smooth muscle actin (SMA)-positive, colonized, and easily differentiated into osteoblastic cells. Calcified aortic valve immunohistochemistry showed that all cells were positive for VEGFR2 and partly α-SMA. Further, VEGFR2-positive cells were more sensitive to tumor necrosis factor-α-induced ectopic calcification with or without α-SMA positivity. We conclude that HAVICs obtained from patients with AVS are VEGFR2-positive undifferentiated mesenchymal cells and may contribute to aortic valve ectopic calcification.

Role of MDA5 in regulating CXCL10 expression induced by TLR3 signaling in human rheumatoid fibroblast-like synoviocytes.

C-X-C motif chemokine 10 (CXCL10) is an inflammatory chemokine and a key molecule in the pathogenesis of rheumatoid arthritis (RA). Melanoma differentiation-associated gene 5 (MDA5) is an RNA helicase that plays a role in innate immune and inflammatory reactions. The details of the regulatory mechanisms of CXCL10 production and the precise role of MDA5 in RA synovitis have not been fully elucidated. The aim of this study was to examine the role of MDA5 in regulating CXCL10 expression in cultured human rheumatoid fibroblast-like synoviocytes (RFLS). RFLS was stimulated with Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA mimetic. Expression of interferon beta (IFN-ß), MDA5, and CXCL10 was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay. A neutralizing antibody of IFN-ß and siRNA-mediated MDA5 knockdown were used to determine the role of these molecules in regulating CXCL10 expression downstream of TLR3 signaling in RFLS. Poly I:C induced IFN-ß, MDA5, and CXCL10 expression in a concentration- and time-dependent manner. IFN-ß neutralizing antibody suppressed the expression of MDA5 and CXCL10, and knockdown of MDA5 decreased a part of CXCL10 expression (p < 0.001). The TLR3/IFN-ß/CXCL10 axis may play a crucial role in the inflammatory responses in RA synovium, and MDA5 may be partially involved in this axis.

Tryptanthrin suppresses double-stranded RNA-induced CXCL10 expression via inhibiting the phosphorylation of STAT1 in human umbilical vein endothelial cells.

Tryptanthrin is a bioactive component of indigo plants such as Polygonum tinctrorium and known to have an anti-inflammatory activity. The aim of this study was to investigate the effects of tryptanthrin on Toll-like receptor 3 (TLR3)-mediated cytokine and chemokine expression in human umbilical vein endothelial cells (HUVEC). Herein, we found that tryptanthrin suppressed the expression of CXCL10 in HUVEC upon stimulation with a TLR3 ligand polyinosinic-polycytidylic acid (poly IC). Tryptanthrin did not inhibit poly IC-induced activation of interferon regulatory factor 3 (IRF3) or the mRNA expression of interferon (IFN)-ß, while it significantly suppressed the expression of RIG-I, MDA5, and classical IFN-stimulated genes (ISGs). Tryptanthrin attenuated the phosphorylation and nuclear translocation of STAT1 in HUVEC stimulated with not only poly IC but also recombinant IFN-ß. These results suggested that tryptanthrin inhibited poly IC-induced expression of CXCL10 and ISGs via suppressing the activation of STAT1 in HUVEC. Our findings indicate that tryptanthrin may be useful for regulating TLR3-mediated vascular inflammation.

IFIT Proteins Are Involved in CXCL10 Expression in Human Glomerular Endothelial Cells Treated with a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.

Expression of IFN-induced transmembrane protein 1 in glomerular endothelial cells.

BACKGROUND: Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre-existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll-like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)-induced transmembrane protein 1 (IFITM1), a member of interferon-stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. METHODS: Cultured, normal human GECs were treated with polyinosinic-polycytidylic acid (poly IC), a synthesized viral double-stranded RNA, then the expression of IFITM1 was examined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. To further elucidate the poly IC-induced signaling pathway, the cells were applied to RNA interference against IFN-ß, nuclear factor-κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. RESULTS: We found that the activation of Toll-like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN-ß, but not nuclear factor-κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. CONCLUSIONS: Antiviral reaction-related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed.

Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer.

PURPOSE: Colorectal cancer (CRC) often recurs after curative resection. Identification of major risk factors for CRC recurrence is important for effective prevention and treatment. In this study, we examined the potential relationship between CRC and TLR3 as this remains unclear. PATIENTS AND METHODS: Correlations between TLR3 immunostaining and clinicopathological factors and prognosis were examined in 50 samples that were randomly extracted from 264 patients with CRC from January 2010 to December 2011. Chemokines induced by TLR3 agonist stimulation were also examined using TLR3-positive human CRC cell lines. Furthermore, the association between TLR3 and chemokine expression was assessed by analyzing the immunohistochemistry of surgical specimens. RESULTS: Of the 50 patients, 14 (28%) were TLR3-negative. In the comparison of clinicopathological factors between the TLR3-negative and -positive groups, there were more lymph node metastasis-positive cases in the TLR3-negative group, and this difference was significant. Furthermore, there was no difference in overall survival rates between the two groups, but the 5-year recurrence-free survival (RFS) was significantly lower in the TLR3-negative group (46.2%) than in the TLR3-positive group (78.1%). Analysis of 5-year RFS using factors thought to be related to recurrence identified a high tumor budding and a TLR3-negative status as independent risk factors for recurrence. TLR3 activation of CRC cell lines induced expression of C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), and interleukin-8. The expressions of CCL2, CCL5, and IL-8 were observed in the TLR3-positive tumor cells of surgical specimens. CONCLUSION: Non-expression of TLR3 in CRC cells was associated with lymph node metastasis and was an independent risk factor for recurrence. These results suggest that TLR3 may not only be used as a prognostic factor and a risk factor for recurrence, but further studies on the involvement of TLR3 with tumor growth may provide new therapeutic strategies.

ISG56 is involved in CXCL10 expression induced by TLR3 signaling in BEAS-2B bronchial epithelial cells.

Purpose and aim of the study: Bronchial epithelial cells play an important role in immune response against viral infections. Toll-like receptor 3 (TLR3) is a pathogen recognition receptor that recognizes viral double-stranded RNA (dsRNA). Activation of TLR3 induces the expression of interferon (IFN)-ß, and newly synthesized IFN-ß exhibits anti-viral activity by upregulating the expression of IFN-stimulated genes (ISGs). ISG56 encodes a multifunctional protein with tetratricopeptide motifs and is involved in anti-viral reactions through various mechanisms. Expression of chemokines such as CXCL10, which induces leukocyte chemotaxis, is essential for defense against airway microbes. However, regulation of chemokine expression by ISG56 in bronchial epithelial cells has not been fully investigated. The aim of this study was to examine the expression of ISG56 and its role in CXCL10 production in BEAS-2B bronchial epithelial cells treated with dsRNA.Materials and methods: BEAS-2B bronchial epithelial cells were treated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 ligand. The mRNA and protein expression levels of ISG 56 were analyzed by quantitative reverse transcription polymerase chain reaction and western blotting. The effect of knocking down TLR3, IFN-ß, and ISG56 was examined using RNA interference. The protein expression of CXCL10 in culture medium was measured using an enzyme-linked immunosorbent assay.Results: Poly IC induced ISG56 expression in a concentration- and time- dependent manner. RNA interference showed that ISG56 induction was inhibited by knockdown of TLR3 or IFN-ß and that ISG 56 knockdown decreased CXCL10 expression.Conclusions: ISG56 was induced by poly IC through TLR3/IFN-ß axis, and ISG56 may positively regulated CXCL10 expression in BEAS-2B cells. ISG56 may modulate anti-viral innate immunity, at least in part, by regulating the expression of CXCL10 in bronchial epithelial cells.

Polyinosinic-Polycytidylic Acid Induces CXCL1 Expression in Cultured hCMEC/D3 Human Cerebral Microvascular Endothelial Cells.

OBJECTIVE: Brain microvascular endothelial cells are integral components of the blood-brain barrier and play a role in protecting the brain from invading microbes. CXC motif chemokine ligand 1 (CXCL1) induces the chemotaxis of neutrophils, and neutrophils are important in host defense in the brain. However, dysregulated neutrophil infiltration leads to brain diseases. Toll-like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double-stranded RNA (dsRNA). The aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endothelial cells. METHODS: hCMEC/D3 human cerebral microvascular endothelial cells were cultured and treated with polyinosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. The production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The expression of CXCL1 was compared with that of CXCL8. The effect of pretreatment of cells with a NF-κB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), an interferon (IFN) regulatory factor 3 inhibitor (MRT67307), and an anti-type I IFN-neutralizing antibody mixture was examined. Phosphorylation of p38 was examined using Western blotting. RESULTS: Treating cultured hCMEC/D3 human cells with poly IC induced the expression of CXCL1 as well as another chemokine CXCL8. Pretreatment of cells with SN50, SB203580, and SP600125 decreased the induction of CXCL1 by poly IC. However, it was not affected by MRT67307 or by an anti-type I IFN-neutralizing antibody mixture. Pretreatment of cells with SN50 decreased the poly IC-induced phosphorylation of p38. CONCLUSIONS: Poly IC induces the expression of CXCL1 in hCMEC/D3 cells. NF-κB, p38 MAPK, and JNK are involved in this reaction. There is a cross-talk between NF-κB and p38, and NF-κB partially regulates phosphorylation of p38. CXCL1 produced by brain microvascular endothelial cells may contribute to the brain's defense against viral infection and various neurological diseases associated with neutrophil accumulation.