Targeting TLR4/3 using chlorogenic acid ameliorates LPS+POLY I:C-induced acute respiratory distress syndrome via alleviating oxidative stress-mediated NLRP3/NF-κB axis.

Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a life-threatening condition caused due to significant pulmonary and systemic inflammation. Chlorogenic acid (CGA) has been shown to possess potent antioxidant, anti-inflammatory, and immunoprotective properties. However, the protective effect of CGA on viral and bacterial-induced ALI/ARDS is not yet explored. Hence, the current study is aimed to evaluate the preclinical efficacy of CGA in lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (POLY I:C)-induced ALI/ARDS models in vitro and in vivo. Human airway epithelial (BEAS-2B) cells exposed to LPS+POLY I:C significantly elevated oxidative stress and inflammatory signaling. Co-treatment with CGA (10 and 50 µM) prevented inflammation and oxidative stress mediated by TLR4/TLR3 and NLRP3 inflammasome axis. BALB/c mice, when chronically challenged with LPS+POLY I:C showed a significant influx of immune cells, up-regulation of pro-inflammatory cytokines, namely: IL-6, IL-1ß, and TNF-α, and treatment with intranasal CGA (1 and 5 mg/kg) normalized the elevated levels of immune cell infiltration as well as pro-inflammatory cytokines. D-Dimer, the serum marker for intravascular coagulation, was significantly increased in LPS+ POLY I:C challenged animals which was reduced with CGA treatment. Further, CGA treatment also has a beneficial effect on the lung and heart, as shown by improving lung physiological and cardiac functional parameters accompanied by the elevated antioxidant response and simultaneous reduction in tissue damage caused by LPS+POLY I:C co-infection. In summary, these comprehensive, in vitro and in vivo studies suggest that CGA may be a viable therapeutic option for bacterial and viral-induced ALI-ARDS-like pathology.

Transcriptome analysis of largemouth bass (Micropterus salmoides) challenged with LPS and polyI:C.

Largemouth bass (Micropterus salmoides) is a worldwide commercially important aquatic species. In recent years, pathogenic diseases cause great economic losses and hinder the industry of largemouth bass. To further understand the immune response against pathogens in largemouth bass, splenic transcriptome libraries of largemouth bass were respectively constructed at 12 h post-challenged with phosphate-buffered saline (PBS), lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (polyI:C) by using RNA sequencing technology (RNA-seq). RNA libraries were constructed using 9 RNA splenic samples isolated from three biological replicates of the three groups and sequenced on the DNBSEQ platform. A total number of 86,306 unigenes were obtained. Through pairwise comparisons among the three groups, we identified 11,295 different expression genes (DEGs) exhibiting significant differences at the transcript level. There were 7, 7, and 13 signal pathways were significantly enriched in LPS-PBS comparison, polyI:C-PBS comparison, and LPS-polyI:C comparison, respectively, indicating that the immune response to different pathogens was distinct in largemouth bass. To the best of our knowledge, this is the first report on the immune response of largemouth bass against different pathogen-associated molecular patterns (PAMPs) stimuli using transcriptomic analysis. Our results provide a valuable resource and new insights to understanding the immune characteristics of largemouth bass against different pathogens.

PolyI:C attenuates transforming growth factor-ß signaling to induce cytostasis of surrounding cells by secreted factors in triple-negative breast cancer.

The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-ß that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-ß (TGF-ß) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-ß signaling by RLR ligands with respect to IFN-ß-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-ß, which inhibits the growth of the surrounding cancer cells. In addition, IFN-ß-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-ß expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-ß that suppresses IFN-ß expression is suggested when RLR-mediated cancer treatment is used in TNBC.

Oral delivery of a dsRNA-Phytoglycogen nanoparticle complex enhances both local and systemic innate immune responses in rainbow trout.

Salmonids are one of the most farmed fish species worldwide. These aquatic vertebrates rely heavily on their innate immune responses as the first line of defense to defend themselves against invading pathogens. Although commercial vaccines are available against some viral and bacterial pathogens affecting salmonids, their protective efficacy varies. Using a prophylactic inducer of local and systemic innate immune responses to limit infection could have significant implications in salmonid aquaculture. A potent inducer of innate immune responses in fish is double-stranded RNA (dsRNA), a molecule that all viruses make during their replicative cycle. Polyinosinic: polycytidylic acid (polyI:C) is a synthetic dsRNA commonly used to induce type I interferons (IFNs), interferon stimulated genes (ISGs) as well as an antiviral state in vertebrate species. Based on in vitro data it was hypothesized that both local and systemic innate immune responses, in salmonids, would be enhanced by orally delivering high molecular weight polyI:C (HMW polyI:C) using cationic phytoglycogen nanoparticles (NPs) as a delivery method. The present study investigates this hypothesis using two feed delivery methods. In the first in vivo study, to ensure an equal distribution of dose, individual rainbow trout (Oncorhynchus mykiss) were orally gavaged with feed moistened with a solution containing HMW-NP (polyI:C complexed with cationic phytoglycogen nanoparticles) or HMW polyI:C alone. In a second in vivo experiment, to better mimic a more realistic feeding scenario, rainbow trout were fed feed pellets to which HMW, or HMW-NP was added. The expression of IFN1 and ISGs (vig-3, Mx1) were quantified using real-time PCR in the intestine (local response) and head kidney (systemic response). The results of these studies indicate that HMW-NP induced a higher level of IFN1 and ISG expression in the intestine and head kidney compared to the HMW fed fish. The results of this study could lead to new advances in therapeutics for the aquaculture industry by utilizing the innate immune response against invading pathogens using an orally delivered stimulant.

Identification, functional characterization and expression pattern of myeloid differentiation factor 88 (MyD88) in Nibea albiflora.

Myeloid differentiation factor 88 (MyD88), composed of an N-terminal death domain and a C-terminal Toll/interleukin (IL)-IR homology domain, is a key connector protein in the TLR signal transduction pathway. In this study a novel isoform of MyD88 in Nibea albiflora (named as NaMyD88) was identified and functionally characterized (GenBank accession no. MN384261.1). Its complete cDNA sequence was 1672 bp and contained an open reading frame of 879 bp encoding 292 amino acid residues, which was similar to its teleost fish counterparts in the length. The theoretical molecular mass was 33.63 kDa and the isoelectric point was 5.24. BLASTp analysis suggested that the deduced amino acids sequence of NaMyD88 shared high identity to the known MyD88, for instance, 94.77% identity with Collichthys lucidus. Sequence analysis showed that NaMyD88 protein was consistent with MyD88 protein of other species at three conserved domains, N-terminal DD, short middle domain and C-terminal TIR, and the TIR domain contained three highly conserved motifs: Box1, Box2, and Box3. NaMyD88 and red fluorescent protein (Dsred) were fused and expressed in the cytoplasm of the epithelioma papulosum cyprini (EPC cells). The NaTLR9-TIR-EGFP fusion protein, which was obtained in our previous studies, showed green fluorescence and mainly distributed in the cytoplasm. After co-transfection, NaMyD88-Dsred and NaTLR9-TIR-EGFP obviously overlapped and displayed orange-yellow color. The results showed that the homologous MyD88-Dsred could interact with NaTLR9-TIR-EGFP. Based on this result pcMV-NaMyD88-TIR-Myc plasmids and the pcDNA3.1-NaTLR9-TIR-flag were constructed and co-transfected into 293T cells for the immunoprecipitation test. According to Western blot, the protein eluted by Flag-beads could be detected by anti-Flag-tag antibody and anti-Myc tag antibody respectively, while the protein without NaTLR9-TIR could not be found, which further proved that TLR and MyD88 could interact each other. The prokaryotic plasmid of MyD88-TIR domain was constructed, expressed in BL21 (DE3) and purified by Ni-NAT super flow resin conforming to the expected molecular weight of 27 kDa with the corresponding active sites for its conferring protein-protein interaction functions. Real-time fluorescence quantitative PCR showed that NaMyD88 could be expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the kidney, and it was up-regulated after being infected with Polyinosinic:polycytidylic acid - Poly (I:C) and Pseudomonas plecoglossicida, which showed that NaMyD88 was involved in the immune response of N.albiflora. These data afforded a basis for understanding the role of NaMyD88 in the TLR signaling pathway of N.albiflora.

Santalum album extract exhibits neuroprotective effect against the TLR3-mediated neuroinflammatory response in human SH-SY5Y neuroblastoma cells.

Neuroinflammation is an inflammatory response in the nervous system that is associated with various neurological diseases including Alzheimer's diseases and others. Many studies evaluated the anti-inflammatory potential of Santalum album (S. album) extract, but none of them analyzed its effects against neuroinflammatory response in vitro. In addition, the precise mechanism underlying the anti-inflammatory effect of the extract has not yet been elucidated. Therefore, in this study, we investigated the effect of S. album extract on modulation of toll-like receptor 3 (TLR3) agonist polyinosnic-polycytidylic acid (PolyI:C)-induced neuroinflammatory response in human neuroblastoma cells. The TLR3-mediated immune response was differentially modulated by S. album extract in SH-SY5Y cells. In addition, treatment of cells with the conditioned medium (CM) of S. album extract significantly increased the mRNA levels of IFN-ß, IFN-α, MxA and OAS-1 and decreased IL-6, CXCL8, CCL2 and IP-10. S. album extract has indirectly affected the expression of IFNs and inflammatory cytokines in SH-SY5Y cells. Furthermore, the extract was able to modulate PolyI:C-induced inflammatory response in Caco2 cells. Overall, S. album was capable to attenuate PolyI:C-induced neuroinflammatory effect through the induction of TLR2, TLR4 and the modulation of TLR negative regulators of the TRAF3, IRF3 and NF-κB pathways.

Dendritic cells modulate c-kit expression on the edge between activation and death.

Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c-kit, the receptor for stem cell factor (SCF), nevertheless c-kit functional role and the regulation of its expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro-survival circuit, and that SCF increases phospho-AKT in c-kit+ mouse bone marrow-derived DCs (BMdDCs). Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c-kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI-/- mice showed that IFN-I pathway was required for c-kit down-regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c-kit mRNA expression. In agreement with the reduced c-kit levels, SCF pro-survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL-6 and IL-12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c-kit levels. Our studies suggest that c-kit down-modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.

LncMSEN1, a mantle-specific LncRNA participating in nacre formation and response to polyI:C stimulation in pearl oyster Pinctada fucata martensii.

Long noncoding RNA (LncRNA) regulates various life processes, including biomineralization and innate immune response through complex mechanisms. In this research, we identified a LncRNA named LncMSEN1 from pearl oyster Pinctada fucata martensii. LncMSEN1 sequence was validated by PCR, and its expression was high in mantle tissues according to qRT-PCR. LncMSEN1 was co-located with the nacre matrix protein N-U8 and fibrinogen domain-containing protein. And LncMSEN1 and N-U8 expression levels in the mantle were positively correlated. RNA interference was used to detect its effect on nacre formation in shells. Results showed that the decreased LncMSEN1 expression in mantle can cause the disordered growth of crystals on the inner surface of nacre in the shells, as well as the decrease expression of N-U8. In addition, the LncMSEN1 expression level significantly increased at 24 h after polyI:C stimulation in the mantle (P < 0.05). These findings suggested the involvement of LncMSEN1 in the formation of nacre in shells and related to innate immune response in pearl oyster, which provided additional insights into the roles of LncRNAs in pearl oysters.

Identification of DNA methylation regulated novel host genes relevant to inhibition of virus replication in porcine PK15 cell using double stranded RNA mimics and DNA methyltransferase inhibitor.

During RNA viruses's replication, double-stranded RNA (dsRNA) is normally produced and induce host innate immune response. Most of gene activation due cytokine mediated but which are due to methylation mediated is still unknown. In the study, DNA methylome was integrated with our previous transcriptome data to investigate the differentially methylated regions and genes using MeDIP-chip technology. We found that the transcriptional expressions of 15, 37 and 18 genes were negatively related with their promoter DNA methylation levels in the cells treated by PolyI:C, Aza-CdR, as well as PolyI:C plus Aza-CdR, respectively, compared with the untreated cells. GO analysis revealed hypo-methylated genes (BNIP3L and CDK9) and a hyper-methylated gene (ZC3HAV1) involved in the host response to viral replication. Our results suggest that these novel genes targeted by DNA methylation can be potential markers relevant to virus replication and host innate immune response to set up a medical model of infectious diseases.

Innate immune activation of astrocytes impairs neurodevelopment via upregulation of follistatin-like 1 and interferon-induced transmembrane protein 3.

BACKGROUND: Polyriboinosinic-polyribocytidylic acid (polyI:C) triggers a strong innate immune response that mimics immune activation by viral infections. Induction of interferon-induced transmembrane protein 3 (Ifitm3) in astrocytes has a crucial role in polyI:C-induced neurodevelopmental abnormalities. Through a quantitative proteomic screen, we previously identified candidate astroglial factors, such as matrix metalloproteinase-3 (Mmp3) and follistatin-like 1 (Fstl1), in polyl:C-induced neurodevelopmental impairment. Here, we characterized the Ifitm3-dependent inflammatory processes focusing on astrocyte-derived Fstl1 following polyI:C treatment to assess the neuropathologic role of Fstl1. METHODS: Astrocytes were treated with PBS (control) or polyI:C (10 µg/mL). The conditioned medium was collected 24 h after the polyI:C treatment and used as astrocyte condition medium (ACM). The expression of Fstl1 mRNA and extracellular Fstl1 protein levels were analyzed by quantitative PCR and western blotting, respectively. For functional studies, neurons were treated with ACM and the effects of ACM on dendritic elongation were assayed. To examine the role of Fstl1, recombinant Fstl1 protein and siRNA for Fstl1 were used. To investigate the expression of Fstl1 in vivo, neonatal mice were treated with vehicle or polyI:C on postnatal day 2 to 6. RESULTS: ACM prepared with polyI:C (polyI:C ACM) contained significantly higher Fstl1 protein than control ACM, but no increase in Fstl1 was observed in polyI:C ACM derived from Ifitm3-deficient astrocytes. We found that the production of Fstl1 involves the inflammatory responsive molecule Ifitm3 in astrocytes and influences neuronal differentiation. In agreement, the levels of Fstl1 increased in the hippocampus of polyI:C-treated neonatal mice. COS7 cells co-transfected with both Fstl1 and Ifitm3 had higher extracellular levels of Fstl1 than the cells transfected with Fstl1 alone. Treatment of primary cultured hippocampal neurons with recombinant Fstl1 impaired dendritic elongation, and the deleterious effect of polyI:C ACM on dendritic elongation was attenuated by knockdown of Fstl1 in astrocytes. CONCLUSIONS: The extracellular level of Fstl1 is regulated by Ifitm3 in astrocytes, which could be involved in polyI:C-induced neurodevelopmental impairment.

Polyriboinosinic-polyribocytidylic acid facilitates interleukin-6, and interleukin-8 secretion in human dermal fibroblasts via the JAK/STAT3 and p38 MAPK signal transduction pathways.

Polyriboinosinic-polyribocytidylic acid (polyI:C) is a viral dsRNA analoguethat promotes wounds healing, accelerates re-epithelialization, granulation and neovascularization, and induces pro-inflammatory cytokine release. Little is known about polyI:C mediated induction of inflammatory mediators in human dermal fibroblast (HDFs), which form the primary scaffold for epithelial cells covering the wound. Here, we found that polyI:C enhances IL-6 and IL-8 mRNA expression and induces of IL-6 and IL-8 production in a concentration-dependent and time-dependent manner in HDFs. PolyI:C treatment rapidly increased phosphorylation level of both STAT3 and p38 mitogen-activated protein kinase (MAPK). Moreover, pretreatment with AG490, a Janus kinase (JAK) inhibitor, inhibited polyI:C-induced STAT3 phosphorylation and subsequent IL-6 and IL-8 release. Conversely, pretreatment with SB203580, a selective inhibitor of p38 MAPK, blocked p38 MAPK phosphorylation and IL-6 and IL-8 expression. In conclusion, polyI:C induces IL-6 and IL-8 production in HDFs via the JAK/STAT3 and p38 MAPK signaling pathways.

Adjuvant immunotherapy for cancer: both dendritic cell-priming and check-point inhibitor blockade are required for immunotherapy.

The immune system eliminates advanced cancer when treated with programmed cell death protein-1 (PD-1) or its ligand (PD-L1) blockade, but PD-1 therapy is effective in only ∼20% of patients with solid cancer. The PD-1 antibody mainly acts on the effector phase of cytotoxic T lymphocytes (CTLs) in tumors but induces no activation of the priming phase of antigen-presenting dendritic cells (DCs). It is reasonable that both DC-priming and PD-1/L1 blocking are mandatory for efficient CTL-mediated tumor cytolysis. For DC-priming, a therapeutic vaccine containing Toll-like receptor (TLR) agonists, namely a priming adjuvant, is a good candidate; however, a means for DC-targeting by TLR adjuvant therapy remains to be developed. TLR adjuvants usually harbor cytokine toxicity, which is a substantial barrier against drug approval. Here, we discuss the functional properties of current TLR adjuvants for cancer immunotherapy and introduce a TLR3-specific adjuvant (ARNAX) that barely induces cytokinemia in mouse models.

Acute Effects of Viral Exposure on P-Glycoprotein Function in the Mouse Fetal Blood-Brain Barrier.

BACKGROUND/AIMS: Viral infection during pregnancy is known to affect the fetal brain. The toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viruses known to elicit adverse fetal neurological outcomes. The P-glycoprotein (P-gp) efflux transporter protects the developing fetus by limiting the transfer of substrates across both the placenta and the fetal blood-brain barrier (BBB). As such, inhibition of P-gp at these blood-barrier sites may result in increased exposure of the developing fetus to environmental toxins and xenobiotics present in the maternal circulation. We hypothesized that viral exposure during pregnancy would impair P-gp function in the placenta and in the developing BBB. Here we investigated whether the TLR-3 ligand, polyinosinic:polycytidylic acid (PolyI:C), increased accumulation of one P-gp substrate in the fetus and in the developing fetal brain. METHODS: Pregnant C57BL/6 mice (GD15.5) were injected (i.p.) with PolyI:C (5 mg/kg or 10 mg/kg) or vehicle (saline). [3H]digoxin (P-gp substrate) was injected (i.v.) 3 or 23h post-treatment and animals were euthanized 1h later. Maternal plasma, 'fetal-units' (fetal membranes, amniotic fluid and whole fetus), and fetal brains were collected. RESULTS: PolyI:C exposure (4h) significantly elevated maternal plasma IL-6 (P<0.001) and increased [3H]digoxin accumulation in the fetal brain (P<0.05). In contrast, 24h after PolyI:C exposure, no effect on IL-6 or fetal brain accumulation of P-gp substrate was observed. CONCLUSION: Viral infection modeled by PolyI:C causes acute increases in fetal brain accumulation of P-gp substrates and by doing so, may increase fetal brain exposure to xenobiotics and environmental toxins present in the maternal circulation.

Maternal immune activation during pregnancy in rats impairs working memory capacity of the offspring.

Maternal immune activation during pregnancy is an environmental risk factor for psychiatric illnesses such as schizophrenia in the offspring. Patients with schizophrenia display an array of cognitive symptoms, including impaired working memory capacity. Rodent models have been developed to understand the relationship between maternal immune activation and the cognitive symptoms of schizophrenia. The present experiment was designed to test whether maternal immune activation with the viral mimetic polyinosinic:polycytidylic acid (polyI:C) during pregnancy affects working memory capacity of the offspring. Pregnant Long Evans rats were treated with either saline or polyI:C (4mg/kg; i.v.) on gestational day 15. Male offspring of the litters (2-3months of age) were subsequently trained on a nonmatching-to-sample task with odors. After a criterion was met, the rats were tested on the odor span task, which requires rats to remember an increasing span of different odors to receive food reward. Rats were tested using delays of approximately 40s during the acquisition of the task. Importantly, polyI:C- and saline-treated offspring did not differ in performance of the nonmatching-to-sample task suggesting that both groups could perform a relatively simple working memory task. In contrast, polyI:C-treated offspring had reduced span capacity in the middle and late phases of odor span task acquisition. After task acquisition, the rats were tested using the 40s delay and a 10min delay. Both groups showed a delay-dependent decrease in span, although the polyI:C-treated offspring had significantly lower spans regardless of delay. Our results support the validity of the maternal immune activation model for studying the cognitive symptoms of neurodevelopmental disorders such as schizophrenia.

Identification of 2 novel type I IFN genes in Japanese flounder, Paralichthys olivaceus.

Two novel type I interferon genes (JfIFN3 and JfIFN4) have been identified in Japanese flounder Paralichthys olivaceus. Open reading frames of JfIFN3 and JfIFN4 were 555bp and 528bp, encoding 184aa and 175aa, respectively. The genomic structures of JfIFN3 and JfIFN4 are composed of 5 exons and 4 introns. JfIFN4 has 2 conserved cysteine residues, while JfIFN3 has 4. JfIFN3 and JfIFN4 showed the highest amino acid sequence identities to turbot IFN1 (74%) and IFN2 (62%), respectively. Interestingly, JfIFN3 and JfIFN4 were clustered in distinct branches with JfIFN1 and JfIFN2, which have reported so far. The mRNA levels of JfIFN4 were apparently increased in the kidney and spleen at 3 h after ployI:C injection, while JfIFN1-3 were not detected by RT-PCR.

Molecular cloning and expression analysis of toll-like receptor genes (TLR7, TLR8 and TLR9) of golden pompano (Trachinotus ovatus).

Toll like receptor (TLR) 7, 8 and 9 are intracellular TLRs which play important roles in host immune defense against bacterial or virus pathogens. In this study, TLR7, 8 and 9 were identified from golden pompano (Trachinotus ovatus), a marine teleost with great economic values. Sequence analysis revealed that the three TLRs contained several conserved characteristic features, including signal peptides, 25 leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motif. These three TLRs shared high sequence identity and similarity with their counterparts from other teleosts. The phylogenetic tree analysis showed the three TLRs were clustered well with their piscine counterparts, confirming the correctness of their nomenclatures and closed relationships during evolution. Quantitative real-time PCR revealed that the three TLRs were ubiquitously expressed in all the tested tissues from normal pompano, with high expression in spleen and head kidney, indicating their role in immune reaction. Further, pompano TLR7 and TLR8 was up-regulated in spleen and head kidney from 12 h to 48 h following polyI:C challenge, but remained no changes to Vibrio alginilyticus infection. In contrast, pompano TLR9 could be induced by V. alginilyticus infection but remained apathetic to polyI:C challenge. These results indicated that pompano TLR7, 8 and 9 might have distinct roles in response to bacterial or virus pathogens. Our results provided the basis for further study on ligand specificity and signaling pathways of fish TLRs which are required for elucidating the immune functions of fish TLRs.

Molecular characterization and expression analysis of toll-like receptors 5 and 22 from natural triploid Carassius auratus.

Innate immunity, as the most primitive and universal host defense in fish, constitutes an efficient first line of defense to combat invading microbes. Toll-like receptors (TLRs) play essential roles in the innate immunity, and TLR5 and TLR22 are two important TLRs that can recognize flagellin and double stranded RNA (dsRNA), respectively. In this study, we identified and characterized two TLRs genes of Qihe crucian carp (Carassius auratus) (designated as CaTLR5 and CaTLR22). The full-length cDNA sequence of CaTLR5 was cloned with 2972 bp including a 140 bp 5'-terminal untranslated region (UTR), a 183 bp 3'-UTR, and a 2649 bp open reading frame (ORF) encoding a deduced protein with 882 amino acids. The full-length cDNA of CaTLR22 was identified to be 3613 bp, consisting of a 228 bp 5'-UTR, a 547 bp 3'-UTR, and a 2838 bp ORF encoding a predicted protein of 945 amino acids. A typical TLR structure (an extracellular leucine-rich repeat domain, a transmembrane domain, and an intracellular Toll/IL-1 receptor domain) was found in CaTLR5 and CaTLR22. For either CaTLR5 or CaTLR22 gene, the mRNA expression levels varied in the different periods during the early stages of development. It was suggested that expression changes of gene CaTLR5 and CaTLR22 at mRNA levels were involved in developmental regulation in the early stages, and it was postulated that CaTLR5 and CaTLR22 play the important roles in immune defense in the early development stages of fish. Quantitative Real-Time PCR (qRT-PCR) revealed that CaTLR5 and CaTLR22 were constitutively expressed in all eleven tissues examined, although the mRNA expression level varied considerably among the different tissues. Following exposure to polyI:C, flagellin, and Aeromonas hydrophila, CaTLR5 and CaTLR22 were up-regulated in different tissues, and it was suggested that CaTLR5 and CaTLR22 were involved in the immune response of Qihe crucian carp against pathogenic invasions. The present findings will provide the valuable information for understanding the structure, function, expression, and the immune defense process of CaTLR5 and CaTLR22 in Qihe crucian carp, and provide new insights for developing the new strategies of disease control to protect fish against pathogens infection.

Live imaging of transforming growth factor-ß activated kinase 1 activation in Lewis lung carcinoma 3LL cells implanted into syngeneic mice and treated with polyinosinic:polycytidylic acid.

Transforming growth factor-ß activated kinase 1 (TAK1) has been shown to play a crucial role in cell death, differentiation, and inflammation. Here, we live-imaged robust TAK1 activation in Lewis lung carcinoma 3LL cells implanted into the s.c. tissue of syngeneic C57BL/6 mice and treated with polyinosinic:polycytidylic acid (PolyI:C). First, we developed and characterized a Förster resonance energy transfer-based biosensor for TAK1 activity. The TAK1 biosensor, named Eevee-TAK1, responded to stress-inducing reagents such as anisomycin, tumor necrosis factor-α, and interleukin1-ß. The anisomycin-induced increase in Förster resonance energy transfer was abolished by the TAK1 inhibitor (5z)-7-oxozeaenol. Activity of TAK1 in 3LL cells was markedly increased by PolyI:C in the presence of macrophages. 3LL cells expressing Eevee-TAK1 were implanted into mice and observed through imaging window by two-photon excitation microscopy. During the growth of tumor, the 3LL cells at the periphery of the tumor showed higher TAK1 activity than the 3LL cells located at the center of the tumor, suggesting that cells at the periphery of the tumor mass were under stronger stress. Injection of PolyI:C, which is known to induce regression of the implanted tumors, induced marked and homogenous TAK1 activation within the tumor tissues. The effect of PolyI:C faded within 4 days. These observations suggest that Eevee-TAK1 is a versatile tool to monitor cellular stress in cancer tissues.

Expression and biological activity of two types of interferon genes in medaka (Oryzias latipes).

Type I interferon (IFN) is one of most important cytokines for antiviral responses in fish innate immunity, after the induction pathway following pattern recognition. In this study, 2 types of type I IFN mRNA from a medaka (Japanese rice fish; Oryzias latipes) were identified and classified (phylogenetic analysis) into subgroup-a and -d by (designated olIFNa and olIFNd, respectively). Both olIFNa and olIFNd (encoding 197 and 187 amino acid residues, respectively) contained 2 cysteines. Gene expression pattern of olIFNa, olIFNd and IFN-stimulated genes (ISGs) was assessed (quantitative real-time reverse transcriptase PCR, qRT-PCR) in various organs (i.e., whole kidney, liver and spleen) of medaka stimulated by polyI:C or infected with nervous necrosis virus (NNV). Expression of olIFNa, olIFNd and ISGs, especially the ISG15 gene, were significantly upregulated after NNV-infection. Furthermore, olIFNa, olIFNd and ISGs mRNAs were sufficiently induced in DIT cells (i.e., medaka hepatoma cell line) transfected with polyI:C or infected with NNV. In addition, in vitro biological activities of recombinant olIFNa and olIFNd (rolIFNa and rolIFNd) produced by mammalian cell line HEK293T were also characterized. Expression of GIG1a and ISG15 genes in kidney cells of adult medaka were induced by rolIFNa or rolIFNd. The olIFNs-overexpressing DIT cells had reduced viral titers following NNV infection. Therefore, we inferred that 2 type I IFNs were involved in innate immunity (antiviral response) in medaka fish.

Adjuvant for vaccine immunotherapy of cancer--focusing on Toll-like receptor 2 and 3 agonists for safely enhancing antitumor immunity.

Immune-enhancing adjuvants usually targets antigen (Ag)-presenting cells to tune up cellular and humoral immunity. CD141(+) dendritic cells (DC) represent the professional Ag-presenting cells in humans. In response to microbial pattern molecules, these DCs upgrade the maturation stage sufficient to improve cross-presentation of exogenous Ag, and upregulation of MHC and costimulators, allowing CD4/CD8 T cells to proliferate and liberating cytokines/chemokines that support lymphocyte attraction and survival. These DCs also facilitate natural killer-mediated cell damage. Toll-like receptors (TLRs) and their signaling pathways in DCs play a pivotal role in DC maturation. Therefore, providing adjuvants in addition to Ag is indispensable for successful vaccine immunotherapy for cancer, which has been approved in comparison with antimicrobial vaccines. Mouse CD8α(+) DCs express TLR7 and TLR9 in addition to the TLR2 family (TLR1, 2, and 6) and TLR3, whereas human CD141(+) DCs exclusively express the TLR2 family and TLR3. Although human and mouse plasmacytoid DCs commonly express TLR7/9 to respond to their agonists, the results on mouse adjuvant studies using TLR7/9 agonists cannot be simply extrapolated to human adjuvant immunotherapy. In contrast, TLR2 and TLR3 are similarly expressed in both human and mouse Ag-presenting DCs. Bacillus Calmette-Guerin peptidoglycan and polyinosinic-polycytidylic acid are representative agonists for TLR2 and TLR3, respectively, although they additionally stimulate cytoplasmic sensors: their functional specificities may not be limited to the relevant TLRs. These adjuvants have been posted up to a certain achievement in immunotherapy in some cancers. We herein summarize the history and perspectives of TLR2 and TLR3 agonists in vaccine-adjuvant immunotherapy for cancer.