Polyinosinic-polycytidylic Acid Research Articles

Molecular Cloning of Toll-like Receptor 2 and 4 (SpTLR2, 4) and Expression of TLR-Related Genes from Schizothorax prenanti after Poly (I:C) Stimulation.

Toll-like receptor (TLR) signaling is conserved between fish and mammals, except for TLR4, which is absent in most fish. In the present study, we aimed to evaluate whether TLR4 is expressed in Schizothorax prenanti (SpTLR4). The SpTLR2 and SpTLR4 were cloned and identified, and their tissue distribution was examined. The cDNA encoding SpTLR4 and SpTLR2 complete coding sequences (CDS) were identified and cloned. Additionally, we examined the expression levels of seven SpTLRs (SpTLR2, 3, 4, 18, 22-1, 22-2, and 22-3), as well as SpMyD88 and SpIRF3 in the liver, head kidney, hindgut, and spleen of S. prenanti, after intraperitoneal injection of polyinosinic-polycytidylic acid (poly (I:C)). The SpTLR2 and SpTLR4 shared amino acid sequence identity of 42.15-96.21% and 36.21-93.58%, respectively, with sequences from other vertebrates. SpTLR2 and SpTLR4 were expressed in all S. prenanti tissues examined, particularly in immune-related tissues. Poly (I:C) significantly upregulated most of the genes evaluated in the four immune organs compared with the PBS-control (p < 0.05); expression of these different genes was tissue-specific. Our findings demonstrate that TLR2 and TLR4 are expressed in S. prenanti and that poly (I:C) affects the expression of nine TLR-related genes, which are potentially involved in S. prenanti antiviral immunity or mediating pathological processes with differential kinetics. This will contribute to a better understanding of the roles of these TLR-related genes in antiviral immunity.

Group I mGluRs positive allosteric modulators improved schizophrenia-related behavioral and molecular deficits in the Poly I:C rat model

RationaleMaternal polyinosinic-polycytidylic acid (Poly I:C) exposure leads to an increase in various proinflammatory cytokines and causes schizophrenia-like symptoms in offspring. In recent years, group I metabotropic glutamate receptors (mGluRs) have emerged as a potential target in the pathophysiology of schizophrenia. ObjectivesThe aim of our study was to investigate the behavioral and molecular changes by using the mGlu1 receptor positive allosteric modulator (PAM) agent RO 67-7476, and the negative allosteric modulator (NAM) agent JNJ 16259685 and the mGlu5 receptor PAM agent VU-29, and NAM agent fenobam in the Poly I:C-induced schizophrenia model in rats. MethodsFemale Wistar albino rats were treated with Poly I:C on day 14 of gestation after mating. On the postnatal day (PND) 34-35, 56-57 and 83-84, behavioral tests were performed in the male offspring. On the PND84, brain tissue was collected and the level of proinflammatory cytokines was determined by ELISA method. ResultsPoly I:C caused impairments in all behavioral tests and increased the levels of proinflammatory cytokines. While PAM agents caused significant improvements in prepulse inhibition (PPI), novel object recognition (NOR), spontaneous alternation and reference memory tests, they brought the levels of proinflammatory cytokines closer to the control group. NAM agents were ineffective on behavioral tests. It was observed that PAM agents significantly improved Poly I:C-induced disruption in behavioral and molecular analyses. ConclusionsThese results suggest that PAM agents, particularly the mGlu5 receptor VU-29, are also promising and could be a potential target in schizophrenia.

Expression of interferon-stimulated gene 20 (ISG20), an antiviral effector protein, in glomerular endothelial cells: possible involvement of ISG20 in lupus nephritis

Background In addition to regulating the antiviral response, increased expression of Toll-like receptor 3 (TLR3) in resident renal cells plays a role in developing some forms of glomerulonephritis. TLR3 activation leads to type I interferon (IFN) production, which induces the expression of IFN-stimulated genes (ISGs). However, the role of ISG20 expression in resident renal cells remains unclear. Methods Cultured normal human glomerular endothelial cells (GECs) were treated with polyinosinic-polycytidylic acid (poly IC), Escherichia coli lipopolysaccharide (LPS), R848, and CpG (TLR3, TLR4, TLR7, and TLR9 agonists, respectively). The mRNA levels of ISG20, CX3CL1/fractalkine, and CXCL10/IP-10 were measured by quantitative reverse transcription-polymerase chain reaction. ISG20 protein expression was assessed by Western blotting. RNA interference was used to knockdown IFN-β and ISG20 expression. CX3CL1 protein levels were assessed by enzyme-linked immunosorbent assay. We performed immunofluorescence to examine endothelial ISG20 expression in biopsy specimens from patients with lupus nephritis (LN). Results In GECs, the expression of ISG20 mRNA and protein was increased by polyIC, not by LPS, R848, or CpG treatment. Moreover, ISG20 knockdown prevented poly IC-induced CX3CL1 expression but had no effect on CXCL10 expression. Intense endothelial ISG20 immunoreactivity was observed in biopsy specimens obtained from patients with proliferative LN. Conclusion In GECs, ISG20 was regulated via TLR3 but not via TLR4, TLR7, or TLR9 signaling. Moreover, ISG20 was involved in regulating CX3CL1 production. In addition to regulating antiviral innate immunity, ISG20 may act as a mediator of CX3CL1 production, thereby inducing glomerular inflammation, particularly in patients with LN.

Alterations in gene expressions of Caco-2 cell responses to LPS and ploy(I:C) stimulation.

The intestinal epithelium barrier serves as a highly dynamic immunologic frontier in the defense against invading pathogenic bacteria and viruses. Hence, understanding of the complicated underlying relationship between enteric pathogens and the intestinal epithelium barrier is vital for developing strategies to improve the intestinal health of farm animals. To this end, Caco-2 cells were stimulated by 1 µg/ml lipopolysaccharide (LPS) for 24 h and 5 µg/ml polyinosinic-polycytidylic acid (ploy(I:C)) for 4 h to imitate bacterial and viral infection processes, respectively. The specific alterations in gene expression of Caco-2 cells after stimulation were characterized by transcriptome sequencing. Seventy differentially expressed genes (DEGs) were identified under LPS exposure, and 17 DEGs were observed under ploy(I:C) exposure. We found that most DEGs were specific, and only one common DEG SPAG7 was observed. Gene Ontology (GO) annotation analysis indicated that all DEGs identified in the different treatments were mainly derived from GO terms related to the maintenance of cellular homeostasis. Moreover, specific DEGs such as SLC39A10, MT2A, and MT1E regulated by LPS treatment, while IFIT2 and RUNX2 mediated by ploy(I:C) treatment, which are derived from immune function modulation related GO terms, were confirmed by both transcriptome sequencing and qRT-PCR. In addition, both transcriptome sequencing and qRT-PCR results verified that LPS specifically down-regulated the DEGs INHBE and ARF6, which are involved in inflammation responses related to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway including the TGF-beta signaling pathways and the Ras signaling pathway. Ploy(I:C) uniquely suppressed the DEGs GABARAP and LAMTOR3, which participated in viral replication-associated pathways including autophagy and mTOR signaling pathway.

Molecular characterization, structural and expression analysis of twelve CXC chemokines and eight CXC chemokine receptors in largemouth bass (Micropterus salmoides).

The chemokine-receptor system plays important roles in the leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. In the present study, the sequence features, structures and expression patterns of twelve CXC chemokine ligands (CXCL8a.1, CXCL8a.2, CXCL8b.1, CXCL8b.2, CXCL12a, CXCL12b, CXCL13.1, CXCL13.2, CXCL14, CXCL18a, CXCL18b and CXCL19) and eight CXC chemokine receptors (CXCR1, CXCR2, CXCR3.1, CXCR3.2, CXCR3.3, CXCR4a, CXCR4b and CXCR5) of largemouth bass (Micropterus salmoides) were analyzed. All the CXCLs and CXCRs of largemouth bass shared high sequence identities with their teleost counterparts and possessed conserved motifs and structures of CXCLs and CXCRs family. Realtime qPCR revealed that these CXCLs and CXCRs were ubiquitously expressed in all examined tissues, with high expression levels in the immune-related tissues (spleen, head kidney, and gill). Following lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (polyI:C) stimulations, most of these CXCLs and CXCRs were significantly up-regulated in spleen. In addition, the potential interacted molecules of these CXCLs and CXCRs were analyzed by protein-protein interaction network analysis. To the best of our knowledge, this is the first study that in detail analyzes the CXCLs and CXCRs of largemouth bass. Our results provide valuable basis for study the function and mechanism of chemokine-receptor system in largemouth bass.

Diverse pathogen-associated molecular patterns affect transcription of genes in the toll-like receptor signaling pathway in goat blood

Pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), Polyinosinic-polycytidylic acid (poly I:C), and CpG Oligodeoxynucleotides (ODN) are recognized by Toll-like receptors (TLR). This study aimed to investigate the effect of diverse PAMPs on the transcription of TLR signaling pathway genes in goat blood. Whole blood was collected from 3 female BoerXSpanish goats and treated with the following PAMPs: 10 µg/ml LPS, PGN, CpG ODN (2216), CpG ODN (2006), and 12.5 µg/ml Poly I:C. Blood-treated PBS served as a control. The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array (Qiagen) was evaluated using real-time PCR. Treatment with PBS affected the expression of 74 genes, Poly I:C affected the expression of 40 genes, t ODN 2006 affected the expression of 50 genes, ODN 2216 affected the expression of 52 genes, LPS affected the expression of 49 genes, while PGN affected the expression of 49 genes. Our results show that PAMPs modulated and increased the expression of genes in the TLR signaling pathway. These results highlight important insights into how the host responds to different pathogens and may help design adjuvants for therapeutics and vaccines that target different.

Efficiency of Interferon-γ in Activating Dendritic Cells and Its Potential Synergy with Toll-like Receptor Agonists.

Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been used as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases or psychoactive compounds. In this study, we aimed to explore the potential of IFN-γ and TLR agonists being applied simultaneously to boost dendritic cell activation and the subsequent antigen presentation. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic-polycytidylic acid (poly I:C), or resiquimod (R848). Next, the dendritic cells were stained for an activation marker, a cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. From the cytometric analysis, IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of dendritic cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 µg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% CD86-positive cells obtained by 10 ng/mL IFN-γ. These results suggested that IFN-γ and TLR agonists could be applied as complementary systems to promote dendritic cell activation and antigen presentation. There might be a synergy between the two classes of molecules, but further investigation is warranted to ascertain the interaction of their promotive activities.

Structural insights of Labeo catla (catla) myxovirus resistance protein,GTP binding recognition and constitutive expression induced with Poly I:C

The Myxovirus resistance (Mx) proteins are critical effectors belonging to the super-family of guanidine triphosphatase, often stimulated by type I interferon (IFN) and mediates antiviral responses to restrict the replication of numerous viral genes in fishes. In teleosts, Mx proteins display diverse and complicated antiviral activity in different species. The present investigation seeks to characterize the Mx gene from Labeo catla upon induction by double-stranded (ds) RNA, polyinosinic-polycytidylic acid, (poly I: C). Molecular modeling and all-atoms molecular dynamics (MD) simulations were employed to understand the architecture of the GTPase domain and its plausible mode of GTP recognition in Mx protein. The full-length L. catla Mx (LcMx) gene sequence (1821 bp nucleotides) encodes an open reading frame of 606 amino acids. Domain search indicated conserved tripartite domain architecture of LcMx and forms a major cluster with the Mx from other teleosts. The positively charged Arginine and polar Glutamine residues from helix 3 and 4 of stalk region LcMx aid in homo-oligomerization. MD simulation portrayed the role of conserved critical residues aid in GTP recognition by the GTPase domain which perfectly corroborates with experimental findings and prior MD studies. After injection of poly I:C, the temporal mRNA profile showed that LcMx expression was significantly elevated in the spleen, brain, kidney, liver, muscle, heart, intestine, and gill tissues. Collectively, these results suggest that the elevated expression of the major innate immune defense gene Mx was able to inhibit the poly I: C mediated virulence in fish. Communicated by Ramaswamy H. Sarma

Behavioral and cellular responses to circadian disruption and prenatal immune activation in mice

Most individuals with neurodevelopmental disorders (NDDs), including schizophrenia and autism spectrum disorders, experience disruptions in sleep and circadian rhythms. Epidemiological studies indicate that exposure to prenatal infection increases the risk of developing NDDs. We studied how environmental circadian disruption contributes to NDDs using maternal immune activation (MIA) in mice, which models prenatal infection. Pregnant dams were injected with viral mimetic poly IC (or saline) at E9.5. Adult poly IC- and saline-exposed offspring were subjected to 4 weeks of each of the following: standard lighting (LD1), constant light (LL) and standard lighting again (LD2). Behavioral tests were conducted in the last 12 days of each condition. Poly IC exposure led to significant behavioral differences, including reduced sociability (males only) and deficits in prepulse inhibition. Interestingly, poly IC exposure led to reduced sociability specifically when males were tested after LL exposure. Mice were exposed again to either LD or LL for 4 weeks and microglia were characterized. Notably, poly IC exposure led to increased microglial morphology index and density in dentate gyrus, an effect attenuated by LL exposure. Our findings highlight interactions between circadian disruption and prenatal infection, which has implications in informing the development of circadian-based therapies for individuals with NDDs.

TLR3 activation by Clonorchis sinensis infection alleviates the fluke-induced liver fibrosis.

Clonorchis sinensis is a zoonotic parasite associated with liver fibrosis and cholangiocarcinoma development. The role of toll-like receptors (TLRs) in C. sinensis infection has not yet been fully elucidated. Here, the TLR3 signaling pathway, cytokine expression and liver fibrosis were examined in C. sinensis-infected wildtype (WT) and TLR3-/- mice. Polyinosinic-polycytidylic acid (Poly (I:C)) was used to treat C. sinensis infections. The results showed that TLR3 deficiency caused severe clonorchiasis with increased parasite burden, exacerbated proinflammatory cytokine expression and liver lesions, promoted the TGF-β1/Smad2/3 pathway and myofibroblast activation, exacerbated liver fibrosis (compared to WT mice). Poly (I:C) intervention increased the body weight, decreased mouse mortality and parasite burden, reduced liver inflammation, and alleviated C. sinensis-induced liver fibrosis. Furthermore, C. sinensis extracellular vesicles (CsEVs) promote the production of IL-6, TNF in WT biliary epithelial cells (BECs) via p38/ERK pathway, compared with control group, while TLR3 deletion induced much higher levels of IL-6 and TNF in TLR3-/- BECs than that in WT BECs. Taken together, TLR3 inhibit IL-6 and TNF production via p38/ERK signaling pathway, a phenomenon that resulted in the alleviation of C. sinensis-induced liver fibrosis. Poly (I:C) is a potential treatment for clonorchiasis.

Activation of the aryl hydrocarbon receptor inhibits the development of experimental autoimmune pancreatitis through IL-22-mediated signaling pathways.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor expressed in hematopoietic and non-hematopoietic cells. Activation of the AhR by xenobiotics, microbial metabolites, and natural substances induces immunoregulatory responses. Autoimmune pancreatitis (AIP) is a chronic fibroinflammatory disorder of the pancreas driven by autoimmunity. Although AhR activation generally suppresses pathogenic autoimmune responses, the roles played by the AhR in AIP have been poorly defined. In this study, we examined how AhR activation affected the development of experimental AIP caused by the activation of plasmacytoid dendritic cells producing IFN-α and IL-33. Experimental AIP was induced in MRL/MpJ mice by repeated injections of polyinosinic-polycytidylic acid. Activation of the AhR by indole-3-pyruvic acid and indigo naturalis, which were supplemented in the diet, inhibited the development of experimental AIP, and these effects were independent of the activation of plasmacytoid dendritic cells producing IFN-α and IL-33. Interaction of indole-3-pyruvic acid and indigo naturalis with AhRs robustly augmented the production of IL-22 by pancreatic islet α cells. The blockade of IL-22 signaling pathways completely canceled the beneficial effects of AhR ligands on experimental AIP. Serum IL-22 concentrations were elevated in patients with type 1 AIP after the induction of remission with prednisolone. These data suggest that AhR activation suppresses chronic fibroinflammatory reactions that characterize AIP via IL-22 produced by pancreatic islet α cells.

Combination adjuvants and antigens provide heterosubtypic protection against lethal influenza infection.

Abstract Influenza A virus (IAV) is a negative strand RNA virus that undergoes antigenic drift and shift, leading to serologically distinct strains that can cause tens of thousands of deaths annually in the US. The wide variety of hemagglutinin (HA) and neuraminidase (NA) proteins necessitates yearly reformulation of the vaccine to protect against novel seasonal and pandemic strains. Current IAV vaccines do not contain adjuvants, however, immune modulators such as pattern recognition receptor (PRR) agonists may enhance antigen presentation to T cells and provide broader protection against IAV strains that differ in HA and NA outer coat proteins. We set out to investigate whether TLR4 (lipopolysaccharide) and TLR3 (poly IC) agonists in combination with HA (H3N2) and nucleoprotein (NP) or NP and matrix (M1) proteins could provide protection against lethal challenge with mismatched IAV strain H1N1. Mice given H3 and NP with LPS + pIC in a prime/boost strategy demonstrated 80% survival after heterosubtypic H1N1 challenge while H3 + NP in PBS showed 20% survival. Similar results were seen using LPS + pIC in combination with NP and M1 protein with 90% survival. High endpoint titers of anti-NP antibodies (that also bound H1N1 virus) correlated with heterosubtypic protection suggesting a role for non-neutralizing antibodies in promoting survival. In addition, LPS and pIC enhanced dendritic cell (DC) function in vitro and increased migratory monocyte DC in the lymph node in vivo, coupled with early upregulation of T cell marker CD69. This strategy may provide increased efficacy over current IAV vaccines by inducing non-neutralizing antibodies and enhanced T cell responses to internal proteins. Supported by Trudeau Institute and Clarkson University.

Abstract 156: Role of Platelets in Maintaining Hemostasis and Coagulation Activation in Viral-induced Acute Respiratory Distress Syndrome (ards) in Mice

Platelets are crucial players during viral infections, as bridge hemostasis, inflammation, and immune response. RNA viruses like IAV are associated with pathologic platelet activation and thrombosis. We are currently investigating how platelet depletion before or post initial IAV infection can affect disease outcome. WT mice were infected with IAV (0.02HAU, intranasally) for 7 days to induce viral ARDS. Mice were injected with platelet depletion antibody R300 or control IgG at day 0 (prior) and day 3 (after infection). Mice treated with R300/control IgG were subjected to dsRNA mimetic poly IC intranasally for 3 days to induce virus-like ARDS. Global lung function was recorded, plasma and BALF were collected 7 or 3 dpi or final poly IC treatment, respectively. BALF cellularity and blood cellular composition was analyzed. Markers of lung injury and cytokines were measured. We observed that IAV infection leads to local lung inflammation, coagulation activation and ARDS-like lung dysfunction. Mice treated with R300 showed severe thrombocytopenia in blood. We observed more impaired alveolar hemostasis and increased hemorrhages into the airspace when platelet depletion was initiated 3 dpi compared to mice where platelets were depleted prior to infection. In addition, in dsRNA-induced ARDS model, platelet depletion resulted in increased signs of lung dysfunction, increased lung injury and local inflammation compared to mice with normal platelet levels. In conclusion, depletion of platelets after IAV infection resulted in increased alveolar hemorrhage and was associated with more pronounced lung inflammation, injury, and lung dysfunction. Our study suggests that interfering with platelet before viral infection can be beneficial as it limits the role of platelets as potential multiplicator of an initial pathologic immune reaction subsequently reducing collateral damage and organ dysfunction/failure. However, at later stages of viral infection, platelet activation is beneficial to facilitate tissue hemostasis as their absence later can result in increased hemorrhagic risk and more pronounced lung dysfunction. We aim to address the mechanism by which platelet contribute later to pathologic immune processes in dsRNA-induced or viral ARDS

Cecropin P1 antimicrobial peptide modulates differential expression of immune relevant genes in rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1

Accumulated evidence indicates that antimicrobial peptides modulate immune activities in fish. In this study, we profiled the differential expression patterns of representative immune relevant genes in an epithelial-like cell line of rainbow trout gill, RTgill-W1, in response to exposure of cecropin P1 antimicrobial peptide. RTgill-W1 cells were treated with synthetic cecropin P1 over time (0, 2, 4 and 24 h) with or without the present of lipopolysaccharide (LPS) or polyinosinic polycytidylic acid (PolyI:C). The relative abundances of each mRNA were measured by real-time quantitative PCR. The dose-response study revealed significant perturbations of mRNA levels of genes related to pro-inflammation, acute phase, surface proteins and transcription factors at 30 μM of cecropin P1. In addition, cecropin P1 altered the differential expression patterns that were induced by LPS or PolyI:C, at different time points in RTgill-W1. Overall, our results indicate that cecropin P1 exhibits pro-inflammation activity, modulate cell-cell interaction and cytokine signal transduction in rainbow trout gill cell, and may suggest a potential application of this peptide as an immune adjuvant for disease control in aquaculture.

Spleen Transcriptome Profiling Reveals Divergent Immune Responses to LPS and Poly (I:C) Challenge in the Yellow Drum (Nibea albiflora).

The yellow drum (Nibea albiflora) is a marine teleost fish with strong disease resistance, yet the understanding of its immune response and key functional genes is fragmented. Here, RNA-Seq was used to investigate the regulation pathways and genes involved in the immune response to infection with lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly (I:C)) on the spleen of the yellow drum. There were fewer differentially expressed genes (DEGs) in the LPS-infected treatment group at either 6 or 48 h. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were mainly significantly enriched in c5-branching dibasic acid metabolic and complement and coagulation cascades pathways. The yellow drum responded more strongly to poly (I:C) infection, with 185 and 521 DEGs obtained under 6 and 48 h treatments, respectively. These DEGs were significantly enriched in the Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Jak-STAT signaling pathway, NOD-like signaling pathway, and cytokine-cytokine receptor interaction. The key functional genes in these pathways played important roles in the immune response and maintenance of immune system homeostasis in the yellow drum. Weighted gene co-expression network analysis (WGCNA) revealed several important hub genes. Although the functions of some genes have not been confirmed, our study still provides significant information for further investigation of the immune system of the yellow drum.

Influence of dose and exposition time in the effectiveness of N-Acetyl-l-cysteine treatment in A549 human epithelial cells

N-Acetyl-l-cysteine (NAC) acts as a precursor of the tripeptide glutathione (GSH), one of the principal cell mechanisms for reactive oxygen species (ROS) detoxification. Chronic obstructive pulmonary disease (COPD) is associated with enhanced inflammatory response and oxidative stress and NAC has been used to suppress various pathogenic processes in this disease. Studies show that the effects of NAC are dose-dependent, and it appears that the efficient doses in vitro are usually higher than the achieved in vivo plasma concentrations. However, to date, the inconsistencies between the in vitro NAC antioxidant and anti-inflammatory in vitro effects, by reproducing the in vivo NAC plasma concentrations as well as high NAC concentrations. To do so, A549 were transfected with polyinosinic-polycytidylic acid (Poly (I:C)) and treated with NAC at different treatment periods. Oxidative stress, release of proinflammatory mediators and NFkB activation were analyzed. Results suggest that NAC at low doses in chronic administration has sustained antioxidant and anti-inflammatory effects, while acute treatment with high dose NAC exerts a strong antioxidant and anti-inflammatory response.

Amanita muscaria extract potentiates production of proinflammatory cytokines by dsRNA-activated human microglia.

Recent interest in mushrooms and their components as potential therapies for mental health, along with recent government and health authority approvals, has necessitated a more comprehensive understanding of their effects on the cellular microenvironment of the brain. Amanita muscaria has been ingested as a treatment for a variety of ailments for centuries, most notably those affecting the central nervous system and conditions associated with neuroinflammation. However, the effects of these extracts on neuroinflammatory cells, such as microglia, are unknown. The effect of commercially-sourced A. muscaria extract (AME-1) on human microglial cell line (HMC3) expression of surface receptors such as CD86, CXCR4, CD45, CD125 and TLR4 was determined by flow cytometry. AME-1 upregulated expression of all of these receptors. The effect of AME-1 on HMC3 production of IL-8 and IL-6 was determined and compared to tumor necrosis factor (TNF), polyinosinic-polycytidylic acid [poly(I:C)], substance P and lipopolysaccharide (LPS), all known activators of HMC-3 and primary microglia. HMC3 produced both IL-8 and IL-6 when activated with LPS, TNF and poly(I:C) but not when they were activated with substance P. Although AME-1 at higher concentrations increased IL-8 production of HMC3 on its own, AME-1 notably potentiated HMC3 production of IL-8 in response to poly(I:C). AME-1 altered expression of toll-like receptor 3 (TLR3) mRNA but not surface protein by HMC3. AME-1 also did not significantly alter expression of retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), both cytosolic sensors of dsRNA. Metabolomics analysis showed that AME-1 contained several metabolites, including the autophagy inducer, trehalose. Like AME-1, trehalose also potentiated HMC3 poly(I:C) mediated production of IL-8. This study suggests that A. muscaria extracts can modify HMC3 inflammatory responses, possibly due to their trehalose content.

An immunoglobulin superfamily member (CgIgIT2) functions as immune inhibitory receptor to inhibit the inflammatory cytokine expressions in Crassostrea gigas

Immune inhibitory receptors are increasingly acknowledged as potent regulators of immune response, which inhibit the overactivation of immune system and play an important role in maintaining immune homeostasis. In the present study, a novel immunoglobulin superfamily member (CgIgIT2) was identified from the Pacific oyster, Crassostrea gigas. The protein sequence of CgIgIT2 contained one signal peptide, four Ig domains, one fibronectin type III domain, one transmembrane domain, and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one immunoreceptor tyrosine-based switch motif (ITSM). The mRNA transcripts of CgIgIT2 were widely expressed in all the tested tissues, including haemolymph, gill, mantle, adductor muscle, labial palp, gonad and hepatopancreas, with the highest expression in haemolymph. The mRNA expressions of CgIgIT2 in haemocytes increased significantly at 24, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgIgIT2 protein were mainly detected in granulocytes of haemocytes, which were 1.27-fold and 2.15-fold (p < 0.05) higher than that of semi-granulocytes and agranulocytes, respectively. And CgIgIT2 was mainly located in the membrane and cytoplasm of haemocytes. The recombinant protein of CgIgIT2-4 × Ig (rCgIgIT2-4 × Ig) exhibited binding activity towards multiple pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN), mannose (MAN) and polyinosinic-polycytidylic acid (Poly (I: C)) with the highest affinity for LPS. rCgIgIT2-4 × Ig could also bind Gram-negative bacteria (V. splendidus, V. anguillarum, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and fungi (Pichia pastoris). In the blocking assay with anti-CgIgIT2 antibody, the mRNA expressions of interleukins (CgIL17-1, CgIL17-3 and CgIL17-6) and tumor necrosis factors (CgTNF-1 and CgTNF-2) in haemocytes all increased significantly at 12 h after V. splendidus stimulation. These results suggested that CgIgIT2 could function as an inhibitor receptor to bind different PAMPs and microbes, as well as inhibit the mRNA expressions of multiple inflammatory cytokines in oysters.

Abstract 3288: A novel and defined TLR3 agonist based anticancer therpay

Abstract Synthetic dsRNA analogs recognized by TLR3 are an attractive adjuvant candidate for fight against intracellular pathogens or tumors, because of their enhanced T cell-mediated and humoral immunities. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been severely limited due to heterogenicity, inconsistent activity, poor stability, and toxicity. In spite of numerous clinical trials, few products have been approved for human use. To overcome these limitations, we try to develop a novel defined form of TLR3 agonist. A single 275-kDa homogeneous molecule was selected as an alternative of poly IC and its derivatives and named NexaVant (NVT). The analytical analyses including reverse phase-HPLC analysis demonstrated that NVT is homologous without a lot-to-lot variation. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without signs of serious toxicity. NVT increases the expression of dsRNA sensors such as TLR3, MDA-5, and RIG-I. Together with a potent apoptotic cell death to several cancer cell lines, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-alpha production. Several poly IC and its derivatives are used as a in situ anticancer agent in clinical trials. NexaVant was tested as an in-situ cancer therapy reagent in several animal models. When NVT is injected into tumors, it induces and recruits diverse immune cells with a potent anti-cancer effect. Combined with anit-PD-1 antibody, it shows the increase number of DC and T-cells, especially CD8 T cells and decrease that of Tregs. Using human PD-1 knock-in mouse model, NVT has shown the synergistic anti-cancer effect with anti-PD-1 antibody. Several animal efficacy data target to head and neck cancer, triple-negative breast cancer, and melanoma would be presented. Citation Format: Dongho Kim, Sungwhan Lee. A novel and defined TLR3 agonist based anticancer therpay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3288.

Comprehensive Analysis of the Expression and Functions of Pattern Recognition Receptors in Differentiated Cytotrophoblasts Derived from Term Human Placentas.

Pregnant women are exposed to various microbes, some of which can harm the mother and/or fetus and can lead to life-long morbidity and even death. The syncytiotrophoblast (STB) covers the placental villi and comes into direct contact with pathogens contained in the maternal blood and plays a key role in placental host defense. However, the precise mechanisms whereby the STB recognizes and responds to pathogenic microbes remain unclear. In this study, we comprehensively analyzed the expression of functional pattern recognition receptors, which are responsible for tissue defense against pathogens, in a primary STB model differentiated from highly purified human term cytotrophoblasts (CTBs). Screening for mRNA expression and multiplex cytokine/chemokine production demonstrated that differentiated CTBs (dCTBs) predominantly expressed dsRNA receptors, including TLR3, MDA5, and RIG-I. We confirmed that term human placentas also expressed TLR3. Transcriptome analysis revealed common and unique responses of dCTBs to a synthetic dsRNA (polyinosinic-polycytidylic acid) compared with human peripheral mononuclear cells. Moreover, polyinosinic-polycytidylic acid induced the release of type I and type III IFNs (IFN-β, IFN-λ1, IFN-λ2, IFN-λ3), as well as mRNA expression of IFN-stimulated genes (IFIT1, MX1, and OAS1). dCTBs underwent apoptosis via the mitochondrial pathway in response to dsRNA stimulation. These results suggest that dsRNA receptors expressed on the STB are key players in antiviral defense in the placenta. Elucidation of the underpinnings of these defense processes can help us better understand the pathophysiology of viral infections during pregnancy.