Induction Of Innate Immune Responses Research Articles

Engineered alphavirus replicon vaccines based on known attenuated viral mutants show limited effects on immunogenicity

The immunogenicity of alphavirus replicon vaccines is determined by many factors including the level of antigen expression and induction of innate immune responses. Characterized attenuated alphavirus mutants contain changes to the genomic 5′ UTR and mutations that result in altered non-structural protein cleavage timing leading to altered levels of antigen expression and interferon (IFN) induction. In an attempt to create more potent replicon vaccines, we engineered a panel of Venezuelan equine encephalitis-Sindbis virus chimeric replicons that contained these attenuating mutations. Modified replicons were ranked for antigen expression and IFN induction levels in cell culture and then evaluated in mice. The results of these studies showed that differences in antigen production and IFN induction in vitro did not correlate with large changes in immunogenicity in vivo. These findings indicate that the complex interactions between innate immune response and the replicon′s ability to express antigen complicate rational design of more potent alphavirus replicons.

Iron deficiency modifies gene expression variation induced by augmented hypoxia sensing

In congenital Chuvash polycythemia (CP), VHL(R200W) homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHL(R200W) homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate of 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHL(R200W) homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL(R200W) for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.

Immune responses to infection with H5N1 influenza virus

Influenza A H5N1 viruses remain a substantial threat to global public health. In particular, the expanding genetic diversity of H5N1 viruses and the associated risk for human adaptation underscore the importance of better understanding host immune responses that may protect against disease or infection. Although much emphasis has been placed on investigating early virus–host interactions and the induction of innate immune responses, little is known of the consequent adaptive immune response to H5N1 virus infection. In this review, we describe the H5N1 virus-specific and cross-reactive antibody and T cell responses in humans and animal models. Data from limited studies suggest that although initially robust, there is substantial waning of the serum antibody responses in survivors of H5N1 virus infection. Characterization of monoclonal antibodies generated from memory B cells of survivors of H5N1 virus infection has provided an understanding of the fine specificity of the human antibody response to H5N1 virus infection and identified strategies for immunotherapy. Human T cell responses induced by infection with seasonal influenza viruses are directed to relatively conserved internal proteins and cross-react with the H5N1 subtype. A role for T cell-based heterosubtypic immunity against H5N1 viruses is suggested in animal studies. Further studies on adaptive immune responses to H5N1 virus infection in both humans and animals are needed to inform the design of optimal immunological treatment and prevention modalities.

Carpesium abrotanoides extract inhibits cyclooxygenase-2 expression induced by toll-like receptor agonists

Toll-like receptors (TLRs) are a group of pattern-recognition receptors that play an important role in the induction of innate immune responses, recognizing conserved microbial structural molecules. The microbial pathogens trigger the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways, leading to proinflammatory cytokine production. Here, we investigated the effect of the ethanol extracts of Carpesium abrotanoides (CAB), a traditional Korean medicine, on the expression of cyclooxygenase-2 (COX-2) by TLR agonists in murine macrophages. CAB suppressed COX-2 expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3 agonist), and macrophage-activating lipopeptide 2-kDa (TLR2 and TLR6 agonist). These results suggest that CAB can regulate the TLR signaling pathways to treat chronic inflammatory diseases.

Drug Resistant Clinical Isolates of Mycobacterium tuberculosis from Different Genotypes Exhibit Differential Host Responses in THP-1 Cells

Mycobacterium tuberculosis (MTB) persistently infects and survives within the host macrophages. Substantial genotypic variation exists among MTB strains which correlate with their interactions with the host. The present study was designed to establish a correlation, if any, between infection and induction of innate immune response by genetically diverse drug resistant MTB isolates from India. For this purpose, three clinical isolates from ancient and modern lineages, along with H37Ra and H37Rv were evaluated for intracellular growth, phagocytic index, induction of proinflammatory cytokines and apoptosis following infection in THP-1 cell line. A wide variation in the induction of cytokines was revealed subsequent to infection with different strains. EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates. Further, the responses induced by LAM-6 from modern lineage 4 were at a moderate level, similar to the laboratory strain H37Rv which also belongs to lineage 4. Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status. Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated. In addition, induction of all pro-inflammatory cytokines correlated well with the host cell apoptosis. A positive correlation was observed between phagocytic index in the category of ‘>10 bacilli/cell’ and induction of apoptosis, only for virulent strains, indicating that initial accumulation of MTB strains inside the host cell may be an important determining factor for different innate responses.

The Stimulated Innate Resistance Event in Bordetella pertussis Infection Is Dependent on Reactive Oxygen Species Production

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 μg) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (10(7) CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.

Triptolide inhibits inducible nitric oxide synthase expression induced by toll-like receptor agonists

Toll-like receptors (TLRs) play an important role in the induction of innate immune responses recognizing many pathogen-associated molecular patterns. The activation of TLRs triggers two downstream signaling pathways; MyD88- (myeloid differential factor 88) and TRIF-(toll-interleukin-1 receptor domain-containing adapter inducing interferon-β) dependent pathways leading to the induction of pro-inflammatory gene products such as inducible nitric oxide synthase (iNOS). The present study investigated the effect of triptolide (TP), a natural component of Tripterygium wilfordii Hook. F, on inflammation by modulating iNOS expression induced by TLR agonists in murine macrophages. TP suppressed iNOS expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3 agonist), and macrophage-activating lipopeptide 2-kDa (TLR2 and TLR6 agonist). All the results suggest that TP can modulate TLR signaling pathways and subsequent chronic inflammatory responses.

Phenethyl isothiocyanate regulates inflammation through suppression of the TRIF-dependent signaling pathway of Toll-like receptors

AimsThe aim of this study was to evaluate the therapeutic potential of the phenethyl isothiocyanate (PEITC) in Toll-like receptor (TLR) signaling pathways. Main methodsTo evaluate the cytotoxic nature of PEITC in RAW 264.7 cells, cytotoxicity was determined using the MTS cell viability assay. RAW264.7 cells were transfected with a nuclear factor-κB (NF-κB), interferon β (IFNβ) PRDIII-I, or interferon inducible protein-10 (IP-10) luciferase plasmid and then luciferase enzyme activities were determined by luciferase assay. The expression of inducible nitric oxide synthase (iNOS) and phosphorylation of interferon regulatory factor 3 (IRF3) were determined by Western blotting. The levels of IP-10 were determined with culture medium by using an IP-10 enzyme-linked immunosorbent assay (ELISA) kit. Key findingsPEITC suppressed the activation of IRF3 and the expression of IP-10 induced by lipopolysaccharide (LPS) or polyinosinic–polycytidylic acid (poly[I:C]). SignificanceTLRs play an important role in the induction of innate immune responses for host defense against invading microbial pathogens. PEITC found in cruciferous vegetables has an effect on treatment of many chronic diseases. Our results suggest that beneficial effects of PEITC on chronic inflammatory diseases are mediated through modulation of Toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent signaling pathway of TLRs.

Study the Toll-like Receptor 2 and Dectin-1 in mice treated with beta‐ glucans and infected with Salmonella Typhimurium

Objective: Toll-like receptors (TLRs) are essential components for the induction of innate immune responses in different tissues including the small intestine, spleen and mesenteric lymph nodes. We investigated the expression of TLR2 in these tissues in mice infected with Salmonella Typhimurium. In this study we have examined how dectin-1, a lectin family receptor for β-glucans, collaborates with TLR-2 in recognition of microbes.Methods :In this study two groups of mice were treated with the β- glucan (local and commercial extract) and infected with(1x10 6 CFU/ml) S. Typhimurium at different intervals group 1 at day zero and group 2 after seven days of the experiment .The immunohistochemistory assay has been performed to detect TLR-2 and dectine-1 in the tissues of small intestine, spleen and mesenteric lymph nodes of the mice; the principle ofthis assay was done according to the manufacturer instructions (U.S.biological) .Results: In the immunohistochemistory assay, positive cells were indicated by cytoplasmic staining and the percentage of positive in 100 cells was recorded and graded. The grades were 1+ (<25%), 2+ (25-75%) and 3+ (>75%). Statistical analysis with Chi- Square tests revealed that there were significant differences (p˂0.05) between groups, also between grades within the same group in the small intestine, spleen with TLR2 expression; also we demonstrated the same results with Dectin-1 expression in the small intestine and mesenteric lymph nodes, except in the spleen which showed that there were no any relevant differences (p˂ 0.05 ) between the groups and the cell- grades.

Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.

Rabies virus glycoprotein is an important determinant for the induction of innate immune responses and the pathogenic mechanisms

Our previous studies have suggested that street and fixed rabies viruses (RABVs) induce diseases in the mouse model via different mechanisms. In the present study, attempts were made to determine if it is the glycoprotein (G) that is responsible for the observed differences in the pathogenic mechanisms. To this end, an infectious clone from fixed virus B2c was established and used as a backbone for exchange of the G from street viruses. The rate of viral replication, expression of viral proteins, and the induction of innate immune responses were compared in cells or in mice infected with each of the viruses. Furthermore, the infiltration of inflammatory cells into the CNS and the enhancement of blood–brain barrier (BBB) permeability were also compared. It was found that fixed viruses induced stronger innate immune responses (expression of chemokines, infiltration of inflammatory cells, and enhancement of BBB permeability) than street RABV or recombinant viruses expressing the G from street RABVs. Fixed viruses induce disease via an immune-mediated pathogenic mechanism while street viruses or recombinant viruses expressing the G from street RABVs induce diseases via a mechanism other than immune-mediated pathogenesis. Therefore, RABV G is an important determinant for the induction of innate immune responses and consequently the pathogenic mechanisms.

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.

Pivotal role of plasmacytoid dendritic cells in inflammation and NK-cell responses after TLR9 triggering in mice

AbstractThe physiologic role played by plasmacytoid dendritic cells (pDCs) in the induction of innate responses and inflammation in response to pathogen signaling is not well understood. Here, we describe a new mouse model lacking pDCs and establish that pDCs are essential for the in vivo induction of NK-cell activity in response to Toll-like receptor 9 (TLR9) triggering. Furthermore, we provide the first evidence that pDCs are critical for the systemic production of a wide variety of chemokines in response to TLR9 activation. Consequently, we observed a profound alteration in monocyte, macrophage, neutrophil, and NK-cell recruitment at the site of inflammation in the absence of pDCs in response to CpG-Dotap and stimulation by microbial pathogens, such as Leishmania major, Escherichia coli, and Mycobacterium bovis. This study, which is based on the development of a constitutively pDC-deficient mouse model, highlights the pivotal role played by pDCs in the induction of innate immune responses and inflammation after TLR9 triggering.

Suppression of TRIF-dependent signaling pathway of toll-like receptors by allyl isothiocyanate in RAW 264.7 macrophages

Toll-like receptors (TLR) play a significant role in the induction of innate immune responses that are essential for host defense against invading microbial pathogens. In general, TLRs have two major downstream signaling pathways: myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways. Allyl isothiocyanate (AITC) found in cruciferous vegetables has an effect on treatment of many chronic diseases. However, the exact molecular targets of AITC are still unidentified. Here, it was investigated whether AITC can modulate TLR signaling pathways and what is the molecular target of AITC in TLRs signaling pathways. AITC suppressed the activation of nuclear factor-κB by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid (poly[I:C]), but not by macrophage-activating lipopeptide of 2kDa (MALP-2) or cytosine-phosphate-guanine dinucleotide (CpG DNA). AITC also suppressed the activation of interferon regulatory factor 3 (IRF3) and the expression of interferon inducible protein-10 (IP-10) induced by LPS or poly[I:C]. These results suggest that AITC can modulate TRIF-dependent signaling pathways of TLRs leading to decreased inflammatory gene expression.

Signal transducers and activators of transcription protein 1 (STAT1) is activated in macrophages by Zymosan through Dectin-1

Zymosan, an insoluble preparation of yeast cell, has been shown to recognized by Dectin-1, a phagocytic receptor expressed on macrophages and dendritic cells, which collaborates with toll-like receptor (TLR) 2 and TLR6 enhancing the immune responses triggered by the recognition of zymosan by each receptor. Although zymosan has been reported as a target molecule for TLR signaling, the role(s) of signal transducers and activators of transcription protein (STAT) 1 on the innate immune responses induced by zymosan remain elusive. In the present study, we investigate the role of STAT1 on the zymosan-induced production of proinflammatory cytokines by murine bone marrow-derived macrophages (BMDMs). Zymosan-induced proinflammatory cytokine production was dependent on the activation of STAT1, because the mRNA of cytokines and chemokines were totally abrogated in STAT1-deficient BMDMs. In addition, zymosan actively induced the activation of STAT1 through both Tyr-701 and Ser-727 phosphorylation by BMDMs.Furthermore, Dectin-1 was required for zymosan-induced STAT1 activation in macrophages. Taken together, these results indicate that STAT1 activation plays an essential role in the induction of innate immune responses to zymosan-induced innate immune responses via Dectin-1.

Abstract LB-4: Preclinical development of spherical nucleic acids, a novel RNAi-based nanomedicinal platform, as anti-glioma therapeutics

Abstract Patients with Glioblastoma (GBM) survive for a mere 14-16 months. Conventional and targeted therapies, foremost inhibitors of receptor tyrosine kinases (RTK-Is) have been ineffective in glioma clinical trials, contributing to a near 100% failure rate of GBM drug development. Impediments to clinical successes are the lack of systematic identification of genes with critical roles in therapy (apoptosis) resistance, and the dearth of innovative technologies to deliver therapeutics to glioma elements, and to effectively target important apoptosis regulators in established gliomas. Here, we describe the development and pre-clinical characterization of a novel RNAi-based nanomedicinal platform (termed spherical nucleic acids, SNAs) to target aberrantly expressed oncogenes in GBM. SNAs consist of polyvalent gold nanoparticles with diameters of ∼13 nm, which are densely functionalized with RNAi molecules via oligoethylene glycol-thiol linkers. Our detailed physico-chemical and cell biological characterization revealed that SNAs exhibit highly efficient cellular uptake and robust serum stability without the use of auxiliary transfection strategies or chemical modifications, less induction of innate immune responses, reduced off-target effects, and minimal systemic toxicity in rodents. In proof-of-priciciple studies, we utilized SNAs to neutralize the expression of anti-apoptotic Bcl-2 proteins, i.e., canonical family members, such as Bcl-2, Bcl-xL, and the atypical member Bcl2L12. We show highly efficient uptake of SNAs into various primary and transformed glial cell lineages in vitro, including patient- and glioma mouse model-derived tumor neurospheres (TNS). Importantly, SNAs provoked robust and persistent knockdown of Bcl-2 family members on mRNA and protein levels, and robustly sensitized glioma cells toward therapy-induced apoptosis. Finally, Magnetic Resonance Imaging and Inductively Coupled Plasma Mass Spectrometry of SNAs co-functionalized with gadolinium revealed that intravenously administered SNAs penetrated the blood-brain- and blood-tumor-barriers, and robustly disseminated xenogeneic glioma explants. High-level accumulation of Bcl2L12-targeting SNAs within glioma elements was associated with increased intratumoral apoptosis, and consequently resulted in reduced glioma formation and prolonged survival of xenografted mice. Thus, silencing of anti-apoptotic signaling driven by members of the Bcl-2 family using nano-RNAi represents a novel therapeutic approach to overcome therapy resistance and to halt progression of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-4. doi:1538-7445.AM2012-LB-4

Toll-like receptors (TLR2 and TLR4) recognize polysaccharides of Pseudallescheria boydii cell wall

Pseudallescheria boydii is an opportunistic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections in both immunocompromised and immunocompetent hosts. The host response to fungi is in part dependent on the activation of evolutionary conserved receptors including Toll-like receptors and phagocytic receptors. This review will discuss the isolation and structural characterization of α-glucans and rhamnomannans from P. boydii cell wall and their roles in the induction of innate immune response.

Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA

BackgroundInnate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse.Methods/Principal FindingsHere we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA.Conclusions/SignificanceThese results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.

Phenethyl Isothiocyanate가 Toll-like Receptor Agonists에 의해 유도된 Nuclear Factor-κB 활성과 Cyclooxygenase-2, Inducible Nitric Oxide Synthase 발현에 미치는 효과

염증의 중요한 분자학적 기전에는 cyclooxygenase-2 (COX-2)에 의한 prostaglandins (PGs) 생성과 inducible nitric oxide synthase (iNOS)에 의한 nitric oxide (NO) 생성이 있다. 많은 종류의 박테리아나 바이러스가 전사요소인 nuclear factor-<TEX>${\kappa}$</TEX>B(NF-<TEX>${\kappa}$</TEX>B)를 활성화시켜 여러 타깃 유전자의 발현을 조절해 PGs나 NO와 같은 염증물질을 유도하게 된다. 우리는 이번 실험을 통하여 phenethyl isothiocyanate (PEITC)가 toll-like receptor(TLR) agonists에 의해 유도된 NF-<TEX>${\kappa}$</TEX>B활성과 COX-2, iNOS 발현에 어떠한 영향을 미치는지 알아 보았다. PEITC는 lipopolysaccharide (LPS)와 polyinosinic-polycytidylic acid (poly[I:C])에 의해 유도된 NF-<TEX>${\kappa}$</TEX>B활성을 억제시켰다. 또한 PEITC는 LPS와 Poly[I:C]에 의해 유도된 iNOS의 발현도 억제시켰다. 하지만 PEITC는 TLR agonists들인 LPS, Poly[I:C], 2 kDa macrophage-activating lipopeptide (MALP-2), oligodeoxynucleotide 1668 (ODN1668)에 의한 COX-2 발현은 억제시키지 못하였다. 즉 PEITC가 TRIF-dependent 신호전달체계만을 조절하여 TRIF-dependent 신호전달체계에 의해 조절되는 iNOS는 억제하지만 MyD88-dependent 신호전달 체계에 의해 조절되는 COX-2는 억제하지 못한다는 것을 설명해준다. 이러한 결과는 iNOS와 COX-2가 서로 다른 메커니즘에 의해 조절된다는 것을 암시하며, PEITC가 여러 병원균들로부터 유도되는 염증반응이나 만성적인 질병들을 조절할 수 있음을 제시하는 중요한 결과이다. Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The activation of TLRs triggers inflammatory responses that are essential for host defense against invading pathogens. Phenethyl isothiocyanate (PEITC) extracted from cruciferous vegetables has an effect on anti-inflammatory therapy. Dysregulated activation of nuclear factor-<TEX>${\kappa}$</TEX>B (NF-<TEX>${\kappa}$</TEX>B), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) has been shown to play important roles in the development of certain inflammatory disease. To evaluate the therapeutic potential of PEITC, NF-<TEX>${\kappa}$</TEX>B activation and COX-2 and iNOS expression induced by lipopolysaccharide (LPS, TLR4 agonist), polyinosinic-polycytidylic acid (Poly[I:C], TLR3 agonist), 2 kDa macrophageactivating lipopeptide (MALP-2, TLR2 and TLR6 agonist) or oligodeoxynucleotide 1668 (ODN1668, TLR9 agonist) were examined. PEITC inhibits the activation of NF-<TEX>${\kappa}$</TEX>B induced by LPS or Poly[I:C] but not by MALP-2 or ODN1668. PEITC also suppressed the iNOS expression induced by LPS or Poly[I:C]. However, PEITC did not suppress COX-2 expression induced by LPS, Poly[I:C], MALP-2, or ODN1668. These results suggest that PEITC has the specific mechanism for antiinflammatory responses.

Induction of Innate Immune Responses by SIV In Vivo and In Vitro: Differential Expression and Function of RIG-I and MDA5

Interferon-β induction occurs during acute simian immunodeficiency virus (SIV) infection in the brain. We have examined expression and function of cytosolic RNA sensors, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5), in vivo in the brain of our consistent, accelerated SIV-macaque model and in vitro in SIV-infected macaque macrophages to identify the pathway of type I interferon (IFN) induction. MDA5 messenger RNA (mRNA) and protein were expressed at higher levels in the brain than RIG-I, with protein expression correlating with the severity of disease from 42 until 84 days post-inoculation. The siRNA experiments reveal that mRNA expression of IFN-inducible gene MxA is dependent on MDA5, but not RIG-I. Finally, we demonstrate that SIV infection leads to the production of double-stranded RNA in vivo, which may act as the MDA5 ligand. We have shown for the first time to our knowledge the functional role of MDA5 in the innate immune response to SIV infection.