Hochu-ekki-to inhibits rhinovirus infection in human tracheal epithelial cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Yamaya M
PROVIDER: S-EPMC2013869 | biostudies-other | 2007 Mar
REPOSITORIES: biostudies-other
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Project description:Rhinovirus (RV) is a primary etiologic agent of common cold that can subsequently acutely exacerbate bronchial asthma or chronic obstructive pulmonary disease. Kakkonto (Ge-gen-tang in Chinese), one of the most frequently prescribed traditional Japanese (Kampo) medicines, is used for treating common cold, shoulder stiffness, or inflammatory diseases of the upper body. Previous experimental studies have indicated that kakkonto exerts antiviral and anti-inflammatory effects on the influenza virus and the human respiratory syncytial virus. However, there is a lack of reports investigating the efficacy of kakkonto in RV infection. Hence, the aim of the current study was to investigate the effects of kakkonto on RV infection of human nasal epithelial (HNE) cells. HNE cells obtained via endoscopic sinus surgery were cultured and infected with RV14, with or without kakkonto treatment. The supernatants from the cells were collected, and the RV14 titer and cytokine levels were assessed. Reverse transcription-polymerase chain reaction was performed to determine the amount of viral RNA, while the level of nuclear factor kappa B (NF-κB) subunits in the nucleus was assessed by enzyme-linked immunosorbent assay. Although kakkonto treatment did not reduce RV14 titer or RNA levels, indicating that it did not inhibit RV14 proliferation, it was found to reduce the production of specific pro-inflammatory cytokines, including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and monocyte chemotactic protein-1 (MCP-1). Unlike that observed with the kakkonto extract, none of the crude drugs contained in kakkonto reduced IL-8 level. Furthermore, though kakkonto treatment significantly reduced p50 levels, it did not impact the p65 subunit of NF-κB. These results indicated that kakkonto can inhibit inflammation caused by RV infection and may exert an immunomodulatory effect on HNE cells. This is the first report to elucidate the effects of kakkonto extract on RV infection in primary cultures of HNE cells, providing evidence that kakkonto may act as an effective therapy for RV infection and subsequent airway inflammation.
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Project description:Human rhinovirus species C (HRV-C) was recently discovered using molecular diagnostic techniques and is associated with lower respiratory tract disease, particularly in children. HRV-C cannot be propagated in immortalized cell lines, and currently sinus organ culture is the only system described that is permissive to HRV-C infection ex vivo. However, the utility of organ culture for studying HRV-C biology is limited. Here, we report that a previously described HRV-C derived from an infectious cDNA, HRV-C15, infects and propagates in fully differentiated human airway epithelial cells but not in undifferentiated cells. We demonstrate that this differentiated epithelial cell culture system supports infection and replication of a second virus generated from a cDNA clone, HRV-C11. We show that HRV-C15 virions preferentially bind fully differentiated airway epithelial cells, suggesting that the block to replication in undifferentiated cells is at the step of viral entry. Consistent with previous reports, HRV-C15 utilizes a cellular receptor other than ICAM-1 or LDLR for infection of differentiated epithelial cells. Furthermore, we demonstrate that HRV-C15 replication can be inhibited by an HRV 3C protease inhibitor (rupintrivir) but not an HRV capsid inhibitor previously under clinical development (pleconaril). The HRV-C cell culture system described here provides a powerful tool for studying the biology of HRV-C and the discovery and development of HRV-C inhibitors.
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Project description:Nasal epithelial cells (NECs) are among the first cells to be exposed to air pollutants and respiratory viruses. Although it is known that air pollution exposure and rhinovirus infections increase the risk for asthma development independently, it is unclear how these risk factors interact on a cellular level. Therefore, we aimed to investigate how exposure to diesel particulate matter (DPM) modifies the response of primary NECs to rhinovirus (RV) infection in vitro. Exposure of re-differentiated, primary NECs (49 healthy children [0-7 years], 12 adults) to DPM modified the mRNA expression of viral cell-surface receptors, pattern recognition receptors, and pro-inflammatory response (also protein levels). After exposure to DPM, we additionally infected the NECs with RV-1b and RV-16. Viral loads (assessed by titration assays) were significantly higher in DPM-exposed compared with non-exposed NECs. Exposure to DPM prior to RV infection resulted in a significant upregulation of pro-inflammatory cytokines (mRNA and protein level) and β-defensins mRNA, and significant downregulation of pattern recognition receptors mRNA and CXCL10 (mRNA and protein levels). There was no difference between all outcomes of NECs from children and adults. We can conclude that exposure to DPM prior to RV infection increases viral loads by downregulation of viral defense receptors and upregulation of pro-inflammatory cytokines. Our findings indicate a strong interaction between air pollution and the antiviral response to RV infection in NECs. We provide mechanistic evidence that exposure to air pollution increases susceptibility to RV infection.
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Project description:Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-?1 and compared it with the previously established protecting effect of IFN-?. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-? (500 IU/ml) or IFN-?1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-?1 and IFN-? treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-? induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-?1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-?1 is comparable to IFN-?. Yet, the long-lasting induction of ISGs by IFN-?1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-?.
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Project description:Human rhinovirus (HRV) is the common virus that causes acute respiratory infection (ARI) and is frequently associated with lower respiratory tract infections (LRTIs). We aimed to investigate whether HRV infection induces a specific gene expression pattern in airway epithelial cells. Alveolar epithelial cell monolayers were infected with HRV species B (HRV-B). RNA was extracted from both supernatants and infected monolayer cells at 6, 12, 24 and 48 hours post infection (hpi) and transcriptional profile was analyzed using Affymetrix GeneChip and the results were subsequently validated using quantitative Real-time PCR method. HRV-B infects alveolar epithelial cells which supports implication of the virus with LRTIs. In total 991 genes were found differentially expressed during the course of infection. Of these, 459 genes were up-regulated whereas 532 genes were down-regulated. Differential gene expression at 6 hpi (187 genes up-regulated vs. 156 down-regulated) were significantly represented by gene ontologies related to the chemokines and inflammatory molecules indicating characteristic of viral infection. The 75 up-regulated genes surpassed the down-regulated genes (35) at 12 hpi and their enriched ontologies fell into discrete functional entities such as regulation of apoptosis, anti-apoptosis, and wound healing. At later time points of 24 and 48 hpi, predominated down-regulated genes were enriched for extracellular matrix proteins and airway remodeling events. Our data provides a comprehensive image of host response to HRV infection. The study suggests the underlying molecular regulatory networks genes which might be involved in pathogenicity of the HRV-B and potential targets for further validations and development of effective treatment.
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Project description:BACKGROUND:Rhinovirus (RV) infection in asthma induces varying degrees of airway inflammation (e.g. neutrophils), but the underlying mechanisms remain unclear. OBJECTIVE:The major goal was to determine the role of genetic variation [e.g. single nucleotide polymorphisms (SNPs)] of Toll-interacting protein (Tollip) in airway epithelial responses to RV in a type 2 cytokine milieu. METHODS:DNA from blood of asthmatic and normal subjects was genotyped for Tollip SNP rs5743899 AA, AG and GG genotypes. Human tracheobronchial epithelial (HTBE) cells from donors without lung disease were cultured to determine pro-inflammatory and antiviral responses to IL-13 and RV16. Tollip knockout and wild-type mice were challenged with house dust mite (HDM) and infected with RV1B to determine lung inflammation and antiviral response. RESULTS:Asthmatic subjects carrying the AG or GG genotype (AG/GG) compared with the AA genotype demonstrated greater airflow limitation. HTBE cells with AG/GG expressed less Tollip. Upon IL-13 and RV16 treatment, cells with AG/GG (vs. AA) produced more IL-8 and expressed less antiviral genes, which was coupled with increased NF-?B activity and decreased expression of LC3, a hallmark of the autophagic pathway. Tollip co-localized and interacted with LC3. Inhibition of autophagy decreased antiviral genes in IL-13- and RV16-treated cells. Upon HDM and RV1B, Tollip knockout (vs. wild-type) mice demonstrated higher levels of lung neutrophilic inflammation and viral load, but lower levels of antiviral gene expression. CONCLUSIONS AND CLINICAL RELEVANCE:Our data suggest that Tollip SNP rs5743899 may predict varying airway response to RV infection in asthma.
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Project description:Polymorphism at the 17q21 gene locus and wheezing responses to rhinovirus (RV) early in childhood conspire to increase the risk of developing asthma. However, the mechanisms mediating this gene-environment interaction remain unclear. In this study, we investigated the impact of one of the 17q21-encoded genes, ORMDL3 (orosomucoid-like 3), on RV replication in human epithelial cells. ORMDL3 knockdown inhibited RV-A16 replication in HeLa, BEAS-2B, A549, and NCI-H358 epithelial cell lines and primary nasal and bronchial epithelial cells. Inhibition varied by RV species, as both minor and major group RV-A subtypes RV-B52 and RV-C2 were inhibited but not RV-C15 or RV-C41. ORMDL3 siRNA did not affect expression of the major group RV-A receptor ICAM-1 or initial internalization of RV-A16. The two major outcomes of ORMDL3 activity, SPT (serine palmitoyl-CoA transferase) inhibition and endoplasmic reticulum (ER) stress induction, were further examined: silencing ORMDL3 decreased RV-induced ER stress and IFN-β mRNA expression. However, pharmacologic induction of ER stress and concomitant increased IFN-β inhibited RV-A16 replication. Conversely, blockade of ER stress with tauroursodeoxycholic acid augmented replication, pointing to an alternative mechanism for the effect of ORMDL3 knockdown on RV replication. In comparison, the SPT inhibitor myriocin increased RV-A16 but not RV-C15 replication and negated the inhibitory effect of ORMDL3 knockdown. Furthermore, lipidomics analysis revealed opposing regulation of specific sphingolipid species (downstream of SPT) by myriocin and ORMDL3 siRNA, correlating with the effect of these treatments on RV replication. Together, these data revealed a requirement for ORMDL3 in supporting RV replication in epithelial cells via SPT inhibition.
| S-EPMC7258826 | biostudies-literature
Project description:Mechanisms underlying the development of virus-induced asthma exacerbations remain unclear. To investigate if epigenetic mechanisms could be involved in virus-induced asthma exacerbations, we undertook DNA methylation profiling in asthmatic and healthy nasal epithelial cells (NECs) during Human Rhinovirus (HRV) infection in vitro.Global and loci-specific methylation profiles were determined via Alu element and Infinium Human Methylation 450 K microarray, respectively. Principal components analysis identified the genomic loci influenced the most by disease-status and infection. Real-time PCR and pyrosequencing were used to confirm gene expression and DNA methylation, respectively.HRV infection significantly increased global DNA methylation in cells from asthmatic subjects only (43.6% to 44.1%, p?=?0.04). Microarray analysis revealed 389 differentially methylated loci either based on disease status, or caused by virus infection. There were disease-associated DNA methylation patterns that were not affected by HRV infection as well as HRV-induced DNA methylation changes that were unique to each group. A common methylation locus stood out in response to HRV infection in both groups, where the small nucleolar RNA, H/ACA box 12 (SNORA12) is located. Further analysis indicated that a relationship existed between SNORA12 DNA methylation and gene expression in response to HRV infection.We describe for the first time that Human rhinovirus infection causes DNA methylation changes in airway epithelial cells that differ between asthmatic and healthy subjects. These epigenetic differences may possibly explain the mechanism by which respiratory viruses cause asthma exacerbations.
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Project description:Human rhinoviruses (HRV) cause the majority of colds and trigger exacerbations of chronic lower airway diseases. Airway epithelial cells are the primary site for HRV infection and replication, and the initiation of host inflammatory responses. At present, the molecular mechanisms that underpin HRV responses in airway epithelial cells are incompletely understood. The aim of this study was to employ microarray profiling, upstream regulator analysis, and siRNA mediated gene silencing to further our understanding of the role of interferon regulatory factor 7 (IRF7) in this response.Primary human bronchial epithelial cells (HBE) where transfected with siRNA that targets IRF7 or a non-silencing control (all-star control) using Lipofectamine. The cells were allowed to recover, and then cultured in the presence or absence of HRV-16 for 24 h. Global patterns of gene expression were profiled on microarrays. A subset of genes identified in the microarray study were validated at the mRNA and/or protein level using real time RT-qPCR, ELISA, and western blots.Hundreds of genes were upregulated in HBE during HRV infection. Pathways analysis demonstrated that these genes were mainly involved in type I and II interferon signaling, RIG-I/MDA5 signaling, antigen processing and presentation, and apoptosis. Upstream regulator analysis of these data suggested that IRF7 was a major molecular driver of this response. Knockdown of IRF7 reduced the HRV-driven upregulation of genes involved in antiviral responses (interferon signaling, Toll-like receptor signaling, NOD-like receptor signaling, RIG-I/MDA5 signaling), and increased the expression of genes that promote inflammation (e.g. CXCL5, IL-33, IL1RL1) and the response to oxidative stress. However, the majority of genes that were perturbed by HRV in HBE cells including those that are known to be regulated by IRF7 were insensitive to IRF7 knockdown. Upstream regulator analysis of the part of the response that was insensitive to IRF7 knockdown suggested it was driven by NF-?B, STAT1, STAT3, and IRF1.Our findings demonstrate that IRF7 regulates the expression of genes involved in antiviral immunity, inflammation, and the response to oxidative stress during HRV infections in HBE cells, and also suggests that other transcription factors play a major role in this response.
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