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Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection.

Yoshikawa T, Hill TE, Yoshikawa N, Popov VL, Galindo CL, Garner HR, Peters CJ, Tseng CT - PLoS ONE (2010)

Bottom Line: Specifically, we found a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-beta, -lambdas, inflammatory mediators, and many IFN-stimulated genes (ISGs).We also showed, for the first time, that IFN-beta and IFN-lambdas were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i.Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-beta, -lambdas, inflammatory mediators, and many IFN-stimulated genes (ISGs). We also showed, for the first time, that IFN-beta and IFN-lambdas were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

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Efficacy of IFN-λs in the host defense against SARS-CoV infection.Confluent cultures of 2B4 cells were treated with rIFN-β, rIFN-λ1, or rIFN-λ2 at the indicated concentrations alone (A) or in combination (B) for 24 hrs prior to their infection with SARS-CoV (MOI = 0.01). Resulting supernatants were harvested at day 2 after infection for assessing yields of infectious progeny virus by the standard Vero E6-based infectivity assay. *, p<0.05 by a two-way ANOVA with Bonferroni's post-hoc test in comparison with IFN-untreated cultures. Data shown were representative of two independent experiments.
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pone-0008729-g009: Efficacy of IFN-λs in the host defense against SARS-CoV infection.Confluent cultures of 2B4 cells were treated with rIFN-β, rIFN-λ1, or rIFN-λ2 at the indicated concentrations alone (A) or in combination (B) for 24 hrs prior to their infection with SARS-CoV (MOI = 0.01). Resulting supernatants were harvested at day 2 after infection for assessing yields of infectious progeny virus by the standard Vero E6-based infectivity assay. *, p<0.05 by a two-way ANOVA with Bonferroni's post-hoc test in comparison with IFN-untreated cultures. Data shown were representative of two independent experiments.

Mentions: Type I IFNs (IFN-α/β) rapidly produced by the infected host serves as the first line of defense against viral infections, in part, via triggering the expressions of many ISGs to combat the invading pathogens. The ability of SARS-CoV to activate MXs, OAS, RIG-I, MDA5, TLR3, STATs, and many other ISGs without stimulating any significant responses of IFN-β, and especially of IFN-αs, prompted us to investigate whether IFN-λs elicited by infected 2B4 cells could potentiate IFN-α/β-like activity against SARS-CoV. To evaluate the potential of IFN-λs alone or in combination with type I IFNs against SARS-CoV infection, we pretreated 2B4 cells with IFN-β, IFN-λ1 and IFN-λ2, either individually or in combination at indicated concentrations prior to infection with SARS-CoV (MOI = 0.01). While IFN-β at the concentration of 5 ng (equivalent to 1,000 IU) alone had a significant antiviral effect, we were unable to reveal any antiviral effect of either IFN-λ1 or -λ2 when used alone at a concentration as high as 1,000 ng (Figure 9A). However, a combinational treatment of both IFN-λ1 and IFN-λ2, even at a concentration as low as 10 ng each, significantly reduced SARS-CoV replication (P<0.05), which suggested to us that both types of IFN-λs are required to effectively limit the replication of SARS-CoV (Figure 9B). Additionally, treatment with either type of IFN-λ, together with an otherwise ineffective low-dose of IFN-β (e.g., 10 IU), drastically hampered the growth of SARS-CoV in 2B4 cells (P<0.05), a finding which may imply that either species of IFN-λ could potentiate the antiviral effect of IFN-β. Surprisingly, we also found that pretreatment of cells with all three types of IFNs together (i.e., IFN-λ1, IFN–λ2, and IFN-β) at a low-dose regimen (i.e., 10 ng each for IFN-λ1 and –λ2 and 0.05 ng for IFN-β) diminished the antiviral effect provided by the combination treatments of IFN-λ1/IFN-β, IFN-λ2/IFN-β, and IFN-λ1/IFN-λ2 at the same concentrations. While the mechanism of this interesting observation remains unknown, our results showed that IFN-λs possess a previously unidentified type I IFN-like activity against SARS-CoV.


Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection.

Yoshikawa T, Hill TE, Yoshikawa N, Popov VL, Galindo CL, Garner HR, Peters CJ, Tseng CT - PLoS ONE (2010)

Efficacy of IFN-λs in the host defense against SARS-CoV infection.Confluent cultures of 2B4 cells were treated with rIFN-β, rIFN-λ1, or rIFN-λ2 at the indicated concentrations alone (A) or in combination (B) for 24 hrs prior to their infection with SARS-CoV (MOI = 0.01). Resulting supernatants were harvested at day 2 after infection for assessing yields of infectious progeny virus by the standard Vero E6-based infectivity assay. *, p<0.05 by a two-way ANOVA with Bonferroni's post-hoc test in comparison with IFN-untreated cultures. Data shown were representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806919&req=5

pone-0008729-g009: Efficacy of IFN-λs in the host defense against SARS-CoV infection.Confluent cultures of 2B4 cells were treated with rIFN-β, rIFN-λ1, or rIFN-λ2 at the indicated concentrations alone (A) or in combination (B) for 24 hrs prior to their infection with SARS-CoV (MOI = 0.01). Resulting supernatants were harvested at day 2 after infection for assessing yields of infectious progeny virus by the standard Vero E6-based infectivity assay. *, p<0.05 by a two-way ANOVA with Bonferroni's post-hoc test in comparison with IFN-untreated cultures. Data shown were representative of two independent experiments.
Mentions: Type I IFNs (IFN-α/β) rapidly produced by the infected host serves as the first line of defense against viral infections, in part, via triggering the expressions of many ISGs to combat the invading pathogens. The ability of SARS-CoV to activate MXs, OAS, RIG-I, MDA5, TLR3, STATs, and many other ISGs without stimulating any significant responses of IFN-β, and especially of IFN-αs, prompted us to investigate whether IFN-λs elicited by infected 2B4 cells could potentiate IFN-α/β-like activity against SARS-CoV. To evaluate the potential of IFN-λs alone or in combination with type I IFNs against SARS-CoV infection, we pretreated 2B4 cells with IFN-β, IFN-λ1 and IFN-λ2, either individually or in combination at indicated concentrations prior to infection with SARS-CoV (MOI = 0.01). While IFN-β at the concentration of 5 ng (equivalent to 1,000 IU) alone had a significant antiviral effect, we were unable to reveal any antiviral effect of either IFN-λ1 or -λ2 when used alone at a concentration as high as 1,000 ng (Figure 9A). However, a combinational treatment of both IFN-λ1 and IFN-λ2, even at a concentration as low as 10 ng each, significantly reduced SARS-CoV replication (P<0.05), which suggested to us that both types of IFN-λs are required to effectively limit the replication of SARS-CoV (Figure 9B). Additionally, treatment with either type of IFN-λ, together with an otherwise ineffective low-dose of IFN-β (e.g., 10 IU), drastically hampered the growth of SARS-CoV in 2B4 cells (P<0.05), a finding which may imply that either species of IFN-λ could potentiate the antiviral effect of IFN-β. Surprisingly, we also found that pretreatment of cells with all three types of IFNs together (i.e., IFN-λ1, IFN–λ2, and IFN-β) at a low-dose regimen (i.e., 10 ng each for IFN-λ1 and –λ2 and 0.05 ng for IFN-β) diminished the antiviral effect provided by the combination treatments of IFN-λ1/IFN-β, IFN-λ2/IFN-β, and IFN-λ1/IFN-λ2 at the same concentrations. While the mechanism of this interesting observation remains unknown, our results showed that IFN-λs possess a previously unidentified type I IFN-like activity against SARS-CoV.

Bottom Line: Specifically, we found a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-beta, -lambdas, inflammatory mediators, and many IFN-stimulated genes (ISGs).We also showed, for the first time, that IFN-beta and IFN-lambdas were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i.Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NF)kappaB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-beta, -lambdas, inflammatory mediators, and many IFN-stimulated genes (ISGs). We also showed, for the first time, that IFN-beta and IFN-lambdas were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

Show MeSH
Related in: MedlinePlus