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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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Characteristics of 2B4 cells clonally derived from human bronchial epithelial Calu-3 cells.Expressions of the viral ACE2 receptor in indicated passages of 2B4 cells and their parental Calu-3 cells were assessed by standard IHC (A) and Western blot analysis (B), whereas the morphological features of polarized 2B4 cells were assessed by TEM (C), as described in Materials and Methods. The images were taken at 6,270 magnifications. The scale bar represents 1 µm. To compare the permissiveness of 2B4 cells to their parental Calu-3 cells, confluent 2B4 cells, at passages #6 and #12, and Calu-3 cells were subjected to SARS-CoV (MOI = 0.1). The growth kinetics of SARS-CoV in culture supernatant and proportion of SARS-CoV-infected 2B4 cells were assessed at indicated time points by the standard Vero E6-based infectivity assay of the resulting cell-free supernatants (D) and infectious center assay (E). Finally, 2B4 cells (passage #6) were infected with SARS-CoV (MOI = 0.1) for 24, 48, and 72 hrs before being fixed with 4% paraformaldehyde for monitoring the morphological changes of infected cells, as visualized by the expression of SARS-CoV NP protein (red) by using the standard IHC (F).
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pone-0008729-g001: Characteristics of 2B4 cells clonally derived from human bronchial epithelial Calu-3 cells.Expressions of the viral ACE2 receptor in indicated passages of 2B4 cells and their parental Calu-3 cells were assessed by standard IHC (A) and Western blot analysis (B), whereas the morphological features of polarized 2B4 cells were assessed by TEM (C), as described in Materials and Methods. The images were taken at 6,270 magnifications. The scale bar represents 1 µm. To compare the permissiveness of 2B4 cells to their parental Calu-3 cells, confluent 2B4 cells, at passages #6 and #12, and Calu-3 cells were subjected to SARS-CoV (MOI = 0.1). The growth kinetics of SARS-CoV in culture supernatant and proportion of SARS-CoV-infected 2B4 cells were assessed at indicated time points by the standard Vero E6-based infectivity assay of the resulting cell-free supernatants (D) and infectious center assay (E). Finally, 2B4 cells (passage #6) were infected with SARS-CoV (MOI = 0.1) for 24, 48, and 72 hrs before being fixed with 4% paraformaldehyde for monitoring the morphological changes of infected cells, as visualized by the expression of SARS-CoV NP protein (red) by using the standard IHC (F).

Mentions: We employed the standard limiting dilution technique to establish clonal derivatives of Calu-3 cells, as described in Materials and Methods. Based on their ACE2 expression and permissiveness to productive SARS-CoV infection, 18 out of a total of 26 clones established exhibited an array of varying intensities of ACE2 expression and permissiveness to SARS-CoV, ranging from intermediate-to-low levels, whereas the remaining eight clones revealed increased ACE2 expression and susceptibility to SARS-CoV infection, when compared to their parental Calu-3 cells (data not shown). Among those clones that were highly permissive to SARS-CoV, we chose cells of the 2B4 clone for a detailed characterization with regard to the stability of ACE2 expression over different passages and the susceptibility to productive SARS-CoV infection. As shown in Figure 1A, results of IHC staining revealed that the ACE2 expression of 2B4 cells (passage #6) was much more intense than that of the parental Calu-3 cells. Such an enhanced ACE2 expression of 2B4 cells, relative to Calu-3 cells, was confirmed by Western blot analysis (Figure 1B). Importantly, the trend of the intense ACE2 expression of 2B4 cells appeared to be stable, as cells derived from two different passages (i.e., #6 and #12) exhibited little difference, if any, in the expression of ACE2 protein. The parental Calu-3 cells, wich originated from a human pulmonary adenocarcinoma, have been well characterized as non-ciliated human bronchial epithelial cells with the expression of many markers of serous gland cells and the formation of tight junction (TJ) complexes [41], [42], [43], [44]. The morphology of 2B4 cells grown in the membrane inserts was subsequently examined by TEM. As shown in Figure 1C, 2B4 cells, like parental Calu-3 cells, appeared to have a morphology resembling that of non-ciliated pseudostratified columnar epithelial cells with the expression of microvilli on the apical surface and the formation of TJ complexes.


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)

Characteristics of 2B4 cells clonally derived from human bronchial epithelial Calu-3 cells.Expressions of the viral ACE2 receptor in indicated passages of 2B4 cells and their parental Calu-3 cells were assessed by standard IHC (A) and Western blot analysis (B), whereas the morphological features of polarized 2B4 cells were assessed by TEM (C), as described in Materials and Methods. The images were taken at 6,270 magnifications. The scale bar represents 1 µm. To compare the permissiveness of 2B4 cells to their parental Calu-3 cells, confluent 2B4 cells, at passages #6 and #12, and Calu-3 cells were subjected to SARS-CoV (MOI = 0.1). The growth kinetics of SARS-CoV in culture supernatant and proportion of SARS-CoV-infected 2B4 cells were assessed at indicated time points by the standard Vero E6-based infectivity assay of the resulting cell-free supernatants (D) and infectious center assay (E). Finally, 2B4 cells (passage #6) were infected with SARS-CoV (MOI = 0.1) for 24, 48, and 72 hrs before being fixed with 4% paraformaldehyde for monitoring the morphological changes of infected cells, as visualized by the expression of SARS-CoV NP protein (red) by using the standard IHC (F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806919&req=5

pone-0008729-g001: Characteristics of 2B4 cells clonally derived from human bronchial epithelial Calu-3 cells.Expressions of the viral ACE2 receptor in indicated passages of 2B4 cells and their parental Calu-3 cells were assessed by standard IHC (A) and Western blot analysis (B), whereas the morphological features of polarized 2B4 cells were assessed by TEM (C), as described in Materials and Methods. The images were taken at 6,270 magnifications. The scale bar represents 1 µm. To compare the permissiveness of 2B4 cells to their parental Calu-3 cells, confluent 2B4 cells, at passages #6 and #12, and Calu-3 cells were subjected to SARS-CoV (MOI = 0.1). The growth kinetics of SARS-CoV in culture supernatant and proportion of SARS-CoV-infected 2B4 cells were assessed at indicated time points by the standard Vero E6-based infectivity assay of the resulting cell-free supernatants (D) and infectious center assay (E). Finally, 2B4 cells (passage #6) were infected with SARS-CoV (MOI = 0.1) for 24, 48, and 72 hrs before being fixed with 4% paraformaldehyde for monitoring the morphological changes of infected cells, as visualized by the expression of SARS-CoV NP protein (red) by using the standard IHC (F).
Mentions: We employed the standard limiting dilution technique to establish clonal derivatives of Calu-3 cells, as described in Materials and Methods. Based on their ACE2 expression and permissiveness to productive SARS-CoV infection, 18 out of a total of 26 clones established exhibited an array of varying intensities of ACE2 expression and permissiveness to SARS-CoV, ranging from intermediate-to-low levels, whereas the remaining eight clones revealed increased ACE2 expression and susceptibility to SARS-CoV infection, when compared to their parental Calu-3 cells (data not shown). Among those clones that were highly permissive to SARS-CoV, we chose cells of the 2B4 clone for a detailed characterization with regard to the stability of ACE2 expression over different passages and the susceptibility to productive SARS-CoV infection. As shown in Figure 1A, results of IHC staining revealed that the ACE2 expression of 2B4 cells (passage #6) was much more intense than that of the parental Calu-3 cells. Such an enhanced ACE2 expression of 2B4 cells, relative to Calu-3 cells, was confirmed by Western blot analysis (Figure 1B). Importantly, the trend of the intense ACE2 expression of 2B4 cells appeared to be stable, as cells derived from two different passages (i.e., #6 and #12) exhibited little difference, if any, in the expression of ACE2 protein. The parental Calu-3 cells, wich originated from a human pulmonary adenocarcinoma, have been well characterized as non-ciliated human bronchial epithelial cells with the expression of many markers of serous gland cells and the formation of tight junction (TJ) complexes [41], [42], [43], [44]. The morphology of 2B4 cells grown in the membrane inserts was subsequently examined by TEM. As shown in Figure 1C, 2B4 cells, like parental Calu-3 cells, appeared to have a morphology resembling that of non-ciliated pseudostratified columnar epithelial cells with the expression of microvilli on the apical surface and the formation of TJ complexes.

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