PMC:7025480 / 6102-11329 JSONTXT

{"project":"TEST0","denotations":[{"id":"32117250-142-148-3633022","span":{"begin":583,"end":585},"obj":"[\"11827991\"]"},{"id":"32117250-91-97-3633023","span":{"begin":4945,"end":4947},"obj":"[\"26483767\"]"}],"text":"P. aeruginosa Inhibits the Antiviral Response of Airway Epithelial Cells\nIn order to analyze whether P. aeruginosa is able to modulate the antiviral response of bronchial epithelial cells, we prepared control medium or conditioned medium (CM) from two different strains of P. aeruginosa, PAO1 (commonly used in research) and Boston (quality control strain), respectively. CM contains soluble factors secreted by P. aeruginosa during growth. We focused on soluble factors since P. aeruginosa is mostly located intraluminally in CF lungs and direct cell-cell contacts are less common (24). Subsequently, we used CM or control medium to treat airway BEAS2B cells and thereafter infected the cells with hRV (strain RV1b) or RSV (Figure 1A). Subsequently, antiviral responses were analyzed after various incubation times. Induction of the antiviral genes MX1 and OAS1 upon virus infection did not show any significant differences between BEAS2B cells pretreated with CM of P. aeruginosa Boston strain (CM-Boston) or control medium (Figure 1B). However, cells treated with CM of P. aeruginosa strain PAO1 (CM-PAO1) showed a significant decrease in the induction of both genes after infection with hRV and RSV, which was most pronounced after 14 h of infection compared to control medium [fold induction (FI) of MX1: 285 vs. 6, p \u003c 0.001; FI OAS1: 49 vs. 1, p \u003c 0.001; Figure 1A]. Since effects were stronger after RSV infection all subsequent experiments were done with RSV. Of note, conditioning with P. aeruginosa medium alone did not affect MX1 or OAS1 expression (not shown).\nFigure 1 P. aeruginosa is able to suppress antiviral response of airway epithelial cells. (A) Cell culture based experimental workflow. (B) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium and subsequently infected with RSV or RV1b. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 6, 10, and 14 h. (C) BEAS2B cells were treated as in (A) and cell culture supernatant of RV1b (human RV) or RSV infected cells (14 h) was transferred to new BEAS2B cells. Levels of RV1b (hRV) or RSV mRNA were analyzed by qRT-PCR and the number of wells displaying RV1b or RSV specific cytopathogenic effects (CPE) was determined. (D) Primary HBE cells were pretreated with conditioned medium of P. aeruginosa (PAO1) or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. (E) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. All experiments n = 3–4, ANOVA and Bonferroni post-test was used for statistical analysis. Significant differences were considered at *p \u003c 0.05, **p \u003c 0.01, and ***p \u003c 0.001 as compared to the control condition. n.s., not significant. As P. aeruginosa might act cytotoxic to eukaryotic cells we chose to use only conditioned medium instead of direct bacterial infection and we also controlled viability by annexin V and propidium iodide staining. Even though we observed some cytotoxic effects at 14 h we did not detect differences in viability of epithelial cells conditioned with medium from either of the two P. aeruginosa strains up to 10 h (viability was 96 ± 1.7% for Boston vs. 96 ± 2.7% for PAO1 after hRV infection and 95 ± 2% and 96 ± 3% after RSV infection, respectively). Therefore, cytotoxic effects can be excluded as an explanation why only PAO1 inhibited the antiviral response. Of note, we did not observe any differences by any CM treatment with respect to the viral load of the primary infected cells indicating that the first infection of the cells was not affected by soluble P. aeruginosa factors (Figure S1). Yet, we speculated that the inhibition of the antiviral response by CM-PAO1 (as indicated by reduced ISG induction) may facilitate spread of the virus infection. To analyze the functional relevance of the decreased antiviral response, we therefore challenged another batch of BEAS2B cells with the supernatant of CM-pretreated and RSV infected cells (Figure 1A). In line with a decrease of the antiviral response in CM-PAO1 treated BEAS2B cells, we observed increases in RSV-RNA and RSV-specific cytopathic effects in cells challenged with the supernatant of CM-PAO1 treated cells as compared to control or CM-Boston treated cells (Figure 1C). These results indicate that the repression of the antiviral response by PAO1 leads to an increase in virus spreading and is therefore of functional relevance. To confirm the effects in primary cells we repeated the main experiment in primary human bronchial epithelial cells (hBrEpC). In line with our observations in BEAS2B cells, PAO1 was also able to repress RSV induced antiviral responses in hBrEpC (Figure 1D). It is known that P. aeruginosa can be classified into three different phylogenetic groups (25), represented by PA14, PAO1, and PA7 for group 1, 2, and 3, respectively. Like PAO1 PA14 but not PA7 was also able to repress the antiviral response toward RSV (Figure 1E) indicating that the inhibition of an antiviral response is not restricted to a specific phylogenetic group."}

{"project":"2_test","denotations":[{"id":"32117250-11827991-35176894","span":{"begin":583,"end":585},"obj":"11827991"},{"id":"32117250-26483767-35176895","span":{"begin":4945,"end":4947},"obj":"26483767"}],"text":"P. aeruginosa Inhibits the Antiviral Response of Airway Epithelial Cells\nIn order to analyze whether P. aeruginosa is able to modulate the antiviral response of bronchial epithelial cells, we prepared control medium or conditioned medium (CM) from two different strains of P. aeruginosa, PAO1 (commonly used in research) and Boston (quality control strain), respectively. CM contains soluble factors secreted by P. aeruginosa during growth. We focused on soluble factors since P. aeruginosa is mostly located intraluminally in CF lungs and direct cell-cell contacts are less common (24). Subsequently, we used CM or control medium to treat airway BEAS2B cells and thereafter infected the cells with hRV (strain RV1b) or RSV (Figure 1A). Subsequently, antiviral responses were analyzed after various incubation times. Induction of the antiviral genes MX1 and OAS1 upon virus infection did not show any significant differences between BEAS2B cells pretreated with CM of P. aeruginosa Boston strain (CM-Boston) or control medium (Figure 1B). However, cells treated with CM of P. aeruginosa strain PAO1 (CM-PAO1) showed a significant decrease in the induction of both genes after infection with hRV and RSV, which was most pronounced after 14 h of infection compared to control medium [fold induction (FI) of MX1: 285 vs. 6, p \u003c 0.001; FI OAS1: 49 vs. 1, p \u003c 0.001; Figure 1A]. Since effects were stronger after RSV infection all subsequent experiments were done with RSV. Of note, conditioning with P. aeruginosa medium alone did not affect MX1 or OAS1 expression (not shown).\nFigure 1 P. aeruginosa is able to suppress antiviral response of airway epithelial cells. (A) Cell culture based experimental workflow. (B) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium and subsequently infected with RSV or RV1b. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 6, 10, and 14 h. (C) BEAS2B cells were treated as in (A) and cell culture supernatant of RV1b (human RV) or RSV infected cells (14 h) was transferred to new BEAS2B cells. Levels of RV1b (hRV) or RSV mRNA were analyzed by qRT-PCR and the number of wells displaying RV1b or RSV specific cytopathogenic effects (CPE) was determined. (D) Primary HBE cells were pretreated with conditioned medium of P. aeruginosa (PAO1) or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. (E) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. All experiments n = 3–4, ANOVA and Bonferroni post-test was used for statistical analysis. Significant differences were considered at *p \u003c 0.05, **p \u003c 0.01, and ***p \u003c 0.001 as compared to the control condition. n.s., not significant. As P. aeruginosa might act cytotoxic to eukaryotic cells we chose to use only conditioned medium instead of direct bacterial infection and we also controlled viability by annexin V and propidium iodide staining. Even though we observed some cytotoxic effects at 14 h we did not detect differences in viability of epithelial cells conditioned with medium from either of the two P. aeruginosa strains up to 10 h (viability was 96 ± 1.7% for Boston vs. 96 ± 2.7% for PAO1 after hRV infection and 95 ± 2% and 96 ± 3% after RSV infection, respectively). Therefore, cytotoxic effects can be excluded as an explanation why only PAO1 inhibited the antiviral response. Of note, we did not observe any differences by any CM treatment with respect to the viral load of the primary infected cells indicating that the first infection of the cells was not affected by soluble P. aeruginosa factors (Figure S1). Yet, we speculated that the inhibition of the antiviral response by CM-PAO1 (as indicated by reduced ISG induction) may facilitate spread of the virus infection. To analyze the functional relevance of the decreased antiviral response, we therefore challenged another batch of BEAS2B cells with the supernatant of CM-pretreated and RSV infected cells (Figure 1A). In line with a decrease of the antiviral response in CM-PAO1 treated BEAS2B cells, we observed increases in RSV-RNA and RSV-specific cytopathic effects in cells challenged with the supernatant of CM-PAO1 treated cells as compared to control or CM-Boston treated cells (Figure 1C). These results indicate that the repression of the antiviral response by PAO1 leads to an increase in virus spreading and is therefore of functional relevance. To confirm the effects in primary cells we repeated the main experiment in primary human bronchial epithelial cells (hBrEpC). In line with our observations in BEAS2B cells, PAO1 was also able to repress RSV induced antiviral responses in hBrEpC (Figure 1D). It is known that P. aeruginosa can be classified into three different phylogenetic groups (25), represented by PA14, PAO1, and PA7 for group 1, 2, and 3, respectively. Like PAO1 PA14 but not PA7 was also able to repress the antiviral response toward RSV (Figure 1E) indicating that the inhibition of an antiviral response is not restricted to a specific phylogenetic group."}

{"project":"MyTest","denotations":[{"id":"32117250-11827991-35176894","span":{"begin":583,"end":585},"obj":"11827991"},{"id":"32117250-26483767-35176895","span":{"begin":4945,"end":4947},"obj":"26483767"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"P. aeruginosa Inhibits the Antiviral Response of Airway Epithelial Cells\nIn order to analyze whether P. aeruginosa is able to modulate the antiviral response of bronchial epithelial cells, we prepared control medium or conditioned medium (CM) from two different strains of P. aeruginosa, PAO1 (commonly used in research) and Boston (quality control strain), respectively. CM contains soluble factors secreted by P. aeruginosa during growth. We focused on soluble factors since P. aeruginosa is mostly located intraluminally in CF lungs and direct cell-cell contacts are less common (24). Subsequently, we used CM or control medium to treat airway BEAS2B cells and thereafter infected the cells with hRV (strain RV1b) or RSV (Figure 1A). Subsequently, antiviral responses were analyzed after various incubation times. Induction of the antiviral genes MX1 and OAS1 upon virus infection did not show any significant differences between BEAS2B cells pretreated with CM of P. aeruginosa Boston strain (CM-Boston) or control medium (Figure 1B). However, cells treated with CM of P. aeruginosa strain PAO1 (CM-PAO1) showed a significant decrease in the induction of both genes after infection with hRV and RSV, which was most pronounced after 14 h of infection compared to control medium [fold induction (FI) of MX1: 285 vs. 6, p \u003c 0.001; FI OAS1: 49 vs. 1, p \u003c 0.001; Figure 1A]. Since effects were stronger after RSV infection all subsequent experiments were done with RSV. Of note, conditioning with P. aeruginosa medium alone did not affect MX1 or OAS1 expression (not shown).\nFigure 1 P. aeruginosa is able to suppress antiviral response of airway epithelial cells. (A) Cell culture based experimental workflow. (B) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa (PAO1 and Boston) or control medium and subsequently infected with RSV or RV1b. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 6, 10, and 14 h. (C) BEAS2B cells were treated as in (A) and cell culture supernatant of RV1b (human RV) or RSV infected cells (14 h) was transferred to new BEAS2B cells. Levels of RV1b (hRV) or RSV mRNA were analyzed by qRT-PCR and the number of wells displaying RV1b or RSV specific cytopathogenic effects (CPE) was determined. (D) Primary HBE cells were pretreated with conditioned medium of P. aeruginosa (PAO1) or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. (E) BEAS2B cells were pretreated with conditioned medium of P. aeruginosa or control medium and subsequently infected with RSV. Induction of MX1 and OAS1 mRNA were analyzed by qRT-PCR after 14 h. All experiments n = 3–4, ANOVA and Bonferroni post-test was used for statistical analysis. Significant differences were considered at *p \u003c 0.05, **p \u003c 0.01, and ***p \u003c 0.001 as compared to the control condition. n.s., not significant. As P. aeruginosa might act cytotoxic to eukaryotic cells we chose to use only conditioned medium instead of direct bacterial infection and we also controlled viability by annexin V and propidium iodide staining. Even though we observed some cytotoxic effects at 14 h we did not detect differences in viability of epithelial cells conditioned with medium from either of the two P. aeruginosa strains up to 10 h (viability was 96 ± 1.7% for Boston vs. 96 ± 2.7% for PAO1 after hRV infection and 95 ± 2% and 96 ± 3% after RSV infection, respectively). Therefore, cytotoxic effects can be excluded as an explanation why only PAO1 inhibited the antiviral response. Of note, we did not observe any differences by any CM treatment with respect to the viral load of the primary infected cells indicating that the first infection of the cells was not affected by soluble P. aeruginosa factors (Figure S1). Yet, we speculated that the inhibition of the antiviral response by CM-PAO1 (as indicated by reduced ISG induction) may facilitate spread of the virus infection. To analyze the functional relevance of the decreased antiviral response, we therefore challenged another batch of BEAS2B cells with the supernatant of CM-pretreated and RSV infected cells (Figure 1A). In line with a decrease of the antiviral response in CM-PAO1 treated BEAS2B cells, we observed increases in RSV-RNA and RSV-specific cytopathic effects in cells challenged with the supernatant of CM-PAO1 treated cells as compared to control or CM-Boston treated cells (Figure 1C). These results indicate that the repression of the antiviral response by PAO1 leads to an increase in virus spreading and is therefore of functional relevance. To confirm the effects in primary cells we repeated the main experiment in primary human bronchial epithelial cells (hBrEpC). In line with our observations in BEAS2B cells, PAO1 was also able to repress RSV induced antiviral responses in hBrEpC (Figure 1D). It is known that P. aeruginosa can be classified into three different phylogenetic groups (25), represented by PA14, PAO1, and PA7 for group 1, 2, and 3, respectively. Like PAO1 PA14 but not PA7 was also able to repress the antiviral response toward RSV (Figure 1E) indicating that the inhibition of an antiviral response is not restricted to a specific phylogenetic group."}