Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify <i>host</i> proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34­(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells

Response of Primary Human Airway Epithelial Cells to Influenza Infection: A Quantitative Proteomic Study

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify <i>host</i> proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34­(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC–MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells

Influenza A Infection of Primary Human Airway Epithelial Cells Up-Regulates Proteins Related to Purine Metabolism and Ubiquitin-Related Signaling

Virus–host interactions are important determinants of virus replication and immune responses, but they are not well-defined. In this study we analyzed quantitative host protein alterations in nuclei-enriched fractions from multiple primary human bronchial airway epithelial (HBAE) cells infected by an H1N1 influenza A virus (A/PR/8/34). We first developed an effective detergent-free nuclear lysis method that was coupled with in-solution digestion and LC–MS/MS. Using SILAC, we identified 837 HBAE nuclear proteins in three different donors and compared their responses to infection at 24 h. Some proteins were altered in all three donors, of which 94 were up-regulated and 13 were down-regulated by at least 1.5-fold. Many of these up-regulated proteins clustered into purine biosynthesis, carbohydrate metabolism, and protein modification. Down-regulated proteins were not associated with any specific pathways or processes. These findings further our understanding of cellular processes that are altered in response to influenza in primary epithelial cells and may be beneficial in the search for host proteins that may be targeted for antiviral therapy

Influenza A Infection of Primary Human Airway Epithelial Cells Up-Regulates Proteins Related to Purine Metabolism and Ubiquitin-Related Signaling

Virus–host interactions are important determinants of virus replication and immune responses, but they are not well-defined. In this study we analyzed quantitative host protein alterations in nuclei-enriched fractions from multiple primary human bronchial airway epithelial (HBAE) cells infected by an H1N1 influenza A virus (A/PR/8/34). We first developed an effective detergent-free nuclear lysis method that was coupled with in-solution digestion and LC–MS/MS. Using SILAC, we identified 837 HBAE nuclear proteins in three different donors and compared their responses to infection at 24 h. Some proteins were altered in all three donors, of which 94 were up-regulated and 13 were down-regulated by at least 1.5-fold. Many of these up-regulated proteins clustered into purine biosynthesis, carbohydrate metabolism, and protein modification. Down-regulated proteins were not associated with any specific pathways or processes. These findings further our understanding of cellular processes that are altered in response to influenza in primary epithelial cells and may be beneficial in the search for host proteins that may be targeted for antiviral therapy

Silencing STAT3 abrogates IL-9 mediated CCL11 expression.

<p><b>A</b>. Efficiency of Lentiviral transduction in human ASM cells. Human ASM cells were transduced by infecting with lentivirus containing scramble sequence or STAT3-shRNA sequence and examined by flow cytometry for tGFP expression. <b>B</b>. Total STAT3 in mock, scramble and STAT3-ShRNA transduced ASM cells as analyzed by Western blot <b>C</b>. ASM cells stably expressing scramble or STAT3-shRNA was transfected with CCL11 promoter luciferase reporter plasmid and stimulated with IL-9 (10 ng/ml) or IL-4 (10 ng/ml) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Methods</a>. The mean ±SEM of three independent experiments are shown. * P<0.05 compared to scramble lentiviral transduced ASM cells stimulated with IL-9.</p

STAT3 inhibitory peptide decreases IL-9 mediated CCL11 transcriptional activity.

<p>Human primary ASM cells were growth-arrested, transfected with CCL11 promoter for 24 h. Cells were then incubated with inhibitory or control peptide for 1 h before stimulation for 12 hour with IL-4 or IL-9 after which transcriptional activation was measured by luciferase activity. Fold induction represents luciferase activity in cytokine treated cells compared to transfected untreated cells, and is the mean of five independent experiments. * P<0.05.</p

TGF-β facilitates Sp1/β-catenin interaction.

<p>Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) for 16 hours. Co-immunoprecipitation was performed as described in the Materials and Methods section. Immunocomplexes and whole cell extracts (WCE) were subjected to western analysis as indicated in the panels.</p

Sp1 is the transcription factor for TGF-β-induced WNT-5A expression in airway smooth muscle cells.

<p>(A-B) Mithramycin A attenuates WNT-5A mRNA and protein expression. (A) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. WNT-5A mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (B) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 48 hours. Whole cell extracts were prepared and WNT-5A protein abundance was evaluated by western analysis. GAPDH was assessed as loading control. (C, D) Cells were transfected with Sp1-specific or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of Sp1 mRNA (C) and WNT-5A mRNA (D) by qRT-PCR. Data represent mean ± SEM of 5 independent experiments. *p<0.05, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (E) Mithramycin A attenuates TGF-β-induced extracellular matrix expression. Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. Collagen IαI and fibronectin mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, #p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (F) Sp1 is recruited to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) for 16 hours. Chromatin was prepared and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers specific for Sp1 binding region on <i>WNT-5A</i> promoter A after immunoprecipitation with anti-Sp1 or control IgG antibody. Input DNA from chromatin preparation before immunoprecipitation was amplified to ascertain the loading. Resulting PCR products were analyzed by DNA PAGE. (G) TAK1 mediates recruitment of Sp1 to <i>WNT-5A</i> promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 hours. ChIP analysis was performed as described above.</p

IL-9 does not induce STAT6 nuclei translocation in human ASM cells.

<p>Growth arrested semi-confluent ASM cells were stimulated with IL-4 (A) or IL-9 (B) both at 10 ng/ml in 8 wells slide. Slides were stained with specific anti- phospho tyrosine STAT-6 mAb or isotype matched control, followed by goat anti-mouse IgG F(ab')₂ AlexaFluor® 488. Nuclear counter-staining was performed using TOTO-1 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Materials and Methods</a>. Original magnification 400X.</p

IL-9 mediated CCL11 expression is not affected in STAT6 silenced ASM cells.

<p><b>A</b>. Human ASM cells were transduced by infecting with lentivirus containing scramble sequence, STAT6-shRNA sequence or mock and examined by flow cytometry for turbo GFP (tGFP) expression. <b>B</b>. Effect of STAT6-shRNA on STAT6 expression by ASM cells. Expression of total STAT6 in mock, scramble or STAT6-shRNA transduced ASM cells was analyzed by western blot. <b>C</b>. Stably silenced STAT6 ASM cells was co-transfected with CCL11 promoter then stimulated with IL-9 or IL-4 (both at 10 ng/ml) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009178#s4" target="_blank">Materials and Methods</a>. The mean ±SE of three independent experiments are shown.</p