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Human coronavirus HKU1 infection of primary human type II alveolar epithelial cells: cytopathic effects and innate immune response.

Dominguez SR, Travanty EA, Qian Z, Mason RJ - PLoS ONE (2013)

Bottom Line: Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults.At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ.These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, United States of America. samuel.dominguez@ucdenver.edu

ABSTRACT
Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease.

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Immunofluorescent staining for HCoV-HKU1 spike protein and selected alveolar type II cell markers.The cells were grown under air/liquid conditions as described in the methods section, inoculated with HCoV-HKU1 and fixed 72 hours post inoculation. Panels A-D show staining for DAPI (A), HCoV-HKU1 (B), TTF-1 (C), and merged (D). Panels E-H show staining for pro DAPI (E), HCoV-HKU1 (F), SP-A (G), and merged (H). Panels I-L show staining for DAPI (I), HCoV-HKU1 (J), proSP-B (K), and merged (L). Panels M-P show staining for DAPI (M), HCoV-HKU1 (N), AT280 (Dobbs) (O), and merged (P). Cells that are infected with HCoV-HKU1 stain for the type II cell markers.
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pone-0070129-g001: Immunofluorescent staining for HCoV-HKU1 spike protein and selected alveolar type II cell markers.The cells were grown under air/liquid conditions as described in the methods section, inoculated with HCoV-HKU1 and fixed 72 hours post inoculation. Panels A-D show staining for DAPI (A), HCoV-HKU1 (B), TTF-1 (C), and merged (D). Panels E-H show staining for pro DAPI (E), HCoV-HKU1 (F), SP-A (G), and merged (H). Panels I-L show staining for DAPI (I), HCoV-HKU1 (J), proSP-B (K), and merged (L). Panels M-P show staining for DAPI (M), HCoV-HKU1 (N), AT280 (Dobbs) (O), and merged (P). Cells that are infected with HCoV-HKU1 stain for the type II cell markers.

Mentions: Human alveolar macrophages and alveolar cells were isolated as previously described [5], [16], [17]. Briefly, de-identified donor lungs that were not suitable for transplant were obtained through the National Disease Research Interchange (Philadelphia, PA) and the International Institute for the Advancement of Medicine (Edison, NJ). The isolation of cells was conducted as previously described with the exception that the type II cells were positively selected using EpCAM (CD326) magnetic beads (Miltenyi, Auburn, CA). Cells were plated in DMEM with 10% FBS on millicell inserts (Millipore, Bedford, MA) coated with a mixture of 80% rat tail collagen and 20% Matrigel (BD Biosciences, San Jose, CA). Cells were allowed to adhere to inserts for 2 days submerged in DMEM with 10% FBS, then cultured at an air-liquid interface in DMEM with 1% charcoal-stripped serum (CS-FBS) supplemented with keratinocyte growth factor (K) for 2 days, and then switched to media that additionally had isobutylmethylxanthine (I), 8-bromo-cAMP (A), and dexamethasone (D) for 2 days to achieve the ATII differentiated phenotype prior to infection (day 6 after plating). The alveolar macrophages and alveolar type I-like cells were cultured and characterized as reported previously [5], [16], [17]. Briefly, to transdifferentiate type II cells to type I–like cells, type II cells were plated on rat tail collagen–coated dishes at a density of 0.5–1.0×105/cm2 in DMEM with 10% FBS (15). After 24–48 h the medium was changed to 5% FBS without additives. Type I-like cell phenotype was documented by positive staining for receptor for advanced glycation end products (RAGE, R & D systems, Minneapolis, MN) and epithelial membrane protein 2 (EMP2, Sigma-Aldrich, Inc, St.Louis, MO). For alveolar macrophages, the middle lobe of the lungs was perfused, and then lavaged with HEPES-buffered saline containing 2 mM EDTA and then HEPES-buffered saline alone. The macrophage purity of the adherent cells was nearly 100% and demonstrated by staining for CD 68 (DakoCytomation, Carpinteria,CA). The purity of the type II cells is demonstrated in Figure 1 and the level of surfactant protein expression has been published [5], [20], [25].


Human coronavirus HKU1 infection of primary human type II alveolar epithelial cells: cytopathic effects and innate immune response.

Dominguez SR, Travanty EA, Qian Z, Mason RJ - PLoS ONE (2013)

Immunofluorescent staining for HCoV-HKU1 spike protein and selected alveolar type II cell markers.The cells were grown under air/liquid conditions as described in the methods section, inoculated with HCoV-HKU1 and fixed 72 hours post inoculation. Panels A-D show staining for DAPI (A), HCoV-HKU1 (B), TTF-1 (C), and merged (D). Panels E-H show staining for pro DAPI (E), HCoV-HKU1 (F), SP-A (G), and merged (H). Panels I-L show staining for DAPI (I), HCoV-HKU1 (J), proSP-B (K), and merged (L). Panels M-P show staining for DAPI (M), HCoV-HKU1 (N), AT280 (Dobbs) (O), and merged (P). Cells that are infected with HCoV-HKU1 stain for the type II cell markers.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722178&req=5

pone-0070129-g001: Immunofluorescent staining for HCoV-HKU1 spike protein and selected alveolar type II cell markers.The cells were grown under air/liquid conditions as described in the methods section, inoculated with HCoV-HKU1 and fixed 72 hours post inoculation. Panels A-D show staining for DAPI (A), HCoV-HKU1 (B), TTF-1 (C), and merged (D). Panels E-H show staining for pro DAPI (E), HCoV-HKU1 (F), SP-A (G), and merged (H). Panels I-L show staining for DAPI (I), HCoV-HKU1 (J), proSP-B (K), and merged (L). Panels M-P show staining for DAPI (M), HCoV-HKU1 (N), AT280 (Dobbs) (O), and merged (P). Cells that are infected with HCoV-HKU1 stain for the type II cell markers.
Mentions: Human alveolar macrophages and alveolar cells were isolated as previously described [5], [16], [17]. Briefly, de-identified donor lungs that were not suitable for transplant were obtained through the National Disease Research Interchange (Philadelphia, PA) and the International Institute for the Advancement of Medicine (Edison, NJ). The isolation of cells was conducted as previously described with the exception that the type II cells were positively selected using EpCAM (CD326) magnetic beads (Miltenyi, Auburn, CA). Cells were plated in DMEM with 10% FBS on millicell inserts (Millipore, Bedford, MA) coated with a mixture of 80% rat tail collagen and 20% Matrigel (BD Biosciences, San Jose, CA). Cells were allowed to adhere to inserts for 2 days submerged in DMEM with 10% FBS, then cultured at an air-liquid interface in DMEM with 1% charcoal-stripped serum (CS-FBS) supplemented with keratinocyte growth factor (K) for 2 days, and then switched to media that additionally had isobutylmethylxanthine (I), 8-bromo-cAMP (A), and dexamethasone (D) for 2 days to achieve the ATII differentiated phenotype prior to infection (day 6 after plating). The alveolar macrophages and alveolar type I-like cells were cultured and characterized as reported previously [5], [16], [17]. Briefly, to transdifferentiate type II cells to type I–like cells, type II cells were plated on rat tail collagen–coated dishes at a density of 0.5–1.0×105/cm2 in DMEM with 10% FBS (15). After 24–48 h the medium was changed to 5% FBS without additives. Type I-like cell phenotype was documented by positive staining for receptor for advanced glycation end products (RAGE, R & D systems, Minneapolis, MN) and epithelial membrane protein 2 (EMP2, Sigma-Aldrich, Inc, St.Louis, MO). For alveolar macrophages, the middle lobe of the lungs was perfused, and then lavaged with HEPES-buffered saline containing 2 mM EDTA and then HEPES-buffered saline alone. The macrophage purity of the adherent cells was nearly 100% and demonstrated by staining for CD 68 (DakoCytomation, Carpinteria,CA). The purity of the type II cells is demonstrated in Figure 1 and the level of surfactant protein expression has been published [5], [20], [25].

Bottom Line: Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults.At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ.These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, United States of America. samuel.dominguez@ucdenver.edu

ABSTRACT
Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease.

Show MeSH
Related in: MedlinePlus