HHS Public Access Author manuscript Author Manuscript
Virology. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Virology. 2016 November ; 498: 128–135. doi:10.1016/j.virol.2016.08.020.
Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge Stacey M. Hartwig1, Margaret Ketterer1, Michael A. Apicella1, and Steven M. Varga1,2,3,* 1Department
of Microbiology, University of Iowa, Iowa City, Iowa, United States of America
Author Manuscript
2Interdisciplinary
Graduate Program in Immunology, University of Iowa, Iowa City, Iowa, United States of America
3Department
of Pathology, University of Iowa, Iowa City, Iowa, United States of America
Abstract
Author Manuscript
Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, nontypeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Coculture of 16HBE14o- cells with NTHi 2019 resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi 2019 failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSVspecific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection.
Keywords Haemophilus influenzae; respiratory syncytial virus; human bronchial epithelial cells
Introduction Author Manuscript
Viral and bacterial co-infections are increasingly detected in patients with respiratory infections. Several studies have examined the impact respiratory viruses have on secondary bacterial infections, showing that respiratory viruses such as influenza A virus (IAV) can
*
[email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Potential conflicts of interest All authors: No conflicts.
Hartwig et al.
Page 2
Author Manuscript
increase susceptibility to a secondary bacterial infection (1-3). However, it is currently unclear how the presence of commensal bacteria may impact a subsequent viral infection. Non-typeable Haemophilus influenza (NTHi) is frequent commensal bacteria found in the upper respiratory tract (4-6). Studies have shown that viral infections of respiratory epithelial cells due to respiratory syncytial virus (RSV) can increase the adherence of NTHi to these cells (7, 8). While NTHi colonization of the upper airways is generally benign, under the proper conditions, it can allow for opportunistic infections to develop, resulting in bacterial conjunctivitis, otitis media, sinusitis, bronchitis or pneumonia (9). RSV is the most common viral respiratory pathogen found in conjunction with NTHi and can often lead to the development of bronchitis, pneumonia or otitis media (10, 11).
Author Manuscript
In this study we sought to determine the impact of prior exposure of NTHi on human bronchial epithelial (16HBE14o-) cells. 16HBE14o- cells co-cultured with NTHi as few as 6 hr prior to an RSV infection exhibited a significant reduction in viral gene expression. Live bacteria were required to elicit this protection, as exposure to heat-killed bacteria failed to protect 16HBE14o- cells from an RSV infection. NTHi 2019ΔlicD, which lacks expression of phosphorylcholine, thus inhibiting its ability to enter the 16HBE14o- cells, exhibited reduced protection indicating that bacteria must successfully invade the epithelial cell to induce optimal protection. Importantly, this inhibition appears to be specific to RSV, as NTHi did not mediate protection of 16HBE14o- cells from an IAV infection.
Material and Methods Virus growth and purification
Author Manuscript Author Manuscript
The A2 strain of RSV was propagated in HEp-2 cells (American Type Culture Collection, Manassas, VA). Infected cells were removed with a cell scraper and both cells and supernatant were centrifuged at 2000 × g for 15 min at 4°C. Pooled supernatants and 50% Polyethylene Glycol (PEG) 8000 were combined at a 1:5 ratio (vol:vol) and slowly mixed for 2 hr at 4°C. The mixture was centrifuged at 9,481 × g for 30 min at 4°C. The supernatant was discarded and the pellet was resuspended in 9 ml of 20% sucrose. A sucrose gradient consisting of 60% and 35% sucrose was prepared and 9 ml of RSV-sucrose was carefully added onto the gradient and centrifuged at 243,050 × g for 1 hr at 4°C. The virus band between the 35% and 60% sucrose layers was removed from each tube, pooled, and aliquoted prior to being flash frozen in liquid nitrogen. RSV was stored at −80°C. A recombinant RSV engineered to express green fluorescent protein (GFP) was obtained from Mark Peeples (Columbus Children's Research Hospital, Columbus, OH) (12). Mouseadapted influenza A virus A/PR/8/34 was grown in the allantoic fluid of 10 day-old embryonated chicken eggs as previously described (13). Bacteria preparation NTHi 2019/S10/V6 (NTHi 2019) is a clinical isolate that has been described previously (14, 15). NTHi 2019 was recently recovered during an NIH sponsored human experimental colonization study, and it has been shown that the majority of the population of organisms in this strain express phosphorylcholine (15). NTHi 2019ΔlicD is a chromosomal mutant in
Virology. Author manuscript; available in PMC 2017 November 01.
Hartwig et al.
Page 3
Author Manuscript
phosphorylcholine transferase (16). Due to this mutation, the organism lacks phosphorylcholine on the surface lipooligosaccharide (LOS) (15). All NTHi strains were grown from frozen stocks onto brain heart infusion agar plates supplemented with 10 μg hemin/ml and 10 μg of NAD/ml at 37°C in 5% CO2. A piliated clinical isolate of Neisseria cinerea, which is a commensal bacteria of the human nasopharnx, was grown as previously described (17). Heat-inactivated bacteria were obtained by incubation of the cultures for 30 mins at 65°C. Human bronchial epithelial (16HBE14o-) cell line infections
Author Manuscript
16HBE14o- cells, which are SV40 transformed human airway epithelial cells, were a gift from Dr. D. C. Gruenert (University of California, San Francisco, CA) (18). 16HBE14ocells were maintained in Minimum Essential Media (MEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FCS; Atlanta Biologicals, Lawrenceville, GA), 2 mM l-glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (all from Gibco). 16HBE14o- cells were seeded in either 6 or 24 well tissue culture plates and cocultured with NTHi at an initial bacterium:cell ratio of 1:1 for either 6 or 24 hr. Following bacteria exposure, the 16HBE14o- cultures were infected with unlabeled purified RSV or purified RSV-GFP (12) at an MOI of either 1 or 10. When indicated, NTHi and RSV were co-incubated together on ice for 30 mins prior to 16HBE14o- infection for 48 hr. In selected experiments the NTHi-exposed 16HBE14o- cells were infected with IAV at an MOI of either 0.1 or 1 for 48 hr. Real-time PCR
Author Manuscript
Total cellular RNA was isolated from 16HBE14o- cells following RSV infections at the indicated time points. 16HBE14o- cells were washed in 1 ml of Trizol (Invitrogen, Carlsbad, CA) and supernatants collected. RNA was purified by chloroform and isopropyl alcohol extractions. cDNA was made using SuperScript VILO cDNA synthesis kit for RT-PCR (Invitrogen). Real-time PCR was performed to detect either the nucleocapsid (N) gene of RSV or the nucleoprotein (NP) gene of IAV. Real-time PCR was performed using a Taqman Universal PCR master mix on an Applied Biosystems 7300 real-time PCR machine (Foster City, CA) under standard thermal cycling parameters. RSV N gene and IAV NP gene primers and probes have been previously described (19, 20). All probes were synthesized to contain a 5’ 6-carboxy-fluorescein reporter dye and 3’ carboxytetramethylrhodamince quencher dye (Integrated DNA Technologies, Coralville, IA). Samples were compared to known standard dilutions of plasmids containing each individual gene. Total copy numbers for each gene were calculated based on the calculated copy numbers in each sample and the total amount of RNA from each well.
Author Manuscript
Microscopy 16HBE14o- cells were grown on collagen-coated coverslips and infected with NTHi and RSV as described above. At 48 hr following RSV infection, the 16HBE14o- cells were fixed in 2% paraformaldehyde. Samples were washed with PBS and then permeabilized in 100% methanol at −20°C for 10 mins. Samples were blocked with 5% normal goat serum for 1 hr at room temperature and labeled with a murine monoclonal antibody that binds to the stable 2-keto-3-deoxyoctulosonic acid epitope on the LOS of Haemophilus influenzae (21, 22) for Virology. Author manuscript; available in PMC 2017 November 01.
Hartwig et al.
Page 4
Author Manuscript
2 hr at room temperature. 16HBE14o- cells were washed three times for 2 mins with PBS and incubated with goat anti-mouse IgG conjugated to AlexaFluor594 (Jackson ImmunoResearch) for 2 hr at room temperature. DAPI was included in the ProLong “Diamond” mounting media (Life Technologies) to label cell nuclei. All specimens were viewed on a Zeiss LSM710 microscope located within the Central Microscopy Research Facility at the University of Iowa (Iowa City). Plaque assay 16HBE14o- cells were cultured as described above. 16HBE14o- cell lysates and supernatants were collected at given time points by scrapping the monolayer with the plunger of a 1 ml syringe, pooled with the supernatant and flash frozen in liquid nitrogen. RSV viral titers were assessed by plaque assay as previously described (23).
Author Manuscript
Statistical analysis All statistical analyses were performed using Prism software (GraphPad Software, Inc., San Diego, CA). Statistical significance was determined by a oneway ANOVA, with a HolmSidak's multiple comparisons post-test; or a nonparametric t-test, with a Mann-Whitney post-test. A value of p
Virology. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Virology. 2016 November ; 498: 128–135. doi:10.1016/j.virol.2016.08.020.
Non-typeable Haemophilus influenzae protects human airway epithelial cells from a subsequent respiratory syncytial virus challenge Stacey M. Hartwig1, Margaret Ketterer1, Michael A. Apicella1, and Steven M. Varga1,2,3,* 1Department
of Microbiology, University of Iowa, Iowa City, Iowa, United States of America
Author Manuscript
2Interdisciplinary
Graduate Program in Immunology, University of Iowa, Iowa City, Iowa, United States of America
3Department
of Pathology, University of Iowa, Iowa City, Iowa, United States of America
Abstract
Author Manuscript
Respiratory syncytial virus (RSV) and the common commensal and opportunistic pathogen, nontypeable Haemophilus influenzae (NTHi) both serve as a frequent cause of respiratory infection in children. Although it is well established that some respiratory viruses can increase host susceptibility to secondary bacterial infections, few studies have examined how commensal bacteria could influence a secondary viral response. Here, we examined the impact of NTHi exposure on a subsequent RSV infection of human bronchial epithelial cells (16HBE14o-). Coculture of 16HBE14o- cells with NTHi 2019 resulted in inhibition of viral gene expression following RSV infection. 16HBE14o- cells co-cultured with heat-killed NTHi 2019 failed to protect against an RSV infection, indicating that protection requires live bacteria. However, NTHi did not inhibit influenza A virus replication, indicating that NTHi-mediated protection was RSVspecific. Our data demonstrates that prior exposure to a commensal bacterium such as NTHi can elicit protection against a subsequent RSV infection.
Keywords Haemophilus influenzae; respiratory syncytial virus; human bronchial epithelial cells
Introduction Author Manuscript
Viral and bacterial co-infections are increasingly detected in patients with respiratory infections. Several studies have examined the impact respiratory viruses have on secondary bacterial infections, showing that respiratory viruses such as influenza A virus (IAV) can
*
[email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Potential conflicts of interest All authors: No conflicts.
Hartwig et al.
Page 2
Author Manuscript
increase susceptibility to a secondary bacterial infection (1-3). However, it is currently unclear how the presence of commensal bacteria may impact a subsequent viral infection. Non-typeable Haemophilus influenza (NTHi) is frequent commensal bacteria found in the upper respiratory tract (4-6). Studies have shown that viral infections of respiratory epithelial cells due to respiratory syncytial virus (RSV) can increase the adherence of NTHi to these cells (7, 8). While NTHi colonization of the upper airways is generally benign, under the proper conditions, it can allow for opportunistic infections to develop, resulting in bacterial conjunctivitis, otitis media, sinusitis, bronchitis or pneumonia (9). RSV is the most common viral respiratory pathogen found in conjunction with NTHi and can often lead to the development of bronchitis, pneumonia or otitis media (10, 11).
Author Manuscript
In this study we sought to determine the impact of prior exposure of NTHi on human bronchial epithelial (16HBE14o-) cells. 16HBE14o- cells co-cultured with NTHi as few as 6 hr prior to an RSV infection exhibited a significant reduction in viral gene expression. Live bacteria were required to elicit this protection, as exposure to heat-killed bacteria failed to protect 16HBE14o- cells from an RSV infection. NTHi 2019ΔlicD, which lacks expression of phosphorylcholine, thus inhibiting its ability to enter the 16HBE14o- cells, exhibited reduced protection indicating that bacteria must successfully invade the epithelial cell to induce optimal protection. Importantly, this inhibition appears to be specific to RSV, as NTHi did not mediate protection of 16HBE14o- cells from an IAV infection.
Material and Methods Virus growth and purification
Author Manuscript Author Manuscript
The A2 strain of RSV was propagated in HEp-2 cells (American Type Culture Collection, Manassas, VA). Infected cells were removed with a cell scraper and both cells and supernatant were centrifuged at 2000 × g for 15 min at 4°C. Pooled supernatants and 50% Polyethylene Glycol (PEG) 8000 were combined at a 1:5 ratio (vol:vol) and slowly mixed for 2 hr at 4°C. The mixture was centrifuged at 9,481 × g for 30 min at 4°C. The supernatant was discarded and the pellet was resuspended in 9 ml of 20% sucrose. A sucrose gradient consisting of 60% and 35% sucrose was prepared and 9 ml of RSV-sucrose was carefully added onto the gradient and centrifuged at 243,050 × g for 1 hr at 4°C. The virus band between the 35% and 60% sucrose layers was removed from each tube, pooled, and aliquoted prior to being flash frozen in liquid nitrogen. RSV was stored at −80°C. A recombinant RSV engineered to express green fluorescent protein (GFP) was obtained from Mark Peeples (Columbus Children's Research Hospital, Columbus, OH) (12). Mouseadapted influenza A virus A/PR/8/34 was grown in the allantoic fluid of 10 day-old embryonated chicken eggs as previously described (13). Bacteria preparation NTHi 2019/S10/V6 (NTHi 2019) is a clinical isolate that has been described previously (14, 15). NTHi 2019 was recently recovered during an NIH sponsored human experimental colonization study, and it has been shown that the majority of the population of organisms in this strain express phosphorylcholine (15). NTHi 2019ΔlicD is a chromosomal mutant in
Virology. Author manuscript; available in PMC 2017 November 01.
Hartwig et al.
Page 3
Author Manuscript
phosphorylcholine transferase (16). Due to this mutation, the organism lacks phosphorylcholine on the surface lipooligosaccharide (LOS) (15). All NTHi strains were grown from frozen stocks onto brain heart infusion agar plates supplemented with 10 μg hemin/ml and 10 μg of NAD/ml at 37°C in 5% CO2. A piliated clinical isolate of Neisseria cinerea, which is a commensal bacteria of the human nasopharnx, was grown as previously described (17). Heat-inactivated bacteria were obtained by incubation of the cultures for 30 mins at 65°C. Human bronchial epithelial (16HBE14o-) cell line infections
Author Manuscript
16HBE14o- cells, which are SV40 transformed human airway epithelial cells, were a gift from Dr. D. C. Gruenert (University of California, San Francisco, CA) (18). 16HBE14ocells were maintained in Minimum Essential Media (MEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FCS; Atlanta Biologicals, Lawrenceville, GA), 2 mM l-glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate (all from Gibco). 16HBE14o- cells were seeded in either 6 or 24 well tissue culture plates and cocultured with NTHi at an initial bacterium:cell ratio of 1:1 for either 6 or 24 hr. Following bacteria exposure, the 16HBE14o- cultures were infected with unlabeled purified RSV or purified RSV-GFP (12) at an MOI of either 1 or 10. When indicated, NTHi and RSV were co-incubated together on ice for 30 mins prior to 16HBE14o- infection for 48 hr. In selected experiments the NTHi-exposed 16HBE14o- cells were infected with IAV at an MOI of either 0.1 or 1 for 48 hr. Real-time PCR
Author Manuscript
Total cellular RNA was isolated from 16HBE14o- cells following RSV infections at the indicated time points. 16HBE14o- cells were washed in 1 ml of Trizol (Invitrogen, Carlsbad, CA) and supernatants collected. RNA was purified by chloroform and isopropyl alcohol extractions. cDNA was made using SuperScript VILO cDNA synthesis kit for RT-PCR (Invitrogen). Real-time PCR was performed to detect either the nucleocapsid (N) gene of RSV or the nucleoprotein (NP) gene of IAV. Real-time PCR was performed using a Taqman Universal PCR master mix on an Applied Biosystems 7300 real-time PCR machine (Foster City, CA) under standard thermal cycling parameters. RSV N gene and IAV NP gene primers and probes have been previously described (19, 20). All probes were synthesized to contain a 5’ 6-carboxy-fluorescein reporter dye and 3’ carboxytetramethylrhodamince quencher dye (Integrated DNA Technologies, Coralville, IA). Samples were compared to known standard dilutions of plasmids containing each individual gene. Total copy numbers for each gene were calculated based on the calculated copy numbers in each sample and the total amount of RNA from each well.
Author Manuscript
Microscopy 16HBE14o- cells were grown on collagen-coated coverslips and infected with NTHi and RSV as described above. At 48 hr following RSV infection, the 16HBE14o- cells were fixed in 2% paraformaldehyde. Samples were washed with PBS and then permeabilized in 100% methanol at −20°C for 10 mins. Samples were blocked with 5% normal goat serum for 1 hr at room temperature and labeled with a murine monoclonal antibody that binds to the stable 2-keto-3-deoxyoctulosonic acid epitope on the LOS of Haemophilus influenzae (21, 22) for Virology. Author manuscript; available in PMC 2017 November 01.
Hartwig et al.
Page 4
Author Manuscript
2 hr at room temperature. 16HBE14o- cells were washed three times for 2 mins with PBS and incubated with goat anti-mouse IgG conjugated to AlexaFluor594 (Jackson ImmunoResearch) for 2 hr at room temperature. DAPI was included in the ProLong “Diamond” mounting media (Life Technologies) to label cell nuclei. All specimens were viewed on a Zeiss LSM710 microscope located within the Central Microscopy Research Facility at the University of Iowa (Iowa City). Plaque assay 16HBE14o- cells were cultured as described above. 16HBE14o- cell lysates and supernatants were collected at given time points by scrapping the monolayer with the plunger of a 1 ml syringe, pooled with the supernatant and flash frozen in liquid nitrogen. RSV viral titers were assessed by plaque assay as previously described (23).
Author Manuscript
Statistical analysis All statistical analyses were performed using Prism software (GraphPad Software, Inc., San Diego, CA). Statistical significance was determined by a oneway ANOVA, with a HolmSidak's multiple comparisons post-test; or a nonparametric t-test, with a Mann-Whitney post-test. A value of p
