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Cited by 113interferon and immune responsesInfluenza Virus Research StudiesRespiratory viral infections research

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Lambda Interferon Restructures the Nasal Microbiome and Increases Susceptibility to Staphylococcus aureus Superinfection

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Planet, Paul J., Parker, Dane, Cohen, Taylor S., Smith, Hannah, Leon, Justinne D., Ryan, Chanelle, Hammer, Tobin J., Fierer, Noah, Chen, Emily I., Prince, Alice S.·2016·mBio
DOI 10.1128/mbio.01939-15PMC PMC4752601
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Abstract

UNLABELLED: Much of the morbidity and mortality associated with influenza virus respiratory infection is due to bacterial coinfection with pathogens that colonize the upper respiratory tract such as methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. A major component of the immune response to influenza virus is the production of type I and III interferons. Here we show that the immune response to infection with influenza virus causes an increase and restructuring of the upper respiratory microbiota in wild-type (WT) mice but not in Il28r(-/-) mutant mice lacking the receptor for type III interferon. Mice lacking the IL-28 receptor fail to induce STAT1 phosphorylation and expression of its regulator, SOCS1. Il28r(-/-) mutant mice have increased expression of interleukin-22 (IL-22), as well as Ngal and RegIIIγ, in the nasal cavity, the source of organisms that would be aspirated to cause pneumonia. Proteomic analysis reveals changes in several cytoskeletal proteins that contribute to barrier function in the nasal epithelium that may contribute to the effects of IL-28 signaling on the microbiota. The importance of the effects of IL-28 signaling in the pathogenesis of MRSA pneumonia after influenza virus infection was confirmed by showing that WT mice nasally colonized before or after influenza virus infection had significantly higher levels of infection in the upper airways, as well as significantly greater susceptibility to MRSA pneumonia than Il28r(-/-) mutant mice did. Our results suggest that activation of the type III interferon in response to influenza virus infection has a major effect in expanding the upper airway microbiome and increasing susceptibility to lower respiratory tract infection. IMPORTANCE: S. aureus and influenza virus are important respiratory pathogens, and coinfection with these organisms is associated with significant morbidity and mortality. The ability of influenza virus to increase susceptibility to S. aureus infection is less well understood. We show here that influenza virus leads to a change in the upper airway microbiome in a type III interferon-dependent manner. Mice lacking the type III interferon receptor have altered STAT1 and IL-22 signaling. In coinfection studies, mice without the type III interferon receptor had significantly less nasal S. aureus colonization and subsequent pneumonia than infected WT mice did. This work demonstrates that type III interferons induced by influenza virus contribute to nasal colonization and pneumonia due to S. aureus superinfection.

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FIG 1 Changes in the upper respiratory microbiome in response to influenza virus infection. WT mice were infected with PR8 or treated with PBS for 3 days. (a) Total bacterial load (CFU) recovered from the upper airway. n = 11. (b, c) Shannon-Weiner diversity of bacteria cultured (b) or identified with culture-independent techniques (c) from the upper airway. n = 11 (b) or 8 (c). (d) Culture-independent analysis of community composition in the upper airways of WT mice infected with PR8 or treated with PBS as a control. Relative abundance plots show differences between PR8-infected and PBS-treated mice. The bar plot shows differences in rarefied reads for the top 10 OTUs (ranked by relative abundance), as indicated by color coding. The top 10 OTUs constitute >99% of the rarefied reads. n = 8. (e) Maximum-parsimony tree based on OTU presence in or absence from the upper airways of mice infected with PR8 or treated with PBS. Branch length is proportional to the number of gain/loss changes in OTUs (scale bar represents 40 changes). Branches representing influenza virus-infected samples are red, and PBS-inoculated control samples are green. (f) Ordination plot of culture-independent analysis demonstrating segregation of PR8-infected mice (red) and PBS-treated controls (green); shaded areas are the minimum convex polygons enclosing the data points in each group. P = 0.004. Data are representative of at least two independent experiments. Graphs display mean values and standard errors. **, P < 0.01; *, P < 0.05.
FIG 1 Changes in the upper respiratory microbiome in response to influenza virus infection. WT mice were infected with PR8 or treated with PBS for 3 days. (a) Total bacterial load (CFU) recovered from the upper airway. n = 11. (b, c) Shannon-Weiner diversity of bacteria cultured (b) or identified with culture-independent techniques (c) from the upper airway. n = 11 (b) or 8 (c). (d) Culture-independent analysis of community composition in the upper airways of WT mice infected with PR8 or treated with PBS as a control. Relative abundance plots show differences between PR8-infected and PBS-treated mice. The bar plot shows differences in rarefied reads for the top 10 OTUs (ranked by relative abundance), as indicated by color coding. The top 10 OTUs constitute >99% of the rarefied reads. n = 8. (e) Maximum-parsimony tree based on OTU presence in or absence from the upper airways of mice infected with PR8 or treated with PBS. Branch length is proportional to the number of gain/loss changes in OTUs (scale bar represents 40 changes). Branches representing influenza virus-infected samples are red, and PBS-inoculated control samples are green. (f) Ordination plot of culture-independent analysis demonstrating segregation of PR8-infected mice (red) and PBS-treated controls (green); shaded areas are the minimum convex polygons enclosing the data points in each group. P = 0.004. Data are representative of at least two independent experiments. Graphs display mean values and standard errors. **, P < 0.01; *, P < 0.05.CC BY-NC-SA · PubMed Central
FIG 2 Effects of type III IFN signaling on upper airway microbiome. (a) WT mice were infected for 3 days with PR8. Nasal lavage fluid was assessed by qRT-PCR for expression of IFN-λ (Il28). n = 5 (PBS) or 4 (PR8). Il28r−/− mutant mice were infected with PR8 or treated with PBS for 3 days. (b, c) Bacterial load (n = 8) (b) and diversity of culture-dependent organisms isolated from upper airways (c). n = 8 (PBS) or 7 (PR8). (d) Diversity analysis from culture-independent data. n = 7. (e) Ordination plot of culture-independent analysis demonstrating lack of segregation of PR8-infected (red) and PBS-treated control (green) Il28r−/− mutant mice. Shaded areas are the minimum convex polygons enclosing the data points in each group. WT mice were treated with 1 µg of recombinant murine IL-28 or with BSA as a control, and the total bacterial load (f) (n = 10) and diversity of culture-dependent organisms (g) were determined the following day. n = 10. (h) Weights of WT and Il28r−/− mutant mice infected with PR8 or treated with PBS. n = 7. (i) qRT-PCR analysis of viral RNA in the lungs of WT and Il28r−/− mutant mice infected with PR8 for 3 or 7 days. n = 6. Data are representative of at least two independent experiments. Graphs display mean values and standard errors. **, P < 0.01.
FIG 2 Effects of type III IFN signaling on upper airway microbiome. (a) WT mice were infected for 3 days with PR8. Nasal lavage fluid was assessed by qRT-PCR for expression of IFN-λ (Il28). n = 5 (PBS) or 4 (PR8). Il28r−/− mutant mice were infected with PR8 or treated with PBS for 3 days. (b, c) Bacterial load (n = 8) (b) and diversity of culture-dependent organisms isolated from upper airways (c). n = 8 (PBS) or 7 (PR8). (d) Diversity analysis from culture-independent data. n = 7. (e) Ordination plot of culture-independent analysis demonstrating lack of segregation of PR8-infected (red) and PBS-treated control (green) Il28r−/− mutant mice. Shaded areas are the minimum convex polygons enclosing the data points in each group. WT mice were treated with 1 µg of recombinant murine IL-28 or with BSA as a control, and the total bacterial load (f) (n = 10) and diversity of culture-dependent organisms (g) were determined the following day. n = 10. (h) Weights of WT and Il28r−/− mutant mice infected with PR8 or treated with PBS. n = 7. (i) qRT-PCR analysis of viral RNA in the lungs of WT and Il28r−/− mutant mice infected with PR8 for 3 or 7 days. n = 6. Data are representative of at least two independent experiments. Graphs display mean values and standard errors. **, P < 0.01.CC BY-NC-SA · PubMed Central
FIG 3 Mutant (Il28r−/−) mice lack STAT1-associated signaling. (a) Immunoblot assays of P-STAT1, STAT1, P-STAT3, STAT3, and actin in WT and Ifnar−/− and Il28r−/− mutant mice infected with PR8 for 3 or 7 days or treated with PBS as a control. Stat1−/− mutant mice were infected with PR8 or treated with PBS as a control for 3 days. (b) Bacterial load isolated from the upper airway and Shannon-Weiner analysis of the diversity of recovered bacteria. n = 4 (WT, PBS), 5 (WT, PR8), 8 (Il28r−/− mutant, PBS), or 7 (Il28r−/− mutant, PR8). (c) WT or Stat1−/− mutant mice were treated with 1 µg of recombinant murine IL-28 or with BSA as a control, and the culture-dependent bacterial load was measured. n = 6. (d) Immunoblot assays of SOCS1 and actin in WT and Ifnar−/− and Il28r−/− mutant mice infected with PR8 for 3 days or treated with PBS as a control. (e) qRT-PCR analysis of Socs1 expression in their upper airways. n = 4 (PBS) or 5 and 6 (left and right; PR8). (f) qRT-PCR analysis of Il22, Ngal, S100a8, and RegIIIγ in the upper airways of WT and Il28r−/− mutant mice. Il22 data are shown for both PBS-treated and PR8-infected mice. Il22, n = 8; Ngal, n = 5 and 9, respectively; S100a8, n = 9 and 8, respectively; RegIIIγ, n = 8 and 7, respectively. Data are representative of at least two independent experiments. Graphs display mean values and standard errors. n.d, not detectable. *, P < 0.05.
FIG 3 Mutant (Il28r−/−) mice lack STAT1-associated signaling. (a) Immunoblot assays of P-STAT1, STAT1, P-STAT3, STAT3, and actin in WT and Ifnar−/− and Il28r−/− mutant mice infected with PR8 for 3 or 7 days or treated with PBS as a control. Stat1−/− mutant mice were infected with PR8 or treated with PBS as a control for 3 days. (b) Bacterial load isolated from the upper airway and Shannon-Weiner analysis of the diversity of recovered bacteria. n = 4 (WT, PBS), 5 (WT, PR8), 8 (Il28r−/− mutant, PBS), or 7 (Il28r−/− mutant, PR8). (c) WT or Stat1−/− mutant mice were treated with 1 µg of recombinant murine IL-28 or with BSA as a control, and the culture-dependent bacterial load was measured. n = 6. (d) Immunoblot assays of SOCS1 and actin in WT and Ifnar−/− and Il28r−/− mutant mice infected with PR8 for 3 days or treated with PBS as a control. (e) qRT-PCR analysis of Socs1 expression in their upper airways. n = 4 (PBS) or 5 and 6 (left and right; PR8). (f) qRT-PCR analysis of Il22, Ngal, S100a8, and RegIIIγ in the upper airways of WT and Il28r−/− mutant mice. Il22 data are shown for both PBS-treated and PR8-infected mice. Il22, n = 8; Ngal, n = 5 and 9, respectively; S100a8, n = 9 and 8, respectively; RegIIIγ, n = 8 and 7, respectively. Data are representative of at least two independent experiments. Graphs display mean values and standard errors. n.d, not detectable. *, P < 0.05.CC BY-NC-SA · PubMed Central
FIG 4 Upper airway proteomic changes in WT and Il28r−/− mutant mice. Two micrograms of protein from nasal lavage fluid was proteolytically cleaved by trypsin and analyzed by LC-MS/MS. (a) The upper airway proteomes of WT and Il28r−/− mutant mice in the resting state were analyzed by an MS-based approach. Relative protein abundance among biological samples is expressed by spectral counts on a log scale. The color scale bar indicates the range of protein expression levels. Each column represents an individual mouse. Significantly different proteins (P < 0.05) are presented and clustered by protein expression profiles among biological samples. (b) Functional annotation of proteins differentially expressed in WT and Il28r−/− mutant mice. Shown are functional categories enriched and statistically significant within the data set. Data were analyzed with David. (c) WT and Il28r−/− mutant mice were infected with PR8 or treated with PBS as a control, and upper airway nasal lavage fluid was analyzed by an MS-based approach. Each column represents an individual mouse. Representative data are shown.
FIG 4 Upper airway proteomic changes in WT and Il28r−/− mutant mice. Two micrograms of protein from nasal lavage fluid was proteolytically cleaved by trypsin and analyzed by LC-MS/MS. (a) The upper airway proteomes of WT and Il28r−/− mutant mice in the resting state were analyzed by an MS-based approach. Relative protein abundance among biological samples is expressed by spectral counts on a log scale. The color scale bar indicates the range of protein expression levels. Each column represents an individual mouse. Significantly different proteins (P < 0.05) are presented and clustered by protein expression profiles among biological samples. (b) Functional annotation of proteins differentially expressed in WT and Il28r−/− mutant mice. Shown are functional categories enriched and statistically significant within the data set. Data were analyzed with David. (c) WT and Il28r−/− mutant mice were infected with PR8 or treated with PBS as a control, and upper airway nasal lavage fluid was analyzed by an MS-based approach. Each column represents an individual mouse. Representative data are shown.CC BY-NC-SA · PubMed Central
FIG 5 Type III IFN signaling contributes to S. aureus colonization of the upper airway and development of bacterial pneumonia. (a) WT C57BL/6J mice were infected intranasally with 50 PFU of influenza virus PR8 or treated with PBS for 7 days prior to the local intranasal application of 108 CFU of S. aureus USA300. Mice were euthanized 3 and 7 days after S. aureus infection. Bacterial counts in mouse nasal and lung homogenates were assessed. n = 6 (3-day data), 4 (PBS, 7 days), or 3 (PR8, 7 days). (b) WT mice were intranasally inoculated locally with 108 CFU of S. aureus USA300 7 days before intranasal infection with 50 PFU of influenza virus PR8 for a further 7 days before euthanasia. Bacterial counts in mouse nasal and lung homogenates were assessed. n = 5. (c) WT and Il28r−/− mutant mice were infected with PR8 or treated with PBS in the lungs for 7 days prior to the local administration of S. aureus (108 CFU). Mice were euthanized 3 days later. Bacterial counts in nasal and lung homogenates are shown. n = 6 (WT and Il28r−/−, PBS) or 4 (Il28r−/−, PR8). (d) Cytokine differences in BALF from mice nasally infected with PR8 and S. aureus (c). n = 3 (uninfected [UN]), 6 (WT), or 4 (Il28r−/−). (e) WT mice were intranasally infected with 107 CFU of P. aeruginosa PAK in the lungs for 24 h after 7 days of PR8 infection or treatment with PBS as a control and 24 h after PAK inoculation. Bacterial counts in the lungs are shown. n = 7. (f) Counts of S. aureus bacteria in the lungs of WT and Il28r−/− mutant mice after 7 days of PR8 infection or treatment with PBS as a control and 24 h after intranasal inoculation with S. aureus USA300 (107 CFU) under anesthesia. n = 13 (WT, PBS), 11 (WT, PR8), or 7 (Il28r−/−). Data are representative of at least two independent experiments. Graphs display mean values and standard errors. ***, P < 0.001; **, P < 0.01; *, P < 0.05.
FIG 5 Type III IFN signaling contributes to S. aureus colonization of the upper airway and development of bacterial pneumonia. (a) WT C57BL/6J mice were infected intranasally with 50 PFU of influenza virus PR8 or treated with PBS for 7 days prior to the local intranasal application of 108 CFU of S. aureus USA300. Mice were euthanized 3 and 7 days after S. aureus infection. Bacterial counts in mouse nasal and lung homogenates were assessed. n = 6 (3-day data), 4 (PBS, 7 days), or 3 (PR8, 7 days). (b) WT mice were intranasally inoculated locally with 108 CFU of S. aureus USA300 7 days before intranasal infection with 50 PFU of influenza virus PR8 for a further 7 days before euthanasia. Bacterial counts in mouse nasal and lung homogenates were assessed. n = 5. (c) WT and Il28r−/− mutant mice were infected with PR8 or treated with PBS in the lungs for 7 days prior to the local administration of S. aureus (108 CFU). Mice were euthanized 3 days later. Bacterial counts in nasal and lung homogenates are shown. n = 6 (WT and Il28r−/−, PBS) or 4 (Il28r−/−, PR8). (d) Cytokine differences in BALF from mice nasally infected with PR8 and S. aureus (c). n = 3 (uninfected [UN]), 6 (WT), or 4 (Il28r−/−). (e) WT mice were intranasally infected with 107 CFU of P. aeruginosa PAK in the lungs for 24 h after 7 days of PR8 infection or treatment with PBS as a control and 24 h after PAK inoculation. Bacterial counts in the lungs are shown. n = 7. (f) Counts of S. aureus bacteria in the lungs of WT and Il28r−/− mutant mice after 7 days of PR8 infection or treatment with PBS as a control and 24 h after intranasal inoculation with S. aureus USA300 (107 CFU) under anesthesia. n = 13 (WT, PBS), 11 (WT, PR8), or 7 (Il28r−/−). Data are representative of at least two independent experiments. Graphs display mean values and standard errors. ***, P < 0.001; **, P < 0.01; *, P < 0.05.CC BY-NC-SA · PubMed Central

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Authors
Planet, P. J., Parker, D., Cohen, T. S., Smith, H., Leon, J. D., Ryan, C., Hammer, T. J., Fierer, N., Chen, E. I., & Prince, A. S.
Affiliations
  • American Museum of Natural History (US)
  • Columbia University (US)
  • Herbert Irving Comprehensive Cancer Center (US)
  • University of Colorado Boulder (US)
  • Cooperative Institute for Research in Environmental Sciences (US)
  • Columbia University Irving Medical Center (US)
Corresponding author
Prince, Alice S.
ORCID
Venue
mBio
Publisher
American Society for Microbiology
Published
2016-03-02
Volume
7
Issue
1
Pages
e01939-15
ISSN
  • 2150-7511 (ISSN-L)
  • 2161-2129
DOI
10.1128/mbio.01939-15
PMC
PMC4752601
OpenAlex
W2261043905
Cited by
113
References
62
Citations by year
2026: 1 · 2025: 4 · 2024: 7 · 2023: 10 · 2022: 9 · 2021: 16 · 2020: 27 · 2019: 13 · 2018: 13 · 2017: 6 · 2016: 7
FWCI
7.87
Type
journal article
Language
en
Open access
Licence
Locations
  • mBio (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
  • CU Scholar (University of Colorado Boulder) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
HHS | National Institutes of Health (K08AI101005, HL079395, P30CA013696, R56HL125653)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Health Sciences › Medicine › Epidemiology › Influenza Virus Research Studies
  • Health Sciences › Medicine › Epidemiology › Respiratory viral infections research
Keywords
interferon lambda, influenza virus, Staphylococcus aureus, MRSA pneumonia, upper respiratory tract microbiome, nasal microbiota, secondary bacterial infection, IL-22, STAT1 signaling, SOCS1, nasal epithelial barrier, cytoskeletal proteins, bacterial co-infection
MeSH terms
Interferon Lambda, Animals, Interleukins, Nasal Cavity, Orthomyxoviridae Infections, Pneumonia, Staphylococcal, Staphylococcus aureus, Superinfection, Cytokines, Mice, Knockout, Mice, Microbiota

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