Loss of circadian protection against influenza infection in adult mice exposed to hyperoxia as neonates
- The Children’s Hospital of Philadelphia, United States
- Institute of Translational Medicine and Therapeutics (ITMAT), University of Pennsylvania, United States
- Systems Pharmacology University of Pennsylvania Perelman School of Medicine, United States
- Chronobiology and Sleep Institute, University of Pennsylvania, United States
- Department of Neuroscience, University of Pennsylvania Perelman School of Medicine, United States
- Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, United States
Figures
Figure 1 with 2 supplements
Overall experimental design and effects of neonatal hyperoxia on circadian regulation of IAV infection adulthood.
(A) Experimental model for neonatal hyperoxia followed by IAV infection: C57Bl6 pups aged <12 hr were exposed to either hyperoxia (>95% O2) or room air (21% O2) from PN0–5. Thereafter, the pups were …
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Experimental model for adult hyperoxia followed by IAV infection: C57Bl6/J mice at least 8 weeks old were exposed hyperoxia (>95% O2) for ~48 hr.
Thereafter, the animals were allowed to recover in room air and acclimatized to revere light–dark cycles for 3–4 weeks. Mice were infected with 25–40 PFU of PR8 at either ZT23 (dawn or just before …
Figure 1—figure supplement 2
Body weights post-hyperoxia exposure.
After the 5 day neonatal hyperoxia exposure, body weight of both the hyperoxia-exposed mice and their room air littermates were measured at 4 weeks and at 8 weeks as a measure of overall well-being …
Figure 2
Loss of temporal gating of IAV infection in adults exposed to hyperoxia as neonates is not mediated through central circadian clock.
Mice pups <12 hr old were exposed to either hyperoxia or room air for 5 days and then recovered in RA into adulthood. As adults, effect on the central (locomotor activity) and peripheral (gene …
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Exposure to hyperoxia as neonates has subtle effects of the circadian regulation of the immune response to influenza infection.
(A) Flow cytometric enumeration of the CD45+ cells from lungs of adult mice exposed to neonatal hyperoxia or room air (n = 5–7/group from three independent experiments). (B) Viral burden measure by …
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Figure 3—source data 1
Criteria for scoring lung injury on histology.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig3-data1-v1.xlsx
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Figure 3—source data 2
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig3-data2-v1.xlsx
Figure 3—figure supplement 1
Viral nucleic acid measured by qPCR after IAV infection in adult animals exposed to hyperoxia as neonates (n = 6–8/group).
Figure 4
Exposure to hyperoxia as neonates reduce the amplitude of circadian oscillations in lung explants from adult animals.
(A) Gene expression of clock genes from whole lungs harvested at 6 hr intervals determined by qPCR (n = 4–6 per time point from three different experiments). (B) Representative bioluminescence …
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig4-data1-v1.xlsx
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Figure 4—source data 2
qPCR primers used to generate data for Figure 4.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig4-data2-v1.xlsx
Figure 5 with 2 supplements
Disrupting the circadian clock in AT2 cells in adults recapitulates the phenotype seen in adult animals exposed to hyperoxia as neonates.
Experimental design: SftpcCreERt2/+::Bmal1fl/fl mice (mice lacking Bmal1 in AT2 cells of the lung epithelium) and their creneg littermates were treated with tamoxifen at 6–8 weeks of age and …
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Figure 5—source data 1
Source data for Figure 5.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Immunofluorescence staining to demonstrate the AT2-specific loss of Bmal1 in SftpcCreERt2/+::Bmal1fl/fl mice, but not in SftpcCreERt2neg::Bmal1fl/fl littermates.
Figure 5—figure supplement 2
Viral nucleic acid measured by qPCR after IAV infection of SftpcCreERt2/+::Bmal1fl/fl mice and SftpcCreERt2neg::Bmal1fl/fl littermates.
(n = 5–8/group).
Figure 6
Disrupting the circadian clock in AT2 cells in adults recapitulates the gene expression pattern and BAL expression in adult animals exposed to hyperoxia.
(A) Whole lung gene expression measured by qPCR on day 5 post-infection in adults exposed to neonatal hyperoxia or room air (left panel) and SftpcCreERt2/+::Bmal1fl/fl mice (mice lacking Bmal1 in …
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/61241/elife-61241-fig6-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Bmal1 (Rabbit monoclonal) | Abcam | ab230822 | IF(1:1000) |
| Antibody | anti-Pro-SPC (Rabbit polyclonal) | EMD Millipore | ab3786 | IF(1:2000) |
| Antibody | Nk1.1 (Mouse monoclonal) | Biolegend | 108716 | Flow cytometry (1:100) |
| Antibody | Ly6C (Mouse monoclonal) | Biolegend | 128024 | Flow cytometry (1:100) |
| Antibody | CD45 (Rat monoclonal) | Biolegend | 103114 | Flow cytometry (1:100) |
| Antibody | CD11c (Hamster monoclonal) | Biolegend | 117324 | Flow cytometry (1:100) |
| Antibody | CD11b (Mouse monoclonal) | EBiosciences | 11-0112-41, 47-0112-82 | Flow cytometry (1:100) |
| Antibody | CD3 (Rat monoclonal) | EBiosciences | 11-0032-82 | Flow cytometry (1:50) |
| Antibody | Ly6G (Rat monoclonal) | Biolegend | 127606, 127608, | Flow cytometry (1:100) |
| Antibody | Siglec F (Rat monoclonal) | BD Pharmingen | 552126 | Flow cytometry (1:100) |
| Software Algorithm | Clocklab | Actimetrics | Rest-activity analyses | |
| Other | DAPI stain | D1306 | (1 µg/mL) | |
| Chemicals strain, strain background (include species and sex here) | Tamoxifen | Sigma | T5648-1G | |
| Strain, strain background (include species and sex here) | C57BL/6J | Jax | Stock No: 000664 | B6 | Both genders and age at infection > 8 weeks |
