High glucose levels increase influenza-associated damage to the pulmonary epithelial-endothelial barrier
- School of Chemistry and Molecular Biosciences, The University of Queensland, Australia
- School of Biomedical Sciences, The University of Queensland, Australia
- Mater Research Institute, The University of Queensland, Translational Research Institute, Australia
- Australian Infectious Diseases Research Centre, The University of Queensland, Australia
- Institute for Molecular Bioscience, The University of Queensland, Australia
Figures
Figure 1 with 2 supplements
High glucose conditions increase IAV-induced barrier independent of cell death.
(A) Schematic representation of the in vitro co-culture model of the alveolar epithelial-endothelial barrier. Image created with Biorender. Micrograph (40x magnification) of transwell membrane. (B) …
Figure 1—figure supplement 1
TEER (Ω) of epithelial-endothelial co-cultures following 3–5 days of different glucose concentrations in the lower compartment of the transwell.
All data are pooled from a minimum of three independent experiments (with 12 biological replicates per group) and are shown as mean ± SEM. Statistical analysis was performed using a Student’s …
Figure 1—figure supplement 2
Tube formation of HPMECs under different media conditions.
Cells were cultured for 5 days in different media conditions and then seeded at a density of 7.5 × 105 cells/mL on reduced growth factor basement membrane matrix. Tube formation was recorded after 3 …
Figure 2 with 1 supplement
IAV-induced barrier damage in high glucose condition is dependent upon the presence of endothelial cells.
(A) Schematic representation of the in vitro mono-culture model of the alveolar epithelial-endothelial barrier. Image created with Biorender. (B) Left: Measurement of epithelial mono-culture barrier …
Figure 2—figure supplement 1
Endothelial cells provide a stabilising effect on the integrity of the epithelial barrier.
Baseline TEER (Ω) of epithelial-endothelial co-cultures and epithelial mono-cultures following 3–4 days of media changes (prior to IAV infection). Data are pooled from a minimum of four independent …
Figure 3 with 2 supplements
IAV-induced barrier damage in infected high glucose co-cultures is associated with a pro-inflammatory response in endothelial cells.
(A) Left: Mean difference (MD) plot depicts the relationship between gene-wise average log expression and the log-fold change comparison between IAV and mock infected cells. DE = differentially …
Figure 3—figure supplement 1
Barrier damage is dependent on active virus.
Measurement of co-culture barrier integrity using TEER (Ω) readings following infection with medium (‘mock’), IAV or ultra-violet inactivated (UVI) IAV (4 × 1 J/cm²). Viral titration of the UV …
Figure 3—figure supplement 2
Barrier damage is and is not reversable via the addition of an IL-1 receptor antagonist.
Measurement of co-culture barrier integrity using TEER (Ω) readings following infection with medium (‘mock’) or IAV in the absence and presence of Anakinra. Anakinra was added to the endothelial …
Figure 4
Barrier damage under high glucose conditions was associated with destruction of the apical junctional complex.
(A–C) Left: Representative immunofluorescence images (63x magnification) of apical junction complex proteins of epithelial cells. Epithelial cells were grown on transwell membrane in co-culture with …
Figure 5
IAV-induced damage to the apical junctional complex is not observed in the absence of endothelial cells.
A–C) Left: Representative immunofluorescence images (63x magnification) of apical junction complex proteins of epithelial cells. Epithelial cells were grown on transwell membrane in mono-culture and …
Figure 6
Mice with hyperglycaemia experience more severe influenza and destruction of the apical junctional complex.
Heterozygous (leprdb/+) and homozygous (leprdb/db) mice were infected with 5.5 × 103 PFU of Auckland/09(H1N1) (n = minimum eight mice per group) or mock infected with PBS (n = minimum three mice per …
Author response image 1
Histopathology scoring of lung sections for activated caspase 3.
Data are pooled from two independent experiments, with mean ± SEM.
Author response image 2
Schematic representation of hypothesised mechanism of tight junction barrier damage under hyperglycaemic conditions in vitro.
Active replicating virus triggers signalling either directly or indirectly acting on the endothelial cells (1). Endothelial cells exposed to a history of hyperglycaemia produce a heat-labile soluble …
Tables
Table 1
Primers used in the present study.
| Gene | Sequence |
|---|---|
| GAPDH | FW: CGAGATCCCTCCAAAATCAA RV: TTCACACCCATGACGAACAT |
| TNF-α | FW: AGCCCATGTTGTAGCAAACC RV: TGAGGTACAGGCCCTCTGAT |
| OAS1 | FW: AGAGACTTCCTGAAGCAGCG RV: GAGCTCCAGGGCATACTGAG |
| IL-6 | FW: CACAGACAGCCACTCACCTC RV: TTTTCTGCCAGTGCCTCTTT |
| CXCL8 | FW: GAATTGGAAAGAGGAGAGTGACAGA RV: GTCTCCACACTCTTTTGGATGCT |
| Influenza A Matrix | FW: AAGACCAATCCTGTCACCTCTGA RV: TCCTCGCTCACTGGGCA |
