Oxygen-independent stabilization of hypoxia inducible factor (HIF)-1 during RSV infection

Abstract

Background: Hypoxia-inducible factor 1 (HIF)-1alpha is a transcription factor that functions as master regulator of mammalian oxygen homeostasis. In addition, recent studies identified a role for HIF-1alpha as transcriptional regulator during inflammation or infection. Based on studies showing that respiratory syncytial virus (RSV) is among the most potent biological stimuli to induce an inflammatory milieu, we hypothesized a role of HIF-1alpha as transcriptional regulator during infections with RSV.

Methodology, principal findings: We gained first insight from immunohistocemical studies of RSV-infected human pulmonary epithelia that were stained for HIF-1alpha. These studies revealed that RSV-positive cells also stained for HIF-1alpha, suggesting concomitant HIF-activation during RSV infection. Similarly, Western blot analysis confirmed an approximately 8-fold increase in HIF-1alpha protein 24 h after RSV infection. In contrast, HIF-1alpha activation was abolished utilizing UV-treated RSV. Moreover, HIF-alpha-regulated genes (VEGF, CD73, FN-1, COX-2) were induced with RSV infection of wild-type cells. In contrast, HIF-1alpha dependent gene induction was abolished in pulmonary epithelia following siRNA mediated repression of HIF-1alpha. Measurements of the partial pressure of oxygen in the supernatants of RSV infected epithelia or controls revealed no differences in oxygen content, suggesting that HIF-1alpha activation is not caused by RSV associated hypoxia. Finally, studies of RSV pneumonitis in mice confirmed HIF-alpha-activation in a murine in vivo model.

Conclusions/significance: Taking together, these studies suggest hypoxia-independent activation of HIF-1alpha during infection with RSV in vitro and in vivo.

Figure 1. HIF-1α during RSV infected of pulmonary epithelia.

(A) ∼1,5×10⁵ A549-cells were seeded on glass slides and infected with RSV (multiplicity of infection, MOI 3). After 24 h they were fixed, permeabilized and incubated with anti-HIF1α and anti-RSV IgG as primary antibodies. Alexa Fluor 488 and Alexa Fluor 594 were used for staining. In addition, slides were counter-stained with Dapi. The cells were visualized with confocal laser scanning microscopy. Uninfected cells were used as controls (B).

Figure 2. HIF-1α protein measurements during RSV infection in vitro.

(A, B) Cultured pulmonary epithelia (A549) were grown to 80% confluency, infected with intact (multiplicity of infection, MOI 1, 3 or 5) or UV-inactivated RSV (MOI 3). In other studies A549 cells were exposed over 24 h to ambient hypoxia (2% oxygen). Cells were lysed and nuclear proteins were isolated, and Western immunoblotting for RSV G-protein (A) or HIF1α was performed. Uninfected cells were used as control (Co). The same blots were probed for β-actin expression as a control for protein loading. A representative blot of 3 is shown, in addition to densitometric analysis of HIF-1α protein levels relative to β-actin (C;*P<.01, different from control, n  =  3).

Figure 3. Transcript levels of HIF-1-dependent genes following RSV infection.

Total RNA was isolated from RSV-infected (multiplicity of infection, MOI 1, 3 and 5) or non-infected A549 cells (control) and (A) CD73, (B) VEGG, (C) FN1, (D) COX2 mRNA levels were determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over uninfected control-cells ±SEM at each infection dose (*P<0.05, different from uninfected control-cells).

Figure 4. Protein levels of HIF-1-dependent genes following RSV infection.

Total protein was isolated from RSV-infected (multiplicity of infection, MOI 1, 3 and 5) or non-infected A549 cells. Protein levels were determined by Western blot. The same blots were probed for β-actin expression as a control for protein loading. In addition, densitometric analysis of protein levels relative to β-actin were performed. Data are expressed as fold increase over uninfected control-cells ±SEM at each infection dose. (A) COX2; (B) FN1 (*P<0.01, different from control, n  =  3).

Figure 5. Transcript levels of HIF-dependent genes following HIF-1α siRNA repression during RSV infection.

(A) HIF-1α protein levels in A549 cells following hairpin siRNA repression of HIF-1α (HIF-/-; A549 cells transfected with control siRNA:SCR). Cells were grown to 80% confluency and exposed to normoxia or hypoxia (2% oxygen) over indicated time period. Nuclear proteins were isolated and Western Blot analysis was performed for HIF-1α. The same blots were probed for β-actin expression as a control for protein loading. A representative blot of 3 is shown, in addition to densitometric analysis of HIF-1α protein levels relative to β-actin (B;*P<0.01, different from control, n  =  3).(C, D, E) Total RNA was isolated from RSV-infected (multiplicity of infection, MOI 3) or non-infected A549 following HIF-1α repression (A549 HIF-/-) or transfection with control siRNA (A549 scr). (C) VEGF, (D) CD73, (E) FN1 transcript levels were determined by RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over uninfected control-cells ±SEM (*P<0.05).

Figure 6. Transcript levels of HIF-dependent genes following infection with inactivated RSV.

Total RNA was isolated from uninfected, RSV-infected or UV-inactivated RSV infected A549 cells. (A) VEGF, (B) CD73, (C) FN1, (D) COX2 transcript levels were determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as fold increase over uninfected control-cells ±SEM at each infection dose (*P<0.05, different from uninfected control-cells).

Figure 7

(a) Measurements of oxygen partial pressures (pO₂) in the supernatants of RSV infected pulmonary epithelia. (A) A549 cells were cultured and infected at a multiplicity of infection (MOI) of 1 or 5 in gas-tight sealed flasks. Oxygen partial pressure was measured in the supernatants at indicated time points following infection. (B) A samples was assessed for HIF-1α protein levels by Western blot 24 h after RSV infection or control conditions. Blots were probed for β-actin expression as a control for protein loading

Figure 8. HIF-1α protein during murine RSV pneumonia in vivo.

Female, 6- to 8-week-old BALB/c mice were inoculated intranasally with purified RSV at 1×10⁷ plaque-forming units (PFUs), diluted in sterile 0.9 % sodium chloride for a total inoculation volume of 50 µl. As mock treatment, control mice were inoculated in the same way with an equivalent volume of sucrose diluted in 0.9 % sodium chloride. Lungs were removed at indicated time-points, and HIF-1α protein levels were determined by Western blot analysis (A) or quantified by densitometry, relative to beta-actin (B; *p<0.01).