HIF-1α Is Essential for Effective PMN Bacterial Killing, Antimicrobial Peptide Production and Apoptosis in <i>Pseudomonas aeruginosa</i> Keratitis

<div><p>Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with <i>Pseudomonas (P.) aeruginosa</i>. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in <i>P. aeruginosa</i>-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.</p></div

In vivo effects of siRNA_HIF-1α treatment.

<p>Bacterial counts (A) were detected from corneas of scrambled siRNA- and siRNA_HIF-1α-treated mice after <i>P. aeruginosa</i> ocular infection. Results showed statistically more bacteria after HIF-1α silencing at 5 days p.i. Nitrite levels (B) were significantly reduced at both 1 and 5 days p.i. after siRNA_HIF-1α treatment when compared to scrambled controls. Corneal levels of MPO (C) were significantly increased at 5 days p.i. in siRNA_HIF-1α versus scrambled siRNA mice. Data represent three individual experiments each with five mice per group per time point. *<i>P</i><0.05, ** <i>P</i><0.01.</p

Immunostaining for HIF-α expression.

<p>Positive staining was detected in the epithelium of normal, uninfected corneas of both scrambled siRNA (A)- and siRNA_HIF-1α-treated (B) mice. At 5 days p.i., increased HIF-1α expression was observed throughout the epithelium and stroma of scrambled siRNA-treated mice (D); while siRNA_HIF-1α-treatment (E) revealed considerably less staining, indicating efficient knock-down of HIF-1α expression. IgG controls (C, F) were negative for staining. Representative results from two independent experiments (three mice per group per time point) are shown. Magnification = 185×.</p

In vitro expression of antimicrobial peptides after HIF-1α inhibition and growth factor treatment.

<p>PMN were incubated +/− DMAG treatment (10 µM) for 18 hours, then treated (4 hours) with either VEGF alone (10 and 50 µg/mL), a growth factor (GF) cocktail (HGF, FGF and EGF) or a combination of VEGF+GF cocktail. After challenge with <i>P. aeruginosa</i> (2.5∶1 ratio of bacteria∶cells), antimicrobial protein levels from supernatants were determined by ELISA. mBD2 (A), mBD3 (B) and CRAMP (C) were significantly up-regulated after VEGF treatment alone and following GF cocktail+VEGF treatment. ** <i>P</i><0.01.</p

Disease response after siRNA_HIF-1α versus siRNA scrambled treatment.

<p>Clinical score (A) indicated statistically significant differences at 3 and 5 days p.i. between the two groups. Representative photographs taken by slit-lamp of <i>P. aeruginosa</i>-infected eyes at 5 days p.i. illustrated less disease (+2) in scrambled siRNA- (B) versus worsened disease (+3) in siRNA_HIF-1α- (C) treated mice. Efficacy of HIF-1α silencing was confirmed at both mRNA (D) and protein (E) levels through 5 days p.i. Representative results from one of three independent experiments are illustrated. *<i>P</i><0.05, ** <i>P</i><0.01; slit lamp magnification = 8×.</p

In vivo expression of antimicrobial peptides after HIF-1α silencing.

<p>siRNA_HIF-1α versus scrambled siRNA mice resulted in significantly decreased mRNA expression for mBD2 (A), mBD3 (C) and CRAMP (E) at 5 days p.i., while there were no differences between the two groups in normal, uninfected corneas. These data were confirmed at the protein level, indicating significant down-regulation of mBD2 (B), mBD3 (D) and CRAMP (F) at both 1 and 5 days p.i. after HIF-1α silencing compared to scrambled controls. Data represent two individual experiments with five mice per group per time point. *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

In vitro effects of HIF-1α inhibition on PMN function.

<p>Phaygocytosis (A), as measured by uptake of GFP⁺<i>P. aeruginosa</i> 19660 by PMN, was not affected by DMAG-induced HIF-1α inhibition. Confocal laser scanning images documented intracellularly located GFP⁺ bacteria (green) with no DMAG treatment (positive control) (B) and after DMAG treatment (10 µM) (C). The negative control (no bacteria, no DMAG) (D) shows no GFP⁺ bacteria associated with the PMN, but stained positive for SYTOX Orange nuclear stain only. Intracellular killing by PMN (E) was analyzed by enumerating viable CFUs in the cell lysates. Significantly more viable bacteria were detected after DMAG treatment and affects were dose-dependent. Apoptotic and necrotic cells were measured (F) after HIF-1α inhibition and indicated that DMAG treatment significantly decreased apoptosis/increased cell necrosis in a dose-dependent response compared to positive controls (bacteria only, no DMAG). Apoptotic and necrotic cells were measured (G) after HIF-1α inhibition (10 µM) using both 19660 (a cytotoxic strain) and PAO1 (an invasive strain) and revealed no significant differences between cell viability, apoptosis and necrosis between the two bacterial strains. Data represent three independent experiments. *<i>P</i><0.05, ** <i>P</i><0.01 for apoptotic cell counts – (+) control versus DMAG treated; # <i>P</i><0.01 for necrotic cell counts – (+) control versus DMAG treated. B, C, D magnification = 1,000×. NS = Not Significant.</p

Pro-inflammatory cytokine/chemokine protein expression after HIF-1α silencing.

<p>Protein levels as detected by ELISA in scrambled siRNA- and siRNA_HIF-1α-treated corneas at 1 and 5 days p.i. IL-1β (A), TNF-α (B) and CXCL2 (MIP-2) (C) levels showed no differences between groups at 1 day p.i. At 5 days p.i. however, all three molecules were significantly increased after HIF-1α silencing when compared to controls. Data represent two individual experiments each with five mice per group per time point. *<i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p

In vitro expression of antimicrobial peptides after HIF-1α inhibition.

<p>PMN were stimulated with <i>P. aeruginosa</i> +/− DMAG treatment (10, 1.0, 0.1 µM) for 18 hours, and antimicrobial protein levels from supernatants were determined by ELISA. mBD2 (A), mBD3 (B) and CRAMP (C) were significantly reduced in a dose-dependent response to DMAG treatment when compared to positive controls (bacteria only, no DMAG). *<i>P</i><0.05, ** <i>P</i><0.01.</p

Immunostaining of T cell infiltrate after siRNA_HIF-1α treatment.

<p>No positive immunostaining for CD3ε, a pan T cell marker, was detected in the corneas of scrambled siRNA-treated mice (A) at 5 days p.i. In contrast, positive immunostaining (red) was observed in the peripheral cornea of HIF-1α silenced animals (B). The control sections shown in (C) and (D) were immunostained with species-specific IgG and were positive for SYTOX Green nuclear stain only. Images shown are representative of three independent experiments each with three mice per group. Magnification = 180×; inset = 335×.</p