Abstract
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 Pseudomonas (P.) aeruginosa. 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 P. aeruginosa-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.
Highlights
While neutrophils (PMN) are essential for the effective clearance of microbial pathogens [1], they are functionally dependent on energetically intensive processes requiring ATP [2]–[][4]
Disease response after siRNAHIF-1a treatment When compared to the normal course of disease in BALB/c mice, knockdown of Hypoxia-inducible factor (HIF)-1a converted the characteristic resistant response to susceptible
Silencing of HIF-1a was assessed at both mRNA (Fig. 1D) and protein levels (Fig. 1E), and both analyses confirmed efficient gene knockdown
Summary
While neutrophils (PMN) are essential for the effective clearance of microbial pathogens [1], they are functionally dependent on energetically intensive processes requiring ATP [2]–[][4]. It is essential that immune cells remain effective at cell migration, pro-inflammatory gene expression, phagocytosis and bacterial killing under such hypoxic conditions, which can be as low as ,1% oxygen [10], [11]. The ability to remain functionally and metabolically competent under low O2 conditions is regulated by hypoxia-inducible factors (HIFs), which activate a hypoxic mode of adaptation [14]. In regards to PMN it has been shown previously by Cramer et al [13] that ATP levels were decreased ,40% in HIF-1a null cells. These results indicate that HIF-1a is required for maintenance of intracellular energy homeostasis in PMN, the extent to which this cell type is functionally influenced by HIF-1a has yet to be examined.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
