Abstract
Recent evidence established a crucial role for mammalian oxygen sensing transcription factor hypoxia inducible factor-1 (HIF-1) in innate immunity against intracellular pathogens. In response to most of these pathogens host phagocytes increase transcription of HIF-1α, the regulatory component of HIF-1 to express various effector molecules against invaders. Leishmania donovani (LD), a protozoan parasite and the causative agent of fatal visceral leishmaniasis resides in macrophages within mammalian host. The mechanism of HIF-1 activation or its role in determining the fate of LD in infected macrophages is still not known. To determine that J774 macrophages were infected with LD and about four-fold increase in HIF-1 activity and HIF-1α expression were detected. A strong increase in HIF-1α expression and nuclear localization was also detected in LD-infected J774 cells, peritoneal macrophages and spleen derived macrophages of LD-infected BALB/c mice. A two-fold increase in HIF-1α mRNA was detected in LD-infected macrophages suggesting involvement of a transcriptional mechanism that was confirmed by promoter activity. We further revealed that LD also induced HIF-1α expression by depleting host cellular iron pool to affect prolyl hydroxylase activity resulting in to stabilization of HIF-1α. To determine the role of HIF-1 on intracellular LD, cells were transfected with HIF-1α siRNA to attenuate its expression and then infected with LD. Although, initial infection rate of LD in HIF-1α attenuated cells was not affected but intracellular growth of LD was significantly inhibited; while, over-expression of stabilized form of HIF-1α promoted intracellular growth of LD in host macrophage. Our results strongly suggest that LD activates HIF-1 by dual mechanism for its survival advantage within macrophage.
Highlights
The oxygen sensing transcription factor hypoxia-inducible factor-1 (HIF-1) is a heterodimer of regulatory subunit HIF-1a and constitutive HIF-1b [1]
Activation of HIF-1 in Macrophages by Leishmania donovani (LD) Infection To determine the influence of LD infection on HIF-1 activation, J774 macrophages were transfected with an active hypoxia response elements (HREs) driven luciferase construct (CpHRE) [22] and infected with virulent LD (MOI-1:10)
To further detect the effect of LD-infection on nuclear localization of HIF-1a of the host macrophages, indirect immunofluorescence assay was performed in J774 and peritoneal macrophages isolated from BALB/c mice (Fig. 2A & 2B)
Summary
The oxygen sensing transcription factor hypoxia-inducible factor-1 (HIF-1) is a heterodimer of regulatory subunit HIF-1a and constitutive HIF-1b [1]. In oxygen deficiency or cellular iron depletion, expression of HIF-1a is regulated by a post-translational protein stability mechanism mediated by a family of prolyl hydroxylases (PHDs) [2]. HIF-1a subunit has a very short half-life (,2 min) because it is targeted by an oxygen-dependent mechanism to the proteasome by the von Hippel-Lindau (VHL) E3 ubiquitin ligation [3]. PHDs hydroxylate HIF-1a using oxygen and 2-oxoglutarate as substrates and iron and ascorbate as essential cofactors [6,7]. HIF-1 activation was reported as a general phenomenon in infections with human pathogens [12]. HIF-1 expression is upregulated through pathways involving key immune response regulator NFkB [10]. The basal expression of HIF-1a is regulated by NF-kB [15] and this evolutionary conserved link between NF-kB and HIF-1 provides a strong innate immunity mechanism to phagocytes against invading pathogens [10,15,16]
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