Research Article: Intracellular Pathogen Leishmania donovani Activates Hypoxia Inducible Factor-1 by Dual Mechanism for Survival Advantage within Macrophage

Date Published: June 12, 2012

Publisher: Public Library of Science

Author(s): Amit Kumar Singh, Chaitali Mukhopadhyay, Sudipta Biswas, Vandana Kumari Singh, Chinmay K. Mukhopadhyay, Ira Blader.


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.

Partial Text

The oxygen sensing transcription factor hypoxia-inducible factor-1 (HIF-1) is a heterodimer of regulatory subunit HIF-1α and constitutive HIF-1β [1]. In oxygen deficiency or cellular iron depletion, expression of HIF-1α is regulated by a post-translational protein stability mechanism mediated by a family of prolyl hydroxylases (PHDs) [2]. HIF-1α 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]. The recognition of HIF-1α by VHL depends on hydroxylation of two proline residues (pro402 and pro564) by three HIF-1α prolyl hydroxylases (PHD1-3) but PHD2 was found as the primary isoform responsible for this hydroxylation mechanism [4]–[6]. In general, PHDs hydroxylate HIF-1α using oxygen and 2-oxoglutarate as substrates and iron and ascorbate as essential cofactors [6], [7]. Upon exposure to hypoxia or iron depletion PHD activity is affected resulting into stabilization of HIF-1α, which in turn translocates to the nucleus and forms a dimer with HIF-1β to activate HIF-1. Once activated, HIF-1 binds to the hypoxia response elements (HREs) of target genes implicated in metabolism, angiogenesis, apoptosis and cellular stress [8]. Recent evidences suggest that HIF-1 plays a novel and important role in infections and inflammatory diseases [9], [10]. HIF-1 activation was reported to be essential for bactericidal capacity of phagocytes by producing several immune effector molecules for host defense [11]. In fact, HIF-1 activation was reported as a general phenomenon in infections with human pathogens [12]. In response to pathogens, HIF-1 expression is upregulated through pathways involving key immune response regulator NFκB [10]. Lipopolysaccharide (LPS), the bacterial membrane component of gram negative bacteria activates HIF-1 in macrophages by NFκB dependent transcriptional mechanism [13], [14]. Interestingly, the basal expression of HIF-1α is also regulated by NF-κB [15] and this evolutionary conserved link between NF-κB and HIF-1 provides a strong innate immunity mechanism to phagocytes against invading pathogens [10], [15], [16].

Mechanism of HIF-1 activation and its role on the outcome of infection of protozoan parasites in macrophage is less understood so far. In this study we demonstrated that unlike most of other infective pathogens, HIF-1 activation in host macrophage is beneficial for survival of the parasite Leishmania donovani. Uniquely, this parasite activates HIF-1 by two distinct mechanisms not reported so far for any other pathogens. This implies a crucial role of HIF-1 for the benefit of this intracellular pathogen.