Hypoxia Inducible Factor Signaling Modulates Susceptibility to Mycobacterial Infection via a Nitric Oxide Dependent Mechanism

et al. (2013) Hypoxia Inducible Factor Signaling Modulates Susceptibility to Mycobacterial Infection via a Nitric Oxide Dependent Mechanism. PLoS Pathog 9(12): e1003789. doi:10.1371/journal.ppat.1003789 Hypoxia Inducible Factor Signaling Modulates Susceptibility to Mycobacterial Infection via a Nitric Oxide Dependent Mechanism Philip M. Elks 0 1 Sabrina Brizee 0 1 Michiel van der Vaart 0 1 Sarah R. Walmsley 0 1 Fredericus J. van Eeden 0 1 Stephen A. Renshaw 0 1 Annemarie H. Meijer 0 1 David S. Schneider, Stanford University, United States of America 0 Current address: Department of Infection and Immunity, The University of Sheffield Medical School , Sheffield , United Kingdom 1 1 Institute of Biology, Leiden University , Leiden , The Netherlands , 2 Academic Unit of Respiratory Medicine, Department of Infection and Immunity, University of Sheffield , Western Bank, Sheffield , United Kingdom , 3 Medical Research Council Centre for Developmental and Biomedical Genetics, University of Sheffield , Western Bank, Sheffield , United Kingdom , 4 Department of Biomedical Science, University of Sheffield , Western Bank, Sheffield , United Kingdom Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor a (HIF-a) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1a prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-a signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1a, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1a signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2a signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1a and Hif-2a have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1a stabilization, and Hif-2a reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1a, or reducing Hif-2a, aids the host during early stages of Mm infection. Stabilization of Hif-1a therefore represents a potential target for therapeutic intervention against tuberculosis. - Funding: PME is the recipient of a European Respiratory Society Fellowship (LTRF fellowship nu176-2011) (http://www.ersnet.org/). SAR is an MRC Senior Clinical Fellowship (G0701932) (http://www.mrc.ac.uk/). AHM is funded by the Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Ministry of Education, Culture and Science (http://www.agentschapnl.nl/subsidies-regelingen/smart-mix). AHM and PME are further supported by the European Commission 7th framework project ZF-HEALTH (HEALTH-F4-2010-242048) (http://zf-health.org/), and AHM and SAR by the European Marie-Curie Initial Training Network FishForPharma (PITN-GA-2011-289209) (http://www.fishforpharma.com/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Pulmonary tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem and is a key priority for infectious disease research. The burden of TB has been exacerbated by the increasing occurrence of Mtb strains with resistance to multiple drug treatments, prioritising the need for understanding of the mechanistic basis of host-pathogen interactions during pathogenesis of disease in order to identify novel therapeutic strategies [1]. Upon infection Mtb are rapidly phagocytosed by host leukocytes, but are able to evade bacterial killing mechanisms and utilize the leukocytes as a niche in which to proliferate and disseminate [2]. Leukocyte infection initiates the recruitment of uninfected macrophages, neutrophils and T-cells, to form highly organised structures known as granulomas [3,4]. Mtb within granulomas can persist for many years and may eventually escape and disseminate during clinical reactivation, causing active disease [5]. The pathogenesis of both initial infection and reactivation of latent infection are not well understood, and further research into host signaling pathways at these stages may uncover novel, host-derived targets for therapeutic intervention against Mtb. Mycobacterial disease and hypoxia are intimately related. Human tuberculous granulomas are hypoxic environments, and it has been suggested that the relative hypoxia of granulomas contributes to the latent infection phenotype and the associated Tuberculosis is a mycobacterial disease that was a major cause of death until the discovery of antibiotics in the midtwentieth century. However, TB is once again on the rise, with the emergence of strains that are multi-drug resistant. Mycobacteria are specialists in evading immune cell killing and use host immune cells as a niche in which they can proliferate and survive latently, until subsequent reactivation and spreading causing life-threatening disease. Pharmaceutical reprogramming of the immune system to kill intracellular mycobacteria would represent a therapeutic strategy, effective against currently untreatable strains and less susceptible to drug resistance. Here we use an in vivo zebrafish model of TB to show that manipulation of the host genetic pathway responsible for detecting low oxygen levels (hypoxia) causes a decrease in mycobacterial infection. This antimicrobial effect was due to a priming of immune cells with increased levels of nitric oxide, a molecule that is used by immune cells to kill bacteria. Here we show in vivo manipulation of a host-signaling pathway aids the host in combatting mycobacteria infection, identifying hypoxic signaling as a potential target for future therapeutics against TB. relative resistance of Mtb to host and pharmacological killing [6,7]. Hypoxia exerts its effects on host cell signaling predominantly through stabilizat (...truncated)