The oxygen-sensing transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) plays a critical role in the regulation of myeloid cell function. The mechanisms of regulation are not well understood, nor are the phenotypic consequences of HIF modulation in the context of neutrophilic inflammation. Species conservation across higher metazoans enables the use of the genetically tractable and transparent zebrafish (Danio rerio) embryo to study in vivo resolution of the inflammatory response. Using both a pharmacologic approach known to lead to stabilization of HIF-1␣, and selective genetic manipulation of zebrafish HIF-1␣ homologs, we sought to determine the roles of HIF-1␣ in inflammation resolution. Both approaches reveal that activated Hif-1␣ delays resolution of inflammation after tail transection in zebrafish larvae. This delay can be replicated by neutrophil-specific Hif activation and is a consequence of both reduced neutrophil apoptosis and increased retention of neutrophils at the site of tissue injury. Hif-activated neutrophils continue to patrol the injury site during the resolution phase, when neutrophils would normally migrate away. Sitedirected mutagenesis of Hif in vivo reveals that hydroxylation of Hif-1␣ by prolyl hydroxylases critically regulates the Hif pathway in zebrafish neutrophils. Our data demonstrate that Hif-1␣ regulates neutrophil function in complex ways during inflammation resolution in vivo. (Blood. 2011;118(3):712-722) IntroductionNeutrophilic inflammation is of fundamental importance in the innate immune response to bacterial and fungal infection in vertebrates, and it can be initiated by sterile tissue injury. Irrespective of its etiology, inflammation must resolve in a timely manner to avoid damage to surrounding tissues. 1 Persisting, noninfectious inflammation is the hallmark of inflammatory diseases, a major cause of morbidity and mortality in the developed world. The resolution phase of inflammation is critical to the restoration of normal tissue function after an inflammatory response, and thus has a central role in determining the outcome of inflammation. 2 Despite the central place of failed resolution in the pathogenesis of inflammatory disease, much remains to be known about the cellular and molecular events involved.Although neutrophil apoptosis, and subsequent uptake and removal by macrophages (efferocytosis), is well documented as a disposal route for inflammatory neutrophils, [3][4][5] there is emerging evidence that other mechanisms also may contribute to certain types of inflammation resolution. In the lung, some neutrophils are lost into the airways and expectorated, 6 and in rheumatoid arthritis, neutrophils may leave the inflammatory site while still alive and re-enter the circulation. 7 Neutrophils also can be removed by migration through tissues away from the infection site; a process termed retrograde chemotaxis, or reverse migration. [8][9][10] This process is most widely characterized in the zebrafish (Danio rerio) embryo and is less well studied in mammalian s...

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 α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α 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-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α 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-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic intervention against tuberculosis.

Key Points Neutrophil lifespan is extended in patients with gain-of-function HIF2A mutations. HIF-2α regulates in vivo neutrophil longevity and thus tissue inflammation and repair.