The oxygen-sensing transcription factor hypoxia-inducible factor-1α (HIF-1α) 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-1α, and selective genetic manipulation of zebrafish HIF-1α homologs, we sought to determine the roles of HIF-1α in inflammation resolution. Both approaches reveal that activated Hif-1α 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. Site-directed mutagenesis of Hif in vivo reveals that hydroxylation of Hif-1α by prolyl hydroxylases critically regulates the Hif pathway in zebrafish neutrophils. Our data demonstrate that Hif-1α regulates neutrophil function in complex ways during inflammation resolution in vivo.

The transcription factors RUNX2 and OSX have been shown to act sequentially to direct mammalian osteoblast differentiation. RUNX2 is required during the early stages of commitment and acts in part to activateOsxtranscription. OSX and RUNX2 then act to direct transcription of bone matrix proteins. Here, we investigate the expression of these genes and others during zebrafish osteoblastogenesis. Using whole‐mount in situ hybridization, we find that, during the formation of a given bone, the zebrafish homologues of mouseRunx2(runx2aandrunx2b) are typically expressed before the onset ofosx.osxexpression is usually followed by up‐regulation of the bone matrix proteins,col1a2andosteonectin. These results suggest that the mammalian pathway is conserved during development of the head and shoulder skeleton of zebrafish. We also analyze the expression of three atypical bone markers (tcf7,cvl2, andcol10a1) in an effort to place them within this canonical hierarchy. Developmental Dynamics 238:459–466, 2009.

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.

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. Tuberculosis is a mycobacterial disease that was a major cause of death until the discovery of antibiotics in the mid-twentieth 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 re-activation 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.

The inappropriate retention of neutrophils at inflammatory sites is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS, and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well-studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signaling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study neutrophilic inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridization and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signaling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signaling axis accelerated resolution of the neutrophil component of inflammation, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signaling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.