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

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

Quantitative Assessment Of The Health Risk For Livestock When Animal Viruses Are Applied in Human Oncolytic Therapy: A Case Study for Seneca Valley Virus

Some viruses cause tumor regression and can be used to treat cancer patients; these viruses are called oncolytic viruses. To assess whether oncolytic viruses from animal origin excreted by patients pose a health risk for livestock, a quantitative risk assessment (QRA) was performed to estimate the risk for the Dutch pig industry after environmental release of Seneca Valley virus (SVV). The QRA assumed SVV excretion in stool by one cancer patient on Day 1 in the Netherlands, discharge of SVV with treated wastewater into the river Meuse, downstream intake of river water for drinking water production, and consumption of this drinking water by pigs. Dose–response curves for SVV infection and clinical disease in pigs were constructed from experimental data. In the worst scenario (four log10 virus reduction by drinking water treatment and a farm with 10,000 pigs), the infection risk is less than 1% with 95% certainty. The risk of clinical disease is almost seven orders of magnitude lower. Risks may increase proportionally with the numbers of treated patients and days of virus excretion. These data indicate that application of wild‐type oncolytic animal viruses may infect susceptible livestock. A QRA regarding the use of oncolytic animal virus is, therefore, highly recommended. For this, data on excretion by patients, and dose–response parameters for infection and clinical disease in livestock, should be studied

Systematic approach towards establishing a National Inventory of Dangerous Pathogens.

International regulations stipulate that countries need to organize their biosafety and biosecurity systems to minimize the risk of accidental (biosafety) or malicious intentional (biosecurity) release of dangerous pathogens. International Health Regulations (IHR) benchmarks from the WHO state that even for a level of limited capacity countries need to ‘Identify and document human and animal health facilities that store/maintain dangerous pathogens and toxins in the relevant sectors and health professionals responsible for them’. This study provides a stepwise, systematic approach and best practices for countries to initiate a national inventory of dangerous pathogens. With a national inventory of dangerous pathogens a country can identify and document information in a dedicated electronic database on institutes that store or maintain dangerous pathogens. The systematic approach for the implementation of a national inventory of dangerous pathogens consists of four stages; identification, preparation, implementation, and maintenance and evaluation. In the identification phase, commitment of the relevant national ministries is to be established, and a responsible government entity needs to be identified. In the preparatory phase, a list of pathogens to be incorporated in the inventory, as well as a list of institutes to include, is to be agreed upon. In the implementation phase, the institutes are contacted, and the collected data is stored safely and securely in a electronical database. Finally, in the maintenance and evaluation phase meaningful insights are derived and reported to the relevant government authorities. Also, preparations for updates and modifications are undertaken, such as modifications of pathogen lists or institute lists. The approach and database, which is available from the authors, have been tested for the implementation of a national inventory of dangerous pathogens in multiple East-African countries. A national inventory of dangerous pathogens helps countries in strengthening national biosafety and biosecurity as well as in their compliance to IHR

<i>phd3</i> is expressed in infected macrophages during early stage Mm pathogenesis in a Hif-1α dependent manner.

<p>(A) Fluorescent confocal micrographs of 2 examples of infected areas prior to granuloma formation of 1 dpi embryos (upper and lower panels). <i>phd3</i> expression was detected by GFP levels, in green, using the <i>Tg(phd3:GFP)i144</i> transgenic line. Mm mCherry is shown in the red channel. Increased levels of <i>phd3</i>:GFP expression were detectable in cells associated with infection. (B) Fluorescent confocal micrographs of 2 granulomas at 6 dpi (upper and lower panels). Only low levels of <i>phd3</i>:GFP expression were detectable in areas of infection. The low level of GFP is illustrated in the upper panel where the auto-fluorescence of a pigment cell (white arrowhead) is brighter than the phd3:GFP expression. (C) Fluorescent confocal micrographs of 2 embryos with Mm infected macrophages at 1 dpi. The <i>phd3</i>:GFP line was outcrossed to the <i>mpeg1</i>:mCherry line to show co-localization with infected macrophages. (D) <i>phd3</i>:GFP embryos were injected at the 1 cell stage with dominant negative <i>hif-1αb</i> RNA (DN1) or phenol red (PR) as a negative control. 60 embryos of each were screened for <i>phd3</i>:GFP expression using confocal microscopy and the 3 brightest areas of <i>phd3</i>:GFP expression were imaged and showed co-localization with Mm infection. In the DN1 group GFP laser levels and confocal settings were increased until background green fluorescence was visible showing no specific co-localisation with Mm.</p

Dominant negative Hif-2α decreases bacterial burden via an iNOS dependent mechanism.

<p>(A) Example fluorescence confocal z-stacks of the caudal vein region of embryos stained with anti-nitrotyrosine antibody, imaged at 1 dpi (2 dpf), in the absence and presence of Mm infection. Embryos were injected with dominant negative <i>hif-2αa</i> (DN2), dominant active <i>hif-2αa</i> (DA2), or phenol red (PR). (B) Corrected fluorescence intensity levels of anti-nitrotyrosine antibody confocal z-stacks in uninfected larvae. Dominant negative <i>hif-2αa</i> (DN2) had significantly increased anti-nitrotyrosine levels in the absence of Mm bacterial challenge compared to phenol red (PR) injected controls. Data shown are mean ± SEM, n = 42–92 cells accumulated from 5 embryos. Graph shown is a representative dataset of 3 independent experiments. (C) Corrected fluorescence intensity levels of anti-nitrotyrosine antibody confocal z-stacks of dominant active <i>hif-2αa</i> (DA2), or phenol red (PR) control injected embryos in the presence or absence of Mm infection at 1 dpi (2 dpf). Data shown are mean ± SEM, n = 46–92 cells accumulated from 5 embryos. Graph shown is a representative dataset of 3 independent experiments. (D) Bacterial burden at 4 dpi after injection of dominant negative <i>hif-2αa</i> (DN2) or phenol red control (PR) and treatment with the pan-NOS inhibitor L-NAME. Data shown are mean ± SEM, n = 67–85 as accumulated from 3 independent experiments. (E) Bacterial burden at 4 dpi after injection of dominant negative <i>hif-2αa</i> (DN2) and treatment with the iNOS inhibitor L-NIL. Data shown are mean ± SEM, n = 52–58 as accumulated from 3 independent experiments. (F) Bacterial burden at 4 dpi after co-injection of dominant negative <i>hif-2αa</i> (DN2) and the <i>nos2a</i> morpholino, using the standard control (SC) morpholino as a negative control. Data shown are mean ± SEM, n = 103–109 as accumulated from 4 independent experiments. (G) Schematic of the effect of early Hif-α modulation during early Mm infection.</p

Hif-1α mediated anti-mycobacterial effect is iNOS dependent.

<p>(A) Fluorescent confocal micrographs of DAF-FM DA stained embryos at 2 dpf in the absence of infection. Neutrophils are identified by <i>lyz</i>:dsRed expression. DAF-FM DA staining between the timepoint of infection and 1 dpi produced varying levels of background and stained cells of the central nervous system (neurons and notochord) as well as having leukocyte-associated staining. The line of staining at the top of each image is DAF-FM DA staining in the notochord. Punctae of DAF-FM DA show upregulation of NO signaling Dominant active <i>hif-1αb</i> (DA1) embryos had more punctae than phenol red (PR) controls. <i>nos2a</i> morpholino (NOS2MO) reduced the punctae in both the PR and DA1 background. (B) Fluorescent confocal micrographs of DAF-FM DA stained embryos at 2 dpf in the presence of Mm infection. In phenol red (PR) controls DAF-FM DA punctae are increased. DAF-FM DA staining is not specific for iNOS (it is a pan-NOS probe), and the <i>nos2a</i> morpholino (NOS2MO) was not able to downregulate all of the DAF-FM DA staining after Mm infection, although punctae number were reduced. The number of punctae was also reduced in the dominant active <i>hif-1αb</i> (DA1) injected embryos after Mm infection. Dominant negative <i>hif-1αb</i> (DN1) caused no change in punctae in the presence of Mm infection compared to PR controls. (C) Fluorescent confocal micrographs of iNOS antibody staining in mpx:GFP embryos. Phenol red (PR) injected controls had very low levels of anti-iNOS antibody staining. Dominant active <i>hif-1αb</i> (DA1) had increased levels of anti-iNOS antibody, a stain which was mainly neutrophil specific. (D) Bacterial burden at 4 dpi after injection of DA <i>hif-1αb</i> (DA1) or phenol red control (PR) and treatment with the pan-NOS inhibitor L-NAME. Data shown are mean ± SEM, n = 62–89 as accumulated from 3 independent experiments. (E) Bacterial burden at 4 dpi after injection of DA <i>hif-1αb</i> (DA1) and treatment with the iNOS inhibitor L-NIL. Data shown are mean ± SEM, n = 60–87 as accumulated from 3 independent experiments. (F) Bacterial burden at 4 dpi after co-injection of DA <i>hif-1αb</i> and the <i>nos2a</i> morpholino, using the standard control (SCMO) morpholino as a negative control. Data shown are mean ± SEM, n = 109–116 as accumulated from 4 independent experiments.</p

Neutrophil-specific stabilization of Hif-1α causes increased neutrophil nitrosylation.

<p>(A) Confocal photomicrographs of expression of <i>lyz</i> driven DA <i>hif-1αb</i> and <i>IRES- nlseGFP</i> (<i>lyz:da-hif-1α</i>) in lyz:dsRed embryos stained with anti-nitrotyrosine antibody at 2 dpf. Cells with nuclear localized eGFP colocalized with lyz:dsRed expression showing neutrophil specificity of the transgenic construct. Mosaic labeled neutrophils (white arrowheads) had a higher level of anti nitrotyrosine signal compared to GFP negative ones (black arrowheads). (B) Confocal photomicrographs of a <i>Tg(lyz:da-hif-1αb:IRES-nlsegfp)</i> (<i>lyz:da-hif-1α</i>) injected ABTL embryo at 2 dpf. TSA staining of endogenous myeloperoxidase was used to stain neutrophils. Due to the nature of the myeloperoxidase staining, the whole cell is not marked, so the cell boundaries have been traced (dotted line) using brightfield z-stacks. The upper panel shows an example of a <i>lyz:da-hif-1α</i> negative neutrophil with low levels of anti-nitrotyrosine. The lower panels show two examples of <i>lyz:da-hif-1α</i> positive neutrophils in the same embryo, exhibiting a greater level of anti-nitrotyrosine compared to the negative neutrophils. (C) Corrected fluorescence intensity levels of anti-nitrotyrosine antibody confocal z-stacks of GFP negative or positive neutrophils in embryos transiently expressing <i>Tg(lyz:da-hif-1αb:IRES-nlsegfp)</i>. Embryos were imaged at 2 dpf. For each GFP positive neutrophil observed, a neighboring GFP negative neutrophil was also imaged. Data shown are mean ± SEM, n = 20 cells per group accumulated from 13 embryos from 4 independent experiments. P values were calculated using a paired T-test.</p

Hif-2α has opposing effects on bacterial burden than Hif-1α.

<p>(A) Bacterial pixel counts of dominant active <i>hif-2αa</i> (DA2) bacterial burden levels in 4 dpi infected embryos compared to phenol red (PR) and dominant active <i>hif-1αb</i> (DA1) injected controls. Data shown are mean ± SEM, n = 52–79 as accumulated from 3 independent experiments. (B) Example fluorescence micrographs of the data shown in (A). (C) Bacterial pixel counts of dominant negative <i>hif-2αa</i> (DN2) bacterial burden levels in 4 dpi infected embryos compared to phenol red (PR) and dominant active <i>hif-1αb</i> (DA1) injected controls. Data shown are mean ± SEM, n = 74–82 performed as 3 independent experiments. (D) Example fluorescence micrographs of the data shown in (C). (E) L-plastin (macrophages and neutrophils) and TSA (neutrophils only) wholebody counts at 30 hpf after injection of dominant active (DA2) and dominant negative (DN2) <i>hif-2αa</i> RNA. No significant difference was observed between groups. Data shown are mean ± SEM, n = 77–82 as accumulated from 3 independent experiments. (F) L-plastin and TSA wholebody counts at 120 hpf after injection of dominant active (DA2) and dominant negative (DN2) <i>hif-2αa</i> RNA. Data shown are mean ± SEM, n = 87–90 as accumulated from 3 independent experiments.</p