A High-Throughput Screen for Tuberculosis Progression

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo

Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model.

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments

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

Beta Interferon Suppresses the Development of Experimental Cerebral Malaria▿ †

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection, particularly in children. The pathogenesis of cerebral malaria involves parasitized red blood cell (RBC)-mediated vascular inflammation, immune stimulation, loss of blood-brain barrier integrity, and obstruction of cerebral capillaries. Therefore, blunting vascular inflammation and immune cell recruitment is crucial in limiting the disease course. Beta interferon (IFN-β) has been used in the treatment of diseases, such as multiple sclerosis (MS) but has not yet been explored in the treatment of CM. Therefore, we sought to determine whether IFN-β also limits disease progression in experimental cerebral malaria (ECM). Plasmodium berghei-infected mice treated with IFN-β died later and showed increased survival, with improved blood-brain barrier function, compared to infected mice. IFN-β did not alter systemic parasitemia. However, we identified multiple action sites that were modified by IFN-β administration. P. berghei infection resulted in increased expression of chemokine (C-X-C motif) ligand 9 (CXCL9) in brain vascular endothelial cells that attract T cells to the brain, as well as increased T-cell chemokine (C-X-C motif) receptor 3 (CXCR3) expression. The infection also increased the cellular content of intercellular adhesion molecule 1 (ICAM-1), a molecule important for attachment of parasitized RBCs to the endothelial cell. In this article, we report that IFN-β treatment leads to reduction of CXCL9 and ICAM-1 in the brain, reduction of T-cell CXCR3 expression, and downregulation of serum tumor necrosis factor alpha (TNF-α). In addition, IFN-β-treated P. berghei-infected mice also had fewer brain T-cell infiltrates, further demonstrating its protective effects. Hence, IFN-β has important anti-inflammatory properties that ameliorate the severity of ECM and prolong mouse survival

HDL decreases endothelial exocytosis.

<p>(A) Dose-response. Endothelial cells were pre-treated with various concentrations of purified HDL-3 within the human serum reference range for 2 h. (B). Time course. Endothelial cells were pre-treated with 0.5 mg/dL HDL-3 for 2 h. Cells were washed and stimulated with thrombin 1 U/ml, and the amount of VWF released over 1 h was measured by an ELISA (n = 3 ± S.D. *P < 0.05 vs. thrombin and 0 HDL-3).</p

Purified apolipoproteins decrease endothelial exocytosis.

<p>(A-D) Dose-response for ApoA-I (human serum reference range is 0.75–1.75 mg/mL), and equivalent concentrations of Apo-AII, ApoC-I, and ApoE. Endothelial cells were pre-treated with various concentrations of purified apolipoproteins for 2 h. Cells were washed and stimulated with thrombin 1 U/ml, and the amount of VWF released over 1 h was measured by an ELISA (n = 3 ± S.D. *P < 0.05 vs. thrombin and 0 aplipoprotein). (E) Purified apolipoproteins are not cytotoxic to endothelial cells. Endothelial cells were pre-treated with IC_50% concentrations of purified apolipoproteins for 2 h. Hydrogen peroxide (H₂O₂) is a positive control for death. Cell viability was measured via formazan release with the MTS assay (n = 3 ± S.D. *P < 0.05 vs. control. **P < 0.01 vs. control.) The dashed line represents control levels.</p

Nitration of apoA-I attenuates apoA-I inhibition of exocytosis.

<p>(A) Purified apoA-I (10⁻⁴ mg/mL) was incubated with increasing concentrations of peroxynitrite (left) or degraded peroxynitrite as a control (right). Mixtures were then fractionated by SDS-PAGE and immunoblotted with antibody to nitrotyrosine in order to assess tyrosine nitration of apoA-I. The position of apoA-I on the gel was confirmed with Coomassie staining (not shown). This experiment was repeated three times with similar results. (B). ApoA-I was treated with increasing concentrations of peroxynitrite or degraded peroxynitrite as a control. Endothelial cells were then incubated with treated or non-treated apoA-I 10⁻⁴ mg/mL for 2 h, washed, and stimulated with thrombin (1 U/ml). The amount of VWF released over 1 h was measured by an ELISA (n = 3 ± S.D. *P < 0.05, peroxynitrite vs. degraded peroxynitrite.</p