The Zebrafish Breathes New Life into the Study of Tuberculosis

Tuberculosis (TB) is a global health emergency. Up to one third of the world’s population is infected with Mycobacterium tuberculosis, and the pathogen continues to kill almost two million people annually. Currently, the means for preventing, diagnosing, and treating TB are unsatisfactory. One of the main reasons for the poor progress in TB research has been a lack of good animal models to study the latency, dormancy and reactivation of the disease. Although sophisticated in vitro and in silico methods suitable for TB research are constantly being developed, they cannot reproduce the complete vertebrate immune system and its interplay with pathogens and vaccines. However, the zebrafish has recently emerged as a useful alternative to more traditional models, such as mice, rabbits, guinea pigs, and non-human primates, for studying the complex pathophysiology of a mycobacterial infection. The model is based on the similarity between Mycobacterium marinum – a natural fish pathogen – and Mycobacterium tuberculosis. In both zebrafish larvae and adult fish, an infection with M. marinum leads to the formation of macrophage aggregates and granulomas, which resemble the M. tuberculosis infections in humans. In this review we will summarize the current status of the zebrafish model in TB research and highlight the advantages of using zebrafish to dissect mycobacterial virulence strategies as well as the host immune responses elicited against them. In addition, we will discuss the possibilities of using the adult zebrafish model for studying latency, dormancy, and reactivation in a mycobacterial infection

A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model

Background Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention. Methods Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles. Results We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD. Conclusions Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention

Tumor Necrosis Factor Induces Tumor Promoting and Anti-Tumoral Effects on Pancreatic Cancer via TNFR1

Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome

Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (<i>Danio rerio</i>)

<div><p>CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (<i>Danio rerio</i>) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity <i>in vivo</i>. Furthermore, some guide RNAs with high <i>in vitro</i> activity possessed poor mutagenesis activity <i>in vivo</i>, suggesting the presence of factors that limit the mutagenesis <i>in vivo</i>. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos.</p></div

Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio)

Abstract CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (Danio rerio) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity in vivo. Furthermore, some guide RNAs with high in vitro activity possessed poor mutagenesis activity in vivo, suggesting the presence of factors that limit the mutagenesis in vivo. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos

Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints.

<p>Correlation between mutagenesis efficiency and chromatin features at different developmental stages and timepoints.</p

Onset of mutagenesis differs between sgRNAs.

<p>Heteroduplex mobility assay to demonstrate the onset of mutagenesis using high efficiency guide RNAs targeting three different genes with different gene expression patterns in early development. Embryos were collected at timepoints 1, 2, 3, 4, 6hpf (15–20 embryos per group). The gene name above the gel image indicates CRISPR-Cas9 injected embryos and control indicates uninjected controls. The legend on the side indicates the positions of wt (wild type) and mutant bands in the gel. Red arrows indicate the point at which first mutations can be detected.</p

In vitro and in vivo CRISPR-Cas9 mutagenesis efficiencies do not correlate for all genes.

<p>a) An in vitro digestion assay shows that sgRNAs differ in their efficiencies. Below the gene name, + and - indicate the presence or absence of Cas9 protein in the reaction. On the right the wild type (wt) and the mutant products are indicated. b) The in vivo CRISPR-Cas9 mutagenesis visualized for ca6, cxcr2 and pycard with a heteroduplex mobility assay, with the wild type (wt) and the mutant products indicated. 5 embryos were collected per sample at 8hpf.</p