ADA2 regulates inflammation and hematopoietic stem cell emergence via the A2bR pathway in zebrafish.

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.

Full-aperture extended-depth oblique plane microscopy through dynamic remote focusing.

Significance: The reprojection setup typical of oblique plane microscopy (OPM) limits the effective aperture of the imaging system, and therefore its efficiency and resolution. Large aperture system is only possible through the use of custom specialized optics. A full-aperture OPM made with off the shelf components would both improve the performance of the method and encourage its widespread adoption. Aim: To prove the feasibility of an OPM without a conventional reprojection setup, retaining the full aperture of the primary objective employed. Approach: A deformable lens based remote focusing setup synchronized with the rolling shutter of a complementary metal-oxide semiconductor detector is used instead of a traditional reprojection system. Results: The system was tested on microbeads, prepared slides, and zebrafish embryos. Resolution and pixel throughput were superior to conventional OPM with cropped apertures, and comparable with OPM implementations with custom made optical components. Conclusions: An easily reproducible approach to OPM imaging is presented, eliminating the conventional reprojection setup and exploiting the full aperture of the employed objective.

Studying Bacteriophage Efficacy Using a Zebrafish Model.

The rise of bacteria resistant to the antibiotics currently in use (multiple drug-resistant, MDR) is a serious problem for patients affected by infections. This situation is even more worrying in the case of chronic bacterial infections, such as those caused by Pseudomonas aeruginosa (Pa), in patients with cystic fibrosis (CF). As an alternative to antibiotic treatments, the use of bacteriophages (phages) to fight bacterial infections has gained increasing interest in the last few years. Phages are viruses that specifically infect and multiply within the bacteria without infecting eukaryotic cells. It is well assumed that phage therapy has a high bacterial specificity, which, unlike antibiotics, should limit the damage to the endogenous microbiome. In addition, phages can kill antibiotic-resistant bacteria and perform self-amplification at the site of the infection.The protocol detailed in this chapter describes how the antimicrobial effect of phages can be studied in vivo in the zebrafish (Danio rerio) model infected with Pa. The same procedure can be applied to test the effectiveness of several different phages killing other bacterial species and for the rapid preclinical testing of phages to be used as personalized medicine.

Lymphatic Defects in Zebrafish sox18 Mutants Are Exacerbated by Perturbed VEGFC Signaling, While Masked by Elevated sox7 Expression.

Mutations in the transcription factor-coding gene SOX18, the growth factor-coding gene VEGFC and its receptor-coding gene VEGFR3/FLT4 cause primary lymphedema in humans. In mammals, SOX18, together with COUP-TFII/NR2F2, activates the expression of Prox1, a master regulator in lymphatic identity and development. Knockdown studies have also suggested an involvement of Sox18, Coup-tfII/Nr2f2, and Prox1 in zebrafish lymphatic development. Mutants in the corresponding genes initially failed to recapitulate the lymphatic defects observed in morphants. In this paper, we describe a novel zebrafish sox18 mutant allele, sa12315, which behaves as a null. The formation of the lymphatic thoracic duct is affected in sox18 homozygous mutants, but defects are milder in both zygotic and maternal-zygotic sox18 mutants than in sox18 morphants. Remarkably, in sox18 mutants, the expression of the closely related sox7 gene is elevated where lymphatic precursors arise. Sox7 could thus mask the absence of a functional Sox18 protein and account for the mild lymphatic phenotype in sox18 mutants, as shown in mice. Partial knockdown of vegfc exacerbates lymphatic defects in sox18 mutants, making them visible in heterozygotes. Our data thus reinforce the genetic interaction between Sox18 and Vegfc in lymphatic development, previously suggested by knockdown studies, and highlight the ability of Sox7 to compensate for Sox18 lymphatic dysfunction.

Generation of a Triadin KnockOut Syndrome Zebrafish Model.

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named "Triadin KnockOut Syndrome" (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.

Phages as immunomodulators and their promising use as anti-inflammatory agents in a cftr loss-of-function zebrafish model.

Cystic Fibrosis (CF), one of the most frequent hereditary diseases due to mutations in the CFTR gene, causes mortality in humans mainly due to infection in the respiratory system. However, besides the massive inflammatory response triggered by chronic bacterial infections, a constitutive pro-inflammatory state associated with the most common CFTR mutations has been reported in paediatric cases before the onset of bacterial colonization. In previous works we isolated and characterized a mix of virulent bacteriophages (phage cocktail) able to efficiently counteract Pseudomonas aeruginosa infection in a zebrafish model with cftr loss-of-function (LOF), but also showing anti-inflammatory effects in zebrafish embryos not infected by bacteria. On these premises, in this work we demonstrated the anti-inflammatory role of the phage cocktail both in the wild-type (WT) and hyper-inflamed cftr LOF zebrafish embryos in terms of reduction of pro-inflammatory markers. We also dissect that only the virion proteinaceous components, but not the phage DNA, are responsible for the immune-modulatory effect and that this action is elicited through the activation of the Toll-like Receptor (TLR) pathway. In the cftr LOF zebrafish embryos, we demonstrated that phages injection significantly reduces neutrophil migration following acute inflammatory induction. The elucidation of the molecular interaction between phages and the cells of vertebrate immune system might open new possibility in their manipulation for therapeutic benefits especially in diseases such as cystic fibrosis, characterized by chronic infection and inflammation.

Animal Models to Translate Phage Therapy to Human Medicine.

Phagotherapy, the use of bacteriophages to fight bacterial infections as an alternative to antibiotic treatments, has become of increasing interest in the last years. This is mainly due to the diffusion of multi-drug resistant (MDR) bacterial infections that constitute a serious issue for public health. Phage therapy is gaining favor due to its success in agriculture and veterinary treatments and its extensive utilization for human therapeutic protocols in the Eastern world. In the last decades, some clinical trials and compassionate treatments have also been performed in the Western world, indicating that phage therapy is getting closer to its introduction in standard therapy protocols. However, several questions concerning the use of phages in human therapeutic treatments are still present and need to be addressed. In this review, we illustrate the state of art of phage therapy and examine the role of animal models to translate these treatments to humans.

Glycogen storage in a zebrafish Pompe disease model is reduced by 3-BrPA treatment.

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.