First person – Vincenzo Torraca

First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping researchers promote themselves alongside their papers. Vincenzo Torraca is first author on ‘ Transcriptional profiling of zebrafish identifies host factors controlling susceptibility toShigella flexneri’, published in DMM. Vincenzo conducted the research described in this article while he was a Marie Skłodowska-Curie Postdoctoral Fellow at Imperial College London, UK and an ISSF (Institutional Strategic Support Fund)-Wellcome Fellow at the London School of Hygiene and Tropical Medicine, UK, where most of the work was carried out in Prof. Serge Mostowy's lab. He has just started his own group at King's College London, investigating host-pathogen interactions and antimicrobial resistance for globally relevant bacterial pathogens, such as Shigella and E. coli, using zebrafish as an in vivo model

Chemokine Receptors and Phagocyte Biology in Zebrafish

Phagocytes are highly motile immune cells that ingest and clear microbial invaders, harmful substances, and dying cells. Their function is critically dependent on the expression of chemokine receptors, a class of G-protein-coupled receptors (GPCRs). Chemokine receptors coordinate the recruitment of phagocytes and other immune cells to sites of infection and damage, modulate inflammatory and wound healing responses, and direct cell differentiation, proliferation, and polarization. Besides, a structurally diverse group of atypical chemokine receptors (ACKRs) are unable to signal in G-protein-dependent fashion themselves but can shape chemokine gradients by fine-tuning the activity of conventional chemokine receptors. The optically transparent zebrafish embryos and larvae provide a powerful in vivo system to visualize phagocytes during development and study them as key elements of the immune response in real-time. In this review, we discuss how the zebrafish model has furthered our understanding of the role of two main classes of chemokine receptors, the CC and CXC subtypes, in phagocyte biology. We address the roles of the receptors in the migratory properties of phagocytes in zebrafish models for cancer, infectious disease, and inflammation. We illustrate how studies in zebrafish enable visualizing the contribution of chemokine receptors and ACKRs in shaping self-generated chemokine gradients of migrating cells. Taking the functional antagonism between two paralogs of the CXCR3 family as an example, we discuss how the duplication of chemokine receptor genes in zebrafish poses challenges, but also provides opportunities to study sub-functionalization or loss-of-function events. We emphasize how the zebrafish model has been instrumental to prove that the major determinant for the functional outcome of a chemokine receptor-ligand interaction is the cell-type expressing the receptor. Finally, we highlight relevant homologies and analogies between mammalian and zebrafish phagocyte function and discuss the potential of zebrafish models to further advance our understanding of chemokine receptors in innate immunity and disease

Macrophage-pathogen interactions in infectious diseases: new therapeutic insights from the zebrafish host model

Studying macrophage biology in the context of a whole living organism provides unique possibilities to understand the contribution of this extremely dynamic cell subset in the reaction to infections, and has revealed the relevance of cellular and molecular processes that are fundamental to the cell-mediated innate immune response. In particular, various recently established zebrafish infectious disease models are contributing substantially to our understanding of the mechanisms by which different pathogens interact with macrophages and evade host innate immunity. Transgenic zebrafish lines with fluorescently labeled macrophages and other leukocyte populations enable non-invasive imaging at the optically transparent early life stages. Furthermore, there is a continuously expanding availability of vital reporters for subcellular compartments and for probing activation of immune defense mechanisms. These are powerful tools to visualize the activity of phagocytic cells in real time and shed light on the intriguing paradoxical roles of these cells in both limiting infection and supporting the dissemination of intracellular pathogens. This Review will discuss how several bacterial and fungal infection models in zebrafish embryos have led to new insights into the dynamic molecular and cellular mechanisms at play when pathogens encounter host macrophages. We also describe how these insights are inspiring novel therapeutic strategies for infectious disease treatment

Frontline Science: Antagonism between regular and atypical Cxcr3 receptors regulates macrophage migration during infection and injury in zebrafish

The CXCR3-CXCL11 chemokine-signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging function, while being unable to signal in the classic G-protein-dependent manner. Two copies of the CXCR3 gene in zebrafish, cxcr3.2 and cxcr3.3, are expressed on macrophages and share a highly conserved ligand-binding site. However, Cxcr3.3 has structural characteristics of ACKRs indicative of a ligand-scavenging role. In contrast, we previously showed that Cxcr3.2 is an active CXCR3 receptor because it is required for macrophage motility and recruitment to sites of mycobacterial infection. In this study, we generated a cxcr3.3 CRISPR-mutant to functionally dissect the antagonistic interplay among the cxcr3 paralogs in the immune response. We observed that cxcr3.3 mutants are more susceptible to mycobacterial infection, whereas cxcr3.2 mutants are more resistant. Furthermore, macrophages in the cxcr3.3 mutant are more motile, show higher activation status, and are recruited more efficiently to sites of infection or injury. Our results suggest that Cxcr3.3 is an ACKR that regulates the activity of Cxcr3.2 by scavenging common ligands and that silencing the scavenging function of Cxcr3.3 results in an exacerbated Cxcr3.2 signaling. In human, splice variants of CXCR3 have antagonistic functions and CXCR3 ligands also interact with ACKRs. Therefore, in zebrafish, an analogous regulatory mechanism appears to have evolved after the cxcr3 gene duplication event, through diversification of conventional and atypical receptor variants

CXCR4 signaling regulates metastatic onset by controlling neutrophil motility and response to malignant cells

Developing tumors interact with the surrounding microenvironment. Myeloid cells exert both anti- and pro-tumor functions and chemokines are known to drive immune cell migration towards cancer cells. It is documented that CXCR4 signaling supports tumor metastasis formation in tissues where CXCL12, its cognate ligand, is abundant. On the other hand, the role of the neutrophilic CXCR4 signaling in driving cancer invasion and metastasis formation is poorly understood. Here, we use the zebrafish xenotransplantation model to study the role of CXCR4 signaling in driving the interaction between invasive human tumor cells and host neutrophils, supporting early metastasis formation. We found that zebrafish cxcr4 (cxcr4b) is highly expressed in neutrophils and experimental micrometastases fail to form in mutant larvae lacking a functional Cxcr4b. We demonstrated that Cxcr4b controls neutrophil number and motility and showed that Cxcr4b transcriptomic signature relates to motility and adhesion regulation in neutrophils in tumor-naïve larvae. Finally, Cxcr4b deficient neutrophils failed to interact with cancer cells initiating early metastatic events. In conclusion, we propose that CXCR4 signaling supports the interaction between tumor cells and host neutrophils in developing tumor metastases. Therefore, targeting CXCR4 on tumor cells and neutrophils could serve as a double bladed razor to limit cancer progression

CXCR4 signaling regulates metastatic onset by controlling neutrophil motility and response to malignant cells

Developing tumors interact with the surrounding microenvironment. Myeloid cells exert both anti- and pro-tumor functions and chemokines are known to drive immune cell migration towards cancer cells. It is documented that CXCR4 signaling supports tumor metastasis formation in tissues where CXCL12, its cognate ligand, is abundant. On the other hand, the role of the neutrophilic CXCR4 signaling in driving cancer invasion and metastasis formation is poorly understood. Here, we use the zebrafish xenotransplantation model to study the role of CXCR4 signaling in driving the interaction between invasive human tumor cells and host neutrophils, supporting early metastasis formation. We found that zebrafish cxcr4 (cxcr4b) is highly expressed in neutrophils and experimental micrometastases fail to form in mutant larvae lacking a functional Cxcr4b. We demonstrated that Cxcr4b controls neutrophil number and motility and showed that Cxcr4b transcriptomic signature relates to motility and adhesion regulation in neutrophils in tumor-naïve larvae. Finally, Cxcr4b deficient neutrophils failed to interact with cancer cells initiating early metastatic events. In conclusion, we propose that CXCR4 signaling supports the interaction between tumor cells and host neutrophils in developing tumor metastases. Therefore, targeting CXCR4 on tumor cells and neutrophils could serve as a double bladed razor to limit cancer progression

Transcriptional profiling of zebrafish identifies host factors controlling susceptibility to Shigella flexneri

Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model to study human infections in vivo. Here we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole animal level. Immune response-related processes dominate the signature of early Shigella infection (6 hours post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24 hours post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 Crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection

The chemokine receptor CXCR4 promotes granuloma formation by sustaining a mycobacteria-induced angiogenesis programme

Animal science

RNAseq Profiling of Leukocyte Populations in Zebrafish Larvae Reveals a cxcl11 Chemokine Gene as a Marker of Macrophage Polarization During Mycobacterial Infection

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model

Macrophages target Salmonella by Lc3-associated phagocytosis in a systemic infection model

Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection