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

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

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response

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

Primer análisis exploratorio sensibilización sobre responsabilidad social universitaria : Desarrollo sostenible en la FCEyA – UNCa. Primer avance exploratorio

El presente trabajo se desarrolla en el marco del Proyecto de Investigación Contabilidad y Universidad dirigido por la Cra. María Beatriz Maza, con vigencia desde el 01/01/2016 al 31/12/2019. En los fundamentos epistemológicos de nuestra tarea de investigación se encuentra el hecho de que, la contabilidad es una disciplina precursora de la escritura y de la abstracción para contar. Por muchos años, la contabilidad se ha limitado a brindar información relacionada con el patrimonio de las organizaciones y sus variaciones, esto ha cambiado. Hoy, una porción importante de investigadores destacados en el mundo y en Argentina, ha comenzado a reconocer e imponer con fuerza la idea de la responsabilidad social empresaria como instrumento de sostenibilidad de los procesos de desarrollo. En tal sentido desde nuestro proyecto de investigación pretendemos ocuparnos puntualmente de la Responsabilidad Social Universitaria (RSU), como sujeto de análisis y espacio de divulgación de procesos metodológicos que colaboren con el desarrollo basado en estas categorías conceptuales.Tema 4: Especialidad, rama o segmento contable social y ambiental.Facultad de Ciencias Económica

Primer análisis exploratorio sensibilización sobre responsabilidad social universitaria : Desarrollo sostenible en la FCEyA – UNCa. Primer avance exploratorio

El presente trabajo se desarrolla en el marco del Proyecto de Investigación Contabilidad y Universidad dirigido por la Cra. María Beatriz Maza, con vigencia desde el 01/01/2016 al 31/12/2019. En los fundamentos epistemológicos de nuestra tarea de investigación se encuentra el hecho de que, la contabilidad es una disciplina precursora de la escritura y de la abstracción para contar. Por muchos años, la contabilidad se ha limitado a brindar información relacionada con el patrimonio de las organizaciones y sus variaciones, esto ha cambiado. Hoy, una porción importante de investigadores destacados en el mundo y en Argentina, ha comenzado a reconocer e imponer con fuerza la idea de la responsabilidad social empresaria como instrumento de sostenibilidad de los procesos de desarrollo. En tal sentido desde nuestro proyecto de investigación pretendemos ocuparnos puntualmente de la Responsabilidad Social Universitaria (RSU), como sujeto de análisis y espacio de divulgación de procesos metodológicos que colaboren con el desarrollo basado en estas categorías conceptuales.Tema 4: Especialidad, rama o segmento contable social y ambiental.Facultad de Ciencias Económica

Primer análisis exploratorio sensibilización sobre responsabilidad social universitaria : Desarrollo sostenible en la FCEyA – UNCa. Primer avance exploratorio

El presente trabajo se desarrolla en el marco del Proyecto de Investigación Contabilidad y Universidad dirigido por la Cra. María Beatriz Maza, con vigencia desde el 01/01/2016 al 31/12/2019. En los fundamentos epistemológicos de nuestra tarea de investigación se encuentra el hecho de que, la contabilidad es una disciplina precursora de la escritura y de la abstracción para contar. Por muchos años, la contabilidad se ha limitado a brindar información relacionada con el patrimonio de las organizaciones y sus variaciones, esto ha cambiado. Hoy, una porción importante de investigadores destacados en el mundo y en Argentina, ha comenzado a reconocer e imponer con fuerza la idea de la responsabilidad social empresaria como instrumento de sostenibilidad de los procesos de desarrollo. En tal sentido desde nuestro proyecto de investigación pretendemos ocuparnos puntualmente de la Responsabilidad Social Universitaria (RSU), como sujeto de análisis y espacio de divulgación de procesos metodológicos que colaboren con el desarrollo basado en estas categorías conceptuales.Tema 4: Especialidad, rama o segmento contable social y ambiental.Facultad de Ciencias Económica

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

[This corrects the article DOI: 10.3389/fimmu.2019.00832.]

Supplementary RNAseq dataset files

Supplementary RNAseq dataset files Supplementary Dataset 1. Supplementary Dataset 2. Supplementary Dataset 3