RNA-Seq of Single Fish Cells - Seeking Out the Leukocytes Mediating Immunity in Teleost Fishes.

The immune system is a complex and sophisticated biological system, spanning multiple levels of complexity, from the molecular level to that of tissue. Our current understanding of its function and complexity, of the heterogeneity of leukocytes, is a result of decades of concentrated efforts to delineate cellular markers using conventional methods of antibody screening and antigen identification. In mammalian models, this led to in-depth understanding of individual leukocyte subsets, their phenotypes, and their roles in health and disease. The field was further propelled forward by the development of single-cell (sc) RNA-seq technologies, offering an even broader and more integrated view of how cells work together to generate a particular response. Consequently, the adoption of scRNA-seq revealed the unexpected plasticity and heterogeneity of leukocyte populations and shifted several long-standing paradigms of immunology. This review article highlights the unprecedented opportunities offered by scRNA-seq technology to unveil the individual contributions of leukocyte subsets and their crosstalk in generating the overall immune responses in bony fishes. Single-cell transcriptomics allow identifying unseen relationships, and formulating novel hypotheses tailored for teleost species, without the need to rely on the limited number of fish-specific antibodies and pre-selected markers. Several recent studies on single-cell transcriptomes of fish have already identified previously unnoticed expression signatures and provided astonishing insights into the diversity of teleost leukocytes and the evolution of vertebrate immunity. Without a doubt, scRNA-seq in tandem with bioinformatics tools and state-of-the-art methods, will facilitate studying the teleost immune system by not only defining key markers, but also teaching us about lymphoid tissue organization, development/differentiation, cell-cell interactions, antigen receptor repertoires, states of health and disease, all across time and space in fishes. These advances will invite more researchers to develop the tools necessary to explore the immunology of fishes, which remain non-conventional animal models from which we have much to learn.

Evidence for a Novel Antiviral Mechanism of Teleost Fish: Serum-Derived Exosomes Inhibit Virus Replication through Incorporating Mx1 Protein.

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.