Editorial: In vitro cellular technologies for advancing research in anamniote vertebrate immunology

In vitro cellular technologies nowadays are indispensable tools, adding tremendous values in the advancement of compara=ve immunology research. These technologies encompass isola=on of primary cells from embryos and mature species, applica=ons of permanent/con=nuous cell lines to study innate and adap=ve immune func=ons/responses, and the development of novel imaging methods to visualize intracellular processes in immune cells. For many decades, cellular technologies have been ac=vely sought and used to respond to specific scien=fic research needs, ul=mately in the pursuit of making discoveries as well as transla=ng knowledge into prac=cal applica=ons. This Research Topic collec=on in the Fron=ers of Immunology journal focuses on teleost fish and amphibian immune systems. Through knowledge dissemina=on in both original research and cri=cal literature review approaches, this collec=on includes five ar=cles from researchers around the world who reported advances in in vitro cellular technologies that provided new insights into our understanding of anamniote vertebrate immunology.The first ar=cle was a contribu=on from Lizhu Tao and colleagues from China and Portugal. Their research focused on the regulatory role of a microRNA named miR-142a-3p in inflammatory responses in grass carp (Ctenopharyngodon idella) infected with Aeromonas hydrophila (a Gram-nega=ve bacterium). A. hydrophila causes serious inflammatory condi=ons in infected grass carp; therefore, unravelling the inflammatory processes involved is important to understand the disease progression. As part of the epigene=c programming, microRNAs bind to the target regions of specific genes, leading to the ac=va=on or suppression of a series of immunological responses. The authors iden=fied the targets of miR-142a-3p to be tnfaip2 and glut3 genes. They found that A. hydrophila infec=on caused increased miR-142a-3p expressions in infected fish =ssues. Using the permanent grass carp kidney CIK cell line, the authors demonstrated that miR-142a-3p promoted cell death. As the kidney is a primary hematopoie=c =ssue in fish, the outcome of cellular death in the kidney could result in severe and generalized pathophysiological altera=ons. The authors also used primary cultures of Kupffer cells (=ssue resident macrophages) to further explore the roles of miR-142a-3p. They revealed that miR-142a-3p down-regulated proinflammatory responses and enhanced an=-inflammatory responses in Kupffer cells. Overall, their work provides new insights into the involvement of microRNAs in general and miR-142a-3p in par=cular in inflammatory responses during bacterial infec=ons in grass carp. To this end, grass carp primary cultures and cell lines are valuable in vitro tools for genera=ng accurate and rapid immunological data that align well with in vivo findings.The second ar=cle was originated from Amro Soliman and colleagues from Canada. Their research was focused on mapping the profile and kine=cs of leukocyte biology and responses during the acute phase of cutaneous inflammatory condi=ons in goldfish (Carassius auratus). Isola=ng leukocytes from connec=ve =ssue-dense organs such as the skin of fish and amphibians is difficult and cumbersome; however, the authors successfully innovated a leukocyte isola=on method specific for fish skin -a major accomplishment to furthering their research. The study revealed that neutrophils and monocytes/macrophages cons=tuted the majority of the inflammatory leukocytes in goldfish skin. This correlated well with the pro-inflammatory gene expression profile. Furthermore, the authors performed a flow cytometry-based imaging technique to visualize the profile and kine=cs of reac=ve oxygen species and nitric oxide produc=on in isolated skin leukocytes. Overall, their research allows us to have a more defined picture of the phagocyte profile that contributes to the induc=on and resolu=on of cutaneous inflamma=on condi=ons in fish.The third ar=cle contributed by Li=ng Wu and colleagues from China and the United States explored plasma cell-like popula=on in the head kidney of Nile =lapia (Oreochromis nilo=cus). They sorted IgM + B cells from a whole head kidney leukocyte prepara=on using flow cytometry and further characterized the cell phenotype with confocal and electron microscopy and cytological staining. Furthermore, they analyzed the spectrum of gene and protein expressions and func=onal assays and confirmed that these IgM + bearing B cells in the head kidney of =lapia resembled plasma cells. Their research enriches our understanding of the role of fish B lymphocytes in the adap=ve immune system.The fourth ar=cle was a comprehensive review paper by Helmut Segner and colleagues from Switzerland, Spain, and China. They discussed in detail the current state of in vitro assays using primary and permanent/con=nuous cell lines to assess immunotoxicological effects of various chemicals in teleost fishes. The authors also iden=fied the limita=ons of in vitro cell-based assays in ecotoxicological risk assessments of fish immune func=ons. While many efforts have been made to advance the field of in vitro immunotoxicology for teleost fishes, the authors emphasized that more collabora=ve works must be done to apply the predic=ve poten=al of results generated in in vitro assays to have as an impacKul contribu=on as the in vivo systems in decision making.The fiph ar=cle contribu=on was from Yun Tian and colleagues from the United States. Their research iden=fied microRNAs and non-coding regulatory elements from the whole transcriptome of an amphibian-infec=ng ranavirus. The authors further showed using in vitro frog kidney A6 cells that some of these viral miRNAs targeted and silenced important genes that were part of the interferon-mediated an=viral immune pathways. Collec=vely, their work demonstrated the existence of an epigene=c co-evolu=on mechanism at play between a frog virus and its host's innate an=viral immune responses.In summary, this Research Topic collec=on highlights the innova=on of in vitro cellular technologies in various areas in anamniote vertebrate immunology. The five ar=cle contribu=ons clearly demonstrate that more than ever these technologies are technical catalysts that are indispensable in research to allow us to shed new lights on immunological func=ons in teleost fishes and amphibians.

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