REVIEW published: 25 February 2021 doi: 10.3389/fimmu.2021.642687 Reflections on the Use of an Invertebrate Chordate Model System for Studies of Gut Edited by: Antonio Figueras, Microbial Immune Interactions Consejo Superior de Investigaciones 1 2,3 3,4 1† Cient´ıficas (CSIC), Spain Assunta Liberti *, Ojas Natarajan , Celine Grace F. Atkinson , Paolo Sordino 2,3* Reviewed by: and Larry J. Dishaw Loriano Ballarin, 1 Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Naples, Italy, 2 Morsani College of University of Padua, Italy Medicine, Department of Pediatrics, University of South Florida, Tampa, FL, United States, 3 Division of Molecular Genetics, M. Carla Piazzon, Children’s Research Institute, St. Petersburg, FL, United States, 4 Department of Cell Biology, Microbiology, and Molecular Torre de la Sal Aquaculture Institute Biology, University of South Florida, Tampa, FL, United States (IATS), Spain *Correspondence: Assunta Liberti The functional ecology of the gastrointestinal tract impacts host physiology, and its [email protected]; dysregulation is at the center of various diseases. The immune system, and specifically [email protected] Larry J. Dishaw innate immunity, plays a fundamental role in modulating the interface of host and microbes [email protected] in the gut. While humans remain a primary focus of research in this field, the use of diverse †Present address: model systems help inform us of the fundamental principles legislating homeostasis in the Paolo Sordino, fi Biology and Evolution of Marine gut. Invertebrates, which lack vertebrate-style adaptive immunity, can help de ne Organisms (BEOM), Stazione conserved features of innate immunity that shape the gut ecosystem. In this context, Zoologica Anton Dohrn, Sicily Marine we previously proposed the use of a marine invertebrate, the protochordate Ciona Centre, Messina, Italy robusta, as a novel tractable model system for studies of host-microbiome interactions. Specialty section: Significant progress, reviewed herein, has been made to fulfill that vision. We examine and This article was submitted to review discoveries from Ciona that include roles for a secreted immune effector interacting Comparative Immunology, a section of the journal with elements of the microbiota, as well as chitin-rich mucus lining the gut epithelium, the Frontiers in Immunology gut-associated microbiome of adults, and the establishment of a large catalog of cultured Received: 16 December 2020 isolates with which juveniles can be colonized. Also discussed is the establishment of Accepted: 20 January 2021 methods to rear the animals germ-free, an essential technology for dissecting the Published: 25 February 2021 Citation: symbiotic interactions at play. As the foundation is now set to extend these studies into Liberti A, Natarajan O, Atkinson CGF, the future, broadening our comprehension of how host effectors shape the ecology of Sordino P and Dishaw LJ (2021) these microbial communities in ways that establish and maintain homeostasis will require fl Re ections on the Use of an “ ” Invertebrate Chordate Model full utilization of multi-omics approaches to merge computational sciences, modeling, System for Studies of Gut and experimental biology in hypothesis-driven investigations. Microbial Immune Interactions. Front. Immunol. 12:642687. Keywords: Ciona robusta, Ciona intestinalis type A, invertebrate model, mucosal immunity, innate immunity, doi: 10.3389/fimmu.2021.642687 gut-microbial interactions, gut microbiota, multi-omics approaches Frontiers in Immunology | www.frontiersin.org 1 February 2021 | Volume 12 | Article 642687 Liberti et al. Ciona Model for Host-Microbiota Studies INTRODUCTION catalog of cultured isolates from which cultured juveniles can be colonized for experimental manipulation and study. And The gut environment includes a dynamic community of finally, we will discuss the establishment of methods to rear microorganisms, consisting mainly of bacteria, but also Archaea, short-term germ-free animals, an essential requirement for the viruses, fungi, protozoans, and occasionally, helminthic worms. In the interrogation of each member of the symbiotic dialogue existing past few decades, the beneficial effects of these microbes on animal within the gut microbiome. As the foundation is now set, future health have become widely recognized, influencing host physiology at studies can be extended further to include large-scale analyses of different levels, i.e., immune system and gut, as well as other organs, host–microbe interactions. Such studies will require the use of development, and metabolic and neurobehavioral functions (1–4). “multi-omics” approaches, thus merging computational sciences, Dysregulation of homeostasis, in terms of both microbial modeling, and experimental biology in hypothesis-driven composition and host capability to regulate interactions with investigations, broadening our comprehension of how host microbes, is most often correlated with intestinal pathologies (5–9). effectors shape the ecology of these microbial communities in The immune system, and more specifically innate immunity, ways that establish and maintain homeostasis, while this plays a fundamental role in modulating the interface of host and equilibrium is challenged by diverse environmental stressors. microorganisms in the gut. It is equally true that microorganisms shape host immunity (10–12) and that host immunity shapes gut microbial communities (13, 14). The innate immune system may have evolved not only for defense but also driven by a necessity to COMPONENTS OF CIONA GUT recognize and tolerate complex communities of beneficial ENVIRONMENT microbes, representing a form of ecosystem management that modulates their composition, diversity, and localization (2, 15, Host defense systems serve bifunctional roles in protecting host 16). To decipher the relationships between microbes and host tissues from pathogenic infection while also supporting the physiology and/or diseases, this field of study has been primarily growth of specific communities of microbes (25–27). This focused on humans. However, fundamental questions remain, dichotomy serves to ensure proper nutritional sustenance for necessitating the use of diverse model systems, including those the host, while training host immunological systems to handle that are “simpler” (17). These model systems can help inform us the load of ingested microbes, resisting those that are pathogenic of the central principles legislating homeostasis in the gut and, or infectious. Phylogenetically, the role of the innate immune specifically, will help refine our recognition of the role(s) of system in maintaining microbial communities in the gut has innate immunity in governing the complex gut ecosystem. ancient origins (25). To ensure this dual function of the immune It was argued previously (18) that the invertebrate chordate, system, the whole gut has evolved distinct anatomical, Ciona robusta, could serve as a novel, informative, model system morphological, and functional characteristics. Indeed, as a for studies of host-microbiome interactions. Ciona has long been filter-feeding invertebrate most closely related to vertebrates, a well-established model for investigating animal development Ciona represents an attractive and useful model for research (19–22) and immune defense (23) due to its experimental into understanding the evolution and diversification of the tractability and its phylogenetic position relative to vertebrates digestive systems in chordates (28, 29). Like other ascidians, (24). These prior studies were focused on what we now recognize Ciona possesses a U-shaped alimentary canal that includes a as C. robusta but were published as C. intestinalis (Type A). pharynx, esophagus, stomach, and intestine (Figure 1D). The Further, with anatomic features of a digestive tract that are easy stomach epithelium is folded and organized with many ciliated to identify and dissect, its reliance on innate immunity, along ridges and grooves, and characterized by four cell types: ciliated with husbandry approaches to rear thousands of transparent mucus cells, gland cells, vacuolated cells, and undifferentiated filter-feeding juveniles, reveals Ciona as a suitable organism for cells. The intestinal epithelium, instead, appears as a smooth studying gut homeostasis (Figures 1A–C). Further, this model layer, with absorptive cells and narrow mucous cells (30–33). helps reveal the essential roles of innate immunity in shaping the Thus, the highly developed and compartmentalized gut of Ciona dynamic interaction between host and microbiome, especially morphologically resembles that of more recently diverged given the unique vantage point of a siphoning filter-feeder that chordates (31, 33). The specific functions of each compartment moves large volumes of microbe-rich seawater across its mucosal have yet to be well defined. Beyond the anatomical organization, epithelium. Significant progress, reviewed herein, has been made an important function of homeostasis in the gut is the to fulfill this vision. establishment of a mucosal environment where immune cells, Here, we will recapitulate recent efforts to characterize the epithelial-mesenchymal cells, and commensal microbes are Ciona gut environment, including the presence of a gut combined to promote the formation and existence of the gut epithelium layered with chitin-rich mucus and roles for a ecosystem (34). Epithelial barriers and associated innate immune secreted immune effector family,