A. Liberti et al. BO REVISION CLEAN

The gastrointestinal tract of Ciona intestinalis, a solitary tunicate that siphon filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona, retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis. Introduction The gastrointestinal tract (GIT) represents an external environment that runs through the otherwise sterile confines of an animal. By ingesting food and water, animals expose the GIT to countless antigens and microorganisms, many of which can be metabolized as part of the diet whereas others become permanent occupants. Gut-dwelling microorganisms that become part of the host microbiota can either remain neutral or serve beneficial or detrimental roles to host health. Owing to the continuous exposure to outer stimuli, the gut has evolved strategies to avoid pathogenic infections and maintain homeostasis, resulting in a balance (i.e. homeostasis) between host immunity and gut microbiota (Hooper and Macpherson, 2010). Dysbiosis, the loss of this balance, is characterized by an inappropriate and aberrant immune response to microbes that is the underlying cause of inflammatory bowel diseases (IBD) pathogenesis, such as ulcerative colitis and Crohn’s disease (Matsuoka and Kanai, 2015; Swidsinski et al., 2002). The experimental induction of colitis has been used as a way to study not just gut inflammatory processes that often contribute to autoimmune phenomena, but also to understand the processes that shape barrier integrity and homeostasis. To induce colitis, a B io lo gy O pe n • A cc ep te d m an us cr ip t by guest on January 11, 2018 http://bio.biologists.org/ Downloaded from variety of approaches have been developed, including the administration of chemicals such as acetic acid, formalin, indomethacin, trinitrobenzene sulfonic acid, oxazolone and nonsteroidal anti-inflammatory drugs. Polysaccharides, such as DSS, that physically and/or functionally disrupts gut barriers also have been employed for this purpose (Gaudio et al., 1999; Randhawa et al., 2014). DSS is by far the most commonly used and has been shown to induce colitis in diverse animal models producing phenotypes and associated inflammatory profiles that resemble human IBD. The addition of DSS to drinking water generates colitis in the mouse (Chassaing et al., 2014; Okayasu et al., 1990; Perse and Cerar, 2012; Rose et al., 2012a; Wirtz et al., 2007; Wirtz et al., 2017), rat (Gaudio et al., 1999), pig (Kim et al., 2009), and the fruit fly, Drosophila melanogaster (Amcheslavsky et al., 2009). The addition of DSS to water for studies involving exposure by immersion has been developed in zebrafish larvae (Oehlers et al., 2012; Oehlers et al., 2013); immersion was found to also result in the induction an IBD-like colitis. The application of diverse model systems for experimentallyinduced colitis offers specific advantages for understanding the phenotypes and physiology associated with IBD-like pathologies. Ciona intestinalis, a well-recognized developmental model system, has been shown to be uniquely informative for studies of host-microbe interactions in the gut (Dishaw et al., 2012; Dishaw et al., 2016). Ciona lacks adaptive immunity (Azumi et al., 2003), relying solely on innate immunity for host defence, and maintains a core microbiome (Dishaw et al., 2014b) that likely is influenced through immune interactions involving chitin-rich mucus that coats the gut epithelium (Dishaw et al., 2016). It was previously hypothesized that the interwoven chitin fibres produced endogenously, and incorporated into gut mucus, could enhance barrier functions in the Ciona gut (Dishaw et al., 2016). Thus, Ciona represents a potentially informative model system to define how barrier integrity shapes the maintenance of homeostasis between host and microbiome. In order to address the roles that endogenous chitin-rich mucus may be serving in barrier defences, we have developed approaches to implement DSS treatment, via immersion, as a means to challenge mucosal barriers and for studying associated inflammatory responses. We find that DSS-treatment in Ciona induces a colitis-like phenotype that is remarkably similar to that seen in mammals. Pre-treatment with exogenous chitin microparticles (CMP) can protect the animal from this DSS-mediated damage. While chitin-rich mucus is not normally found in mammals, it was shown previously that administration of CMPs to mice, prior to DSS treatment, could afford protection from inflammatory colitis (Nagatani et al., 2012). Collectively these findings suggest that endogenous chitin likely serves a role in B io lo gy O pe n • A cc ep te d m an us cr ip t by guest on January 11, 2018 http://bio.biologists.org/ Downloaded from enhancing barriers and underscore the utility of the Ciona model system in the identification of evolutionarily conserved features as well as taxa-specific innovation that shape mucosal barriers and associated innate immune responses. Materials and Methods Ethics statement The research described herein was performed on Ciona intestinalis (subtype A, more recently recognized as Ciona robusta), a marine invertebrate collected in the Gulf of Napoli (Italy) or in San Diego, CA (M-Rep), in locations that are not privately-owned nor protected in any way, according to the authorization of Marina Mercantile (DPR 1639/68, 09/19/1980, confirmed on 01/10/2000). The study did not involve mammalian or vertebrate subjects, or endangered or protected species, and was carried out in strict accordance with European (Directive 2010/63) and Italian (Decreto Legislativo n. 116/1992) legislation for the care and use of animals for scientific purposes. Ciona is considered an invasive species and is not regulated or protected by environmental agencies in the United States or Italy. The collection services contracted in this study maintain current permits and licenses for collection and distribution of marine invertebrates to academic institutions; special permission was not required to collect Ciona. Handling of live animals was in accordance with the guidelines of our academic institutions. Animals were recovered and brought to the laboratory alive and maintained in clean water with aeration. In accordance with general animal protocols, the least number of animals required per experiment were utilized. Animal waste products were disposed of appropriately. Treatment with DSS Adult Ciona were immersed in filtered seawater (FSW) containing 0.5% or 1% DSS (40 kDA, TdB, Uppsala, Sweden) and maintained at 18°C overnight; viability was determined by verifying the response to external stimuli, such as gentle poking with a Pasteur pipette and the presence of a heartbeat. Animals were sacrificed and stomach samples were collected. In recovery experiments, DSS-treated animals were transferred to circulating seawater for one week. Stomachs from controls, as well as animals treated with DSS and animals recovered from DSS treatment were fixed and processed for transmission electron microscopy (TEM) or other histology procedures (see below). Ciona stage 7/8 juveniles, developed from a single batch of gametes as described previously (Liberti et al., 2014), were divided into two samples, and maintained as a control B io lo gy O pe n • A cc ep te d m an us cr ip t by guest on January 11, 2018 http://bio.biologists.org/ Downloaded from or treated with 0.05% DSS for 3 hrs. These experimental conditions, which produced the same phenotype(s) observed in adults treated with 1% DSS, were refined to minimize the high mortality observed in higher dose DSS treatment of the younger, more experimentally labile, 7/8 juveniles.