Gallbladder microbiota in early vertebrates provides evolutionary insights into mucosal homeostasis.

The gallbladder (GB) microbiota plays critical roles in mammalian metabolism and immune homeostasis, and its relationship with human disease has been extensively studied over the past decade. However, very little is known about the interplay between GB microbiota and the immune functions of teleost fish, the earliest bony vertebrate with a GB. Therefore, this study sought to investigate the composition of the teleost GB microbiota and the potential mechanisms through which it affects mucosal immunity. In our results, we found that the GB mucosa (GM) and bile bacterial community shared a similar microbiological composition with that of the gut mucosa in naïve individuals. IHNV infection induced a profound GB inflammation and disrupted their microbial homeostasis followed by a strong anti-bacterial response. Interestingly, beneficial bacteria from the Lactobacillales order showed a significant increase in the abundance of the bile microbial community, whereas the structure of the Mycoplasmatales order in the gut microbial community was markedly changed. All in all, our study characterized the structure of the GB microbial ecosystem in teleost fish, and the fish GB microbiome shared a high similarity with the gut microbiota. More importantly, our findings offer solid evidence that the teleost GB evolved immune functions to preserve its mucosal microbial homeostasis, suggesting that both the microbiota and mucosal immunity of the GB might have co-evolved in early vertebrates.

Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

Mammalian studies have demonstrated that B cell immune responses are regulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling. Teleost fish represent the oldest living bony vertebrates that contain bona fide B cells. So far, whether the regulatory mechanism of mTORC1 signaling in B cells occurred in teleost fish is still unknown. In this study, we developed a fish model by using rapamycin (RAPA) treatment to inhibit mTORC1 signaling and demonstrated the role of mTORC1 signaling in teleost B cells. In support, we found inhibition of mTORC1 signaling by RAPA decreased the phagocytic capacity, proliferation, and Ig production of B cells. Critically, Flavobacterium columnare induced specific IgM binding in serum, and these titers were significantly inhibited by RAPA treatment, thus decreasing Ab-mediated agglutination of F. columnare and significantly increasing the susceptibility of fish upon F. columnare reinfection. Collectively, our findings elucidated that the mTORC1 pathway is evolutionarily conserved in regulating B cell responses, thus providing a new point for understanding the B cells functions in teleost fish.

Dynamic Interaction Between Mucosal Immunity and Microbiota Drives Nose and Pharynx Homeostasis of Common Carp (Cyprinus carpio) After SVCV Infection.

The crosstalk between the immune system and microbiota drives an amazingly complex mutualistic symbiosis. In mammals, the upper respiratory tract acts as a gateway for pathogen invasion, and the dynamic interaction between microbiota and mucosal immunity on its surface can effectively prevent disease development. However, the relationship between virus-mediated mucosal immune responses and microbes in lower vertebrates remains uncharacterized. In this study, we successfully constructed an infection model by intraperitoneally injecting common carp (Cyprinus carpio) with spring viremia of carp virus (SVCV). In addition to the detection of the SVCV in the nose and pharynx of common carp, we also identified obvious histopathological changes following viral infection. Moreover, numerous immune-related genes were significantly upregulated in the nose and pharynx at the peak of SVCV infection, after which the expression levels decreased to levels similar to those of the control group. Transcriptome sequencing results revealed that pathways associated with bacterial infection in the Toll-like receptor pathway and the Nod-like receptor pathway were activated in addition to the virus-related Rig-I-like receptor pathway after SVCV infection, suggesting that viral infection may be followed by opportunistic bacterial infection in these mucosal tissues. Using 16S rRNA gene sequencing, we further identified an upward trend in pathogenic bacteria on the mucosal surface of the nose and pharynx 4 days after SVCV infection, after which these tissues eventually reached new homeostasis. Taken together, our results suggest that the dynamic interaction between mucosal immunity and microbiota promotes the host to a new ecological state.

Molecular Characterization and Expression Analysis of Intercellular Adhesion Molecule-1 (ICAM-1) Genes in Rainbow Trout (Oncorhynchus mykiss) in Response to Viral, Bacterial and Parasitic Challenge.

The intercellular adhesion molecule-1 (ICAM-1), known as CD54, is a transmembrane cell surface glycoprotein that interacts with two integrins (i.e., LFA-1 and Mac-l) important for trans-endothelial migration of leukocytes. The level of ICAM-1 expression is upregulated in response to some inflammatory stimulations, including pathogen infection and proinflammatory cytokines. Yet, to date, our knowledge regarding the functional role of ICAM-1 in teleost fish remains largely unknown. In this study, we cloned and characterized the sequence of ICAM-1 in rainbow trout (Oncorhynchus mykiss) for the first time, which exhibited that the molecular features of ICAM-1 in fishes were relatively conserved compared with human ICAM-1. The transcriptional level of ICAM-1 was detected in 12 different tissues, and we found high expression of this gene in the head kidney, spleen, gills, skin, nose, and pharynx. Moreover, upon stimulation with infectious hematopoietic necrosis virus (IHNV), Flavobacterium columnare G4 (F. columnare), and Ichthyophthirius multifiliis (Ich) in rainbow trout, the morphological changes were observed in the skin and gills, and enhanced expression of ICAM-1 mRNA was detected both in the systemic and mucosal tissues. These results indicate that ICAM-1 may be implicated in the mucosal immune responses to viral, bacterial, and parasitic infections in teleost fish, meaning that ICAM-1 emerges as a master regulator of mucosal immune responses against pathogen infections in teleost fish.

Prevailing Role of Mucosal Igs and B Cells in Teleost Skin Immune Responses to Bacterial Infection.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Mediation of Mucosal Immunoglobulins in Buccal Cavity of Teleost in Antibacterial Immunity.

The buccal mucosa (BM) of vertebrates is a critical mucosal barrier constantly exposed to rich and diverse pathogens from air, water, and food. While mammals are known to contain a mucosal associated lymphoid tissue (MALT) in the buccal cavity which induces B-cells and immunoglobulins (Igs) responses against bacterial pathogens, however, very little is known about the evolutionary roles of buccal MALT in immune defense. Here we developed a bath infection model that rainbow trout experimentally exposed to Flavobacterium columnare (F. columnare), which is well known as a mucosal pathogen. Using this model, we provided the first evidence for the process of bacterial invasion in the fish BM. Moreover, strong pathogen-specific IgT responses and accumulation of IgT+ B-cells were induced in the buccal mucus and BM of infected trout with F. columnare. In contrast, specific IgM responses were for the most part detected in the fish serum. More specifically, we showed that the local proliferation of IgT+ B-cells and production of pathogen-specific IgT within the BM upon bacterial infection. Overall, our findings represent the first demonstration that IgT is the main Ig isotype specialized for buccal immune responses against bacterial infection in a non-tetrapod species.