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.

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.

Pharyngeal Immunity in Early Vertebrates Provides Functional and Evolutionary Insight into Mucosal Homeostasis.

The pharyngeal organ is located at the crossroad of the respiratory and digestive tracts in vertebrate, and it is continuously challenged by varying Ags during breathing and feeding. In mammals, the pharyngeal mucosa (PM) is a critical first line of defense. However, the evolutionary origins and ancient roles of immune defense and microbiota homeostasis of PM are still unknown. In this study, to our knowledge, we are the first to find that diffuse MALT is present in PM of rainbow trout, an early vertebrate. Importantly, following parasitic infection, we detect that strong parasite-specific mucosal IgT and dominant proliferation of IgT+ B cell immune responses occurs in trout PM, providing, to our knowledge, the first demonstration of local mucosal Ig responses against pathogens in pharyngeal organ of a nonmammal species. Moreover, we show that the trout PM microbiota is prevalently coated with secretory IgT and, to a much lesser degree, by IgM and IgD, suggesting the key role of mucosal Igs in the immune exclusion of teleost pharyngeal bacteria. Overall, to our knowledge, our findings provide the first evidence that pharyngeal mucosal immunity appear earlier than tetrapods.

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.

Interactions Between Commensal Microbiota and Mucosal Immunity in Teleost Fish During Viral Infection With SVCV.

The mucosa of vertebrates is a particularly complex but dynamic environment in which the host constantly interacts with trillions of commensal microorganisms and pathogens. Although the internal and external mucosal microbiomes with immune defense of mammals have been well investigated, the relationship between mucosal microbes and their host's immune responses has not been systematically understood in the early vertebrates. In this study, we compared the composition and distribution of mucosal microbiota in common carp (Cyprinus carpio), and found that there were significant differences of microbiota between in the internal (gut) and external mucosal (buccal mucosa, gills and skin) tissues. Next, we successfully constructed an infection model with spring viremia of carp virus (SVCV). Specifically, following viral infection, the immune and antiviral related genes showed different up-regulation in all selected mucosal tissues while significant morphological changes were only found in external tissues including buccal mucosa, gills and skin. Using 16S rRNA gene sequence, we revealed that the abundance of Proteobacteria in mucosal tissues including buccal mucosa, gills and gut showed increased trend after viral infection, whereas the abundance of Fusobacteria significantly decreased in gut. In addition, the loss of dominant commensal microorganisms and increased colonization of opportunistic bacteria were discovered in the mucosal surfaces indicating that a secondary bacterial infection might occur in these mucosal tissues after viral infection. Overall, our results firstly point out the distribution of internal and external mucosal microbiota and analyze the changes of mucosal microbiota in common carp after SVCV infection, which may indicated that the potential role of mucosal microbiota in the antiviral process in early vertebrates.

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.

Viral-Infected Change of the Digestive Tract Microbiota Associated With Mucosal Immunity in Teleost Fish.

The digestive tract is a unique series of organs that is inhabited by a range of commensal microbes while also exposed to an overwhelming load of dietary antigens. It is widely known that mammals have evolved complex and efficient immune strategies to protect the mucosa of the digestive tract. However, in the early vertebrates, the roles of mucosal immune defense and microbial communities in the different segments of the digestive tract are not well-understood. Here, we constructed a bath infection model with infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Importantly, following viral infection, we found that the IHNV distribution and the reactions of immune-related genes had similar trends that decreased across the digestive tract. Hematoxylin and eosin (H & E) and alcian blue (A & B) staining of the trout digestive tract showed that the pathological changes only occurred in the buccal and pharyngeal mucosal tissues. Moreover, the increased diversity of the microbial community was only detected in the buccal mucosa through 16S rRNA gene sequencing, suggesting that the magnitude of the immune response and microbial community changes are related to the IHNV load and the original microbial diversity. In addition, the loss of digestive tract dominant species and increased colonization of opportunistic bacteria were discovered in the buccal mucosal surface indicating that a secondary bacterial infection occurred in this mucosal tissue.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

The buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.