Identification of the First Teleost CD5 Molecule: Additional Evidence on Phenotypical and Functional Similarities between Fish IgM+ B Cells and Mammalian B1 Cells.

Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells.

The BAFF / APRIL axis plays an important role in virus-induced peritoneal responses in rainbow trout.

IgM+ B cells have been recently demonstrated to be key regulators of peritoneal inflammation in teleost, as a large number of them occupy the peritoneal cavity after 48 h of antigenic stimulation. Despite this, the number of studies addressing the mechanism through which this cell population expands and differentiates in response to stimuli has been scarcely addressed. Because the BAFF/APRIL axis is known to play a major role in B cell survival and differentiation in mammals, we hypothesized that it could be affected in the peritoneal cavity in response to an inflammatory stimulus. To verify this hypothesis, we studied how BAFF, APRIL and the fish-specific related cytokine BALM as well as their putative receptors are regulated in rainbow trout after intraperitoneal (i.p.) injection of viral hemorrhagic septicemia virus (VHSV). When the transcriptional analysis was performed in total cells from the peritoneum, we observed that VHSV provoked an up-regulation of both BAFF and BAFF receptor (BAFF-R) mRNA levels. However, when we examined how isolated peritoneal IgM+ B cells were transcriptionally affected by VHSV i.p. injection, we found that APRIL, BALM and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) were also up-regulated in response to the virus. IgM- cells, on the other hand, only up-regulated BALM transcription in response to VHSV. Finally, to gain further insight on the role that these cytokines play in the peritoneum, we have studied their effect on the survival of peritoneal IgM+ B cells. This work demonstrates a key role for the BAFF/APRIL axis in the peritoneal inflammatory response and contributes to further understanding how IgM+ B cells are regulated at this specific peripheral site.

Identification of Teleost Skin CD8α+ Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α(+) in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103(+) DCs and the human CD141(+) DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α(+) MHC II(+) DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8(+) DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.

Early activation of teleost B cells in response to rhabdovirus infection.

UNLABELLED: To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM(+)) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM(+) cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM(+) cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM(+) cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM(+) cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM(+) cell proliferation, a consistent effect on IgM(+) cell survival was detected. IMPORTANCE: Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects on B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-κB and increased IgM(+) cell survival. Interestingly, VHSV activated B lymphocytes toward an antigen-presenting profile, suggesting an important role of IgM(+) cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells.

CCR7 is mainly expressed in teleost gills, where it defines an IgD+IgM- B lymphocyte subset.

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyer's patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7(+) cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7(+) cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7(+) cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD(+) cells in the gills expressed CCR7. Intriguingly, the IgD(+)CCR7(+) population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7(+) cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD(+)memIgM(-) B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD(+)IgM(-) subsets in mammals are discussed.

Single-cell atlas of rainbow trout peripheral blood leukocytes and profiling of their early response to infectious pancreatic necrosis virus.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.

Endoplasmic reticulum expansion throughout the differentiation of teleost B cells to plasmablasts.

The differentiation of B cells into antibody-secreting cells is fundamental for the generation of humoral immunity. In mammals, this process involves a series of metabolic and intracellular changes, not studied to date in teleost fish, where a clear distinction between naive B cells and plasmablasts/plasma cells (PCs) is still missing. Thus, in the current study, we have established that upon activation, teleost B cells undergo an expansion of the endoplasmic reticulum (ER) but experience no significant changes in mitochondria content. In parallel, the transcription of genes implicated in B cell differentiation increases, while that of mitochondrial genes decreases. In this context, ER monitoring has allowed us to distinguish between small cells with low amounts of ER (FSCloERlo B cells), that correspond to undifferentiated cells, and large cells with expanded ER (FSChiERhi B cells), characterized as plasmablasts. The results shed new light on the B cell differentiation process in teleosts and provide us with novel tools to study B cell function in these species.

Bacillus subtilis Expressing the Infectious Pancreatic Necrosis Virus VP2 Protein Retains Its Immunostimulatory Properties and Induces a Specific Antibody Response.

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout (Oncorhynchus mykiss) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM+ B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.

Human MR1 expression on the cell surface is acid sensitive, proteasome independent and increases after culturing at 26°C.

Mucosal-associated invariant T (MAIT) cells are a population of non-conventional T-lymphocytes which are restricted by the MHC-related 1 (MR1) molecule. MR1 is a non-classical member of the MHC class I family of proteins, it is unknown if MR1 presents any kind of antigens to MAIT cells. In the present manuscript we describe that detection of MR1 on the cell surface by conformation-dependent monoclonal antibodies is enhanced upon culture the cells at 26°C; we also show that detection of MR1 on the cell surface is lost after treating the cells at pH 3.3 as in the case of classical MHC class I molecules. Finally, the re-expression of MR1 on the cell surface is independent of proteasome. Taken together these results strongly suggest that MR1 needs to bind proteasome-independent ligands in order to properly reach the cell surface.

Pro-Inflammatory and B Cell Regulating Capacities of TWEAK in Rainbow Trout (Oncorhynchus mykiss).

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.

CD38 Defines a Subset of B Cells in Rainbow Trout Kidney With High IgM Secreting Capacities.

CD38 is a multifunctional molecule that functions both as a transmembrane signaling receptor and as an ectoenzyme with important roles in cell adhesion, calcium regulation and signal transduction. Within the B cell linage, CD38 is expressed in diverse murine B cell subsets, with highest levels in innate B cell subpopulations such as marginal zone (MZ) B cells or B1 cells. In humans, however, CD38 is transiently expressed on early lymphocyte precursors, is lost on mature B cells and is consistently expressed on terminally differentiated plasma cells. In the present work, we have identified two homologues of mammalian CD38 in rainbow trout (Oncorhynchus mykiss), designating them as CD38A and CD38B. Although constitutively transcribed throughout different tissues in homeostasis, both CD38A and CD38B mRNA levels were significantly up-regulated in head kidney (HK) in response to a viral infection. In this organ, after the generation of a specific monoclonal antibody (mAb) against CD38A, the presence of CD38A+ populations among IgM+ B cells and IgM- leukocytes was investigated by flow cytometry. Interestingly, the percentage of IgM+CD38A+ B cells increased in response to an in vitro stimulation with inactivated Aeromonas salmonicida. Finally, we demonstrated that HK IgM+CD38A+ B cells had an increased IgM secreting capacity than that of cells lacking CD38A on the cell surface, also showing increased transcription levels of genes associated with B cell differentiation. This study strongly suggests a role for CD38 on the B cell differentiation process in teleosts, and provides us with novel tools to discern between B cell subsets in these species.

Distinct modes of action of CD40L and adaptive cytokines IL-2, IL-4/13, IL-10 and IL-21 on rainbow trout IgM+ B cells.

In mammals, conventional B (B2) cells are activated within lymphoid follicles through a close relationship with T follicular helper (Tfh) cells. The interaction between CD40 expressed on B cells and its ligand (CD40L) expressed on Tfh cells is a key signal that regulates the formation of germinal centers (GCs), B cell survival, proliferation and differentiation to plasma cells (PCs) or memory cells. Additionally, certain soluble cytokines produced by T cells also strongly condition the outcome of this interaction. Despite the many differences found between fish B cells and mammalian B2 cells, and the lack of conventional GCs, rainbow trout IgM+ B cells have been shown to be stimulated by CD40L, however, whether cytokines commonly produced by T cells can further modulate this response has never been addressed to date. Thus, in this study, we determined the effects of recombinant rainbow trout adaptive cytokines interleukin 2B (IL-2B), IL-4/13A, IL-4/13B, IL-10 and IL-21 (cytokines known to activate B cells in mammals) on splenic IgM+ B cells alone or in combination with CD40L. We studied how these cytokines and CD40L cooperated to promote IgM+ B cell survival, proliferation and IgM secretion. The results obtained provide valuable information for the first time in teleost fish on how different T cell signals cooperate to activate B cells in the absence of GCs.

Rainbow trout mount a robust specific immune response upon anal administration of thymus-independent antigens.

Despite the strong demand for orally-delivered fish vaccines and the deficient response of those currently available in the market, little is known about how teleost B cells differentiate to antibody secreting cells (ASCs) in response to antigens delivered to the intestinal mucosa. To fill this gap, in the current study, we have studied the dynamics of B cell differentiation in spleen and kidney of rainbow trout (Oncorhynchus mykiss) anally immunized with antigens catalogued in mammals as thymus dependent (TD) or thymus-independent (TI). Our results show that, in the absence of additional adjuvants, rainbow trout preferentially responded to a model TI antigen such as TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The anal administration of TNP-LPS elicited TNP-specific serum antibodies, and a significant increase in the number of total and TNP-specific ASCs in both spleen and kidney, being the kidney the site where most ASCs are found at later time points. In the spleen, a proliferative response of both IgM+ B and T cells was also clearly visible, while the proliferative response was weaker in the kidney. Finally, TNP-LPS also provoked a transcriptional regulation of some immune genes in the spleen and the intestine, including a decreased transcription of foxp3a and foxp3b in intestine that suggests a breach in tolerogenic responses in response to TI stimulation. These results contribute to a better understanding of how intestinal immunity is regulated in teleost and will aid in the future design of effective oral strategies for aquaculture.

Monocyte-Derived Dendritic Cells Differentiated in the Presence of Lenalidomide Display a Semi-Mature Phenotype, Enhanced Phagocytic Capacity, and Th1 Polarization Capability.

Lenalidomide is an analog of thalidomide, with potent anticancer activity demonstrated in several hematological malignancies. It has immunomodulatory properties, being able to enhance the activation of different types of immune cells, which results in antitumor activities. Dendritic cells (DCs) are pivotal in the immune response, and different immunotherapeutic approaches targeting these cells are being developed. Since little is known about the effect of lenalidomide on DCs, the goal of the present work was to investigate the phenotype and function of human monocyte-derived DCs differentiated in the presence of lenalidomide (L-DCs). Our results showed that L-DCs display a unique phenotype, with increased cell surface expression of some maturation markers such as CD1d, CD83, CD86, and HLA-DR. This phenotype correlates with a lower expression of the E3 ubiquitin-ligase MARCH-I in L-DCs, upregulating the cell surface expression of CD86 and HLA-DR. In addition, immature L-DCs express higher amounts of DC-SIGN on the cell surface than control immature DCs. After LPS stimulation, production of IL-6 and TNF-α was severely decreased, whereas IL-12 and IL-10 secretion was dramatically upregulated in L-DCs, compared to that in the controls. Functionally, L-DCs are more effectively recognized by NKT cells in cytotoxicity experiments. Furthermore, L-DCs display higher opsonin-independent antigen uptake capability than control DCs. Mixed lymphocyte reaction experiments showed that L-DCs could stimulate naïve CD4 T-cells, polarizing them toward a predominant Th1 phenotype. In summary, DCs derived from monocytes in the presence of lenalidomide present a semi-mature phenotype, increased phagocytic capacity, reduced production of proinflammatory cytokines, and the ability to polarize T-cells toward predominant Th1-type responses; these are qualities that might be useful in the development of new immunotherapeutic treatments.

Dysregulation of B Cell Activity During Proliferative Kidney Disease in Rainbow Trout.

Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout (Oncorhynchus mykiss) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae. We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT+ B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD, they point to a major involvement of IgT in the immune response to the parasite. These results provide further insights into the pathology of PKD that may facilitate the future development of control strategies.

Expansion and differentiation of IgM+ B cells in the rainbow trout peritoneal cavity in response to different antigens.

To date, intraperitoneal (i.p.) injection seems to be the most effective vaccination route in aquaculture, as many i.p. administered fish vaccines are capable of conferring strong and long-lasting immune responses. Despite this, how peritoneal leukocytes are regulated upon antigen encounter has only been scarcely studied in fish. Although, in the past, myeloid cells were thought to be the main responders to peritoneal inflammation, a recent study revealed that IgM+ B cells are one of the main cell types in the teleost peritoneal cavity in response to pathogenic bacteria. Thus, in the current work, we have focused on establishing how IgM+ B cells are recruited into the peritoneum in rainbow trout (Oncorhynchus mykiss) comparing different antigens: Escherichia coli as a bacterial model, E. coli-derived lipopolysaccharide (LPS) or viral hemorrhagic septicemia virus (VHSV). In addition to studying their capacity to dominate the peritoneal cavity, we have established how these IgM+ B cells are regulated in response to the different antigens, determining their levels of IgM secretion, surface MHC II expression, cell size and phagocytic abilities. Our results reveal that IgM+ B cells are one of the main cell types amplified in the peritoneum in response to either bacterial or viral antigens and that these immunogenic stimulations provoke a differentiation of some of these cells towards plasmablasts/plasma cells whereas others seem to be implicated in antigen presentation. These findings contribute to a better understanding of the immune processes that regulate peritoneal inflammation in teleost fish.

Rainbow trout CK9, a CCL25-like ancient chemokine that attracts and regulates B cells and macrophages, the main antigen presenting cells in fish.

CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 is chemoattractant for antigen presenting cells (APCs) expressing major histocompatibility complex class II (MHC II) on the cell surface. Among these APCs, CK9 has a strong chemotactic capacity for both B cells (IgM+ and IgT+) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM+ cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT+ lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte trafficking in rainbow trout, able to modulate innate functions of teleost B lymphocytes and macrophages.

Distinct Differentiation Programs Triggered by IL-6 and LPS in Teleost IgM(+) B Cells in The Absence of Germinal Centers.

Although originally identified as a B cell differentiation factor, it is now known that mammalian interleukin-6 (IL-6) only regulates B cells committed to plasma cells in response to T-dependent (TD) antigens within germinal centers (GCs). Even though adaptive immunity is present in teleost fish, these species lack lymph nodes and GCs. Thus, the aim of the present study was to establish the role of trout IL-6 on B cells, comparing its effects to those induced by bacterial lipopolysaccharide (LPS). We demonstrate that the effects of teleost IL-6 on naïve spleen B cells include proliferation, activation of NF-κB, increased IgM secretion, up-regulation of Blimp1 transcription and decreased MHC-II surface expression that point to trout IL-6 as a differentiation factor for IgM antibody-secreting cells (ASCs). However, LPS induced the secretion of IgM without up-regulating Blimp1, driving the cells towards an intermediate activation state in which antigen presenting mechanisms are elicited together with antibody secretion and expression of pro-inflammatory genes. Our results reveal that, in trout, IL-6 is a differentiation factor for B cells, stimulating IgM responses in the absence of follicular structures, and suggest that it was after follicular structures appeared that this cytokine evolved to modulate TD responses within the GC.

First in-depth analysis of the novel Th2-type cytokines in salmonid fish reveals distinct patterns of expression and modulation but overlapping bioactivities.

IL-4 and IL-13 are closely related canonical type-2 cytokines in mammals and have overlapping bioactivities via shared receptors. They are frequently activated together as part of the same immune response and are the signature cytokines produced by T-helper (Th)2 cells and type-2 innate lymphoid cells (ILC2), mediating immunity against extracellular pathogens. Little is known about the origin of type-2 responses, and whether they were an essential component of the early adaptive immune system that gave a fitness advantage by limiting collateral damage caused by metazoan parasites. Two evolutionary related type-2 cytokines, IL-4/13A and IL-4/13B, have been identified recently in several teleost fish that likely arose by duplication of an ancestral IL-4/13 gene as a consequence of a whole genome duplication event that occurred at the base of this lineage. However, studies of their comparative expression levels are largely missing and bioactivity analysis has been limited to IL-4/13A in zebrafish. Through interrogation of the recently released salmonid genomes, species in which an additional whole genome duplication event has occurred, four genomic IL-4/13 loci have been identified leading to the cloning of three active genes, IL-4/13A, IL-4/13B1 and IL-4/13B2, in both rainbow trout and Atlantic salmon. Comparative expression analysis by real-time PCR in rainbow trout revealed that the IL-4/13A expression is broad and high constitutively but less responsive to pathogen-associated molecular patterns (PAMPs) and pathogen challenge. In contrast, the expression of IL-4/13B1 and IL-4/13B2 is low constitutively but is highly induced by viral haemorrhagic septicaemia virus (VHSH) infection and during proliferative kidney disease (PKD) in vivo, and by formalin-killed bacteria, PAMPs, the T cell mitogen PHA, and the T-cell cytokines IL-2 and IL-21 in vitro. Moreover, bioactive recombinant cytokines of both IL-4/13A and B were produced and found to have shared but also distinct bioactivities. Both cytokines rapidly induce the gene expression of antimicrobial peptides and acute phase proteins, providing an effector mechanism of fish type-2 cytokines in immunity. They are anti-inflammatory via up-regulation of IL-10 and down-regulation of IL-1ß and IFN-γ. They modulate the expression of cellular markers of T cells, macrophages and B cells, the receptors of IFN-γ, the IL-6 cytokine family and their own potential receptors, suggesting multiple target cells and important roles of fish type-2 cytokines in the piscine cytokine network. Furthermore both cytokines increased the number of IgM secreting B cells but had no effects on the proliferation of IgM+ B cells in vitro. Taken as a whole, fish IL-4/13A may provide a basal level of type-2 immunity whilst IL-4/13B, when activated, provides an enhanced type-2 immunity, which may have an important role in specific cell-mediated immunity. To our knowledge this is the first in-depth analysis of the expression, modulation and bioactivities of type-2 cytokines in the same fish species, and in any early vertebrate. It contributes to a broader understanding of the evolution of type-2 immunity in vertebrates, and establishes a framework for further studies and manipulation of type-2 cytokines in fish.

Molecular characterization of CD9 and CD63, two tetraspanin family members expressed in trout B lymphocytes.

Tetraspanins are a family of membrane-organizing proteins, characterized by the presence of four highly conserved transmembrane regions that mediate diverse physiological functions. In the current study, we have identified two novel tetraspanin members in rainbow trout (Oncorhynchus mykiss), homologs to mammalian CD9 and CD63. Both genes were expressed in muscle, skin, gills, hindgut, gonad, liver, spleen, head kidney, thymus and peripheral blood leukocytes. Throughout the early life cycle stages, CD9 mRNA levels significantly increased after first feeding, whereas CD63 transcription remained constant during all the developmental stages analyzed. In response to an experimental bath infection with viral hemorrhagic septicemia virus (VHSV), CD9 transcription was down-regulated in the gills, while CD63 mRNA levels were down-regulated in the head kidney. Instead, when the virus was intraperitoneally injected, the transcription of both genes was significantly up-regulated in peritoneal cells at several days post-infection. Additionally, both genes were transcriptionally up-regulated in the muscle of trout injected with a VHSV DNA vaccine. To gain insight on the relation of these tetraspanins with B cell activity we determined their constitutive expression in naive IgM(+) populations from different sources and observed that both molecules were being transcribed by IgM(+) cells in different tissues. Furthermore, CD9 transcription was significantly down-regulated in splenic IgM(+) cells in response to in vitro VHSV exposure. Our results provide insights on the potential role of these tetraspanins on teleost B cell and antiviral immunity.