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In the original article, there was a mistake in Figure 1 as published. Although the numbers were correct, all dot plots in Figure 1A were the same. The corrected Figure 1 appears below. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. © 2021 Benedicenti, Wang, Morel, Secombes, Soleto, Dı́az-Rosales and Tafalla. This corrects the article DOI: 10.3389/fimmu.2020.01494.

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.

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.

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.

In fish, intramuscular (i.m) injection of plasmid DNA encoding viral proteins has proved a highly effective vaccination strategy against some viral pathogens. The efficacy of DNA vaccination in teleost fish is based on the high level of viral antigen expression in muscle cells inducing a strong and long-lasting protection. However, the mechanisms through which this protection is established and effectuated in fish are still not fully understood. Moreover, similarities to mammalian models cannot be established since DNA vaccination in mammals usually induces much weaker responses. In this work, we have focused on the characterization of the immune cells that infiltrate the muscle at the site of DNA injection in vaccinated fish and the chemokines and chemokine receptors that may be involved in their infiltration. We have demonstrated through diverse techniques that B lymphocytes, both IgM+ and IgT+ cells, represented a major infiltrating cell type in fish vaccinated with a viral haemorrhagic septicaemia virus (VHSV) glycoprotein-encoding DNA vaccine, whereas in control fish injected with an oil adjuvant mainly granulocyte/monocyte-type cells were attracted. Among twelve chemokine genes studied, only CXCL11_L1, CK5B and CK6 mRNA levels were up-regulated in DNA vaccinated fish compared to fish injected with the corresponding vector backbone. Furthermore, the transcription of CXCR3B, a possible receptor for CXCL11_L1 was also significantly up-regulated in vaccinated fish. Finally, experiments performed with recombinant trout CK5B and CK6 and chemokine expression plasmids revealed that these chemokines have chemotactic capacities which might explain the recruitment of B cells to the site of DNA injection. Altogether, our results reveal that there is an early chemokine-related B cell recruitment triggered by i.m. DNA vaccination against VHSV which might play an important role in the initial phase of the immune response.

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

QC 20190211

Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i. e., induction of interleukin-1 beta(1) [IL-1 beta(1)], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglanding E-2 (PGE(2)) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE(2) was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-gamma] and the IFN-gamma-inducible protein [gamma-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development., QC 20150109. Correction in: Infection and Immunity, Volume 83, Issue 1, 2015, Page 454. DOI: 10.1128/IAI.02821-14. ScopusID: 2-s2.0-84919465565.

The mononuclear phagocyte system is composed of monocytes, macrophages and dendritic cells and has crucial roles in inflammation, autoimmunity, infection, cancer, organ transplantation and in maintaining organismal homeostasis. Interleukin-34 (IL-34) and macrophage colony stimulating factor (MCSF), both signalling through the MCSF receptor, regulate the mononuclear phagocyte system. A single IL-34 and MCSF gene are present in tetrapods. Two types of MCSF exist in teleost fish which is resulted from teleost-wide whole genome duplication. In this report, we first identified and sequence analysed six IL-34 genes in five teleost fish, rainbow trout, fugu, Atlantic salmon, catfish and zebrafish. The fish IL-34 molecules had a higher identity within fish group but low identities to IL-34s from birds (27.2–33.8%) and mammals (22.2–31.4%). However, they grouped with tetrapod IL-34 molecules in phylogenetic tree analysis, had a similar 7 exon/6 intron gene organisation, and genes in the IL-34 loci were syntenically conserved. In addition, the regions of the four main helices, along with a critical N-glycosylation site were well conserved. Taken together these data suggest that the teleost IL-34 genes described in this report are orthologues of tetrapod IL-34. Comparative expression study of the three trout MCSFR ligands revealed that IL-34, MCSF1 and MCSF2 are differentially expressed in tissues and cell lines. The expression of MCSF1 and MCSF2 showed great variance in different tissues and cell lines, suggesting a role in the differentiation and maintenance of specific macrophage lineages in specific locations. The relatively high levels of IL-34 expression across different tissues suggests a homeostatic role of IL-34 for the macrophage lineage in fish. One striking observation in the present study was the lack of induction of MCSF1 and MCSF2 expression but the quick induction of IL-34 expression by PAMPs and inflammatory cytokines in cell lines and primary head kidney macr

This report describes the cloning, characterization and gene expression pattern of two Th17 cytokines, interleukin (IL)-17A/F and -22, in turbot Scophthalmus maximus. The turbot IL-17A/F cDNA contains a 516 bp open reading frame encoding a deduced IL-17A/F protein of 171 amino acid (aa) residues, containing a predicted signal peptide of 31 aa. Turbot IL-22 had a 564 bp ORF coding for a 187 aa protein with a 33 aa signal peptide. The turbot IL-22 protein contained a typical IL-10 family signature. Both cytokines had highest expression levels in the intestine followed by head kidney and gills. Stimulation with the Gram negative bacterium Aeromonas salmonicida was able to modulate IL-17A/F and IL-22 expression in head kidney, spleen and liver but not the intestine. PMA and PHA were also able to induce the expression of both cytokines, suggesting that, as expected, T-cells are likely the main producers of these molecules in turbot as in mammals.

Themammalian gamma-chain (gC) cytokine family consists of interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. They signal through a receptor complex containing the common gC and a private alpha chain, and in the case of IL-2 and IL-15 an additional common IL-2/15Rb chain. Deficiency of gC signalling inmammals prevents CD4þ T cells from developing effector functions and CD8þ T cells from developing immunological memory. Thus gC cytokines are critical for the generation and peripheral homeostasis of naïve and memory T cells. This review will give an update on the gC ligands and receptor subunits in fish, and also present some new data on the cloning and expression of a second gC and two IL-2Rb chains in rainbow trout Oncorhynchus mykiss. In recent years, aided by the availability of sequenced fish genomes and expressed sequence tag databases, five of the six mammalian gC cytokines and their cognate receptors have been discovered in fish, with only the IL-9/IL-9R homologues apparently absent. Paralogues have been discovered in diploid fish and all the receptors described in the tetraploid rainbowtrout, including gC itself, IL-2Rb, IL-4Ra, IL-13Ra1, IL-13Ra2 and IL-2/15Ra, have duplicates. As a consequence of the teleost and salmonid whole genome duplications, even more paralogues may yet be discovered. Some of the paralogues have changes in domain structures and show differential expression and modulation, suggesting the potential for a change in function. Functional characterisation of fish gC cytokines is beginning butmademore difficult by the co-existence of somany paralogues of the ligands and their receptors. Initial functional studies have shown that fish gC cytokines can modulate the expression of key cytokines (e.g. interferon-g, IL-10 and IL-22) of the adaptive immune response, andmay thus have promise as adjuvants to improve vaccination efficiency in fish.