Neutrophils are essential to the acute inflammatory response, where they serve as the first line of defense against infiltrating pathogens. We report that, on receiving the necessary signals, teleost (Carassius auratus) neutrophils leave the hematopoietic kidney, enter into the circulation, and dominate the initial influx of cells into a site of inflammation. Unlike mammals, teleost neutrophils represent <5% of circulating leukocytes during periods of homeostasis. However, this increases to nearly 50% immediately after intraperitoneal challenge with zymosan, identifying a period of neutrophilia that precedes the peak influx of neutrophils into the challenge site at 18 h after injection). We demonstrate that neutrophils at the site of inflammation alter their phenotype throughout the acute inflammatory response, and contribute to both the induction and the resolution of inflammation. However, neutrophils isolated during the proinflammatory phase (18 h after injection) produced robust respiratory burst responses, released inflammation-associated leukotriene B(4), and induced macrophages to increase reactive oxygen species production. In contrast, neutrophils isolated at 48 h after infection (proresolving phase) displayed low levels of reactive oxygen species, released the proresolving lipid mediator lipoxin A(4), and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A(4) was a significant contributor to the uptake of apoptotic cells by teleost macrophages and also played a role, at least in part, in the downregulation of macrophage reactive oxygen species production. Our results highlight the contributions of neutrophils to both the promotion and the regulation of teleost fish inflammation and provide added context for the evolution of this hematopoietic lineage.

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.

It is now widely recognized that neutrophils are sophisticated cells that are critical to host defense and the maintenance of homeostasis. In addition, concepts such as neutrophil plasticity are helping to define the range of phenotypic profiles available to cells in this group and the physiological conditions that contribute to their differentiation. Herein, we discuss key features of the life of a teleost neutrophil including their development, migration to an inflammatory site, and contributions to pathogen killing and the control of acute inflammation. The potent anti-microbial mechanisms elicited by these cells in bony fish are a testament to their long-standing evolutionary contributions in host defense. In addition, recent insights into their active roles in the control of inflammation prior to induction of apoptosis highlight their importance to the maintenance of host integrity in these early vertebrates. Overall, our goal is to summarize recent progress in our understanding of this cell type in teleost fish, and to provide evolutionary context for the contributions of this hematopoietic lineage in host defense and an efficient return to homeostasis following injury or infection.

Phagocytosis is a cellular mechanism that is important to the early induction of antimicrobial responses and the regulation of adaptive immunity. At an inflammatory site, phagocytes serve as central regulators for both pro-inflammatory and homeostatic anti-inflammatory processes. However, it remains unclear if this is a recent evolutionary development or whether the capacity to balance between these two seemingly contradictory processes is a feature already displayed in lower vertebrates. In this study, we used murine (C57BL/6) and teleost fish (C. auratus) in vitro and in vivo models to assess the evolutionary conservation of this dichotomy at a site of inflammation. At the level of the macrophage, we found that teleost fish already displayed divergent pro-inflammatory and homeostatic responses following internalization of zymosan or apoptotic bodies, respectively, and that these were consistent with those of mice. However, fish and mice displayed significant differences in vivo with regards to the level of responsiveness to zymosan and apoptotic bodies, the identity of infiltrating leukocytes, their rate of infiltration, and the kinetics and strength of resulting antimicrobial responses. Unlike macrophages, significant differences were identified between teleost and murine neutrophilic responses. We report for the first time that activated murine, but not teleost neutrophils, possess the capacity to internalize apoptotic bodies. This internalization translates into reduction of neutrophil ROS production. This may play an important part in the recently identified anti-inflammatory activity that mammalian neutrophils display during the resolution phase of inflammation. Our observations are consistent with continued honing of inflammatory control mechanisms from fish to mammals, and provide added insights into the evolutionary path that has resulted in the integrated, multilayered responses that are characteristic of higher vertebrates.

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 IgMpositive (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 In mammals, Toll-like receptors (TLRs) recognize highly conserved structures of viral (TLR3, -7, -8, and -9) and bacterial (TLR1, -2, -4, -5, -6, -7, -8, and -9) origins. While TLR1, -2, -4, -5, and -6, together with TLR11 and TLR12 in mice and TLR10 in humans, are mostly expressed on the cell surface, a second group of TLRs, including TLR3, -7, -8, and -9, are localized within endosomal compartments and detect foreign nucleic acids (1). Recognition of pathogen-associated molecular patterns (PAMPs) through TLRs and other pattern recognition receptors (PRRs) leads to the activation and maturation of innate immune cells such as macrophages or dendritic cells (DCs). Additionally, once the presence of several TLR receptors on distinct populations of human and murine B cells was verified, further investigations concluded that B cells have evolved to directly sense microbes and that this TLR-mediated activation of B cells contributes to the establishment of an adequate humoral response (2). However, controversy remains as to what degree TLR signaling in B cells conditions the antibody response. On one hand, early studies showed that mice lacking B cell TLR signaling failed to mount an efficient antibody response (3). However, subsequent studies suggested a slightly different model in which these receptors play a role in the regulation of antibody class switching and in sustaining antibody secretion at late times after immunization in B cells (4), contributing to the amplification of the humoral response but not being completely responsible for it (5). In support of these observations, further studies demonstrated that the primary responses of some immunoglobulin (Ig) subclasses (i.e., IgG2a or IgG2c) were absolutely dependent on signaling through the adaptor protein MyD88, used by most TLRs, whereas other Ig classes were not (IgG1 and IgG3) or were much less (IgG2b and IgA) dependent on the MyD88 signaling cascade (6, 7). Interestingly, the conditional deletion of MyD88 in either DCs or B cells revealed that the antibody response to virus-like particles required TLR signaling in B cells, while the response to a soluble antigen was dependent on TLR signaling on DCs (8). This result reveals an ability of B cells to discriminate among antigens based on their physical form.