Jeffrey J. Havixbeck

Fish and Mammalian Phagocytes Differentially Regulate Pro-Inflammatory and Homeostatic Responses In Vivo

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.

Neutrophil contributions to the induction and regulation of the acute inflammatory response in teleost fish

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 B4, 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 A4, and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A4 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.

Neutrophil Development, Migration, and Function in Teleost Fish.

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.

Early Activation of Teleost B Cells in Response to Rhabdovirus Infection

ABSTRACT 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.

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.

Teleost soluble CSF-1R modulates cytokine profiles at an inflammatory site, and inhibits neutrophil chemotaxis, phagocytosis, and bacterial killing

Soluble colony stimulating factor-1 receptor (sCSF-1R) is a novel bony fish protein that contributes to the regulation of macrophage proliferation. We recently showed that this soluble receptor is highly upregulated by teleost macrophages in the presence of apoptotic cells. Further, recombinant sCSF-1R inhibited leukocyte infiltration into a challenge site in vivo. Herein, we characterized the mechanisms underlying these changes as a platform to better understand the evolutionary origins of the CSF-1 immune-regulatory axis and inflammation control in teleosts. Using an in vivo model of self-resolving peritonitis, we show that sCSF-1R downregulates chemokine expression and inhibits neutrophil chemotaxis. Soluble CSF-1R also inhibited gene expression of several pro-inflammatory cytokines and promoted the expression of an anti-inflammatory mediator, IL-10. Finally, the phenotype of infiltrating neutrophils changed significantly in the presence of sCSF-1R. Both a reduced capacity for phagocytosis and pathogen killing were observed. Overall, our results implicate sCSF-1R as an important regulator of neutrophil responses in teleosts. It remains unclear whether this represents an inflammation regulatory factor that is unique to this animal group or one that may be evolutionarily conserved and continues to contribute to the regulation of antimicrobial processes at inflammatory sites in higher vertebrates.

UV and hydrogen peroxide treatment restores changes in innate immunity caused by exposure of fish to reuse water

The purpose of this study was to assess the innate immunity of goldfish exposed to reuse water, and UV/H2O2-treated reuse water, using a real-time flow-through exposure system. The reuse water generated by ultrafiltration of finished wastewater from the municipal wastewater treatment plant was analyzed for the presence of a panel of 20 herbicides/fungicides and 46 pharmaceuticals and personal care products (PPCP). There was a seasonal variation in the profile and concentrations of xenobiotics in reuse water with lowest levels occurring in the summer. The innate immunity parameters assessed were cytokine (IFNγ, IL-1β, IL-10, TNFα2), and cytokine receptor (TNFR1, TNFR2, IFNGR1, IFNGR2) gene expression, and phagocytosis of kidney leukocyte subpopulations. Assessment of innate immunity parameters was done after acute (7 days) and sub chronic (30 and 60 days) exposure to reuse water, UV/H2O2-treated reuse water, and activated carbon-treated reuse water (ACT; control), during spring, summer and fall of 2012. Temporal (acute versus sub chronic) as well as seasonal differences in innate immunity of fish exposed to reuse water were observed. The acute exposure of fish to reuse water caused significant down-regulation in cytokine gene expression in different organs of fish (kidney, spleen, liver) and phagocytic ability of different kidney leukocyte subpopulations. The immune gene expression and phagocytosis of kidney leukocytes of fish returned to ACT control levels after sub chronic exposure suggesting that fish have habituated to the reuse water exposure. The changes in gene expression after acute exposure were related to variations in the profile of xenobiotics in reuse water during different seasons. The efficiency of xenobiotic removal using UV/H2O2 ranged between 1.6 and 100% indicating that treatment of reuse water using high dose UV/H2O2 was only partially effective in removing the xenobiotics, as assessed by both chemical analyses and measurement of innate immune responsiveness of the fish. Furthermore, exposure of fish to reuse water and UV/H2O2-treated reuse water generated in the spring and fall caused greater changes in innate immunity after acute exposure, compared to fish exposed to ACT reuse water, indicating that the remediation of reuse water, should be considered in order to protect aquatic and public health.

Evolutionary Conservation of Divergent Pro-Inflammatory and Homeostatic Responses in Lamprey Phagocytes

In higher vertebrates, phagocytosis plays a critical role in development and immunity, based on the internalization and removal of apoptotic cells and invading pathogens, respectively. Previous studies describe the effective uptake of these particles by lower vertebrate and invertebrate phagocytes, and identify important molecular players that contribute to this internalization. However, it remains unclear if individual phagocytes mediate internalization processes in these ancient organisms, and how this impacts the balance of pro-inflammatory and homeostatic events within their infection sites. Herein we show that individual phagocytes of the jawless vertebrate Petromyzon marinus (sea lamprey), like those of teleost fish and mice, display the capacity for divergent pro-inflammatory and homeostatic responses following internalization of zymosan and apoptotic cells, respectively. Professional phagocytes (macrophages, monocytes, neutrophils) were the primary contributors to the internalization of pro-inflammatory particles among goldfish (C. auratus) and lamprey (P. marinus) hematopoietic leukocytes. However, goldfish showed a greater ability for zymosan phagocytosis when compared to their jawless counterparts. Coupled to this increase was a significantly lower sensitivity of goldfish phagocytes to homeostatic signals derived from apoptotic cell internalization. Together, this translated into a significantly greater capacity for induction of antimicrobial respiratory burst responses compared to lamprey phagocytes, but also a decreased efficacy in apoptotic cell-driven leukocyte homeostatic mechanisms that attenuate this pro-inflammatory process. Overall, our results show the long-standing evolutionary contribution of intrinsic phagocyte mechanisms for the control of inflammation, and illustrate one effective evolutionary strategy for increased responsiveness against invading pathogens. In addition, they highlight the need for development of complementary regulatory mechanisms of inflammation to ensure continued maintenance of host integrity amidst increasing challenges from invading pathogens.

Development of an in vitro model system to study the interactions between Mycobacterium marinum and teleost neutrophils.

The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates.

A soluble form of the CSF-1 receptor contributes to the inhibition of inflammation in a teleost fish

We previously reported on the identification of a novel soluble form of the CSF-1 receptor (sCSF-1R) in goldfish that induced dose-dependent down-regulation of macrophage proliferation. Herein, we report that sCSF-1R has a role beyond macrophage development, which extends into the control of cellular antimicrobial inflammatory responses in this lower vertebrate. Using an in vivo model of self-resolving peritonitis coupled to in vitro characterization of sCSF-1R activity, we show that sCSF-1R plays a role in the inhibition of inflammation which follows an initial acute phase of innate antimicrobial responses within an inflammatory site. In vitro, mature goldfish primary kidney macrophages but not monocytes up-regulated sCSF-1R expression upon direct contact with apoptotic cells. In vivo, sCSF-1R expression coincided with an increase in macrophage numbers that resulted from administration of apoptotic cells into the goldfish peritoneal cavity. This contrasted the decrease in sCSF-1R expression during zymosan-induced inflammatory responses in vivo. Subsequent experiments showed an anti-inflammatory effect for sCSF-1R. Leukocyte infiltration and ROS production decreased in a dose-dependent manner compared to zymosan-stimulated controls upon addition of increasing doses of recombinant sCSF-1R. Among others, sCSF-1R may contribute to the dual role that phagocytic macrophages play in the induction and regulation of inflammation. Overall, our results provide new insights into ancient mechanisms of inflammation control and, in particular, the evolutionary origins of the CSF-1 immune regulatory axis.