Counteracting immunotyrosine-based signaling motifs augment zebrafish leukocyte immune-type receptor-mediated phagocytic activity.

Leukocyte immune-type receptors (LITRs) represent a polymorphic and polygenic family of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs). Belonging to the immunoglobulin superfamily (IgSF), IpLITRs are generally classified as stimulatory or inhibitory types based on their utilization of various intracellular tyrosine-based signaling motifs. While research has shown that IpLITRs can activate as well as abrogate different immune cell effector responses including phagocytosis, recent identification of LITRs within the zebrafish genome (Danio rerio; DrLITRs) revealed the existence of fish LITR-types uniquely containing counteracting stimulatory and inhibitory cytoplasmic tail (CYT) region motifs (i.e., an immunoreceptor tyrosine-based activation motif; ITAM, and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that then co-engage to fine-tune cellular signaling and effector responses. Using a flow cytometric-based phagocytosis assay, we show here that engagement of DrLITR 1.2-expressing cells with antibody coated 4.5 µm beads causes a robust ITAM-dependent phagocytic response and reveal that its tandem ITIM motif surprisingly enhances the DrLITR 1.2-induced phagocytic activity while simultaneously decreasing the receptors ability to bind the beads. Confocal microscopy studies also revealed that the ITIM-associated inhibitory signaling molecule SHP-2 is localized to the phagocytic synapse during the phagocytic response. Overall, these results provide the first functional characterization of teleost immune receptors containing a tandem ITAM and ITIM and allow for the proposal of an intracytoplasmic tail signaling model for ITIM-mediated enhancement of ITAM-dependent cellular activation.

Examining the immunotoxicity of oil sands process affected waters using a human macrophage cell line.

Oil sands process affected water (OSPW) is produced during the surface mining of the oil sands bitumen deposits in Northern Alberta. OSPW contains variable quantities of organic and inorganic components causing toxic effects on living organisms. Advanced Oxidation Processes (AOPs) are widely used to degrade toxic organic components from OSPW including naphthenic acids (NAs). However, there is no established biological procedure to assess the effectiveness of the remediation processes. Our previous study showed that human macrophage cells (THP-1) can be used as a bioindicator system to evaluate the effectiveness of OSPW treatments through examining the proinflammatory gene transcription levels. In the present study, we investigated the immunotoxicological changes in THP-1 cells following exposure to untreated and AOP-treated OSPW. Specifically, using proinflammatory cytokine protein secretion assays we showed that AOP treatment significantly abrogates the ability of OSPW to induce the secretion of IL-1ß, IL-6, IL-8, TNF-α, IL-1Ra and MCP-1. By measuring transcriptional activity as well as surface protein expression levels, we also showed that two select immune cell surface markers, CD40 and CD54, were significantly elevated following OSPW exposure. However, AOP treatments abolished the immunostimulatory properties of OSPW to enhance the surface expression of these immune proteins. Finally, a transcriptome-based approach was used to examine the proinflammatory effects of OSPW as well as the abrogation of immunotoxicity following AOP treatments. Overall, this research shows how a human macrophage cell-based biomonitoring system serves as an effective in vitro tool to study the immunotoxicity of OSPW samples before and after targeted remediation strategies.

Teleost leukocyte immune-type receptors.

Leukocyte immune-type receptors (LITRs) are a large family of teleost immunoregulatory receptor-types belonging to the immunoglobulin superfamily. These immune genes are phylogenetically and syntenically related to Fc receptor-like protein genes (fcrls) present in other vertebrates, including amphibians, birds, mice, and man. In vitro-based functional analyses of LITRs, using transfection approaches, have shown that LITRs have diverse immunoregulatory potentials including the activation and inhibition of several innate immune effector responses such as cell-mediated killing responses, degranulation, cytokine secretion, and phagocytosis. The purpose of this mini review is to provide an overview of fish LITR-mediated immunoregulatory potentials obtained from various teleost model systems, including channel catfish, zebrafish, and goldfish. We will also describe preliminary characterization of a new goldish LITR-specific polyclonal antibody (pAb) and discuss the significance of this tool for further investigation of the functions of fish LITRs.

Using immune cell-based bioactivity assays to compare the inflammatory activities of oil sands process-affected waters from a pilot scale demonstration pit lake.

In this study, we provide evidence that oil sands process-affected waters (OSPW) contain factors that activate the antimicrobial and proinflammatory responses of immune cells. Specifically, using the murine macrophage RAW 264.7 cell line, we establish the bioactivity of two different OSPW samples and their isolated fractions. Here, we directly compared the bioactivity of two pilot scale demonstration pit lake (DPL) water samples, which included expressed water from treated tailings (termed the before water capping sample; BWC) as well as an after water capping (AWC) sample consisting of a mixture of expressed water, precipitation, upland runoff, coagulated OSPW and added freshwater. Significant inflammatory (i.e. macrophage activating) bioactivity was associated with the AWC sample and its organic fraction (OF), whereas the BWC sample had reduced bioactivity that was primarily associated with its inorganic fraction (IF). Overall, these results indicate that at non-toxic exposure doses, the RAW 264.7 cell line serves as an acute, sensitive and reliable biosensor for the screening of inflammatory constituents within and among discrete OSPW samples.

Expression analysis of Carassius auratus-leukocyte-immune-type receptors (CaLITRs) during goldfish kidney macrophage development and in activated kidney leukocyte cultures.

Carassius auratus leukocyte immune-type receptors (CaLITRs) were recently discovered immunoregulatory receptors in goldfish that have diverse immunoglobulin-like (Ig-like) ectodomains and intracellular signaling motifs. Genomic analysis shows that CaLITR-types are also located as distinct gene clusters across multiple goldfish chromosomes. For example, CaLITR1 (unplaced) is a functionally ambiguous receptor having two Ig-like domains, a transmembrane domain (TM), and a short cytoplasmic tail (CYT) devoid of any recognizable signaling motifs. CaLITR2 (Chr47) is a putative inhibitory receptor containing four Ig-like domains, a TM, and a long CYT with an immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM). A putative activating receptor-type, CaLITR3 (Chr3), has four Ig-like domains, a TM, and a short CYT containing a positively charged histidine residue and CaLITR4 (ChrLG28B) is a receptor with putative multifunctional signaling potential as well as five Ig-like domains, a TM, and a long tyrosine-motif containing CYT region. The variable genomic locations of the CaLITRs suggest that they are likely under the influence of different cis- and/or trans-regulatory elements. To better understand the transcriptional activities of select CaLITRs from variable genomic regions, we used an RT-qPCR-based approach to examine the expression of CaLITR1, CaLITR2, CaLITR3, and CaLITR4 during goldfish primary kidney macrophage (PKM) development and in mixed leukocyte reaction cultures (MLRs) of the goldfish. Our results showed that the select CaLITRs are differentially expressed during PKM development and in goldfish MLRs exposed to T-cell mitogens/immunosuppressive drugs, supporting that the transcription of these CaLITRs is likely regulated by distinct cis- and/or trans-regulatory elements.

Abiotic and biotic constituents of oil sands process-affected waters.

The oil sands in Northern Alberta are the largest oil sands in the world, providing an important economic resource for the Canadian energy industry. The extraction of petroleum in the oil sands begins with the addition of hot water to the bituminous sediment, generating oil sands process-affected water (OSPW), which is acutely toxic to organisms. Trillions of litres of OSPW are stored on oil sands mining leased sites in man-made reservoirs called tailings ponds. As the volume of OSPW increases, concerns arise regarding the reclamation and eventual release of this water back into the environment. OSPW is composed of a complex and heterogeneous mix of components that vary based on factors such as company extraction techniques, age of the water, location, and bitumen ore quality. Therefore, the effective remediation of OSPW requires the consideration of abiotic and biotic constituents within it to understand short and long term effects of treatments used. This review summarizes selected chemicals and organisms in these waters and their interactions to provide a holistic perspective on the physiochemical and microbial dynamics underpinning OSPW .

Solar-activated zinc oxide photocatalytic treatment of real oil sands process water: Effect of treatment parameters on naphthenic acids, polyaromatic hydrocarbons and acute toxicity removal.

Oil sands process water (OSPW) is an industrial process effluent that contains organic compounds such as naphthenic acids (NAs) and polyaromatic hydrocarbons (PAHs), as well as large quantities of inorganic compounds in its mixture. OSPW requires effective treatment for successful reclamation and water reuse. This study investigated the impact of solar-activated zinc oxide (ZnO) photocatalysis on the degradation and removal of NAs and PAHs in OSPW, as well as the elimination of its acute toxicity. With catalyst particles suspended in the effluent (at 1 g/L) under simulated solar radiation of steady irradiance of ~278 W/m2, more than 99% removal of NAs was achieved after 4 h of treatment, while nearly all PAHs were simultaneously oxidized within the same reaction time. The photocatalytic treatment appeared to selectively convert classical NAs faster than oxidized NAs. Additionally, NAs with higher double-bond equivalents (DBEs) and higher carbon numbers seemed more susceptible to photocatalytic destruction than others. An overall pseudo first-order rate constant of 1.14 × 10-2 min-1, and a fluence-based rate constant of 6.81 × 10-1 m2/MJ were recorded in apparently hydroxyl radicals (OH) and superoxide (O2-) radicals mediated NAs degradation mechanisms. Assessment of the toxicity levels in raw and treated OSPW samples by using Microtox® bioassay indicated that the photocatalytic treatment resulted in ~50% reduction in acute toxicity. Furthermore, we showed that by monitoring the expression levels of key proinflammatory genes using qPCR that treated OSPW significantly reduced the ability of raw OSPW to activate the inflammatory response of immune cells. This indicates that at acute sub-lethal exposure doses, photocatalytic treatment also reduces immunotoxicity. Overall, our results suggest that the ZnO-based photocatalytic degradation of these NAs and PAHs in OSPW could be a significant treatment process aimed at detoxifying OSPW.

An Imaging Flow Cytometry Protocol for Studying Immunoregulatory Receptor-Mediated Regulation of Phagocytosis.

Advances in flow cytometry have allowed for innovative functional investigations of innate immune cell responses. Imaging flow cytometers combine the imaging capabilities of microscopy with rapid, high-throughput data acquisition attributes of standard flow cytometers. Here, we describe a detailed method for co-expressing stimulatory and inhibitory immunoregulatory receptor-types in AD293 cells and then measuring receptor cross-talk during the regulation of the phagocytic response. Information on reagent selection, imaging flow cytometry calibration, and automated template analyses are included.

Isolation of Skin Leukocytes Uncovers Phagocyte Inflammatory Responses During Induction and Resolution of Cutaneous Inflammation in Fish.

Leukocytes offer a critical layer of protection to the host following skin infections. Delineating the kinetics of cutaneous leukocyte recruitment as well as their anti-microbial and regulatory profiles is challenging since it requires the isolation of adequate cell numbers and maintenance of their functional properties. Herein, we took advantage of a modified procedure to gain insights into the contributions of fish phagocytes through induction and resolution phases of acute cutaneous inflammation in goldfish (Carassius auratus). Our data shows early upregulation of pro-inflammatory cytokines and chemokines, which was paired with neutrophil-dominant leukocyte migration of neutrophils from circulation to the injury site. Recruited neutrophils were associated with high levels of reactive oxygen species (ROS). Following pathogen elimination, a reduction in ROS levels and pro-inflammatory cytokines expression preceded the resolution of inflammation. These results provide a better understanding of the cutaneous immune responses in fish. Moreover, the increased viability and functionality of isolated skin leukocytes opens the door to better understand a range of additional skin diseases.

Effect of amino acid composition of elastin-like polypeptide nanoparticles on nonspecific protein adsorption, macrophage cell viability and phagocytosis.

Development of elastin-like polypeptide (ELP) biomaterials is widespread, but information critical for clinical deployment is limited, with biocompatibility studies focused on a narrow cross-section of ELP sequences. Macrophages can impair biomaterial systems by degrading or isolating the biomaterial and by activating additional immune functions. Their phagocytic response will reveal early immune biocompatibility of ELP nanoparticles (NPs). This study examines that response, induced by the adsorbed protein corona, as a function of ELP guest amino acid, chain length and NP diameter. The breadth of proteins adsorbed to ELP NPs varied, with valine-containing ELP NPs adsorbing fewer types of proteins than leucine-containing constructs. Particle diameter was also a factor, with smaller leucine-containing ELP NPs adsorbing the broadest range of proteins. Macrophage viability was unaffected by the ELP NPs, and their phagocytic capabilities were unimpeded except when incubated with a 500 nm valine-containing 40-mer. This NP significantly decreased the phagocytic capacity of macrophages relative to the control and to a corresponding 500 nm leucine-containing 40-mer. NP size and the proportion of opsonin to dysopsonin proteins likely influenced this outcome. These results suggest that certain combinations of ELP sequence and particle size can result in an adsorbed protein corona, which may hinder macrophage function.

Identification of distinct LRC- and Fc receptor complex-like chromosomal regions in fish supports that teleost leukocyte immune-type receptors are distant relatives of mammalian Fc receptor-like molecules.

Leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor-types originally identified in the channel catfish (Ictalurus punctatus (Ip)LITRs). Phylogenetic analyses of LITRs show that they share distant evolutionary relationships with important mammalian immunoregulatory receptors belonging to the Fc receptors family and the leukocyte receptor complex (LRC), but their syntenic relationships with these immunoglobulin superfamily members have not been investigated. To further examine the possible evolutionary connections between teleost LITRs and various mammalian immunoregulatory receptor-types, we surveyed the genomic databases of representative vertebrate taxa and our results show that teleost LITRs generally exist in large genomic clusters, which are linked to vangl2, arhgef11, and slam family genes, features that are also shared by amphibian and mammalian Fc receptor-like molecules (FCRLs). Moreover, detailed phylogenetic comparisons between the individual Ig-like domains of LITRs and mammalian FCRLs shows that these receptors share related Ig-like domains indicative of their common ancestry. However, contrary to our previous reports, no supportive evidence for phylogenetic relationships between the Ig-like domains of LITRs with the Ig-like domains of LRC-encoded mammalian immunoregulatory receptors was found. We also identified an LRC-like region in the zebrafish genome, but no expanded litr-related genes were located in this region. Similarly, no lilr-related genes were found in spotted gar, a representative basal ray-finned fish. Finally, two distantly related fcrls and an LRC-like gene were identified in the elephant shark genome, suggesting that the loss of an immunoregulatory receptor-containing LRC region may be unique to ray-finned fish.

Polymer-coated nanoparticle protein corona formation potentiates phagocytosis of bacteria by innate immune cells and inhibits coagulation in human plasma.

Nanoparticles (NPs) that are exposed to blood are coated with an assortment of proteins that establish their biological identity by forming the interface between the NP and the cells and tissues of the body. The biological relevance of this protein corona is often overlooked during toxicological assessments of NPs. However, accurate interpretation of biological outcomes following exposure to NPs, including activation of coagulation, opsonization of pathogens, and cellular phagocytosis, must take this adsorbed proteome into account. In this study, we examined protein coronas on the surface of five poly(acrylic acid) (PAA) metal-oxide NPs (TiO2, CeO2, Fe2O3, ZnO, and PAA-capsules) following exposure to human plasma for key markers of various host response pathways, including humoral immunity and coagulation. We also evaluated the impacts of pre-exposing serum proteins to PAA-NPs on the opsonization and phagocytosis of bacteria by two immune cell lines. Results demonstrated that each PAA-NP type adsorbed a unique profile of blood proteins and that protein-coated PAA-NPs significantly inhibited human plasma coagulation with PAA-zinc oxide NPs and their associated proteome fully abrogating clotting. Protein-coated PAA-NPs also resulted in a 50% increase in phagocytic activity of RBL-2H3 cells and a 12.5% increase in phagocytic activity in the RAW 264.7 cell line. We also identified numerous structural, coagulation, and immune-activating proteins in the adsorbed protein corona, which resulted in altered biological function. Overall, our findings demonstrate that the formation of protein coronas on the surface of NPs plays an important role in directing the biological outcomes of opsonization, cell phagocytosis, and blood coagulation.

A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins shown to regulate several innate immune cell effector responses, including phagocytosis. The precise mechanisms of IpLITR-mediated regulation of the phagocytic process are not entirely understood, but we have previously shown that different IpLITR-types use classical as well as novel pathways for controlling immune cell-mediated target engulfment. To date, all functional assessments of IpLITR-mediated regulatory actions have focused on the independent characterization of select IpLITR-types in transfected cells. As members of the immunoglobulin superfamily, many IpLITRs share similar extracellular Ig-like domains, thus it is possible that various IpLITR actions are influenced by cross-talk mechanisms between different IpLITR-types; analogous to the paired innate receptor paradigm in mammals. Here, we describe in detail the co-expression of different IpLITR-types in the human embryonic AD293 cell line and examination of their receptor cross-talk mechanisms during the regulation of the phagocytic response using imaging flow cytometry, confocal microscopy, and immunoprecipitation protocols. Overall, our data provides interesting new insights into the integrated control of phagocytosis via the antagonistic networking of independent IpLITR-types that requires the selective recruitment of inhibitory signaling molecules for the initiation and sustained cross-inhibition of phagocytosis.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification.

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

Inorganic fraction of oil sands process-affected water induces mammalian macrophage stress gene expression and acutely modulates immune cell functional markers at both the gene and protein levels.

The focus of the present study was to examine the acute immunotoxic properties of oil sands process-affected waters (OSPW) using the RAW 264.7 macrophage cell line. Specifically, we used a quantitative PCR assay to monitor changes in the expression of stress, cytokine, and antimicrobial enzyme genes in activated macrophages following acute (i.e. < 24 h) exposure of the cells to whole OSPW and its fractions. Overall, our data shows that OSPW inorganic fraction (IF) significantly induces the expression of genes associated with oxidative stress and DNA damage and that the OSPW-IF also significantly augmented cytokine gene expression. These effects are similar to what was observed following whole OSPW exposures, which contrasts the minimal effects observed when cells were treated with equivalent doses of the OSPW organic fraction (OF). Surprisingly, OSPW-IF had reciprocal effects on gene and protein expression levels of two key macrophage enzymes (e.g. inducible nitric oxide (iNOS) synthase and arginase), which indicates that components within OSPW-IF have the unique ability to alter the overall functional states of macrophage by polarizing them towards an alternatively activated status; concomitant with the reciprocal depression of iNOS levels and enhanced expression and activity of arginase. Collectively, these findings show that at sub-lethal exposure doses, the inorganic constituents of OSPW have significant immunotoxicological properties that could potentially affect innate cellular defense responses of exposed animals.

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions.

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.

Exposure to Organic Fraction Extracted from Oil Sands Process-Affected Water Has Negligible Impact on Pregnancy and Lactation of Mice.

Dissolved organic compounds are major contaminants in oil sands process-affected water (OSPW), of which naphthenic acids (NAs) are one of the main persistent toxicants. In the present study, we explore the toxic effects of the organic fraction extracted from OSPW (OSPW-OF) in mice during pregnancy and lactation. Here, we report that acute oral exposure of female Balb/c mice during gestation, and subchronic exposure throughout gestation and lactation to OSPW-OF (containing naturally occurring levels of NAs found in tailings ponds), had negligible effects on their reproductive performance. Specifically, mating behavior, pregnancy success, embryonic implantation, gestation period, litter size, and offspring viability were not affected by OSPW-OF containing up to 55 mg/L NAs. OSPW-OF exposure also did not affect plasma concentrations of pregnancy-associated hormones or pro- and anti-inflammatory cytokines, and it had minimal effects on liver stress gene expression. This study presents the first comprehensive in vivo analysis of mammalian toxicity associated with OSPW-OF exposure. Overall, our results suggest that the risk of acute and subchronic toxicity to mice exposed to OSPW-OF at environmentally relevant concentrations of NAs in contaminated drinking water is likely negligible.

Imaging flow cytometry and confocal microscopy-based examination of F-actin and phosphoinositide dynamics during leukocyte immune-type receptor-mediated phagocytic events.

Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish.

Selective Regulation of Cytoskeletal Dynamics and Filopodia Formation by Teleost Leukocyte Immune-Type Receptors Differentially Contributes to Target Capture During the Phagocytic Process.

Phagocytosis evolved from a fundamental nutrient acquisition mechanism in primitive unicellular amoeboids, into a dynamic and complex component of innate immunity in multicellular organisms. To better understand the cellular mechanisms contributing to phagocytic processes across vertebrates, our research has focused on characterizing the involvement of innate immune proteins originally identified in channel catfish (Ictalurus punctatus) called leukocyte immune-type receptors (IpLITRs). These unique teleost proteins share basic structural as well as distant phylogenetic relationships with several immunoregulatory proteins within the mammalian immunoglobulin superfamily. In the present study, we use a combination of live-cell confocal imaging and high-resolution scanning electron microscopy to further examine the classical immunoreceptor tyrosine-based activation motif (ITAM)-dependent phagocytic pathway mediated by the chimeric construct IpLITR 2.6b/IpFcRγ-L and the functionally diverse immunoreceptor tyrosine-based inhibitory motif-containing receptor IpLITR 1.1b. Results demonstrate that IpLITR 1.1b-expressing cells can uniquely generate actin-dense filopodia-like protrusions during the early stages of extracellular target interactions. In addition, we observed that these structures retract after contacting extracellular targets to secure captured microspheres on the cell surface. This activity was often followed by the generation of robust secondary waves of actin polymerization leading to the formation of stabilized phagocytic cups. At depressed temperatures of 27°C, IpLITR 2.6b/IpFcRγ-L-mediated phagocytosis was completely blocked, whereas IpLITR 1.1b-expressing cells continued to generate dynamic actin-dense filopodia at this lower temperature. Overall, these results provide new support for the hypothesis that IpLITR 1.1b, but not IpLITR 2.6b/IpFcRγ-L, directly triggers filopodia formation when expressed in representative myeloid cells. This also offers new information regarding the directed ability of immunoregulatory receptor-types to initiate dynamic membrane structures and provides insights into an alternative ITAM-independent target capture pathway that is functionally distinct from the classical phagocytic pathways.

Comparison of the Acute Immunotoxicity of Nonfractionated and Fractionated Oil Sands Process-Affected Water Using Mammalian Macrophages.

OSPW is a complex mixture of inorganic and organic substances and its principal toxic components have yet to be fully characterized. Previously, we showed in vitro that the oil sands process-affected water (OSPW) organic fraction (OF) caused a concentration-dependent immunotoxicity in mammals. In the present study we further explore the immunotoxicological properties of OSPW in mammals using a series of in vitro bioassays. Specifically, using the RAW 264.7 mouse macrophage cell line we show that whole OSPW containing naphthenic acid (NA) concentrations ranging from 12 to 18 mg/L, significantly inhibited cell proliferation, reduced cell viability, and was directly cytotoxic, whereas the exposure of cells to equivalent doses of the OSPW-OF had no measurable effects. Whole OSPW exposures also caused morphological changes in RAW 264.7 cells, and at sublethal doses (i.e., 10 mg/L) it induced the early expression of the stress genes hmox1 and gadd45. In addition, at NA concentrations of 10 mg/L, whole OSPW but not the OSPW-OF had significant effects on pro-inflammatory cytokine mRNA levels and cytokine protein secretion activities. Finally, whole OSPW also impaired the ability of RAW 264.7 cells to perform phagocytosis. Overall, we demonstrate that exposure to whole OSPW (at NA doses ranging from 10 to 20 mg/L), but not the OSPW-OF caused both cytotoxic and immunomodulatory changes in mouse macrophages. This suggests that the complex mixture of inorganic and organic components found in whole OSPW are acutely toxic at much lower doses than we previously reported for the OSPW-OF (i.e., 50 mg/L) due to unknown additive and/or synergistic interactions that likely occur between the various components present in whole OSPW.