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.

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.

Connected component masking accurately identifies the ratio of phagocytosed and cell-bound particles in individual cells by imaging flow cytometry.

Innate immune cell-mediated recognition, capture, and engulfment of large particulate targets such as bacteria is known as phagocytosis. This highly dynamic cellular process involves a series of steps including receptor-mediated target binding, phagocytic cup formation, pseudopod extension, and phagosome closure, which depend on distinct actin polymerization events. Using flow cytometry, precise determination of target locations relative to cell membranes (i.e., surface-bound vs. fully engulfed/internalized) during the phagocytic process is difficult to quantify. Here, we describe the application of new analysis features within the IDEAS® software to distinguish internalized and surface-bound particles on individual cells with a high degree of accuracy and reproducibility. Through the use of connected component masks, the accurate discrimination of surface-bound beads versus those internalized is clearly demonstrated. In addition, we were able to further analyze the ratio of beads that had been surface-bound or internalized within individual cells. This novel method of analyzing the phagocytic process provides more accurate determination of target-cell interactions that will assist in examination of the signalling events that occur during the various stages of phagocytosis. © 2017 International Society for Advancement of Cytometry.

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.

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.

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.

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 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.

Imaging flow cytometry and GST pulldown assays provide new insights into channel catfish leukocyte immune-type receptor-mediated phagocytic pathways.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) control various innate immune cell effector responses including the phagocytic process. This large immunoregulatory receptor family also consists of multiple receptor-types with variable signaling abilities that is dependent on their inherent or acquired tyrosine-containing cytoplasmic tail (CYT) regions. For example, IpLITR 2.6b associates with the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor molecule IpFcRγ-L, and when expressed in mammalian cells it activates phagocytosis using a similar profile of intracellular signaling mediators that also regulate the prototypical mammalian Fc receptor (FcR) phagocytic pathway. Alternatively, IpLITR 1.1b contains a long tyrosine-containing CYT with multifunctional capabilities including both inhibitory and stimulatory actions. Recently, we demonstrated that IpLITR 1.1b activates a unique phagocytic pathway involving the generation of multiple plasma membrane extensions that rapidly capture extracellular targets and secure them on the cell surface in phagocytic cup-like structures. Occasionally, these captured targets are completely engulfed albeit at a significantly lower rate than what was observed for IpLITR 2.6b. While this novel IpLITR 1.1b phagocytic activity is insensitive to classical blockers of phagocytosis, its distinct target capture and engulfment actions depend on the engagement of the actin polymerization machinery. However, it is not known how this protein translates target recognition into intracellular signaling events during this atypical mode of phagocytosis. Using imaging flow cytometry and GST pulldown assays, the aims of this study were to specifically examine what regions of the IpLITR 1.1b CYT trigger phagocytosis and to establish what profile of intracellular signaling molecules likely participate in its actions. Our results show that in stably transfected AD293 cells, the membrane proximal and distal CYT segments of IpLITR 1.1b independently regulate its phagocytic activities. These CYT regions were also shown to differentially recruit various SH2 domain-containing intracellular mediators, which provides new information about the dynamic immunoregulatory abilities of IpLITR 1.1b. Overall, this work further advances our understanding of how certain immunoregulatory receptor-types link extracellular target binding events to the actin polymerization machinery during a non-classical mode of phagocytosis.

Trypsin differentially modulates the surface expression and function of channel catfish leukocyte immune-type receptors.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins that control innate immune cellular responses. Previously, we demonstrated that two representative IpLITR forms, IpLITR 2.6b and IpLITR 1.1b, engage distinct components of the phagocytic machinery resulting in unique target capture and engulfment phenotypes. IpLITR-induced phagocytic mechanisms were also differentially susceptible to temperature and pharmacological inhibitors of canonical signaling mediators. In the present study, we examined the sensitivity of IpLITR-mediated phagocytosis to the endogenous serine-protease trypsin, a well-known mediator of immunoregulatory receptor functions. Trypsin selectively reduced IpLITR 1.1b cell surface expression and phagocytic activity in a dose-dependent manner. We also observed a significant alteration of the IpLITR 1.1b phagocytic phenotype post-trypsin exposure; whereas, the IpLITR 2.6b-mediated target engulfment phenotype was unchanged. Recovery experiments suggested that trypsin-induced inhibition of IpLITR 1.1b-dependent phagocytosis was reversible and that the re-establishment of phagocytic function was associated with a recovery of receptor surface expression. Cell-surface biotinylation and immunoprecipitation studies demonstrated that IpLITR 1.1b normally exists as a mature (∼70 kDa) protein on the cell surface. However, trypsin treatment reduced expression of the mature receptor and processed IpLITR 1.1b into an ∼60 kDa form. The trypsin-generated and putative immature IpLITR 1.1b form was not present on the cell surface; suggesting that the cleaved receptor may have been internalized, post-processing, by regulated endocytosis. Taken together, these results reveal a unique role for trypsin as a selective modulator of IpLITR-mediated phagocytosis and highlight a conserved role for serine proteases as potent immunomodulatory factors.

Biochemical and Functional Insights into the Integrated Regulation of Innate Immune Cell Responses by Teleost Leukocyte Immune-Type Receptors.

Across vertebrates, innate immunity consists of a complex assortment of highly specialized cells capable of unleashing potent effector responses designed to destroy or mitigate foreign pathogens. The execution of various innate cellular behaviors such as phagocytosis, degranulation, or cell-mediated cytotoxicity are functionally indistinguishable when being performed by immune cells isolated from humans or teleost fishes; vertebrates that diverged from one another more than 450 million years ago. This suggests that vital components of the vertebrate innate defense machinery are conserved and investigating such processes in a range of model systems provides an important opportunity to identify fundamental features of vertebrate immunity. One characteristic that is highly conserved across vertebrate systems is that cellular immune responses are dependent on specialized immunoregulatory receptors that sense environmental stimuli and initiate intracellular cascades that can elicit appropriate effector responses. A wide variety of immunoregulatory receptor families have been extensively studied in mammals, and many have been identified as cell- and function-specific regulators of a range of innate responses. Although much less is known in fish, the growing database of genomic information has recently allowed for the identification of several immunoregulatory receptor gene families in teleosts. Many of these putative immunoregulatory receptors have yet to be assigned any specific role(s), and much of what is known has been based solely on structural and/or phylogenetic relationships with mammalian receptor families. As an attempt to address some of these shortcomings, this review will focus on our growing understanding of the functional roles played by specific members of the channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs), which appear to be important regulators of several innate cellular responses via classical as well as unique biochemical signaling networks.

Teleost leukocyte immune-type receptors activate distinct phagocytic modes for target acquisition and engulfment.

Channel catfish (Ictalurus punctatus) IpLITRs belong to the Ig superfamily and regulate innate immune cell effector responses. This study tested the hypothesis that ITAM-dependent and ITAM-independent phagocytic pathways are engaged by different subtypes of the IpLITR family. When stably expressed in RBL-2H3 cells, the ITAM-containing fusion-construct IpLITR 2.6b/IpFcRγ-L stimulated phagocytic responses that were abrogated at suboptimal incubation temperatures and by pharmacological inhibitors of the classic signaling components of the mammalian FcR-dependent phagocytic pathway. Interestingly, the ITIM-containing receptor IpLITR 1.1b also induced phagocytosis through an actin-dependent mechanism, but this process was insensitive to the pharmacological inhibitors tested and remained functional at temperatures as low as 22°C. The IpLITR 1.1b also displayed a unique target-acquisition phenotype that consisted of complex, membranous protrusions, which captured targets in phagocytic cup-like structures but often failed to completely engulf targets. Taken together, these findings suggest that teleost immunoregulatory receptors that associate with ITAM-containing adaptors can engage conserved components of the phagocytic machinery to engulf extracellular targets akin to the classic FcR-mediated response in mammals. Alternatively, IpLITR 1.1b displays a stalled phagocytic phenotype that is likely dependent on the selective recruitment of the minimal molecular machinery required for target capture but results in incomplete target engulfment. Overall, this study demonstrates that IpLITRs can selectively engage distinct components of the phagocytic process and provides important new information regarding the target acquisition as well as internalization mechanisms involved in controlling phagocytic responses across vertebrates.

Induction of phagocytosis and intracellular signaling by an inhibitory channel catfish leukocyte immune-type receptor: evidence for immunoregulatory receptor functional plasticity in teleosts.

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.