Transcriptome Profiling of Atlantic Salmon Adherent Head Kidney Leukocytes Reveals That Macrophages Are Selectively Enriched During Culture.

The Atlantic salmon (Salmo salar) is an economically important fish, both in aquaculture and in the wild. In vertebrates, macrophages are some of the first cell types to respond to pathogen infection and disease. While macrophage biology has been characterized in mammals, less is known in fish. Our previous work identified changes in the morphology, phagocytic ability, and miRNA profile of Atlantic salmon adherent head kidney leukocytes (HKLs) from predominantly "monocyte-like" at Day 1 of in vitro culture to predominantly "macrophage-like" at Day 5 of culture. Therefore, to further characterize these two cell populations, we examined the mRNA transcriptome profile in Day 1 and Day 5 HKLs using a 44K oligonucleotide microarray. Large changes in the transcriptome were revealed, including changes in the expression of macrophage and immune-related transcripts (e.g. csf1r, arg1, tnfa, mx2), lipid-related transcripts (e.g. fasn, dhcr7, fabp6), and transcription factors involved in macrophage differentiation and function (e.g. klf2, klf9, irf7, irf8, stat1). The in silico target prediction analysis of differentially expressed genes (DEGs) using miRNAs known to change expression in Day 5 HKLs, followed by gene pathway enrichment analysis, supported that these miRNAs may be involved in macrophage maturation by targeting specific DEGs. Elucidating how immune cells, such as macrophages, develop and function is a key step in understanding the Atlantic salmon immune system. Overall, the results indicate that, without the addition of exogenous factors, the adherent HKL cell population differentiates in vitro to become macrophage-like.

Deciphering the messages carried by extracellular vesicles in hematological malignancies.

Extracellular vesicles (EVs) are nanosized membrane-bound particles released from all living cells examined thus far. EVs can transfer information in the form of proteins, nucleic acids, and lipids from donor cells to recipient cells. Here we summarize recent advances in understanding the role(s) EVs play in hematological malignancies (HM) and outline potential prognostic and diagnostic strategies using EVs. EVs have been shown to promote proliferation and angiogenesis, and alter the bone marrow microenvironment to favour the growth and survival of diverse HM. They also promote evasion of anti-cancer immune responses and increase multi-drug resistance. Using knowledge of EV biology, including HM-specific packaging of cargo, EV based diagnostics and therapeutic approaches show substantial clinical promise. However, while EVs may represent a new paradigm to solve many of the challenges in treating and/or diagnosing HM, much work is needed before they can be used clinically to improve patient outcomes.

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily "monocyte-like" at Day 1 to primarily "macrophage-like" at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

Characterization of miRNAs in Cultured Atlantic Salmon Head Kidney Monocyte-Like and Macrophage-Like Cells.

Macrophages are among the first cells to respond to infection and disease. While microRNAs (miRNAs) are involved in the process of monocyte-to-macrophage differentiation in mammals, less is known in teleost fish. Here, Atlantic salmon head kidney leukocytes (HKLs) were used to study the expression of miRNAs in response to in vitro culture. The morphological analysis of cultures showed predominantly monocyte-like cells on Day 1 and macrophage-like cells on Day 5, suggesting that the HKLs had differentiated from monocytes to macrophages. Day 5 HKLs also contained a higher percentage of phagocytic cells. Small RNA sequencing and qPCR analysis were applied to examine the miRNA diversity and expression. There were 370 known mature Atlantic salmon miRNAs in HKLs. Twenty-two miRNAs (15 families) were downregulated while 44 miRNAs (25 families) were upregulated on Day 5 vs. Day 1. Mammalian orthologs of many of the differentially expressed (DE) miRNAs are known to regulate macrophage activation and differentiation, while the teleost-specific miR-2188, miR-462 and miR-731 were also DE and are associated with immune responses in fish. In silico predictions identified several putative target genes of qPCR-validated miRNAs associated with vertebrate macrophage differentiation. This study identified Atlantic salmon miRNAs likely to influence macrophage differentiation, providing important knowledge for future functional studies.

A Comparison of the Innate and Adaptive Immune Systems in Cartilaginous Fish, Ray-Finned Fish, and Lobe-Finned Fish.

The immune system is composed of two subsystems-the innate immune system and the adaptive immune system. The innate immune system is the first to respond to pathogens and does not retain memory of previous responses. Innate immune responses are evolutionarily older than adaptive responses and elements of innate immunity can be found in all multicellular organisms. If a pathogen persists, the adaptive immune system will engage the pathogen with specificity and memory. Several components of the adaptive system including immunoglobulins (Igs), T cell receptors (TCR), and major histocompatibility complex (MHC), are assumed to have arisen in the first jawed vertebrates-the Gnathostomata. This review will discuss and compare components of both the innate and adaptive immune systems in Gnathostomes, particularly in Chondrichthyes (cartilaginous fish) and in Osteichthyes [bony fish: the Actinopterygii (ray-finned fish) and the Sarcopterygii (lobe-finned fish)]. While many elements of both the innate and adaptive immune systems are conserved within these species and with higher level vertebrates, some elements have marked differences. Components of the innate immune system covered here include physical barriers, such as the skin and gastrointestinal tract, cellular components, such as pattern recognition receptors and immune cells including macrophages and neutrophils, and humoral components, such as the complement system. Components of the adaptive system covered include the fundamental cells and molecules of adaptive immunity: B lymphocytes (B cells), T lymphocytes (T cells), immunoglobulins (Igs), and major histocompatibility complex (MHC). Comparative studies in fish such as those discussed here are essential for developing a comprehensive understanding of the evolution of the immune system.

CD24 is required for regulating gene expression, but not glucose uptake, during adipogenesis.

Adipose tissue dysfunction in obesity and lipodystrophy results in major health complications such as heart disease and stroke, and is associated with an increased risk of some cancers. We have previously found that the cell surface receptor CD24 regulates adipogenesis as measured by lipid accumulation and gene expression in mature adipocytes. How CD24 regulates these processes remains unknown. To begin answering this question, we first determined that CD24 does not affect glucose uptake in differentiating adipocytes in vitro. We then examined changes in global gene expression via DNA microarray in 3T3-L1 adipocytes with siRNA-mediated knock-down of CD24 expression. We found that CD24 expression is necessary for upregulation of up to 134 genes. We validated the CD24-mediated regulation of 4 of these genes during in vitro adipogenesis of 3T3-L1 and primary cells isolated from the inguinal white adipose tissue depots of CD24 knockout mice. Surprisingly, we found that only 1 of these genes was also regulated by CD24 in cells from the epididymal depot. Overall, these data suggest that CD24 is necessary for select gene expression in a depot-specific manner during adipogenesis in vitro. These findings could help elucidate the mechanisms regulating lipid accumulation in adipocytes thereby aiding in the development of novel treatment strategies for obesity and lipodystophy.

Immune modulatory properties of 6-gingerol and resveratrol in Atlantic salmon macrophages.

The innate immune system provides the first barrier to infection and disease through various processes including activation of inflammation. Inflammation is a biological process whereby immune cells, including macrophages, respond to, and work together to eliminate, damage from injury and disease. Chronic or sustained inflammation, however, can be detrimental to the health and growth of animals, including fish. Immune modulating functional plant compounds, supplemented as feed additives, may be safe and natural approaches to controlling inflammation and disease. This project employed an in vitro cell model to assess the effect of two plant phytoextracts, 6-gingerol and resveratrol, on Atlantic salmon macrophages. To determine if these plant compounds can modify Atlantic salmon macrophage function, primary macrophages were isolated from the head kidney, cultured and pre-treated with the compound for 24 hours. The cells were then challenged with a bacterial pathogen-associated molecular pattern (PAMP), lipopolysaccharide (LPS). To determine if the plant compound pre-treatment modifies macrophage response to LPS stimulation, the mRNA expression of anti-bacterial and cytokine genes (COX2, TNFα, IL-1ß, IL-8, Lect-2 and sTLR5) was analyzed by real-time quantitative polymerase chain reaction (qPCR). In addition, the effects of compound pre-treatment on macrophage ROS production and phagocytic responses were analyzed via flow cytometry. We found that both 6-gingerol and resveratrol modified macrophage function, with 6-gingerol having an overall greater effect on expression of the genes analyzed, suggesting that these functional plant compounds may be good candidates as feed additives and should be investigated further.

Transcriptome profiling of antiviral immune and dietary fatty acid dependent responses of Atlantic salmon macrophage-like cells.

BACKGROUND: Due to the limited availability and high cost of fish oil in the face of increasing aquaculture production, there is a need to reduce usage of fish oil in aquafeeds without compromising farm fish health. Therefore, the present study was conducted to determine if different levels of vegetable and fish oils can alter antiviral responses of salmon macrophage-like cells (MLCs). Atlantic salmon (Salmo salar) were fed diets containing 7.4% (FO7) or 5.1% (FO5) fish oil. These diets were designed to be relatively low in EPA + DHA (i.e. FO7: 1.41% and FO5: 1%), but near the requirement level, and resulting in comparable growth. Vegetable oil (i.e. rapeseed oil) was used to balance fish oil in experimental diets. After a 16-week feeding trial, MLCs isolated from fish in these dietary groups were stimulated by a viral mimic (dsRNA: pIC) for 6 h (qPCR assay) and 24 h (microarray and qPCR assays). RESULTS: The fatty acid composition of head kidney leukocytes varied between the two dietary groups (e.g. higher 20:5n-3 in the FO7 group). Following microarray assays using a 44K salmonid platform, Rank Products (RP) analysis showed 14 and 54 differentially expressed probes (DEP) (PFP < 0.05) between the two diets in control and pIC groups (FO5 vs. FO7), respectively. Nonetheless, Significance Analysis of Microarrays (SAM, FDR < 0.05) identified only one DEP between pIC groups of the two diets. Moreover, we identified a large number (i.e. 890 DEP in FO7 and 1128 DEP in FO5 overlapping between SAM and RP) of pIC-responsive transcripts, and several of them were involved in TLR-/RLR-dependent and cytokine-mediated pathways. The microarray results were validated as significantly differentially expressed by qPCR assays for 2 out of 9 diet-responsive transcripts and for all of the 35 selected pIC-responsive transcripts. CONCLUSION: Fatty acid-binding protein adipocyte (fabp4) and proteasome subunit beta type-8 (psmb8) were significantly up- and down-regulated, respectively, in the MLCs of fish fed the diet with a lower level of fish oil, suggesting that they are important diet-responsive, immune-related biomarkers for future studies. Although the different levels of dietary fish and vegetable oils involved in this study affected the expression of some transcripts, the immune-related pathways and functions activated by the antiviral response of salmon MLCs in both groups were comparable overall. Moreover, the qPCR revealed transcripts responding early to pIC (e.g. lgp2, map3k8, socs1, dusp5 and cflar) and time-responsive transcripts (e.g. scarb1-a, csf1r, traf5a, cd80 and ctsf) in salmon MLCs. The present study provides a comprehensive picture of the putative molecular pathways (e.g. RLR-, TLR-, MAPK- and IFN-associated pathways) activated by the antiviral response of salmon MLCs.

Transcriptome profiling of the antiviral immune response in Atlantic cod macrophages.

A study was conducted to determine the transcriptome response of Atlantic cod (Gadus morhua) macrophages to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC), using a 20K Atlantic cod microarray platform and qPCR. We identified 285 significantly up-regulated and 161 significantly down-regulated probes in cod macrophages 24 h after pIC stimulation. A subset of 26 microarray-identified transcripts was subjected to qPCR validation using samples treated with pIC or phosphate-buffered saline (control) over time (3, 6, 12, 24, 48 h), and 77% of them showed a significant response to pIC. The microarray and qPCR analyses in this study showed that pIC induced the expression of cod macrophage transcripts involved in RLR- and TLR-dependent pathogen recognition (e.g. tlr3, tlr7, mda5 and lgp2), as well as signal transducers (e.g. stat1 and nfkbia) and transcription activators (e.g. irf7 and irf10) in the MyD88-independent and dependent signalling pathways. Several immune effectors (e.g. isg15s, viperin, herc4, mip2 and ccl13) were significantly up-regulated in pIC-stimulated cod macrophages. The expression of some transcripts (e.g. irf7, irf10, viperin) was significantly up-regulated by pIC as early as 12 h. All pIC-induced transcripts had peak expression at either 24 h (e.g. tlr7, irf7, mip2) or 48 h (e.g. tlr3, lgp2, stat1). This study suggests possible roles of both vertebrate-conserved (e.g. tlr3 as an up-regulated gene) and fish-specific (tlr22g as a down-regulated gene) receptors in dsRNA recognition, and the importance of conserved and potentially fish-specific interferon stimulated genes in cod macrophages.

Dynamic upregulation of CD24 in pre-adipocytes promotes adipogenesis.

The development of mature adipocytes from pre-adipocytes is a highly regulated process. CD24 is a glycophosphatidylinositol-linked cell surface receptor that has been identified as a critical cell surface marker for identifying pre-adipocytes that are able to reconstitute white adipose tissue (WAT) in vivo. Here, we examined the role and regulation of CD24 during adipogenesis in vitro. We found that CD24 mRNA and protein expression is upregulated early during adipogenesis in the 3T3-L1 pre-adipocytes and in murine primary pre-adipocytes isolated from subcutaneous and visceral WAT, followed by downregulation in mature adipocytes. CD24 mRNA expression was found to be dependent on increased transcription due to increased promoter activity in response to activation of a pre-existing transcriptional regulator. Furthermore, either intracellular cAMP or dexamethasone were sufficient to increase expression in pre-adipocytes, while both additively increased CD24 expression. Preventing the increase in CD24 expression, by siRNA-mediated knock-down, resulted in fewer mature lipid-laden adipocytes and decreased expression of mature adipogenic genes. Therefore, conditions experienced during adipogenesis in vitro are sufficient to increase CD24 expression, which is necessary for differentiation. Overall, we conclude that the dynamic upregulation of CD24 actively promotes adipogenesis in vitro.

Dynamic regulation of CD24 expression and release of CD24-containing microvesicles in immature B cells in response to CD24 engagement.

The glycophosphatidylinositol-anchored cell surface receptor CD24 (also called heat-stable antigen) promotes the apoptosis of progenitor and precursor B-lymphocytes. However, the immediate proximal events that occur after engagement of CD24 in B cells are not precisely understood. Using a bioinformatics analysis of mouse (Mus musculus) gene expression data from the Immunological Genome Project, we found that known vesicle trafficking and cellular organization genes have similar expression patterns to CD24 during B-cell development in the bone marrow. We therefore hypothesized that CD24 regulates vesicle trafficking. We first validated that antibody-mediated engagement of CD24 induces apoptosis in the mouse WEHI-231 cell line and mouse primary bone marrow-derived B cells. We next found that CD24 surface protein expression is rapidly and dynamically regulated in both WEHI-231 cells and primary immature B cells in response to engagement of CD24. The change in surface expression was not mediated by classical endocytosis or exocytosis. However, we found that CD24-bearing plasma membrane-derived extracellular microvesicles were released in response to CD24 engagement. Furthermore, in response to CD24 engagement we observed a clear exchange of CD24 between different populations of B cells. Hence, we show that engagement of CD24 in immature B cells results in a dynamic regulation of surface CD24 protein and a redistribution of CD24 within the population.

Genetic reduction of insulin-like growth factor-1 mimics the anticancer effects of calorie restriction on cyclooxygenase-2-driven pancreatic neoplasia.

Risk of pancreatic cancer, the fourth deadliest cancer in the United States, is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of IGF-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 levels and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing COX-2 [bovine keratin-5 promoter (BK5.COX-2)]. These findings in CR mice correlated with reductions in Ki-67-positive cells, vascular luminal size, VEGF expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101 cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1-deficient (LID) mice. The approximate 65% reduction of serum IGF-1 levels in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy.

Obesity accelerates mouse mammary tumor growth in the absence of ovarian hormones.

Obesity increases incidence and mortality of breast cancer in postmenopausal women. Mechanisms underlying this association are poorly understood. Suitable animal models are needed to elucidate potential mechanisms for this association. To determine the effects of obesity on mammary tumor growth, nonovariectomized and ovariectomized C57BL/6 mice of various body weights (lean, overweight, and obese) were implanted subcutaneously with mammary tumor cells from syngeneic Wnt-1 transgenic mice. In mice, the lean phenotype was associated with reduced Wnt-1 tumor growth regardless of ovarian hormone status. Ovariectomy delayed Wnt-1 tumor growth consistent with the known hormone responsiveness of these tumors. However, obesity accelerated tumor growth in ovariectomized but not in nonovariectomized animals. Diet-induced obesity in a syngeneic mouse model of breast cancer enhanced tumor growth, specifically in the absence of ovarian hormones. These results support epidemiological evidence that obesity is associated with increased breast cancer incidence and mortality in postmenopausal but not premenopausal women. In contrast, maintaining a lean body weight phenotype was associated with reduced Wnt-1 tumor growth regardless of ovarian hormone status.

Low-carbohydrate diet versus caloric restriction: effects on weight loss, hormones, and colon tumor growth in obese mice.

Our objective was to compare the effects of a low-carbohydrate diet to a high-carbohydrate/calorie-restricted diet on weight loss, hormones, and transplanted colon tumor growth. Eighty male C57BL/6 mice consumed a diet-induced obesity regimen (DIO) ad libitum for 7 weeks. From Weeks 8 to 14, the mice consumed a 1) DIO diet ad libitum (HF); 2) low-carbohydrate diet ad libitum (LC); 3) high-carbohydrate diet ad libitum (HC); or 4) HC calorie restricted diet (HC-CR). MC38 cells were injected at Week 15. At the time of injection, the HC-CR group displayed the lowest body weight (25.5 +/- 0.57 g), serum insulin-like growth factor I (IGF-I; 135 +/- 56.0 ng/ml), and leptin (1.0 +/- 0.3 ng/ml) levels. This group also exhibited the longest time to palpable tumor (20.1 +/- 0.9 days). Compared to the HF group, the HC group exhibited lower body weight (39.4 +/- 1.4 vs. 32.9 +/- 0.7 g, respectively), IGF-I (604 +/- 44.2 vs. 243.4 +/- 88.9 ng/ml, respectively), and leptin (15.6 +/- 2.2 vs. 7.0 +/- 0.7 ng/ml, respectively) levels but similar tumor growth. IGF-I levels were lower in the LC group (320.0 +/- 39.9 ng/ml) than the HF group, but tumor growth did not differ. These data suggest LC diets do not slow colon tumor growth in obese mice.

Carbohydrate versus energy restriction: effects on weight loss, body composition and metabolism.

BACKGROUND/AIMS: To compare weight loss, body composition, and metabolic changes in response to carbohydrate versus dietary energy restriction (DER) in obese mice. METHODS: One hundred C57BL/6 mice were randomized into five groups of 20. The group of high-carbohydrate (HC) mice consumed an HC diet ad libitum and the group of high-fat (HF) mice consumed an HF diet ad libitum for 14 weeks. Additional groups consumed the HF diet for 7 weeks ad libitum and during weeks 8-14 were switched to either a low-carbohydrate diet (LC) consumed ad libitum, the HC diet pair-fed (PF) to the energy intake of the LC group, or an HC DER regimen providing 70% of the energy intake of the HF group. RESULTS: At 14 weeks, the LC and HF groups weighed more and exhibited higher percent fat mass and lower bone mineral density than the HC, PF, and DER groups. Relative to the DER group, the LC group displayed comparable serum ketone bodies but higher serum glucose, triglycerides, cholesterol, leptin, insulin, and insulin-like growth factor-1. CONCLUSIONS: In contrast to DER, the LC diet did not cause weight loss or reduce serum markers associated with obesity-related diseases other than diabetes in obese mice, suggesting that carbohydraterestriction without reduced energy intake does not induce weight loss.

Increased tumor growth in mice with diet-induced obesity: impact of ovarian hormones.

Obesity increases the risk of many cancers in both males and females. This study describes a link between obesity, obesity-associated metabolic alterations, and the risk of developing cancer in male and female mice. The goal of this study was to evaluate the relationship between gender and obesity and to determine the role of estrogen status in obese females and its effect on tumor growth. We examined the susceptibility of C57BL/6 mice to diet-induced obesity, insulin resistance/glucose intolerance, and tumors. Mice were injected sc with one of two tumorigenic cell lines, Lewis lung carcinoma, or mouse colon 38-adenocarcinoma. Results show that tumor growth rate was increased in obese mice vs. control mice irrespective of the tumor cell type. To investigate the effect of estrogen status on tumor development in obese females, we compared metabolic parameters and tumor growth in ovariectomized (ovx) and intact obese female mice. Obese ovx female mice developed insulin resistance and glucose intolerance similar to that observed in obese males. Our results demonstrate that body adiposity increased in ovx females irrespective of the diet administered and that tumor growth correlated positively with body adiposity. Overall, these data point to more rapid tumor growth in obese mice and suggest that endogenous sex steroids, together with diet, affect adiposity, insulin sensitivity, and tumor growth in female mice.