Progress 02/01/16 to 01/31/20
Outputs
Target Audience:Academic researchers in aquatic animal disease and basic immunology. Changes/Problems:The changes/problems in approach are specified above in the section describing "what was accomplished under these goals". In short, we were unable to distinguish a definitive population of dendritic cells in primary cultures of trout head kidney cells. This precluded efforts to reintroduce presumptive DCs into live fish in ordert to identify sites of antigen-presentation and ultimately validate the presumptive pool of head kidney-derived dendritic cells in fish. To address this, we contemplated a workaround to Aims 2 and 3 of the proposed studies. Indeed, this workaround was described in the original aims. Briefly, we planned to inject fish directly with bacterial superantigens to initiate polyclonal T-cell proliferation and at varying times thereafter (1, 3 and 7 days) inject them with either BrdU or EdU labeling solutions to identify sites of rapid cell division. Animals would be humanely euthanized 1, 3 and 7 days post injection with BrdU or EdU, tissues isolated and then flash frozen. Click-iTTM EdU reagent chemistry or BrdU immunocytochemistry would then be used to reveal sites of T-cell proliferation in tissue sections from experimental versus control animals. This approach would obviate the need to identify DCs in cell culture or use syngeneic MHC-matched fish which would have been required in the original aims. While this workaround was entirely straightforward and is practiced routinely in rodent models, we hit a roadblock with our local Institutional Biosafety Committee who ruled that we would have to prove that fish were not leaching bacterial supertoxins into the surrounding water (a potential hazard to staff and the local water supply). We were asked to either determine the amount of toxin present in the water (not feasible even at the highest concentrations of toxins possible - submicromole per liter - given the sensitivity of existing tests) or decontaminate the effulent from our continuous flow tanks (which would have required treating thousads of gallons of water with hundreds of gallons of chlorine bleach over a prolonged periods of time - also not possible). In the end, what we considered (and still believe to be) a safe and plausible workaround had to be abandoned. As indicated above, the availability of next generation (single-cell) sequencing methods should enable completion of the proposed studies at some future time. What opportunities for training and professional development has the project provided?We trained two postdoctoral reserach associates (Drs. Wisler Charles and Cengiz Akkale) as well as a visiting Assistant Professor from the PRC (Dr. Fei Zhao) during the course of these studies. In the first instance, Drs. Charles and Akkale were trained in the isolation of fish tissues, preparation of single-cell suspensions and primary cell culture of hematopoietic stem cells from rainbow trout. Dr. Zhao became familiar with gene cloning, transformaton and analysis of Mycobacterium marinum. How have the results been disseminated to communities of interest?We reported our findings at joint meetings of the Conference of Research Workers in Animal Disease (CRWAD) and USDA NIFA nimal Health and Well-Being Project Directors between 2016-2019. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our long-term goal is the development of better vaccines for the aquaculture industry. To a large extent, this requires a better understanding of how immune responses are initiated in fish. From work in mammals, we know that initiation of acquired or adaptive immune responses are shaped by the interaction of pathogens and pathogen-associated molecular patterns (PAMPs) with so-called antigen-presenting cells, the most important of which are dendritic cells or DCs. We know little about the nature of these cells in fish, nor do we know much about the anatomical sites at which antigen presenting takes place. To address these fundamental questions, we sought to isolate dendritic cells from rainbow trout and characterize their response to microbial pathogens and their respective PAMPs. This basic knowledge is intended to provide insight into improved vaccine design, increase yield and profitability on fish farms, and limit the use of antibiotics which can contaminate groundwaters and lead to antimicrobial resistance in human pathogens. To begin to characterize trout DCs, we excised regions of the anterior kidney (the primary hematopoietic organ) from adult fish and prepared single cell suspensions that were placed in plastic dishes containing L-15 medium supplemented with fetal bovine serum. Over time (1-2 weeks), cells proliferated and formed hematopoietic foci that continuously spun off loosely adherent cells that were actively motile and were morphologically similar to mammalian DCs. Eventually, these cells became the predominant cells in culture. Our primary goal was to characterize the response of the presumptive trout DCs to pathogens and pathogen associated molecular patterns (alternatively referred to as toll-like receptor agonists) namely, single-stranded RNA (ssRNA); double-stranded RNA (polyI:C); imiquimod (R387); and bacterial flagellin. In mammals, immature DCs are adept at phagocytosing particulate matter and processing foreign antigens for subsequent display on MHC class I and II. When they encounter PAMPs, DCs become activated, terminate phagocytosis and traffic to local lymph nodes where they present antigens. Using fluorescently-tagged latex beads, we were able to show that presumptive trout DCs were in fact capable of phagocytosing particles establishing an important criterion for calling these cells DCs. Additionally, we found a dramatic inhibition of bead uptake (phagocytosis) after treatment with ssRNA and bacterial flagellin consistent with changes to mammalian DCs upon activation. Unexpectedly, imiquimod appeared somewhat toxic to the trout DCs. Our next aim was to determine the precise changes to these cells undergo at the molecular level by transcriptional profiling of mRNA before and after exposure to either toll-like receptor ligands or microbial pathogens. Although our original goal was to include two different pathogens (one intracellular and one extracellular) in our study, in the end we limited the study to a well known intracellular pathogen of freshwater fish, Mycobacterium marinum. We engineered two different bacterial cell lines, one expressing the fluorescent marker, mCherry, in order to visualize their uptake into phagocytic vacuoles of trout DCs, and the second, a conditional lethal strain that we intended to use in evaluating trout DCs for their ability to induce antigen-specific immune responses and protection against bacterial infection following transplantation into live fish. In the first instance, competent M. marinum were transformed by electroporation with varying concentrations of the autonomously replicating plasmid, pVV16, harboring a codon-optimized gene for mCherry under the control of a constitutive M. marinum promoter. To generate a conditional lethal strain, competent M. marinum were transformed with the integrative plasmid, pMT02, harboring coding sequences for Cas9 and a guide RNA that recognizes a breakage site within the plasmid, both under the control of inducible (Tet-ON) promoters. Following integration of plasmid DNA and treatment of cells with anhydrotetracycline, Cas9 mediates bacterial cell death through cleavage of the bacterial chromosome. Although we had planned to use these bacteria in transcriptional profiling studies, we were unable to establish a reliable dose that could be detected following uptake into presumptive trout DCs in culture but that did not kill cells within the time frame of the planned studies. Based on that result, and the fact only a subset of cells in primary cultures took up fluorescently-labelled latex beads, we decided to change our original approach towards the transcriptional profiling studies. With the acquisition of new flow cytometry equipment at Cornell we decided to use fluorescence activated cell sorting to isolate presumptive DCs (based on their ability to phagocytose fluorescent beads) prior to activation with toll-receptor agonists and RNA isolation. We reasoned that an enriched population of cells would off the best opportunity to distinguish changes in gene expression in response to toll-like receptor agonists or pathogen-associated molecular patterns, respectively. Flow cytometry experiments were initially encouraging (we could identify populations of cells that had taken up beads) but yielded a completely unexpected finding in the end. Rather than a single population of phagocytic cells, we found that there were two distinct populations of these cells differing in size (based on forward/side scattering) that arose at different times in culture, one supplanting the other over time. On the one hand, this validated nagging suspicions we had had that cells were changing in appearance over time in culture (getting smaller) based on microscopic observations. At the same time it left us in a quandary regarding which of these two populations were the real DCs and therefore which to focus on. With the idea that we might be able to distinguish these populations and identify a true presumptive DC subset, we did an exhaustive search for antibodies against DC markers from the broader community of fish immunologists in the U.S. and abroad but were unable to identify a source for specific DC markers in fish. This was particularly disappointing since we thought these sources might exist. Needless to say, without these reagents we reached an impasse in our studies. Although we were unable to accomplish what was the principal goal of this work, we may yet be positioned to answer this question using new single-cell sequencing methods (10 X Genomic Chromium platform for single-cell RNAseq) available through Cornell's core sequencing facilities. This approach would permit us to use next generation sequencing methods to obtain transcriptional profiling data from cells within these primary cultures and bin them according to the similarity of gene expression. We fully intend to pursue this goal outside the scope of our original USDA NIFA proposal and using discretionary funds under the control of the PI. Finally, we considered workarounds to Aims 2 and 3 of the original proposal that involved injection of bacterial superantigens directly into fish (to identify sites of T-cell proliferation) but were thwarted by our local Institutional Biosafety Committee who we were unable to convince that the experiments could be done safely despite having been approved for the studies by IACUC.
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Progress 02/01/17 to 01/31/18
Outputs
Target Audience:USDA NIFA award recipients Changes/Problems:We have faced two challenges in accomplishing the goals of the project. The first is defining and enriching the population of cultured cells that we identify as trout dendritic cells. Based on the work we have accomplished to date we are not satisfied that our original conditions are sufficient to provide a highly enriched population of the cells we are most interested in. To address this issue, we intend to use fluroescence activated cell sorting to isolate a population of non-adherent cells that are capable of phagocytosis as the starting population for our RNAseq analysis. A second major challenge we have faced has been recruiting outstanding personnel (graduate students and postdoctoral trainees) to the project. This has affected our overall productivity but should still allow us to complete our major goals. What opportunities for training and professional development has the project provided? Dr. Wisler Charles, a postdoctoral trainee, has conducted the bulk of these experiments and has learned how to culture trout DCs and teach others in the laboratory to do this. How have the results been disseminated to communities of interest?We reported our results at the most recent USDA NIFA Animal Health and Well-Being project directors meeting. What do you plan to do during the next reporting period to accomplish the goals?Our primary goals for the next period are to 1) complete RNAseq analysis of gene expression profiles of trout DCs in the presence and absence of bacterial infection and toll-like receptor agonists including bioinformatics analysis; 2) determine the correct dosing of bacterial superantiges and determine whether these induce lymphocyte proliferation in vivo; and, 3) secure isogenic lines of trout and begin efforts to track the migration of cultured trout DCs in recipient animals.
Impacts
What was accomplished under these goals?
The goal of this work along with its underlying impact will be the design of more effective vaccines for the aquaculture industry. The development of such vaccines requires a detailed understanding of the biology around how microbial pathogens induce robust immune responses in the host. It is clear from studies on mammals that this largely depends on the recognition of molecular patterns on microbes (sometimes referred to as adjuvants) by cells of the host immune system, the most important of which are dendritic cells. To date, dendritic cells (DCs) are not well characterized in fish. This study aims to better undertand DCs in the rainbow trout, and to determine precisely how they respond to adjuvat substance to elicit robust response to vaccines. The long-term impact of these studies will be to improve farm efficiency and profitability, while at the same time reducing potential release of antibiotics and antimicrobials into the environment, which can be highly problematic to the human population. As per our last Progress Report, we have reproduced our initial findings that cells cultured from rainbow trout head kidney continuously produce non-adherent monocytic cells that take on a morphology highly reminiscent of mammalian dendritic cells. We have now completed a detailed analysis of the effects of a number of adjuvant substances (the the toll-like receptor ligands ssRNA; dsRNA (polyI:C); imiquimod (R387); and bacterial flagellin) on these cells, specifically with respect to phagocytosis. Using fluorescently-tagged latex beads, we have found a dramatic inhibition of bead uptake (phagocytosis) after treatment with ssRNA and bacterial flagellin consistent with changes to mammalian DCs upon activation. Unexpectedly, imiquimod appeared somewhat toxic to the trout DCs. Our next aim is to determine the precise changes to these cells at the molecular level by transcriptional profiling of mRNA before and after exposure to either toll-like receptor ligands or microbial pathogens. With regard to the latter, we have constructed two engineered cell lines of Mycobacterium marinum, a well known pathogen of freshwater fish including rainbow trout. One of these cell lines expresses the fluorescent marker, mCherry, that allows us to visualize their uptake into phagocytic vacuoles of trout DCs, and the second, is a conditional lethal strain that we will use in evaluating trout DCs for their ability to induce antigen-specific responses in fish lymphocytes and protection against bacterial infection following transplantation into live fish. In the first case, competent M. marinum were transformed by electroporation with varying concentrations of the autonomously replicating plasmid, pVV16, harboring a codon-optimized gene for mCherry under the control of a constitutive M. marinum promoter. To generate a conditional lethal strain, competent M. marinum were transformed with the integrative plasmid, pMT02, harboring coding sequences for Cas9 and a guide RNA that recognizes a breakage site within the plasmid, both under the control of inducible (Tet-ON) promoters. Following integration of plasmid DNA and treatment of cells with anhydrotetracycline, Cas9 mediates bacterial cell death through cleavage of the bacterial chromsosme. We are in the process of determining the optimal dosages of the two bacterial cell lines for infection of trout DCs in culture prior to transcriptional profiling and lymphocyte activation studies. We have made a change to the original proposed approach towards transcriptional profiling of trout dendritic cells based on results obtained in the current reporting period. Specifically, we will attempt to use fluorescence activated cell sorting to purify populations of DC-like cells that are capable of phagocytosing either latex beads or fluorescently-tagged bacteria prior to RNA isolation. We believe this approach will help enrich the DC-like cell populations and offer the best chance of identifying transcriptional changes in response to toll-like receptor agonists or pathogen-associated molecular patterns, respectively. Lastly, we are continuing to identify and determine optimal levels of bacterial superantigens in injected fish. This aim is directed at inducing MHC class II-dependent polyclonal T-cell activation with the goal of identifying where in fish antigen-presentation takes place.
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Progress 02/01/16 to 01/31/17
Outputs
Target Audience:Academic researchers; Agricultural extension agents; Fish farmers; USDA panel members Changes/Problems:The only major challenge associated with this project has been in staffing. We had recruited a highly qualified Research Associate as the primary person to do the experimental work but that person took another job at the start of the initial funding period. This set us back a couple of months in terms of identifying and hiring a new person to fill this role. We have done this and are now on track to perform the necessary studies. What opportunities for training and professional development has the project provided?Dr. Wisler Charles, a post-doctoral trainee, has been conducting the bulk of these experiments and has learned how to culture rainbow trout dendritic cells, and teach others in the laboratory how to do this. How have the results been disseminated to communities of interest?Poster presentation at the recent meeting for USDA-NIFA Project Directors. What do you plan to do during the next reporting period to accomplish the goals?By the next reporting period we expect to have constructed and analyzed mRNA expression patterns from cultured rainbow trout DCs by RNAseq; contructed and/or obtained fluorescent reporter strains of Mycobacterium marinum and Yersinia ruckeri;determined the effects of bacterial infection on trout DCs; obtained isogenic lines of rainbow trout and cultured dendritic cells from the head kidneys of these animals; begun to test whether bacterial superantigens loaded on trout DCscan induce T-cell responses in cells in culture; begun to examine the migration patterns of trout cells in live fish.
Impacts
What was accomplished under these goals?
To date we have made substantial progress towards completion of Aim 1 of our proposal. Specifically, we have reproduced our initial findings and been able to culture cells that appear morphologically similar to mammalian dendritic cells starting with primary cultures of rainbow trout head kidney. Under the appropriate conditions, we find that stem cells from head kidney explants continuously producenon-adherent monocytic cells in culture that take on the appearanceof dendritic cells (DCs) over time. We are currently testing these cells with a range of toll-receptor agonists in order to determine their effects on phagocytic activity (maturation state) and mRNA expression of putative DCs. In addition, we are culturing the bacterial pathogen, Mycobacterium marium, to examine the examine the effects of this agent on dendritic cell activity. As part of that effort, bacteria are being transformed with genes encoding fluorescent reporter proteins to track the presence of infected cells in live fish. We anticipate the construction and sequencing of libraries of mRNA from rainbow trout dendritic cell cultures in the next 60-90 days. We have just begun Aim 2 of our original proposal that seeks to usebacterial superantigens to test the ability of rainbow trout DCs to stimulate T-cell proliferation in culture and determine sites of antigen-presentation in vivo.
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