Source: MISSISSIPPI STATE UNIV submitted to
DIRECT BINDING OF NON-SPECIFIC CYTOTOXIC CELLS (NCCS) TO BACTERIA IS AN IMPORTANT INNATE IMMUNE MECHANISM IN CATFISH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1027934
Grant No.
2022-67016-36333
Project No.
MISV-371080
Proposal No.
2021-06905
Multistate No.
(N/A)
Program Code
A1221
Project Start Date
Jan 1, 2022
Project End Date
Dec 31, 2024
Grant Year
2022
Project Director
Petrie-Hanson, L.
Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
Dept of Comparative Biomed Sci
Non Technical Summary
Non-specific cytotoxic cells (NCCs) are a unique group of cells that are present in fish and amphibians, but not mammals. These cells are innate immune cells and have functions similar to Natural Killer (NK) cells and T cytotoxic cells. We have determined that catfish NCCs directly bind bacteria. Direct bacterial killing by cytotoxic cells has been demonstrated in one mammalian cell model but not in any fish model. We hypothesize that direct NCC:bacteria binding and killing is an important immune mechanism in fish. This project has two objectives: 1. Determine how NCCs directly kill bacteria after they bind. We will use two bacteria: Edwardsiella ictaluri and Edwardsiella piscicida. 2. Visualize how NCCs and bacteria bind. Because hybrid catfish are important in the industry, these experiments will be repeated with hybrids. Rag1-/- mutant zebrafish will be used for adoptive NCC transfers under a different IACUC protocol. Completion of this project will provide key information about how catfish NCCs kill bacteria. These results will increase our understanding of how catfish fight bacterial diseases and will lead to useful methods to prevent or reduce disease loss in the catfish industry.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31137101090100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3710 - Catfish;

Field Of Science
1090 - Immunology;
Goals / Objectives
Determine the basic mechanisms required for direct bacterial binding and killing by catfish NCCs.Is complement mediated direct binding and killing used by NCCsIs antibody dependent cellular cytotoxicity (ADCC) another mechanism involved in direct bacterial binding by catfish NCCs?Are accessary cells required for direct NCC bacteria binding to occurDoes direct binding occur with bacteria other than E. ictaluri?Do these mechanisms occur in hybrid catfish?Determine if direct NCC:bacteria binding and formation of the immune synapse involves cell membrane changes and cytoskeletal rearrangement.How does the NCC membrane directly bind and engage bacteria?What are the cytoskeletal characteristics of direct NCC:bacteria binding?Do hybrid catfish NCCs do the same thing?
Project Methods
Aim 1: Determine the basic mechanisms required for direct bacterial binding and killing by catfish NCCsWorking hypotheses:Direct binding and killing of bacteria by catfish NCCs is complement mediated.Direct binding and killing of bacteria by catfish NCCs is antibody mediated.Specific Aim 1.1: Does direct bacterial binding require complement proteins? Ak cells will be isolated as described below (combined leukocyte populations)Add sera with complement proteins (treatment 1) or heat inactivated sera without complement proteins (treatment 2); five replicates in each treatmentIncubate cells with bacteria, E. ictaluri, then label cells and dead/live bacterial stainPerform flow cytometry (with controls and compensations) to analyze for number of NCCs, and number of direct bound live bacteria/NCC and number of direct bound dead bacteria/NCCRepeat 1-6 above with magnetic bead purified NCCs instead of combined leukocytes to determine if accessory cells are required for NCCs to bind/kill bacteriaRepeat with E. piscicidaYear 2: Repeat all of above with hybrid catfishSpecific Aim 1.2: Does direct NCC:bacteria binding and killing involve Antibody Dependent Cellular Cytotoxicity (ADCC)? This aim will be completed by following the same experiments in 1.1 with the exception of step 2. Instead of 1. normal or 2. heat-inactivated sera being used; the two treatments are 1. sera from vaccinated fish (that will contain specific antibodies against the bacteria the NCCs will be incubated with) and 2. sera from sham vaccinated fish that will not contain specific antibodies against the bacteria the NCCs will be incubated with. The experimental design will be the same as described in 1.1.Aim 2: Visualize the NCC:bacteria immune synapse by confocal microscopy and determine if cytoskeletal changes occur.Working hypothesis: NCC surface binding of bacteria involves cell membrane ruffling and cytoskeletal rearrangement.Specific Aim 2.1: Signaling and modulation of the NCC cytoskeletonSignalingAk cells will be isolated as described above (combined leukocyte populations)Treatment 1: to inhibit calcium-dependent mechanisms, incubate cells in EDTA (1 mM) as in Hohn et al 2009; Treatment 2: cells in HBSS; five replicates in each treatmentIncubate cells with bacteria, E. ictaluriAdd 5C6:FITC to label NCCsPerform flow cytometry and repeat as described in 1.1Experimental design will be the same as described in 1.1.Cytoskeletal rearrangement, or modulationAk cells will be isolated as described above (combined leukocyte populations)Treatment 1: to inhibit cytoskeletal rearrangement, Cytochalasin D (CCD) (Sigma-Aldrich Inc., St. Louis, MO) will be used (Hohn et al 2009). Treatment 2: cells in HBSS; five replicates in each treatmentIncubate cells with bacteria, E. ictaluriAdd 5C6:FITC to label NCCsPerform flow cytometry and repeat as described in 1.1Experimental design will be the same as described in 1.1.Specific Aim 2.2: Visualize NCC:bacteria immune synapse by confocal microscopy; determination of cell membrane morphological changesAk cells will be isolated and NCCs purified by magnetic bead sorting with mAb 5C6.Treatment 1: incubate cells with E. ictaluri, treatment 2: cells alone; five replicates in each treatmentRinse cellsNCCs will be loaded with actin or tubulin dye by incubation with CellLight® Actin-GFP, and BacMam 2.0CellLight® Tubulin-RFP, BacMam 2.0NCCs and mcherry:E. ictaluri will be co-incubated while being viewed on a Leica DMi8 confocal microscope with LAS X Navigator software for cell visualization, time lapse experiments and analysis.Repeat above with magnetic bead purified NCCsRepeat above with E. piscicidaYear 2: Repeat 1 through 9 above with hybrid catfishExperimental design is the same as described in 1.1.How data will be analyzedData collected will be flow cytometry and confocal images. For flow cytometry, One-way analysis of variance (ANOVA) will be used for data analysis. Statistical analyses will be conducted using SPSS statistical package version 25.0 (SPSS Inc., Chicago, IL, USA). In all statistical tests, values will be considered significantly different at p<0.05. Power analysis: using phagocytosis assays as a reference for assay variability, 5 replicate assays will detect a 10% change in phagocytosis at p<0.05 at a power of 0.89 (type II error rate of 0.11).

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The target audience are fish immunology researchers and fish producers. Changes/Problems:During year 1, the graduate student resigned from the graduate program and the Research Associate/Technical support resigned and relocated to another job. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The major activities completed include the initiation of the research project. None of the objectives have been completed yet. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will accomplish Aim 2, Visualizing the immune synapse and cytoskeletal rearrangements.

Impacts
What was accomplished under these goals? The initiation of the research project was completed. Actin & Tubulin Staining Test 2- Removespleen, liver, kidney fromfish. Place each tissue in a C tube with 3 ml of FACS buffer. Run the samples on designated programs. Pour off supernatant-add 4 ml of FACS buffer-add 500 microliters of cells-add 1 microliter of each stain. Incubate for 30 or 60 minutes at 37 degrees Celsius.Centrifuge at 300xg for 10 mins. Pour off supernatant, cells should stay at the bottom of tube. Add 1 ml FACS buffer. Place in well plate. Spleen and kidney tissues were run in the normal program. The liver tissues were dissociated using 36, 36, 36. Alot of background was generated with these steps. Actin & Tubulin Staining Test 3-Removespleen, liver, kidney. Place each tissue intube with 3 ml of FACS buffer. Run the samples. Filter cells into tube-centrifuge for 10 minutes. Pour off supernatant-add 2 ml of FACS buffer-count cells. Dilute cells to 105/ml. Add 500 ul of cells. Add 1 ul of each stain. Incubate for 30 or 60 minutes at 37 degrees C.Centrifuge-pour off supernatant-add 1 ml of FACS buffer. Place in well plate. Run on dissociator using program 9,15,9 for spleen & kidney and program 36,36,36 for liver.We were able to see both colors in catfish and zebrafish cells. Not much background present compared to previous procedure. Octodissociator modifications & associated survival trial 1-Removespleen-cut into 3 sections. Place each section in C tube with 3 ml of FACS buffer. Remove the posterior kidney-cut into 3 sections. Place each section of in a C tube with 3 ml of FACS buffer. Perfuse the liver with 20 ml of PBS. Cut into 3 sections, place in a C tube with 3 ml of RPMI. Run samples. Filter samples with a 70 um MACS SmartStrainer into a 50 ml tube. Count the cells-centrifuge-Pour off supernatant-Add 2 ml of FACS buffer. Layer cells ongradient and centrifuge.Remove buffy layer-put in new tube-add 1 ml of FACS buffer-centrifuge at 500xg for 5 mins. Pour off supernatant-add 1 ml of FACS buffer. Count the cells. Before gradient Octodissociator Trial 1-In liver tissue, normal treatment resulted in 3.35 x 107, with 59% live and 41% dead. The '36, 36, 36' treatment resulted in 1.65 x 107 cells/mL with 24% live and 76% dead. 2x normal treatment resulted in 2.21 x 107 cells/mL with 30% live and 70% dead. In kidney tissue, normal treatment resulted in 3.00 x 107 cells/mL with 50% live and 50% dead, the '9,15,9' treatment resulted in 2.65 x 107 cells/mL with 36% live and 64% dead and 2x normal treatmentresulted in 2.82 x 107 cells/mL with 52% live and 48% dead. In spleen tissue, normal treatment resulted in 1.00 x 107 cells/mL with 18% live and 82% dead. The '9,15,9' treatment resulted in 1.13 x 107 cells/mL with 13% live and 87% dead, and2x normal treatment resulted in 9.31 x 107 cells/mL with 20% live and 80% dead. After gradient Octodissociator Trial 1- In liver tissue, normal treatment resulted in 3.03 x 106, with 13% live and 87% dead. The '36, 36, 36' treatment resulted in 4.39 x 106 cells/mL with 16% live and 84% dead. 2x normal treatment resulted in 2.79 x 106 cells/mL with 12% live and 88% dead. In kidney tissue, normal treatment resulted in 7.54 x 106 cells/mL with 82% live and 18% dead, '9,15,9' treatment resulted in 6.77 x 106 cells/mL with 83% live and 17% dead, and 2x normal treatment resulted in 1.21 x 106 cells/mL with 24% live and 76% dead. In spleen tissue, normal treatment resulted in 5.10 x 106 cells/mL with 23% live and 77% dead. The '9,15,9' treatment resulted in 1.79 x 106 cells/mL with 26% live and 74% dead, and2x normal treatment resulted in 2.28 x 106 cells/mL with 14% live and 86% dead. Octodissociator modifications & associated survival trial 2- Perfusespleen with 5 ml of PBS. Remove fromfish-cut into 3 sections. Placein a C tube with 3 ml of FACS buffer. Remove posterior kidney-cut into 3 sections. Place in a C tube with 3 ml of RPMI. Perfuse the liver with 20 ml of PBS. Remove the liver-cut into 3 sections. Placein a C tube with 3 ml of FACS buffer. Add 60 ul of serum to the normal run time samples. Add 150 ul of seru to the 1.5 run time samples. Runsamples. Filterwith a 40 um strainer into a 50 ml tube. Count cells-centrifuge-pour off supernatant-add 2 ml of FACS buffer. Layercells onto gradient-centrifuge at 800xg for 15 mins. Remove buffy layer-put in new tube. Add 1 ml of FACS buffer. Centrifuge at 300xg for 5 mins. Pour off supernatant. Add 1 ml of FACS buffer. Count cells. Before gradient Octodissociator Trial 2-In liver tissue, normal treatment resulted in 3.35 x 107, with 59% live and 41% dead. The '36, 36, 36' treatment resulted in 3.26 x 107 cells/mL with 57% live and 43% dead. 2x normal treatment resulted in 2.80 x 107 cells/mL with 42% live and 58% dead. In kidney tissue, normal treatment resulted in 1.55 x 107 cells/mL with 20% live and 80% dead, the '9,15,9' treatment resulted in 2.09 x 107 cells/mL with 18% live and 82% dead-2x normal treatment resulted in 2.19 x 107 cells/mL with 30% live and 70% dead. In spleen tissue, normal treatment resulted in 1.05 x 107 cells/mL with 82% live and 18% dead. The '9,15,9' treatment resulted in 1.76 x 107 cells/mL with 31% live and 69% dead, and 2x normal treatment resulted in 7.59 x 106 cells/mL with 78% live and 22% dead. After gradient Octodissociator Trial 2-In liver tissue, normal treatment resulted in 3.06 x 106, with 24% live and 76% dead. The '36, 36, 36' treatment resulted in 3.04 x 106 cells/mL with 24% live and 76% dead. The 2x normal treatment resulted in 2.99 x 106 cells/mL with 24% live and 76% dead. In kidney tissue, normal treatment resulted in 2.55 x 106 cells/mL with 86% live and 14% dead, the '9,15,9' treatment resulted in 6.38 x 106 cells/mL with 88% live and 12% dead, and 2x normal treatment resulted in 7.24 x 106 cells/mL with 89% live and 11% dead. In spleen tissue,normal treatment resulted in 1.23 x 106 cells/mL with 17% live and 83% dead. The '9,15,9' treatment resulted in 1.05 x 106 cells/mL with 36% live and 64% dead, and2x normal treatment resulted in 1.44 x 106 cells/mL with 69% live and 31% dead. Octodissociator & associated survival trial 3-Remove the spleen-cut into 3 sections. Placein a C tube with 3 ml of RPMI. Perfuse the liver with 20 ml of PBS. Remove the liver-cut into 3 sections. Place in tube with 2,940 ml of RPMI. Add 60 ul of serum tosamples. Runsamples. Filter with a 70 um and 30 um smartstrainer into a 50 ml tube. Count the cells-centrifuge at 300xg for 10 mins. Pour off supernatant-add 2 ml of FACS buffer. Layer the cells ongradient and centrifuge. Remove buffy layer-put innew 14ml tube. Add 1 ml of FACS buffer. Centrifuge.Pour off supernatant-cells should stay at the bottom. Add 1 ml of FACS buffer. Count cells. Before gradient Octodissociator Trial 3-In liver tissue, normal treatment resulted in 1.10 x 107, with 27% live and 73% dead. The '36, 36, 36' treatment resulted in 2.87 x 107 cells/mL with 60% live and 40% dead. The 2x normal treatment resulted in 2.53 x 107 cells/mL with 34% live and 66% dead. In spleen tissue, normal treatment resulted in 8.34 x 106 cells/mL with 86% live and 14% dead. The '9,15,9' treatment resulted in 8.75 x 106 cells/mL with 4% live and 96% dead, and 2x normal treatment resulted in 1.17 x 107 cells/mL with 86% live and 14% dead. After gradient Octodissociator Trial 3-In liver tissue, normal treatment resulted in 3.33 x 106, with 18% live and 82% dead. The '36, 36, 36' treatment resulted in 8.43 x 106 cells/mL with 28% live and 72% dead. 2x normal treatment resulted in 3.21 x 106 cells/mL with 22% live and 78% dead. In spleen tissue, normal treatment resulted in 1.16 x 106 cells/mL with 12% live and 83% dead. The '9,15,9' treatment resulted in 5.51 x 105 cells/mL with 36% live and 64% dead, and 2x normal treatment resulted in 1.44 x 106 cells/mL with 24% live and 76% dead.

Publications