ENHANCING IMMUNITY IN PIGS THROUGH NKT-CELLS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0228943
• Project Start Date: Apr 1, 2012
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611

Performing Department
Animal Sciences

Non Technical Summary
This project is focused on strengthening the immune system of young animals. Enhancing immunity to microbial pathogens has wide ranging benefits for both humans and domestic animals during early life when the immune system is underdeveloped and often incapable of efficiently controlling infection. The vulnerability of young animals to microbes and their ability to respond to vaccinations depends largely on how quickly the developing immune system colonizes lymphoid organs. This can vary significantly between individuals due to a multitude of genetic and environmental effects. This study seeks to understand these factors in order to develop strategies to improve the resistance of young animals to infection and to promote the development of a robust immune system that will endure in adults. We will focus on a particular type of white blood cell called natural killer T (NKT) cells that profoundly affects how cells of the immune system identify and destroy pathogens. The development of NKT cells is strongly influenced by specific nutrients the young animal is exposed to during gestation and lactation. One of these is vitamin D. Indeed young mice that do not receive vitamin D during pregnancy and while nursing do not develop normal NKT cells and this defect cannot be corrected even by feeding high levels of vitamin D later in life. This proposal seeks to identify how NKT cells can be used to improve the health of young pigs. First we will characterize these cells in different pig lines to determine how much variation there is in the numbers and function of these cells between breeds. We will then determine whether we can improve immunity in pigs by treating them with specially designed synthetic molecules that activate NKT cells. Lastly, we will determine whether the relationship between vitamin D and NKT cells reported for mice also holds true for pigs. We expect that the outcomes from this study will demonstrate that NKT cell activation is a powerful tool with which to enhance immunity in commercial swineherds and that vitamin D supplementation is important to maintain this cell population in pigs.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1090	60%
315	3510	1090	40%

Knowledge Area
311 - Animal Diseases; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1090 - Immunology;

Keywords

pig

nkt cell

vitamin d

immunity

Goals / Objectives
This project is focused on strengthening the immune system of young animals. Enhancing immunity to pathogens has wide ranging benefits for both humans and domestic animals during early life when the immune system is underdeveloped and often incapable of efficiently controlling infection. The vulnerability of young animals to microbes and their ability to respond to vaccinations depends largely on how quickly the developing immune system colonizes lymphoid organs. This can vary significantly between individuals due to a multitude of genetic and environmental effects. This study seeks to understand these factors in order to develop strategies to improve the resistance of young animals to infection and to promote the development of a robust immune system that will endure in adults. We will focus on a particular type of leukocyte called natural killer T (NKT) cells that profoundly affects how cells of the immune system identify and destroy pathogens. The development of NKT cells is strongly influenced by specific nutrients the young animal is exposed to during gestation and lactation. Mouse studies have identified vitamin D as a nutrient of particular importance for the development of NKT cells. One of our principle objectives is to establish how early vitamin D exposure affects the maturation of NKT cells in pigs. This work may help to identify mechanisms that predispose some animals and humans to infectious agents, and hence reveal dietary and pharmaceutical interventions that prevent disease. To accomplish these goals we propose to: Aim 1: Determine the frequency of NKT cells amongst pig strains and whether their adjuvant effects may be harnessed to augment vaccine responses using the NKT cell-superagonist alpha-galactosylceramide. Aim 2: Determine whether dietary vitamin D supplementation during pregnancy and early postnatal life enhances NKT cell development in pigs. We expect that the outcomes from this study will demonstrate that NKT cell activation is a powerful tool with which to enhance immunity in commercial swineherds and that vitamin D supplementation is important to maintain this cell population in pigs.

Project Methods
For experiments to determine the frequency of natural killer T (NKT) cells in pigs at different ages, 2 ml blood samples will be collected at monthly intervals from birth to 1 year of age. In addition to blood samples, lymphoid organs from pig cadavers will be analyzed for NKT cells. This is necessary because the frequency of NKT cells in peripheral blood and other lymphoid organs is poorly correlated. Additional tissues sampled will include thymus, spleen, liver and lymph nodes. In total, tissue samples will be collected from up to 30 pigs from birth to 1 year of age. In other experiments pigs will be treated with 3 different types of NKT cell agonist (alpha-galactosylceramide, OCH [(2S, 3S, 4R)-1-O-(alpha-D-galactopyranosyl)-N-tetracosanoyl-2 amino-1,3,4-nonanetriol] and c-glycoside) dissolved in phosphate buffered solution (PBS) at concentrations ranging from 0.1 to 10 ug/kg body weight. Studies in mice, humans and non-human primates have demonstrated that these concentrations are well tolerated. The NKT-agonists will be administered once or twice (2 weeks apart) by intramuscular (IM) and intravenous (IV) injection between 1 and 4 weeks of age. The IM and IV injections will be administered on the side of the neck and through the ear vein, respectively. Control animals will be injected with PBS alone. Pigs will be bled once before and three times after administration of the agonists, at 12 h increments. It is estimated that 6 pigs per treatment will be required to accurately measure NKT cell responses. Half of these pigs will be sacrificed 48 h post-treatment to determine NKT cell activation status in peripheral lymphoid organs. Additional studies will be performed in which NKT-agonists (10 ug/kg) will be administered IV or IM with the neoantigen hen egg lysozyme (HEL; 2.5 mg/kg) at one and again at three weeks of age. Blood will be collected once before treatment and again at three and five weeks of age. Pigs treated with HEL peptide alone or PBS will serve as controls. Five pigs per treatment group are required for these studies. In another study pregnant sows will be fed a vitamin D deficient diet and maintained in farrowing facilities that preclude exposure to UV radiation. Sows will be maintained under vitamin D deficient conditions from 45 days post conception until their piglets are weaned at 8 weeks of age (a total of 16 weeks). Piglets from these dams will be maintained in the same facility until weaning. Sows will be bled once per month to monitor plasma vitamin D levels. Piglets will be bled at one, three and five weeks after birth. At five weeks half of the piglets will be sacrificed and their lymphoid organs removed for analysis. All remaining piglets will be place on a complete diet with adequate levels of vitamin D and maintained in outside pens until 4 months of age. They will be bled once per month during this period. At four months of age these pigs will be sacrificed and their tissues collected for analysis. Piglets from dams fed a complete diet during gestation and lactation or lactation only will be used as controls. Ten piglets per treatment group will be used to allow for adequate statistical comparisons.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:NKT cell biologists Swine immunologists Vaccine researchers Industry specialists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals?We will establish whether pigs lacking NKT cell are more susceptible to influenza infections. We will also determine whether pigs infected with influenza can be protected by intranasal administration of therapeutic NKT cell agonists.

Impacts
What was accomplished under these goals? Experiments were carried out to determine the natural role of natural killer T cells for swine immunity against influenza infections. Further studies were conducted to determine how NKT cell agonists can be used to improve swine influenza vaccines and to clear established infections.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: 1. Artiaga, B. L., G. Yang, T. E. Hutchinson, J. C. Loeb, J. A. Richt, J. A. Lednicky, S. Salek-Ardakani, and J. P. Driver. 2016. Rapid control of pandemic H1N1 influenza by targeting NKT-cells. Sci Rep 6: 37999. 2. Artiaga, B. L., G. Yang, T. J. Hackmann, Q. Liu, J. A. Richt, S. Salek-Ardakani, W. L. Castleman, J. A. Lednicky, and J. P. Driver. 2016. ?-Galactosylceramide protects swine against influenza infection when administered as a vaccine adjuvant. Sci Rep 6: 23593.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target auduences are swine immunologists, veterinary immunologists, biomedical researchers, clinical and translational scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training was taken to develop infection models in pigs. How have the results been disseminated to communities of interest?Journal articles What do you plan to do during the next reporting period to accomplish the goals?Use our model for a variety of therapeutic interventions.

Impacts
What was accomplished under these goals? In 2015 we repeated vaccinations studies to show that NKT cell agonists can be used as a powerful adjuvant to prevent infection for preventing influenza infections in pigs. We also completed a study to show that NKT cell agonists can be used to improve the course of ongoing influenza infections. Results of our studies have been submitted to Nature's Scientific Reports. Our results have great potential to significantly limit the spread of influenza infections in pigs and zoonotic infections in humans.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Yang G, Artiaga BL, Hackmann TJ, Samuel MS, Walters EM, Salek-Ardakani S, and Driver JP. 2015. Targeted disruption of CD1d prevents NKT cell development in pigs. Mammalian Genome. 26(5-6):264-270.
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Artiaga BL, Yang G, Hackmann TJ, Lui Q, Richt JA, Salek-Ardakani S, Castleman W, Lednicky JA, and Driver JP. ?-Galactosylceramide protects swine against influenza infection when administered as a vaccine adjuvant. Sci. Report.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience:The target audience includes vaccine developers, veterinary immunologists, NKT cell biologists and clinicians seeking to adapt NKT cell therapies for human applications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Our data have been published in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?test whether NKT cell therapy can be used to enhance swine influenza vaccines and to clear established swine influenza infections when used as an anti-viral therapy.

Impacts
What was accomplished under these goals? NKT cell subsets were characterized in pigs NKT cell frequencies in different tissues were evaluated NKT cells were targeted with lipid antigens to enhance immune responses that can be used to improve swine immunity and vaccines CD1d KO pigs lacking NKT cells were imported and bred at UF to test whether these cells are capable of protecting pigs from influenza challenge.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Artiaga BL, Whitener RL, Staples CR, Driver JP. 2014. Adjuvant effects of therapeutic glycolipids administered to a cohort of NKT cell-diverse pigs. Vet Immunol Immunopathol 162:1-13.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: 14. Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O'Gorman C, Walters EM, Murphy CN, Driver JP, Mileham A, McLaren D, Wells KD, and Prather RS. 2014. Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from In Vitro-Derived Oocytes and Embryos. Biol Reprod. 9/3: 78.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target auduences are swine immunologists, veterinary immunologists, biomedical researchers, clinical and tramslational scienctists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training was taken to develop infection models in pigs. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Use our model for a variety of therapeutic interventions.

Impacts
What was accomplished under these goals? The major goal achieved was developing a swime model that can be used to study how natural killer T cells can be targeted for a wide range of therapeutic purposes, in humans and in pigs.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Adjuvant effects of therapeutic glycolipids administered to a cohort of NKT cell-diverse pigs. Artiaga BL, Whitener RL, Staples CR, Driver JP. Vet Immunol Immunopathol. 2014 Nov 15;162(1-2):1-13. doi: 10.1016/j.vetimm.2014.09.006. Epub 2014 Oct 5. PMID: 25441499 [PubMed - in process] Related citations Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O'Gorman C, Walters EM, Murphy CN, Driver J, Mileham A, McLaren D, Wells KD, Prather RS. Biol Reprod. 2014 Sep;91(3):78. doi: 10.1095/biolreprod.114.121723. Epub 2014 Aug 6. PMID: 25100712 [PubMed - in process]

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: This past year we have: 1)Determined the frequency of natural killer T (NKT) cells in different lymphoid organs in pigs. 2)Characterized the different subsets of NKT cells expressed by pigs. 3)Tracked the development of NKT cells in young post-natal pigs. 4)Performed functional assays using pigs to test their ability to produce immune signaling molecules upon stimulation 5)Treated pigs with an NKT cell-activating agent to show that stimulation of these cells boosts immunity to the neo-antigen hen-egg lysozyme. This was to demonstrate that the same agents could be used to increase vaccine responses. PARTICIPANTS: Ms. Bianca Libanori my MS graduate student performed experiments and compiled data for this project. Dr. Jeurgen Richt at the University of Kansas is a swine influenza expert and is collaborating with us to test whether NKT cells can be harnessed to protect against influenza infection. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This past year we have made considerable progress characterizing natural killer T cells from pigs. This minor lymphocyte population has been well studied in mice due to the potential applications these cells have for a multitude of human diseases. However, so far NKT cell therapeutics have not been widely used for clinical applications because of safely concerns based largely on the finding that mouse and human NKT cells are very dissimilar. Because they share very many immune similarities to humans, we selected pigs as an alternative model to investigate how NKT cells could be used to benefit human health. The work we completed this year demonstrated that indeed pig NKT cells share many common characteristics with those from humans. We also demonstrated that activating NKT cells in pigs with NKT cell agonists boosts immune responses to a simultaneously injected neo-antigen. This finding confirms similar results in mice and suggests that NKT cells will be useful to enhance immunity in humans and commercial swineherds.

Publications

• No publications reported this period