Source: MONTANA STATE UNIVERSITY submitted to
ENHANCING INNATE IMMUNITY IN BOVINE CALVES BY STIMULATING GAMMA DELTA T CELLS WITH PLANT-DERIVED POLYSACCHARIDES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0215217
Grant No.
2008-35204-04653
Project No.
MONB00420
Proposal No.
2008-00926
Multistate No.
(N/A)
Program Code
44.0A
Project Start Date
Sep 1, 2008
Project End Date
Aug 31, 2010
Grant Year
2008
Project Director
Hedges, J. F.
Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717
Performing Department
Immunology & Infectious Diseases
Non Technical Summary
We recently determined that several common plant-derived products with anecdotal health benefits stimulate cells that function early in protection from disease, or in innate immunity. We sought products that specifically stimulated gamma delta T cells, which are an enigmatic cell with innate immune properties. Unlike in humans and rodents, gamma delta T cells are predominant cells in cattle and likely serve important roles in disease protection, particularly in very young animals. We identified one dietary supplement called Yamoa that contained bioactive components comprised of complex polysaccharides (carbohydrates) that stimulate aspects of innate immune function and, potentially, protect from Salmonella enterica serotype Typhimurium (ST) infection, a common cause of intestinal disease, in mice and cattle. Thus, Yamoa and similar dietary supplements have potential to be effective for disease amelioration, but their modes of action and appropriate application in veterinary medicine are unknown. The experiments in this proposal are designed to begin to determine the utility of compounds derived from dietary supplements to enhance innate immunity in bovine calves. We hypothesize that ingestion of polysaccharides increases resistance of calves to ST infection. Given that enhancement of innate immunity could potentially protect against a wide range of agents that cause disease, identification of new methods to enhance immunity are critical to animal protection and biosecurity. Characterization of compounds derived from dietary supplements (such as polysaccharides) may lead to a formulation that could be combined with standard mineral supplements and used to enhance the immunological health of newborn to six month old calves, shipped animals, and feedlot cattle.
Animal Health Component
100%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113399109017%
3113499109017%
3114010109018%
3153399109016%
3153499109016%
3154010109016%
Goals / Objectives
We recently determined that several common plant-derived products with anecdotal immune benefits stimulate innate immunity by affecting the innate lymphocytes, gamma delta T cells. Yamoa (ground bark of Funtumia elastica tree) has distinct priming effects, very similar to that of lipopolysaccharide (LPS), on bovine, mouse and human gamma delta T cells. Unlike in humans and rodents, gamma delta T cells are a major subset in cattle and likely serve important roles in the innate immunity, particularly in neonates. Gene expression patterns in bovine cells induced by Yamoa are very similar to those induced by LPS, but the agonists in Yamoa signal through a distinct pathway without toxic negative effects. The bioactive component of Yamoa is comprised of complex polysaccharides that stimulate human cells to produce innate cytokines, and in animal models induce rapid increases in circulating neutrophils, expansion of gamma delta T cells in the intestinal mucosa, and, potentially, protect from Salmonella enterica serotype Typhimurium (ST) infection in bovine calves and mice. Yamoa has potential to be a potent innate immunostimulant, for which mechanisms and appropriate application in veterinary medicine are unknown. The experiments in this proposal are designed to begin to determine the utility of polysaccharides derived from Yamoa to enhance innate immunity in bovine calves. We hypothesize that ingestion of polysaccharides in Yamoa increase innate resistance of calves to ST infection. Given that enhancement of innate immunity could potentially protect against a wide range of pathogens, identification of novel innate adjuvants are critical to animal protection and biosecurity. Characterization of immunostimulatory polysaccharides in Yamoa may lead to a formulation that could be combined with standard mineral supplements and used to enhance the immunological health of newborn to six month old calves, shipped animals, and feedlot cattle. Three Specific Aims are proposed: Specific Aim 1. Optimize oral dose of Yamoa-derived polysaccharides (Yam-I). a. Identify the optimal oral dose of Yamoa-derived polysaccharides in calves. b. Monitor changes in peripheral blood leukocytes in response to the optimal oral dose. Specific Aim 2. Analyze in vivo target tissue response to the optimal oral dose of Yam-I. a. Determine effect of oral Yam-I on innate immune cells in mucosal derived lymphatic fluid. b. Determine effect of Yam-I on tissue cells at the target site. Specific Aim 3. Evaluate effect of orally delivered Yam-I in ST challenge in calves. a. Assess the capacity of prophylactic use of Yam-I to protect from ST challenge. b. Determine if Yam-I can be effective therapeutically after ST infection.
Project Methods
We propose to better characterize the action of Yamoa in an effort to stimulate the innate immune system for protection against a broad range of pathogens in bovine calves. We will confirm the in vivo effect of oral delivery of the polysaccharide fraction derived from Yamoa (Yam-I) by analysis of innate cell population changes and Salmonella challenge experiments. We will first determine the optimal oral dose of Yam-I. Calves will be given various doses of Yam-I daily and markers of innate immune activation will be measured following standard flow cytometry protocols, and temperatures will be taken daily. Sera will be collected and haptoglobin, an acute phase protein elevated in cattle as a result of infection, will be measured. In response to oral doses of Yam-I we expect to observe little change in temperature, similar to i.v. delivery of Yamoa, slight increases in serum haptoglobin, as was observed after CpG treatment in cattle and changes in other innate immune populations. An ideal treatment regimen is one that leads to the maximum innate immune stimulation in the absence of overt signs of toxicity. We expect to identify an optimal maximal, non-toxic dose of Yam-I for use in calves. We will then better characterize the specific action of Yam-I on lymphocyte subsets derived from target tissue and immunohistological analyses of changes in the tissue. Using our cannulation protocol that allows collection of mesenteric efferent lymphatic fluid, we will monitor changes in percentage, number, protein and gene expression in mucosal derived lymphocytes as we have previously described (Hedges et al. 2007). We will also investigate intestinal mucosal tissue sections from calves fed Yam-I and Yamoa using standard histochemical techniques. We expect that ingestion of Yam-I will mediate changes in gene and protein expression in cells in the gut mucosa detectable either in mesenteric derived lymphocytes found in lymphatic fluid or in the tissue directly. It is of interest to determine if oral delivery can result in protection from Salmonella infection, either by prophylactic or therapeutic use of Yam-I. To assess prophylactic Yam-I use, calves will be fed the optimal dose of Yam-I or vehicle, and closely monitored, then infected with a stock calf isolate of ST. Calves will be closely monitored and blood collected daily for flow cytometric and haptoglobin evaluation. Daily fecal samples will be tested for the input strain of ST and for other infectious agents. We will also assess therapeutic treatment of calves with the optimal does of Yam-I, providing it after infection. We expect to detect delayed or diminished fever responses and disease as measured by diarrhea and shed bacteria, and/or accelerated recovery in calves fed Yam-I, compared to those fed vehicle only.

Progress 09/01/08 to 08/31/10

Outputs
OUTPUTS: We have conducted and analyzed experiments to determine the immunomodulatory effects of Yamoa in bovine calves. The results of the first 2 aims were discussed in the annual report submitted in November 2009. Since that time we have thoroughly analyzed data resulting from collection of blood and lymphatic cells derived from calves ingesting Yamoa compared to control calves, compared the responses of calves fed Yamoa or vehicle only control (milk) during Salmonella challenge, and, initiated studies to confirm the anti-inflammatory nature of Yamoa in bovine monocytes. PARTICIPANTS: Jodi F. Hedges, Ph.D. Assistant Research Professor. Dr. Hedges is the PD and will be responsible for the overall administration of this project. Her specific day-to-day participation in the experimental aspect of the project will be directing the proposed animal studies and supervision of the research technicians associated with the program. Kerri Rask, B.S. Large Animal Facility Manager and Laboratory Technician. has the critical role of directing the care and monitoring of calves during experimental procedures and will interact with Dr. Hedges in all aspects associated with this animal-intensive project. Amy Robison, B.S. is a Laboratory Technician who has been added to the project since its initiation. Ms. Robison is a key technical support member of the research team. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of the experiments have resulted in both a change in knowledge and a change in approach to the studies. The results suggested very subtle changes in cell populations. It appeared that calves that underwent cannulation surgery, but were fed milk only had subtle but consistent increases in inflammatory markers compared to Yamoa fed calves. This result suggested that ingesting Yamoa may have had an anti-inflammatory effect that dampened the inflammation involved in surgery. This anti-inflammatory effect would be consistent with the main use of Yamoa in humans, to alleviate asthma. We then proceeded to assess the effects of calves fed Yamoa in Salmonella challenges. There was a clearly significant delay in Salmonella-induced fever onset in some settings. This potential anti-inflammatory capacity of Yamoa was confirmed in vitro and experiments are now underway to examine the effect in vivo. Outcomes of these investigations suggest that contrary to our hypothesis, Yamoa and related polysaccharides may have anti-inflammatory capacity that may alleviate such severe inflammatory conditions as Johne's disease. These data are currently being compiled in a manuscript in preparation. A new project to study the expression of a novel anti-inflammatory cytokine in bovine gamma delta T cells is also providing a change in knowledge. This project resulted in a new grant application.

Publications

  • No publications reported this period


Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: Three Specific Aims were proposed: Specific Aim 1. Optimize oral dose of Yamoa-derived polysaccharides (Yam-I). Specific Aim 2. Analyze in vivo target tissue response to the optimal oral dose of Yam-I. Specific Aim 3. Evaluate effect of orally delivered Yam-I in Salmonella challenge in calves. We have involved 29 calves thus far in our investigation of the immunological changes induced by oral delivery of Yamoa. We quickly determined that it was too costly and time consuming to produce sufficient quantities of Yam-I, the purified polysaccharide fraction from Yamoa for oral delivery to cattle. However, we have since published our case that Yam-I is the active fraction in Yamoa (Graff et al 2009) and proceeded with experiments using the equivalent of the human dose of crude Yamoa in calves (1g/day). Only very minor changes to surface markers on peripheral blood cells were noted in calves fed Yamoa. A consistent increase in B cells was observed, suggesting some changes did occur at this dose, and the calves were healthy, so cannulation surgeries were performed to observe lymphatic-derived cell subsets. Seven successful cannulation surgeries have been performed to date. We are now capable of performing 2 surgeries on the same day for greatly increased data collection capacity and the same amount of labor. Again, very few consistent changes in surface protein expression were observed in cells derived from lymphatic fluid. However, using real time RT-PCR analyses with RNA derived from sorted subsets of lymphatic cells, consistent changes in gene expression with implications for innate immunity were observed. Many RNA samples are frozen for additional analyses; negative control cannulation experiments, in which calves were fed vehicle only (not Yamoa), have been performed and peripheral blood cell subsets from calves eating Yamoa for 48 hours have also been collected for real time RT-PCR analyses. Tissues for histochemical analyses 48 hours after Yamoa or vehicle only (milk) have also have also been collected for analyses. The results collected as of last spring were presented in an oral presentation and poster at the 2009 American Association of Immunologists Meeting in Seattle, WA in May 2009. PARTICIPANTS: Jodi F. Hedges, Ph.D. Assistant Research Professor. Dr. Hedges is the PD and will be responsible for the overall administration of this project. Her specific day-to-day participation in the experimental aspect of the project will be directing the proposed animal studies and supervision of the research technicians associated with the program. Kerri Rask, B.S. Large Animal Facility Manager and Laboratory Technician. has the critical role of directing the care and monitoring of calves during experimental procedures and will interact with Dr. Hedges in all aspects associated with this animal-intensive project. Amy Robison, B.S. is a Laboratory Technician who has been added to the project since its initiation. Ms. Robison is a key technical support member of the research team. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A substantial increase in IL-17 transcripts (average fold change of 7.3, n=3) was observed in gammadelta T cells and not in alphabeta or B cells derived from lymphatic fluid after 48 hours of Yamoa ingestion. This change in gene expression in the absence of changes in cell surface proteins is indicative of priming of gammadelta T cells after oral ingestion of Yamoa. IL-17 is an inflammatory cytokine shown to be expressed early from gammadelta T cells in many infectious disease settings. This change was not observed in other analyses in vitro , suggesting that the change is specific to the in vivo situation, and supports the importance of observing changes in cells derived from lymphatic vessels. These data will be included in a manuscript in preparation. As part of transcript analyses of bovine cells subsets, we have amplified a novel transcript with specific robust expression in bovine gammadelta T cells. Gas6 is an agonist for the newly described suppressors of inflammation, the TAM receptor family. We have determined transcripts encoding gas6 are unusually highly expressed in purified bovine gammadelta T cells, compared to alphabeta and B cells. Functional gas6 is dependent on Vitamin K for post-translational gamma-carboxylation. Vitamin K-dependent proteins are known for roles in the blood coagulation cascades and in bone growth. As such there are well known human drugs that affect Vitamin K pathways developed to modulate coagulation, the most common of which is Warfarin (Coumadin). We hypothesized that functional gas6 expressed by gammadelta T cells in the presence of Vitamin K would be immunosuppressive and addition of Warfarin would be pro-inflammatory. Observations to date are consistent with these assumptions. As part of a new collaboration with Dr. Jay Radke (Veterinary Molecular Biology, MSU), some of these ideas have also been applied to in vitro experiments with Mycobacterium avium infection. Importantly, an immunomodulatory role for gammadelta T cells has been observed in some models of Mycobacterium infection. Thus, expression of gas6 by gammadelta T cells maybe critical to observed immunomodulatory function, and the potential to modulate its function may have important implications for mucosal health. These data will be included in a manuscript and as part of a new grant proposal under development to support these studies.

Publications

  • Graff J.C., Kimmel E.M., Freedman B., Schepetkin I.A., Holderness J., Quinn M.T., Jutila M.A., Hedges, J.F. (2009) Polysaccharides derived from Yamoa (Funtumia elastica) prime gammadelta T cells in vitro and enhance innate immune responses in vivo. Int Immunopharmacol. 9(11):1313-22.
  • Hedges J.F., Rask, K.M., Jackiw, L.O., Jutila, M.A. 2009 Enhanced immunity following ingestion of plant derived polysaccharides AAI 2009 Annual Meeting Abstracts Book.