Source: OKLAHOMA STATE UNIVERSITY submitted to
IMMUNE BOOSTING DIETARY COMPOUNDS FOR GROWTH PROMOTION AND DISEASE CONTROL AND PREVENTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
ANIMAL HEALTH
Reporting Frequency
Annual
Accession No.
1009879
Grant No.
(N/A)
Project No.
OKL03025
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2016
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Project Director
Zhang, GU.
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Animal Science
Non Technical Summary
Routine use of antibiotics as growth promoters in food animal industry poses increasing public health concerns worldwide. Such practice has been criticized for the contamination of food products and the environment with unwanted antibiotic residues and rapid emergence and dissemination of antibiotic-resistant pathogens. Medically important antibiotics will be phased out for use in healthy animals by December 2016. To ensure optimal animal health and food security, alternatives to antibiotics are urgently needed for livestock applications. Broadly effective against virtually all classes of pathogens, antimicrobial host defense peptides (HDPs) offer a new concept of disease control. These peptides are naturally produced by immune cells of the animals, and the level of their synthesis was shown recently to be strongly augmented by certain dietary compounds such as short-chain fatty acids, vitamins, and essential amino acids, providing the rationale for boosting animal growth performance, immunity, disease resistance, and preharvest food safety through convenient dietary modulation of endogenous HDP production. Our group has identified several classes of natural and synthetic compounds with a strong capacity to induce HDP synthesis individually and in combination in chickens.In this proposal, we will explore the potential for dietary modulation of natural HDPs ingrowth promotion as well as thecontrol and prevention of important foodborne pathogens in chickens. It is ourgoal to develop a truly innovative antibiotic-freestrategy to augment animal growth,immunity and disease resistance for livestock applications.
Animal Health Component
100%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3153220109080%
3113999101020%
Knowledge Area
311 - Animal Diseases; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3999 - Animal research, general;

Field Of Science
1090 - Immunology; 1010 - Nutrition and metabolism;
Goals / Objectives
The overall goal of this project is to evaluate the in vitro and in vivo efficacy of several selected different combinations of natural dietary compounds in HDP induction, growth promotion, and bacterial clearance in chickens in both healthy and Salmonella-infected chickens, paving the way for their development as next-generation alternatives to antibiotics. The central hypothesis is that strategies to enhance HDP synthesis will confer on animals the capacity to fight off infections and promote growth. We plan to test this hypothesis and accomplish the overall goal by pursuing the following two specific objectives: 1) Identify the most efficient combinations of dietary compounds in inducing the synthesis of multiple endogenous HDPs without causing inflammation, and 2) Demonstrate selected compounds are as efficient as antibiotics in growth promotion and bacterial clearance in live animals.
Project Methods
In Objective 1, we will attempt to identify the most efficient combinations of dietary compounds in inducing the synthesis of multiple endogenous HDPs without causing inflammation. Several compounds have been found to synergistically interact with each other in HDP induction in chickens. A minimum of five compounds such as butyrate, propionate, lactose, galactose, and forskolin will be selected for quick evaluation of their HDP-inducing activities in a stable luciferase reporter cell line that we recently established. Combinations of 2-3 different natural products will be tested, with a major focus on the synergy between histone deacetylase inhibitors (butyrate and propionate) with 1-2 other classes of compounds. Top 10 combinations of the compounds will be further examined for their ability to induce HDPs and cytokines in multiple different chicken cell types. The top 5 combinations of the compounds with the strongest HDP-inducing activity and a minimum impact on the inflammatory gene expression will then be pursued for their efficacy in HDP induction in live animals.In Objective 2, we will demonstrate selected compounds are as efficient as antibiotics in growth promotion and bacterial clearance in live animals.Topthree combinations of natural compounds with the highest HDP-inducing activity and a minimum influence on the inflammatory response will be tested further for their efficacy in growth promotion, intestinal health, and disease control in both healthy and experimentally infected broiler chickens, in comparison with a commonly used in-feed antibiotic, bacitracin methylene disalicylate (BMD).

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:Livestock producers, animal feed additives companies, veterinary immunologists, and animal nutritionists have been reached by the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students, three undergraduate students, and the PD have attended the university, regional, and national research conferences and workshops multiple times throughout the duration of the project. The students are exposed to the latest advances in the field and had great networking opportunities. How have the results been disseminated to communities of interest?PD and his graduate students and undergraduate research scholars attended several professional and university conferences each year and presented their results there. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have identified a number of small-molecule compounds with the ability to induce the synthesis of host defense peptides (HDP), which are a critical component of the animal's innate immunity. In addition, we have also revealed several of these HDP-inducing compounds can synergize with each other to induce HDP gene expression and enhance barrier function in vitro and in vivo without triggering inflammation. A few can be used to protect chickens from necrotic enteritis and coccidiosis. Moreover, some combinations of the compounds have been shown to have a strong tendency to increase feed efficiency without affecting the growth rate of broilers. In addition, we have also developed and employed a cell-based high throughput screening assay to identify HDP-inducing compounds. After screening hundreds of natural and synthetic small-molecule compounds, we have identified several classes with the ability to induce HDP gene expression. A series of in vitro and in vivo characterizations are underway to evaluate their potential as novel antimicrobial alternatives for disease control and prevention.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Xiao, K., Q. Xu, C. Liu, P. He, Q. Qin, H. Zhu, J. Zhang, A. Gin, G. Zhang, and Y. Liu. 2020. Docosahexaenoic acid alleviates cell injury and improves barrier function by suppressing necroptosis signaling in TNF-?-challenged porcine intestinal epithelial cells. Innate Immunity 26: 653-665.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wang, J., W. Lyu, W. Zhang, Y. Chen, F. Luo, Y. Wang, H. Ji, and G. Zhang. 2021. Discovery of natural products capable of inducing porcine host defense peptide gene expression using a cell-based high throughput screening. Journal of Animal Science and Biotechnology 12: 14.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Liu. J., S.N. Stewart, K. Robinson, Q. Yang, W. Lyu, M.A. Whitmore, and G. Zhang. 2021. Linkage between the intestinal microbiota and residual feed intake in broiler chickens. Journal of Animal Science and Biotechnology 12: 22.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Xu, Q., X. Li, Y. Wang, D. Wang, K. Xiao, H. Zhu, X. Wang, C.-A. A. Hu, G. Zhang, and Y. Liu. 2021. Necroptosis underlies hepatic damage in a piglet model of lipopolysaccharide-induced sepsis. Frontiers in Immunology 12: 633830.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Yang, Q., M. Whitmore; K. Robinson, W. Lyu, and G. Zhang. 2021. Butyrate, forskolin, and lactose synergistically enhance disease resistance by inducing the expression of the genes involved in innate host defense and barrier function. Antibiotics 10: 1175.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Robinson, K., Q. Yang, H. Li, L. Zhang, B. Aylward, R.J. Arsenault, and G. Zhang. 2021. Butyrate and forskolin augment host defense, barrier function, and disease resistance without eliciting inflammation. Frontiers in Nutrition 8: 778424.


Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Livestock producers, animal feed additives companies, veterinary immunologists, and animal nutritionists have been reached bythe outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students, one undergraduate student, and the PI have attended virtual research conferences and workshops multiple times in the past year. The students were exposed to the latest advances in the field. How have the results been disseminated to communities of interest?PI and his graduate students and undergraduate research scholars attended several professionaland university conferences online and presented their results there. What do you plan to do during the next reporting period to accomplish the goals?Weplan to further characterize the synergy amongnewly-identified host defense peptide (HDP)-inducing compounds in the induction of host defense peptide and barrier function genes both in vitro and in vivo. Their abilityfor disease control and prevention will be further evaluated.

Impacts
What was accomplished under these goals? We investigated the ability of butyrate, a short-chain fatty acid,and forskolin, a phytochemical,to induce the expression of chicken host defense peptide(HDP) genes along with barrier function genes, mucin-2 and claudin-1. Chicken HD11 macrophage cells or day-of-hatch chicks were treated with butyrate and forskolin independently and in combination. Gene expression was then evaluated in cells or tissues using real-time PCR and RNAseq analysis. Protein phosphorylation and signaling pathways were analyzed using a kinome peptide array analysis. We report the ability of butyrate and forskolin to induce avian β-defensin 10 (AvBD10) and mucin 2 expression in chicken macrophage cells. We also discovered a synergistic induction of these genes when cells were stimulated with butyrate and forskolin simultaneously. Furthermore, dietary supplementation of forskolin or butyrate alone produced a weak induction of AvBD10 in chicken jejunum, while a combination of both ingredients produced a desirably strong synergistic effect. Expression of mucin 2 and the tight junction protein claudin-1 was also induced by forskolin alone. A weak, but significant, synergistic induction of these genes was also seen in the jejunum of chickens fed butyrate and forskolin in combination. Further screening of additional chicken HDPs revealed a synergistic induction of AvBD3, 4, and 8 as well. Measuring the changes in kinase activity and peptide phosphorylation revealed butyrate and forskolin synergize to produce a unique kinome profile not observed in either of the separate treatments. This profile is more similar to forskolin than butyrate but is a unique non-additive phosphorylation pattern. Butyrate, forskolin, and the combination result in unique changes in MAPK and tight junction signaling. Overall, these results indicate a possible role of butyrate and forskolin in HDP induction, intestinal barrier function, and disease resistance in animals.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, X., W. Wang, L. Wang, C. Yu, G. Zhang, H. Zhu, C. Wang, S. Zhao, C.-A. Hu, and Y. Liu. 2019. Lentinan modulates intestinal microbiota and enhances barrier integrity in a piglet model challenged with lipopolysaccharide. Food & Function 10: 479-489.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists will be interested in the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students, one undergraduate student, and the PIhave attended the university, regional, and national research conferences and workshops multiple times in the past year. The students were exposed to the latest advances in the field and hadgreat networking opportunities. How have the results been disseminated to communities of interest?PIand his graduate students and undergraduate research scholars attended multiple national and regional conferences and presented their results there. What do you plan to do during the next reporting period to accomplish the goals?We are in the process of and plan to further characterize a number of newly-identified host defense peptide (HDP)-inducing compounds and further studytheir possible synergies. Different combinations of compounds will be evaluated for their synergistic actions in HDP gene induction and barrier function improvement as well as in disease control and prevention.

Impacts
What was accomplished under these goals? To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1-Kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1-5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24-h exposure to HDAC inhibitors. In conclusion, a cell-based high throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Deng, Z., J. Wang, W. Lyu, X. Wieneke, R. Matts, X. Ma, and G. Zhang. 2018. Development of a cell-based high throughput screening assay to identify porcine host defense peptide-inducing compounds. Journal of Immunology Research 5492941.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang, L., X. Wang, S. Chen, S. Wang, Z. Tu, G. Zhang, H. Zhu, X. Li, J. Xiong, and Y. Liu. 2018. Medium-chain triglycerides attenuate liver injury in lipopolysaccharide-challenged pigs by inhibiting necroptotic and inflammatory signaling pathways. International Journal of Molecular Science 19: 3697.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Robinson, K., S. Becker, Y. Xiao, W. Lyu, Q. Yang, H. Zhu, H. Yang, J. Zhao, and G. Zhang. 2019. Differential impact of subtherapeutic antibiotics and ionophores on intestinal microbiota of broilers. Microorganisms 7: 282.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists will be interested in the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students, one undergraduate student, and the PD have attended the university, regional, and national research conferences and workshops multiple times in the past year. The students were exposed to the latest advances in the field and had great networking opportunities. How have the results been disseminated to communities of interest?PD and his graduate students and undergraduate research scholars attended multiple national and regional conferences and presented their results there. What do you plan to do during the next reporting period to accomplish the goals?We plan to further characterize a number of newly-identified host defense peptide (HDP)-inducing compounds and further study their possible synergies. Different combinations of compounds will be evaluated for their synergistic actions in HDP gene induction and barrier function improvement as well as in disease control and prevention.

Impacts
What was accomplished under these goals? To facilitate the identification of natural products with a strong capacity to induce host defense peptide (HDP) synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian β-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Robinson, K., X. Ma, Y. Liu, S. Qiao, Y. Hou, and G. Zhang. 2018. Dietary modulation of endogenous host defense peptide synthesis as an alternative approach to in-feed antibiotics. Animal Nutrition. 4: 160-169.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lyu, W., Z. Deng, L.T. Sunkara, S. Becker, K. Robinson, R. Matts, and G. Zhang. 2018. High throughput screening for natural host defense peptide-inducing compounds as novel alternatives to antibiotics. Frontiers in Cellular and Infection Microbiology 8: 191.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ma, N., P. Guo, J. Zhang, T. He, S.W. Kim, G. Zhang, and X. Ma. 2018. Nutrients mediate intestinal bacteriamucosal immune crosstalk. Frontiers in Immunology 9:5.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu, H., J, Wang, T. He, S. Becker, G. Zhang, D. Li, and X. Ma. 2018. Butyrate: a double-edged sword for health? Advances in Nutrition 9: 21-29.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Nie, C., T. He, W. Zhang, G. Zhang, X. Ma. 2018. Branched chain amino acids: Beyond nutrition metabolism. International Journal of Molecular Science. 19: 954.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Livestock producers, veterinary doctors, veterinary immunologists, and animal nutritionists will be interested in the outcomes of the research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students, one undergraduate student, and the PD have attended the university, regional, and national research conferences and workshops multiple times in the past year. The students were exposed to the latest advances in the field and has great networking opportunities. 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?We will plan to develop a high throughput system in attempt to identify additional host defense peptide (HDP)-inducing compounds and further study their possibly synergies. Different combinations of compounds will be evaluated for their synergistic actions in HDP gene induction and barrier function improvement.

Impacts
What was accomplished under these goals? We showed previously that butyrate, a short chain fatty acid, and forskolin, a natural adenylate cyclase agonist, are capable of upregulating host defense peptide (HDP) gene expression in chickens. To follow up, we investigated the ability of butyrate and forskolin to induce the expression of multiple chicken HDPs along with the barrier protective genes such as mucin-2 and claudin-1. Chicken HD11 macrophage cells or day-of-hatch chicks were treated with butyrate and forskolin independently and in combination. Gene expression was then evaluated in cells or tissues using real-time PCR and RNAseq analysis. Protein phosphorylation and signaling pathways were analyzed using a kinome peptide array analysis. We report the ability of butyrate and forskolin to induce avian β-defensin 10 (AvBD10) and mucin-2 expression in chicken macrophage cells. We also discovered a synergistic induction of these genes when cells were stimulated with butyrate and forskolin simultaneously. Furthermore, dietary supplementation of forskolin or butyrate alone produced a weak induction of AvBD10 in chicken jejunum while a combination of both ingredients produced a desirably strong synergistic effect. Expression of mucin 2 and the tight junction protein claudin-1 was also induced by forskolin alone, while a weak, but significant, synergistic induction of these genes was also in the jejunum of chickens fed butyrate and forskolin in combination. Further screening of additional chicken HDPs revealed a synergistic induction of AvBD3, 4, and 8 as well. Measuring the changes in kinase activity and peptide phosphorylation revealed butyrate and forskolin synergize to produce a unique kinome profile not observed in either of the separate treatments. This profile is more similar to forskolin than butyrate but is a unique non-additive phosphorylation pattern. Butyrate, forskolin, and the combination result in unique changes in MAPK and tight junction signaling. Overall, these results indicate a possible role of butyrate and forskolin in the induction of host defense peptides, intestinal barrier function, and disease resistance in animals and humans.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhang, L., L. Lu, S. Li, G. Zhang, L. Ouyang, K. Robinson, Y. Tang, Q. Zhu, D. Li, Y. Hu, and Y. Liu. 2016. 1,25-Dihydroxyvitamin-D3 induces avian ?-defensin gene expression in chickens. PLoS ONE 11: e0154546.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, J., M. Han, G. Zhang, S. Qiao, D. Li, X. Ma. 2016. The signaling pathway of antibiotic alternatives on intestinal microbiota and immune function. Current Protein and Peptide Science 17: 785-796.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu, H., C. Hou, G. Wang, H. Jia, H. Yu, X. Zeng, P.A. Thacker, G. Zhang, and S. Qiao. 2017. Lactobacillus reuteri I5007 modulates intestinal host defense peptide expression in the model of IPEC-J2 cells and neonatal piglets. Nutrients 9: 559.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Zhang, G., L.-A. Fong, and L.T. Sunkara. 2016. Synergistic induction of chicken host defense peptide gene expression by sugars and butyrate. Symposium on Gut Health in Production of Food Animals, November 14-16, 2016, St. Louis, MO.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Lyu, W., Z. Deng, and G. Zhang. 2017. Identification of natural host defense peptide-inducing compounds using a cell-based high throughput screening assay. Poultry Science Association Annual Meeting, July 17-20, 2017, Orlando, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Stewart, S., K. Robinson, and G. Zhang. 2017. Tissue and developmental expression patterns of claudin-1 and claudin-2, two major tight junction proteins, in chickens. 4th Annual Research Retreat of the Oklahoma Center for Respiratory and Infectious Diseases, April 4, 2017, Stillwater, OK.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Robinson, K., H. Li, L. Zhang, B. Aylward, R.J. Arsenault, L. Sunkara, B. Couger, and G. Zhang. 2017. Molecular mechanisms of synergistic enhancement of chicken innate immunity and barrier function by butyrate and forksolin. Poultry Science Association Annual Meeting, July 17-20, 2017, Orlando, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Deng, Z., W. Lyu, and G. Zhang. 2017. High throughput screening of histone deacetylase inhibitors that induce host defense peptide expression. Poultry Science Association Annual Meeting, July 17-20, 2017, Orlando, FL