Source: UNIVERSITY OF NEBRASKA submitted to NRP
BIOAVAILABILITY AND DISTRIBUTION OF DIETARY EXOSOMES AND THEIR RNA CARGOS FROM BOVINE MILK IN NON-BOVINE SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1011996
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 11, 2017
Project End Date
Sep 30, 2021
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Nutritional & Health Sciences
Non Technical Summary
This five-year project studies the roles of nanoparticles (exosomes) and exosomes cargos in cow's milk and their roles in the regulation of human genes and metabolism. The transfer of exosomes and their cargos from cows to humans is a paradigm-shifting discovery made by this project's director. The project has major implications for human health because endogenous, non-dietary exosomes have been implicated in virtually all physiological and pathological conditions in humans. Consistent with the roles of endogenous exosomes in health and disease the project director has already disseminated preliminary data at conferences that implicate dietary exosomes in fecundity, immune function, composition of the gut microbiome, cognitive performance, protein and DNA metabolism, and resistance to chemically induced seizures.
Animal Health Component
5%
Research Effort Categories
Basic
95%
Applied
5%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70234501010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3450 - Milk;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
This proposal will test the central hypotheses that cow's milk exosomes are bioavailable and deliver functional RNA species to the intestinal mucosa, macrophages, and other tissues. Our long-term goal is to assess pathways that regulate the bioactivity and distribution of dietary exosomes and their cargos. As a first step toward this goal, we will complete the following objectives.Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. This aim will test the hypothesis that milk exosomes are absorbed after oral administration and accumulate primarily in macrophages in the intestinal mucosa, liver, and spleen.Objective 2. Assess the delivery of functional milk miRNAs in mice. This aim will test the hypothesis that milk miRNAs, encapsulated in exosomes, rescue tamoxifen-inducible conditional Drosha and Dicer knockout mice, which cannot synthesize mature miRNAs, and are delivered to tissues in miR-155-5p sensor mice.Objective 3. Assess the delivery of functional milk mRNAs in mice. This aim will test the hypothesis that milk mRNAs, encapsulated in exosomes, can be translated into functional protein in wild-type and Cre reporter mice.
Project Methods
The strengths of the experimental plan are: 1) experiments will be conducted in whole organisms, 2) procedures are well established in our laboratories, 3) transgenic models (as opposed to inhibitors) minimize the occurrence of off-target effects, 4) tamoxifen-inducible KO mice permit exercising spatial/temporal control over gene KO, 5) unique milk exosomes endogenously labeled with ZsGreen1 will be used, 6) exosomes (as opposed to milk) will be used in "feeding" studies, eliminating milk compounds other than exosomes as confounders, and 7) the researchers' considerable bioinformatics expertise.In objective 1, nanoparticles ("exosomes") will be isolated from bovine milk. Exosome membranes will be labeled with a fluorescent dye, DiR and the bioavailability and tissue distribution will be assessed following oral delivery of DiR-labeled exosomes by gavage. Exosomes will be imaged in live animals using an iBox small animal imaging sytem and in excised tissues using a Licor imaging systemIn objective 2, the bioavailability and distribution of microRNAs (encapsulated in exosomes) will be assessed using miR-155 and miR-29b sensor mice. Bovine exosomes will be delivered by gavage. If miR-155 or miR-29b are delivered to tissues, the fluorescence signal in these tissues will decrease. MicroRNA delivery will be imaged using an iBox imaging system.In obejctive 3, the bioavailability and distribution of mRNAs (encapsulated in exosomes) will be assessed in Cre reporter mice. Briefly if mRNA coding for Cre is delivered to tissues in Cre reporter mice, the color in these tissues will change from red to green. mRNA delivery will be assessed using an iBox imaging system. In addition, the entire mRNA cargo in bovine milk exosomes will be assessed using RNA sequencing and their translation into protein in non-bovine organisms will be assessed by using an in vitro translation kit, mass spectrometry, cell cultures models, and wild-type mice.Endpoints, pitfalls and alternative strategies, and statistical methods and power calculations are documented in a detailed research plan.

Progress 01/11/17 to 09/30/21

Outputs
(N/A)

Impacts
What was accomplished under these goals? Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. In previous years we reported that bovine milk exosomes and their microRNA cargos are bioavailable across species boundaries and accumulate primarily in the intestinal mucosa, liver, brain, spleen and kidneys. In this reporting period, we expanded these studies to show that the fraction of exosomes that goes unabsorbed causes changes in bacterial communities in the murine gut. We also developed three transgenic mouse models that we used to demonstrate that maternal exosomes are transferred to offspring through milk and loss of exosomes and microRNA cargos impair mucosal integrity and barrier function in the gut. Maternal exosomes accumulated primarily in the intestinal mucosa, liver and brain in pups. We also reported that glycoproteins on the surface of milk exosomes play an essential role in exosome absorption, and we identified more than 200 microRNAs in human milk exosomes. We further showed that 1) cold storage of milk leads to a loss of exosomes, 2) ultrasonication of bovine milk causes a near complete loss of bioavailable exosomes and their microRNA cargos in cow's milk, and 3) cow's milk exosomes contain microbial and bovine mRNAs which are bioavailable in humans and mice. In the final reporting period, we identified the transporter that facilitates the uptake of milk exosomes by macrophages, demonstrated that flow filtration does not enrich milk exosomes from the run off in cottage cheese making, and reported that lectins in soy infant formula prevent absorption of milk exosomes. Objective 2. Assess the delivery of functional milk miRNAs in mice. In previous years we reported that bovine milk exosomes do not elicit an immune response if administered orally to humans and in ex vivo cultures of human immune cells. This is an important discovery, because milk exosomes are considered an attractive for delivery cancer drugs to patients. We also discovered that milk exosomes and microRNA cargos added to soy infant formula are not bioavailable, probably due to the precipitation of milk exosomes by lectins in the formula. We have published a paper that identified potential confounders of microRNA analysis in plasma, e.g., improper sample storage, use of heparin as anti-coagulant, and hemolysis of red blood cells. We advanced scientific knowledge by providing evidence that microbial mRNAs in cow's milk exosome activate anti-viral pathways in mice. We also discovered that depletion of cow's milk exosomes selects mutations in bacteria that increase the virulence of gut pathogens. We demonstrated that bovine mRNAs in cow's milk exosomes cannot be translated to peptides and proteins in non-bovine cell cultures. In final reporting period, we demonstrated that dietary depletion of milk exosomes impairs brain health and cognitive function in mice. Objective 3. Assess the delivery of functional milk mRNAs in mice. We have discovered that bovine milk exosomes contain both bovine and bacterial mRNAs. This discovery sparked two lines of investigation. First, we demonstrated that bovine mRNAs are translated into peptides in vitro, suggesting that mRNAs in milk exosomes may contribute to food allergies or immune tolerance in a protein-free environment. Second, we demonstrated that microbial mRNAs bind to Toll-like receptors in mammalian cells in culture. Activation of Toll-like receptors by bacterial mRNAs improved survival and body weight regain in mice challenged with influenza A. In the final reporting period, we reported that bovine mammary alveolar cells secrete milk exomes and therefore afford a tool that allows researchers to genetically engineer milk exosomes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Liu, B, Lu Y, Chen X, Muthuraj, PG, Li X, Pattabiraman M, Zempleni J, Kachman SD, Natarajan SK, Yu J. Protective role of shiitake mushroom-derived exosome-like nanoparticles in D-galactosamine and lipopolysaccharide-induced liver injury in mice. Nutrients 12:477, 2020
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sadri M, Shu J, Kachman SD, Cui J, Zempleni J. Milk exosomes and microRNAs cross the placenta and promote embryo survival in mice. Reproduction 160:501-509, 2020
  • Type: Other Status: Published Year Published: 2021 Citation: Zempleni J. Letter to the editor (regarding Perspective: The Role of Human Breast-Milk Extracellular Vesicles in Child Health and Disease by D. OReilly et al., published in AN on 24 August 2020). Adv Nutr 12:280, 2021
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Sukreet S, Pereira Braga C, An TT, Adamec J, Cui J, Trible B, Zempleni J. Isolation of extracellular vesicles from byproducts of cheese making by tangential flow filtration yields heterogeneous fractions of nanoparticles. J Dairy Sci 104:9478-9493, 2021
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Khanam A. Yu J, Zempleni J. Class A scavenger receptor-1/2 facilitates the uptake of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice. Am J Physiol Cell Physiol 321:C606-C614, 2021
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ogunnaike M, Wang, H, Zempleni J. Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery. Int J Pharm 610:121263, 2021
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Sadri M, Wang H, Kuroishi T, Li Y, Zempleni J. Holocarboxylase synthetase knockout is embryonic lethal in mice (submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Zhou, F, Dogan H, Shu J, Fernando SC, Cui J, Zempleni J. Evolution of gut bacteria through the selection of genetic variations by extracellular vesicles in milk (submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Mutai E, Ngu AKH, Zempleni J. Preliminary evidence that lectins in infant soy formula apparently bind bovine milk exosomes and prevent their absorption in healthy adults (submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Wang H, Wu D, Sukreet S, Delaney A, Belfort MB, Zempleni J. Quantitation of exosomes and their microRNA cargos in frozen human milk (revision submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Sukreet S, Pereira Braga C, An, TT, Adamec J, Cui J, Zempleni J. Modification of the AIN-93G diet by substituting ultrasonicated milk for casein yields a rodent diet deplete of bioavailable milk exosomes and microRNAs (submitted)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. Biological activities of milk exosomes and their RNA cargos within and across species boundaries. Laureate Institute for Brain Research, Tulsa, OK, January 22, 2020 [talk]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health during lactation in murine pups. Keystone Virtual Symposia Optimizing Nutrition for Maternal, Newborn and Child Health, October 22, 2020 [ePoster]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health during lactation in murine pups. Keystone Virtual Symposia Optimizing Nutrition for Maternal, Newborn and Child Health, October 22, 2020 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. Exosomes and microRNAs in maternal milk are important for growth and gut health during weaning in murine pups. Chapman University, CA. November 11, 2020 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. The role of milk exosomes and their RNA cargos in neonatal health. Life Span Diseases Mini Summit in the Child Health Research Institute, University of Nebraska Medical Center, November 13, 2020 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. Resources in the Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules. Life Span Diseases Mini Summit in the Child Health Research Institute, University of Nebraska Medical Center, November 13, 2020 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health in neonate mice. American Society for Cell Biology Virtual, December 14-16, 2020 [ePoster]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J, Zhou F, Dogan H, Cui J. Divergence of gut bacteria through the selection of genetic variations by milk exosomes. Keystone Virtual Symposia The Microbiome: From Mother to Child, January 17-21, 2021 [video poster, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Invited presentation titled W-4002 progress report Zempleni lab: milk exosomes as part of the remote annual W-4002 Multistate group meeting; Ohio State University, Columbus, OH, January 27th, 2021 [Zoom talk]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Milk exosomes and their microRNA cargos: infants, gut and brain. Department of Nutritional Sciences, University of Michigan, MI. February 10, 2021 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Biological Activities of Natural Nanoparticles (Exosomes) in Milk. Department of Ophthalmology, Penn State Hershey Medical Center, Hershey, PA. February 17, 2021 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Novel bioactive compounds in milk: exosomes. Department of Animal Science, University of Nebraska-Lincoln, NE. April 6, 2021 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. NPODs transition from Phase I to Phase II. Rural Drug Addiction Research Center, University of Nebraska-Lincoln, NE. April 8, 2021 [talk, delivered by using Zoom]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Khanam A, Yu J, Zempleni J. Class A scavenger receptor-1/2 facilitates the uptake and clearance of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice. NPOD 12th Annual Spring Research Retreat, April 14th, 2021, Lincoln, NE [video poster]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Zempleni J. Milk exosome-driven evolution of antibiotic-resistant gut pathogens. NIFA Program Directors Meeting, Kansas City, KS. May 4, 2021 [talk, delivered by using Zoom]


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

Outputs
Target Audience:1) Peer scientists (information disseminated through invited seminars, presentations at national conferences, publications in science journals) 2) Policy makers (federal agencies such as USDA, NIH, CDC) 3) Students (hands-on training in laboratory, class room teaching, national and international conferences) 4) Lay public (newspaper and magazine interviews) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?To date, the project has supported the training of five postdoctoral fellows, 10 graduate students, and a research technologist. Students and postdocs presented at local and national conferences. How have the results been disseminated to communities of interest?1) Journal articles 2) Publications in the lay press 3) Conference presentations 4) Invited seminars 5) Annual reports (NIFA grantee meetings, USDA multistate meetings) 6) Workshop sponsored by NIH and USDA 7) NIFA blogs, twitter What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. Publish our research on bovine and microbial mRNAs in cow's milk exosomes and their bioavailability. Publish a research article on the transporter that facilitates the uptake of milk exosomes in macrophages. Publish a research article on the role of glycoproteins in the intestinal uptake of cow's milk exosomes. Establish bovine mammary epithelial MAC-T cells as a tool for producing genetically altered milk exosomes. Objective 2. Assess the delivery of functional milk miRNAs in mice. Assess interactions of miRNAs, encapsulated in bovine milk exosomes, with the human and murine microbiome. Objective 3. Assess the delivery of functional milk mRNAs in mice. Assess whether cow's milk exosomes select genetic variations in gut bacteria that alter the availability of precursors for neurotransmitter synthesis in the host. Identify modifications of cow's milk exosomes that enhance the delivery of siRNA to brain tumors.

Impacts
What was accomplished under these goals? Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. In previous years we reported that bovine milk exosomes and their microRNA cargos are bioavailable across species boundaries and accumulate primarily in the intestinal mucosa, liver, brain, spleen and kidneys. In this reporting period, we expanded these studies to show that the fraction of exosomes that goes unabsorbed causes changes in bacterial communities in the murine gut. We also developed three transgenic mouse models that we used to demonstrate that maternal exosomes are transferred to offspring through milk and loss of exosomes and microRNA cargos impair mucosal integrity and barrier function in the gut. Maternal exosomes accumulated primarily in the intestinal mucosa, liver and brain in pups. We also reported that glycoproteins on the surface of milk exosomes play an essential role in exosome absorption, and we identified more than 200 microRNAs in human milk exosomes. In this reporting period, we advanced scientific knowledge by demonstrating that 1) cold storage of milk leads to a loss of exosomes, 2) ultrasonication of bovine milk causes a near complete loss of bioavailable exosomes and their microRNA cargos in cow's milk, and 3) cow's milk exosomes contain microbial and bovine mRNAs which are bioavailable in humans and mice. Objective 2. Assess the delivery of functional milk miRNAs in mice. In previous years we reported that bovine milk exosomes do not elicit an immune response if administered orally to humans and in ex vivo cultures of human immune cells. This is an important discovery, because milk exosomes are considered an attractive for delivery cancer drugs to patients. We also discovered that milk exosomes and microRNA cargos added to soy infant formula are not bioavailable, probably due to the precipitation of milk exosomes by lectins in the formula. We have published a paper that identified potential confounders of microRNA analysis in plasma, e.g., improper sample storage, use of heparin as anti-coagulant, and hemolysis of red blood cells. In this reporting period, we advanced scientific knowledge by providing evidence that microbial mRNAs in cow's milk exosome activate anti-viral pathways in mice. We also discovered that depletion of cow's milk exosomes selects mutations in bacteria that increase the virulence of gut pathogens. We demonstrated that bovine mRNAs in cow's milk exosomes cannot be translated to peptides and proteins in non-bovine cell cultures. Objective 3. Assess the delivery of functional milk mRNAs in mice. We have discovered that bovine milk exosomes contain both bovine and bacterial mRNAs. This discovery sparked two lines of investigation. First, we demonstrated that bovine mRNAs are translated into peptides in vitro, suggesting that mRNAs in milk exosomes may contribute to food allergies or immune tolerance in a protein-free environment. Second, we demonstrated that microbial mRNAs bind to Toll-like receptors in mammalian cells in culture. Activation of Toll-like receptors by bacterial mRNAs improved survival and body weight regain in mice challenged with influenza A.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J. Biological activities of milk exosomes and their RNA cargos within and across species boundaries. Laureate Institute for Brain Research, Tulsa, OK, January 22, 2020 [talk]
  • Type: Other Status: Published Year Published: 2020 Citation: NIFA blog regarding research sponsored by NIFA/USDA (2020-67017-30834) Milk exosome-driven evolution of antibiotic-resistant gut pathogens: National Institute of Food and Agriculture website: https://nifa.usda.gov/blog/milk-findings-may-help-infants
  • Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: NIFA twitter regarding research sponsored by NIFA/USDA (2020-67017-30834) Milk exosome-driven evolution of antibiotic-resistant gut pathogens: National Institute of Food and Agriculture Twitter: https://twitter.com/usda_nifa
  • Type: Other Status: Other Year Published: 2020 Citation: USDA blog regarding research sponsored by NIFA/USDA (2020-67017-30834) Milk exosome-driven evolution of antibiotic-resistant gut pathogens: USDA website: https://www.usda.gov/media/blog/2020/06/01/milk-findings-may-help-infants-worldwide
  • Type: Other Status: Published Year Published: 2020 Citation: Nature Outlook Do the microRNAs we eat affect gene expression?: https://www.nature.com/articles/d41586-020-01767-x and https://www.nature.com/collections/hibdgeeijf. Nature. June 17, 2020 Nature 582, S10-S11 (2020), doi: 10.1038/d41586-020-01767-x
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Liu, B, Lu Y, Chen X, Muthuraj, PG, Li X, Pattabiraman M, Zempleni J, Kachman SD, Natarajan SK, Yu J. Protective role of shiitake mushroom-derived exosome-like nanoparticles in D-galactosamine and lipopolysaccharide-induced liver injury in mice. Nutrients 12:477, 2020
  • Type: Journal Articles Status: Other Year Published: 2020 Citation: Sadri M, Shu J, Kachman SD, Cui J, Zempleni J. Milk exosomes and microRNAs cross the placenta and promote embryo survival in mice. Reproduction 160:501-509, 2020
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Zempleni J. Letter to the editor (regarding Perspective: The Role of Human Breast-Milk Extracellular Vesicles in Child Health and Disease by D. OReilly et al., published in AN on 24 August 2020). Adv Nutr 12:280, 2021
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Sadri M, Wang H, Kuroishi T, Li Y, Zempleni J. Engineering of a conditional holocarboxylase synthetase knockout mouse (submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Sukreet S, Pereira Braga C, An TT, Adamec J, Cui J, Trible B, Zempleni J. Isolation of extracellular vesicles from byproducts of cheese making by tangential flow filtration yields heterogenous fractions of nanoparticles (submitted)
  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Zhou, F, Dogan H, Shu J, Fernando SC, Cui J, Zempleni J. Evolution of gut bacteria through the selection of genetic variations by extracellular vesicles in milk (submitted)
  • Type: Books Status: Published Year Published: 2020 Citation: Penberthy WT, Sadri M, Zempleni J. Chapter 23: Biotin. In: Present Knowledge in Nutrition. Marriott BP, Birt DF, Stallings VA, Yates AA (eds.), 11th edition. Elsevier & International Life Sciences Institute, London, UK, 2020, 289-303
  • Type: Book Chapters Status: Published Year Published: 2020 Citation: Zempleni J. MicroRNAs and exosomes in human milk. In: Human Milk: Sampling and Measurement of Energy-Yielding Nutrients and Other Macromolecules. McGuire S. (ed), Elsevier, Amsterdam, The Netherlands, 2020, 339-346
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhou F, Zempleni J. Loss of maternal microRNA biogenesis impairs gut health in wild-type pups fostered to Dicer knockout dams. Nutrition 2020 Conference, American Society for Nutrition; Seattle, WA, 5/30  6/2, 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zhao W, Zempleni J. Adipocyte-derived exosomes increase the growth of breast tumors in obese mice. Nutrition 2020 Conference, American Society for Nutrition; Seattle, WA, 5/30  6/2, 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zempleni J, Sadri M, Zhou F. Exosomes and microRNAs in maternal milk are important for growth and gut health during lactation in murine pups. American Society for Exosomes and Microvesicles, November 17, 2020 (virtual meeting) [short talk, delivered by using Zoom]. Extracellular Vesicle and Circulating Nucleic Acid (EVCNA) Journal. 1:33-34, 2020


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

Outputs
Target Audience:1) Peer scientists (information disseminated through invited seminars, presentations at national conferences, publications in science journals) 2) Policy makers (federal agencies such as USDA, NIH, CDC) 3) Students (hands-on training in laboratory, class room teaching, national and international conferences) 4) Lay public (newspaper and magazine interviews) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of three postdoctoral fellows, nine graduate students, and a research technologist.Students and postdocs presented at local and national conferences. How have the results been disseminated to communities of interest?1) Journal articles 2) Publications in the lay press 3) Conference presentations 4) Invited seminars 5) Annual reports (NIFA grantee meetings, USDA multistate meetings) 6) Workshop sponsored by NIH and USDA What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. Focus the exosome studies on the delivery to immune cells such as macrophages and distinct regions in the brain. Assess the effects of membrane lipids on exosome bioavailability and distribution. Determine whether intracellular degradation of exosomes and their cargos in lysosomes can be altered by manipulating the exosome surface glycoproteins and membrane lipids. Assess interactions of bovine milk exosomes with the human and murine microbiome (genetic makeup and pathogenicity). Objective 2. Assess the delivery of functional milk miRNAs in mice. Assess interactions of miRNAs, encapsulated in bovine milk exosomes, with the human and murine microbiome. Objective 3. Assess the delivery of functional milk mRNAs in mice. Determine whether mRNAs in bovine milk can be translated in vivo and have effects on immune tolerance or food allergies. Assess the role of miRNAs, encapsulated in bovine milk exosomes, in the activation of anti-viral responses through Toll-like receptor signaling. Overall: Continue the dissemination of results from these studies through publications in peer-reviewed journals and other outlets as described above. Pursue external research funding.

Impacts
What was accomplished under these goals? Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. In previous years we reported that bovine milk exosomes and their microRNA cargos are bioavailable across species boundaries and accumulate primarily in the intestinal mucosa, liver, brain, spleen and kidneys. In this reporting period, we expanded these studies to show that the fraction of exosomes that goes unabsorbed causes changes in bacterial communities in the murine gut. We also developed three transgenic mouse models that we used to demonstrate that maternal exosomes are transferred to offspring through milk and loss of exosomes and microRNA cargos impair mucosal integrity and barrier function in the gut. Maternal exosomes accumulated primarily in the intestinal mucosa, liver and brain in pups. We have continued our studies assessing the roles of glycoproteins in the absorption and distribution of milk exosomes. Briefly, we have identified approximately 200 glycoproteins on the surface of bovine milk exosomes. Among the various classes of glycoproteins, N-glycans were of particular importance for the intestinal transport of exosomes and their microRNA cargos in humans and mice. These studies form the basis for new partnerships with industry in which we seek to use milk exosomes for drug delivery and exosome-optimized diets in livestock. We have published a paper in which we describe protocols for exosome isolation, storage and microRNAs in small samples of human milk, and identified more than 200 microRNAs in human milk. Objective 2. Assess the delivery of functional milk miRNAs in mice. We have published a paper (in press) showing that bovine milk exosomes do not elicit an immune response if administered orally to humans and in ex vivo cultures of human immune cells. This is an important discovery, because milk exosomes are considered an attractive for delivery cancer drugs to patients. We have discovered that milk exosomes and microRNA cargos added to soy infant formula are not bioavailable, probably due to the precipitation of milk exosomes by lectins in the formula. We have published a paper that identified potential confounders of microRNA analysis in plasma, e.g., improper sample storage, use of heparin as anti-coagulant, and hemolysis of red blood cells. We have discovered that milk exosomes select for polymorphisms in gut bacteria, thereby changing the metabolism of bacteria. We are testing the novel hypothesis that phenotypic changes elicited by feeding bovine exosomes are not solely due to changes in microRNA cargos, but that other exosome cargos also contribute to these phenotypes. Specifically, we are in the process of testing whether mRNA in bovine exosomes can be translated into peptides and proteins in non-bovine species, thereby causing food allergies or promoting immune tolerance. We have identified about 3,600 bovine mRNAs in bovine exosomes, most of which are truncated. However, 107 of these transcript s contain an ATG start codon and therefore can be translated into protein. Thirteen of these 107 mRNAs encode amino acid sequences that are not found in the proteins' human or murine orthologs and therefore are of interest in the context of immune response. We demonstrated that about 10 of the 107 ATG-containing mRNAs are translated into protein by rabbit reticulocyte lysates. We currently determine whether macrophage-like cells can translate the mRNAs into protein. Objective 3. Assess the delivery of functional milk mRNAs in mice. We have discovered that bovine milk exosomes contain both bovine and bacterial mRNAs. This discovery sparked two lines of investigation. First, we demonstrated that bovine mRNAs are translated into peptides in vitro, suggesting that mRNAs in milk exosomes may contribute to food allergies or immune tolerance in a protein-free environment. Second, we demonstrated that microbial mRNAs bind to Toll-like receptors in mammalian cells in culture. Activation of Toll-like receptors by bacterial mRNAs improved survival and body weight regain in mice challenged with influenza A.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Mutai E, Ramer-Tait AE, Zempleni J. MicroRNAs in bovine milk exosomes are bioavailable in humans but do not elicit a robust pro-inflammatory cytokine response. BMC exRNA
  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Wu D, Kittana H, Shu J, Kachman SD, Cui J, Ramer-Tait AE, Zempleni J. Dietary Depletion of milk exosomes and their microRNA cargos elicits a depletion of miR-200a-3p and elevated intestinal inflammation and CXCL9 expression in Mdr1a-/- mice. Curr Develop Nutr
  • Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Sadri M, Shu J, Kachman SD, Cui J, Zempleni J. Cow milk exosomes and microRNAs accumulate in placenta and embryos and promote placentation in mice
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Casavale KO, Ahuja J, Wu X, Li Y, Quam J, Olson R, Pehrsson P, Allen L, Balentine D, Hanspal M, Hayward D, Hines EP, McClung JP, Perrine C, Brown Belfort M, Dallas D, German B, Kim J, McGuire M, McGuire M, Morrow A, Neville M, Nommsen-Rivers L, Rasmussen KM, Zempleni J, Lynch CJ. NIH workshop on human milk composition: summary and visions. Am J Clin Nutr 110:769-779, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited lecture titled Dietary exosomes in the graduate class Exosomes: Molecular Mechanisms and Biomedical Applications (ME 340.714) at Johns Hopkins University. Baltimore, MD, March 27th, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Biological activities of milk exosomes and their RNA cargos across and within species boundaries in the Department of Biological Chemistry at Johns Hopkins University. Baltimore, MD, March 27th, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J, Sadri M, Zhou F. Knockout of maternal Tsg101 and Dicer impair gut health in suckling wild-type pups. American Society for Exosomes and Microvesicles, Asilomar, CA, October 6-10, 2019
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Zempleni J. Milk exosomes. Session Exosomes & microRNA at the 7th International Conference on Food Factors, Kobe, Japan, from 1st to 5th December, 2019
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Zempleni J. Future outlook. Session Exosomes & microRNA at the 7th International Conference on Food Factors, Kobe, Japan, from 1st to 5th December, 2019
  • Type: Other Status: Published Year Published: 2019 Citation: McCann M. Milk miracle: New Zealand AgResearch scientists eye new baby booster. New Zealand Herald, 2/10/2019
  • Type: Other Status: Published Year Published: 2019 Citation: Dance, A. The bodys tiny cargo carriers. The Knowable Magazine (from Annual Reviews), April 30, 2019. https://www.knowablemagazine.org/article/living-world/2019/bodys-tiny-cargo-carriers
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Biological activities of milk exosomes and their RNA cargos. In the symposia titled Nutrition, Extracellular Vesicles and RNA Cargos, Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 9, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. UNL Spring Research Fair; Lincoln, NE, April 15, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sukreet S, Adamec J, Cui J, Zempleni J. Galactose and sialo-galactose modifications in glycoproteins on the surface of bovine milk exosome are essential for exosome uptake in non bovine species. UNL Spring Research Fair, Lincoln, NE, April 15, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by murine bone marrow derived macrophages. UNL Spring Research Fair, Lincoln, NE, April 15, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by murine bone marrow derived macrophages. NPOD 11th Annual retreat 2019, Lincoln, NE, April 17, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. NPOD 11th Annual retreat 2019, Lincoln, NE, April 17, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mutai E, Ramer-Tait A, Zempleni J, Immunomodulatory microRNAs in bovine milk exosomes are bioavailable and depend on co-stimulation with concanavalin A to elicit cytokine secretion by peripheral blood mononuclear cells ex vivo in humans. University of Nebraska Graduate Poster Session, April 17th at the and NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhou F, Sadri M, Zempleni J. Impaired maternal milk exosomes and microRNAs biogenesis decrease gut health and food intake in mice pups. Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 8-11, 2019
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Parry HA, Mobley CB, Mumford PW, Romero MA, Haun CT, Zhang Y, Roberson PA, Zempleni J, Ferrando AA, Vecchetti IJ Jr., McCarthy JJ, Young KC, Roberts MD, Kavazis AN. Bovine milk extracellular vesicles (EVs) modification elicits skeletal muscle growth in rats. Front Physiol 10:436, 2019
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Penberthy WT, Sadri M, Zempleni J. Chapter 23: Biotin. In: Present Knowledge in Nutrition. Birt D (ed.), 11th edition. International Life Sciences Institute, Washington, DC
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zempleni J, Sukreet S, Zhou F, Wu D, Mutai E. Milk-derived exosomes and metabolic regulation. Ann Rev Anim Biosci 7:245-262, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Shu J, Braga CP, Cui J, Adamec J, Zempleni J. Bovine mRNAs in cow's milk exosomes are bioavailable and translated into peptides in non-bovine species. Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 8-11, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J, Upadhyaya B, Xia M, Moriyama H, Ohtsuka M. Milk exosomes accumulate in the intestinal mucosa and peripheral tissues in wild-type pups nursed by exosome and cargo tracking dams. International Society for Extracellular Vesicles, International Society for Extracellular Vesicles, Miyakomesse, Kyoto, Japan, April 24-28, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. UNL & PureTech Health, Inc: milk exosomes in drug delivery. Presentation to the NUtech Ventures Board of Directors, March 1, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. Nutrition 2019 Conference, American Society for Nutrition; Baltimore, MD, June 8-11, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Effects of milk exosomes and their RNA cargos on anti-viral response and postnatal growth as part of the annual W-4002 Multistate group meeting; UC-Davis, Davis, CA. February 7th, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited lecture titled Dietary exosomes in the graduate class Exosomes: Molecular Mechanisms and Biomedical Applications (ME 340.714) at Johns Hopkins University. Baltimore, MD, March 27th, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J. Invited presentation titled Biological activities of milk exosomes and their RNA cargos across and within species boundaries in the Department of Biological Chemistry at Johns Hopkins University. Baltimore, MD, March 27th, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. Nebraska Spring Research Fair; Lincoln, NE, April 15, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by murine bone marrow derived macrophages. Nebraska Spring Research Fair; Lincoln, NE, April 15, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ebea P, Sukreet S, Zempleni J, Bovine milk exosomes and miR-34a cargo cross the blood brain barrier and elimination by microglial accumulation is minimal. NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Development of an exosome and cargo tracking mouse model. NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Mutai E, Ramer-Tait A, Zempleni J, Immunomodulatory microRNAs in bovine milk exosomes are bioavailable and depend on co-stimulation with concanavalin A to elicit cytokine secretion by peripheral blood mononuclear cells ex vivo in humans. University of Nebraska Graduate Poster Session, April 17th at the and NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhou F, Dogan H, Shu J, Fernando S, Cui J, Zempleni J, Bovine milk exosomes select polymorphisms in murine intestinal microbiome ex vivo. NPOD 12thAnnual Retreat, September 11th, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by murine bone marrow derived macrophages. NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Mutai E, Cui J, Zempleni J. Microbial mRNAs in bovine milk exosomes are bioavailable in humans and mice and increase survival of mice challenged with influenza A. NPOD 11th Annual Retreat, April 17, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhou F, Sadri M, Zempleni J, Loss of exosome and microRNA biogenesis in lactating dams impairs gut health, food intake and postnatal growth in suckling C57BL/6 pups. NPOD 11thAnnual Retreat, April 17th, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by murine bone marrow-derived macrophages. NPOD 12thAnnual Retreat, September 11th, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zhao W, Zempleni J. A mouse for tracking and cargo analysis in adipose-derived exosomes. NPOD 12thAnnual Retreat, September 11th, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sadri M, Zempleni J, loss of exosome biogenesis in lactating dams impairs gut health and postnatal growth in C57/BL6 pups. NPOD 12th Annual Retreat, September 11th, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wu D, Zhou F, Upadhyaya B, Shu J, Pereira C, Mutai E, Cui J, Adamec J, Zempleni J, Bioavailability and the potential functions of bovine and microbial mRNAs in bovine milk exosomes. NPOD 12thAnnual Retreat, September 11th, 2019, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Parry HA, Mobley CB, Mumford PW, Romero MA, Zhang Y. Zempleni J, McCarthy JJ, Young KC, Roberts MD, Kavazis AN. Bovine Milk Exosome Depletion Affects Skeletal Muscle and Liver in Young Growing Rats. American College of Sports Medicine, Orlando, FL, May 28  June 1, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J, Sadri M, Zhou F. Knockout of maternal Tsg101 and Dicer impair gut health in suckling wild-type pups. American Society for Exosomes and Microvesicles, Asilomar, CA, October 6-10, 2019 [talk]
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Zempleni J, Sadri M, Zhou F. Knockout of maternal Tsg101 and Dicer impair gut health in suckling wild-type pups. American Society for Exosomes and Microvesicles, Asilomar, CA, October 6-10, 2019 [poster]


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

Outputs
Target Audience:1) Peer scientists (information disseminated through invited seminars, presentations at national conferences, publications in science journals) 2) Policy makers (federal agencies such as USDA, NIH, CDC) 3) Students (hands-on training in laboratory, class room teaching, national and international conferences) 4) Lay public (newspaper and magazine interviews) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of three postdoctoral fellows, nine graduate students, two undergraduate students, a visiting scientist and a research technologist. How have the results been disseminated to communities of interest?1) Journal articles 2) Publications in the lay press 3) Conference presentations 4) Invited seminars 5) Annual reports (NIFA grantee meetings, USDA multistate meetings) 6) Workshop sponsored by NIH and USDA What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. Focus the exosome studies on the delivery to immune cells such as macrophages and distinct regions in the brain. Assess the effects of membrane lipids on exosome bioavailability and distribution. Determine whether intracellular degradation of exosomes and their cargos in lysosomes can be altered by manipulating the exosome surface glycoproteins and membrane lipids. Assess interactions of bovine milk exosomes with the human and murine microbiome (communities and genetic makeup). Objective 2. Assess the delivery of functional milk miRNAs in mice. Assess interactions of miRNAs, encapsulated in bovine milk exosomes, with the human and murine microbiome. Objective 3. Assess the delivery of functional milk mRNAs in mice. Determine whether mRNAs in bovine milk can be translated in vivo and have effects on immune tolerance or food allergies. Assess the delivery of microbial mRNAs through bovine milk exosomes in humans and mice. Assess the role of miRNAs, encapsulated in bovine milk exosomes, in the activation of anti-viral responses through Toll-like receptor signaling.

Impacts
What was accomplished under these goals? Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. We have demonstrated that milk exosomes are bioavailable and accumulate in liver, spleen and brain by using the following protocols. 1) Bovine milk exosomes were labeled with a fluorescent dye, DiR, and administered orally and intravenously to mice. DiR accumulated in the liver, but we demonstrated that DiR is not a reliable label in exosome distribution studies. 2) Exosomes were transfected with fluorophore-labeled microRNAs and mRNAs. The RNAs accumulated primarily in liver and brain. 3) We used transgenic pigs that produce milk in which exosomes are endogenously labeled with a fluorescent protein. This model was used to collect exosomes for oral and intravenous administration to mice, and for monitoring the transfer of milk exosomes through the natural route (suckling). The exosomes accumulated in brain. 4) We have developed a transgenic mouse that secretes milk in which exosomes are endogenously labeled with a fluorescent protein. To date, this mouse was used to assess the transfer of milk exosomes through the natural route (suckling). The exosomes accumulated primarily in liver and brain. We have used mass spectrometry protocols, bioinformatics protocols, transgenic mice and enzyme-based protocols to assess the roles of glycoproteins in the absorption and distribution of milk exosomes. Briefly, we have identified approximately 200 glycoproteins on the surface of bovine milk exosomes. Among the various classes of glycoproteins, N-glycans were of particular importance for the intestinal transport of exosomes and their microRNA cargos in humans and mice. These studies form the basis for new partnerships with industry in which we seek to use milk exosomes for drug delivery and exosome-optimized diets in livestock. We have submitted a paper in which we describe protocols for exosome isolation, storage and microRNAs in small samples of human milk, and identified more than 200 microRNAs in human milk. Objective 2. Assess the delivery of functional milk miRNAs in mice. We have demonstrated that microRNAs in bovine milk, if encapsulated in biological nanoparticles (exosomes) are absorbed in human milk feeding studies. In these studies, we have adopted a novel PCR technology (RNase H2 PCR) that allows distinguishing bovine and human microRNAs that differ by as little as one nucleotide. In addition to this milk feeding study, we have completed the sample collection in humans who consumed the following milk-based treatment: 1) Infant soy formula (zero bovine microRNAs, negative control) 2) Sonicated bovine milk (microRNA depleted) 3) Infant soy formula fortified with bovine milk exosomes and their RNA cargos 4) Sonicated bovine milk fortified with bovine milk exosomes and their RNA cargos 5) Bovine milk (positive control) The samples (plasma) from these studies have been analyzed for the plasma concentrations of immune-relevant microRNAs before and at timed intervals after milk consumption. The postprandial plasma microRNA concentrations were not higher than baseline concentrations in groups 1, 2 and 3, whereas postprandial plasma concentrations were significantly higher than baseline concentrations in groups 4 and 5. We propose that lectins in soy formulas precipitate exosomes and therefore fortification was not successful in group 3, whereas fortification was successful in a milk-based matrix (group 4), consistent with studies of glycan features in Objective 1. We have published a paper that identified potential confounders of microRNA analysis in plasma, e.g., improper sample storage, use of heparin as anti-coagulant, and hemolysis of red blood cells. Dietary depletion of milk exosomes and their microRNA cargos elicits phenotypes such as aberrant purine metabolism, impaired cognitive performance and moderate loss of muscle grip strength in human adults and infants and mice. We have developed protocols for assessing the bioavailability of bovine milk exosomes and their RNA cargos in mice. Briefly, exosomes or their microRNA cargos were labeled with fluorophores and administered intravenously or orally to mice. The exosomes accumulated in liver and spleen, whereas different microRNAs had unique distributions: miR-320a accumulated primarily in the liver, miR-155 accumulated primarily in the spleen, miR-34a accumulated primarily in the brain and miR-375 accumulated in the intestine. A fraction of dietary exosomes was not absorbed and entered the large where it caused a change in microbial communities. We are testing the novel hypothesis that phenotypic changes elicited by feeding bovine exosomes are not solely due to changes in microRNA cargos, but that other exosome cargos also contribute to these phenotypes. Specifically, we are in the process of testing whether mRNA in bovine exosomes can be translated into peptides and proteins in non-bovine species, thereby causing food allergies or promoting immune tolerance. We have identified about 3,600 bovine mRNAs in bovine exosomes, most of which are truncated. However, 107 of these transcript s contain an ATG start codon and therefore can be translated into protein. Thirteen of these 107 mRNAs encode amino acid sequences that are not found in the proteins' human or murine orthologs and therefore are of interest in the context of immune response. We demonstrated that about 10 of the 107 ATG-containing mRNAs are translated into protein by rabbit reticulocyte lysates. We currently determine whether macrophage-like cells can translate the mRNAs into protein. Objective 3. Assess the delivery of functional milk mRNAs in mice. In previous studies we demonstrated that bovine milk microRNAs are delivered to human immune cells and alter gene expression therein (see 2015 report). These studies did not focus on cytokines. For this NIFA project, we have enrolled new subjects to assess the secretion of pro-inflammatory cytokines by human lymphocytes ex vivo. Feeding groups were the same as described in Goal 1. The following cytokines were assessed using a cytokine multiplex system:IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, IFN-gamma and TNF-alpha. Consumption of sonicated cow's milk caused a 50% to 900% increase in secretion of IL-1b, TNA-alpha, and IL-6 by concanavalin A-stimulated lymphocytes, whereas consumption of regular (non-sonicated) cow's milk caused a 10% to 300% increase. Consumption of unsonicated bovine milk, soy infant formula and exosome-fortified soy formula elicited only a minor increase in cytokine secretion. We are conducting additional studies in which human peripheral blood monouclear cells are treated ex vivo with bovine milk exosomes transfected with immune-relevant microRNAs. This experiment will allow us to draw a causal link between milk exosome microRNA cargos and immune response. Of particular interest is the observation that about 15-20% of the mRNAs in bovine milk are of microbial origin. We have demonstrated that microbial mRNAs in milk exosomes activate anti-viral responses in mice, and enhance the survival of mice challenged with influenza A.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sukreet S, Silva B, Wu Di, Shu J, Adamec J, Cui J, Zempleni J. Sonication and incubation of milk causes a moderate loss of exosomes and substantial changes in exosomal RNA, lipid and protein cargos. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mutai E, Ramer-Tait A, Zempleni J. Bioavailability of immunomodulatory microRNAs in bovine milk and cytokine secretion by peripheral blood mononuclear cells ex vivo in humans. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhou F, Giraud D, Wu D, Brown DM, Zempleni J. Microbial mRNAs in bovine milk exosomes activate interferon-beta in mice. Nebraska Center for the Prevention of Obesity Diseases, April 11th, 2018, Lincoln, NE. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Generation of transgenic exosome and cargo tracking mice. NPOD 10th Annual Retreat, April 11, 2018, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d cargos cross the placenta contribute toward embryonic development and survival in C57BL/6 mice. NPOD 10th Annual Retreat, April 11, 2018, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J, Zhou F, Shu J, Wu D, Upadhyaya B, Cui J, Paz H, Fernando S. Milk exosomes interface with the microbiome. Keystone symposium Exosomes/Microvesicles: Heterogeneity, Biogenesis, Function and Therapeutic Developments, June 4-8, 2018, Beaver Run Resort, Breckenridge, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu D, Kittana H, Shu J, Ramer-Tait A, Cui J, Zempleni J. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. Keystone symposium Exosomes/Microvesicles: Heterogeneity, Biogenesis, Function and Therapeutic Developments, June 4-8, 2018, Beaver Run Resort, Breckenridge, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Exosome and cargo tracking mice. Keystone symposium Exosomes/Microvesicles: Heterogeneity, Biogenesis, Function and Therapeutic Developments, June 4-8, 2018, Beaver Run Resort, Breckenridge, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu D, Kittana H, Shu J, Ramer-Tait AE, Cui J, Zempleni J. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Fratantonio D, Shu J, Cui J and Zempleni J. Chicken egg exosomes and their cargo are bioavailable and dietary depletion affects the hippocampus gene expression in mice. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ebea P, Sukreet S, Zempleni J. Uptake of RNA cargos in bovine milk exosomes by murine brain endothelial cells. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by bone marrow derived macrophages. Nebraska Center for the Prevention of Obesity Diseases, Spring Retreat. Lincoln, NE, April 11th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J, Wu D, Ramer-Tait A. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. NIFA Program Directors meeting; Boston, MA, June 9th, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sukreet S, Adamec J, Cui J, Zempleni J. Identification of Glycoproteins on the Surface of Cows Milk Exosomes that Mediate the Uptake of Exosomes into Human Cells. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhou F, Shu J, Fernando S, Cui J, Zempleni J. Bovine milk exosomes select polymorphisms in murine intestinal microbiome in vitro. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d and miR-21-5p cargos cross the placenta and contribute toward embryonic development and survival in C57BL/6 mice. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wei Z, Zempleni J. The role of adipose derived-exosomes in breast tumorigenesis in obese mice. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parry HA, Mobley CB, Mumford PW, Romero MA, Zhang Y. Zempleni J, Young KC, Roberts MD, Kavazis AN. Dietary Exosomes Affect Growth and Skeletal Muscle Physiology in Young Male and Female Rats. American College of Sports Medicine, San Diego, CA, September 5-8. 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Upadhyaya B, Xia M, Moriyama H, Ohtsuka M, Zempleni J. Development of an exosome and cargo tracking mouse. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ebea P, Sukreet S, Zempleni J. Uptake of RNA cargos in bovine milk exosomes by murine brain endothelial cells. NPOD 4th Annual Symposium, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Khanam A, Yu J, Zempleni J. Uptake of bovine milk exosomes by bone marrow derived macrophages. NPOD 4th Annual Symposium 2018, Lincoln, NE, September 12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Exosomes in milk. Grand Challenges Meeting, sponsored by the Gates Foundation, Berlin, Germany, October 15-18, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Visiting Professor delivering the following lecture: Dietary exosomes and their RNA cargos as novel bioactive food compounds. November 5, 2018; Yogyakarta, Indonesia
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Visiting Professor delivering the following lecture: Biotin metabolism. November 2, 2018; Yogyakarta, Indonesia
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Visiting Professor delivering the following lecture: Pursuing a graduate education in the US. November 2, 2018; Yogyakarta, Indonesia
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Visiting Professor delivering the following lecture: Obesity research in the United States. November 3, 2018; Yogyakarta, Indonesia
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang L, Sadri M, Giraud D, Zempleni J. RNase H2-dependent PCR and elimination of confounders in sample collection, storage and analysis strengthen evidence that microRNAs in bovine milk are bioavailable in humans. J Nutr 148:153-159, 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Manca S, Upadhyaya B, Mutai E, Desaulniers AT, Cederberg RA, White BR, Zempleni J. Milk exosomes are bioavailable and distinct microRNA cargos have unique distribution patterns. Sci Rep 8:11321, 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Camara Teixeira D, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins. J Nutr Biochem 6:48-54, 2018
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. J Nutr Biochem 12:123-128, 2018
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Cui J. Zempleni J. RNase H2-dependent PCR Detects Bovine MicroRNAs in Human Plasma. [reply: letter to the editor] J Nutr 148:1508, 2018
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Aguilar-Lozano A, Baier SR, Grove R, Shu J, Giraud D, Mercer KE, Cui J, Badger TM, Adamec J, Andres A, Zempleni J. Concentrations of purine metabolites are elevated in fluids from adults and infants and in livers from mice fed diets depleted of bovine milk exosomes and their RNA cargos. J Nutr (in press)
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Swanson B, Zempleni J, Nordgren T, Romberger D, Heires A. Bovine milk-derived exosomes enhance inflammation and promote M1 polarization following agricultural dust exposure in mice. J Nutr Biochem (in press)
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Fratantonio D,* Shu J,* Howard K, Baier SR, Giraud D, Cui J, Zempleni.MicroRNAs from chicken eggs are bioavailable and alter gene expression in peripheral blood mononuclear cells in humans (submitted, under revision). *These authors contributed equally
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Zhou F, Paz HA, Shu J, Sadri M, Cui J, Fernando SC, Zempleni J. Dietary bovine milk exosomes and their RNA cargos elicit changes in the composition of the intestinal microbiome and the hepatic transcriptome in C57BL/6 mice (under revision)
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Zempleni J, Sukreet S, Zhou F, Wu D, Mutai E. Milk-derived exosomes and metabolic regulation. Ann Rev Anim Biosci (in press)
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2018 Citation: Zempleni J. MicroRNAs and exosomes in human milk. In: Human Milk: Sampling and Measurement of Energy-Yielding Nutrients and Other Macromolecules. McGuire S. (ed), Elsevier (invited chapter)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Parry HA, C. Mobley B, Mumford PW, Romero MA, Zhang Y, Zempleni J, Young KC, Roberts MD, Kavazis AN. Dairy Exosome Effects on Mitochondria Function and Antioxidant Enzymes in Growing Male and Female Rats. Experimental Biology 2018, April 21-25, 2018, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Nordgren TM, Zempleni J, Heires AJ, Romberger DJ. Dietary exosomes amplify the inflammatory effects of agricultural dust exposure in mice. 2018 American Thoracic Society International conference, May, 18-23, 2018, San Diego, CA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d cargo cross the placenta and contribute toward embryo development in C57BL/6 mice. Nutrition 2018 Conference, American Society for Nutrition; Boston, MA, June 9-12, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Bioavailability, distribution and phenotypes of bovine milk exosomes in non-bovine species. The American Dairy Science Association, Annual Meeting, Knoxville, TN, June 24-27, 2018 (presentation date 6/25/2018)
  • Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Milk exosomes. Program Officials of the Bill and Melinda Gates Foundation. August 7, 2018 [delivered remotely by Zoom]
  • Type: Other Status: Published Year Published: 2018 Citation: Zempleni J. Bovine milk exosomes: bioavailability, distribution and interactions with the gut microbiome. PureTech Health, Inc., Boston, MA, August 23, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J, Upadhyaya B, Xia M, Moriyama H, Ohtsuka M. Milk exosomes accumulate in the intestinal mucosa and peripheral tissues in wild-type pups nursed by exosome and cargo tracking dams. American Society for Exosomes and Microvesicles, Baltimore Marriott Waterfront Conference Center, Baltimore, MD, October 20-24, 2018
  • Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Leiferman A, Shu J, Upadhyaya B, Cui J, Zempleni J. Storage stability of extracellular vesicles in human and bovine milk, and microRNA profiles in human milk exosomes and infant formulas. (submitted)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species. W-3002 Multistate group meeting. February 8/9, 2018,Tucson, AZ
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zempleni J. Cross-kingdom communication: bovine milk exosomes talk to the gut microbiome talk to the host. Invited seminar in the Interdepartmental Nutrition seminar series. Speaker chosen by the Nutrition Science Graduate Student Organization as Spring Seminar 2018 Speaker. Purdue University, Lafayette, IN, March 9, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu D, Kittana H, Shu J, Ramer-Tait AE, Cui J, Zempleni J. Effects of diets defined by their content of bovine milk exosomes and their RNA cargos on inflammatory bowel disease in Mdr1a-/- mice. University of Nebraska-Lincoln Graduate Student Spring Research Fair, April 10, 2018, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sukreet S, Silva B, Wu Di, Shu J, Adamec J, Cui J, Zempleni J. Sonication and short term incubation causes specific cargos loss in bovine milk exosomes. University of Nebraska-Lincoln Graduate Student Spring Research Fair, April 10, 2018, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhou F, Paz H, Fernando S, Sadri M, Zempleni J. A diet defined by its content of bovine milk exosomes alters the composition of the intestinal microbiome in C57BL/6 mice. University of Nebraska-Lincoln Graduate Student Spring Research Fair, April 10, 2018, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Sadri M, Shu J, Cui J, Zempleni J. Bovine milk exosomes and their miR-30d cargos cross the placenta contribute toward embryonic development and survival in C57BL/6 mice. UNL Research Fair, April 10, 2018, Lincoln, NE


Progress 01/11/17 to 09/30/17

Outputs
Target Audience:1) Peer scientists (information disseminated through invited seminars, presentations at national conferences, publications in science journals) 2) Policy makers (federal agencies such as USDA, NIH, CDC) 3) Students (hands-on training in laboratory, class room teaching, national and international conferences) 4) Lay public (newspaper and magazine interviews) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has supported the training of three postdoctoral fellows, nine graduate students, two undergraduate students, a visiting scientist and a research technologist. How have the results been disseminated to communities of interest?Dissemination 1) Journal articles 2) Publications in the lay press 3) Conference presentations 4) Invited seminars 5) Annual reports (NIFA grantee meetings, USDA multistate meetings) 6) Workshop sponsored by NIH and USDA What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. Focus the exosome studies on the delivery to immune cells such as macrophages and distinct regions in the brain. Assess interactions of bovine milk exosomes with the human and murine microbiome. Objective 2. Assess the delivery of functional milk miRNAs in mice. Assess the role of miRNAs, encapsulated in bovine milk exosomes, in the activation of anti-viral responses through Toll-like receptor signaling. Assess interactions of miRNAs, encapsulated in bovine milk exosomes, with the human and murine microbiome. Objective 3. Assess the delivery of functional milk mRNAs in mice. Determine whether mRNAs in bovine milk can be translated in vivo and have effects on immune tolerance or food allergies. Assess the delivery of microbial mRNAs through bovine milk exosomes in humans and mice. Overall: Continue the dissemination of results from these studies through publications in peer-reviewed journals and other outlets as described above. Pursue external research funding.

Impacts
What was accomplished under these goals? Objective 1: Assess the bioavailability and distribution of cow's milk exosomes in mice. We have demonstrated that milk exosomes are bioavailable and accumulate in liver, spleen and brain by using the following protocols. 1) Bovine milk exosomes were labeled with a fluorescent dye, DiR, and administered orally and intravenously to mice. 2) We used transgenic pigs that produce milk in which exosomes are endogenously labeled with a fluorescent protein. This model was used to collect exosomes for oral and intravenous administration to mice, and for monitoring the transfer of milk exosomes through the natural route (suckling). 3) We have developed a transgenic mouse that secretes milk in which exosomes are endogenously labeled with a fluorescent protein. To date, this mouse was used to assess the transfer of milk exosomes through the natural route (suckling). Finally, we are assessing the roles of glycoproteins on the surface of bovine exosomes in the intestinal uptake and bioavailability of bovine exosomes. These studies provided compelling evidence that the uptake of milk exosomes into cells depends on glycoproteins on the surface of exosomes and cells. Objective 2. Assess the delivery of functional milk miRNAs in mice. We have demonstrated that microRNAs in bovine milk, if encapsulated in biological nanoparticles (exosomes) are absorbed in human milk feeding studies. In these studies, we have adopted a novel PCR technology (RNase H2 PCR) that allows distinguishing bovine and human microRNAs that differ by as little as one nucleotide. In addition to this milk feeding study, we have completed the sample collection in humans who consumed the following milk-based treatment: 1) Infant soy formula (zero bovine microRNAs, negative control) 2) Sonicated bovine milk (microRNA depleted) 3) Infant soy formula fortified with bovine milk exosomes and their RNA cargos 4) Sonicated bovine milk fortified with bovine milk exosomes and their RNA cargos 5) Bovine milk (positive control) Sample collection is complete and these samples are currently analyzed for the plasma concentrations of immune-relevant microRNAs before and at timed intervals after milk consumption. Preliminary data suggest that the postprandial plasma concentrations were not higher than baseline concentrations in groups 1, 2 and 3, whereas postprandial plasma concentrations were significantly higher than baseline concentrations in groups 4 and 5. We propose that lectins in soy formulas precipitate exosomes and therefore fortification was not successful in group 3, whereas fortification was successful in a milk-based matrix (group 4). This theory is being tested in studies in cell cultures. We have published a paper that identified potential confounders of microRNA analysis in plasma, e.g., improper sample storage, use of heparin as anti-coagulant, and hemolysis of red blood cells. We have developed protocols for assessing the bioavailability of bovine milk exosomes and their RNA cargos in mice. Briefly, exosomes or their microRNA cargos were labeled with fluorophores and administered intravenously or orally to mice. The exosomes accumulated in liver and spleen, whereas different microRNAs had unique distributions: miR-320a accumulated primarily in the liver, miR-155 accumulated primarily in the spleen, miR-34a accumulated primarily in the brain and miR-375 accumulated in the intestine. A fraction of dietary exosomes was not absorbed and entered the large where it caused a change in microbial communities. We are testing the novel hypothesis that phenotypic changes elicited by feeding bovine exosomes are not solely due to changes in microRNA cargos, but that other exosome cargos also contribute to these phenotypes. Specifically, we are in the process of testing whether mRNA in bovine exosomes can be translated into peptides and proteins in non-bovine species, thereby causing food allergies or promoting immune tolerance. We have identified about 3,600 bovine mRNAs in bovine exosomes, most of which are truncated. However, 107 of these transcript s contain an ATG start codon and therefore can be translated into protein. Thirteen of these 107 mRNAs encode amino acid sequences that are not found in the proteins' human or murine orthologs and therefore are of interest in the context of immune response. We demonstrated that about 10 of the 107 ATG-containing mRNAs are translated into protein by rabbit reticulocyte lysates. We currently determine whether macrophage-like cells can translate the mRNAs into protein. Of particular interest is the observation that about 15-20% of the mRNAs in bovine milk are of microbial origin. We currently seek to identify the molecular targets of the microbial mRNAs in mice. Objective 3. Assess the delivery of functional milk mRNAs in mice. In previous studies we demonstrated that bovine milk microRNAs are delivered to human immune cells and alter gene expression therein (see 2015 report). These studies did not focus on cytokines. For this NIFA project, we have enrolled new subjects to assess the secretion of pro-inflammatory cytokines by human lymphocytes ex vivo. Feeding groups were the same as described in Goal 1. The following cytokines were assessed using a cytokine multiplex system:IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, IFN-gamma and TNF-alpha. Consumption of sonicated cow's milk caused a 50% to 900% increase in secretion of IL-1b, TNA-alpha, and IL-6 by concanavalin A-stimulated lymphocytes, whereas consumption of regular (non-sonicated) cow's milk caused a 10% to 300% increase. Consumption of unsonicated bovine milk, soy infant formula and exosome-fortified soy formula elicited only a minor increase in cytokine secretion. We are conducting additional studies in which human peripheral blood monouclear cells are treated ex vivo with bovine milk exosomes transfected with immune-relevant microRNAs. This experiment will allow us to draw a causal link between milk exosome microRNA cargos and immune response. We have shown that microRNAs in bovine milk bind to Toll-like receptors (TLRs) and activate anti-viral response pathways. We are currently testing the activation of TLRs by milk microRNAs in TLR reporter cell lines and TLR reporter mice. The latter is done in conjunction with an influenza A virus challenge of mice to determine whether pro-inflammatory effects of milk, mediated by binding to TLRs (unpublished), has beneficial effects regarding anti-viral response.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Non-canonical pathways of signaling and metabolic regulation by extracellular vesicles from bovine milk. Baylor University College of Medicine & Nutrition Research Center; April 6, 2017, Houston, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Are dietary RNAs bioavailable? RNA Group of Houston, Baylor University College of Medicine, UT Southwestern, University of Houston, and MD Anderson; April 6, 2017, Houston, TX
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Fratantonio D, Zempleni J. MicroRNAs in chicken egg exosomes are bioavailable in humans and contribute toward spatial learning and memory in mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sadri M, Mutai E, Zempleni J. Analysis of plasma by RNase H2 PCR provides evidence that microRNAs in bovine milk are bioavailable in humans. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manca S, Zempleni J. The bioavailability and distribution of bovine milk exosomes is distinct from that of their cargos in mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sukreet S, Silva-Resende B, Wu D, Shu J, Adamac J, Cui J, Zempleni J. Sonication and short-term incubation causes a specific loss of RNA cargos in bovine milk exosome. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Invited expert at the NIH-sponsored workshop titled Workshop on Human Milk Composition-Biological, Environmental, Nutritional, and Methodological Considerations Meeting. Bethesda, MD, November 16-17, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wu D, Shu J, Upadhyaya B, Cui J, Zempleni J. Bioavailability of microbial RNAs in bovine milk exosomes in mice. University of Nebraska, Fall 2017 Research Fair, November 8, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos affects gene expression but not amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mutai E, Ramer-Tait A, Zempleni J. Immunomodulatory microRNAs in Bovine Milk are Bioavailable and Synergize with Concanavalin A in the Stimulation of Cytokine Secretion by Peripheral Blood Mononuclear Cells ex vivo in Humans. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017, Lincoln, NE
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Wu D, Shu J, Upadhyaya B, Cui J, Zempleni J. Bioavailability of microbial RNAs in bovine milk exosomes in mice. University of Nebraska, NPOD 3rd Annual Fall Symposium/10th Annual Retreat, September 20, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Opening ceremony speaker at the meeting of the American Society for Exosomes and Microvesicles. Zempleni J, Zhou F, Wu D, Upadhyaya B, Shu J, Paz H, Fernando S, Cui J. Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species. Asilomar Conference Center, Pacific Beach, CA, October 8-12, 2017
  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Wang L, Sadri M, Giraud D, Zempleni J. RNase H2-dependent PCR and elimination of confounders in sample collection, storage and analysis strengthen evidence that microRNAs in bovine milk are bioavailable in humans. J Nutr
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Camara Teixeira D, Cordonier EL, Wijeratne SSK, Huebbe P, Jamin A, Jarecke S, Wiebe M, Zempleni J. A cell death assay for assessing the mitochondrial targeting of proteins
  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Leiferman A, Shu J, Grove R, Cui J, Adamec J, Zempleni J. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Cui J, Zhou B, Ross S, Zempleni J. Nutrition, microRNAs and human health. Minireview based on the symposium Nutrition, microRNAs and human health held 5 April 2016 at the ASN Scientific Sessions and Annual Meeting at Experimental Biology 2016 in San Diego, CA. The symposium was sponsored by the American Society for Nutrition (ASN), and the ASN Nutrition Science Council. Adv Nutr 8:105-112, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Debate #1: Are dietary RNA/EVs detected in circulation? Do they have any functional role? Pros and cons. 2017 ISEV (International Society for Extracellular Vesicles) Workshop on Diet, Environment and Extracellular Vesicles, January 28, 2017, La Trobe University, Melbourne, Australia
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zempleni J, Aguilar-Lozano A, Sadri M, Manca S, Wu D, Zhou F, Mutai E, Sukreet S. Biological activities of extracellular vesicles and their cargos from bovine and human milk in humans and implications for infants. J Nutr 147:3-10, 2017
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zempleni J. Milk exosomes: beyond dietary microRNAs. Nutr Genes 12:12, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Zhou F, Wu D, Manca S, Sadri M, Fernando S, Paz H, Shu J, Cui J. Bovine milk exosomes and their cargos may regulate metabolism through non-canonical pathways in non-bovine species. 2017 ISEV (International Society for Extracellular Vesicles) Workshop on Diet, Environment and Extracellular Vesicles, January 27, 2017, La Trobe University, Melbourne, Australia
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Leiferman AL, Aguilar A, Mutai E, Adamec J, Zempleni J. Dietary depletion of bovine milk exosomes elicits changes in amino acid metabolism in C57BL/6 mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zhou F, Paz AH, Sadri M, Fernando CS, Zempleni J. A diet defined by its content of bovine milk exosomes alters the composition of the intestinal microbiome in C57BL/6 mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manca S, Giraud D, Zempleni J. The bioavailability and distribution of bovine milk exosomes is distinct from that of their cargos in mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mutai E, Zhou F, Zempleni J. Depletion of dietary bovine milk exosomes impairs sensorimotor gating and spatial learning in C57BL/6 mice. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Sukreet S, Zhang H, Adamec J, Cui J, Zempleni J. Identification of glycoproteins on the surface of bovine milk exosomes and intestinal cells that facilitate exosome uptake in human colon carcinoma Caco-2 cells. Experimental Biology Meeting; Chicago, IL, April 22-26, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Belak EE, Nordgren TM, Heires AJ, Zempleni J, Romberger DJ. Serum exosomes modulate monocyte and macrophage inflammatory responses. American Thoracic Society, May 2017, Washington, DC
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Zhou F, Wu, D, Manca S, Sadri M, Fernando S, Paz H, Shu J, Cui J. Bovine milk exosomes and their cargos may regulate metabolism through non-canonical pathways in non-bovine species, San Diego, CA, March 25/26, 2017. Abstract published in: Functional Foods and bioactive compounds in health and disease. Martirosyan D (ed.), Volume 21, Proceedings of the 21st International Conference, Food Science Publisher, Dallas, TX, p. 144-145
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manca S, Zhou F, Sadri M, Shu J, Jarecke J, Aguilar-Lozano A, Leiferman A, Grove R, Paz H, Adamec J, Fernando S, Cui J, Zempleni J.* Biological activities of extracellular vesicles and their cargos from bovine milk in non-bovine species. International Society for Extracellular Vesicles, Toronto, Canada, May 18-21, 2017. *Presenter
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J,* Zhou F, Wu D, Shu J, Paz H, Cui J, Fernando S. The communication of animal and bacterial kingdoms through exosomes and their RNA cargos in bovine milk. 22. International Conference. Functional Foods Center, September 22-23, 2017, Harvard Medical Center, Boston, MA. *Presenter
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Fratanotonio D,* Shu J, Cui J, Zempleni J. MicroRNAs in chicken egg exosomes: content and bioavailability in healthy humans. 22. International Conference. Functional Foods Center, September 22-23, 2017, Harvard Medical Center, Boston, MA. *Presenter
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Zhou F, Wu D, Upadhyaya B, Shu J, Paz H, Fernando S, Cui J. Delivery and alterations of microbial signals by bovine milk exosomes in non-bovine species. Opening ceremony speaker. American Society for Exosomes and Microvesicles, Asilomar Conference Center, Pacific Beach, CA, October 8-12, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Non-canonical pathways of signaling and metabolic regulation by extracellular vesicles from bovine milk. University of Nebraska Medical Center, Department of Internal Medicine; March 9, 2017, Omaha, NE
  • Type: Other Status: Published Year Published: 2017 Citation: Zempleni J. Invited presentation titled Biological activities of bovine milk exosomes and their cargos in non-bovine species as part of the annual W-3002 Multistate group meeting; Oregon State University, Portland, OR. May 23th, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Molecular signatures of exosomes and their RNA cargos from bovine milk in humans and mice. NIFA Program Directors meeting; Las Vegas, NV. June 24th, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Bioavailability of immune-relevant RNAs from bovine milk exosomes and its implications for pro-inflammatory responses in humans and mice. NIFA Program Directors meeting; Las Vegas, NV. June 24th, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules. Agricultural Research Division at the University of Nebraska-Lincoln, August 11, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J. Invited seminar titled Biological activities of bovine milk exosomes and their RNA cargos in non-bovine species. University of Nebraska Medical Center, Department of Cellular and Integrative Physiology, Omaha, NE, September 1, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Zempleni J, Sadri M, Mutai E, Manca S. The bioavailability and distribution of exosomes and their RNA cargos from bovine and porcine milk in mice. 9th exRNA Communications Consortium Investigators Meeting. Rockville, MD. November 6/7, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Leiferman AL, Aguilar A, Grove R, Shu J, Cui J, Adamec J, Zempleni J. Dietary depletion of bovine milk exosomes elicits changes in amino acid metabolism in C57BL/6 mice. UNL Spring Research Fair; Lincoln, NE, April 5, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mutai E, Ramer-Tait A, Zempleni J. Effects of extracellular vesicles and their cargos from cows milk on the release of cytokines by human peripheral blood mononuclear cells ex vitro. UNL Spring Research Fair; Lincoln, NE, April 5, 2017