Source: SOUTH DAKOTA STATE UNIVERSITY submitted to NRP
CONTROLLING INFLAMMATION AND PROMOTING REPAIR OF THE EPIDERMIS OF FOOD ANIMALS.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1009595
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jun 1, 2016
Project End Date
May 31, 2021
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Biology & Microbiology
Non Technical Summary
Skin is the first line of defense to the outside world providing a very large surface area for entry of pathogens which are always present. Lesions in the epidermis of food animals lead to many problems such as digital dermatitis in cloven hoof food animals including beef and dairy cattle, swine and sheep, and also dermatitis in turkey foot pads. Digital dermatitis is a primary factor leading to animal lameness which results in poor weight gain, increased animal care costs, loss of milk production and even early culling of animals. In feedlots, lameness has accounted for 16% of the health problems and 5% of deaths as well reduced average weight gain and reduced longevity of dairy cows giving rise to significant economic loss (1-3). The research proposed here is specifically directed toward finding solutions to control the inflammatory responses associated with epidermal lesions and to speed the repair of the epidermis in order to promote animal welfare and well-being. Despite the common occurrence of epidermal or epithelial damage there are only few treatments for reducing inflammation in food animals e.g. oral aspirin or shots of anti-inflammatories, and none for promoting healing. Most common treatments are to administer antibiotics after the inflammation is present with the assumption that the major cause of the inflammation is always microbial infection. However, not all cases of bovine digital dermatitis (BDD) are the result of bacteria or viruses (4) and in other cases bacterial infection goes undetected via both passive and active mechanisms, whereby the pathogen is purposely evading the immune system of the infected host (5, 6). Despite preventative measures and treatment with antibiotics epidermal lesions and inflammation continue to cost the meat and dairy industry billions each year (1-3, 7). Our significant lack of understanding regarding the signaling pathways that are activated during epidermal injury and that are used during epidermal repair has limited our ability to understand the healing process and has prevented us from developing treatments for promoting healing. The epidermis is both a first line barrier for defense and also a first line sensory organ. Excess physical or chemical stimulation of epidermal cells leads to release of inflammatory signals, signaling of pain to sensory neurons and poor healing. We are only beginning to learn of the physical and chemical receptors leading to these inflammatory and immunological signals. Continued inflammation, a first response to injury, prevents repair processes and wounds do not heal. Once we have characterized these receptors and their downstream pathways we will have targets for inhibiting them and attenuating the inflammatory response. Similarly, we can use this information to provoke defense responses to fight off organisms which impair the natural defense responses. We have taken an integrated approach to studying epidermal injury and repair by characterizing both the physical and chemical stimulation of keratinocytes and fibroblasts as we think it will provide significant advances to generating useful treatments for promoting epidermal wound healing. The overall strategy is to identify components of the pathways which are unique to inflammation but do not impair wound healing. We think that advances from our research may lead to additions to bedding material or topical treatments that will stimulate the innate immune system or lead to epidermal care that will profoundly reduce inflammation, lead to more rapid wound healing and strengthen epidermal integrity. Our efforts to improve healing have been directed toward understanding and exploiting endogenous mechanisms involved with tissue repair as well as expanding and optimizing use of natural materials that have displayed evidence for promoting epidermal repair.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3153899102080%
3153899109020%
Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3899 - Other animals, general;

Field Of Science
1090 - Immunology; 1020 - Physiology;
Goals / Objectives
The long-term goals of these studies are to identify new solutions for treatments of epidermal lesions including measures to enhance the innate immune system and speed repair of livestock wounds.Objective 1. Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain.Objective 2. Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier.Objective 3. Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system.
Project Methods
Objective 1. Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain.Spirochete Lysates Spirochete infections are common to digital dermatitis and other epidermal lesions in food animals. We are interested in identifying the macromolecules from the spirochetes that activate inflammatory responses and increase expression of inflammatory genes in fibroblasts but do not activate inflammatory responses in keratinocytes. Representative spirochetes will be obtained from ATCC and cultured in modified OTEB media (Anaerobe systems) (18) under anaerobic conditions, pelleted, rinsed and then sonicated to isolate bacterial fragments which have been used for inflammatory assays (6). Culture conditions of this anaerobe will be performed in collaboration with the expertise of Dr. Joy Scaria at South Dakota State University.Culture of Porcine Epidermal Cells We will extract and culture porcine keratinocytes and fibroblasts from the epidermis of freshly deceased pigs, proximal to the interdigital cleft using standard methods (6). We will use single electrode dielectrophoresis under aseptic conditions, developed by the PI (19), to pick single cells and transfer them to multiwell plates in order to start clonal lines. Lines will be identified by expression markers, basally expressed keratin for keratinocytes and Col1A or CD90 for fibroblasts. The choice of porcine cell lines has been made in order to maintain future possibilities of animal studies which will be significantly easier with swine than with cattle. A differential transcriptomic screen will be performed between the keratinocytes and fibroblasts after exposure to spirochete lysates with special emphasis on surface receptors, as discussed above, in attempts to identify the differences between the two cell types which provides the fibroblasts the ability to sense spirochete infections. A list of receptors will be identified from the differential screen in collaboration with Dr. Winyoo Chowanadisai, and candidates will be knocked down in the fibroblasts or increased in expression in the keratinocytes to test the hypothesis regarding their importance in sensing spirochete infection. Real time PCR will be performed after exposure of genetically altered cells to spirochete lysates to determine whether the cells have lost sensitivity (fibroblast) or have increased sensitivity (keratinocyte) to the spirochetes. In addition to the assay above we will attempt to identify the macromolecules from the spirochetes that are activating the inflammatory pathway in fibroblasts. Spirochete lysates will be first separated based on macromolecule type e.g. protein, lipid, carbohydrate nucleic acid and then, when necessary separated further based on hydrophobicity, size and charge with column chromatography. We will use real time PCR of an increased transcript identified above to determine whether a reduced pool of macromolecules continues to enable an inflammatory response when introduced to fibroblasts. We will use mass spectrometry to help identify the macromolecules in the reduced pool with the help of Dr. Eduardo Callegari at University of South Dakota. These experiments will help us to identify a weakness of the spirochete infections with the aim of exploiting it.Objective 2. Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier.We aim to study the family of TRP receptors and TLRs to elucidate their roles in sensing of physical and chemical damage as well as the mechanisms by which they promote repair of the epidermis. We will use the broad range of pharmacological inhibitors or molecular knockdown methods available to us. We will follow the pharmacological screen with knockdown of specific TRPs and TLRs if their pharmacological antagonists show supporting evidence that those specific receptors are involved. Additionally, we think that mechanotransduction and inflammatory pathways are leading to release of a number of extracellular signaling molecules including ATP, ROS and cytokines. ATP is used by keratinocytes to communicate cellular damage with other keratinocytes, pain to sensory neurons and extracellular matrix release by fibroblasts (20-22). For these experiments extracellular ATP and ROS will be measured with the chemiluminescent assays during physical stimulation. We propose that ATP enables communication and propagation of the wounding response between keratinocytes (20). However, we propose that overstimulation of ATP release leads to exocytosis of storage lysosomes and the release of hydrolytic enzymes which leads to greater inflammation, pain and chronic wounds. Both ATP and ROS may promote chemotaxis of other cells however, excess release could generate chronic inflammation and impair healing. We will determine the normal concentration range of ATP and ROS and treat cells with higher levels to test our hypothesis. First ATP will be used to monitor release of procollagen in dermal fibroblasts using the Clontech Procollagen ELISA kit. Cells will be loaded in a 96 well plate with a concentration range of ATP for 1 hour and samples will be developed. The chromogenic indicator for the ELISA reaction will be measured at 450 nm absorbance with a plate reader in our Genomics core facility. Inhibition of extracellular ATP and ROS production will also be performed to assess their roles during the injury response. Cellular signaling by stressed or damaged epithelial cells may also occur through secretion of cytokines using classic exocytosis and nontraditional pathways (25). We will assess the secretion of cytokines during mechanical stress and chemical activation of the receptors listed above. It is important to understand which signaling pathways are activated and what other cell types are involved in the wound injury and repair responses as over stimulated injury responses may be giving rise to long term injury and lameness. Identification of these important receptors give us targets to enhance or impair in order to promote healing, reduce inflammation and reduce pain.Objective 3. Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system.Epidermal keratinocytes and fibroblasts will be grown on tissue culture plastic dishes. Cellular morphology, growth rates, migration and cellular signaling will be assessed through imaging with either transmitted light or epifluorescence illumination as previously reported by the PI (11). Keratinocyte morphology, migration speed, persistence of migration direction and changes in cytosolic Ca2+ will be assessed (11) in the presence of chitin oligosaccharides with chain length of 1-6 NAGs, as well as in the presence of long polymeric chitin and chitosan obtained from diatom extracellular filaments, shrimp shells and squid pens. Statistical analysis of each of these parameters will enable identification of very subtle differences in normal and altered migration or signaling patterns and enable identification of biologically active extracts. Cytokine release from epithelial cells occurs in response to classic exocytosis pathways. Chitin oligosaccharides are elicitors of inflammatory responses for some cells. We will test the ability of chitin oligos and polymers in eliciting cytokine release from keratinocytes and fibroblasts using standard ELISA kits in multi-well plates. A careful comparison between oligos, chitin and chitosan is very important in identifying the signaling role of this interesting polymer and how it may be used to improve repair of epidermal wounds.

Progress 06/01/16 to 05/31/21

Outputs
Target Audience:The primary target audiences this period included local and national scientists in the research fields of veterinary science, biomedical physiology, immunology, biophysics, and engineering. Presentations were made to faculty and students comprising the next generation of scientists, animal producers, and animal care providers. We have engaged 2 industry partners as suppliers of raw materials for more extensive experimentation and generation of commercial end products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate Student Training Brian Kobylkevich, MS Student Defended in August 2019, accepted job at Research Technology Innovation in Brookings SD and is now working for ThermoFisher in St. Louis, MO. He learned techniques, data analysis, and writing and presentation skills to publish 2 first author papers, 1 coauthored paper, an abstract, and an MS thesis. He presented 1 national lecture and 1 national poster, 1 regional poster, and 1 local poster. Anyesha Sarkar, PhD student Defended in August 2021 and accepted a postdoctoral research position at Washington University in St. Louis, MO. He learned techniques, data analysis, and writing and presentation skills to publish 3 first author papers, 4 abstracts, and a PhD thesis. He presented 4 national posters, 3 regional posters, and 1 local poster. M. Jahir Raihan, MS student Defended in December 2020 and is now a PhD student at SDSU, Brookings SD. He learned techniques, data analysis, and writing and presentation skills to publish 2 co-authored papers and an MS thesis. He presented 1 regional poster and 1 local poster. Undergraduate Student Training Jonah DeVries, BS student He started as USD medical student 2018, Vermillion SD He learned techniques and presentation skills to present 1 local poster Austin Benson, BS student He started as USD medical student 2018, Vermillion SD He learned techniques and presentation skills to publish 1 co-authored paper and presented 2 local posters Brady Carlberg, BS student He started as U of Minnesota medical student 2019, Minneapolis, MN He learned techniques and presentation skills to publish 1 co-authored publication complete an Honors thesis project and report present 1 local poster and 1 national poster Kristi Bruening, BS student She started as a Masters Student in Physician Assistant Studies 2019, Missouri State, MO, and now practices as a PA in Rapid, SD. She learned techniques and presentation skills to publish 1 co-authored publication complete an Honors thesis project and report present 1 local poster Jessica Scheer, BS student She started as a USD medical student 2021, Vermillion SD. She learned techniques and presentation skills to completed an Honors thesis project and report present 2 local posters and 1 national poster How have the results been disseminated to communities of interest?Research articles and abstracts have been published in international journals including Veterinary Immunology and Immunopathology, Physical Biology, Journal of Theoretical Biology, Tissue Engineering, Biophysical Journal and Molecular Biology of the Cell. Public presentations of these results have been made at: national and international conferences including the Autumn Immunology Conference, American Society for Cell Biology, and Biophysical Society, regional conferences of the Nebraska Physiological Society, Eastern South Dakota Research Symposia, and Avera Symposia, and several local meetings on the campus of SDSU, Brookings SD. We have engaged commercial partners to use chitin in promoting growth by stimulating the innate immune systems of agricultural products. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. (100% Accomplished) Impact: Our initial goals were to identify the inflammatory pathways used by epidermal cells so that we could block inflammation and speed wound repair in farm animals. In our non-technical summary we wrote, "Once we have characterized these receptors and their downstream pathways, we will have targets for inhibiting them and attenuating the inflammatory response." We first characterized inflammatory responses to microbial stimuli. We found that we could significantly inhibit these inflammatory responses by adding low cost, inert polysaccharides that could be made from industrial waste products. The results are reported in a peer review journal (Vet. Immunology and Immunopathology). These findings have two significant impacts; first, that recycled waste materials could be used to make anti-inflammatory materials, and second, that these lower cost materials could be used to increase productivity and growth of farm animals by reducing the use of general antibiotics for treating superficial infection and inflammation. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. (100% Accomplished) Impact: Cells use surface receptors to sense extracellular chemical and physical cues that promote distinctive behaviors. These receptors sense chemical cues reflecting cellular damage and invasion by infectious microbes. Our goal was to identify the sensory receptors on keratinocytes and fibroblasts that were involved with directed migration and rebuilding of the epidermal barrier. Rather than use an expensive and time consuming proteomic or genetic screen to identify these receptors and signaling pathways for a relatively understudied tissue type, we developed a combination of bioinformatic approaches (computer programs). This method enables us to identify surface receptors from the cells of interest from our organism, using previously published data sets and molecular similarity with other related organisms. This is especially useful for rare or understudied organisms or cell types that are not maintained in stock collections, including cells from farm animals and other agriculturally related organisms. The method and the programs that were developed and used were reported in a peer reviewed journal (Journal of Theoretical Biology) so others could make use of them. Our method reduces the time and cost for identifying cellular receptors and signaling pathways on less commonly studied cell populations and can be adapted for use in identifying intracellular or organellar receptors. It will also help shorten research time and reduce costs for more targeted drug/receptor screens or expression profile comparison in response to chemical or physical stimuli. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. (100% Accomplished) Impact: Chitin and chitosan are naturally occurring sugars found in bacteria and fungi. The immune systems of both plants and animals can identify these sugars and will mount defense responses against the pathogens that may be built with them to prevent infection. Therefore, chitin as an industrial waste product, can be introduced to stimulate defensive responses prior to infection by native microbes or parasites and give the organism a greater chance for success in defeating the pathogen. We have explored the interaction of these polysaccharides with bovine fibroblasts, the primary source of inflammation during bovine digital dermatitis (BDD), an inflamed lesion leading to lameness in dairy cattle. We report that chitin alone, is relatively inert to the bovine fibroblasts and does not increase the death rate. In fact, chitin also reduces the inflammatory response of bovine fibroblasts in response to a component of bacterial cell walls that stimulates strong inflammatory responses. These results are published in a peer reviewed journal (Vet. Immunology and Immunopathology). By identifying low cost materials that stimulate immune defense responses and reduce inflammation, we can speed healing in farm animals and promote defense responses to reduce infection in the absence of antibiotics.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Sarkar, A. and M.A. Messerli Electrokinetic perfusion through three-dimensional culture reduces cell mortality. Tissue Engineering. Epub ahead of print. https://www.liebertpub.com/doi/abs/10.1089/ten.tea.2021.0008
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Scheer, J. 2020. Promoting tissue growth in the absence of blood flow. Undergraduate Research, National Collegiate Honors Council (NCHC) Annual Conference. (virtual). online starting Sept 30
  • Type: Journal Articles Status: Accepted Year Published: 2021 Citation: Kobylkevich, B.M., M.J. Raihan, T. Uprety, R.S. Kaushik, J.S. Shore, J.J. Sohn, M.A. Messerli. 2020. Linear polysaccharides reduce production of inflammatory cytokines by LPS-stimulated bovine fibroblasts. Veterinary Immunology and Immunopathology. 234:110220 Linear polysaccharides reduce production of inflammatory cytokines by LPS-stimulated bovine fibroblasts - ScienceDirect


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

Outputs
Target Audience:The primary target audiences this period included local and national scientists in the research fields of veterinary science, biomedical physiology, immunology, biophysics, and engineering. Presentations were made to faculty and students comprising the next generation of scientists, animal producers, and animal care providers. We have engaged 2 industry partners as suppliers of raw materials for more extensive experimentation and generation of commercial end products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate Student Training Anyesha Sarkar, PhD student. (Oct-Sept. 2020). Ms. Sarkar published: 1) a paper as first author, Anyesha, Sarkar, Brian M.Kobylkevich, David M.Graham, Mark A.Messerli, "Electromigration of cell surface macromolecules in DC electric fields during cell polarization and galvanotaxis" in the Journal of Theoretical Biology, and 2) an abstract as first author, Anyesha Sarkar and Mark A Messerli "Competing Forces Redistribute Surface Macromolecules during EF?directed Cell Migration" at the Annual American Society for Cell Biology Meeting in Washington, DC. Molecular Biology of the Cell 30:257-258a. M. Jahir Raihan, MS graduate student Mr. Raihan presented his results at a Regional Meeting, Nebraska Physiological Society, 2019. He is coauthor on a submitted manuscript about the polysaccharide bandages. He continues to learn and apply bioinformatic tools and chemical separations to characterize other sources for chitin bandages. Undergraduate Student Training Undergraduate research opportunities were provided to Jessica Scheer. Ms. Scheer learned 3D tissue culture and helped to identify low cost macromolecules that help these 3D cultures to mimic native conditions found in the skin. Her honors research project is still in progress. How have the results been disseminated to communities of interest?Public presentations of these results have been made at: 1) the annual conference for the American Society for Cell Biology in Washington D.C. in 2019, 2) the Nebraska Physiological Society Meeting in Omaha, NE, 3) and several local meetings on the campus of SDSU, Brookings SD. In addition, a research article regarding Objective 2 has been published in the Journal of Theoretical Biology. A second article on Objectives 1 and 3 is currently under review. We have engaged commercial partners to use chitin in promoting growth and stimulating the innate immune systems of agriculture products. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. A manuscript has been submitted to an international journal but COVID has slowed the review process which has been going on since the middle of April, 2020. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. We are exploring the use of linear chain polysaccharides in reconstruction of 3D tissues and organs, most specifically in maintaining cellular viability during poor or absent blood flow. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. The anti-inflammatory properties of chitin have been characterized and compared to another linear chain polysaccharide. Subtle modifications to chitin will improve water solubility and expand its applications for tissue repair. We are exploring functionalization of chitin and chitin/polysaccharide mixtures to expand commercial applications.

Impacts
What was accomplished under these goals? Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. (100% Accomplished) We report that only 1ug/mL of lipopolysaccharides (LPS) from cell walls of gram negative bacteria, including treponemes, increase expression of inflammatory cytokines IL-1a, IL-6, and Il-8 by factors of 8, 128 and 256, respectively. Relatively inert polysaccharides, including oxidized regenerated cellulose (ORC) and chitin nanofibers, reduce the increase in expression of inflammatory cytokines in response to LPS after 24 h. Chitin (2mg/mL) reduced expression of IL-6 and IL-8 by 10.2 and 4.7 fold, while ORC (2mg/mL) reduced IL-6 expression by 4.7 fold but did not significantly affect IL-8 expression. IL-6 and IL-8 promote inflammation and scarring. Chitin is predicted to reduce both in vivo. After 48 h, cytokine expression was not different than after 24 h. Fibroblasts have been reported as the primary site of inflammation by treponemes in bovine digital dermatitis (BDD) and not keratinocytes. Therefore, we focused our attention on the fibroblasts and identified 2 low cost blockers of LPS induced inflammation in bovine fibroblasts. (This is described further in objective 3). Impact: Low cost, inert polysaccharides made from cellulose or chitin waste may be useful in reducing inflammation in farm animals by preventing elicitors from stimulating inflammation. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. (100% Accomplished) We have developed a combination of bioinformatic approaches (computer programs) to screen databases containing an organism's genetic or proteomic information to identify all surface exposed proteins of the cells of that organism. In order to restrict that pool of proteins to epidermal cells of our organism of interest, we have used further bioinformatic programs to identify proteins with greatest homology to epidermal cells of a similar organism that have already been characterized. This combination of programs sort surface exposed proteins into 3 categories including transmembrane, integral membrane, and lipid anchored. The programs also help to characterize the physical characteristics of the proteins, including distribution of surface charge and net charge, and also predicts the level of protein glycosylation. This is the first time that such a large protein screen has been performed in this manner. We have we have identified that 2,370 gene products in our model migratory cell are surface proteins. These results and methods are published in Sarkar et al., Journal of Theoretical Biology 2019, https://www.sciencedirect.com/science/article/pii/S0022519319302504. Impact: Our approach reduces the time and cost for identifying cellular receptors and signaling pathways on less commonly studied cell populations, such as those that exist on farm animals and other agriculturally related organisms. The linear chain polysaccharides under study in this project mimic physical characteristics of extracellular matrix in tissues and organs. We have found other uses for these materials in rebuilding the epidermal barrier. An NSF grant has been submitted to continue this work. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. (100% Accomplished) Chitin and chitosan (deacetylated chitin) are linear chain (polysaccharides) found in bacteria and fungi. Immune systems of plants and animals recognize these polysaccharides and mount defense responses against them to prevent infection by any other nearby microbes or parasites. We have explored the interaction of these polysaccharides with bovine fibroblasts, the primary source of inflammation during bovine digital dermatitis (BDD), an inflamed lesion leading to lameness in dairy cattle. We report that two polysaccharides, including a chitin nanofiber and oxidized regenerated cellulose (ORC), do not increase mortality of bovine fibroblasts when used at low (2mg/mL) or high concentrations (5 mg/ml). We also report that the materials are relatively inert to the bovine fibroblasts that generate weak inflammatory responses to the two materials. In fact, the materials are so inert that they reduce the inflammatory response of bovine fibroblasts in response to lipopolysaccharide (LPS), a component of bacterial cell walls that stimulates strong inflammatory responses at 1,000 fold lower concentration than these polysaccharides. These results are published in a graduate MS thesis https://openprairie.sdstate.edu/etd/3369/ and have been submitted for publication in Veterinary Immunology and Immunopathology. Impact: By identifying low cost materials that stimulate immune defense responses and reduce inflammation, we can speed healing in farm animals and promote defense responses to reduce infection in the absence of antibiotics.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Raihan, M.J., B.M. Kobylkevich, J.S. Shore, M.A. Messerli. 2019. Innate response and biodegradation of linear chain polysaccharide bandages. Annual Meeting of the Nebraska Physiological Society. Oct 26. Omaha, NE.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Sarkar, A., B.M. Kobylkevich, D.M. Graham, M.A. Messerli. 2019. Electromigration of cell surface macromolecules in DC electric fields during cell polarization and galvanotaxis. J. Theoretical Biology. 478:58-73.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Sarkar, A., M.A. Messerli. 2019. Competing forces redistribute surface macromolecules during EF?directed cell migration. Annual American Society for Cell Biology. Washington, DC. Molecular Biology of the Cell. 30:257-258a.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Scheer, J. 2019. Promoting tissue growth in the absence of blood flow. SDSU Day of Scholars. December 3. Brookings, SD.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Scheer, J. 2020. Promoting tissue growth in the absence of blood flow. Undergraduate Research, Scholarship, and Creative Activity Day (URSCAD). April 22. Brookings, SD.
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Kobylkevich, B.M., M.J. Raihan, T. Uprety, R.S. Kaushik, J.S. Shore, J.J. Sohn, M.A. Messerli. 2020. Linear polysaccharides reduce production of inflammatory cytokines by LPS-stimulated bovine fibroblasts. Veterinary Immunology and Immunopathology. (Submitted)


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

Outputs
Target Audience:The primary target audiences this period included local and national scientists in the research fields of veterinary science, biomedical physiology, immunology, biophysics, and engineering. Presentations were made to faculty and students comprising the next generation of scientists, animal producers, and animal care providers. We are also engaging industry partners as suppliers of raw materials for more extensive experimentation and generation of commercial end products. Changes/Problems:We have had a great deal of difficulty in promoting bovine keratinocyte growth over bovine epidermal fibroblast growth. We have tried chemical and mechanical methods to select for keratinocytes over fibroblasts, including adhesion differences to collagen, selective UV irradiation, and mechanical scraping. The growth of the keratinocytes is very slow and in sheets while the growth of the fibroblasts is rapid and they spread out. We have tried isolating keratinocytes to grow as clones with no success, as keratinocytes tend to differentiate which slows proliferation. We have decided to direct our emphasis to the fibroblasts which tend to be the primary source of inflammation for BDD. What opportunities for training and professional development has the project provided?Graduate Student Training Anyesha Sarkar, PhD student. (June-Sept. 2019). Ms. Sarkar published: 1) a paper as first author, Anyesha, Sarkar, Brian M.Kobylkevich, David M.Graham, Mark A.Messerli, "Electromigration of cell surface macromolecules in DC electric fields during cell polarization and galvanotaxis" in Journal of Theoretical Biology epub ahead of print, 2) an abstract as first author, Anyesha Sarkar, Brian M Kobylkevich, David M Graham, Mark A Messerli "Electromigration of Cell Surface Macromolecules during Galvanotaxis" in Biophysical Journal 116:207a-208a, and 3) a paper as coauthor, Messerli, Mark and Sarkar, Anyesha "Advances in Electrochemistry for Monitoring Cellular Chemical Flux" in Current Medicinal Chemistry epub ahead of print. She was also able to present her work at a national conference (Annual Biophysical Society Meeting, Baltimore, MD) and local conference (Avera Symposium, Brookings, SD). Brian Kobylkevich, MS graduate student (Oct. 2018 - Aug. 2019). This year Mr. Kobylkevich presented his work at both a regional (Nebraska Physiological Society, Omaha, NE) and a national meeting (Autumn Immunology Conference, Chicago, IL). At the national meeting he was also invited to present a talk about his work. He published his Thesis entitled "Bovine Cellular and Innate Immune Response to Hemostatic Polysaccharides" https://openprairie.sdstate.edu/etd/3369/. He is also coauthor on two publications and an abstract from a second national meeting. He successfully defended his Masters thesis this summer. M. Jahir Raihan, MS summer graduate student (June-Aug. 2019) Mr. Raihan presented his results for the first time at a local meeting (SDSU Day of Scholars, Brookings SD). He is coauthor on one publication this year and coauthor on a meeting abstract, as listed above. He continues to learn and apply Bioinformatic tools and chemical separations to characterize other sources for chitin bandages. Undergraduate Student Training Undergraduate research opportunities were provided to Kristi Bruening and Jessica Scheer. Ms. Bruening learned protein extraction and identification and chitin extraction and characterization. She has completed her honors research project and graduated. Ms. Scheer learned 3D tissue culture and helped to identify low cost macromolecules that help these 3D cultures to mimic native conditions found in the skin. Her honors research project is still in progress. How have the results been disseminated to communities of interest?Public presentations of these results have been made at 2 national meetings: the Biophysical Society Conference in Baltimore, MD and the Autumn Immunology Conference in Chicago, IL. Presentations were also made at 2 regional meetings: Avera Symposium in Brookings, SD and the Nebraska Physiological Society Meeting in Omaha, NE. Presentations were also made at local meetings on the campus of SDSU, Brookings SD. In addition, a research article has been published in Journal of Theoretical Biology, regarding Objective 2. National and local news reports have been published for these topics as well. The title and initial web addresses for the articles are listed below. We have not included information for the other news agencies that reprinted the articles. "New tool predicts how electrical stimulation promotes healing" www.newswise.com/articles/how-electricity-stimulates-healing What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. We have been most successful in characterizing the innate immune response in bovine fibroblasts cells. We completed some additional experiments last year to support a more robust story and are in the process of completing the manuscript for publication. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. We published results this year and presented them at a national meeting. During this next year we plan to explore the redistribution of these surface receptors in conditions found in vivo, i.e. conditions in 3D culture which mimic native conditions in the skin. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. Our understanding of chitin and chitosan has grown this last year. We are now considering that most samples in the literature are more chitosan than chitin. Our research confirms that we have been using chitin and not chitosan. We are working to isolate a similar chitin rich material from an alternative source that will lower the cost of the material. After extracting this chitin we will use CP-NMR to determine its level of acetylation and then expose cells to it and compare the cellular response to the chitin nanofibers in our earlier study.

Impacts
What was accomplished under these goals? Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. (40% Accomplished) We report that only 1ug/mL of lipopolysaccharides (LPS) from cell walls of gram negative bacteria, including treponemes, increase expression of inflammatory cytokines IL-1a, IL-6, and Il-8 by factors of 8, 128 and 256, respectively. Relatively inert polysaccharides including oxidized regenerated cellulose (ORC) and chitin nanofibers reduce the increase in expression of inflammatory cytokines in response to LPS after 24 hours. After 48 hours, cytokine expression was not different than after 24 hours. Impact: Low cost, inert polysaccharides made from cotton or chitin waste, may be useful in reducing inflammation in farm animals by preventing elicitors from stimulating inflammation. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. (95% Accomplished) We have developed a combination of bioinformatic approaches (computer programs) to screen databases containing an organism's genetic or proteomic information to identify all surface exposed proteins of the cells of that organism. In order to restrict that pool of proteins to epidermal cells of our organism of interest, we have used further bioinformatic programs to identify proteins with greatest homology to epidermal cells of a similar organism that has already been characterized. This combination of programs sorts surface exposed proteins into 3 categories including transmembrane, integral membrane, and lipid anchored. The programs also help to characterize the physical characteristics of the proteins, including distribution of surface charge and net charge, and also predicts the level of protein glycosylation. This is the first time that such a large protein screen has been performed in this manner. We have we have identified that 2,370 gene products in our model migratory cell are surface proteins. These results and methods are published in Sarkar et al., Journal of Theoretical Biology 2019, https://www.sciencedirect.com/science/article/pii/S0022519319302504. Impact: Our approach reduces the time and cost for identifying cellular receptors and signaling pathways on less commonly studied cell populations, such as those that exist on farm animals and other agriculturally related organisms. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. (85% Accomplished) Chitin and chitosan (deacetylated chitin) are linear chain (polysaccharides) found in bacteria and fungi. Immune systems of plants and animals recognize these polysaccharides and mount defense responses against them to prevent infection by any other nearby microbes or parasites. We have explored the interaction of these polysaccharides with bovine fibroblasts, the primary source of inflammation during bovine digital dermatitis (BDD), an inflamed lesion leading to lameness in dairy cattle. We report that two polysaccharides, including a chitin nanofiber and oxidized regenerated cellulose (ORC), do not increase mortality of bovine fibroblasts when used at low (2mg/mL) or high concentrations (5 mg/ml). We also report that the materials are relatively inert to the bovine fibroblasts that generate weak inflammatory responses to the two materials. In fact, the materials are so inert that they reduce the inflammatory response of bovine fibroblasts in response to lipopolysaccharide (LPS) a component of bacterial cell walls that stimulates strong inflammatory responses at 1,000 fold lower concentration than these polysaccharides. These results are published in a graduate MS thesis https://openprairie.sdstate.edu/etd/3369/ and are being formatted for publication in a research journal. Impact: By identifying low cost materials that stimulate immune defense responses and reduce inflammation, we can speed healing in farm animals and promote defense responses to reduce infection in the absence of antibiotics.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: B.M. Kobylkevich, M.J. Raihan, M.A. Messerli. 2018. Skin cellular response to hemostatic bandages. Annual Meeting of the Nebraska Physiological Society. Omaha, NE. Oct, 20th.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: A. Sarkar, B.M Kobylkevich, M.A. Messerli. 2018. Soft tissue repair by electrical stimulation. Avera Symposium. Brookings, SD. Oct., 24th
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: B.M. Kobylkevich, M.J. Raihan, M.A. Messerli. 2018. Comparing stimulation of inflammation regulatory cytokines and receptors in mammalian cells exposed to polysaccharide hemostatics. Autumn Immunology Conference. Chicago IL, Nov, 16-19.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: A. Sarkar, B.M. Kobylkevich, D.M. Graham, M.A. Messerli. 2019. Electromigration of cell surface macromolecules during galvanotaxis. Annual Biophysical Society Meeting. Baltimore, MD. Biophysical Journal 116 (3), 207a-208a.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: M.J. Raihan, B.M. Kobylkevich, J.S. Shore, M.A. Messerli. 2019. Innate response and biodegradation of linear chain polysaccharide bandages. Biology and Microbiology Day of Scholars. Brookings, SD. April, 23rd.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: A. Sarkar, B.M. Kobylkevich, D.M. Graham, M.A. Messerli. 2019. Electromigration of cell surface macromolecules in DC electric fields during cell polarization and galvanotaxis. E-pub ahead of print. Journal of Theoretical Biology https://www.sciencedirect.com/science/article/pii/S0022519319302504?via%3Dihub
  • Type: Theses/Dissertations Status: Accepted Year Published: 2019 Citation: B.M. Kobylkevich. 2019. Bovine cellular and innate immune response to hemostatic polysaccharides. MS T thesis. South Dakota State University, Brookings, SD. https://openprairie.sdstate.edu/etd/3369/


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

Outputs
Target Audience:The primary target audiences this period included local and national scientists in the research fields of veterinary science, biomedical science, pharmacy, biology, and engineering. Presentations were made to faculty and students comprising the next generation of scientists, animal producers, and animal care providers. We have also started to engage potential industry partners as suppliers of raw materials for more extensive experimentation and generation of commercial end products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate Student Training Anyesha Sarkar, PhD student. (Jan-July 2018 - current) 49% of a graduate research assistantship. Developed 20 of her own computer programs and used 7 other computer programs to sort and screen databases of DNA and proteins to characterize unknown or poorly characterized cell lines. Student also performed mathematical modeling to understand the biophysics of natural wound repair. Student disseminated work through presentations at national and regional meetings. Brian Kobylkevich, MS graduate student (Oct. 2017 - Aug. 2018). Continued to gain experience in collection and primary culture of farm animal epidermal cells, and molecular techniques by screening for changes in gene expression in response to elicitors. Brian took a course covering strategies for public presentations and presented his results at a local meeting. M. Jahir Raihan, MS summer graduate student assistantship (June-August, 2018). Learned culturing of mammalian cell lines for chemiluminescent assays of cell function. Also learned to perform experiments with elicitors, mRNA extraction, and conversion to cDNA and qPCR analysis to identify changes in gene expression in response to chemical elicitors. Undergraduate Student Training Undergraduate research opportunities were provided to Brady Carlberg and Kristi Bruening. Brady learned aseptic technique, primary cell culture, and transmitted light imaging. Brady completed his honors project, published a paper related to this research and presented his work at a national and local meeting. Kristi is learning protein extraction and identification and chitin extraction and characterization. She will be completing her honors project in the next year. How have the results been disseminated to communities of interest?Public presentations at national, regional and local meetings have been used to disseminate results. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. Our emphasis has been to promote characterization of the innate immune response in epidermal cells. We have been most successful characterizing responses by the bovine fibroblasts and plan to publish our results this next year. We will direct our attention toward the keratinocytes when we have completed with the fibroblasts. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. Results from the prior year will be presented at both a local and national meeting. We plan to publish our results of this experimental method so that others may put it to use. We will also identify the top candidates for surface proteins that we think are controlling directional migration. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. The results from our prior year will be presented at both a regional and national meeting. We are in the process of repeating this year's results using bovine fibroblasts from 3 different specimens. We will also experiment to determine the role that the polysaccharides play in interfering with inflammation by known elicitors. We are also exploring methods to promote chitin fiber extraction from other materials.

Impacts
What was accomplished under these goals? Objective 1: Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. 40% Accomplished a. We have found that lipopolysaccharides (LPS) from cell walls of gram negative bacteria including treponemes activate overexpression of the Tol-like receptors (TLR) 2 and 4 by a factor of 15 and 7, respectively, in bovine fibroblasts, and increase expression of inflammatory cytokines IL-6 and Il-8 by a factor of 6 and 8, respectively. This identifies surface receptors that control inflammation that may prevent healing if over stimulated. We have found that LPS also stimulates an anti-inflammatory signal. Further experiments are required to determine the time course of the inflammatory and anti-inflammatory signals. Impact: By characterizing receptors that promote inflammation we identified targets that could be used to reduce or block inflammation. b. The mammalian epidermis makes use of reactive oxygen species (ROS) or "super oxygen" which damages the anaerobic machinery of these microbes ultimately killing them. We have constructed a photon counting system for sensitive detection of ROS release in response to pathogenic stimuli. The LPS elicitor promotes ROS production. However, while testing the sensitivity of the innate immune system to exogenous polysaccharides, we found that the polysaccharide materials do not promote measureable ROS production. Further experiments revealed that in combination with LPS, polysaccharides prevented the LPS from producing ROS. Impact: This may indicate that inert polysaccharide materials reduce inflammation by preventing elicitors from stimulating inflammation. After finding this result we placed more emphasis into Objective 3. Objective 2: Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. 60% Accomplished In order to promote this second objective as quickly as possible and at low cost, we have been assembling and using computer programs to screen electronic databases to identify sensory receptors on epidermal cells and identify their physical characteristics including surface charge and level of glycosylation. Many electronic databases contain the genetic information of understudied organisms including farm animals. By comparing similar mammalian organisms we can quickly obtain a picture of the cellular proteins in the understudied organisms without repeating decades of research originally performed to characterize the more commonly studied organisms. We have employed 4 programs to help identify all plasma membrane surface proteins from epidermal keratinocytes, 3 programs to identify surface charge and level of glycosylation, and 20 of our own programs to manage the large datasets and remove redundant sequences. To our knowledge this is the first time that such a large protein identification has been performed in this manner. Impact: Our method could be applied to many different organisms in only a few weeks to characterize targets and pathways by which cells function. In our experimental epidermal cell line we have identified that 2370 out of 20-25K gene products are surface proteins that we have arranged into 3 different categories of surface attachment. We have arranged them in order of size and charge as those characteristics are important for controlling cell polarity during epidermal repair. Objective 3: Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. 65% Accomplished a. The mammalian immune system recognizes long chain sugars (polysaccharides) from bacteria and fungi and mounts a defense response against these chemical elicitors to prevent infection. However, mammalian wound repair is also dependent on polysaccharides with similar structure. We are characterizing the ability of the mammalian immune system to sense low cost, exogenous polysaccharides currently approved by the FDA as hemostatic agents (materials that prevent bleeding). Using bovine epidermal fibroblasts, we confirm that fragments of bacterial cell walls (LPS) and large fragments of the polysaccharide, chitin, promote inflammatory responses. However, in contrast, we show that long nanofibers of chitin and oxidized regenerated cellulose (ORC, recently added to the project), promote anti-inflammatory responses. Further experiments have identified a cell surface receptor by which the inflammatory elicitors are detected. However the chitin nanofiber is not detected by this receptor. Also, by using mammalian epidermal keratinocytes we have identified a mechanism by which the chitin nanofibers are degraded and therefore avoid promoting the inflammatory response. Impact: By identifying polysaccharides that promote healing and control inflammation we can speed healing in farm animals or can promote defense responses to reduce infection.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Sarkar, A., Messerli, M. A. 2018. "How does electrical stimulation promote epidermal wound repair?" Sanford Symposium, Sanford Research, Sioux Falls, SD. May 30th.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Sarkar, A., Kobylkevich, B. M., Carlberg, B. R., Messerli, M. A. 2017. "Cellular Polarity is Directed by electrophoresis and electrically driven water flow," American Society for Cell Biology, Philadelphia, PA. Published in Molecular Biology of the Cell vol. 28, 8120.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Kobylkevich, B., Benson, A., Messerli, M. A. 2017. "Optimizing chitin polymers for promoting wound healing and enhancing the innate immune system," SDSU Day of Scholars, Brookings, SD. November 28th


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

Outputs
Target Audience:The target audience this period waslocal and national, including scientists in the research fields of veterinary science, pharmacy, biology and engineering and also animal care providers. Presentations were made to faculty and students comprisingthe next generation of scientists, animal producers and animal care providers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate Student Training Anyesha Sarkar, PhD student. (June-Sept 2017 - current) 49% of a graduate research assistantship. Learned protein extraction and gel electrophoresis, use of bioinformatics tools and programming to sort and screen databases of DNA and proteins. Student disseminated work through lecture and poster presentations. Brian Kobylkevich, research assistant (Oct. 2016 - Jan. 2017) converted to MS student (49% of a graduate research assistantship Jan 2017-Sept. 2017). Learned primary keratinocyte extraction from bovine and porcine skin, plating and screening of cells for clonal cell extraction, RNA extraction and cDNA production for PCR and qPCR methods. Undergraduate Student Training Undergraduate research opportunities were provided to Brady Carlberg, Jonah DeVries, Austin Benson regarding different components of wound healing and epithelial physiology. Students learned aseptic technique, primary and clonal cell culture, transmitted light and fluorescence imaging and electrophysiology. Each student was able to present their results in a local format while one student went on to present results at a regional meeting with plans to attend a national meeting the following year. How have the results been disseminated to communities of interest? Results have been disseminated to target audiences through public presentations, a submitted publication and scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Continue the characterization of changes in gene expression in bovine epidermal cells in response to pathogenic stimuli. Objective 2. Rebuild the mathematical model describing changes in cell polarity. Perform time course experiments to follow redistribution of the cell surface receptors in order to test the model. Identify the physical properties of the cell surface polarity receptor and screen all cell surface receptors to create a short list of receptors that are the best candidates for directing cell migration. Objective 3. Characterize the response of keratinocytes to different sizes of chitin, including its ability to stimulate the oxidative burst as well as the long term enhancement of innate defense response by changes in protein expression for different sensors and signals of the innate immune system.

Impacts
What was accomplished under these goals? Objective 1. Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain. (15% accomplished) Short-term and long-term responses to spirochete infection are being explored. a. Culture of bovine keratinocytes requires separation of the epidermis from the dermis. We have identified a simple and short culture procedure for removing the dermis from the epidermis. This has enabled us to enrich cultures for keratinocytes and enhance our ability for cloning bovine epidermal cells. We are in the process of growing up, selecting and testing the different clonal cell lines for experiments described below. b. Host cells are thought to identify Spirochete infection through sensing of lipoproteins (LP) and lipopolysaccharides (LPS). Changes in defense gene expression patterns in response to infectious agents can be detected from 3-48 hours after introduction of the agent. We are using commercially available LPS, a known elicitor of the inflammatory and immune responses in mammalian keratinocytes, to optimize conditions for screening changes in cultured bovine epidermal cells using qPCR. We have successfully isolated RNA, converted it to cDNA and run a test screen for common genes, from control and LPS stimulated keratinocytes. These experiments will enable us to determine the sensors that spirochetes are able to avoid during infection. c. Cell to cell signaling, which promotes inflammation, is being characterized by collecting and identifying secreted proteins in response to external infectious stimuli and stress. Intracellular signaling pathways begin with sensing of an infectious agent, which activates the innate immune system, and causes cells to respond by secreting cytokines and other extracellular signaling molecules through second messenger pathways including cytosolic Ca2+ signaling. We have directly stimulated the Ca2+ pathway and found that it leads to secretion of 546 different proteins while mechanical perturbation of keratinocytes with shear stress results in release of 345 proteins. There is a good deal of overlap in the secreted proteins between these two stimuli. We have identified that some of the proteins are similar to those secreted in exosomes, secreted sacs of membranes carrying different protein pools. This is the first time that chemical and mechanical perturbation have been found to use a similar extracellular signaling mechanism. These experiments are helping us understand extracellular communication between keratinocytes and surrounding cells in the epidermis and dermis that are responsible for chronic inflammation. d. Many infectious microbes thrive in the absence of O2 (i.e. anaerobic conditions). Many organisms have developed a defense response by releasing reactive oxygen species (ROS) or "super oxygen" which damages the anaerobic machinery of these microbes ultimately killing them. We are exploring the role of infectious agents in eliciting ROS from keratinocytes in order to determine the effectiveness of this pathway in removing pathogens. We have constructed a photon counting system for sensitive detection of ROS release in response to pathogenic stimuli. Objective 2. Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier. (25% accomplished) a. We have submitted a manuscript entitled "Reversing direction of galvanotaxis by controlled increases in boundary layer viscosity." We provide evidence for controlled reversal of cell migration, and position the field closer to identifying the putative receptor involved with directing cells to the center of a wound. b. We are building on the submitted manuscript described above by rebuilding the mathematical model originally proposed to direct cell migration and using that revised model to narrow down the identity of different sensory receptors based on their physical properties to fit the parameters identified in the manuscript. Using numerical simulations and bioinformatic screening methods, along with a keratinocyte transcriptome, we have narrowed the protein candidates to 25% of the original protein pool. Further screening will enable us to continue narrowing the possibilities further and may ultimately lead to identification of chemical pathways for promoting wound repair. Identification of the biochemical basis by which a physical stimulus is sensed to promote wound repair will enable us to target that pathway through chemical means to promote wound repair. Objective 3. Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. (15% accomplished) a. Chitin is a natural elicitor of the defense response in mammalian cells. However, chitins of different sizes stimulate very different responses. In leukocytes, chitin polymers larger than the cells stimulate an inflammatory response while those the size of the cell or slightly smaller stimulate an immune response but not inflammatory response. Short chain chitin oligomers have not been carefully tested in mammalian cells however they are the most potent activators of the innate immune system in plants. We are characterizing the short-term and long-term chitin stimulated defense responses of keratinocytes to chitins of different sizes. We have successfully worked out a method of sorting chitin into different sizes and have confirmed that chitin polymers of similar size to keratinocytes, stimulate the cytosolic Ca2+ signaling pathway. We have also confirmed that the chitin is pure with immeasurable amounts of contaminating proteins, and have identified a low cost source for chitin nanofibers. Overall Impacts After the first full year of this award the lab has advanced each of the 3 objectives. While much is known about the bovine extracellular matrix proteins which are used for leather production and as the basis for skin grafts on humans, relatively little is known about the living cell material or how to enhance its defensive and healing capacity. Objective 1 will advance our understanding of the bovine epidermal defense responses and enable us to elucidate mechanisms for enhancing them especially against infectious organisms that are capable of camouflage and hide from the immune system. Advancement of Objective 2 has enabled us to provide the first evidence to support a 40 year old hypothesis describing the natural mechanism by which keratinocytes migrate to the center of a wound to reseal the epidermis. We continue to advance this knowledge with the aim of finding the surface receptor that directs cell migration into the wound bed with the aim of identifying a chemical pathway to enhance healing. Our investigations in Objective 3 make use of a natural product stimulator of defense responses in plants and animals. While its use has long been under development for use in promoting defense of crops against fungal infection, little consideration has been given to animals because of the success of the adaptive immune system. However, Treponemes and other microbes are known to evade the adaptive immune system. Enhancing the innate immune system of the epidermis in animals will strengthen their first line of defense to fight off microbial, fungal and viral pathogens.

Publications


    Progress 06/01/16 to 09/30/16

    Outputs
    Target Audience:The primary stakeholders include livestock owners, both small and large scale business owners. Intellectual properties obtained from this work may be presented to commercial suppliers of livestock wound care products. Presentations will also be used to excite and recruit the next generation of young scientists through outreach opportunities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Anyesha Sarkar, PhD student. (June 2016 - current) 49% of a summer graduate research assistantship. Learned aseptic technique during eukaryotic cell culture, live cell confocal fluorescence microscopy, and electrical resistance measurements of epithelia. Brian Kobylkevich, research assistant, (June 2016 - current) Learned aseptic technique during eukaryotic cellculture, time-lapse live cell imaging using transmitted light and confocal fluorescence microscopy, image processing and analysis, transcriptome analysis, protein extraction, purification and gel electrophoresis. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Goal 1. Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain Arrange keratinocyte and fibroblast cultures for exposure to spirochete lysate. RNA will be extracted before and after exposure in order to perform differential transcriptome analysis to identify signaling pathways stimulated by the spirochete. Goal 2. Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier Directed migration results will be submitted for publication. Continuing experiments will involve modeling and imaging of live cell surface proteins to identify the physical parameters (e.g. mass, charge, etc.) ofthe cell surface macromolecule that isdirecting migration. Targeted pharmacological screens will be used to identify additional Ca2+-permeable channels that sense shear and tensile stresses in keratinocytes. Cellular secretions will be isolated from mechanically stressed cells and screened for signaling molecules and inflammatory mediators. Goal 3. Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system. After exposure to chitin, cellular secretions will be isolated and screened for signaling molecules and inflammatory mediators to identify activated pathways of the innate immune system.

    Impacts
    What was accomplished under these goals? Goal 1. Identify the difference in receptors and signaling pathways of keratinocytes and fibroblasts leading to spirochete inflammatory signals and pain (5% accomplished). Advances in achieving this goal require culturing of both eukaryotes and anaerobic prokaryotes. Extraction, isolation and long term culture of porcine keratinocytes and fibroblasts are being optimized. A research assistant with a degree in microbiology was hired to help perform the anaerobic culture and preparation of spirochete extracts required for the experiments. The researcher is being trained in other methods required to perform these experiments (see below, training provided by the project). Goal 2. Identify the sensory receptors and signaling pathways of keratinocytes and fibroblasts leading to directional migration and rebuilding of the epidermal barrier (10% accomplished). Significant progress has been made toward proving a decades old hypothesis by which epidermal cells sense the wound barrier and migrate toward the wound center to close the wound and rebuild epithelial integrity. This is the first time that this hypothesis has been proven on migrating cells. Methods developed during these experiments and the results that have been acquired have placed us closer to identifying the minimal sensory machinery required to promote wound closure and rebuildepithelial integrity by keratinocytes. Identification of this primary receptor will enable its characterization in chronic wounds and how the conditions of a chronic wound mayimpactthis critical receptor. Manuscript submission for this work is anticipated for spring of 2017. Additional experiments have been started to elucidate the cellular receptors for shear stressthat promote the inflammatory response and promote epidermal repair. One member of a family of mechanosensitive Ca2+ channels has been linked to shear stress. Further experiments will be performed to identify the role(s) of other members. Pharmacological blockers of this ion channel will act not only as analgesics but also as inhibitors of inflammation. Goal 3. Elucidate the interactions between epidermal cells and chitin/ chitosan as a low cost, biodegradable material for stimulating the innate immune system (5% accomplished).The first part of this goal, proving a Ca2+ signaling pathway for chitin/chitosan in keratinocytes, has been achieved. The signaling pathway is stimulated regardless of polymer size which contradicts an earlier report. Additionally, the pathway is only active in the presence of chitin and does not linger in the absence of chitin. These results are consistent with a chitin stimulated innate immune response in keratinocytes.

    Publications