Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Students, producers, veterinarians and faculty involved in Animal Health and Microbiology research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two MS graduate students Chithra Sreenivasan and Jocelin Joseph worked on the project. These students wrote their MS thesis based on the work done under this project and graduated in 2012. Ph.D. graduate student Milton Thomas worked on this project and is continuing in the program. MS graduate student Pratik Katwal worked on this project and is continuing in the program. Five undergraduate students Alexandra Moffitt, Andrew Nelson, Brittany Poelaert, Max Pierson, and Ethan Snow were trained and worked on the project during 2009-14. How have the results been disseminated to communities of interest? The findings of research studies were presented at following conferences and scientific meetings by undergraduate and graduate students and Principal investigator during 2013-14. 73rd Annual Meeting of NCB-ASM held on Oct 11-12, 13. Abstract #: Session 2.2-OP4. SDSU Dept. of Biology and Microbiology Scholarship Day on Nov18, 13. Sanford Health-SDS biomedical Research symposium held at Sanford building in Sioux Falls, SD on Nov 19, 2013. P# 64. 94th Annual CRWAD meeting held on Dec 8-10, 2013. Abstract # 065P. Annual Meeting of American Society of Virology, June 21-25, 2014 Fort Collins, CO, USA. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Under objective 1 the main goal was to study the innate immune responses of IPEC-1 cells derived from one day old pig small intestine in response to lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin (FLA), and CpG oligonucleotide (ODN). We stimulated IPEC-1 cells with these bacterial ligands and gene expression of various toll-like receptors (TLRs), NOD proteins, antimicrobial peptides (AMPs), cytokines, and chemokines were quantified using real time RT-PCR. We found that expression of various TLRs, NOD proteins, AMPs, cytokines and chemokines significantly changed in response to these bacterial ligands stimulation (details given in 2010 report). Under objectives 2 and 3, two porcine macrophage cell lines (Cdelta+ and Cdelta-) were characterized for the constitutive expression of pathogen-recognition receptors (PRRs), AMPs, cytokines, and chemokines using real time RT-PCR. We showed that various PRRs, AMPs, cytokines and chemokine genes were constitutively expressed by both macrophage cell lines. Both macrophage cell lines were also tested for the presence of various surface sugars including sialic acid using different lectins. Under objectives 2 and 3, another goal was to study the innate immune responses of porcine intestinal epithelial cells (IPEC-J2) and porcine macrophage cell line (Cdelta+) to virus-associated molecular patterns. We stimulated both porcine IPEC-J2 and Cdelta+ cells with viral ligands imiquimod, Poly IC and Poly IC with Lyovec. We determined that all three viral ligands induced significant changes in gene expression of various TLRs, RLRs, AMPS, cytokines and chemokines genes in both IPEC-J2 and Cdelta+ cells (Details given in 2010 and 2011 annual reports). These studies showed that both IPEC-J2 and Cdelta2+ cell lines may serve as a good cell culture models for studying the viral ligand interactions and virus pathogenesis. In another study, we further characterized macrophage cell lines (Cdelta+ and Cdelta-) for the protein expression of TLRs 2, 4, 5, and 6 in response to LPS. The results showed that LPS stimulation induced some significant changes in various TLR proteins expression in these cells. In continuation to previous studies, the main goals under objective 5 were to 1. determine the infectivity of Cdelta+ and Cdelta- cell lines to influenza viruses and to study the innate immune responses of Cdelta+ cells to influenza A and influenza B viruses and 2. To develop porcine intestinal epithelial cells (IPEC-J2) as an in vitro model for porcine influenza studies. We found that both influenza A and B viruses were able to replicate in both Cdelta2+ and Cdelta2- cells. We further infected Cdelta2+ cells and showed that both virus strains induced significant changes in various TLRs, RLRs, cytokines and chemokines genes expression. In another study we also developed a cloned cell line from IPEC-J2 cells (SD-PJEC) for studying the pathogenesis of influenza viruses. Our results demonstrated that this cell line was permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly supported the growth of human influenza B viruses. We reported detailed results from these studies in 2012 report. Through these studies, we showed that both Cdelta2+ and Cdelta2- porcine macrophage cell lines and SD-PJEC cell line may serve as good cell culture models for studying the porcine influenza virus pathogenesis. In 2013, we expanded the scope of our objective 5 to study the role of porcine respiratory epithelial cells (MK1-OSU cell line) in porcine influenza pathogenesis and the main research goals were: 1. To biochemically characterize MK1-OSU cell line (clonally derived from tracheal and bronchial epithelial cells of 5 week old piglet), and determine its susceptibility to Influenza A, B, and C viruses and 2. To study the lectin binding profile of MK1-OSU and SD-JPEC cell lines. The MK1-OSU cells expressed various influenza virus specific sialic acid receptors and were susceptible to various influenza viruses. We characterized and compared the lectin binding profile of MK1-OSU and SD-JPEC cell lines and found that both cell lines expressed a large number of sugar moieties on their surfaces and had very similar lectin binding profiles. In further studies conducted in 2013 and 2014, TLR7, RIG-I, and MDA5 protein expressions of MK1-OSU cells at 24h post-infection with Influenza A and B strains were quantified. SD-PJEC cell line was also infected with Influenza A. All three proteins showed few significant changes in both these cell lines in response to influenza virus infection. Under objective 4, one of the major goals was to establish pure primary cultures of bovine intestinal epithelial cells from mixed cultures of bovine intestinal epithelial and myofibroblasts cultures established earlier from young calves. To achieve this objective in 2014, bovine mixed ileal cultures were used to obtain a clone of bovine ileal epithelial cells which was further amplified and characterized. The selected clone was cytokeratin positive and expressed low levels of vimentin. These primary bovine intestinal epithelial cell cultures in future studies will be immortalized and used for studying the role of intestinal epithelial cells in innate immunity and pathogenesis of enteric pathogens.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Milton Thomas, Zhao Wang, Chithra C. Sreenivasan, Ben M. Hause,Gourapura J. Renukaradhya, Feng Lib,, David H. Francis, Radhey S. Kaushik, Mahesh Khatri. 2015. Poly I:C adjuvanted inactivated swine influenza vaccine inducesheterologous protective immunity in pigs. Vaccine 33 (2015) 542548.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Student and faculty involved in Animal Health and Microbiology research Changes/Problems: In addition to study the immunobiology of porcine intestinal epithelial cellsas proposed in the original project, we have also included porcine respiratory epithelial cell line in our proposed studies as these cells play important role in influenza and other infectious disease pathogenesis. What opportunities for training and professional development has the project provided? Trained one graduate student and one undergraduate student. How have the results been disseminated to communities of interest? These findings were submitted for presentation at 2013 SD Academy of Science Meeting held on April 12-13, 2013. These studies were also presented at SDSU Biology and Microbiology Scholarship Day on April 19, 2013, & SDSU Undergraduate Research Day on April 22, 2013. What do you plan to do during the next reporting period to accomplish the goals? 1. To study the innate immune responses of porcine respiratroy epithelial OSU-MK1 and porcine intestinal epithelial SD-PJEC cell line to influenza virus infection. 2. To study the innate immune repsonses of porcine macrophage cell line to bacterial ligands. 3. To establish and characterize bovine intestinal epithelial and myofibroblast-like cell culture models for studying the pathogenisis of enteric infection.
Impacts
What was accomplished under these goals?
In this reporting period (Jan-Sept 2013) the main research goals were: 1. To biochemically characterize MK1-OSU cell line (clonally derived from tracheal and bronchial epithelial cells of 5 week old piglet), and determine its susceptibility to Influenza A, B, and C viruses. 2. To study the lectin binding profile of MK1-OSU and SD-JPEC cell lines. In our previous report (Jan -Dec 2012) we showed that porcine influenza viruses infected two Cdelta2+ and Cdelta2- porcine macrophage cell lines and mounted innate immune response to influenza infection. As Influenza viruses mainly infect respiratory epithelial cells; we also attempted to establish a porcine respiratory epithelial cell culture model for studying influenza pathogenesis. We obtain MK1-OSU cell line-clonally derived from tracheal and bronchial epithelial cells of 5 week old piglet, from Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH. We biochemically characterized this cell line and determined its susceptibility to Influenza A, B, and C viruses. The expression of cell marker proteins in these cells was determined by immunocytochemistry. The presence of α-2,3- and α-2,6-linked sialic acid receptors for Influenza viruses on MK1-OSU cells was detected by flow cytometry based lectin binding assay. Growth kinetic of five Influenza viruses -A/swine/Iowa/0855/2007(H3N2), A/swine/Minnesota/2073/2008(H1N1), B/Florida/4/2006, B/Brisbane/60/2008, and C/Swine/Oklahoma/1334/2011 in MK1-OSU cell line was also determined. All MK1-OSU cells expressed epithelial cell marker cytokeratin and some of these cells also expressed vimentin. The MK1-OSU cells expressed both α -2, 3- and α-2, 6-linked sialic acid receptors and were infected with all the five viruses used in this study. A larger percentage of MK1-OSU cells 24h post-infection were infected by both Influenza A strains as determined by flow cytometry and immunofluorescence assay. Hence, this epithelial cell line could serve as an excellent model for studying the innate immune responses and pathogenesis of Influenza viruses. In our previous report we also showed that the SD-PJEC cell line, a subclone of the IPEC-J2 cell line which was originally derived from newborn piglet jejunum, can be infected with influenza viruses. Both respiratory and intestinal epithelial cells express many sugar moieties as part of various surface receptors involved in pathogen recognition. In another study conducted by an undergraduate student Max Pierson and graduate student Milton Thomas, we characterized and compared the lectin binding profile of MK1-OSU and SD-JPEC cell lines. MK1-OSU and SD-PJEC cells were incubated with biotinylated lectins followed by staining with streptavidin-FITC. The stained cells were analyzed by flow cytometry. Twenty one lectins were used to stain both cell types and the specificity of lectin binding to the cell surfaces was confirmed by using specific lectin inhibitors. Fifteen different lectins stained a large percentage of MK1-OSU cells. However, SJA, and GSL-2 lectins did not bind and DBA, UEA-1 and VVA lectins showed lower binding to MK1-OSU cells. Sixteen different lectins stained a large percentage of SDJPEC cells. However, UEA-1 and GSL-2 lectins did not bind and CON-A and DBA lectins showed lower binding to SD-PJEC cells. These findings were submitted for presentation at 2013 SD Academy of Science Meeting. These results were also presented at SDSU Biology and Microbiology Scholarship Day on April 19, 2013, and SDSU Undergraduate Research Day on April 22, 2013. These studies suggest that both cell lines express similar lectin binding profiles and may be successfully used for infectious disease pathogenesis studies. Through these studies, we showed that both MK1-OSU and SD-PJEC cell lines may serve as good cell culture models for studying the pathogenesis of porcine influenza virus and other infections.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
C. P. Guevara, W. B. Luiz, A. Sierra, C. Cruz , F. Qadri , R. S. Kaushik, L. C.S. Ferreira, O. G. G�mez-Duarte. 2013. Enterotoxigenic Escherichia coli (ETEC) CS21 pilus contributes to adhesion to intestinal cells and to pathogenesis under in vivo conditions. Microbiology. 159, 17251735.
3.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Peter Z. Fekete, Kristina S. Mateo, Weiping Zhang , Rodney A. Moxley , Radhey S. Kaushik, David H. Francis. 2013. Both enzymatic and non-enzymatic properties of heat-labile enterotoxin are responsible for LT-enhanced adherence of enterotoxigenic Escherichia coli to porcine IPEC-J2 cells. Veterinary Microbiology. 164: 330-335.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Sreevidya Santha, Ajay Bommareddy, Brittny Rule, Ruth Guillermo, Radhey S Kaushik, Alan Young, Chandradhar Dwivedi. 2013. Antineoplastic Effects of a-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis. PLoS ONE 8(2): e56982. doi:10.1371/journal.pone.0056982.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: In this reporting period (Jan-Dec 2012) the main research goals were: 1. To determine the infectivity of porcine macrophage cell lines (Cdelta+ and Cdelta-) to influenza viruses and to study the innate immune responses of Cdelta+ cells to influenza A and influenza B viruses and; 2. To develop porcine intestinal epithelial cells (IPEC-J2) as an in vitro model for porcine influenza studies. We first characterized both Cdelta+ and Cdelta- cell lines for the presence of sialic acid based surface receptors responsible for the influenza virus infection. Both cell types mainly expressed sialic acid receptor Sial-2,6-Gal as determined by SNA lectin binding using flow cytometry. Then we checked if influenza viruses (swine H1N1/A/SW/IOWA and human B/FLORIDA/4/2006 strains) were able to infect and replicate in these cells. Using immunoflourescence assays, we showed that these viral strains infected both cell lines. We also determined the percentage of virus infected cells using flow cytometry. We also showed, using hemagglutination (HA) assays, that both influenza A and B viruses were able to replicate in both Cdelta2+ and Cdelta2- cells. We further infected Cdelta2+ cells with these two influenza viruses and studied the changes in gene expression of different pathogen-recognition receptors (PRRs), cytokines, chemokines and anti-microbial peptides. Both swine influenza A virus and human influenza B virus strains induced significant changes in various toll-like receptors (TLRs), RIG1-like receptors (RLRs), type 1 interferons, pro-inflammatory cytokines and chemokines genes expression. These findings were included in the thesis of MS graduate student Joseph Jocelin, and also presented as poster at the 93nd CRWAD meeting held on Dec 2-4, 2012 at Chicago, IL. In another study conducted in collaboration with Dr. Ying Fang, we developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrated that SD-PJEC cell line preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly supports the growth of human-origin influenza B viruses. Propagation of swine influenza viruses in these cells results in a rapid growth rate within 24 h post-infection. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin-Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 per ml, respectively. The findings of this study were published in 2012 J. Gen Virol. 93(9):2008-16. Through these studies, we showed that both Cdelta2+ and Cdelta2- porcine macrophage cell lines and SD-PJEC cell line may serve as good cell culture models for studying the porcine influenza virus pathogenesis. PARTICIPANTS: Individual who worked on the project: Chithra Sreenivasan: Graduate student; Jocelin Joseph: Graduate Student; Max Pierson: Undergraduate student; Brittany Poelaert: Undergraduate student; Radhey S. Kaushik: Principal investigator. Partner Organization: South Dakota State University. Collaborators and Contracts: Carol G. Chitko-McKown: USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933, USA; Ying Fang: Dept. of Veterinary and Biomedical Sciences, SDSU. Training and professional development: Trained two graduate students and two undergraduate students. Two graduate students Chithra Sreenivasan and Jocelin Joseph graduated with Masters in Science (MS)in 2012. TARGET AUDIENCES: Target audience: Student and faulty involved in Animal Health and Microbiology research. Efforts: The research findings obtained from porcine macrophage Cdelta+ and Cdelta- cell lines were presented as poster presentation at 93nd CRWAD Meeting, Chicago, Illinois on Dec 2-4, 12. The research findings obtained from porcine intestinal epithelial SD-PJEC cell line were published in 2012 J. Gen Virol.93(9:2008-16. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Porcine influenza is an acute respiratory disease of pigs caused by Type A influenza viruses. Porcine influenza virus is an important player in the porcine respiratory disease complex. Porcine influenza virus infection causes significant morbidity and mortality in swine herds and result in huge economic losses in the swine industry. Respiratory epithelial cells are the first cells which are infected with influenza virus and these cells play a major role in influenza pathogenesis. However, many studies have shown that alveolar macrophages also play a very important role in the pathogenesis and immunity to influenza infection. Until recently, useful porcine macrophage cell lines were not available for viral/influenza studies. Recently two monocyte-derived porcine macrophage cell lines (Cdelta2+ and Cdelta2-) have been developed and characterized for their phagocytic ability and biochemical properties. In our last year's report we studied the innate immune responses of these macrophage cell lines to various viral ligands and speculated that these cell lines would be suitable to study innate immune responses to viral infections. In the present study we checked these macrophage cell lines for the infectivity to influenza viruses and used one of these cell lines Cdelta2+ to study innate immune responses to influenza virus. The findings obtained in the present studies clearly indicate that both porcine macrophage cell lines are effectively infected with influenza A and B viruses and result in changes in the expression of various TLRs, RLRs, antimicrobial peptides, cytokines and chemokines which are important immune mediators. Although respiratory epithelial cells play important role in the pathogenesis of influenza virus, no porcine respiratory epithelial cultures are currently available. Thus, in this study we also cloned previously commonly used porcine intestinal epithelial IPEC-J2 cells and named the cell line as SD-PJEC. We checked if SD-PJEC cell line could be infected with the influenza viruses. Our findings clearly indicate that SD-PJEC cell line serves as an excellent in vitro model for studying porcine influenza pathogenesis. Overall, these findings suggest that both porcine macrophage Cdelta2+ and porcine intestinal SD-PJEC cell lines serve as good cell culture models for studying the porcine influenza pathogenesis and innate immune response to influenza infection. The findings of this study also support the use of porcine intestinal epithelial and macrophage cell cultures as an alternative or replacement for many in vivo studies and use of animals in research.
Publications
- Preety Sahdev, Satheesh Podaralla, Radhey S. Kaushik, and Omathanu Perumal. 2013. Calcium Phosphate Nanoparticles for Transcutaneous Vaccine Delivery. Journal of Biomedical Nanotechnology. 9(1):132-141.
- Sajan George, Katherine Circle, Stacy Lindblom, Sebastien Vilain, Artur J.M. Rosa, David Francis, Volker Brozel and Radhey S. Kaushik. 2012. Assessment of Toll-like Receptors in the Ileum of Weanling Pigs - Responses to Feed Antibiotic Chlortetracycline and Gnotobiotic Conditions. J Clin Cell Immunol. 3:125.
- Guillermo, R.F., Zhang, X., Kaushik, R.S., Zeman, D., Ahmed, S., Khalifa, S., Fahmy, H., Dwivedi, C. 2012. Dose-response on the chemopreventive effects of sarcophine-diol on UVB-induced skin tumor development in SKH-1 hairless mice. Marine Drugs. 10: 2111-25.
- Sun Z, Huber VC, McCormick K, Kaushik RS, Boon AC, Zhu L, Hause B, Webby RJ, Fang Y. 2012. Characterization of a porcine intestinal epithelial cell line for influenza virus production. J. Gen Virol. 93(Pt 9):2008-16.
- Wei Chen, Teresa Seefeldt, Alan Young, Xiaoying Zhang, Yong Zhao, John Ruffolo, Radhey S. Kaushik, Xiangming Guan. 2012. Microtubule S-Glutathionylation as a Potential Approach for Antimitotic Agents. BMC Cancer. 12:245.
- Qiji Deng, Minxun Song, Andrew Demers, Yuejin Weng, Wuxun Lu, Dan Wang, Radhey S. Kaushik, Qingzhong Yu, and Feng Li. 2012. Biochemical characterization of the small hydrophobic protein of avian metapneumovirus. Virus Research.167:297-301.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: In this reporting period (Jan-Dec 2011) the main research goal was to study the innate immune responses of porcine intestinal epithelial cells (IPEC-J2) and porcine macrophage cell line (Cdelta+) to virus-associated molecular patterns. We stimulated both porcine IPEC-J2 and Cdelta+ cells for 3 and 24h with imiquimod, Poly IC and Poly IC with Lyovec. Cells with media alone were used as negative control. Cell lysates were processed for total RNA extraction which was used to prepare cDNA. The gene expressions for toll-like receptors (TLRs) and RIG-1 like receptors (RLRs) and various cytokines were quantified using real time RT-PCR relative to cyclophilin-A for IPEC-J2 cells and Ribosomal Protein L4 for Cdelta+ cells. The results from IPEC-J2 cell line study showed that the expression of TLR-3, -7 and -9 genes did not change at 3h and 24h under any condition. TLR-4,-5,-6 and -8 was downregulated at 24h by imiquimod and poly IC. TLR-2 was upregulated at 3h by Poly IC with Lyovec. RIG-1 gene expression was upregulated by imiquimod and poly IC at 3h but downregulated at 24h by imiquimod. Poly IC and poly IC with Lyovec upregulated MDA-5 gene expression at 3h. NOD-1 and NOD-2 genes were upregulated at 24 and 3h respectively by all three ligands. The expression of IFN-alpha in response to imiquimod and IFN-beta in response to all three viral ligands showed upregulatory trend at 3h; however, these data were not statistically significant. Cytokine IL-1a was upregulated by imiquimod at 3h and IL-1b was down regulated by Poly IC and imiquimod at 24h. IL-8 was upregulated at 3h by imiquimod. TNF-alpha was up regulated by Poly IC at 3h and IL-12p40 was upregulated by imiquimod at 3h. Both IL-15 and IL-18 were upregulated at 3h. Beta-defensin (BD)-1 was up regulated at 24h and BD-2 was up regulated at 3h by all three ligands. These findings were presented as posters at 96th Meeting of the SD Academy of Science, Oacoma, SD on Apr 8-9, 11 and 92nd CRWAD Meeting, Chicago, Illinois on Dec 4-6, 11. In another study conducted using Cdelta+ cells, the expression of TLR-2 was downregulated by poly IC and poly IC with lyovec at 3h. TLR-3 gene was upregulated at 3 and 24h while TLR-7 was upregulated at 3h with all three ligands. TLR-8 was upregulated by imiquimod and Poly IC at 3h. Poly IC with Lyovec upregulated RIG-1 gene but downregulated MDA-5 at 3h. All three ligands upregulated RIG-1 gene but only poly IC upregulated MDA-5 gene at 24h.The expression of IFN-alpha was upregulated at 3h by all three ligands while IFN-beta was upregulated at 3h by imiquimod and Poly IC with Lyovec. IL-1alpha gene was upregulated at 3 and 24h while IL-6 was upregulated at 3h by all three ligands. TNF-alpha expression was upregulated by imiquimod at 3 and 24h. IL-12p35 and IL-8 were upregulated at 3h by all three ligands. BD-1 and -2 were upregulated by Poly IC at 3h. These findings were presented as poster at the 71th Meeting of North Central Branch of the ASM on Oct 7-8, 11 at Des Moines, IA. Based on these studies, it is expected that both IPEC-J2 and Cdelta2+ cell lines may serve as a good cell culture model for studying the viral ligand interactions and the virus pathogenesis. PARTICIPANTS: Individual who worked on the project: Jocelin Joseph: Graduate Student; Chithra Sreenivasan: Graduate student; Andrew Nelson: Undergraduate student; Brittany Poelaert: Undergraduate student; Radhey S. Kaushik: Principal investigator. Partner Organization: South Dakota State University. Collaborators and Contracts: Carol G. Chitko-McKown: USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933, USA. Training and professional development: Trained two graduate students and two undergraduate students. TARGET AUDIENCES: Target audience: Student and faculty involved in Animal Health and Microbiology research. Efforts: The research findings obtained from porcine intestinal epithelial IPEC-J2 cells were presented as poster presentation at 96th Meeting of the SD Academy of Science, Oacoma, SD on Apr 8-9, 11 and 92nd CRWAD Meeting, Chicago, Illinois on Dec 4-6, 11. The research findings obtained from porcine macrophage Cdelta2+ cell line were presented as poster presentation at the 71th Annual Meeting of North Central Branch of the ASM on Oct 7-8, 11 at Des Moines, IA by the graduate student Jocelin Joseph. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Intestinal enteric pathogens present many bacteria or virus-associated molecular patterns to intestinal epithelial cells and macrophages and these cells effectively respond to these ligands and mediate effective innate immune responses; however, immune responses of porcine intestinal epithelial cells and macrophages to various viral ligands have not been studied in sufficient detail. The findings obtained in the present studies indicate that porcine intestinal epithelial cells (IPEC-J2 cells) effectively respond to stimulation with various virus-associated molecular patterns (imiquimod, Poly IC and Poly IC with Lyovec) and result in changes in the expression of various TLRs, RLRs, antimicrobial peptides, cytokines and chemokines which are important immune mediators. Thus, the findings of this study will help in understanding the basic biology of porcine intestinal epithelial cells and viral pathogenesis for developing effective preventive or therapeutic measures for enteric viral infections in pigs. Macrophages also play important role in mediating innate immune responses to various bacterial and viral pathogens. Macrophages express various pathogen-recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) and activate genes responsible for host defense. In the present study we found that porcine macrophage cell line (Cdelta+) constitutively expressed mRNAs for various TLRs, antimicrobial peptides, cytokines and chemokines at variable levels and this cell line effectively respond to various virus-associated molecular patterns. These findings suggest that the porcine macrophage Cdelta2+ cell line can act as a good cell culture model for studying the innate immune response to viral ligands. The findings of this study also support the use of porcine intestinal epithelial and macrophage cell cultures as an alternative or replacement for many in vivo studies and use of animals in research.
Publications
- Qiji Deng, Dan Wang, Xiaoxiao Xiang, Xiaofei Gao, Philip R. Hardwidge, Radhey Kaushik, Thorsten Wolff, Suvobrata Chakravarty, and Feng Li, 2011. Nuclear localization of influenza B polymerase proteins and their binary complexes. Virus Research. 156: 49-53.
- Qiji Deng , Dan Wang , Xiaoxiao Xiang , Xiaofei Gao, Philip R. Hardwidge, Radhey S. Kaushik, Thorsten Wolff, Suvobrata Chakravarty, Feng Li. 2011. Application of a split luciferase complementation assay for the detection of viral protein:protein interactions. Journal of Virological Methods. 176(1-2):108-11.
- Chandeshwari Chilampalli, Ruth Guillermo, Radhey S Kaushik, Alan Young, Gudiseva Chandrasekher, Hesham Fahmy and Chandradhar Dwivedi. 2011. Honokiol, a chemopreventive agent against skin cancer, induces cell cycle arrest and apoptosis in human epidermoid A431 cells. Experimental Biology and Medicine 2011: 1-9.
- Chandeshwari Chilampalli, Ruth Guillermo, Xiaoying Zhang, Radhey S. Kaushik, Alan Young, David Zeman, Michael B. Hildreth, Hesham Fahmy and Chandradhar Dwivedi. 2011. Effects of Magnolol on UVB-induced Skin Cancer Development in Mice and its Possible Mechanism of Action. BMC Cancer 2011, 11:456.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In this reporting period (Jan-Dec 2010) the main research goal was to study the innate immune responses of IPEC-1 cells in response to lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin (FLA), CpG oligonucleotide (ODN) and GpC ODN. IPEC-1 cells were derived by others from one day old pig small intestine and maintained for a number of passages as primary cultures. We stimulated IPEC-1 cells with LPS, PGN, FLA, CpG ODN and GpC ODN for 3 and 24 hrs. Total RNA was extracted from cell lysates at each time-point and used to prepare cDNA. The gene expressions of various toll-like receptors (TLRs), NOD proteins, antimicrobial peptides (AMPs), cytokines, and chemokines were quantified using real time RT-PCR relative to cyclophilin-A gene. The expression of TLRs and NOD proteins did not change upon stimulation with LPS, FLA, CpG and GpC at 3 hrs; however, PGN increased TLR-1, -2 and -6 expressions at 3 hrs. LPS decreased TLR-4,-5,-6 and NOD-1; PGN decreased TLR-8 and -10; FLA decreased TLR-4, -8 and -10; CpG decreased TLR-4 and MD-2; and GpC decreased TLR-6 expression at 24 hrs. LPS stimulation induced increase in IL-1 alpha and CCL-20 expression at 3 hrs and increased IL-1 alpha and decreased IL-10 and MCP-1 expression at 24 hrs. PGN increased IL-1beta and osteopontin (OPN) expression at 3 hrs; and increased IL-1 beta and decreased IL-10 expression at 24 hrs. FLA increased IL-1beta, CCL-20, OPN and MIF expression at 3hrs; and increased IL-1beta and decreased IL-10 expression at 24 hrs. CpG induced increase in IL-12p40, IL-18 and MIF expression at 3 hrs; and increased MCP-1 and decreased IL-10 at 24 hrs compared to normal cells; however, no significant changes were induced by CpG when compared to GpC. These findings were presented as poster presentation at the 91st CRWAD Annual Meeting on Dec 5-7, 2010 held at Chicago, Illinois. In another preliminary study, two porcine macrophage cell lines (Cdelta+ and Cdelta-) were characterized for the constitutive expression of pathogen-recognition receptors (PRRs), beta-defensins (BD), cytokines, and chemokines. Gene expressions of TLRs, retinoic acid-inducible gene 1 (RIG-1), melanoma differentiation-associated antigen 5 (MDA-5), nucleotide oligomerization domain (NOD) proteins, BD, cytokines, and chemokines were quantified using real time RT-PCR relative to cyclophilin-A. The results from a single experiment showed that TLR-5, TLR-10, IL-1β, TNF-α, IL-7, IL-15, IL-18, CCL-20, and BD-1 genes were expressed at higher levels while TLR-7, TLR-9, MD-2, NOD-1, NOD-2, IL-6, IL-10, IL-12p40, osteopontin, BD-2, RIG-1 and MDA-5 genes were expressed at lower levels in Cdelta2+ cells compared to Cdelta2- cells. TLR-1, 2, 3, 4, 6, and 8, IL-1alpha, IL-12p35, MCP-1, and MIF were expressed at similar levels by both cell types. We also tested both cell lines for the presence of surface sialic acid by flow cytometry using Maackia amurensis lectin (MAL-II) and Sambucus nigra agglutinin (SNA). Both cell types showed ~3-6% MAL-II and 62-78% SNA positive cells. These findings were presented as poster presentation at the 70th Annual Meeting of North Central Branch of the ASM on Oct 1-2, 2010 held at Mankato, MN. PARTICIPANTS: Individual who worked on the project: Chithra Sreenivasan: Graduate student; Jocelin Joseph: Graduate Student; Alexandra Moffitt: Undergraduate student; Radhey S. Kaushik: Principal investigator. Partner Organization: South Dakota State University. Collaborators and Contracts: Carol G. Chitko-McKown: USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933, USA. Training and professional development: Trained two graduate students and on undergraduate student. TARGET AUDIENCES: Target Audiences: Student and faculty involved in Animal Health and Microbiology research. Efforts: The research findings obtained from porcine intestinal epitheilal IPEC-1 cells were presented as poster presentation at the 91st CRWAD Annual Meeting on Dec 5-7, 2010 held at Chicago, Illinois. The research findings obtained from porcine macrophage cell lines were presented as poster presentation at the 70th Annual Meeting of North Central Branch of the ASM on Oct 1-2, 2010 held at Mankato, MN by the graduate student Jocelin Joseph. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Intestinal normal flora and enteric pathogens present many viral or bacteria-associated molecular patterns to intestinal epithelial cells and macrophages and these cells effectively respond to these bacterial ligands and mediate effective innate immune responses; however, immune responses of porcine intestinal epithelial cells and macrophages to various bacterial and viral ligands have not been studied in sufficient detail. The findings obtained in the present studies indicate that porcine intestinal epithelial cells (IPEC-1 cells) effectively respond to stimulation with various bacterial ligands and result in changes in the expression of various TLRs, antimicrobial peptides, cytokines and chemokines which are important immune mediators. Thus, the findings of this study will help in understanding the basic biology of porcine intestinal epithelial cells and for developing effective preventive or therapeutic measures for enteric bacterial infections in pigs. Macrophages also play important role in mediating innate immune responses to various bacterial and viral pathogens. Macrophages express various pathogen-recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) and activate genes responsible for host defense. In the present study we found that two porcine macrophage cell lines (Cdelta+ and Cdelta-) constitutively expressed mRNAs for various TLRs, antimicrobial peptides, cytokines and chemokines at variable levels. Both these macrophage cells lines also expressed sialic acids on their surface which act as receptors for various viruses including swine influenza virus (SIV). These findings suggest that these porcine macrophage cell lines can act as a good cell culture model for studying the innate immune response to bacterial and viral ligands and SIV and can be used in the development of possible antiviral therapies against SIV. The findings of this study also support the use of porcine intestinal epithelial and macrophage cell cultures as an alternative or replacement for many in vivo studies and use of animals in research.
Publications
- Amber M. Johnson, Radhey S. Kaushik, Philip R. Hardwidge. 2010. Disruption of transepithelial resistance by enterotoxigenic Escherichia coli. Veterinary Microbiology. 141:115-119.
- Xiaoying Zhang, Wei Chen, Ruth Guillermo, Gudiseva Chandrasekher, Radhey S. Kaushik, Alan Young, Hesham Fahmy, Chandradhar Dwivedi. 2010. Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in bothp53-mutated human epidermoid carcinoma A431cells and p53 wild-type human melanoma UACC-62 cells. BMC Research Notes. 3:220.
- Chilampalli, S., Zhang, X., Fahmy, H., Kaushik, R.S., Zeman, D., Hildreth, M. B., and Dwivedi, C. 2010. Chemopreventive effects of honokiol on UVB-induced skin cancer development, Anticancer Res. 30:777-83.
- Ajay Bommareddy, Xiaoying Zhang, Radhey S. Kaushik, Chandradhar Dwivedi. 2010. Effects of components present in flaxseed on human colon adenocarcinoma Caco-2 cells: Possible mechanisms of flaxseed on colon cancer development in animals. Drug Discoveries & Therapeutics. 4(3):184-189.
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