Progress 09/01/05 to 08/31/08
Outputs
OUTPUTS: During the life of this project, we were able to address the specific aims of the project. First, the following bovine Toll-like receptor-4 (TLR-4) signaling molecules were cloned and sequenced: interleukin-1 receptor-associated kinase 1 (IRAK1), MD-2 protein (LY96), toll-like receptor adaptor molecule 2 (TICAM2), toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), toll interacting protein (TOLLIP), TNF receptor-associated factor 6 (TRAF6), and caspase 8 (CASP8). In addition, the chromosomal locations of these genes, as well as myeloid differentiation primary response gene 88 (MyD88) and toll-like receptor adaptor molecule 1 (TICAM1), were determined by radiation hybrid mapping. Second, we developed the dominant-negative reagents necessary for addressing the specific aims, which proposed to identify the bovine Toll-like receptor-4 (TLR-4) signaling molecules that are operative in bacterial lipopolysaccharide-induced NF-kappaB activation, apoptosis, and endothelial permeability. We used a retroviral-based delivery system to induce stable expression of these dominant-negative constructs in two types of primary bovine endothelial cells, aortic and pulmonary artery endothelial cells. We confirmed expression of these constructs in the endothelial cells at both the mRNA and protein level. In addition, we developed a reporter system for measuring NF-kappaB activation in bovine endothelial cells, a step that was required for functional analysis of the bovine Toll-like receptor-4 (TLR-4) signaling molecules. Third, we conducted functional studies to establish roles for bovine TLR-4 signaling molecules in mediating bacterial lipopolysaccharide-induced NF-kappaB activation and apoptosis. Collaborations with another research group enabled the identification of downstream signaling molecules involved in bacterial lipopolysaccharide-induced changes in endothelial permeability. The results from this work have been disseminated through presentations at the annual Conference of Research Workers in Animal Diseases (CRWAD) and through the publication of the results in peer-reviewed journal articles. PARTICIPANTS: Douglas D. Bannerman served as the Principal Investigator and to direct the overall research project. Erin E. Connor served as a Co-Investigator and provide technical direction/expertise on the molecular biology aspects of the project. Elisabeth Cates, a graduate student from the University of Maryland at College Park, conducted the experiments described in this report, under the direction and guidance of Drs. Bannerman and Connor. TARGET AUDIENCES: Scientists engaged in comparative studies of innate immunity and those investigating the pathogenesis of infectious diseases in cattle will benefit from the knowledge generated from this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results of this work indicate differences with a previously published bovine sequence for LY96 and a predicted sequence in the GenBank database for TIRAP based on the most recent assembly of the bovine genome. In addition, discrepancies between actual and predicted chromosomal map positions based on the Btau_2.0 genome assembly release were identified, although map positions were consistent with predicted locations based on the current bovine-human comparative map. Alignment of the bovine amino acid sequences with human and murine sequences showed a broad range in conservation, from 52 to 93%. These findings will: 1) assist in the assembly and annotation of the bovine genome sequence; 2) enable the identification of variations in genes critically involved in host innate immunity; and 3) facilitate further studies of bovine Toll-like receptor signaling pathways in cattle. Based on comparative immunological studies in humans and mice, the findings of the current research should facilitate studies into the role of Toll-like receptors in regulating the host immune responses of cattle to infectious diseases.
Publications
- Kim, F., M. Pham, I. Luttrell, D.D. Bannerman, J. Tupper, J. Thaler, T.R. Hawn, E.W. Raines, and M.W. Schwartz. 2007. Toll-like receptor-4 mediates vascular inflammation and insulin resistance in diet-induced obesity. Circ. Res. 100(11):1589-1596. Connor, E.E., E.A. Cates, J.L. Williams, and D.D. Bannerman. 2006. Cloning and radiation hybrid mapping of bovine toll-like receptor-4 (TLR-4) signaling molecules. Vet. Immunol. Immunopathol. 112(3-4):302-308.
- Cates, E.A., E.E. Connor, D.M. Mosser, and D.D. Bannerman. 2008. Functional characterization of bovine TIRAP and MyD88 in mediating bacterial lipopolysaccharide-induced endothelial NF-kappaB activation and apoptosis. Comp. Immunol. Microbiol. Infect. Dis. In Press.
- Gong, P., D.J. Angelini, S. Yang, G. Xia, A.S. Cross, D. Mann, D.D. Bannerman, S.N. Vogel, and S.E. Goldblum. 2008. TLR4 signaling is coupled to src family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. J. Biol. Chem. 283(19):13437-13449.
- Ibeagha-Awemu, E.M., J.W. Lee, A.E. Ibeagha, D.D. Bannerman, M.J. Paape, and X. Zhao. 2008. Bacterial lipopolysaccharide induces increased expression of toll-like receptor (TLR) 4 and downstream TLR signaling molecules in bovine mammary epithelial cells. Vet. Res. 39(2):(article #11)1-12.
- De Schepper, S., A. De Ketelaere, D.D. Bannerman, M.J. Paape, L. Peelman, and C. Burvenich. 2008. The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle. Vet. Res. 39(1):(article #5)1-23.
|
Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: During the past year, significant progress was made in addressing specific aims #1, #3, and #4. First, we have developed the dominant-negative reagents necessary for addressing these 3 specific aims, which propose to identify the bovine Toll-like receptor-4 (TLR-4) signaling molecules that are operative in bacterial lipopolysaccharide-induced NF-kappaB activation, apoptosis, and endothelial permeability, respectively. Specifically, we have developed dominant-negative constructs for bovine interleukin-1 receptor-associated kinase 1 (IRAK1), myeloid differentiation primary response gene 88 (MyD88), toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), and TNF receptor-associated factor 6 (TRAF6). We have also used a retroviral-based delivery system to induce stable expression of these dominant-negative constructs in two types of primary bovine endothelial cells, aortic and pulmonary artery endothelial cells. We have confirmed expression of these constructs in
the endothelial cells at both the mRNA and protein level. In addition, we have developed a NF-kappaB reporter system for measuring NF-kappaB activation in bovine endothelial cells, a step that is required for functional analysis of these proteins as proposed in specific aim #1. Functional studies are now under way to assess the roles of these signaling molecules in bacterial lipopolysaccharide-induced NF-kappaB activation, apoptosis, and endothelial permeability. The results from this work are being disseminated through their presentation at the annual Conference of Research Workers in Animal Diseases (CRWAD).
PARTICIPANTS: Douglas D. Bannerman continued to serve as the Principal Investigator and to direct the overall research project. Erin E. Connor continued to serve as a Co-Investigator and provide technical direction/expertise on the molecular biology aspects of the project. Elisabeth Cates, a graduate student from the University of Maryland at College Park, conducted the experiments described in this progress report, under the direction and guidance of Drs. Bannerman and Connor.
Impacts
The progress that we have made will now enable us to proceed with functionally characterizing the roles of bovine TLR signaling molecules in mediating endothelial responses to bacterial lipopolysaccharide and will facilitate studies into the role of Toll-like receptors in regulating the host immune responses of cattle to infectious diseases.
Publications
- Kim, F., Pham, M., Luttrell, I., Bannerman, D.D., Tupper, J., Thaler, J., Hawn, T.R., Raines, E.W., and Schwartz, M.W. (2007). Toll-like receptor-4 mediates vascular inflammation and insulin resistance in diet-induced obesity. Circulation Research. 100(11):1589-1596.
- De Schepper, S., De Ketelaere, A., Bannerman, D.D., Paape, M.J., Peelman, L., and Burvenich, C. (2008). The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle. Veterinary Research. 39(1):(article #5)1-23.
|
Progress 09/01/05 to 08/31/06
Outputs
During the past year, significant progress was made in completing the first aim of the project. The following bovine Toll-like receptor-4 (TLR-4) signaling molecules were cloned and sequenced: interleukin-1 receptor-associated kinase 1 (IRAK1), MD-2 protein (LY96), toll-like receptor adaptor molecule 2 (TICAM2), toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), toll interacting protein (TOLLIP), TNF receptor-associated factor 6 (TRAF6), and caspase 8 (CASP8). In addition, the chromosomal locations of these genes, as well as myeloid differentiation primary response gene 88 (MyD88) and toll-like receptor adaptor molecule 1 (TICAM1), were determined by radiation hybrid mapping. Results of this work indicate differences with a previously published bovine sequence for LY96 and a predicted sequence in the GenBank database for TIRAP based on the most recent assembly of the bovine genome. In addition, discrepancies between actual and predicted chromosomal
map positions based on the Btau_2.0 genome assembly release were identified, although map positions were consistent with predicted locations based on the current bovine-human comparative map. Alignment of the bovine amino acid sequences with human and murine sequences showed a broad range in conservation, from 52 to 93%.
Impacts
These findings will: 1) assist in the assembly and annotation of the bovine genome sequence; 2) enable the identification of variations in genes critically involved in host innate immunity; and 3) facilitate further studies of bovine Toll-like receptor signaling pathways in cattle. Based on comparative immunological studies in humans and mice, the findings of the current research should facilitate studies into the role of Toll-like receptors in regulating the host immune responses of cattle to infectious diseases.
Publications
- Connor, E.E., Cates, E.A., Williams, J.L., Bannerman, D.D. 2006. Cloning and radiation hybrid mapping of bovine toll-like receptor-4 (TLR-4) signaling molecules. Veterinary Immunology and Immunopathology. 112(3-4):302-308.
|
|