Project Leader:

Dr. Philip Griebel

Background:
Immunity is generally considered to have two major arms: innate immunity and adaptive immunity. We are working to find ways of enabling the host to capitalize on its innate immune mechanisms to clear infections.

Adaptive immunity includes the humoral (antibody-based) and cellular (activated T-cell based) immune responses. It takes time for adaptive immunity to be triggered (at least 3-7 days), but this response is highly effective in dealing with specific pathogens. In contrast, innate immunity is either immediately available or rapidly activated, hinges on pattern recognition receptors (PRR) that recognize conserved microbial signature molecules, and is relatively non-specific.

The innate and adaptive immune systems are interconnected in two ways: (A) the innate immune system directs the development of adaptive immune responses; and (B) there are mechanisms for ensuring a transition to adaptive immunity, if innate immunity fails to control infections.

Innate immunity can be boosted to become more effective, but this can lead to potentially harmful inflammation, and in extreme cases, sepsis (a syndrome afflicting 700,000 North Americans annually with 120,000 deaths). We are proposing new approaches to infectious disease prevention based on the modulation of innate immune mechanisms. To do this effectively, we must understand the stages in innate immunity at which one can intervene. We have obtained, through our own and other researchers’ biological studies with cationic host defence peptides (HDP) and bacterial signature DNA (CpG), strong evidence that it is possible to boost innate immunity while not causing inflammation, or while actually suppressing inflammation.

We will use a variety of approaches to identify key genes that regulate innate immunity and inflammation at mucosal surfaces. The identification of individual genes which function as either key regulators of innate immunity or inflammation will provide targets which can be used to screen potential therapeutic compounds to either enhance or suppress innate immunity.

Objectives:
Long-term:
Characterization of the innate mucosal immune system to identify critical control points at which we can intervene to resolve infections without causing excessive inflammation. This will enable us to modulate disease pathogenesis and possibly enhance mucosal vaccine formulation and delivery.

Short-term:

• To conditionally knock down genes key to the regulation of innate immunity. The genes selected are based on research performed in the Functional Pathogenomics of Mucosal Immunity (FPMI) project, funded in 2002 and completed in 2006. We were successful in a subsequent Genome Canada competition and thus the FPMI findings formed the foundation for this project.
• To use microarrays, peptide chips, and functional assays to study the interaction between Salmonella and host cells and tissues in which innate immune genes are silenced.
• To build a bioinformatics database named Innate DB for the genes and biomolecular interactions involved in critical components of the innate immune response.
• To apply gene knock-down and over-expression techniques in cells isolated from humans and cattle to enable a comparative analysis of knowledge generated in the mouse model.
• To use proteomics techniques to characterize ligand-receptor interactions that play critical roles in host-pathogen interactions.
  

Progress:

Recent publications:

• Kindrachuk, J. Potter, J.E., Brownlie, R., Ficzycz, A.D., Griebel, P.J., Mookherjee, N., Mutwiri, G.W., Babiuk, L.A., Napper, S. 2007. Nucleic acids exert a sequence-independent cooperative effect on sequence-dependent activation of TLR9. J Biol. Chem. 282(19): 13944-13953.
• Wilson HL, Kaushik RS, Begg AA, Jalal, S., Conner, W., Mirakhur, K., Van den Hurk, J., Hokamp, K., Roche, F.M., Brinkman, F.S.L., Potter A, Babiuk, L.A. Griebel, P.J., and Aich, P. 2007. Comparative functional genomic analysis of rotavirus and coronavirus infection in neonatal calves. J. gen Virol 88 (Pt10), 2749-61.
• Aich, P., Jalal, S., Czuba, C., Schatte, G., Herzog, K., Olson, D., Ross, A., Potter, A., Babiuk, L.A., and Griebel, P.J. 2007. Comparative Approaches to the Investigation of Responses to Stress and Viral Infection in Cattle. OMICS A Journal of Integrative Biology 11(4), 413-34.
• Kaushik RS, Begg AA, Wilson HL, Aich P, Abrahamsen MS, Potter A, Babiuk LA, and Griebel PJ. 2008. Establishment of fetal bovine intestinal epithelial cell cultures susceptible to bovine rotavirus infection. J. Virol. Methods 148, 182-196.
  

During the last 10 years, there has been an explosion of knowledge in innate immunity and the regulation of inflammation, and its role in the development of protective immune responses following infection and vaccination. The Pathogenomics project has established the expertise at VIDO to analyze host-pathogen interactions at the level of gene expression. This has been an important advance since the limited availability of tools to analyze host responses in a variety of domestic species has been a major limitation in studying disease pathogenesis.

The analysis of host responses at the level of gene expression or transcription provides insight into the complex pathways by which host cells respond to infectious agents or regulate the responses to these agents to ensure that immune-mediated pathology does not result. During the last three years, the Pathogenomics project has identified over 50 genes which may play a pivotal role in the induction and regulation of innate immune responses. Understanding the role of these individual genes in the regulation of immune responses will be the focus of research during the coming years.

A more detailed analysis of each gene may also identify key regulatory genes that can be used as novel targets for rapid screening of immunotherapeutic compounds or vaccine adjuvants. Thus, the Pathogenomics project is expected to provide basic knowledge in a variety of domestic species, including cattle and pigs, which will facilitate a more rapid development of effective therapies for disease control.

VIDO has focused specifically on the generation of methodologies to target gene expression in cattle. A major objective within the last year was to develop gene silencing methods for bovine cells through the delivery of small RNA molecules (shRNA). A lentivirus vector was designed for the delivery of shRNA. This vector system is now being used to knock-down (KD) the expression of innate immune genes in bovine intestinal epithelial cell lines and intestinal macrophage cell lines. To date we have validated the KD of two genes thought to play a critical role in the response of epithelial cells and macrophages following infection. These cell lines are being used to characterize the role of these genes in cellular response to infection by both viruses (rotavirus, coronavirus, bovine herpesvirus-1) and bacteria (Salmonella).Going forward, we will focus on the analysis of mucosal immune responses and inflammation in a variety of gene knock-out mice following bacterial (Salmonella) and viral (rotavirus) infections.

Toll-like receptors (TLRs) are an important family of molecules which enable animals to detect the presence of pathogens and activate innate immune defences. Understanding how TLRs activate cellular responses will provide insight into the mechanisms by which innate and adaptive immune responses are activated and regulated. We developed and validated a unique peptide chip technology to analyze the regulation of cellular signaling by an important family of molecules known as protein kinases. These peptide chips were used during the last year to analyze the responses of bovine monocytes following stimulation with either LPS (TLR4 ligand) or CpG ODN (TLR9 ligand).

These analyses have confirmed that many of the cell signaling pathways associated with these TLRs in a variety of cells in other species are also conserved in bovine monocytes. Furthermore, these analyses identified a number of distinct differences in kinase activity when cells were stimulated with either LPS or CpG ODN. These observations provide significant insight into how bovine macrophages discriminate between various pathogens and tailor the immune response during infection.

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