Project Leader:

Dr. Philip Griebel

Background:
Immunity is generally considered to have two major arms, innate immunity and adaptive immunity. Innate immunity is either immediately available or rapidly activated, works through non-rearranging receptors (e.g., Toll-like receptors - TLR), that recognize conserved microbial signature molecules, and is relatively non-specific.

In terms of disease control, current therapies based on antibiotics are under severe threat due to resistance and the lack of new antibiotic classes on the market. We are proposing a new approach, namely to boost immunity and encourage the host to utilize its innate immune mechanisms to clear an infection. Innate immunity can be boosted to become more effective, but this can lead to a double-edged sword with the coincident effect of potentially harmful inflammation, and in extreme cases septic shock and death. To boost immunity effectively, we must understand the stages in innate immunity at which one can intervene.

We have obtained, through our own and other researcher’s biological studies with cationic host defence peptides (HDP) and bacterial signature DNA (CpG), strong evidence that it is possible to boost innate immunity a) without causing, or b) in fact suppressing, inflammation. This knowledge creates a unique opportunity to enhance disease protection without the risk of immune pathology.

This program’s precursor (the Functional Pathogenomics of Mucosal Immunity – FPMI – program), was successful in starting to define genes involved in innate immune responses to a variety of pathogens and innate-immunity-stimulating compounds. This project is continuing to explore the basic innate immune mechanisms required to clear or control infection.

This exploration of innate immunity will involve the use of a gene knock-out mouse model to target a number of critical innate immune genes that we have identified. The role of these targeted genes in innate mucosal immunity will be investigated by performing detailed infection studies using Salmonella as a model enteric pathogen. These studies will be complimented by studying the modulation of gene and protein responses in isolated cell types and comparing this to siRNA (or dominant negative) knockdowns of the same genes in human and bovine cells to better understand the role of specific genes in regulating responses to infection and immunomodulatory stimuli.

We will also attempt to establish in vivo siRNA delivery methods or pharmacological methods to inhibit specific cell signalling pathways. These methodologies will be used to determine if key immune regulatory genes identified in the gene-knockout mice and in vitro cell studies also play a key role in either immune protection or the immune pathology which occurs during Salmonella infection in cattle.

Objectives:
To fully exploit this disease control opportunity, it will be critical to fully understand the regulation of innate immunity and inflammation. Our primary objective is to use a variety of functional genomic and proteomic tools to characterize the role of selected genes in the regulation of innate mucosal immune responses. The model system for this analysis of innate immunity during host-pathogen interactions will be Salmonella infection. This enteric pathogen occurs in a wide variety of mammalian species and infection is characterized by an acute inflammatory response. Thus, this infection model offers a unique opportunity to test the hypothesis that it is possible to identify key regulators of inflammation and utilize these as therapeutic targets to modify disease pathogenesis.

The specific aims of the project include:

1. To knock down at least 50 genes involved in innate immunity, using those novel genes identified in the FPMI project
2. To study the interaction between Salmonella and the mice, tissues and cells that have these genes knocked out. The impact of these knock-outs will be studied using microarrays, protein chips and functional assays.
3. To build a bioinformatics database of the genes and biomolecular interactions involved in innate immunity.
4. To utilize knock-down, over-expression techniques or pharmacological reagents in isolated cells from humans and cattle to permit comparative genomic extrapolation of these studies to other species. The role of specific genes in the innate immune response following Salmonella infection will also be explored in young calves. The development of in vivo siRNA techniques in cattle will be a component of these studies.

Progress:
Over the course of the Functional Pathogenomics of Mucosal Immunity (FPMI) program, we progressed from information-intensive functional genomics to providing a window into novel biology.

Recently, specific achievements have included completion of a comparative analysis of gene expression following enteric infection of neonatal calves with bovine rotavirus and coronavirus. This analysis has revealed a remarkable absence of antiviral effector responses, such as interferon (IFN)-α and IFN–γ, following infection with either virus. It has become increasingly apparent that viruses have evolved a variety of strategies to circumvent these potent anti-viral responses and experiments are ongoing to determine the relevance of our functional genomic data. The susceptibility of bovine rotavirus and coronavirus to IFN-induced antiviral effector molecules is now being evaluated to test the hypothesis that infection by these viruses requires effective strategies to circumvent the production of either IFN-α or IFN–γ.

Functional genomic analysis of toll-like receptor (TLR4) and CD14 expression indicated that a primary BHV-1 respiratory infection was linked to an increased susceptibility to a fatal secondary bacterial infection. We hypothesized that the IFN-γ production during BHV-1 infection was responsible for this apparent increase in host response to the lipopolysaccharides (LPS) present in Gram negative bacteria, such as Mannheimia haemolytica. We repeated the BHV-1 infection studies and performed a variety of functional studies with monocytes isolated from the blood of infected calves. These functional studies confirmed that IFN-γ but not IFN-α significantly enhanced the production of the potent pro-inflammatory cytokine, TNF, by bovine monocytes. Furthermore, we confirmed that during the course of BHV-1 infection, the monocytes acquired a significantly enhanced capacity to produce TNF in response to LPS stimulation. Thus, these functional studies provided further evidence to support the hypothesis generated from the functional genomic studies.

Our characterization of the role of TLR9 in the recognition of its synthetic ligand, CpG ODN, continued at both the cellular and protein level. Our analysis of CpG ODN responses was extended using a variety of MACS purified cell populations. These analyses revealed that effective stimulation of cells by CpG ODN requires a variety of co-signals. Thus, although the expression of TLR9 is required for CpG ODN-induced activation of innate immune responses, expression of this receptor is, in itself, not sufficient for cell activation. These observations have led us to hypothesize that the induction of innate immune responses will require multiple signals to ensure that both the amplitude and duration of responses can be regulated. Understanding the mechanisms which regulate innate immune responses will be critical if we are to develop synthetic ligands or molecules which can be used to effectively induce innate immunity for either immune protection or the development of vaccine adjuvants.

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