Influenza virus causes a respiratory infection that leads to morbidity and mortality worldwide. The virus infects the respiratory epithelium and replicates rapidly to peak levels in two days. It is eventually cleared from the lungs by a strong cellular immune response. In humans and mice, influenza virus triggers a strong cellular response. Surprisingly influenza virus is a very poor stimulator of DC maturation in vitro because the viral NS1 protein inhibits the maturation process. However, if the DCs are pre-exposed to interferon, the cells can overcome the viral antagonism and respond strongly. To try to better understand the process by which immunity is generated in vivo, we began to explore the cellular movements that occurred in mice infected by aerosol. We were unable to detect any inflammatory response in the animals until almost 48 hours after infection when virus replication was reaching its apex. At this point, a sudden rapid generation of chemokines and cytokines that we refer to as the inflammatory burst occurs in the lungs. Cytokines and chemokines reach high levels in the serum. Cells in distal lymphoid compartments, particularly the bone marrow, begin to express an interferon signature characterized by the up-regulation of antiviral genes. Our data suggests that the bone marrow derived monocytes migrate to the draining lymph nodes and participate in both innate and adaptive immunity. We hypothesize that the preactivation allows the cells to respond strongly and overcome the antagonism of the viral NS1 protein leading to their subsequent migration to the draining lymph nodes where they stimulate T cell activation. In this application we will test the tenets of this hypothesis by studying the events that occur in the lungs during influenza virus infection. A kinetic analysis of chemokine/cytokine/growth factor release will be performed to determine the exact timing and sequence of events that occur during the inflammatory burst in influenza virus infected mice. The effect of inoculum size on the kinetics will be evaluated based upon evidence that suggests an altered kinetics and severity of response in such situations. The cell populations responsible for initiating the inflammatory burst will be identified by the collection of lungs at time points after infection and using a number of sensitive methods to isolate purified populations and probe for the production of inflammatory substances.
In Aim 2 we will study the preactivation of monocytes in the bone marrow. Using a PCR panel we will determine if they express an interferon signature and how this may affect their sensitivity to infection, response to virus challenge, and their ability to present antigen to T cells. Finally, a microarray analysis of the bone marrow compartment using mice competent or incompetent to respond to interferon signaling during influenza virus infection will be performed to more comprehensively map the activation profile of the bone marrow. This will allow us to observe both the interferon dependent and independent activation that occurs in the bone marrow during virus infection.
The work should provide a new understanding of the mechanisms by which the immune system responds to virus infection. We propose to try to speed up the initiation of immunity by a number of mechanisms. If this succeeds it could be a viable immunotherapeutic strategy.
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