This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ***Please note Dr. Oliver Dienz joined our program as a junior faculty PI for Project 5 on 1/1/10.*** Influenza virus infections are a major cause for morbidity and mortality each year especially in the young and elderly. Due to the high mutation rate of influenza, vaccines need to be administered every year to achieve protection against the season's most prevalent strains. Emergences of new, highly pathogenic subtypes of influenza are of even greater concern as they can cause widespread pandemics with potentially high mortality rates. Victims of the H5N1 avian flu and the recent H1N1 pandemic displayed significant lung tissue damage with acute alveolitis, pulmonary edema and destruction of the respiratory epithelium. Studies in animal models of flu indicate that the cytopathic effects of the virus itself contribute less to the pathology than an excessive inflammatory host response. Especially expression of TNFa and TNF-related ligands (e. g. TRAIL) by macrophages and CD8 T cells are thought to play a major role in lung pathogenesis after influenza infection, while negative regulators of inflammation such as IL-10 and CD200 protect from excessive lung damage and lethality. Retinoic acid (RA) is an important factor in embryonic lung development and has been linked to alveolar regeneration in chronic obstructive pulmonary disease (COPD). All-trans retinoic acid (ATRA) and 9-cis RA are the best known physiological active isoforms of RA. ATRA activates the RAR family of transcription factors but 9-cis RA is able to bind additionally to members of the RXR transcription factor family. Members of both receptor families can form homo- and heterodimers with each other. The complex network of the different ligands and their receptors in promoting specific physiological responses is far from being understood. In addition to its effect in lung development and lung regeneration, RA plays a role in the immune reponse. Several recent studies make a strong argument for ATRA, but not 9-cis RA, in the induction of T regulatory (Treg) cells on the expense of Th17 cells. In contrast, both isoforms have been shown to reduce inflammatory cytokine expression such as TNFa production in macrophages. Given the recently gained knowledge about the molecular effects of RA we hypothesize that RA treatment reduces the inflammatory response after influenza infection and promotes lung tissue repair thereby ameliorating the disease. By infecting mice with mouse-adapted H1N1 (PR8) influenza virus, we will address this hypothesis in the following specific aims: Specific Aim 1: To determine the inflammatory response to influenza in the presence of RA. With our increased knowledge about the molecular mechanisms of the anti-inflammatory properties of RA, we will examine the production of pro-inflammatory and anti-inflammatory cytokines such as TNFa and IL-10 by a screen of bronchoalveolar lavage fluid and gene expression of lung tissue during administration of ATRA and 9-cis RA after influenza virus infection. The cellular sources of these cytokines will then be identified by intracellular cytokine stain to investigate the molecular mechanisms of RA in controlling cytokine gene expression in Specific Aim 2. Specific Aim 2: To examine the contribution of RAR and RXR family members in the inflammatory response after influenza infection. Activation of RAR and RXR transcription factors in immune cells and their role in the expression of specific inflammatory and regulatory cytokines will be analyzed. Selective agonists and antagonists as well as chromatin immunoprecipitation assays will be employed to investigate the role of the different receptor isoforms in cytokine gene expression after influenza infection. Specific Aim 3: To demonstrate the effect of RA treatment on lung tissue and lung function after influenza infection. As RA has been shown to induce alveolar regeneration in mouse models of COPD, we will administer ATRA and 9-cis RA to mice infected with influenza virus and assess lung tissue damage and respiratory functions. We will examine the effects of ATRA and 9-cis RA on apoptosis and proliferation of lung epithelial cells after influenza infection in vitro as well as in vivo. Significance: Given the long lead time for the development of a vaccine and the increasing resistance of certain influenza subtypes to antivirals, new strategies are needed to combat potential influenza pandemics. Controlling the inflammatory response and improving respiratory functions could be a new therapeutic strategy independent of the specific influenza strain. The proposed research builds upon our work with IL-6 in the immune response to influenza. If successful it will give important insights on how inflammation contributes to lung pathogenesis after influenza infection and ways to control it.
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