HRF Transgenic Mice Phenotype Figure 4. Transgenic Protein Expression for Mice in Protocol II. Panel A depicts the relative amount of HRF protein based on densitometric analysis of Western blots. Panel B is the relative amount of GFP protein based on Western blots. Relative protein levels are expressed as a function of the positive control of each protein. In all cases mice were sensitized with PBS or OVA, then all mice were challenged with OVA. Each group had between 6 and 9 mice (backcrosses 3–6). doi:10.1371/journal.pone.0011077.g004 sensitized with OVA, perhaps due to the robust levels induced by the sensitization and challenge. OVA specific IgE was significantly increased following OVA sensitization and challenge, but not changed by HRF expression in the transgene++ mice (data not shown). These data suggest that HRF exerts its effect independently of sensitization and suggest a role in the response phase. Furthermore, BAL IgE levels were significantly elevated in transgene++ mice compared to CC10 littermate controls in both OVA (p = 0.02) and PBS (p = 0.05) sensitized groups (Figure 9, Panel B). While OVA sensitization and challenge elevated the BAL IgE as expected, HRF over expression in the lung appears to have a greater effect on these levels. Of note, all mice received Figure 5. Immunoflourescent Staining for HRF in Mouse Lung. The bronchi of 3 mice fed Dox to induce over expression of HRF are shown. The genotype and sensitization reagent for each mouse is shown in the top left corner of each panel. Panels A, C and D are stained with rabbit anti-HRF antibody. Panel B illustrates the nonspecific staining with secondary antibody alone. doi:10.1371/journal.pone.0011077.g005 antigen to the lung and the combination of HRF and antigen gave a strong local effect on BAL IgE. These data are consistant with HRF being important in the response phase and support HRF’s role in the allergic inflammation in the lung, given that native HRF is seen in increased levels in the lung following OVA sensitization and challenge (Figure 4). Cytokine levels. Asthma is a chronic inflammatory disorder of the lung and TH2 inflammation of the airway is a major component of asthma. It has been demonstrated in animal models of asthma that allergens can elicit TH2 inflammation and that IL- 4 is essential in this response. For example, in the presence of IL-4, na¨ıve CD4+ T cells differentiate into TH2 cells [31]. In order to investigate the underlying molecular mechanism of the augmented pulmonary inflammation in the HRF-inducible transgenic mouse we investigated several cytokine and chemokine expression profiles in the lung. As shown in Figure 10, when mice were sensitized and challenged with OVA, transgene++ (++/OVA) mice had greater mRNA levels of IL-4 (p = 0.04) than CC10 littermate controls treated the same way (CC10/OVA). There were no differences observed in mRNA levels for eotaxin, IL-5 or IL-13 between these groups (Figure 10). Protein levels for IL-4 were also measured from BAL supernatants (Figure 10 Panel B). IL-4 protein levels were Figure 6. Expression of HRF Protein in BAL. Transgenic HRF mice (++) and littermate control (CC10) mice were treated with doxycycline water, sensitized and challenged with OVA, as previously described. BAL supernatants were collected and pooled with 9 mice per pool, the IgG was precleared by incubation with Protein A beads, and then the supernatants were concentrated with a Centricon to 50-fold above the original pool/volume. Concentrated samples were run a tris-glycine gel with a recombinant HRF positive control. Western blotting was completed using a monoclonal anti-HRF, as described for the whole lung lysates. doi:10.1371/journal.pone.0011077.g006 PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11077