Figure 7. Mouse Lung Histology after OVA Challenge. The mouse lungs were fixed, sectioned and stained with H and E. The genotype and sensitization reagent of each mouse is shown above the corresponding panel. doi:10.1371/journal.pone.0011077.g007 consistent with the mRNA levels and were significantly increased in both CC10 and transgene++ mice following OVA sensitization and challenge. Moreover, following OVA sensitization and challenge transgenic mice (++/OVA) had significantly more IL-4 protein than CC10 mice (CC10/OVA) (p = 0.05). The up regulation of IL-4 as evidenced by both mRNA and protein in the presence of HRF expression illustrates HRF’s role in stimulating other cytokines central to the disease process. Airway hyperreactivity (AHR). Airway physiology abnormalities are a hallmark of asthma in human and animal models of asthma. Methylcholine challenge was used to measure airway reactivity. We saw an increase in airway reactivity following sensitization and challenge with OVA, however, there were no significant differences between littermate controls, except at the top dose (50 mg/ml) between CC10/OVA vs. CC10/PBS sensitized (data not shown). The over expression of HRF either by Dox induced over expression, or by natural allergen induced up- regulation did not appear to significantly effect the airway reactivity to methylcholine compared to that seen with control CC10 mice. This result is not unexpected since it is known that AHR is IL-13 dependent [26] and IL-13 was not elevated in this transgenic mouse model (Figure 10). Furthermore, IL-4, which we do see increased in this model, does not induce AHR to methylcholine. [32]. Since we did not see any changes in AHR using Penh, we choose not to pursue invasive measurements of AHR. OVA Sensitization followed by OVA (or PBS) Challenge (Protocol III) To further explore the role of HRF in the allergic diathesis we designed additional experiments that compare effects of HRF on mice that are sensitized with OVA and then challenged with OVA (as previously done in Protocol II) or with sham challenge of PBS (n = 628 per group). The absence of OVA challenge would further dissect the role of HRF in the sensitization and response phases. The protocol and phenotypic analysis were otherwise the same as previously described. Transgene expression. Transgene expression was confirmed in each mouse as previously described and HRF protein was up regulated following Dox administration on HRF Transgenic Mice Phenotype transgene++ mice compared to CC10 littermate controls (p = 0.03). Of note again, is that HRF protein levels are increased following OVA sensitization and challenge compared to OVA sensitized PBS challenged litter mate controls in both transgene++ (p = 0.03) and CC10 mice (p = 0.02, data not shown). This indicates that OVA challenge increases naturally occurring HRF, not unlike the results in Protocol II (Figure 4, Panel A) where OVA sensitization increase HRF protein levels. GFP protein levels were not significantly different following OVA challenge (data not shown). This was previously shown in Protocol II and indicates that endogenous HRF, not promoter driven transgenic HRF, accounts for the increase in HRF protein. Phenotype Analysis. Overall, results seen in the Protocol III were similar to the data shown in the previous Protocol II between transgenic++ and CC10 control mice OVA sensitized and challenged. Namely, an increase in airway reactivity to methylcholine challenge following sensitization and challenge with OVA was seen, however, there were no significant difference between littermate controls between transgene++ OVA versus PBS challenge. Also, IL-4 levels and eosinophil numbers were elevated (data not shown). However, total cells and macropahges were not significantly increased as seen in both Protocol I, which are antigen na¨ıve mice, and Protocol II which were not sensitized with antigen. These data indicate that HRF does not affect macrophage recruitment during OVA sensitization, but HRF may initiate inflammation via macrophage recruitment in the absence of sensitization. IgE levels. As expected, serum IgE levels significantly increased following OVA sensitization and challenge in both the CC10 controls and the transgene++ group when compared to littermate controls challenged with PBS (Figure 11, Panel A). Transgene++ mice sham challenged with PBS (++/OVA-PBS) had greater serum IgE levels than CC10 controls (CC10/OVA- PBS)[p = .04]. Based on these data, as well as serum IgE levels from Protocol II (Figure 9), HRF can increase serum IgE levels in conjunction with either OVA sensitization or challenge. Interestingly, transgene++ mice sensitized and challenged with OVA (++/OVA-OVA) also had higher IgE levels than CC10 mice treated the same way (CC10/OVA-OVA)[p = .01]. Unlike the data in Protocol II (Figure 9), this difference could be due to increase in strain purity between the protocols i.e. increased strain purity revealed a previously unappreciated significance. Additionally, OVA challenge is necessary for HRF to exert an effect in the response phase as seen in the BAL levels (Figure 11, Panel B). Specifically, there was no increase in BAL IgE in the sham-sensitized transgene++ mice compared to the CC10 mice treated the same way. This is in contrast to the results in Protocol II, where sham-sensitized and OVA-challenged mice showed a significant increase in BAL/IgE in the presence of HRF (Figure 9, Panel B). BAL IgE levels between transgene++ and CC10 mice sensitized and challenged with OVA had the same trend as seen in Protocol II, but did not reach statistical significance (p = .07). Discussion In our transgenic HRF mouse model, we observed an induction of HRF in the lung and corresponding increased numbers of macrophages as well as levels of MCP-1 levels in the lungs. These data were shown in Protocol I (Figures 2 and 3). MCP-1 expression is associated with many inflammatory conditions and notably is elevated in the bronchial epithelium of asthmatics [28]. MCP-1 also is known to be involved with the initiation of inflammation and is a chemoattractant for monocytes and macrophages to areas of inflammation. Additionally, cultured PLoS ONE | www.plosone.org 8 June 2010 | Volume 5 | Issue 6 | e11077