*3.4. BLT2 Knockout Attenuates Both G-CSF Production and Neutrophilic Airway Inflammation*

To further investigate the role of BLT2 in neutrophilic airway inflammation, BLT2−/<sup>−</sup> mice were tested. As expected, we found that BLT2−/<sup>−</sup> mice showed the suppression of inflammation and mucus secretion in the airways (Figure 4A,B). Clearly, G-CSF, as well as MPO levels after LPS/OVA treatment, were also attenuated in BLT2−/<sup>−</sup> mice (Figure 4C,D). The numbers of total cells and neutrophils in BLT2−/<sup>−</sup> mice were reduced compared to those in WT mice (Figure 4E). IF staining showed decreased levels of G-CSF and MPO in the lung tissue of BLT2−/<sup>−</sup> mice after LPS/OVA treatment compared to the lung tissue of WT mice (Figure 4F,G). These results also suggest that BLT2 mediates the production of G-CSF and neutrophilic airway inflammation.

**Figure 4.** BLT2 knockout attenuates both G-CSF production and neutrophilic airway inflammation. BLT2−/<sup>−</sup> mice received the same schedule for sensitization and challenge (BLT2−/<sup>−</sup> + LPS/OVA).

Wild-type negative controls (WT + NC) and BLT2−/<sup>−</sup> negative controls (BLT2−/<sup>−</sup> + NC) were not treated. (**A**,**B**) H&E and PAS staining of mice lung tissues. Perivascular and peribronchial inflammation in addition to mucus secretion, was examined and scored (400×). Inflammation scores are shown as the mean ± SD (*n* = 5 per group). (**C**,**D**) G-CSF and MPO levels in BALF were analyzed by ELISA. (**E**) The number of immune cells in BALF was estimated using a CytoSpin and staining with Diff-Quik. The results are shown as the mean ± SD (*n* = 5 per group). (**F**,**G**) IF staining of mice lung tissues for BLT2 (green, Alexa Flour 488), G-CSF (red, Alexa Flour 647), and MPO (magenta, Alexa Flour 594). The nuclei were counterstained with DAPI (blue; 400×). The IF images are representatives of three independent trials with similar results. All experiments were performed in triplicate. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\*\* *p* < 0.0001, ns: not significant versus each control group.
