**4. Discussion**

In this study, we demonstrated the critical mediatory role of BLT2 in the production of G-CSF in steroid-resistant neutrophilic airway inflammation. The results showed that the blockade of BLT2 by antagonist treatment or genetic ablation suppressed the production of G-CSF, thus alleviating neutrophilic inflammation in the murine model. We also found that 12-LO, an enzyme that synthesizes 12(*S*)-HETE, which is a ligand for BLT2, was necessary for the production of G-CSF. Taken together, our results point to BLT2 as a potential therapeutic target in G-CSF-associated neutrophilic airway inflammation.

The contributory role of myeloid hematopoietic growth factors such as G-CSF has been reported in a variety of neutrophilic inflammatory diseases. G-CSF was shown to play roles in the pathogenesis of neutrophilic inflammatory diseases such as inflammatory arthritis, allergic encephalomyelitis, and cigarette smoke-induced chronic obstructive pulmonary disease (COPD) [33–36]. In patients with smoke-induced COPD, single nucleotide polymorphisms (SNPs) of G-CSF were suggested to protect against low lung function [37]. Recently, the contributory roles of G-CSF in inducing neutrophilic influx were demonstrated in severe asthma [10,38,39].

In addition to G-CSF, interleukin-17 (IL-17) and interleukin-1β (IL-1β) have been reported to be strongly related to severe neutrophilic asthma [40–44]. Th17 cells secrete inflammatory cytokines such as IL-17 to communicate with other cells in the immune system and were shown to be involved with the neutrophil influx into bronchial airways and asthma severity [45,46]. NLRP3 inflammasome-dependent IL-1β production also acts as a major chemoattractant of neutrophils and contributes to the development of neutrophilic airway inflammation [43,47]. We previously reported the mediating roles of BLT2 in regulating the production of IL-17 and NLRP3-dependent IL-1β in neutrophilic airway inflammation [29–31]. Thus, we were curious about the signaling network linking BLT2-mediated G-CSF production to IL-17 or IL-1β production. To test this, we examined whether G-CSF depletion affected the production of IL-17/IL-1β in neutrophilic airway inflammation. We observed the suppression of IL-17 levels in BALF and IL-1β in lung lysates by anti-G-CSF treatment (Figure S1A,B). These results suggest that G-CSF is necessary for the production of IL-17/IL-1β in the development of neutrophilic airway inflammation.

In addition to BLT2, BLT1 was also reported to play a role in mediating the recruitment of neutrophils in inflammatory responses [48]. Therefore, we investigated whether LTB4 and its receptor BLT1 played roles in the production of G-CSF in our experimental conditions. Quite interestingly, no reduction in the levels of G-CSF in BALF was detected by treatment with the BLT1 antagonist U75302 (Figure S2A). We also did not observe increases in the levels of LTB4 in BALF (data not shown). The reason why BLT1 did not mediate the production of G-CSF in the present study is not clear, but we suspect that it may be due to the different cell types targeted by LTB4-BLT1 and 12(*S*)-HETE-BLT2 signaling. BLT1 is highly expressed in leukocytes [16], whereas BLT2 is broadly expressed in other cell types, including airway epithelial cells [20]. Therefore, we suspect that BLT2 activation in airway epithelial cells mainly mediated the production of G-CSF at the early time point (6 h) following the LPS/OVA challenge in our murine experimental model. Then, G-CSF was, in turn, likely to trigger the production of IL-17 and IL-1β at the delayed time point by activating BLT1 and BLT2 on other cell types (e.g., macrophages) in the asthmatic airway microenvironment. Indeed, the level of G-CSF in BALF was reduced by the antagonist of BLT1 as well as BLT2 at the delayed time point (24 h) following the last challenge (Figure S2B). Further studies are necessary to elucidate the detailed mechanism of how BLT1 contributes to the synthesis of these cytokines in the development of severe neutrophilic asthma.
