Results

TLR2 Is Required for OVA-Induced Murine Allergic Airway Inflammation
We first set out to confirm the role of TLR2 in murine allergic airway disease. WT and TLR2−/− mice were sensitized and challenged with OVA following the protocol showed in Figure 1A. Immunohistochemistry and western blot results showed that TLR2 protein expression was significantly increased in OVA-challenged WT mice in comparison with that of control mice (Figures 1B,C), and TLR2 was expressed on various types of cells, such as epithelial cells and leukocytes. Concomitantly, lung histology showed an increase in leukocyte recruitment to peribronchial and mucous cell metaplasia in OVA-challenged WT mice (Figures 1D–G). In sharp contrast, OVA-challenged TLR2−/− mice showed reductions in inflammatory cells recruitment (Figures 1D,F) and airway PAS+ cells (Figures 1E,G) in comparison with that of OVA-challenged WT mice.
Figure 1  TLR2 is required for OVA-induced murine allergic airway inflammation. (A) Protocol of establishing allergic airway inflammation, and comparison of resolution of WT and TLR2−/− mice. (B) The expression of TLR2 in lung tissue from vehicle and OVA-challenged mice was analyzed by immunohistochemistry. (C) The protein expression of TLR2 in OVA-challenged WT mice analyzed by western blot and quantification of the protein expression of TLR2. (D) Histological evaluation of the airway inflammation by staining lung sections with H&E, arrows indicates infiltrated leukocytes. (E) Histological examination of mucus production in the lung sections stained with PAS, arrow heads indicates goblet cells. (F) Quantitative analysis of airway inflammation. (G) Quantitative of mucus production. Scale bar: 50 μm. **p < 0.01, ***p < 0.001.

TLR2 Is Required for OVA-Induced Inflammatory Cells Infiltration, IgE, and Th2 Cytokines Production
In addition to lung histological changes, airway challenged with OVA induced a significant increase in total BALF cellularity in comparison with that of control mice (Figure 2A). Further morphologic assessments of differentially stained BALF samples revealed that the increase in cellularity was resulted from a significant influx of neutrophils, lymphocytes, monocytes and eosinophils (Figures 2B–E). However, in comparison with WT mice, the total number or composition of the BALF cellularity in TLR2−/− mice post OVA challenge was significantly decreased except for monocytes, which trended to increase but did not reach statistical significance (Figures 2A–E). Meanwhile, the level of OVA-specific IgE in TLR2−/− mice was significantly lower than that of WT mice (Figure 2F). Furthermore, significant increase in the levels of Th2-associated cytokines including IL-4 and IL-13 was observed in OVA-challenge WT mice (Figures 2E,F). Similarly, significant differences between WT and TLR2−/− mice were observed that TLR2 deficiency significantly decreased the levels of these two Th2-associated cytokines post OVA challenge (Figures 2G,H). Together, these data supported the role of TLR2 in the development of allergic airway inflammation in this OVA model.
Figure 2  TLR2 is required for OVA-induced inflammatory cells infiltration, IgE and Th2 cytokines production. (A) Total cell counts in the BALF of WT and TLR2−/− mice. (B–E) Differential cell counts in BALF of WT and TLR2−/− mice. (F) The level of OVA-specific IgE in serum. (G,H) Productions of IL-4 and IL-13 in BALF of WT and TLR2−/− mice were analyzed by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001.

OVA-Induced Activation of NLRP3 Inflammasome and Decrease of Melatonin Biosynthesis Are TLR2 Dependent
We next questioned how TLR2 regulated allergic airway inflammation. It has been shown that NLRP3 inflammasome is associated with allergic airway disease in response to OVA (10). We next assessed the link between TLR2 and NLRP3 inflammasome activity. Our results showed that NLRP3, cleaved form of IL-1β and caspase 1(p20) were increased in OVA-challenged WT mice in comparison with those of control mice, while such increase was completely abrogated in TLR2−/− mice following OVA challenge (Figures 3A–D). Similarly, productions of NLRP3-associated IL-1β and IL-18 were markedly decreased in OVA-challenged TLR2−/− mice, comparable to those of control mice (Figures 3E,F). Taken together, in this OVA model, NLRP3 inflammasome activated by OVA required licensing through TLR2, suggesting that TLR2-NLRP3 axis mediated OVA-allergic airway inflammation.
Figure 3  OVA-induced activation of NLRP3 inflammasome and decrease of melatonin biosynthesis are TLR2 dependent. (A) NLRP3, mature IL-1β, pro-IL-1β, and caspase1(p20) western blot analysis. (B–D) Relative ratio of NLRP3 to GAPDH, IL-1β to pro-IL-1β, and caspase1(p20) to GAPDH. (E,F) ELISA analysis of IL-1β and IL-18 in BALF. (G) Western blot analysis of AANAT and ASMT in lung tissues. (H,I) Relative ratio of AANAT or ASMT to GAPDH. (J,K) The levels of 5-HT in BALF and melatonin in the lung homogenate were analyzed by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001. Consequently, we sought to investigate the regulatory networks how TLR2-NLRP3 axis mediated allergic airway diseases. Previous study has shown that TLR9 negatively regulates melatonin production in response to OVA challenge, and this endogenous synthesized melatonin may regulate airway inflammation (22). Here, our present study showed that OVA notably suppressed the protein expression of ASMT but not AANAT in lung tissues (Figures 3G–I), and lowered the level of 5-HT in BALF and melatonin in lung homogenate in WT mice (Figures 3J,K), while these reductions were significantly restored by TLR2 deficiency (Figures 3H–K). These data confirmed that besides TLR9, TLR2, another member of TLRs family suppressed endogenous melatonin biosynthesis in OVA-induced allergic airway inflammation, therefore suggesting that TLR2-NLRP3 -mediated allergic airway inflammation was associated with decreased endogenous melatonin biosynthesis.

The Effect of Melatonin or Luzindole on OVA-Induced Airway Inflammation
Another important question arises whether endogenous melatonin increased by TLR2 deficiency directly suppresses NLRP3 inflammasome activity or feedback controls TLR2-NLRP3 signal. To address this question, melatonin or its receptor antagonist luzindole was applied as illustrated in Figure 4A. First, we found that administration of melatonin significantly attenuated the protein level of OVA-induced TLR2 in WT mice (Figures 4B,C). Meanwhile, melatonin-treated mice showed reductions in leukocyte recruitment and mucus productions compared with vehicle-treated WT mice post OVA challenge (Figures 4D–G). However, administration of luzindole only significantly promoted mucus productions in comparison with vehicle-treated TLR2−/− mice post OVA challenge (Figures 4D–G).
Figure 4  The effect of melatonin or luzindole on OVA-induced airway inflammation. (A) Protocol of administration of melatonin or luzindole during establishing allergic airway diseases model. (B,C) Protein expression of TLR2 in lung tissues of OVA-challenged WT mice treated with melatonin or not. (D) Lung histopathology based on H&E staining to evaluate inflammatory cell infiltration, arrows indicates infiltrated leukocytes. (E) Mucus secretion was analyzed based on PAS staining, arrow heads indicates goblet cells. (F) Histological scoring of lung inflammation. (G) Quantification of mucus secretion. *p < 0.05, **p < 0.01, ***p < 0.001.

The Effect of Exogenous Melatonin or Luzindole on OVA-Induced Inflammatory Cells Infiltration, IgE, and Th2 Cytokines Production
Moreover, upon OVA challenge, melatonin treatment dramatically reduced the total number or composition of the BALF cellularity except for monocytes (Figures 5A–E), lowered the concentration of IgE in serum (Figure 5F), and decreased the productions of IL-4 and IL-13 in BALF (Figures 5G,H) in comparison with those in vehicle-treated WT mice. Interestingly, no significant difference was found between luzindole-treated and vehicle-treated TLR2−/− mice in BALF cellularity or production of IgE and Th2-associated cytokines following OVA challenge (Figures 5A–C,E,F). We only found significant increase in monocytes infiltration in luzindole-treated TLR2−/− mice (Figure 5D). These results demonstrated that melatonin remarkably promoted resolution of allergic airway inflammation by down-regulating TLR2 signaling. However, blocking the effect of melatonin in TLR2−/− mice by luzindole only aggravated allergen-induced mucus hyper-secretion, but had null effect on leukocyte infiltration and Th2 cytokines production.
Figure 5  The effect of exogenous melatonin or luzindole on OVA-induced inflammatory cells infiltration, IgE and Th2 cytokines production. (A–E) Analysis of total and differential cells found in BALF of OVA-challenged WT or TLR2−/− mice after melatonin or luzindole treatment, respectively. (F) The level of OVA-specific IgE in serum. (G,H) The levels of IL-4 and IL-13 in BALF. **p < 0.01, ***p < 0.001.

The Effect of Exogenous Melatonin or Luzindole on NLRP3 Inflammasome Activation and Endogenous Melatonin Synthesis
Subsequently, we showed that administration of melatonin dramatically decreased the protein expressions of NLRP3, mature IL-1β and caspase 1(p20), lowered the productions of IL-1β and IL-18 in comparison with those in vehicle-treated WT mice following OVA challenge (Figures 6A–F). Importantly, NLRP3 inflammasome activity was not significantly different between vehicle-treated and luzindole-treated TLR2−/− mice post OVA challenge (Figures 6A–F). These data suggested that melatonin elicited its effect on NLRP3 inflammasome activity via TLR2 signaling, when TLR2 was deficient, blocking the effect of melatonin had null effect on NLRP3 inflammasome activity.
Figure 6  The effect of exogenous melatonin or luzindole on NLRP3 inflammasome activation and endogenous melatonin synthesis. (A) Representative blots showing the protein expression of NLRP3, mature IL-1β, pro-IL-1β, and caspase1(p20) in lung homogenate. (B–D) Relative band densities of NLRP3 and caspase1(p20) were analyzed by normalizing to GAPDH, relative band densities of IL-1β was analyzed by normalizing to pro-IL-1β. (E,F) The levels of IL-1β, IL-18 in BALF were detected by ELISA. (G) Representative blots showing the levels of AANAT and ASMT in lung homogenate. (H,I) Relative band densities of AANAT and ASMT were analyzed by normalizing to GAPDH. (J,K) The level of 5-HT in BALF and melatonin in the lung homogenate were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001. Finally, we found that treatment of melatonin resulted in dramatic increase in the protein expression of ASMT, marked elevation in the levels of 5-HT and melatonin, while did not affect the AANAT expression in comparing with vehicle-treated WT mice (Figures 6G–K). The protein expression of ASMT in lung tissues, the levels of 5-HT in BALF and melatonin in lung homogenate in melatonin-treated WT mice were comparable to that in vehicle-treated TLR2−/− mice post OVA challenge. However, TLR2−/− mice treated with luzindole showed notably reduced expressions of both AANAT and ASMT (Figures 6G–I), and lowered the level of melatonin in lung homogenate but not 5-HT in BALF in comparison with that of vehicle-treated TLR2−/− mice following OVA challenge (Figures 6J,K). Therefore, our study suggested that administration of melatonin further promoted endogenous melatonin biosynthesis, while treatment of luzindole significant inhibited endogenous melatonin synthesis in TLR2−/− mice.