JNK–TLR9 signal pathway mediates allergic airway inflammation through suppressing melatonin biosynthesis

Abstract

Toll-like receptors (TLRs) play pivotal role in the pathogenesis of allergic airway diseases such as asthma. TLR9 is one of the most extensively studied TLRs as an approach to treat asthma. In this study, we investigated the role of TLR9 in the allergic airway inflammation and the underlying mechanism. Wild-type (WT) mice and TLR9(-/-) mice were sensitized and challenged with OVA to establish allergic airway disease model. We found that the expression of TLR9 was elevated concomitantly with airway inflammation post-OVA challenge, and TLR9 deficiency effectively inhibited airway inflammation, including serum OVA-specific immunoglobulin E (IgE), pulmonary inflammatory cell recruitment, mucus secretion, and bronchoalveolar lavage fluid (BALF) inflammatory cytokine production. Meanwhile, the protein expression of hydroxyindole-o-methyltransferase (HIOMT) in lung tissues, the level of melatonin in serum, and BALF were reduced in OVA-challenged WT mice, while these reductions were significantly restored by TLR9 deficiency. Additionally, we showed that although TLR9 deficiency had no effect on OVA-induced phosphorylation of JNK, inhibition of JNK by specific inhibitor SP600125 significantly decreased OVA-induced expression of TLR9, suggesting that JNK is the upstream signal molecular of TLR9. Furthermore, SP600125 treatment promoted resolution of allergic airway inflammation in OVA-challenged WT mice, but not further ameliorated allergic airway inflammation in OVA-challenged TLR9(-/-) mice. Similarly, SP600125 significantly restored the protein expression of HIOMT and the level of melatonin in OVA-challenged WT mice, while such effect was not further enhanced by TLR9 deficiency. Collectively, our results indicated that JNK-TLR9 signal pathway mediates allergic airway inflammation through suppressing melatonin biosynthesis.

TLR2-Melatonin Feedback Loop Regulates the Activation of NLRP3 Inflammasome in Murine Allergic Airway Inflammation

“…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).…”

“…Exogenous administration of melatonin has been reported to pronouncedly ameliorate airway inflammation (19,20). Similarly, endogenous melatonin, which is produced from tryptophan (Trp) by converting 5hydroxytryptamine (5-HT) successively by two key enzymes AANAT and ASMT, is also tightly associated with the pathogenesis of asthma (21,22). It has been reported that endogenous melatonin synthesis is suppressed by activation of TLR9 (22), while other study shows that melatonin is able to inhibit TLRs-mediated inflammation (23), suggesting there may be a feedback loop between TLRs system and endogenous melatonin synthesis.…”

“…Similarly, endogenous melatonin, which is produced from tryptophan (Trp) by converting 5hydroxytryptamine (5-HT) successively by two key enzymes AANAT and ASMT, is also tightly associated with the pathogenesis of asthma (21,22). It has been reported that endogenous melatonin synthesis is suppressed by activation of TLR9 (22), while other study shows that melatonin is able to inhibit TLRs-mediated inflammation (23), suggesting there may be a feedback loop between TLRs system and endogenous melatonin synthesis. More notably, it has been recently identified that a novel molecular target for melatonin is NLRP3 in murine model of septic response, liver injury and acute lung injury (24)(25)(26).…”

A critical review of melatonin assays: Past and present

“…This is particularly important when melatonin is measured in mice since the majority of laboratory mouse strains have pineal enzyme mutations which result in an inability to produce melatonin . There are nevertheless published reports of extremely high melatonin levels in Balb/c and other melatonin‐deficient strains using commercial kits (eg, Refs …”

“…Clearly to determine DLMO, an assay that produces basal levels less than 2 pg/mL will give the true result other melatonin-deficient strains using commercial kits (eg, Refs. [83][84][85]. It is particularly risky to use assays (especially unextracted assays) without appropriate validation as for example was found with domestic pigs 86 and melatonin proficient mice.…”