LL-37 showed additive and synergistic inhibition with budesonide on lung and serum TGF-β levels (lung TNF-α: 15.9 ± 1.7 pg/mL in LL37 group, and 9.7 ± 2.9 pg/mL in Bud+LL37 group; serum TNF-α: 8.2 ± 2.8 pg/mL in LL37 group, and 5.4 ± 0.8 pg/mL in Bud+LL37 group; Lung TGF-β: 69.6 ± 10.0 pg/mL in LL37 group, and 39.4 ± 11.8 pg/mL in Bud+LL37 group; serum TGF-β: 22.3 ± 4.7 pg/mL in LL37 group, and 13.9 ± 2.0 pg/mL in Bud+LL37 group; Figure 1). Furthermore, the combination of LL-37 and budesonide could significantly improve the destruction of lung tissue induced by smoke and LPS [Figure 2].
COPD is a chronic inflammatory disorder, in which innate immune responses are not a relevant component. Cigarette smoking is the most commonly encountered risk factor for COPD. Animal models of cigarette smoke-induced COPD reliably reflect the inflammatory and pathogenic mechanisms of the disease. Using a guinea pig or murine, it usually takes 6-month exposure period to establish the COPD model. Combining induction agents could shorten the modeling period and induce severer stage. In present study, COPD model was established by exposing male Wistar rats to cigarette smoke combined with intratracheal instillation of LPS. Consequently, elevated inflammatory cytokines levels and destruction of lung structure were observed confirming the presence of COPD-like inflammation in the exposed animals. However, COPD is a complex disease and no such animal model has completely replicated the inflammatory response of COPD to date. Our models, with the shortcomings of short modeling time, could only partially reflect the characteristics of COPD.
LL-37 is the only peptide of the cathelicidin family found in the human body, that acts as an effector molecule of the innate immune system. Apart from broad antibacterial effects, it also serves as a potent immunoregulator having a delicate role in inflammatory/anti-inflammatory balance in infectious and inflammatory diseases. Significantly higher levels of LL-37 have been observed in sputum, airway epithelium and BALF samples in COPD patients compared to healthy individuals.[32–36] In addition, LL-37 expression in airway epithelium has shown to be positively correlated with airway wall thickness and collagen deposition. Moreover, high sputum hCAP18/LL-37 levels were associated with increased risk of exacerbation, non-typeable Haemophilus influenzae colonization, higher age and higher levels of inflammatory markers. Overall, these studies have suggested that LL-37 was involved in the pathogenesis of COPD, which was somewhat contradictory with our results. Yet, compared to healthy individuals, significantly higher LL-37 levels were found in bronchoalveolar lavage fluid (BALF) in patients with early stages of COPD (GOLD I–II), and significantly lower LL-37 levels in patients with advanced COPD (GOLD III-IV). Although there were also some contradictory reports,[33,38] these data suggested that the role of LL-37 and its regulation in COPD was a complicated process, especially in patients with the advanced stage of COPD.
Moreover, protein structures are closely related to their functions. An increased presence of peptidylarginine deiminase (PADIs) and citrullinated proteins have been found in the lungs of smokers and COPD patients, which led to post-translational modification of proteins like LL-37 by converting cationic peptidylarginine residues to neutral peptidylcitrulline.[39,40] Previous study has reported that citrullinated LL-37 was less efficient at neutralizing LPS, and more prone to degradation by proteases. Cell death events were crucial for balancing inflammatory reactions. LL-37 facilitated clearance of apoptotic neutrophils (ie, non-functional cells with intact membranes) from the system by surrounding macrophages in an immunologically silent manner; this process in turn resulted in a massive secretion of anti-inflammatory mediators.[41,42] The secondary necrosis of apoptotic neutrophils represents was an important immunomodulatory function of LL-37, that was abolished by citrullination, which could potentially explain the contradictory reports concerning the role of LL-37 in COPD. Immunomodulatory function of LL-37 relies on normal structure, which is altered in COPD and smokers. Our study proved that exogenous treatment of naïve LL-37 could regulate the abnormal inflammation, thus supporting this point of view.
Glucocorticoid resistance is a main barrier of ICS implication in COPD. A body of evidence has verified the important role of HDAC2 in the induction of steroid resistance. The levels of HDAC2 were decreased in lung parenchyma, bronchial biopsies and alveolar macrophages in patients with COPD and in smokers, as well as in macrophages and lungs of mice exposed to cigarette smoke. They were also correlated with disease severity and exacerbation. In patients with very severe COPD, the expression of HDAC-2 was less than 5% of the expression observed in normal lung. In our study, HDAC2 expression was significantly decreased in COPD model rats, along with unsatisfactory response to ICS, which was consistent with previous reports.[27,46]
In conclusion, this study indicated that LL-37 improved the anti-inflammatory activity of budesonide in cigarette smoke and LPS-induced COPD model rats by enhancing the expression and activity of HDAC2. The mechanism of this function of LL-37 may involve the inhibition of PI3K/Akt pathway.
This study was supported by a grant from National Natural Science Foundation of China (No. 81300033).
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