Cathelicidin LL-37 Restoring Glucocorticoid Function in Smoking and Lipopolysaccharide-Induced Airway Inflammation in Rats
Chronic obstructive pulmonary disease (COPD) remains a leading cause of global morbidity and mortality, characterized by persistent airway inflammation and progressive airflow limitation. Despite the widespread use of glucocorticoids as anti-inflammatory agents, their efficacy in COPD is limited due to corticosteroid resistance. This resistance has been linked to reduced histone deacetylase-2 (HDAC2) activity, a critical regulator of glucocorticoid receptor function, and activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. The antimicrobial peptide cathelicidin LL-37, known for its immunomodulatory properties, was investigated in this study for its potential to restore glucocorticoid sensitivity in a rat model of COPD.
Establishment of the COPD Rat Model
Male Wistar rats were exposed to cigarette smoke (1 cigarette/rat, twice daily for 28 days) and intratracheal lipopolysaccharide (LPS, 200 µL of 1 g/L) to induce COPD-like inflammation. Control rats were maintained under specific-pathogen-free conditions. The COPD model exhibited hallmark features of the disease, including elevated inflammatory cytokines and pathological lung changes. Lung tissue homogenate and serum levels of tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) were significantly higher in COPD rats compared to controls (lung TNF-α: 45.7 ± 6.1 vs. 20.1 ± 3.8 pg/mL, P < 0.01; serum TNF-α: 8.9 ± 1.2 vs. 6.7 ± 0.5 pg/mL, P = 0.01; lung TGF-β: 122.4 ± 20.8 vs. 81.9 ± 10.8 pg/mL, P < 0.01; serum TGF-β: 38.9 ± 8.5 vs. 20.6 ± 2.3 pg/mL, P < 0.01). Histopathological analysis revealed bronchial smooth muscle thickening, inflammatory cell infiltration, and emphysematous alveolar destruction in COPD rats, consistent with human COPD pathology.
Corticosteroid Resistance and HDAC2 Dysregulation
COPD rats demonstrated glucocorticoid insensitivity, as evidenced by the limited efficacy of inhaled budesonide (2 mg/20 mL per rat). While budesonide reduced TNF-α levels (lung: 30.2 ± 4.0 pg/mL; serum: 6.2 ± 1.2 pg/mL, P < 0.01 vs. COPD group), it failed to significantly lower TGF-β (lung: 114.0 ± 13.4 pg/mL; serum: 30.9 ± 8.4 pg/mL, P > 0.05 vs. COPD group). This resistance correlated with reduced HDAC2 expression and activity in lung tissue (expression: 13.1 ± 0.4 vs. 17.4 ± 1.1 mmol/mg in controls, P < 0.01; activity: 1.1 ± 0.1 vs. 1.4 ± 0.1 units, P < 0.01). Furthermore, phosphorylation of Akt (p-Akt), a marker of PI3K pathway activation, was elevated in COPD rats (0.5 ± 0.1 vs. 0.2 ± 0.1 fold of control, P = 0.04), suggesting a mechanistic link between PI3K/Akt signaling and HDAC2 impairment.
LL-37 Enhances Glucocorticoid Efficacy
Intratracheal administration of LL-37 (1.5 mg/kg) alone reduced inflammatory cytokines, but its combination with budesonide produced synergistic effects. In the Bud+LL-37 group, TNF-α and TGF-β levels in lung and serum were significantly lower than in the budesonide monotherapy group (lung TNF-α: 9.7 ± 2.9 vs. 30.2 ± 4.0 pg/mL; serum TNF-α: 5.4 ± 0.8 vs. 6.2 ± 1.2 pg/mL; lung TGF-β: 39.4 ± 11.8 vs. 114.0 ± 13.4 pg/mL; serum TGF-β: 13.9 ± 2.0 vs. 30.9 ± 8.4 pg/mL; P < 0.01 for all comparisons). Histopathological improvements included attenuated inflammatory infiltration and alveolar wall destruction, approaching near-normal lung architecture.
Mechanistic Insights: HDAC2 Restoration and PI3K/Akt Inhibition
LL-37’s ability to reverse glucocorticoid resistance was mediated through HDAC2 upregulation and PI3K/Akt suppression. In the Bud+LL-37 group, HDAC2 expression and activity increased significantly compared to budesonide alone (expression: 15.7 ± 0.4 vs. 14.1 ± 0.9 mmol/mg; activity: 1.3 ± 0.1 vs. 1.0 ± 0.1 units; P < 0.01). Concurrently, p-Akt levels decreased to 0.1 ± 0.0 fold of control, lower than both the COPD (0.5 ± 0.1) and budesonide monotherapy (0.3 ± 0.1) groups (P < 0.01). These findings suggest that LL-37 restores glucocorticoid sensitivity by augmenting HDAC2 function via PI3K/Akt pathway inhibition.
Discussion and Implications
The study highlights LL-37 as a potential adjunct therapy to overcome corticosteroid resistance in COPD. By targeting the PI3K/Akt-HDAC2 axis, LL-37 enhances the anti-inflammatory effects of glucocorticoids, addressing a critical limitation in current COPD management. The observed reduction in TGF-β levels with combination therapy is particularly notable, as TGF-β drives airway remodeling and fibrosis—key processes in COPD progression.
The dual action of LL-37—direct anti-inflammatory effects and glucocorticoid sensitization—positions it as a multifunctional therapeutic agent. Its ability to inhibit PI3K/Akt aligns with previous studies showing that PI3Kδ inhibitors improve HDAC2 activity and corticosteroid responsiveness. However, this study is the first to demonstrate that an endogenous antimicrobial peptide can achieve similar effects, offering a novel strategy to modulate immune responses in chronic inflammation.
Limitations and Future Directions
While the COPD model replicated major features of human disease, the short exposure period (6 weeks) may not fully capture chronic progression. Additionally, the study focused on HDAC2 and PI3K/Akt, but other pathways, such as JNK or NF-κB, could contribute to LL-37’s effects. Future research should explore LL-37’s efficacy in human COPD cohorts and investigate its interactions with other signaling pathways.
In conclusion, this study provides compelling evidence that cathelicidin LL-37 restores glucocorticoid function in COPD by enhancing HDAC2 activity through PI3K/Akt inhibition. These findings open new avenues for combination therapies to improve outcomes in corticosteroid-resistant inflammatory diseases.
doi.org/10.1097/CM9.0000000000000107
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