Role of antimicrobial peptides in tuberculosis and respiratory tract infections : clinical and mechanistic studies

Abstract: Antimicrobial peptides (AMPs) are effector molecules of the innate immune system in multicellular organisms. They are mainly expressed in epithelial cells and immune cells, providing the first line of defense against a wide range of pathogens. AMPs are able to kill pathogens and show additional important functions such as chemotaxis, angiogenesis, and wound healing. These peptides are constitutively expressed; however, their expression can also be induced or suppressed by different stimuli in a cell and tissue specific manner. The overall aim of the present thesis was to elucidate the role of AMPs in tuberculosis and respiratory tract infections by clinical and mechanistic studies. Vitamin D3 (vitD3) is known as a potent inducer of AMPs. Low levels of serum vitD3 are associated with an increased risk of respiratory tract infections (RTIs). One of our objectives was to elucidate whether supplementation with vitD3 could reduce infectious symptoms and antibiotic consumption in patients with antibody deficiency or frequent RTIs. Patients (n=140) were included with symptoms of respiratory tract infections for more than 42 days over a 12-month period. They were randomized and received either vitD3 (4000 IU) or placebo daily for one year. The primary endpoint was an infectious score based on five parameters: symptoms from the respiratory tract, sinuses, and ears, malaise, and antibiotic consumption. Secondary endpoints were serum 25-hydroxyvitamin D3 [25(OH)D3] levels, microbiological outcomes, and the levels of the antimicrobial peptides LL-37 and Human Neutrophil Peptides (HNP) 1-3 in nasal fluids. We observed that vitD3 supplementation reduced infectious score, antibiotic consumption, and increased serum 25(OH)D3 concentrations in the patients compared to placebo control group. However, no major changes were observed for LL-37 and HNP 1-3. To control the global spread of tuberculosis (TB) and multi-drug resistance TB, development of new anti-tuberculosis drugs and alternative treatment strategies are urgently required. PBA is a potent inducer of AMPs, and together with 1,25-dihydroxyvitamin D3, it synergistically induces the expression of LL-37 in lung epithelial cell line. Thus, we aimed to estimate a therapeutic dose of PBA alone, or in combination with vitD3 for the induction of LL-37 expression in immune cells, and enhanced antimycobacterial activity in monocyte-derived macrophages (MDMs). Healthy volunteers were enrolled in an 8-days open trial (n=15). The expression of the CAMP (cathelicidin antimicrobial peptide) gene encoding LL-37 was measured in immune cells both in mRNA and peptide levels. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed. From this trial, we demonstrated that 500 mg PBA twice daily with 5000 IU vitD3 once daily was the optimal dose for the induction of LL-37 in MDMs and lymphocytes, and the enhancement of intracellular killing of Mtb by MDMs. Using these findings, we further investigated if oral adjunctive therapy with 5000 IU vitD3 and/or 2x500 mg PBA along with standard anti-TB therapy would lead to an enhanced recovery in sputum smear-positive pulmonary TB patients. Adult TB patients (n=288) were enrolled in a randomized, double-blind, placebo-controlled trial. Primary endpoints were the proportion of patients with a negative sputum culture at week 4, and the reduction in clinical symptoms at week 8. Secondary endpoints included sputum smear conversion time, radiological findings, concentrations of 25(OH)D3 in plasma, expression of the antimicrobial peptide LL-37 in immune cells and intracellular killing of Mtb by MDMs. We found that the adjunct therapy with PBA and vitD3 treatment significantly reduced the sputum culture conversion time together with better clinical recovery in pulmonary tuberculosis patients. Additionally we observed that PBA and vitD3 treatment enhanced the expression of LL-37 in immune cells and increased intracellular killing of Mtb by MDMs. Next, we explored the potential mechanisms of PBA and LL-37-induced intracellular killing of Mtb in macrophages by autophagy. We observed that Mtb infection of MDMs downregulated the expression of LL-37 and certain autophagy-related genes (Beclin1 and ATG5) at both mRNA and protein levels. We also found that PBA and/or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were able to overcome the Mtb-induced suppression of LL-37 expression. In addition, autophagy process was activated by stimulation of MDMs with PBA and promoted co-localization of LL-37 and LC3-II (a marker of autophagy) in autophagosomes. When LL-37 expression was silenced, PBA treatment failed to induce autophagy in Mtb-infected THP-1 cells. On the other hand, when LL-37 knockdown cells were supplemented with synthetic LL-37, autophagy was restored. Additionally, we found that LL-37-induced autophagy was mediated via the P2RX7 receptor and intracellular free Ca2+, the AMPK and the PI3K pathways. These results suggest that PBA induces autophagy in LL-37-dependent manner and promotes intracellular killing of Mtb in human MDMs. In summary, vitD3 supplementation could beneficial for the patients with antibody deficiency or frequent RTIs. PBA and vitD3 supplementation improves the clinical outcomes through the increased expression of LL-37, indicating that this supplementation might be an alternative strategy to treat pulmonary TB patients. The enhanced expression of LL-37 activates the cellular host defense mechanism autophagy, and subsequent killing of Mtb in human macrophages.