Pathogenesis of antimicrobial peptides LL-37 and CpG-ODN in ANCA associated vasculitis

Abstract

Objectives: Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) are a group of systemic autoimmune disorders characterized by necrotizing inflammation of small- to medium-sized blood vessels. The pathogenesis of patients with AAV are still in investigation. In this study, we explored the involvement of LL-37 and nucleic acids in AAV.

Methods: 15 patients with AAV diagnosed according to the Chapel Hill definition between October 2014 and July 2015 in the department of Nephrology of Huangdao, affiliated Hospital of Qingdao University were enrolled. 16 patients with chronic bronchitis (CB) were selected as disease control group. 15 cases of healthy people from Medical Healthy Center were as healthy control group. Peripheral blood mononuclear cells (PBMCs) were collected from these groups and stimulated by LL-37and (or) two types of CpG-ODN for 7 days. The IFN-α and ANCA in vitro were measured by ELISA. The serum IFN-α, LL-37 and ANCA were measured also.

Results: The serum level of IFN-α in AAV group was much higher than that in CB group (692.13 ± 407.28 vs 397.07 ± 211.62 pg/ml, p = 0.019), and that in healthy control group (692.13 ± 407.28 vs 251.54 ± 190.46 pg/ml, p < 0.001). The serum level of LL-37 in AAV group was much higher than that in CB group (101.18 ± 66.59 vs 40.23 ± 13.51 ng/ml, p < 0.001, and that in healthy control group (101.18 ± 66.59 vs 27.80 ± 16.86 ng/ml, p < 0.001). Also the level of IFN-α showed a significant positive relationship with ANCA in AAV group both in serum and in supernatant of cultured PBMCs stimulated by LL-37 and (or) CpG-ODN (r = 0.783, p = 0.001; r = 0.575, p = 0.064; r = 0.649, p = 0.031; r = 0.806, p = 0.003). In patients with AAV, the supernatant levels of IFN-α in cultured PBMCs stimulated by LL-37 and (or) CpG-ODN were higher than that without stimulating factor (p < 0.05). The supernatant level of IFN-α in cultured PBMC stimulated by LL-37 alone was lower than that stimulated by CpGA alone (699.57 ± 476.26 vs 2342.63 ± 2025.11 pg/ml, p = 0.001). But the supernatant level of IFN-α in cultured PBMCs stimulated by LL-37 alone was higher than in that stimulated by CpGB alone (699.57 ± 476.26 vs 153.35 ± 78.08 pg/ml, p < 0.001). The supernatant level of IFN-α in cultured PBMCs stimulated by both LL-37 and CpG-ODN was higher than that stimulated by LL-37 or CpG-ODN alone (2550.57 ± 2217.41 vs 699.57 ± 476.26 pg/ml, p = 0.003; 2550.57 ± 2217.41 vs 153.35 ± 78.08 pg/ml, p = 0.001; 2660.95 ± 391.31 vs 699.57 ± 476.26 pg/ml, p < 0.001; 2660.95 ± 391.31 vs 153.35 ± 78.08 pg/ml, p < 0.001). Either it is stimulated by LL-37 or CpG-ODN or both, the supernatant level of IFN-α in cultured PBMCs in AAV patients was the highest, that in healthy controls was the lowest. Either stimulated by LL-37 or CPG-ODN, or both, the levels of ANCA production in vitro in AAV groups were statistically significantly higher than those in CB group and healthy control group.

Conclusions: There were higher serum levels of LL-37 and IFN-α in patients with AAV. IFN-α could reach a higher level stimulated by LL-37 and nucleic acids both of which are related to infection. Patients with AAV have ANCA-producing B lymphocytes in the circulation even in remission stage. Infections could induce the relapse of AAV.

Keywords: ANCA associated vasculitis; CpG-ODN; Interferon-α; LL-37.