AUTOIMMUNITY

Sepsis is a major cause of death for hospitalized patients, and is characterized by massive overreaction of immune responses to invading pathogens which is mediated by cytokines. For decades, there has been no effective treatment for sepsis. Sialic acid-binding, Ig-like lectin-9 (Siglec-9), is an immunomodulatory receptor expressed primarily on hematopoietic cells which is involved in various aspects of inflammatory responses and is a potential target for treatment of sepsis. The aim of the present study was to develop a human anti-Siglec-9 Fab fragment, which was named hS9-Fab03, and investigated its immune activity in human macrophages. We began by constructing the hS9-Fab03 prokaryotic expression vector from human antibody library and phage display. Then we utilized a multitude of assays, including SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay to evaluate the binding affinity and specificity of hS9-Fab03. Results demonstrated that hS9-Fab03 specifically bind to Siglec-9 antigen with high affinity, and pre-treatment with hS9-Fab03 could attenuated LPS-induced TNF-α, IL-6, IL-1β, IL-8 and IFN-β production in human PBMCs derived macrophages, but slightly increased IL-10 production in an early time point. We also observed similar results in human THP-1 differentiated macrophages. Collectively, we prepared the hS9-Fab03 with efficient activity for blocking LPS-induced proinflammatory cytokines production in human macrophages. These results indicated that ligation of Siglec-9 with hS9-Fab03 might be a novel anti-inflammatory therapeutic strategy for sepsis.

Lactoferrin (Lf) is one of the antigens of anti-neutrophil cytoplasmic antibodies (ANCA) and functions as an endogenous suppressor of neutrophil extracellular trap (NET) formation. However, the prevalence and pathogenicity of anti-lactoferrin antibodies (aLf) in ANCA-associated vasculitis (AAV) remain unrevealed. This study aimed to examine the significance of aLf in AAV, initially. Sixty-five sera from AAV patients, including 41 microscopic polyangiitis (MPA), 5 granulomatosis with polyangiitis (GPA), and 19 eosinophilic granulomatosis with polyangiitis (EGPA) patients, were subjected to aLf detection using enzyme-linked immunosorbent assay. Clinical characteristics were compared between aLf-positive and aLf-negative patients. Neutrophils from healthy donors were exposed to suboptimal dose (10 nM) of phorbol myristate acetate (PMA) with aLf followed by evaluation of NET formation. Results demonstrated that four out of 65 AAV sera (6.2%) were positive for aLf. All of them were EGPA sera (4/19, 21.1%). In EGPA, the frequency of renal involvement, serum CRP levels, and Birmingham Vasculitis Activity Score (BVAS) in the aLf-positive patients were significantly higher than those in the aLf-negative patients, and the aLf titer correlated positively with the serum CRP level and BVAS. The NET formation was particularly enhanced by combined stimulation of 10 nM PMA and 1 µg/mL aLf. IgG isolated from sera of the aLf-positive EGPA patients (250 µg/mL) enhanced NET formation induced by 10 nM of PMA, and the effect was abolished completely by absorption of the aLf. This pilot study suggests that aLf enhance NET formation induced by PMA and are associated with disease activity of EGPA.