The Fab Fragment of a Human Anti-Siglec-9 Monoclonal Antibody Suppresses LPS-Induced Inflammatory Responses in Human Macrophages.

Sasa Chu,Yiwen Wang,Feng Zheng,Xuhui Zhu,Wei Zhang,Qiannan Sun,Shinan Nie,Changjun Wang,Maorong Wang,Xin Zhang,Binggang Cai,Tingting Zhou,Zhiguo Yang,Na You,Jin Zhu

doi:10.3389/fimmu.2016.00649

Abstract

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 investigate 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 pretreatment with hS9-Fab03 could attenuate lipopolysaccharide (LPS)-induced TNF-α, IL-6, IL-1β, IL-8, and IFN-β production in human PBMC-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 pro-inflammatory 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.

Highlights

Sepsis is a leading cause of mortality in intensive care units; recent statistics have indicated the occurrence about 19 million cases worldwide per year [1]
We found that hS9-Fab03 could obviously inhibit LPS-initiated production of pro-inflammatory cytokines, such as TNF-α, IL-6, and IFN-β, no effect on the expression of IL-1β, IL-8, and IL-10 in THP-1-differentiated macrophages (Figures 3 and 4)
The screening strategy was applied by the repeated panning with coated recombinant Siglec-9 protein in microtiter plates to assure the efficiency of specific Siglec-9 binding phage

Summary

Introduction

Sepsis is a leading cause of mortality in intensive care units; recent statistics have indicated the occurrence about 19 million cases worldwide per year [1]. Anti-hSiglec-9 Fab Inhibits TLR4 Responses and persistent critical illness [3]. Lethality caused by sepsis arises from a massive hyperinflammatory immune response to pathogens, such as Gram-negative organisms, Gram-positive organisms, and fungi [4]. The uncontrolled pro-inflammatory responses that lead to organ dysfunction in sepsis are primarily initiated by the toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns (PAMPs), such as bacterial lipoproteins, lipopolysaccharide (LPS), and non-methylated CpG DNA [5]. Our recent studies indicated that anti-TLR4 Fab fragment could reduce the inflammatory responses by inhibiting LPS-induced TLR4 signaling pathway in mouse primary macrophages and human THP-1-differentiated macrophages [8, 9]. It is intriguing that macrophages and inflammatory cytokines could be potential therapeutic targets in patients with sepsis

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