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Abstract
Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.
Highlights
Maltose-binding protein (MBP) is a high-affinity maltose/maltodextrin-binding protein encoded by malE gene, and functions in capture and transportation of maltodextrins in Escherichia coli (E. coli) [1].When fused with a recombinant protein, MBP can increase the solubility of the fusion protein by a poorly understood mechanism, and is commonly used to improve the yield, facilitate the purification and enhance the stability of fusion proteins [2,3]
To explore the effect of MBP on production of nitric oxide (NO) in RAW264.7 macrophage cells, we examined the NO levels in the culture supernatants of cells stimulated with various concentrations of MBP (0.1–10 μg/mL) for 48 h and those with 5 μg/mL MBP for 12 to 72 h
The results showed that MBP significantly increased NO production in RAW264.7 cells in a dose and time-dependent manner (Figure 1A,B), suggesting that MBP induced activation and potentiates M1 polarity of RAW264.7 macrophage cells
Summary
Maltose-binding protein (MBP) is a high-affinity maltose/maltodextrin-binding protein encoded by malE gene, and functions in capture and transportation of maltodextrins in Escherichia coli (E. coli) [1]. The immunoadjuvant effect of MBP has been further demonstrated in our follow-up study of mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS), and the activity of MBP is likely to be mediated by toll-like receptor 2 (TLR2) and TLR4 signaling pathways [9]. These findings suggest that MBP itself possesses potent immune enhancing properties. Our previous studies have shown that MBP enhances the production of inflammatory mediators and nitric oxide (NO) in mouse peritoneal macrophages and in macrophage cell lines [8,9,10]. Molecules involved in the nuclear factor-κB (NF-κB) and p38 MAPK pathways were profiled in RAW264.7 cells with MBP stimulation
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