Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

Malgorzata Teodorowicz; Olaf Perdijk; Iris Verhoek; Coen Govers; Huub F J Savelkoul; Yongfu Tang; Harry Wichers; Kerensa Broersen

doi:10.1371/journal.pone.0173778

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

PLoS One. 2017 Mar 29;12(3):e0173778. doi: 10.1371/journal.pone.0173778. eCollection 2017.

Authors

Malgorzata Teodorowicz¹, Olaf Perdijk¹, Iris Verhoek², Coen Govers³, Huub F J Savelkoul¹, Yongfu Tang³, Harry Wichers³, Kerensa Broersen²

Affiliations

¹ Department of Cell Biology and Immunology, Wageningen University and Research, Wageningen, the Netherlands.
² Nanobiophysics Group, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands.
³ Food and Biobased Research, Wageningen University and Research, Wageningen, the Netherlands.

Abstract

Scope: Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for β-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays.

Methods and results: Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination.

Conclusion: The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

MeSH terms

Animals
Cattle
Cell Line
Detergents / chemistry*
Detergents / isolation & purification
Food Analysis
Gene Expression / drug effects
HEK293 Cells
Humans
Interleukin-1beta / genetics
Interleukin-1beta / immunology
Interleukin-6 / genetics
Interleukin-6 / immunology
Interleukin-8 / genetics
Interleukin-8 / immunology
Lactoglobulins / chemistry
Lactoglobulins / pharmacology*
Lipopolysaccharides / isolation & purification*
Lipopolysaccharides / pharmacology
Liquid-Liquid Extraction / methods*
Macrophage Activation / drug effects
Macrophages / cytology
Macrophages / drug effects*
Macrophages / immunology
Octoxynol
Polyethylene Glycols / chemistry*
Polyethylene Glycols / isolation & purification
Toll-Like Receptor 2 / genetics
Toll-Like Receptor 2 / immunology
Toll-Like Receptor 4 / genetics
Toll-Like Receptor 4 / immunology
Tumor Necrosis Factor-alpha / genetics
Tumor Necrosis Factor-alpha / immunology

Substances

Detergents
IL1B protein, human
IL6 protein, human
Interleukin-1beta
Interleukin-6
Interleukin-8
Lactoglobulins
Lipopolysaccharides
TLR2 protein, human
TLR4 protein, human
Toll-Like Receptor 2
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha
Polyethylene Glycols
Octoxynol
Nonidet P-40

Grants and funding

Malgorzata Teodorowicz was supported by European Seventh Framework Program FP7-PEOPLE-2011-IEF, grant number PIEF-GA-2011-302295. Presented work was a part of the FibeBiotics EU project supported by the European Community’s Seventh Framework Programme [FP7/2007-2013]’ and ‘Nutricia Research Foundation’ project number 2012-35. Kerensa Broersen is supported by a UTWIST fellowship.