ScopeInvestigations 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 resultsOptimization 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.ConclusionThe 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.
Various
mechanisms for regulated cell death include the formation of oxidative mediators
such as lipid peroxides and nitric oxide (NO). In this respect, 15-lipoxygenase-1
(15-LOX-1) is a key enzyme that catalyzes the formation of lipid peroxides.
The actions of these peroxides are interconnected with nuclear factor-κB
signaling and NO production. Inhibition of 15-LOX-1 holds promise
to interfere with regulated cell death in inflammatory conditions.
In this study, a novel potent 15-LOX-1 inhibitor, 9c (i472), was developed and structure–activity relationships
were explored. In vitro, this inhibitor protected cells from lipopolysaccharide-induced
cell death, inhibiting NO formation and lipid peroxidation. Thus,
we provide a novel 15-LOX-1 inhibitor that inhibits cellular NO production
and lipid peroxidation, which set the stage for further exploration
of these mechanisms.
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