Recombinant human HSP60 produced in ClearColi™ BL21(DE3) does not activate the NFjB pathway Cynthia Planesse a,1 , Brice Nativel a,b,1 , Thomas Iwema b , Philippe Gasque b , Christine Robert-Da Silva a,2 , Wildriss Viranaïcken b,2,⇑ a GEICO, EA4516, Groupe d’Etude sur l’Inflammation Chronique et l’Obésité, Université de La Réunion et plateforme CYROI, 15, Avenue René Cassin, BP 7151, 97715 Saint Denis Messag. Cedex 9, Reunion b GRI, EA4517, Groupe de Recherche Immunopathologie et maladies Infectieuses, Université de La Réunion et plateforme CYROI, 15, Avenue René Cassin, BP 7151, 97715 Saint Denis Messag. Cedex 9, Reunion article info Article history: Received 7 November 2014 Received in revised form 14 January 2015 Accepted 22 January 2015 Keywords: HSP60 recombinant protein ClearColi BL21(DE3) Cytokine NFjB abstract HSP60, an intracellular molecular chaperone has been largely described as an alarmin or damage-associ- ated molecular pattern when released outside the cell. HSP60 has been reported as a possible ligand of TLR2 or TLR4 inducing NFjB-dependant signaling pathway leading to cytokine secretion. However, recent publications suggested that HSP60 could not act as an activator of TLR4 by itself. The observed effect could be due to the presence of endotoxin in HSP60 preparation especially LPS. In order to clarify the controversy, we produced recombinant human HSP60 in two different strains of Escherichia coli, stan- dard strain for protein overproduction, BL21(DE3), and the new ClearColi BL21(DE3) strain which lacks LPS-activity through TLR4. Undoubtedly, we have shown that recombinant HSP60 by itself was not able to induce NFjB-dependant signaling pathway in a model of THP1 monocyte cell line. Our data suggest that HSP60 needs either pathogen-associated molecules, specific post-translational modification and/or other host factors to activate immune cells via NFjB activation. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Heat shock proteins (HSPs) are a group of proteins highly con- served during evolution. HSPs are molecular chaperones which assist the refolding of non-native or misfolded proteins. Thus, HSPs help proteins to recover their complete function or provoke their proteolytic degradation [1,2]. Mitochondrial 60 kDa-heat shock proteins (HSP60) belong to the family of type I chaperonins. HSP60 plays a key role as a shuttle of newly translated, translocat- ed and stress-denatured or misfolded proteins [3]. HSP60 is able to stimulate innate and adaptive immunity [4,5]. During stress in prokaryotic or eukaryotic cells, it has been shown that the synthesis of HSP60 was up-regulated. This up-regulation occurred also under viral and bacterial infections, autoimmune or metabolic disorders [6–9]. Although HSP60 localizes primarily within mitochondria, its localization can also change when its level of expression is dramatically increased. These proteins can be exposed on the cell surface or released into the extracellular space [10–14]. Since 1993, HSP60 preparations from diverse sources (bacteria, mammalian and human tissues) were shown to activate the innate immune system. Indeed, extracellular HSP60 (exHSP60) has been considered as an alarmin or a Damage-Associated Molecular Pattern (DAMP) [15–19]. Interestingly, HSP60 cytokine effect was mediated by Toll-Like Receptor (TLR) 2 (TLR2) and TLR4 signal transduction pathway which triggers the activation of Nuclear Factor kappa B (NFjB) in different cells types [20–22]. CD14, which was required for the interaction between lipopolysac- charide (LPS) and TLR4, was shown to be necessary for exHsp60- mediated inflammatory signaling [23]. http://dx.doi.org/10.1016/j.cyto.2015.01.028 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved. Abbreviations: APC, antigen presenting cells; CD14, cluster of differentiation 14; DAMP, damage-associated molecular pattern; exHSP60, extracellular 60 kDa-heat shock protein; GST, glutathion-S-transferase; HSP, heat shock proteins; HSP60, 60 kDa-heat shock protein; hTNF-a, human tumor necrosis factor a; LPS, lipopolysaccharide; MD2, myeloid differentiation 2; NFjB, nuclear factor kappa B; PMA, phorbol 12-myristate 13-acetate; PMB, polymixin B; rhHSP60, recombinant human 60 kDa-heat shock protein; RPMI medium, Roswell Park Memorial Institute medium; SEAP, secreted alkaline phosphatase; TLR2, toll-like receptor 2; TLR4, toll-like receptor 4. ⇑ Corresponding author at: Laboratoire GRI, Université de La Réunion and plateforme CYROI, 15, Avenue René Cassin, BP 7151, 97715 Saint Denis Messag. Cedex 9, Reunion. Tel.: +262 262 93 88 29. E-mail address: wildriss.viranaicken@univ-reunion.fr (W. Viranaïcken). 1 These two authors contribute equally to this work. 2 Co-authors seniorship. Cytokine 73 (2015) 190–195 Contents lists available at ScienceDirect Cytokine journal homepage: www.journals.elsevier.com/cytokine