CD14‐independent responses induced by a synthetic lipid A mimetic

Abstract

CRX-527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates, which are considered as potential vaccine adjuvants or standalone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane-bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX-527. First, we found that CRX-527 induces NF-jB and interferon regulatory factor-3 (IRF-3) activation in human embryonic kidney cells transfected with TLR4 and MD-2 genes alone, whereas the responses to LPS require either co-transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte-derived DC, which are devoid of mCD14 respond to CRX-527 but not to LPS in serum-free medium. Furthermore, we found that, in contrast to LPS, CRX-527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14-dependent responses are defective. Finally, we demonstrated that splenocytes from CD14-deficient mice produce cytokines in response to CRX-527 but not to LPS. We conclude that the lipid A mimetic CRX-527 does not require the CD14 co-receptor to elicit TLR4-mediated responses.Key words: CD14 . Lipid A . TLR4 Supporting Information available online IntroductionAminoalkyl glucosaminide phosphates lipid A mimetics are currently developed as synthetic Toll-like receptor 4 (TLR4) agonists with possible applications as vaccine adjuvants or standalone immunostimulants [1][2][3][4]. The hexa-acylate compound CRX-527, composed of three myristic and three decanoic acyl chains, has been described as one of the most powerful members of this aminoalkyl glucosaminide 4-phosphate family [5]. CD14, a GPI-anchored protein expressed at the surface of phagocytes, has been shown to be involved in the induction of TLR4-mediated responses by bacterial LPS [6,7]. Indeed, LPS was shown to bind the LPS binding protein facilitating its transfer to membranebound CD14 (mCD14), which allows LPS to engage TLR4 associated with MD-2 [6]. There is evidence that CD14 is especially important for TLR4 response mediated by the TRIF adaptor molecule, which culminates in activation of the transcription factor interferon regulatory factor-3 (IRF3) required for the production of type I interferon [8]. Cells which express TLR4 but not mCD14, such as endothelial cells and monocytederived DC (MoDC), can use the soluble form of CD14 (sCD14) to mount TLR4-mediated responses to LPS [9]. sCD14 was recently shown to discriminate slight structural differences between distinct TLR4 ligands [10]. The present study was therefore SHORT COMMUNICATION Results and discussionExpression of TLR4 and MD-2 is sufficient for the induction of NF-jB and IRF3 activities by CRX-527In preliminary experiments using transfected human embryonic kidney (HEK) cells expressing different TLR receptors, we confirmed that CRX-527 is a TLR4 agonist devoid of activity o...

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