Abstract
Synthetic forms of E. coli monophosphoryl lipid A (sMLA) weakly activate the MyD88 (myeloid differentiation primary response protein) branch of the bifurcated TLR4 (Toll-like receptor 4) signaling pathway, in contrast to diphosphoryl lipid A (sDLA), which is a strong activator of both branches of TLR4. sMLA’s weak MyD88 signaling activity is apparent downstream of TLR4/MyD88 signaling as we show that sMLA, unlike sDLA, is unable to efficiently recruit the TNF receptor-associated factor 6 (TRAF6) to the Interleukin-1 receptor-associated kinase 1 (IRAK1). This reduced recruitment of TRAF6 explains MLA’s lower MAPK (Mitogen Activated Protein Kinase) and NF-κB activity. As further tests of sMLA’s ability to activate TLR4/Myeloid differentiation factor 2 (MD-2), we used the antibody MTS510 as an indicator for TLR4/MD-2 heterotetramer formation. Staining patterns with this antibody indicated that sMLA does not effectively drive heterotetramerization of TLR4/MD-2 when compared to sDLA. However, a F126A mutant of MD-2, which allows lipid A binding but interferes with TLR4/MD-2 heterotetramerization, revealed that while sMLA is unable to efficiently form TLR4/MD-2 heterotetramers, it still needs heterotetramer formation for the full extent of signaling it is able to achieve. Monophosphoryl lipid A’s weak ability to form TLR4/MD-2 heterotetramers was not restricted to synthetic E. coli type because cells exposed to a biological preparation of S. minnesota monophosphoryl lipid A (MPLA) also showed reduced TLR4/MD-2 heterotetramer formation. The low potency with which sMLA and MPLA drive heterotetramerization of TLR4/MD-2 contributes to their weak MyD88 signaling activities.
Highlights
New vaccine development by pharmaceutical companies is focused on non-infectious subunit vaccines, but the increase in safety seen with these vaccines sacrifices the usefulness of naturally occurring adjuvant compounds such as bacterial cell wall components and nucleic acids normally present in attenuated or whole killed vaccine preparations [1,2]
To expand on the mechanism of weak MyD88 signaling from sMLA we sought to determine the level of recruitment of TNF receptor-associated factor 6 (TRAF6) to the MyD88 branch of the TLR4 signaling complex because TRAF6 is necessary for strong downstream signaling via MAP kinase (MAPK) and nuclear factor-kB (NF-kB) [32,33]
And D, sMLA drives weaker MAPK and NF-kB activation as demonstrated by lower phospho-activation of ERK1/2 and weaker degradation of the NF-kB inhibitor, IkBa. Since both TRIF and MyD88 can contribute to activation of MAPKs and NF-kB, it is unclear what level of sMLA’s residual activity is due to weak MyD88 signaling versus what is due to TRIF signaling
Summary
New vaccine development by pharmaceutical companies is focused on non-infectious subunit vaccines, but the increase in safety seen with these vaccines sacrifices the usefulness of naturally occurring adjuvant compounds such as bacterial cell wall components and nucleic acids normally present in attenuated or whole killed vaccine preparations [1,2]. The first adjuvant approved by the FDA since alum is a modified bacterial cell wall component, monophosporyl lipid A, MPL adjuvantTM. MPL adjuvantTM is made from the endotoxin lipopolysaccharide (LPS), removal of the 1 phosphate from the diphosphoryl active component of endotoxin, lipid A, renders it .2,000 fold less toxic in rabbits [3]. We have long been interested in discovering the molecular mechanism of MPLA’s low toxicity because understanding it will help with rational design of new classes of adjuvants such as generation mimetics of MPLA
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