Both human and mouse macrophage-derived cell lines were transfected with wild-type and different mutant LPS species and their capacity to induce cell death, IL-1β and TNF-α production and caspase-1 activity were measured. Additionally, the direct caspase-4 binding capability of wild-type and different mutant LPSs was investigated by an in vitro oligomerization assay. Wild-type meningococcal LPS provoked strong caspase-mediated inflammatory signaling in terms of cell death and cytokine production. Penta-acylated LPS mutants showed decreased caspase-mediated responses, similar to what was found for TLR4. Expression of PagL deacylase in LptA or LpxL1 mutants, leading to loss of the 3-OH-linked acyl chain, further decreased and increased LPS activity, respectively, in terms of IL-1β production and caspase-1 activation. Incubation in vitro of the different LPS species with purified caspase-4 resulted in aggregation to a variety of molecular sizes, with all LPS structures inducing at least dimerization of caspase-4. Wild-type and LptA LPS induced the largest aggregates, relating to their effect on IL-1β and TNF-α secretion. Overall, these results highlight that the mutant meningococcal LPSs mediate graded non-canonical inflammasome activation, with maximal activity for wildtype LPS and reduced or absent activity for penta- or tetra-acylated LPS species.
LPS is also an important component of outer membrane vesicle (OMV) - based vaccines. The overall TLR4 activation by purified mutant LPS is similar to LPS as presented in OMVs, at least in the sense that the order of activity from high to low activity forms is the same. However, important differences exist, as LPS generally becomes less active when incorporated in membranes like OMVs. In addition, OMVs can be taken up and processed differently compared to purified LPS, and they have been reported to be efficient intracellular and cytosolic delivery vehicles for LPS. OMVs containing the various mutant LPS forms have therefore also been used to investigate uptake into human and mouse cell lines, and their caspase activation properties were determined using the methods established for LPS.
Labeled OMVs were abundantly present in both mouse and human macrophage cell lines, showing that highly efficient uptake without transfection was indeed the case. No difference in uptake between OMVs containing wildtype or mutant LPS was observed, but caspase-mediated responses were decreased for the mutants. Further to our research on purified mutant meningococcal LPS, these results highlight that the OMVs containing mutant meningococcal LPS also mediate graded non-canonical inflammasome activation.
Two manuscripts have been written based on results of the project and will be submitted for publication.
