Periodic Reporting for period 1 - skILL (Intracellular signalling by modified lipopolysaccharide)
Periodo di rendicontazione: 2019-04-25 al 2021-04-24
Neisseria meningitidis typically produces a very potent hexa-acylated lipopolysaccharide (LPS) which is too toxic for therapeutic applications. An important determinant of this endotoxic activity is recognition of the membrane-anchoring lipid A moiety of LPS by the TLR4/MD-2 receptor. We have previously developed an extensive set of meningococcal lipid A mutants which cover the whole range of TLR4 activation, from wildtype level to practically zero. However, determining only their TLR4 recognition properties will give an incomplete picture of biological activity, as recent studies have identified the non-canonical inflammasome pathway as an additional recognition system for LPS. However, there is very limited information on how structural alterations in LPS mutants affect activation of this newly identified intracellular caspase pathway. A major aim of the project has been to obtain this information. In vitro assays have been applied with cell lines into which LPS is introduced intracellularly. Methods have been introduced and used to specifically measure the non-canonical caspase pathway, in both mouse and human cells and with a large panel of LPS variants. Overall, the obtained results demonstrate 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. In comparison to the TLR4/MD-2 complex signalling pathway much less is known about the detailed structure-function relationships of modified LPS structures and their degree of caspase activation. Our results are among the first to provide this information, which is important for the practical application of LPS variants as adjuvants, vaccine components and immunomodulators.
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.
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.
In comparison to the TLR4/MD-2 complex signalling pathway much less is known about the detailed structure-function relationships of modified LPS structures and their degree of caspase activation. Our results are among the first to systematically analyze LPS mutants with regard to activation of this non-canonical inflammasome pathway. We conclude that combinatorial engineering of LPS can be exploited to generate a spectrum of bioactive variants of immunostimulatory molecules which can be used as vaccine adjuvants or for other therapeutic applications. It is anticipated that caspase recognition is also important for in vivo adjuvant and reactogenicity properties of mutant LPS, so the obtained information will be important for these applications. The LPS mutants are used by Intravacc as novel adjuvant candidates and as components of our outer membrane vesicle vaccine platform. Further understanding of the basis of their biological activity will contribute the further development and application of these vaccine platforms by Intravacc and its customers. See our website https://www.intravacc.nl