Modulation of outer membrane vesicle-based immune responses by cathelicidins.

Antibiotic resistance is increasing and one strategy to prevent resistance development is the use of bacterial vaccines. For Gram-negative bacteria, natural outer membrane vesicles (OMVs) could be used for vaccine development. These vesicular structures are naturally produced by all Gram-negative bacteria and contain several antigens in their native environment. However, despite that the presence of lipopolysaccharide (LPS) may aid as intrinsic adjuvant, there is a risk that it may also cause undesired immune responses. Therefore, molecules to dampen LPS-induced toll-like receptor (TLR) 4 activation may be needed. Here host defense peptides (HDPs), like cathelicidins, can play an important role. They have been shown to interact with LPS and thereby neutralize LPS-induced TLR4 activation. However, there is currently no knowledge about neutralization in an OMV-based setting. Therefore, in this paper the immune modulating capacity of HDPs was investigated after macrophage stimulation with either spontaneous or heat-induced B. bronchiseptica OMVs. This revealed that the cathelicidins LL-37, CATH-2, PMAP-36 and K9CATH were able to modulate immune responses. Interestingly, immune modulation by these cathelicidins was different for spontaneous compared to heat-induced OMVs. Interaction studies revealed that the mode of binding of cathelicidins to OMVs slightly differed between OMV classes. Furthermore, TLR screening revealed that TLR2, 4, 5 and 9 were involved in stimulation of macrophages by OMVs, with TLR4-mediated activation being the most important pathway. Uptake of OMVs did not play a major role in macrophage activation. Taken together, this study shows how OMVs can activate macrophages and how cathelicidins may modulate these immune responses.

PMAP-36 reduces the innate immune response induced by Bordetella bronchiseptica-derived outer membrane vesicles.

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines.