Porcine Cathelicidins Efficiently Complex and Deliver Nucleic Acids to Plasmacytoid Dendritic Cells and Can Thereby Mediate Bacteria-Induced IFN-α Responses

Abstract

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine–rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide–DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid–complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.

Importance of Endosomal Cathelicidin Degradation To Enhance DNA-Induced Chicken Macrophage Activation

“…This enhanced response against DNA-HDP complexes has also been reported for other mammalian cell types, such as pDCs, B cells, and monocytes (18,25,(29)(30)(31)), but appears to be limited to professional phagocytes. Keratinocyte responses toward DNA are even inhibited when DNA is presented in complex with LL-37 (32,33).…”

“…Keratinocyte responses toward DNA are even inhibited when DNA is presented in complex with LL-37 (32,33). Of the previously described responses, macrophage activation by DNA-CATH-2 complexes appears to be most similar to B cell activation by DNA-LL-37 complexes (30), which also respond by upregulation of proinflammatory cytokines, such as IL-6, whereas reported responses of monocytes and pDCs are limited to enhanced type I IFN production (18,25,31), which was unaffected in our study. Interestingly, although many cathelicidins were able to enhance DNAinduced activation in chicken macrophages, increasing DNA uptake might not be the only way by which they can enhance endosomal TLR activation (34,35).…”

“…In mammalian models, cellular activation in response to DNA-HDP complexes has so far been attributed to either activation of a TLR9-independent pathway, such as the activation of a cytosolic receptor in monocytes (31), or alternative downstream signaling of TLR9, leading to IRF7-phosphorylation and IFN-a production instead of NF-kB phosphorylation and proinflammatory cytokine production (18,25,28). In chickens, comparatively little is known yet about cellular responses toward DNA.…”

“…The three CATHs, CATH-1, -2, and -3, were all able to increase DNAinduced HD11 activation, as measured by means of NO production, with CATH-2 being the most potent. Other cathelicidins with known DNA-enhancing abilities, including human LL-37 (18), porcine PR-39 and PMAP-23 (25), and murine CRAMP (19), increased DNA-induced NO production as well. In addition, equine cathelicidin-1 and -2 also enhanced activation, whereas equine cathelicidin-3 and canine K9 did not significantly increase activation.…”

Interspecies cathelicidin comparison reveals divergence in antimicrobial activity, TLR modulation, chemokine induction and regulation of phagocytosis

“…Since, LL-37, CRAMP, chCATH-2, and PMAP-36 have been described to enhance DNA-induced activation22233637, the effect of all cathelicidins on DNA-induced TNFα release in RAW264.7 cells was analyzed (Fig. 3C).…”