Novel Cathelicidins From Pigeon Highlights Evolutionary Convergence In Avain Cathelicidins And Functions In Modulation Of Innate Immunity

Cathelicidins are a family of short cationic host defense peptides and play a central role as an essential component of innate immune system. Here we identified two novel cathelicidins, designated as Cl-CATH2 and 3, from a bursa of fabrieius cDNA library of Columba livia using RACE-PCR. Sequence alignments demonstrate that unlike the mammalian cathelicidins that are highly divergent, avian cathelicidins are remarkably conserved among different orders of birds. The evolutionary relationship of the avian cathelicidins was analyzed via the phylogenetic tree combined with Ka/Ks calculations between pairs of genes. The results supported the positive selection that prompted evolution of CATH2s to CATH1s and CATH3s with smaller size and better antimicrobial activity. Besides, CATH1 and 3 families are proposedly originated from a common ancestor through gene duplication. Cl-CATH2 and 3 both adopt amphipathic a-helical comformations, a structural prerequisite for disrupting the membrane integrity, as identified by circular dichroism (CD) and the 3D structures built by Rosetta ab initio. Since CATH2 family is the most abundantly expressed cathelicidin in bird, Cl-CATH2 was selected to study the functions in innate immunity modulation. Cl-CATH2 exhibited broad but weaker antimicrobial activities than CATH1s and CATH3s, and acted instead on the innate immune response. In mouse macrophages primed by lipopolysaccharide (LPS), Cl-CATH2 significantly down-regulated the gene and protein expressions of inducible nitric oxide synthase (INOS) and pro-inflammatory cytokines, such as tumor necrosis factor (TNF-a), interleukin-6 (IL-6), and IL-1β, whilst enhanced the levels of anti-inflammatory cytokine, IL-10, without showing undesirable toxicities. The mechanism whereby Cl-CATH2 balanced the innate immune response was investigated by molecular docking and western blotting. For the first time cathelicidin was demonstrated to bind exterior of the LPS-binding pocket on MD-2 of TLR4 (toll-like receptor 4)-MD-2 complex, which in turn interferes the dimerization of the TLR4-MD-2 complex caused by LPS and finally block the TLR4 pathway. Furthermore, Cl-CATH2 acted through several mitogen-activated protein kinase signaling pathways, and suppressed the activation of the downstream transcription factors like NF-κB. Collectively, Cl-CATH2 may defend host against infections by selective modulation of innate immunity in addition to direct killing of pathogens, and meanwhile can be exploited as novel natural antibiotics and immunoregulatory agent.